PACCON18 ProceedingBK Fullpaper

Proceedings
The Pure and Applied Chemistry International

Conference 2018 (PACCON 2018)
“Chemistry toward a Sustainable Future”

February 7-9, 2018
at
The 60th Anniversary of His Majesty the King’s Accession
to the Throne International Convention Center,
Hat Yai, Songkhla, Thailand
Organized by
The Chemical Society of Thailand under the
Patronage of Professor Dr. HRH Princess Chulabhorn
In association with
Department of Chemistry, Faculty of Science
Prince of Songkla University
Proceedings of the Pure and Applied Chemistry
International Conference 2018 (PACCON 2018)
Editor-in-Chief Vatcharin Rukachaisirikul
ISBN (E-book) 978-616-271-465-8
Prepared by Department of Chemistry, Faculty of Science

Prince of Songkla University
1st Edition May 2018 (available in PDF file only)
© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) (2)
Editorial Information:
Editor-in-Chief Vatcharin Rukachaisirikul

Assistant Editors Chittreeya Tansakul
Dhassida Sooksawat
Panwadee Wattanasin
Sirinya Chantarak
Uraiwan Sirimahachai
Editorial Board see PACCON2018 Scientific Committee
Editorial Staff Apon Numnuam
Chongdee Buranachai
Juthanat Kaeobamrung
Kanda Panthong
Khamphe Phoungthong
Kwanruthai Tadpetch
Laemthong Chuenchom
Nararak Leesakul
Neeranuch Phusunti
Opas Bunkoed
Panote Thavarungkul
Pongsaton Amornpitoksuk
Proespichaya Kanatharana
Saowanit Saithong
Sumetha Suwanboon
Surajit Tekasakul
Teerapong Pirojsirikul
Thitima Rujiralai
Watchanida Chinpa
Yaowapa Sukpondma
Scientific Sessions
AN Analytical Chemistry EE Environmental

Renewable Energy
Chemistry and
Chair: Chair:
Assoc. Prof. Dr. Proespichaya Kanatharana Prof. Dr. Navadol Laosiripojana
Co-Chair: Co-Chair:
Assoc. Prof. Dr. Panote Thavarungkul Asst. Prof. Dr. Surajit Tekasakul
Asst. Prof. Dr. Chongdee Buranachai Dr. Neeranuch Phusunti
Asst. Prof. Dr. Apon Numnuam Dr. Khamphe Phoungthong
FA Food and Agricultural Chemistry IN Inorganic Chemistry
Chair: Chair:
Prof. Dr. Soottawat Benjakul Prof. Dr. Thawatchai Tuntulani
Co-Chair: Co-Chair:
Prof. Dr. Sompong Techato Asst. Prof. Dr. Nararak Leesakul
Asst. Prof. Dr. Opas Bunkoed Dr. Dhassida Sooksawat
Dr. Thitima Rujiralai
MN Material Chemistry and

Nanotechnology NP Natural Products, Biological Chemistry
and Chemical Biology
Chair: Chair:
Prof. Dr. Santi Maensiri Prof. Dr. Vatcharin Rukachaisirikul
Co-Chair: Co-Chair:
Assoc. Prof. Dr. Sumetha Suwanboon Assoc. Prof. Dr. Kanda Panthong
Assoc. Prof. Dr. Pongsaton Amornpitoksuk Asst. Prof. Dr. Yaowapa Sukpondma
Dr. Laemthong Chuenchom
OR Organic and Medicinal Chemistry PH Physical and Theoretical Chemistry

Chair: Chair:
Prof. Dr. Tirayut Vilaivan Assoc. Prof. Dr. Vudhichai Parasuk
Co-Chair: Co-Chair:
Asst. Prof. Dr. Kwanruthai Tadpetch Assoc. Prof. Dr. Yuthana Tantirungrotechai
Asst. Prof. Dr. Juthanat Kaeobamrung Asst. Prof. Dr. Saowanit Saithong
Dr. Teerapong Pirojsirikul
PO Polymer Chemistry
Chair:
Prof. Dr. Suwabun Chirachanchai
Co-Chair:
Asst. Prof. Dr. Watchanida Chinpa
Dr. Sirinya Chantarak
Contents
AN: Analytical Chemistry
Glucose biosensors based on nickel ferrite composite materials modified glassy AN1-5
carbon electrode
Chantarattana Nontapha, Chochanon Moonla, Anchana Preechaworapun and
Tanin Tangkuaram
Extraction efficiency of lead(II) from seaweed Gracilaria fisheri with food additive AN6-10
extractants
Charuwan Khamkaew and Sontaya Manaboot
Detection of polycyclic aromatic hydrocarbons by specific thiol-functionalized silver AN11-16
nanoparticle films using surface-enhanced raman scattering
Sathita Taksadej, Kanet Wongravee, Sanong Ekgasit and Prompong Pienpinijtham
The synthesis of Metal/PDDA/rGO catalyst for detection of nitrite AN17-21
Aunyarut Paisanpisuttisin, Chaiya Prasittichai and Wilai Siriwatcharapiboon
Global calibration model for determination of glucose in non-alcoholic beverages AN22-27
using near infrared spectroscopy combined with chemometrics
Sureerat Makmuang, Nontawat Sricharoen, Prompong Pienpinijtham, Sanong Ekgasit
and Kanet Wongravee
Extraction of phenol using poly(styrene-co-divinylbenzene) monolithic bead AN28-32
Yaowalak Wimanthong and Orapin Chienthavon
Determination of mercury (II) by using 3-dimentional microfluidic paper-based AN33-37
analytical device
Hikmanita Lisan Nashukha, Jirayu Sitanurak, Hermin Sulistryarti, Duangjai Nacapricha
and Kanchana Uraisin
Highly sensitive electrochemical capsaicin sensor based on polyaniline-derived AN38-43
N-doped mesoporous carbon
Pathaporn Temcheon, Wilai Siriwatcharapiboon, Orapin Chienthavorn and
Panitat Hasin
Facile immunoassay for colorimetric detection of parathyroid hormone based on AN44-47
aggregation of gold nanoparticles
Aurachat Lert-itthiporn, Tipachai Vatanavicharn and Nathawut Choengchan
Label-free gold nanoparticles for spectrophotometric determination of creatinine in AN48-53
urine
Arjnarong Mathaweesansurn, Kanisa Tipanantakun, Parinya Thaothong,
Pimmada Ooybumrung and Nathawut Choengchan
Paper-based ammonia gas sensor using zinc complex of Eriochrome Black T and AN54-57
digital image analysis
Supa Sangdang, Pongsak Lowmunkhong and Pakawadee Sutthivaiyakit
A ‘contact stamping’ microfluidic paper-based analytical device for determination AN58-62
of creatinine using camera phone and standard addition approach
Suthathip Thongrod, Arjnarong Mathaweesansurn and Nathawut Cheongchan
A Low cost device for colorimetric determination of salicylic acid AN63-68
Anukool Khammona and Veeramol Vailikhit
Fiber optical sensor based on surface plasmon resonance spectroscopy for AN69-73
determination of atrazine
Chidchanok Tabtimhin and Pakorn Varanusupakul
Perchlorate analysis of vegetables grown in Chiang Rai, Thailand AN74-78
Sodkaneung Dachpanya, Phuangpetch Saysanguan, Prachak Inkaew, Siwaporn Praman
and Thaneeya Hawiset
Development of microfluidic device for determination of total cholesterol in blood AN79-82
Suphakorn Katib, Chalermpong Saenjum, Plaipol Dedvisitsakul and
Kanchana Watla-iad
An effective enrichment method using catanionic surfactant couple to high perfor- AN83-88
mance liquid chromatographic determination of polycyclic aromatic hydrocarbons
Sophon Hem, Suthasinee Boonchiangma and Supalax Srijaranai
Extraction and preconcentration method for trace Pb determination by UV-Visible AN89-93
spectrophotometry
Apinya Navakhun
Aqueous copper(II) measurement by smart phone photometry AN94-98
Tharaphan Youkhong, Rungwalee Suklim and Veeramol Vailikhit
Modified silica gel for practical preparative chromatographic resolution of AN99-103
D, L-aspartic acid derivatives
Varaporn Paradamit, Chanakan Maungrong, Kittisak Thammapichai,
Kantamanee Pantang and Punlop Kuntiyong
Analysis of volatile components in Arabica coffee roasted at different temperatures AN104-109
by using solid phase microextraction-gas chromatography-mass spectrometry
Suchawadee Insawang and Patcharee Pripdeevech
Non-enzymatic electrochemical detection of glucose based on copper foam modified AN110-115
on copper wire
Suntisak Khumngern, Ratchaneekorn Jirakunakorn, Ira Musarafa, Jittima Choosang,
Panote Thavarungkul, Proespichaya Kanatharana and Apon Numnuam
A polyaniline/pencil lead solid phase microextractor for trace phthalate esters AN116-120
Nichapat Chunin, Morakot Kaewpet, Panwadee Wattanasin, Proespichaya Kanatharana,
Panote Thavarungkul and Chongdee Thammakhet-Buranachai
Synthesis of carbon nanotubes as a sorbent for solid-phase extraction of AN121-125
formaldehyde in water sample
Patthama Yenanong and Netnapit Kaewchuay
Hydrophilic interaction liquid chromatography (HILIC) with diode array detection AN126-130
for determination of phenylalanine and tyrosine in dietary supplements
Woraphot Wanichalanant and Warawut Tiyapongpattana
A polyaniline-chitosan bead sorbent for parabens extraction AN131-135
Titiwan Changsan, Proespichaya Kanatharana, Panote Thavarungkul and
Chongdee Thammakhet-Buranachai
Colorimetric sensor for hydrogen sulfide AN136-139
Supitcha Thammajinno, Chongdee Thammakhet-Buranachai, Proespichaya Kanatharana
and Panote Thavarungkul
Development of latent fingerprints with Lawsone/PVP on thermal paper AN140-144
Benjawan Timdee and Gedsirin Eksinitkun
Novel charged iridium(III) complexes for colorimetric butylamine sensor AN145-148
Panida Seetawan, Witsanu Sombat, Wimonsiri Amornchai and Kittiya Wongkhan
Construction of sensor network on pontoon for monitoring of water quality in AN149-154
coastal area of Samut Sakorn Province
Paithoon Prasertying, Khongpan Rungprateepthaworn, Sommai Chokrung,
Prachumpong Dangsakul, Thanika Duangtanoo, Kamol Kaemarungsri,
Rachaporn Kienprasit, Phoonthawee Saetear, Prawpan Inpota, Thitirat Mantim,
Warawut Tiyapongpattana, Nathawut Choengchan, Kanchana Uraisin,
Nuanlaor Ratanawimarnwong and Duangjai Nacapricha
Downscaling ion-association solvent extraction for tetracycline determination with AN155-159
RGB color detection system
Saiphon Chanpaka, Orapan Intharaksa, Nopbhasinthu Patdhanagul, Thitikul Boonsri,
Khotchaporn Chankaew and Kate Grudpan
Solid phase extraction of uranium and thorium from various multi-element standard AN160-163
solution and geological sample
Kalaya Changkrueng, Saowaluck Thong-in and Harinate Mungpayaban
Development of an online flow analysis system for determination of phosphate AN164-169
Dechen Pelden, Duangjai Nacapricha, Shoji Motomizu and Kanchana Uraisin
Simultaneous determination of p-coumaric acid and naringenin in honey using AN170-174
dispersive liquid-liquid microextraction and high performance liquid chromatography
Korbkarn Khukitirat, Pongsak Lowmunkhong, Somyote Sutthivaiyakit and
Pakawadee Sutthivaiyakit
EE: Environmental Chemistry and Renewable Energy
Optimization of oleaginous yeast production by central composite design EE1-6
Supatsara Rujanant, Sarun Keansaior and Sasithorn Kongruang
Effect of the Pd-Mg/SiO2 on catalytic behavior for partial hydrogenation of soybean EE7-12
oil biodiesel
Jakkrapong Jitjamnong and Chachchaya Thunyaratchatanon
Adsorption of methylene blue dye from water using Cymbopogon citrates EE13-16
Vanida Chairgulprasert and Sakeeyah Masamae
Simulation of in situ removal of ethanol from culture broth using microbubbles EE17-22
Rungrote Kokoo and Matthana Khangkhamano
Effects of the processing on properties of biochars from rice husk and rice straw EE23-28
pyrolyzed between 500-1800 °C
Pranuda Jivaganont, Phontip Tammawat, Kritkaew Somton and Pimpa Limthongkul
Surface and cross-sectional comparative study of natural indigo dyed cotton fabrics EE29-33
using Na2S2O4 and monosaccharides as reducing agents in different alkaline solutions
Laksanawadee Saikhao, Jantip Setthayanond, Thitinun Karpkird and Potjanart Suwanruji
Effect of Molybdenum disulfide (MoS2) on carbon support from sawdust via EE34-38
hydrothermal carbonization for jatropha oil residue upgrading
Peerawith Sumtong, Vituruch Goodwin, Nuwong Chollacoop and Apiluck Eiad-ua
Lewis base poly(ethylene glycol) immobilized solid amine sorbent for carbon EE39-44
dioxide capture
Pathompong Janetaisong, Buppa Somchoam and Somsak Supasitmongkol
The methanol synthesis from glycerol in a one-step over basic oxide catalysts EE45-49
Rujira Jitrwung, Jiraporn Chalorngtham, Mukrawee Maneewuthiworasakul,
Kamonrat Leeheng, Kuntima Krekkeitsakul and Anantachai Wannajampa
Preparation of activated carbon from Salak seeds for dye removal EE50-53
Supattra Raksaphort, Nuntaporn Moonrungsee, Wiparat Srimueng and
Jatupong Pampaisong
Extraction and characterization of humic substances derived from Thailand’s leonardite EE54-59
Buntita Jomhataikool, Sanchai kuboon, Wasawat Kraithong and Apiluck Eiad-ua
Modification of electrodes by diamond-like carbon for electrochemical advanced EE60-65
oxidation of diuron
Nimit Kantiyawong, Boonchoat Paosawatyanyong and Varong Pavarajarn
Carbon dioxide capture on solid amine sorbent: breakthrough behavior and kinetic EE66-71
analysis
Buppa Shomchoam, Pathompong Janetaisong and Somsak Supasitmongkol
Performance comparison of different membrane micro-channel reactors for methanol EE72-77
production from biogas and hydrogen
Khunnawat Ountaksinkul, Tara Jiwanuruk, Nattapong Kasempremchit,
Paravee Vas-Umnuay, Palang Bumroongsakulsawat, Sompong Putivisutisak and
Suttichai Assabumrungrat
Catalytic dehydration of ethanol to diethyl ether over ZSM-5 catalysts with different EE78-82
Si/Al molar ratios
Nattawat Nampipat, Peangpit Glinrun, Thongchai Glinrun, Nattaya Comsup and
Bunjerd Jongsomjit
Decarboxylation of free fatty acid over Ni-MCM-41 EE83-88
Orhathai Jirarattanapochai, Duangkamon Jiraroj, Joseph Samec and
Duangamol N. Tungasmita
Systematic investigation of biofilm formation of acetogens and methanogens derived EE89-94
from agro-industrial wastewater using microfluidic devices and fabricated biointerface
Arslan Siddique, Benjaphon Suraraksa, Sukunya Oaew and Sarawut Cheunkar
The improvement in the properties of fatty acid methyl ester using partial EE95-98
hydrogenation reaction in a continuous fixed-bed reactor
Atichat Khongngam, Chaiya Roopsung, Thanita Sonthisawate, Yoothana Thanmongkhon,
Piyanan Sreesiri and Weerawat Patthaveekongka
Production processes and characterization of high -cellulose pulp from sugar cane EE99-103
bagasse and oil palm empty fruit bunch for development in cellulose fiber
Sappasit Winthachai, Wallop Arirob and Vittaya Punsuvon
The effect of contaminated gases in syngas on biomethanol production EE104-109
Rujira Jitrwung, Kuntima Krekkeitsakul, Kamonrat Leeheng, Jiraporn Chalorngtham
and Anantachai Wannajampa
Halogen-free catalysts for the cycloaddition reaction of CO2 to aziridines EE110-114
Prapussorn Yingcharoen, Sunatda Arayachukiat and Valerio D’Elia
The utilization of biomass waste from the palm oil industry using hydrothermal EE115-120
carbonization process
Theerapat Nangam, Chayut Pornprasitpol, Amornrat Suemanotham, Lalita Attanatho
and Weerawat Patthaveekongka
Electrochromic properties of polyaniline on sputtered crystalline TiO2 on fluorine- EE121-124
doped tin oxide substrate prepared by electropolymerization for solar cell applications
Thanapat Chaipitisiri and Palang Bumroongsakulsawat
Thermodynamic analysis of the chemical-looping water splitting and sorption enhanced EE125-130
reforming process for hydrogen production from biogas
Natthaporn Saithong, Karittha Im-orb and Amornchai Arpornwichanop
Effect of the electrolyte imbalance on performance of a vanadium redox flow EE131-136
battery: dynamic simulation and analysis
Tossaporn Jirabovornwisut, Yong-Song Chen and Amornchai Arpornwichanop
Removal of copper in aqueous solution by an alternative adsorbent modified EE137-142
napier grass stem
Wimonrat Tongpoothorn, Pitchayapa Kampeaw and Chittinan Kamjung
The optimization of esterification reaction for biodiesel production derived from EE143-148
high FFA non-edible oils
Nichaphat Sitthisuk, Charoenporn Lertsatitthanakorn, Vittaya Punsuvon,
Kaokanya Sudaprasert, Rewadee Anuwattana and Chanakan Puemchalad
Production of biofuels in Fischer-Tropsch synthesis using mono- and bi-metallic EE149-154
Fe-based catalysts
Napaporn Chancharoenlap, Borisut Chantrawongphaisal, Patthanant Natpinit,
Phavit Thapnui, Phongsak Hongcharoensri, Phichai Wongharn, Chotika Kongsomboon
and Manoo Boonsae
Oxidative dehydrogenation of ethanol to acetaldehyde over AgLi/SSP catalyst EE155-159
Narawich Mukda, Jakrapan Janlamool and Bunjerd Jongsomjit
Conductivity of Nb-, V- doped Sr2FeTiO6- materials for solid oxide fuel cells EE160-164
Ausa Potong and Soamwadee Chaianansutcharit
Influence of minerals in crude glycerol from biodiesel production on products EE165-169
distribution of bio-oil via co-pyrolysis with palm oil residues
Teerawit Laosombut, Lalita Attanatho, Amornrat Suemanotham, Warunyoo Phondate,
Panida Thepkhun and Yoothana Thanmongkhon
Parametric study of CZTS thin film solar cells by convective deposition method EE170-175
Thanawat Anantamongkolchai, Sojiphong Chatraphorn and Paravee Vas-Umnuay
Design and performance evaluation of sustainable process combining hydrotreating EE176-181
and hydrogen generation for biojet fuel production from palm oil
Siriporn Boonsuk, Worapon Kiatkittipong, Kanokwan Ngaosuwan,
Doonyapong Wongsawaeng and Suttichai Assabumrungrat
Development of lignocellulose sugarcane bagasse as a cooperative catalyst in EE182-186
cycloaddition reaction with CO2
Kawisa Chaipojjana, Mutsee Termtanun, Worapon Kiatkittipong, Kunlanan Kiatkittipong,
Sushil Adhikari, Navadol Laosiripojana and Suttichai Assabumrungrat
The studies of ethane dehydrogenation over supported gallium catalysts EE187-191
Kittipong Prakobtham, Korawich Trangwachirachai, Kittisak Choojun and
Tawan Sooknoi
Nitrogen-enriched activated carbon from shrimp shells as the electrodes for EE192-196
supercapacitors
Weerawit Luewanichwong, Parinya Inthasuwan, Chakorn Tangsirisatian,
Nopachote Sandhu and Khanin Nueangnoraj
Hydrotreating of pyrolysis oil model compounds with fatty acid and high water EE197-201
content over molybdenum sulfide catalyst supported on titania
Vituruch Goodwin, Paweekasit Sitthanawaitin, Tanakorn Ratana and
Sabaithip Tungkamani
The effect of carbon, nitrogen, and CuSO4 on laccase production by Bacillus sp. EE202-206
strain LA1
Prasert Nimnual and Sinthuwat Ritthitham
Methanol to gasoline conversion over ZSM-5 zeolites: influence of catalyst property EE207-212
and operating conditions
Lalita Attanatho, Wanchana Sisuthog, Yoothana Thanmongkhon, Amornrat Suemanotham,
Teerawit Laosombut and Siriporn Larpkiattaworn
Effect of operating conditions on synthesis of methanol and dimethyl ether from EE213-217
syngas over solid catalysts
Amornrat Suemanotham, Lalita Attanatho, Yoothana Thanmongkhon and
Teerawit Laosombut
Preparation and properties of carboxylated styrene-butadiene rubber/cassava starch/ EE218-222
cellulose fiber composite
Kanokwan Chantawee and Sa-Ad Riyajan
Investigation of the operation parameters affected on biofuel production via EE223-228
Fischer-Tropsch synthesis in milli-tubular reactor
Warunyoo Phondate, Lalita Attanatho, Amornrat Suemanotham, Teerawit Laosombut
and Yoothana Thanmongkhon
Bio-oils from palm oil residue via hydrothermal liquefaction process in the EE229-233
environmental impact aspect
Panida Thepkhun and Amornrat Suemanotham
Adsorptive removal of methyl red dye using eggshell particles EE234-237
Tanadta Archvapisith and Wanida Wattanakaroon
Synthesis of biodiesel by transesterification reaction of used palm oil using modified EE238-242
calcium oxide doped strontium ion as a catalyst
Apisit Prokaew, Siwaporn Meejoo Smith, Apanee Luengnaruemitchai and
Supakorn Boonyeun
Isolation and screening of gluconic acid producing microorganisms from flowers EE243-247
and fruits
Bongkot Chuenpraphai and Phimchanok Jaturapiree
Simulation and analysis of a novel dual fluidized-bed biomass gasification integrated EE248-253
with CO2 capture for high-purity hydrogen production
Aranyakorn Sampavapon, Pimporn Ponpesh and Amornchai Arpornwichanop
Enhancement of biodiesel properties via hydrogenation process: a pilot plant study EE254-259
Thanita Sonthisawate, Lalita Attanatho, Panida Thepkhun, Chiraphat Kumpidet,
Nattawee Teerananont, Piyanan Sreesiri, Chanakan Puemchalad, Yuji Yoshimura,
Takehisa Mochizuki, Yohko Abe, Shih-Yuan Chen and Makoto Toba
Neural network control of the direct methanol fuel cell for household applications EE260-265
Kornkamol Eamsiri, Soorathep Kheawhom and Amornchai Arpornwichanop
Utilization of zeolites synthesized from water sludge for heavy metal treatment in EE266-270
wastewater
Issaraporn Saenglam, Uthai Donkwang, Kotchakorn Yotyiamkrae,
Warangkana Kittiwongwisan, Bongkotchawan Pakamwong, Nittaya Phankon,
Orawan Simpan, Malinee Rangkatkij, Suphawan Pimdee, Thimpika Promprom,
Malee Prajuabsuk, Saisamon Lamlong, Janpen Intaraprasert, Daungdao Sattayakul,
Pharit Kamsri, Khemmakorn Gomonsirisuk, Parjaree Thavorniti and Pornpan Pungpo
The equilibrium study of methylene blue from aqueous solution using zeolite EE271-275
synthesized as highly potential adsorbent from water sludge
Warangkana Kittiwongwisan, Kotchakorn Yotyiamkrae, Uthai Donkwang,
Naruedon Phusi, Chayanin Hanwarinroj, Malee Prajuabsuk, Saisamorn Lumlong,
Duangdao Sattayakul, Jitlada Deshativong, Pisichanun Srisuwan, Pharit Kamsri,
Auradee Punkvang, Pajaree Thavorniti, Khemmakorn Gomonsirisuk and
Pornpan Pungpo
Effect of doped Al2O3 support in Ni-based catalyst for ammonia decomposition EE276-280
Mooklada Chaisorn, Teerapat Jerawattanakaset, Nutnicha Singhapunt,
Suparoek Henpraserttae and Pisanu Toochinda
Activated carbon derived from rice husk char and its application for dye adsorption: EE281-286
kinetics and isotherm studies
Nureesan Deemae, Ruethaithip Wisedsri and Nareerat Kitisripanya
Effect of doped support and urea on catalytic activity and coke prevention from Ni EE287-290
catalyst for ethanol steam reforming reaction
Disorn Maneewongvatana, Methichai Manatmethikun, Pornapat Tiyapornsuwan,
Sirintra Arayawate, Pisanu Toochinda and Pumiwat Vachrapong
Effect of immobilization materials and shapes on biohydrogen production from EE291-295
green alga Tetraspora sp. CU2551
Cherdsak Maneeruttanarungroj
Reforming of residue gas from CBG process to synthetic gas for bio-methanol EE296-300
production
Rujira Jitrwung, Kamonrat Leeheng, Kuntima Krekkeitsakul, Jiraporn Chalorngtham
and Anantachai Wannajampa
The optimization of biohydrogen production from green alga KS03 EE301-306
Thanaporn Sirawattanamongkol and Cherdsak Maneeruttanarungroj
Reduction of toxic gases from hydrolysis reaction of secondary aluminium dross EE307-311
Satamongkon Trekitwattanakul, Achiraya Kamwongsa, Mananya Pounglamjeak,
Lida Simasatitkul, Sukanya Thepwatee, Siriporn pranee and Samithichai Seeyangnok
Ketalization of glycerol with acetone by using aluminum sulfate as a solid acid EE312-316
catalyst
Tidarat Wahamongkol and Jirdsak Tscheikuna
Preparation of silk fibroin/chitosan film and its ability to remove Rhodamine B EE317-321
dye in aqueous solution
Peeravat Tanpadid, Nareerat Kittisripanya, Thanonchat Imsombat, Rapeepan Mahem,
Tianchai Panpojan, Sittichai Prapan and Ruethaithip Wisedsri
FA: Food and Agricultural Chemistry

The relationship between free radical scavenging activities and germination time of FA1-4
peanut (Arachis hypogaea L.) extracts
Kwanyuen Leamsamrong, Wathida Thornpho, Butsayamat Rattanadon and
Sarin Thongthummachat
Effect of infrared drying on phytochemical content, antioxidant activity and FA5-10
physicochemical properties of germinated brown rice
Hareesah Kohing, Jutarat Tasara and Weeraya Khummueng
Application of irradiated chitosan for controlling chili fruit anthracnose control and FA11-13
yield in chili field in Sukhothai
Prartana Kewsuwan, Penchan Suthanukul and Vichai PuriPunyavanich
Effects of extraction solvents ratio and methylation time for the determination of FA14-18
fatty acids from rambutan seeds kernel by gas chromatography-flame ionization
detector
Sirirat Chanvaivit and Ticha Caechua
The effects of EM-Bokashi compost and vegetable wastes compost on the growth FA19-24
of Hibiscus esculentus L. (Lady’s finger) plant
Min Aung, Kyaw Zan Aung and Nay Thwe Kyi
Chemical and physical properties of sago starch from tradition and machine method FA25-29
Rattiya Saradit and Nutthawadee Prabtan
Effects of subcritical water treatment on functional properties of soy protein isolate FA30-33
Chutikarn Chuchaisakpichit, Issara Slamala, Thongchai R. Srinophakun and
Kittiwut Kasemwong
Leucaena leaves: an oyster adjective FA34-38
Rattana Wongchuphan, Supaporn Apirattananusorn, Patcharee Lungmann and
Naengnoi Saengsane
Effect of subcritical water treatment on physicochemical properties of rice bran FA39-43
protein
Imboon Wongthaweewatan, Issara Sramala, Thongchai R. Srinophakun and
Kittiwut Kasemwong
Optimization of extraction condition of microcrystalline cellulose from rice bran FA44-49
residue
Supatsorn Chupong, Pakorn Opaprakasit and Kittiwut Kaseamwong
The effects of lime lemongrass and oregano essential oils treatment on quality FA50-55
preservation of salted fish (Rastrelliger brachysoma)
Lanchakon Chanudom, Ausnee Sakarasa, Nursofwanee Uma and Nutwara Ongsara
Comparison between mimic olfactory tool and sensory evaluation for grading of FA56-60
jasmine rice
Wimonsiri Tanomsridachchai, Siriwit Buajarern, Adisak Romputtal and
Yuthana Tantirungrotechai
Antioxidation, antibacterial and cytotoxicity activities of Hom tong banana flower FA61-64
extract
Atchara Patamakhachorn, Piyaporn Nongharn, Supakorn Boonyuen, Pariya Na Nakorn,
Audjima Poomkleang and Papassorn Kindklao
The preliminary study of volatile oil in Melaleuca cajuputi by GC-MS FA65-68
Mayoon Lamsub, Sirirat Phaisansuthichol and Orasa Choola-Aied
Free radical scavenging activity, total phenolic and flavonoid contents of herbal FA69-72
extracts
Jirapa Rattpan, Narumon Wuttiprom, Panadda Kulla and Supaporn Sangsrichan
Antioxidant activity and stability of total carotenoid and lycopene in coconut oil FA73-76
with extracts from gac fruit aril and tomato
Pensri Penprapai and Supamas Intharit
Survey of benzophenones residues contaminated in food packaging paper in FA77-82
Bangkok; Thailand; using GC-MS and IDMS techniques
Neungrutai Saesaengseerung
The detection of organophosphate insecticide using immobilized whole cells from FA83-87
the clone carrying mpdB gene expressing methyl parathion hydrolase
Chadaporn Pootawee, Witsanu Senbua, Chaiyong Taechapairoj and
Jesdawan Wichitwechkarn
Comparative study of total phenolic content and antioxidant activity of coconut oil FA88-91
with Curcuma zedoaria Roscoe and Curcuma longa L. extracts
Norasing Penprapai, Amporn Ratanamusik, Pensri Penprapai and
Manoch Chumchareon
Nanoemulsion of Syzygium aromaticum (L.) Merr. & L.M.Perry. oil extract in FA92-95
water prepared by emulsification process
Rungnapa Pimsen, Thiprada Thongrak, Paweena Porawatkul, Arnannit Kuyyogsuy,
Nongyao Tepaya, Monthakharn Thongsom and Sudkamon Lasopa
IN: Inorganic Chemistry

A reactor design for the synthesis of aluminum phosphide for insects elimination IN1-5
in rice mill
Tanawat Sakoltanaviroj, Samitthichai Seeyangnok and Kraipat Cheenkachorn
Synthesis, characterization and anticancer studies of acesulfame mixed with IN6-10
triphenylphosphine silver(I) complexes
Bussaba Boonseng, Jaruwan Chatwichien, Ratanon Chotima, Teerawat Khudkam and
Taweesak Pila
Synthesis, ESR study, and antibacterial activity of Cu(II) and Ag(II) IN11-16
tetrakis(4-alkyloxyphenyl)porphyrin complexes
Tossapon Prohmsatit, Autthavit Nuchthanom, Ausjima Poomkleang, Pariya Na Nakorn,
Soraya Pornsuwan and Supakorn Boonyuen
Fe2O3 particles deposition onto stainless steel IN17-19
Taweechai Watcharapiboon and Varong Pavarajarn
Studies on the preparation and bioactivity of highly porous bioactive glass IN20-25
Monthita Sasana and Radchada Buntem
A dinuclear cadmium(II) complex based on 2-hydroxybenzoate and IN26-30
2-aminopyrimidine ligand: synthesis, characterization, and crystal structure
Sudarat Thummatudtho, Natthakorn Phadungsak, Sukanya Mingphimai,
Supakorn Boonyuen and Kittipong Chainok
Effect of additional phosphine in n-butanol production catalyzed by IN31-35
Fe(II)-n-hetero-cyclic carbene complexes
Nattapong Siengdang, Watsapon Yimkaew, Bussaba Boonseng and Ratanon Chotima
A three-dimensional cobalt(II) coordination polymer with formate ligands; synthesis IN36-41
and crystal structure
Sukanya Mingphimai, Natthakorn Phadungsak, Supakorn Boonyeun and
Kittipong Chainok
Synthesis, crystal structure and alcohol sensing properties of a cobalt(II) containing IN42-46
by benzoylacetonate and imidazole ligands
Pornsan Lueangsriprech, Supakorn Boonyuen, Kittipong Chainok,
Natthakorn Phadungsak and Sumana Kladsomboon
MN: Material Chemistry and Nanotechnology
Ag2O-ZnO photocatalyst supported on hydroxyapatite from bovine bones for MN1-6
photo-reforming of glycerol
Damrong Adam, Netnapid Ongsuwan and Saowapa Chotisuwan
A new solid state sensor using rhodamine derivative for copper ion detection in MN7-11
aqueous solution
Nilobol Tungsombatvisit, Thitirat Inprasit and Penwisa Pisitsak
Amino-functionalized porphyrinic catalysts for electrochemical reduction of carbon MN12-17
dioxide
Piyawat Sangpittayatorn and Patchanita Thamyongkit
The preparation and characterization Ag and Ag alloy catalysts supported on MN18-21
treated carbon
Jennarong Jaikang, Anukorn Phuruangrat, Thapanee Sarakonsri, Aphiruk Chaisena and
Siwat Thungprasert
The study of using nanostructured lipid carrier for turmeric encapsulation in MN22-27
skincare application
Sukanya Thepwatee, Warunya Tienpratan, Nuanlawong Sumanojitraporn and
Jayanant Iemsam-arng
Crosslinking of Thai silk fibroin and bioactive calcium silicate glass for development MN28-32
of bone scaffolds
Pimnara Wattanachai and Peerapat Thongnuek
The effect of nucleating agent (paraffin) on the supercooling in encapsulated MN33-38
microcapsule for building application
Piyalak Ngernchuklin, Priyagorn Pholsrimuang, Nestchanok Yongpraderm,
Arjin Boonrung, Preeyaporn Chaiyasat and Amorn Chaiyasat
Development of natural rubber based composite foam containing magnetite MN39-43
nanoparticles for oil spill removal
Arisa Jaiyu, Julaluk Pannoi, Passakorn Sueprasit and Siriporn Thongon
Fabrication of superhydrophobic gold microstructure on polymer substrates MN44-48
Siriwan Boonmeewiriya, Supeera Nootchanat, Sanong Ekgasit and Kanet Wongravee
MXene/polymer based electrode for enhanced sensor performances in the detection MN49-53
of H2O2
Supawat Neampet, Jiaqian Qin, Nadnudda Rodthongkum and Orawon Chailapakul
Effects of natural fibers on morphology and acoustic properties of natural rubber MN54-59
foam composites
Julaluk Phunnoi, Pracha Lao-auyporn, Laksana Wangmooklang, Borwon Narupai,
Arisa Jaiyu and Passakorn Sueprasit
Nitrogen-doped porous carbon derived from cattail flower waste via hydrothermal MN60-63
treatment with chemical activation
Napat Kaewtrakulchai, Gasidit Panomsuwan, Kajornsak Faungnawakij and
Apiluck Eiad-ua
Improving the efficiency of dye-sensitized solar cells with spray-coated TiO2 MN64-69
electrode modified by CuO
Sakun Preedavijitkul and Akawat Sirisuk
Effect of chemical pretreatment on surface modification of carbon supports derived MN70-75
from Cattail flower
Parncheewa Udomsap, Nuwong Chollacoop, Nilubon Jong-anurakkun, Yuji Yoshimura
and Apiluck Eiad-Ua
Synthesis and photophysical properties of tetra-substituted pyrene derivatives as MN76-80
blue emitting materials for electroluminescent devices
Patchareepond Panoy, Taweesak Sudyoadsak, Pichaya Pattanasattayavong and
Vinich Promarak
Preparation and phase characterization of composites based on dispersion of gold MN81-86
(III) chloride in dipolar discotic liquid crystal
Sawitree Juabram, Chanaiporn Danvirutai and Suwat Nanan
One step synthesis of PtCo/TiO2 catalysts by flame spray pyrolysis for selective MN87-92
hydrogenation of furfural to furfuryl alcohol
Kitima Kruachao, Okorn Mekasuwandumrong, Piyasan Praserthdam and
Joongjai Panpranot
Fundamental study of carbon materials from oil male palm flowers via hydrothermal MN93-97
carbonization
Kanogpan Guntagerng, Gasidit Panomsuwan and Apiluck Eiad-ua
Photocatalytic degradation of methylene blue over polydopamine-Ag/TiO2 MN98-103
Kamonthip Tammarakwattana and Akawat Sirisuk
Synthesis and photophysical properties of carbazole and triphenylamine dendrons MN104-108
end capped-phenanthrocarbazole for light-emitting applications
Jirat Chatsirisupachai, Taweesak Sudyoadsuk and Vinich Promarak
Simple hole-transporting materials based on bis(2,4-dimethoxyphenyl)carbazole for MN109-113
perovskite solar cell
Thanyarat Chawanpunyawat, Duangratchaneekorn Muenmart, Taweesak Sudyoadsuk,
Pichaya Pattanasattayavong and Vinich Promarak
TADF materials based on carbazole substituted on triphenyl-triazines; the effect of MN114-118
acceptor strength
Pattarawadee Therdkatanyuphong, Taweesak Sudyoadsuk and Vinich promarak
CO2 methanation over NiO/NiAl2O4 spinel catalysts MN119-124
Chanya Thamma, Piyasan Praserthdam and Joongjai Panpranot
Hole-transporting thermally activated delayed fluorescence (TADF) materials based MN125-129
on carbazole dendronized anthraquinone derivatives for organic light-emitting
diodes
Namthip Khammultri, Rossatorn Muangpaisal, Taweesak Sudyoadsuk and
Vinich Promarak
Thermally activated delayed fluorescence (TADF) materials based on triphenylamine MN130-135
end capped benzothiadiazole for OLEDs
Jakkapan Kumsampao, A-monrat Thangthong, Taweesak Sudyoadsuk and
Vinich Promarak
Liquid-phase selective hydrogenation of furfural to furfuryl alcohol on RuCo/TiO2 MN136-140
catalysts
Natdanai Nanthasanti, Piyasan Praserthdam and Joongjai Panpranot
Synthesis of boron carbide powder from cellulose and boric acid using MN141-146
freezing-thawing technique
Wiphawi Jansuda and Sujarinee Sinchai
Styrene/stearyl methacrylate foams as oil absorbent MN147-151
Voratida Chuensukum, Orathai Boondamnoen, Pakorn Opaprakasit, Atitsa Petchsuk
and Mantana Opaprakasit
Preparation of silica supported zirconium-based heterogeneous catalysts by surface MN152-157
organometallic chemistry strategy for the conversion of CO2 to cyclic carbonates
under mild conditions
Ounjit Sodpiban, Silvano Del Gobbo and Valerio D’Elia
Process optimization and characterization of activated carbon from cattail weed MN158-162
waste prepared by chemical activation for efficient CO2 removal
Nardnutda Katunyoo, Araya Smuthkochorn, Napat Kaewtrakulchai, Duangduen Atong,
Kanit Soongprasit and Apiluck Eiad-ua
Conversion of CO2 to cyclic carbonates catalyzed by lanthanide salen complexes MN163-168
Vatcharaporn Aomchad and Valerio D’Elia
Preparation and characterization of Pb(Mg1/3Nb2/3)1-xTixO3 ceramics to fabricate MN169-172
flexible piezoelectric composites
Sukanya Samadoloh, Napatporn Promsawat, Ekwipoo Kalkornsurapranee,
Kowit Lertwittayanon, Soodkhet Pojprapai and Methee Promsawat
Preparation of complex hydrogels composed of chitosan, sodium tripolyphosphate MN173-177
and -carrageenan as xanthone encapsulating material
Apisara Yimprayul, Nataporn Sowasod and Wiwut Tanthapanichakoon
Perylene diimide functionalized with fused-thiophenes for organic field effect MN178-183
transistors: synthesis and electronic properties
Anna Pachariyangkun, Wiyada Saennawa, Taweesak Sudyoadsuk,
Pichaya Pattanasattayavong and Vinich Promarak
Removal of Cd2+ and Pb2+ ions by aluminium oxide and the prepared aluminium MN184-189
oxide nanoparticles
Saytanar Tun, Nay Thwe Kyi and Kyaw Naing
Fabrication and characterization of fouling-resistant membrane based on polyamide MN190-193
thin film composite incorporated with functionalized graphene oxide
Nuntanat Worrakunpiset, Chavakorn Samthong, Anongnat Somwangthanaroj and
Chalida Klaysom
The morphology of zeolite 13X/PDMS mixed matrix membrane for flue gas separation MN194-198
Nampetch Padungpetch, Chavakorn Samthong, Kajornsak Faungnawakij,
Anongnat Somwangthanaroj and Chalida Klaysom
Surface transformation of porous bioactive glass MN199-204
Thanatcha Kanthamoon and Radchada Buntem
Development organic coolants for electronics industry MN205-208
Rattikierati Tnuputdhanapun, Duangkamon Jiraroj, Sukkaneste Tungasmita,
Sarapong Choumwong, Chakkrit Supavasuthi and Duangamol Tungasmita
The effect of film-forming amines (FFAs) on flow-accelerated corrosion (FAC) of MN209-214
carbon steels in single-phase water
Salinla Charintara, Chutima Kongvarhodom, Derek H. Lister and Shintarou Mori
The green synthesis of gold nanoparticles using Crinum moorei extract MN215-220
Warinda Marujiwat and Nongnuj Muangsin
Effects of silver sulfadiazine on crosslinking and properties of Thai silk fibroin/ MN221-225
gelatin films
Patchara Umprasert, Siriporn Damrongsakkul and Juthamas Ratanavaraporn
Role of -silver sulfide solid electrolyte on Ag nanofilament fabrication for MN226-230
surface-enhanced Raman spectroscopy activity
Phichaya Fueaimi, Pannee Leeladee, Thawatchai Tuntulani, Annop Klamcheun,
Alongkot Treetong, Tuksadon Wutikhun and Panita Kasamechonchung
Corrosion resistance properties of poly(acrylic acid-co-sodium styrene sulfonate) MN231-235
inhibitors on low carbon steel AISI 1018 in recirculating cooling water
Parinya Jitreewas, Siriporn Pranee, Sukanya Thepwatee, Narong Pungwiwat and
Samitthichai Seeyangnok
A top-down fabrication of Thai silk fibroin/gelatin composited microparticles MN236-241
Orathai Suphawannawiboon, Jutarat Jamkratoke and Sorada Kanokpanont
Conjugated polycarbazole nanoparticles dispersion in aqueous solution for printed MN242-247
organic light-emitting diodes
Patteera Funchien, Duangratchaneekorn Muenmart, Taweesak Sudyoadsuk and
Vinich Promarak
The effect of NaCl on SiC formation from sugarcane bagasse ash via SHS method MN248-253
Matthana Khangkhamano and Rungrote Kokoo
Preparation and application of poly(acrylamide) based polymers as corrosion MN254-257
inhibitor
Phanita Tansiri, Parinya Jitreewas, Siriporn Pranee, Narong Pungwiwat,
Sukanya Thepwatee and Samitthichai Seeyangnok
Synthesis of hydrophobically-modified poly(ethyleneimine) as surfactant for cationic MN258-263
CdSe/ZnS quantum dots with high colloidal stability
Jamornpan Yangcharoenyuenyong, Tirayut Vilaivan and Numpon Insin
Preservation of nanodroplets by the colloid-electrospinning technique MN264-267
Kusuma Thongchaivetcharat, Ratchapol Jenjob and Daniel Crespy
Influence of monetite on photocatalytic activity of titanium dioxide P–25 MN268-271
Pakpassagun Somwong, Neeranut Kuanchertchoo and Dujreutai Pongkao Kashima
Nano-zinc oxide-doped activated carbon for feed additive from popped rice MN272-276
Duangporn Saramas and Sanong Ekgasit
Magnetic nanoparticles for organic synthesis MN277-279
Parintip Rattanaburi, Vachira Choommongkol, Khemmaphat Chudam and
Nussaba Juntorn
Physical properties of gelatin and silk fibroin microspheres MN280-285
Aviga Soonmongkol, Jutarat Jamkratoke and Sorada Kanokpanont
Bacteria removal in wastewater by using silver nanoclusters materials MN286-291
Nittaya Foansoongnoen, Wittaya Ngeontae and Pornsawai Praipipat
Development of AgVO3/C electrocatalysts for an ethanol-tolerant in alkaline MN292-297
electrolyte for direct ethanol fuel cells
Chakkrapong Chaiburi and Sudkate Chaiyo
Influence of Ce ions in the La1-xCexCoO3+ studied by XAS and XPS MN298-302
Sutasinee Kityakarn, Anupap Pungsomchit, Suphannipha Thara,
Rachadaporn Supruangnet, Hideki Nakajima and Yingyot Poo-arporn
Effects of surfactants as additives in electronic-grade lubricant for magnetic recording MN303-308
head lapping processes
Chuenkamol Khongphow, Duangamol Tungasmita, Benjie L. Fernandez,
Chakkrit Supavasuthi and Sukkaneste Tungasmita
Effects of corrosion inhibitors in lubricant for microelectronic lapping processes MN309-313
Natkritta Maipul, Duangamol Tungasmita, Benjie L. Fernandez, Chakkrit Supavasuthi
and Sukkaneste Tungasmita
Investigation of purification procedures for MIL-101(Cr) metal-organic framework MN314-319
obtained from HF-free synthesis
Ratchadaporn Srikhong, Somsak Supasitmongkol and Ramida Rattanakam
Colourtuning of organic light emitting diodes based on heteroleptic iridium (III) MN320-323
complexes
Thanyanat Saiboh, Laongdao Kangkeaw, Somboon Sahasithiwat and Rukkiat Jitchati
A comparative study on glutaraldehyde crosslinking methods of three-dimensional MN324-328
Thai silk fibroin/gelatin scaffolds for medical application
Wanwarang Wannaborvorn, Anita Manassakorn, Rath Itthipanichpong,
Prin Rojana-pongpun, Visanee Tantisevi, Sunee Chansangpetch,
Siriporn Damrongsakkul and Juthamas Ratanavaraporn
A simple and cost-effective bimetallic cobalt/iron biosensing catalyst on activated MN329-333
carbon support for non-enzymatic glucose detection in human urine
Metini Janyasupab, Chen-Wei Liu, Chamras Promptmas and Werasak Surareungchai
The effect of phosphoric acid concentration in crude oil pretreatment process on MN334-338
the phosphorus content of crude palm oil
Boonruen Sunpetch and Oangkana Champanon
Preservation of nicotine by nanoencapsulation technique using -cyclodextrin MN339-343
Nathasak Sinlikhitkul, Harid Pataveepaisit, Waritsara Noodang, Arayaporn Krissawong,
Suriyawut Kulatee, Anotai Suksangpanomrung, Luckhana Lawtrakul and Pisanu
Toochinda
Improvement of mechanical properties of jute fabric by bio-scouring and MN344-349
bio-polishing process with enzymes
Chatdanai Thongsamut, Chutimon Satirapipathkul and Rattanapol Mongkholrattanasit
Characterization of epoxidized castor oil blended with epoxy phenol novolac resin MN350-353
for adhesive application
Thanapat Supannasud, Chavakorn Samthong and Anongnat Somwangthanaroj
Electrochemical oxidation of ethanol on palladium electrodeposited on nickel catalyst MN354-358
Wilaiwan Supap, Thaneeya Hawiset and Prachak Inkaew
The antibacterial activities of silver nanomaterials against gram positive and gram MN359-363
negative bacteria
Romrawin Rooblert, Sujittra Youngme and Pornsawai Praipipat
Effect of the sample preparation on the composition of hydroxyapatite derived MN364-368
from waste anchovy fish bone
Hasan Daupor, Pateeroh Kuwae, Anugrah Ricky Wijaya, Isma-ae Chelong and
Abd Naser Haji Samoh
Synthesis of mesoporous silica with site-isolated acid and base-based catalysts and MN369-374
their bifunctional application in a one-pot cascade reaction
Apichat Klayanon and Wipark Anutrasakda
NP: Natural Products, Biological Chemistry and Chemical Biology

Formulation of facial cream and body mist from avocado oil NP1-5
Thitiphan Chimsook and Dorngporn Amornlerdpisan
Depsidones and a -butenolide from Simplicillium sp. PSU-H168, an endophytic NP6-11
fungus from Hevea brasiliensis leaves
Supaporn Chinpha, Souwalak Phongpaichit, Jariya Sakayaroj and
Vatcharin Rukachaisirikul
Phytochemical screening and antioxidant activity of 10 Araceae rhizomes in the NP12-16
Dong Phayayen, Khao Yai forest
Sawittree Rujitanapanich, Duangchai Sookchaloem, Pannee Denrungruang and
Chaleaw Petchthong
Volatile compositions of Clausena harmandiana essential oil NP17-20
Chalermporn Thongpoon, Chalida Phungpanya and Theeraphan Machan
Evaluation of chemical constituents from the leaves of Oroxylum indicum as NP21-25
histone deacetylase inhibitors
Laor Somsakeesit, Pakit Kumboonma, Somprasong Saenglee, Thanaset Senawong and
Chanokbhorn Phaosiri
Cytotoxic and antimalarial constituents from the roots of Atalantia monophylla NP26-31
Awat Wisetsai, Ratsami Lekphrom and Florian T. Schevenels
Semi-synthetic studies of 2-substituted huperzine A analogues as acetylcholinesterase NP32-37
inhibitors
Wareepat Tiyabhorn, Pornkanok Pongpamorn, Richard Sessions, Somsak Ruchirawat
and Nopporn Thasana
Chemical constituents from the leaves of Artocapus chama Buch.-Ham. NP38-41
Suthida Rattanaburi and Wilawan Mahabusarakam
Extraction of phenolic compounds from fenugreek seeds using liquefied dimethyl NP42-47
ether (DME)
Apinya Thungoen, Artiwan Shotipruk and Panatpong Boonnoun
Synthesis of cationic lipids with spermine as a polar head and its analogues for NP48-52
gene delivery
Chopaka Thongbamrer, Boon-ek Yingyongnarongkul, Uthai Sakee and
Widchaya Radchatawedchakoon
Fungal metabolites from the endophytic fungus Xylaria sp. PSU-STD70 NP53-58
Wipapan Kongyen, Vatcharin Rukachaisirikul, Souwalak Phongpaichit and Jariya Sakayaroj
Synthesis of novel neutral helper lipids with amino acids as polar heads NP59-63
Prutchaya Kumpairee, Nattisa Niyomtham, Nuttapon Apiratikul and
Boon-ek Yingyongnarongkul
Phenolic content and antioxidant activity in four edible green algae from Tha Li NP64-68
district, Loei province
Suwatchai Misuna, Thananan Machai, Niramol Srichana, Krittipong Choojit,
Bussabavadee Puttanu and Chanchaem Duang-u-pa
Antioxidant and cytotoxic activities and total phenolic content of Mesua ferrea NP69-73
Sukanya Keawsa-ard, Montakarn Boonyakarn, Piranchana Phanusan,
Oranuch Thammasorn and Samart Kongtaweelert
Pretreatment of water hyacinth (Eichhornia crassipes (Mart.) Solms) for NP74-77
biodegradable polyhydroxybutyrate (PHB) production
Napassorn Wititsuwankul and Phimchanok Jaturapiree
Radical scavenging activity and total phenolic content of Lumnitzera littorea fruit NP78-82
and flower
Luksamee Vittaya, Sunanta Khongsai, Sutkanung Na Ranong, Sittichoke Janyong and
Nararak Leesakul
OR: Organic and Medicinal Chemistry

Tribenzo[a,c,k,l]xanthene: a novel fluorophore from an intramolecular Scholl reaction OR1-3
Thikhamporn Uppalabat and Rukkiat Jitchati
Photooxidation of organosulfur compounds catalyzed by iodo-BODIPY derivatives OR4-9
under visible light irradiation
Piyamaporn Tangkasemsamran, Mongkol Sukwattanasinitt and Sumrit Wacharasindhu
The study of ene-yne-oxazolone intramolecular cyclization using hydrogen- OR10-15
phosphine oxide
Sirikan Deesiri, Somsak Ruchirawat and Nopporn Thasana
Anti-cervical cancer activities of 3-(2-hydroxyphenyl)propanoic acid and OR16-20
3-(2-hydroxy phenyl)acrylic acid derivatives on cervical cancer cell lines
Kraikrit Utama, Puttinan Meepowpan, Thiti Janpirom, Vachira Choommongkol,
Paitoon Aobchey, Jiraporn Kantapan and Padchanee Sangthong
Synthetic small molecules for isolation of Gram-positive bacteria from contaminated OR21-26
samples
Krittapas Jantarug, Sakol Warintaraporn and Chutima Jiarpinitnun
Antidiabetic activity of synthetic furofuran lignans containing multiphenolic groups OR27-31
Titiruetai Doungwichitrkul, Nantaporn Surachaitanawat, Wisuttaya Worawalai and
Preecha Phuwapraisirisan
Vanillic acid-resorcinol esters with potent anti-oxidation and tyrosinase inhibitory OR32-37
activities
Jiraporn Kara, Jindaporn Puripattanavong, Teerapat Nualnoi and Luelak Lomlim
N-(2-(Dimethylamino)ethyl)-4-oxo-4H-chromene-2-carboxamide analogues as OR38-43
acetylcholinesterase inhibitors
Paptawan Suwanhom, Teerapat Nualnoi, Vannajan Sanghiran Lee and Luelak Lomlim
Bromodiethylsulfonium bromopentachloroantimonate (BDSB) as a novel activator OR44-48
for chemical glycosylation
Ladawan Punrum and Panuwat Padungros
A new method for the preparation of aryl halides from phenols bearing electron- OR49-52
withdrawing groups using PPh3/halogenating agent
Korakot Saowalak, Parinthorn Temyarasilp, Anchulee Pengsook and
Wanchai Pluempanupat
Hybrid molecule: combination between sulfonamides and trimethoprim to inhibit OR53-58
growth of methicillin-resistant Staphylococcus aureus (MRSA)
Taechin Nowwarat, Napon Nilchan and Chutima Jiarpinitnun
Synthesis of cationic lipids with guanidine and cyclic guanidine polar head OR59-63
Charoen Posa, Nattisa Niyomtham, Saowanee Kumpun and Boon-ek Yingyongnarongkul
Sulfonamidation for the synthesis of trisubstituted arene intermediates OR64-69
Pongsun Jhangthong and Pitak Chuawong
Synthesis and cytotoxicity evaluation of tetrahydro- -carboline derivatives OR70-75
Koonchira Buaban, Thongchai Taechowisan, Weerachai Phutdhawong and
Waya Phutdhawong
Synthesis of pyrrole-azepine core of stemoamide OR76-81
Nantachai Inprung, Sathita Thaosiriwong and Punlop Kuntiyong
Ring opening of epoxides catalyzed by PdCl2 impregnated on Al2O3-pillared clay OR82-86
Sauwalak Wassanapadit and Piyarat Trikittiwong
Design, synthesis and antibacterial activities of a series of bis(indolyl)arylmethanes OR87-91
Chayamon Chantana, Onnicha Khaikate, Nannaphat Chumsri, Umaporn Thathaisong
and Jaray Jaratjaroonphong
Gold nanoparticles and pyrrolidinyl peptide nucleic acid-based platform for the OR92-97
detection of RNA
Kriangsak Faikhruea and Tirayut Vilaivan
Synthesis and optical properties of blue emitters from pyrene derivatives OR98-103
Tanika Khanasa and Vinich Promarak
PH: Physical and Theoretical Chemistry
Molecular docking investigation on antimicrobial mechanism of plumbagin derivatives PH1-5
Issaree Charoenpinyoying, Boworn Sriyaphai, Angsina Kongdee and Luckhana Lawtrakul
Determination of ammonia using Pt/PANI ES nanocomposite: a DFT study PH6-10
Duangporn Ngampring and Chinapong Kritayakornupong
Optical and electronic properties of Ag/PANI ES interacting with ammonia: PH11-15
a theoretical investigation
Taksaphorn Nopkaew and Chinapong Kritayakornupong
Theoretical study of photoinduced intramolecular electron transfer in Ru(II)-Re(I) PH16-21
mixed-valence complex
Rangsiman Ketkaew, Jen-Shiang K. Yu and Yuthana Tantirungrotechai
DFT calculation, biological activity, anion sensing application studies and crystal PH22-27
structure of (E)-4-methoxy-2-((4-methylbenzo[d]thiazol-2-ylimino)methyl)phenol
H seyin Unver, Mustafa Y ld z, Nuray Y ld r m, G khan Alpaslan, Bahadir Boyacioglu
and Ayhan Elmali
In-silico modification of recombinant interleukin-18 for binding affinity prediction PH28-33
towards its binding receptor
Kaweeyut Poonkaeo, Surapong Chatpun, Surasak Sangkhathat and Varomyalin Tipmanee
Adsorption study of chloroform and hexane by using silver nanoparticles supported PH34-36
on reduced graphene oxide
Nattawadee Wisitruangsakul, Kanyapat Teekayupak and Tiwaporn Kruk
Theoretical study of twisted intramolecular charge transfer and intermolecular PH37-42
double proton transfer in the excited state of N,N’-bis(salicylidene)-p-phenylene-
diamine Schiff base
Nichakan Miengmern, Supa Hannongbua and Songwut Suramitr
Substituent effect on photophysical properties of salicylidene Schiff base and its PH43-48
derivatives as colorimetric dyes: a TD-DFT study
Amonchat Koonwong, Suwannee Sriyab, Supa Hannongbua and Songwut Suramitr
In-silico aided inhibitor design of benzoimidazole-containing derivatives as novel PH49-53
direct InhA inhibitors
Pharit Kamsri, Auradee Punkvang, Patchreenart Saparpakorn, Supa Hannongbua,
Sanya Sureram, Prasat Kittakoop, Nitima Suttipanta, Khomson Suttisintong,
Adrian Mulholland, James Spencer, Potjanee Srimanote and Pornpan Pungpo
Molecular modeling of InhA inhibitors in the class of benzimidazole derivatives as PH54-59
a novel anti-tuberculosis agents
Bunrat Tharat, Bongkochawan Pakanwong, Paptawan Thongdee, Kotchakorn Yotyiamkrae,
Bundit Khamsri, Thitima Chuansupak, Naruedon Phusi, Chayanin Hanwarinroj,
Kampanart Chayajaras, Thimpika Pornprom, Nitima Suttipanta, Pharit Kamsri,
Auradee Punkvang, Patchreenart Saparpakorn, Supa Hannongbua, Prasat Kittakoop,
Khomson Suttisintong, Adrian Mulholland, James Spencer and Pornpan Pungpo
Key structural basis of nitrothiazolyl carboxamide analogues as DNA gyrase subunit PH60-63
B inhibitors for anti-tuberculosis agents : molecular docking calculations
Thitima Chuansupak, Bongkochawan Pakamwong, Paptawan Thongdee,
Kotchakorn Yotyiamkrae, Bunrat Tharat, Bundit Khamsri, Naruedon Phusi,
Chayanin Hanwarinroj, Nitima Suttipanta, Pharit Kamsri, Auradee Punkvang,
Patchreenart Saparpakorn, Supa Hannongbua, Prasat Kittakoop, Khomson Suttisintong,
Ubolsree Leartsakulpanich, Adrian Mulholland, James Spencer and Pornpan Pungpo
Structure based drug design of 2-(1H-benzo[d]imidazol-2-yl)-2-cyanovinyl as DNA PH64-68
gyrase subunit B inhibitors for anti-tuberculosis agents
Kotchakorn Yotyiamkrae, Bongkochawan Pakanwong, Paptawan Thongdee,
Bundit Khamsri, Thitima Chuansupak, Naruedon Phusi, Chayanin Hanwarinroj,
Thimpika Pornprom, Pharit Kamsri, Auradee Punkvang, Patchreenart Saparpakorn,
Supa Hannongbua, Prasat Kittakoop, Khomson Suttisintong, Nitima Suttipanta,
Adrian Mulholland, James Spencer and Pornpan Pungpo
Elucidating structural basis of 4-aminoquinoline derivatives as novel M. tuberculosis PH69-74
DNA GyrB inhibiters: molecular docking study
Bandit Khamsri, Bongkochawan Pakamwong, Bunrat Tharat, Kotchakorn Yotyiamkrae,
Paptawan Thongdee, Thitima Chuansupak, Naruedon Phusi, Chayanin Hanwarinroj,
Kampanart Chayajarus, Pharit Kamsri, Auradee Punkvang, Nitima Suttipanta,
Patchreenart Saparpakorn, Supa Hannongbua, Khomson Suttisintong, Prasat Kittakoop,
James Spencer, Adrian Mulholland and Pornpan Pungpo
Computer aided molecular design of ATPase inhibitors in a series of PH75-79
7-chloro-4-piperazinoquinoline derivatives: molecular docking calculations
Bongkochawan Pakamwong, Kotchakorn Yotyiamkrae, Paptawan Thongdee,
Bunrat Tharat, Bandit Khamsri, Thitima Chuansupak, Naruedon Phusi,
Chayanin Hanwarinroj, Kampanart Chayajarus, Pharit Kamsri, Auradee Punkvang,
Nitima Suttipanta, Patchreenart Saparpakorn, Supa Hannongbua, Prasat Kittakoop,
Ubonsree Leartsakulpanich, Khomson Suttisintong, James Spencer, Adrian Mulholland
and Pornpan Pungpo
Rational design of PknB inhibitors in class of 4-oxo-crotonic acid derivatives as PH80-84
highly potent anti-tuberculosis agents
Chayanin Hanwarinroj, Naruedon Phusi, Kampanart Chayajarus, Pharit Kamsri,
Auradee Punkvang, Nitima Suttipanta, Patchreenart Saparpakorn, Supa Hannongbua,
Khomson Suttisintong, Prasat Kittakoop, James Spencer, Adrian Mulholland and
Pornpan Pungpo
Molecular docking study of pyrrolyl benzohydrazide derivatives to explore the PH85-88
structure requirements of InhA inhibitors
Paptawan Thongdee, Bongkochawan Pakamwong, Kotchakorn Yotyiamkrae,
Bunrat Tharat, Bundit Khamsri, Thitima Chuansupak, Naruedon Phusi,
Chayanin Hanwarinroj, Kampanart Chayajarus, Pharit Kamsri, Auradee Punkvang,
Nitima Suttipanta, Patchreenart Saparpakorn, Supa Hannongbua, Prasat Kittakoop,
Ubonsree Leartsakulpanich, Khomson Suttisintong, James Spencer, Adrian Mulholland
and Pornpan Pungpo
Determination of antioxidant properties and total phenolic compound of various PH89-92
pigmented Thai rice varieties grown of Thailand
Sudarat Chanchoie, Chayanin Hanwarinroj, Naruedon Phusi, Duangdao Sattayakul,
Jitlada Dechatiwong, Saisamorn Lumlong, Kampanart Chayajaras,
Somjintana Taweepanich, Janpen Intaraprasert, Nuchanaporn Pijarn, Pharit Kamsri,
Nitima Suttipanta and Pornpan Pungpo
Studies of sildenafil and phosphodiesterase type 5 binding properties by using PH93-96
computational methods
Bunpot Intarathat, Autchara Namkhaw, Supawadee Srinimnuan, Supa Hannongbua
and Patchreenart Saparpakorn
Molecular docking study of the alkaloids from Tilicora triandra against acetyl- PH97-102
cholinesterase for the treatment of Alzheimer’s disease
Pathumwadee Yotmanee, Kanokporn Lehboon and Apichart Suksamrarn
PO: Polymer Chemistry

Comparative study on two polymerization methods for synthesis of copper(II) ion PO1-6
imprinted polymers
Angkana Chaipuang, Chalida Phungpanya, Chalermporn Thongpoon,
Kanchana Watla-iad, Orawan Suwantong, Prachak Inkaew and Theeraphan Machan
The removal of cationic dye from wastewater with polymethacrylic acid grafted PO7-12
silica gel
Praethong Laopa and Duangruthai Puksachat
PLA foams for scaffold application PO13-18
Pasuta Sungsee and Varaporn Tanrattanakul
Poly(lactic acid)/epoxidized natural rubber blends: effect of processing condition PO19-24
Jenjira Klinkajorn, Varaporn Tanrattanakul and Ekwipoo Kalkornsurapranee
Novel unsaturated polyester copolymers based on poly(butylene succinate) for PO25-30
using as toughening agent for PLA
Vachiravit Chalermpanaphan and Chantiga Choochottiros
High recovery stress obtained in benzoxazine-epoxy shape memory polymers PO31-35
reinforced with carbon fiber
Jutamart Plylaharn, Manunya Okhawilai and Sarawut Rimdusit
Tensile behaviors of multi-walled carbon nanotube filled polybenzoxazine/urethane PO36-40
alloys reinforced with aramid fiber
Jusmin Daungkumsawat, Manunya Okhawilai, Tewarak Parnklang and
Sarawut Rimdusit
Characterization of novel shape memory polymer from green-polybenzoxazine/ PO41-45
epoxy alloy
Phakakrong Hombunma, Manunya Okhawilai and Sarawut Rimdusit
Supramolecular self-assembling for enhanced oil recovery PO46-51
Saran Banthong, Chutima Kongvarhodom and Laura Romero-Zer n
Effect of drinking water treatment sludge on the properties of rubber compounds PO52-56
Wasana Khongwong, Nittaya Keawprak, Punthinee Somwongsa, Duriyong Tattaporn
and Piyalak Ngernchuklin
Relationship between crosslink structures and tensile properties of sulphur- PO57-62
vulcanized natural rubbers
Watcharin Sainumsai
Effect of acrylic-typed processing aid on thermal and mechanical properties of PO63-66
poly(lactic acid) blown films
Tanchanok Wongkhan, Pollawat Charoeythornkhajhornchai and
Anongnat Somwangthanaroj
Toughness improvement of poly(lactic acid) blown films PO67-71
Sawanont Puttagoon, Pollawat Charoeythornkhajhornchai and
Anongnat Somwangthanaroj
Effects of curing conversions on properties of benzoxazine-urethane shape memory PO72-76
polymer alloys
Wassika Prasomsin, Manunya Okhawilai and Sarawut Rimdusit
Preparation of magnetic molecularly imprinted polymer for glutathione PO77-81
Monnapas Wongtipakorn, Angkana Chaipuang, Chalida Phungpanya,
Chalermporn Thongpoon, Theeraphan Machan and Kanchana Watla-iad
Preparation of Camellia oleifera oil loaded chitosan nanoparticles and their PO82-86
release characteristics
Atchara Kaolaor and Orawan Suwantong
In situ preparation of corn starch-filled epoxidized natural rubber composites and PO87-92
its properties
Pajaree Chooprayoon and Ploenpit Boochathum
Preparation of poly(lactic acid) filled with cellulose/SiO2 composite PO93-98
Kittithorn Lertphirun and Kawee Srikulkit
Electrospinning of PVP/KOH/PAN blended: effects of PAN on PVP/KOH alkaline PO99-104
polymer system
Tanissara Pinijmontree and Santi Maensiri
Functional polyurethanes containing active agents and their hydrolytic stability PO105-110
Butsabarat Klahan, Farzad Seidi and Daniel Crespy
Graft copolymerization of butyl acrylate/methyl methacrylate onto waterborne PO111-116
polyurethane from blend formulation with hydroxyl telechelic natural rubber and
modified rubber seed oil
Wanida Raksanak, Nitinart Saetung and Anuwat Saetung
Electrospun tamarind seed polysaccharide combined with polyethylene glycol-based PO117-122
nanofibers development
Khanittha Chawananorasest, Jate Panichapakdee, Chatporn Klaykaew,
Punthinee Somwongsa, Patsuda Seangthongdee and Praphakorn Kaemchantuek
Fabrication and characterization of composite films of bacterial cellulose and silk PO123-127
cocoon fragment
Junchanok Chaikhunsaeng and Muenduen Phisalaphong
Study of low cost adsorbent from waste natural fiber for removal of crystal violet dye PO128-131
Adisak Keereeruk and Watchanida Chinpa
Mechanical and thermal properties of reprocessed ABS with its improved impact PO132-137
property by blending with NBR
Poonsub Threepopnatkul, Pathuthip Tongtun, Rawipreeya Suesuan, Saikhim Panawes
and Chanin Kulsetthanchalee
Effect of blowing agent on the physical and mechanical properties of styrene- PO138-142
butadiene rubber for shoe application
Romnanat Charoensawat, Narunat Saraboot, Ratchadapron Maithai and
Atiwat Wiriyaamornchai
Preparation and properties of modified polystyrene and amino alcohol rubber blends PO143-148
Amnard Sittattrakul, Poonsub Threeponakul, Kanyapat Peevalaisirikorn, Piraya Sriklum
and Wanvipa Majchacheep
Author Index Index 1-10
Glucose biosensors based on nickel ferrite composite materials modified
glassy carbon electrode
Chantarattana Nontapha1, Chochanon Moonla2, Anchana Preechaworapun3,
Tanin Tangkuaram2*
1
Chemistry Program, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand
2
Applied Chemistry Program, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand
3
Chemistry Program, Faculty of Science and Technology, Pibulsongkram Rajabhat University,
Phitsanulok 65000, Thailand
*E-mail: tanin.tang@gmail.com
Abstract:
The development of glucose biosensors using poly (diallydimethylammonium
chloride) capped gold nanoparticles (PDDA-AuNPs) with nickel ferrite nanoparticles
(NiFe2O4NPs) and poly(o-phenylenediamine) (PoPD) in the scaffold of chitosan linked
carbon nanotube (CNT-CHIT) and glucose oxidase was developed. The electroanalytical
potential was applied at -0.35 V for glucose determination in 0.05 M phosphate buffer
solution pH 7.0. The figure of merits of the glucose biosensors was linearity in the range of 5
µM to 6 mM (R2 = 0.999) with a detection limit of 1.21 µM glucose. The biosensors exhibit
excellent repeatability (% RSD = 3.38 %, n = 5), and reproducibility (%RSD = 3.76%, n = 5)
without interfered from common electroactive species such as ascorbic acid, aspirin, caffeine,
cholesterol, dopamine and uric acid.
1. Introduction conductivity, high surface area, and catalytic

Diabetes is a major health problem in properties4 and NiFe2O4NPs is highly
Thailand and all around the world that electrochemically stable and capable of
affects family, society, and economy.1 This catalyzing the chemical reaction.5 Moreover,
disease can be diagnosed using various carbon nanotubes (CNTs) also has a high
advanced techniques such as High- electrocatalytic effect, a fast electron-
Performance Liquid Chromatography transfer rate and great promise as oxidase-
(HPLC)2 and Ultraviolet-visible spectro- based amperometric sensor owing to its
scopy (UV-Vis).3 These provide very ability to promote the electron transfer of
precise and accurate results but there are hydrogen peroxide (H2O2).6 Thus, it is
some disadvantages due to long time widely used for application of
consuming and high expense. In this work, a electrochemical sensors. However, CNTs
biosensor for glucose determination was has some limit applications because of its
developed that a popular sensitive and poor solubility and chemical inertness in an
selective enzymatic method was applied. In aqueous solution that needs to improve.7
this case, glucose oxidase (GOx) was Recently, there are many researchers which
operated by immobilization on the electrode reported that chitosan (CHIT), a natural
surface. The interesting materials were biocompatible polymer, can solubilize CNTs
modified on glassy carbon electrode surface in aqueous solution. The composite of
to enhance the effectiveness of the CNTs-CHIT exhibits robust film-forming
developed biosensor. According to their ability, good conductivity, high stability and
good physical and chemical properties, gold good biocompatibility.8 Additionally, some
nanoparticles (AuNPs) and nickel ferrite polymer materials are applied for the
nanoparticles (NiFe2O4NPs) were applied. It combination with other kinds of materials.
is found that AuNPs has an excellent Poly(diallydimethyl ammonium chloride)
© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) AN1
(PDDA) was utilized to synthesize PDDA 3 grams of purified CNTs powder
capped AuNPs (PDDA-AuNPs) and Poly(o- were dissolved in CHIT-GA cross-linked
phenylene diamine) (PoPD), a polymer with solution (Mole ratio of CHIT:GA as 1:200).
high electrical conductivity and ionic After that, it was sonicated for 3 hrs. Then,
strength, was electrochemically deposited on the obtained solution was ready for further
the electrode surface. This method is an use.
interesting alternative way in terms of 2.3 Preparation of poly(diallyldimethyl-
permselectivity, reproducibility, thickness ammonium chloride) capped gold
control and the uniforming of the excellent nanoparticles (PDDA-AuNPs)
polymer film that can eliminate possible The PDDA-AuNPs solution was
interferences. These materials are used for successfully prepared by the following
the development of glucose biosensors method. First, 60 µL PDDA (35 wt.%), 10
which become portable, accurate, mL UHP water, 50 µL NaOH (0.5 M) and
inexpensive, simple and fast for analysis. 75 µL HAuCl4 (1%) were mixed and stirred
for few minutes. Then, the solution was
2. Materials and Methods heated at 90 oC for half hour. Finally, its
2.1 Materials and apparatus color changed to watermelon red of PDDA-
All the experiments used an AuNPs.
analytical grade of reagents and ultra-high 2.4 Synthesis of nickel ferrite
purity water (UHP). Hydrochloric acid from nanoparticles (NiFe2O4NPs)
Ajax Finechem (NSW, Australia), sodium Firstly, Ni(NO3)2·6H2O, FeSO4·
hydroxide (97%) from RCI Labscan 7H2O and Fe(NO3)3·9H2O (Stoichiometric
(Bangkok, Thailand), and ethanol from ratio of 3:5:1) were dissolved in N2-
Merck (Darmstadt, Germany) were applied. saturated UHP water and adjusted to pH 12
Carbon nanotube (Multi-walled), chitosan, by using 1.5 M NaOH solution. Then,
glucose, gold(II) chloride trihydrate and Suspension was obtained after the solution
poly(diallyldimethyl-ammonium chloride) was heated at 100 oC for half hour under a
35 wt.% in H2O were provided from Sigma- continuously stirred condition in the N2
Aldrich Co. LLC (St. Louis, USA). Iron(II) atmosphere. The solid was filtered, washed
sulfate heptahydrate, iron(II) nitrate nona- with N2-saturated UHP water and ethanol,
hydrate, and nickel(II) nitrate hexahydrate respectively. After that, it was dried
were purchased by QRëC, New Zealand. overnight at room temperature and calcined
Glutaraldehyde solution (50.0%) and o- at 700oC for 2 hours to afford a pure
Phenylenediamine (98%) were obtained NiFe2O4NPs. As same as prepared CNTs-
from Fluka (St. Louis, USA). All measure- CHIT powder, the synthesized NiFe2O4NPs
ments, voltammetry and amperometry were was dispersed in PDDA-AuNPs solution by
operated with CH instrument (CH 1230, ultrasonic for 30 minutes.
USA) with three-electrode system including 2.5 Electropolymerization of PoPD film
a glassy carbon electrode (GCE, 3 mm The PoPD film was performed by
diameter) as a working electrode, a platinum using cyclic voltammetry. The PDDA-
wire electrode (0.2 mm diameter) as a AuNPs-NiFe2O4NPs-CNTs-CHIT/GCE was
counter electrode and a Ag/AgCl electrode electropolymerized in 1 mM o-phenyl
(3M KCl) as reference electrode. The enediamine containing 0.1 M HCl by
electrochemical experiments in this work potential cycling between -0.4 and 1.3 V vs.
were examined in 5 mL of 0.05 mol L-1 Ag/AgCl at a scan rate of 50 mV s-1 with 10
phosphate buffer solution (pH 7.4) at room cycles. Ultimately, the obtained PoPD
temperature. /PDDA-AuNPs-NiFe2O4NPs-CNTs-CHIT
2.2 Preparation of carbon nanotubes– /GCE was dried at room temperature.
chitosan (CNTs-CHIT) scaffold
2.6 Preparation of glucose biosensor dominant response curve occurred in the
First, 20 µL the PDDA-AuNPs- potential range of -0.5 to 0.0 V resulting
NiFe2O4NPs-CNTs-CHIT solution was from H2O2 oxidation reaction via
dropped casting on the cleaned surface of NiFe2O4NPs as an electrocatalytic mediator,
the glassy carbon electrode and dried at a product from the enzymatic reaction
room temperature. Then, the PDDA-AuNPs- between glucose oxidase and glucose that
NiFe2O4NPs-CNTs-CHIT/GCE was actually the oxidation is higher than 0.4 V.
polymerized with an o-phenylenediamine These indicated that the modified electrode
solution by electropolymerization. Lastly, it provided obvious response current which
was dropped with 20 µL of 3 mg L-1 glucose then this active applied potential range was
oxidase (GOx) and dried at room optimized for the further experiment.
temperature, respectively. The modified 3.2 Optimization of experimental
electrode denoted as GOx/PoPD/PDDA- parameters
AuNPs-NiFe2O4NPs-CNTs-CHIT/GCE. As the active applied potential range
resulting from an electrochemical reaction
3. Results and Discussion of hydrogen peroxide led to an obvious
This work, the electrochemical oxidation curve by cyclic voltammetric
performance of the modified electrode was studies, the modified electrode was
studied by using cyclic voltammetric examined at different potential values from -
technique and the optimization of 0.45 to -0.25 V versus Ag/AgCl to gain the
experimental parameters such as applied highest current response for glucose
potential and pH loading was investigated determination as shown in Figure 2. It
by using amperometric technique. After that, showed that the current response slightly
the effect of interferences and linearity were increased from -0.45 to 0.40 V. Then, it
operated under the obtained optimal readily increased at -0.35 V. After that, the
condition. current response clearly decreased when the
potential values were -0.30 and -0.25 V.
300 These results also corresponded to 3.1 cyclic
200 b
a voltammetric studies. Thus, -0.35 V was
100
chosen in this work.
I / µA
0
-100
-200 5.0
-300 4.5
-400 4.0
-1.0 -0.5 0.0 0.5 1.0
I / µA
E/V 3.5
Figure 1. Cyclic voltammograms of the 3.0

2.5
GOx/PoPD/PDDA-AuNPs-NiFe2O4NPs
2.0
CNTs -CHIT/GCE in a) 0.05 M PBS pH 7.0 -0.50 -0.45 -0.40 -0.35 -0.30 -0.25 -0.20
and b) 10 mM glucose at scan rate of 50 mV E/V
s-1 Figure 2. Response current of the modified
electrode at a different applied potential in
3.1 Cyclic voltammetric studies 0.05 M PBS pH 7.0 with spiking 1 mM
The electrochemical performance of glucose solution under continuous stirring
the GOx/PoPD/PDDA-AuNPs-NiFe2O4NPs- condition
CNTs-CHIT/GCE was operated by using
cyclic voltammetry. As shown in Figure 1, it Each pH value of the solution for
presented cyclic voltammograms of the electrochemical analysis affected H2O2
modified electrode in 0.05 M PBS pH 7.0 oxidation of modified electrode. Therefore,
(a) and (b) with containing 10 mM glucose. pH is one of the important parameters that
These results showed that there was a its influence was also studied to obtain a
proper value for the determination of by successive addition of glucose solution in
glucose in this work. In Figure 3, the results continuous stirring 5 mL of 0.05 M PBS, pH
showed that the current response increased 7.0 under optimal condition by
from pH 6.8 to 7.0. However, the current amperometry. The results exhibited two
response was visibly decreased at higher pH wide linear ranges from 0.5 µM to 100 µM
value. It was because the neutral pH is (R2 = 0.9999) and 100 µM to 6,000 µM
similar to the physiological environment and (R2 = 0.9985) with low detection limit (1.21
can stabilize CNTs-CHIT for a long lifetime µM at S/N = 3) as shown in Figure 4. This
as well. So, pH 7.0 was selected for this suggested that the modified materials coated
study. on GCE enhance sensitivity and selectivity
of the biosensor.
5.5 Besides the study of linearity,
5.2 repeatability was determined by spiking
4.9 1 mM glucose solution under optimal
I / µA
4.6 condition using amperometry. The obtained

4.3 values from the same modified electrode
4.0 each time presented a satisfying result of
3.7 3.38 %RSD (n = 5). The modified electrodes
6.7 6.9 7.1 7.3 7.5 7.7 7.9 8.1
(n = 5) were also tested its reproducibility as
pH
same as its repeatability. They all provided
Figure 3. Effect of pH loading on glucose
acceptably indifferent values with 3.76
analysis
%RSD.
3.3 Effect of interferences 14
The effect of some possible 12 y = 0.0015x + 3.5723
interfering substances including ascorbic 10 R2 = 0.9985
acid, aspirin, caffeine, cholesterol and uric
I / µM
8 4.0
y = 0.0326x + 0.3916
acid were estimated because of their abilities 3.0 R² = 0.9999
I / µM
6
2.0
to interfere the analysis as a positive false 4 1.0
LOD = 1.21 µM
signal. In Table 1, the results of the modified 2
0.0
0 50 100
electrode showed that the ascorbic acid was 0
Concentration / µM
no interfered at ≤ 0.1 mM while the others 0 2000 4000 6000

no interfered at ≤ 0.5 mM. As the Concentration / µM
permselectivity of PoPD membrane on the Figure 4. The linear calibration plot of

PDDA-AuNPs-NiFe2O4NPs-CNTs-CHIT/GCE, glucose. Inset is the calibration at low
these interferences can be eliminated by concentration
PoPD.
4. Conclusion
Table 1. Study of interferences In this research, a glucose biosensor
Interferences Concentration / mM was successfully developed. The result of all
Ascorbic acid ≤ 0.1 modified materials including CNTs-CHIT-
Aspirin ≤ 0.5 PDDA-AuNPs-NiFe2O4NPs, PoPD and
Caffeine ≤ 0.5 GOx coated on glassy carbon electrode
Cholesterol ≤ 0.5 presented excellent repeatability (%RSD =
Dopamine ≤ 0.5
Uric acid ≤ 0.5
3.38%, n = 5), reproducibility (%RSD =
3.76%, n = 5), low detection limit (1.21 µM)
3.4 Linearity and selectivity with no interference from
The linear range of this glucose electroactive species. The glucose
biosensor was examined. The modified biosensors will be applied to analyze
electrode, GOx/PoPD/PDDA-AuNPsNi- glucose in the blood sample in near future.
Fe2O4NPs-CNTs-CHIT/GCE, was operated
Acknowledgements Spectrochim. Acta A. 2016, 153, 212-
The authors would like to 215.
acknowledge Faculty of Science, Maejo 4. Wei, Y.; Li, Y.; Liu, X.; Xian, Y.; Shi,
University for partial financial support. G.; Jin, L. Biosens. Bioelectron. 2010,
26 (1), 275-278.
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Hussain, H.; Khan, A.; Perveen, S.
Extraction efficiency of lead(II) from seaweed Gracilaria fisheri with food
additive extractants
Charuwan Khamkaew1*, Sontaya Manaboot 2
1
Program of Chemistry and Applied Chemistry, Faculty of Science and Technology,
Songkhla Rajabhat University, Muang, Songkhla 90000, Thailand
2
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand
*E-mail: csuitcharit@gmail.com
Abstract:
Food additive extractants, namely acetic acid (HOAc), citric acid (CTA), sodium
chloride (NaCl), sodium bicarbonate (NaHCO3), ethylenediaminetetraacetic acid (EDTA),
and chitosan (CTS) were examined to evaluate the maximized extraction efficiency of
lead(II) from seaweed G. fisheri. The performance under batch system was operated by
impregnating a 1.0-g clean dried-seaweed into 75 mL individual extractant then shaking at
100 rpm for 2 h at ambient temperature. It was found that EDTA was the most effective
extractant to remove lead(II) from such seaweed with the extraction efficiency up to 82.2%.
Afterward, the extraction efficiency of lead(II) in the sample using EDTA extractant was
maximized by optimizing extraction conditions, including concentration, pH and contact
time. Results showed that the highest efficiency values obtained by using the extraction
conditions under 0.1 M EDTA at pH 2.0 for 120 min. shaking were found at 83.0, 92.4 and
93.0% respectively. The extraction efficiency of the method was showed at 93.2% that the
initial and final concentrations of lead(II) in real sample were found at 6.17+0.13 and
0.42+0.06 µgL-1, respectively. Therefore, these results provide strong evidence of the
promise to apply for removal of lead(II) from G. fisheri by batch operation.
1. Introduction the observation in seaweed salad processing,

Gracilaria fisheri is agarophytic it was found that no procedures showed the
seaweed which mainly serves as a raw removal of lead(II) from the seaweed prior
material producing agar. It is widely used cooking. Therefore, the consumption of
for various purposes such as in medical, seaweed G. fisheri products has increased as
pharmaceutical, industrial and laboratory hazard to health causing from bound lead(II)
experiment, primarily due to its excellent contamination.3-5
gelling ability.1,2 It is abundant seaweed Although, several strong acids such
found along the coastal gulf of Thailand, as HCl, HNO3, H2SO4 and chelating agent
especially Pattani province and Songkhla such as EDTA demonstrated as high
lake. The previous studies have been effective extractants for removal of heavy
reported that seaweed G. fisheri can be metals from different types of seaweed.5-8
contaminated with heavy metals,3,4 However, those inorganic acids are inedible
particularly of lead(II) which was found at extractants and they are unsuitable
somewhat higher concentrations in fresh and extracting agents for removal of
dried ones.3 Because of seaweed contained contaminated heavy metals from edible
significant amounts of nutrient elements,2 seaweeds which were further consumed for
therefore, fresh and dried seaweed G. fisheri food products. Therefore, this work
have been applied for food products. One of determined which food additive extractants,
well known seaweed product in Songkhla namely, acetic acid (HOAc), citric acid
province is seaweed salad called as “Yam (CTA), sodium chloride (NaCl), sodium
Salai Phom-Ngang” which is extensively bicarbonate (NaHCO3), chitosan (CTS), and
consumed by local people and tourists. From ethylenediaminetetraacetic acid (EDTA) is
the most effective extractant to remove from the filtered seaweed sample
lead(II) from seaweed G. fisheri by batch impregnated into DI water, whereas the Cf
method with considering in extraction was obtained from the filtered seaweed
efficiency of extractants for lead(II). sample after extraction with food additive
Afterward, extraction behaviour of lead(II) extractants. All filtered seaweed samples
using the optimal extractant in batch system were subjected to digestion before
under various conditions, i.e. concentration, measurement as described in section 2.4.
pH and contact time was also further 2.3 Effect of food additive extractant,
investigated. EDTA concentration, pH and contact
time
2. Materials and Methods In these studies, the same procedure
2.1 Chemicals and materials under batch extraction experiments as
The chemicals used, i.e. Na2EDTA. described earlier was performed.
2H2O (Rankem; India), NaHCO3 (Unilab; For the study effect of extractant,
Australia), HOAc (Labscan; Thailand), CTA different types of them, i.e. HOAc, CTA,
(Univar; Australia), HNO3 (Merck, NaCl, NaHCO3, EDTA and CTS were
Germany), were of analytical grade except performed at actual pH. The concentration
for CTS (α-chitosan powder; Ebase; of extractant was controlled at 0.1 M except
Thailand) were of commercial grade. De- for 0.2% (w/v) of CTS.
ionized (DI) water (18 MΩ) obtained from a For the study effect of concentration,
Milli-Q water purification system was used different EDTA concentrations; 0.0001,
through-out. All glassware was cleaned by 0.001, 0.01 and 0.1 M at the actual pH were
soaking for 24 hours in 2 M HNO3 and carried out.
rinsed with DI water until no acid remained. For the study effect of pH, the pH
Dried seaweed G. fisheri samples range from 2.0 to 8.0 of 0.1 M EDTA
taken from Pattani Province were purchased solution was investigated.
from KhoYor market, Songkhla Province. For the study effect of contact time,
The seaweed samples were initially cleaned the shaking time started from 15 to 1440
by triplicate washing with DI water until no minutes with 0.1 M EDTA extractant at pH
foreign things found. Then, cleaned seaweed 2.0 was carried out.
samples were dried at 60 oC in hot air oven All studies, three replicates for each
for 2 hours and finally kept in zipped plastic treatment were conducted. Percentage of
bag. extraction efficiency for lead(II) from the
2.2 Batch extraction experiment seaweed at each effect studied was
A 1.0 g clean dried-seaweed weighed calculated.
into 250 mL flask with 75-mL individual 2.4 Lead(II) measurement
extractant was impregnated. The seaweed- A portion of 1.0 g filtered seaweed
extractant mixtures were left on 100 rpm sample was subjected to microwave-assisted
orbital shaker at room temperature. After 2 acid digestion9 using 5 mL of 65% HNO3
hours, each mixture was filtered. Then the acid in Teflon vessels which was placed in a
filtrated seaweed was taken to digest for pressure cooker until clear solution was
measurement. Extraction efficiency (%) for obtained.10 Quality control for the digestion
lead(II) from seaweed was calculated with method was monitored by recovery from 10
the following equation: µgL-1 of lead(II) standard solution.
C - Cf The concentration of lead(II) in clear
% Extraction efficiency  i  100 (1) solution after digestion were measured by
Ci
differential pulse anodic stripping
where Ci and Cf were the concentrations of
voltammetry (DPASV) at a hanging
lead(II) in seaweed before and after
mercury drop electrode (HMDE) as working
extraction, respectively. The Ci was obtained
electrode, a double junction Ag/AgCl/3 M
KCl as reference electrode, and a Pt wire as dominated such as the ion exchange7
auxiliary electrode. The high pure mercury interaction between Pb2+ and H+ ions from
(99.9999%) was used. The votammetric HOAc or CTA, the metal sorption of lead(II)
measurement made using computrace and through the amine group (-NH2)13,14 of the
connected to an electrode standard under CTS, and the complexed metal ion
797 VA series (Metrohm, Switzerland). All formations between Pb2+ ion and
experiments were carried out at ambient bicarbonate (HCO3-) or chloride (Cl-) of
temperature. NaHCO3, and NaCl, respectively.5
The operating parameters used for
100
lead(II) measurement by DPASV were listed
Extraction efficiency (%)

in Table 1.11 80
60
Table 1. Operating parameters for lead(II)
measurement 40
Parameters Operating value
Initial purging time (s) 300 20
Stirring speed (rpm) 200
0
Start potential (V) -0.65 NaCl HOAc NaHCO3 EDTA CTA
End potential (V) -0.25 CTS
Deposition time (s) 90 Extractants
Equilibration time (s) 5.0 Figure 1. Effect of food additive extractant
Voltage step (mV) 6.0 on the extraction process
Pulse amplitude (mV) 50
Pulse time (s) 0.04
Sweep rate (mV/s) 14 3.2 Effect of EDTA concentration
Peak potential lead(II) (V) -0.38 In this experiment, the following
EDTA concentrations were tested at the
In determination, a portion of 1.0 mL actual pH, i.e. 0.0001, 0.001, 0.01 and 0.1 M
clear solution after digestion was pipetted and the results obtained are shown in Figure
into measuring vessel contained a 10-mL DI 2.
water and a 1mL 0.2 M CH3COONH4 pH 100
7.5.11 Mixture solution was purged with high 90

80
pure N2 gas to remove oxygen before 70
measurement. 60
50
40
3. Results and Discussion 30
20
3.1 Effect of food additive extractant 10
According to removal of lead(II) 0
0.0001 0.001 0.01 0.1
from seaweed G. fisheri is based on the
EDTA concentration (M)
extraction mechanism, the extraction
Figure 2. Effect of EDTA concentration on
efficiency of different types of extractants
the extraction process
was investigated. The obtained results
(Figure 1) show that EDTA solution was the It can be seen that the percent
most effective extractant to remove lead(II) extraction efficiency increased with EDTA
from seaweed with extraction efficiency concentration7 reaching at the highest
over 82.2%. The way that EDTA is the concentration of 0.1 M with extraction
optimal chelating agent for binding Pb2+ ion efficiency over 83.0%. Indicating that such
by complexed metal ion and being stable concentration of EDTA is enough for Pb-
formation of Pb-EDTA complex7,12 during EDTA complex formation completely.
extraction process. Meanwhile, the 3.3 Effect of pH
interactions between lead(II) and other For this investigated, the pH ranged
extractants were much more lower between 2 to 8 were tested. The results
obtained are shown in Figure 3. This figure approximately 93% with 0.1 M EDTA at pH
reveals that percentage of lead(II) removal 2.0. The removal of lead(II) rapidly
in extraction process increased with increased for the first 120 minutes and
decreasing pH reaching at the pH 2 with remained nearly constant thereafter. The
extraction efficiency over 92.4%. At the necessary time to reach this constant or
lower pH, especially at pH 2, the higher equilibrium of desorption was about 2 hours.
amount of protons in EDTA dissociation These observations were in agreement with
preferred to suggest an ion exchange the works of Hammini et al.,7 Niinae et al.,12
between protons and Pb2+ ion retaining at Vaudrias et al.,16 and Bermond and
the cell wall of seaweed. The results were in Ghestem.17
agreement with these reports by Hammini et 3.5 Quantification method, linear range,
al.7 and Covelo et al.15 limit of detection, precision and accuracy
The quantification of lead(II) using
100 the operating parameters (Table 1) by
90
DPASV was measured. The standard
80
addition method was used. The voltammo-
70
60 grams obtained from DPASV system for a
50 portion solution of seaweed sample in
40 supporting electrolyte of 0.2 M NH4OAc pH
30 7.5 containing various concentrations of
20
lead(II) which was supported to compute
10
0
from the standard addition method were
2 3 4 5 6 7 8 depicted in Figure 5.
pH 120n
Figure 3. Effect of pH on the extraction

Cd
process 100n
Pb(II)
3.4 Effect of contact time 80.0n
The treatment time to remove

I (A)
60.0n
lead(II) by extraction with 0.1 M EDTA at
pH 2.0 under batch experiments were 40.0n
performed at 15 to 440 minutes.

20.0n
100
90
-5.00e-1 -4.50e-1 -4.00e-1 -3.50e-1
Extraction effiency (%)
80 U (V)
70
Figure 5. Voltammograms obtained from
60
50
DPASV system for lead(II) measurement in
40 seaweed G. fisheri
30
20 In the voltammograms, peak currents
10 2+
of Pb ion were proportional to its lead(II)
0
0 180 360 540 720 900 1080 1260 1440 concentration.
Contact time (min) For quantification of lead(II), the
Figure 4. Effect of contact time on the standard additions method was achieved.
extraction process The calibration curve in the range from 0.83
to 12.50 µgL-1 of lead(II) was then
Figure 4 shows the relationship constructed by plotting peak current against
between the extraction efficiency of lead(II) lead(II) concentration and the plot was
from contaminated seaweed and the depicted in Figure 6. The curve linearity
treatment time. The extract of lead(II) is with a correlation (r2) of 0.9999 and a slope
of 5.6975 was excellent demonstrated. and Technology, Songkhla Rajabhat
Under this system for triplicate University are gratefully acknowledged.
measurements using 1.8 µgL-1 of lead(II),
the reproducibility was showed the RSD of References
1.22%. The LOD (3σ) obtained by 1. Romero, J. B.; Villanueva, R. D.;
undertaking 10-replicate aliquots of Milli-Q Montaño, M. N. E. Bioresource
water was found at 0.600 µgL-1. Technol. 2008, 99, 8151-8155.
2. Noriziah, M. H.; Ching, Y. C. Food
80.00 Chem. 2000, 68, 69-76.
3. Netten C. V.; Cann S. A. H.; Morley D.
Peak curent (nA)
60.00 R.; Netten, J.P.V. Sci. Total. Environ.

2000, 255, 169-175.
40.00 4. Chaisuksant, Y. Environ. Technol.
y = 5.6975x + 6.3727
2003, 24, 1501-1508.
r2 = 0.9999
20.00 5. Stirk, W. A.; Staden, J. V. Bot. Mar.
2002, 45, 9-16.
0.00 6. Zhou, J. L.; Huang, P. L.; Lin, R. G.
-3 0 3 6 9 12 15 Environ. Pollut. 1998, 101, 67-75.
Concentration of Pb(II) 7. Hammaini, A.; González, F.; Ballester,
Figure 6. Standard addition calibration A.; Blázquez, M.; Muñoz, J. A. J.
curve for the remained amount of lead(II) in Environ. Manage. 2007, 84, 419-426.
seaweed G. fisheri 8. Gong, R.; Ding, Y.; Liu, H.; Chen, Q.;
Liu, Z. Chemosphere. 2005, 58, 125-
The accuracy of digestion method for 130.
quality control of lead(II) extraction was 9. Loring, D. H.; Rantala, R. T. T. Earth-
evaluated on recovery. The recovery Sci. Rev. 1992, 32, 235-283.
obtained was 92.3%. The extraction 10. Khamkaew, C.; Manaboot, S. Appl.
efficiency of the method was showed at Mech. Mater. 2016, 855, 3-8.
93.2% that the initial and final 11. Suitcharit, C.; Mohamed, N.; Lim, P.
concentrations of lead(II) in real sample E.; Sirinawin, W. J. Phy. Sci. 2005, 16,
were found at 6.17+0.13 and 0.42+0.06 69-83.
µgL-1, respectively. 12. Niinae, M.; Nishigaki, K.; Aoki, K.
Mater.Trans. 2008, 49, 2377-2382.
4. Conclusion 13. Cervera, M. L.; Arnal, M. C. Anal
The use of food additive extractants Bioanal. Chem. 2003, 375, 820-825.
for removal of lead(II) from seaweed G. 14. Verbych, S.; Bryk, M.; Chornokur, G.;
fisheri by batch method was examined. Fuhr, B. Sep.Sci.Technol. 2005, 40,
EDTA shown excellent ability for lead(II) 1749-1759.
removal with an extraction efficiency of 15. Cavelo, E .F.; Vega, F. A.; Andrade, M.
82.2%. The variations of effect on extraction L. J. Hazard. Mater. 2008, 159, 342-
behaviour of lead(II) in EDTA solution was 347.
also investigated. The range of highest 16. Vaudrias, E.; Fytianos, K.; Bozani, E.
efficiency obtained from 0.1 M EDTA at pH Globalnest: the Int. J. 2002, 4, 75-83.
2.0 for 120 minutes shaking was found 17. Bermond, A.; Ghestem, J. P. in Heavy
between 83.0 to 93.0%. The efficiency of Metals release in Soils: Chapter 6
the extraction method was showed at 93.2%. Kinetic Study of Trace Metal EDTA-
Desorption from Contaminated Soils;
Acknowledgements Selim, H.M.; Spark, D.L.1st Eds. Taylor
Research facilities provided by the & Francis group, 2001, 131-147.
Program of Chemistry, Faculty of Science
Detection of polycyclic aromatic hydrocarbons by specific
thiol-functionalized silver nanoparticle films using surface-enhanced
Raman scattering
Sathita Taksadej1,2, Kanet Wongravee2, Sanong Ekgasit2, Prompong Pienpinijtham2,*
1
Program in Petrochemistry and Polymer Science, Faculty of Science, Chulalongkorn University,
254 Phyathai Road, Patumwan, Bangkok 10330, Thailand
2
Sensor Research Unit (SRU), Department of Chemistry, Faculty of Science,
Chulalongkorn University, 254 Phyathai Road, Patumwan, Bangkok 10330, Thailand
*E-mail: prompong.p@chula.ac.th
Abstract:
Surface-enhanced Raman scattering (SERS) is widely used to detect trace analytes by
using metal nanoparticles with plasmonic oscillation as a substrate where the analytes are
adsorbed and affected by the enhanced electromagnetic field resulting in the enhancement of
Raman intensity. Although metal nanoparticle colloid can promote high Raman intensity,
they are unstable and inconvenient to use. Metal nanoparticle film is another way to create
SERS substrate that is stable, portable, and easy to use. In this study, we use silver
nanoparticle film fabricated by a Langmuir-Blodgett method as a SERS substrate to detect
polycyclic aromatic hydrocarbons (PAHs). In order to detect PAHs by SERS, the adsorption
of PAHs on the silver nanoparticle film is required. Therefore, the surface of silver
nanoparticle film is functionalized with various types of thiols (aromatics and aliphatics) to
create a hydrophobic layer for trapping PAHs. The fabricated films are stable as they can be
kept for, at least, 1 month before measurements. The results indicate that chrysene,
pentacene, pyrene, perylene, and triphenylene can penetrate into the surface of functionalized
silver nanoparticle film as Raman spectra show their own characteristic peaks and can be
detected with a limit of detection reaching to 10-8 M.
1. Introduction methods used to detect PAHs e.g., gas

Polycyclic aromatic hydrocarbons chromatography with mass spectroscopy
(PAHs) are compounds built from two or (GC-MS), gas chromatography with flame
more aromatic rings. They are neutral and ionization detector (GC-FID), high
non-polar. PAHs are mostly produced from performance liquid chromatography (HPLC),
incomplete combustion of organic and fluorescence spectroscopy.
substances and can also be produced when Raman spectroscopy is one type of
organic matters are transformed into fossil vibrational spectroscopies which investigate
fuels. The main sources of PAHs in vibrations of chemical bonds in compounds.
environments are from human activities e.g., Raman spectroscopy observes the shift in
wood burning, biofuel combustion, wavelength of scattered light after
emissions from vehicles, and smoking. interacting with the molecules. These
Burning solid fuel, such as coal in scattered lights are inelastic as they lose or
household, is the major PAHs emission in gain energy into the vibrations of sample
developing countries. PAHs usually stay in a molecules. The resulted bands correspond to
complex form with heavy metals, which the specific molecular vibration. Thus, each
makes them difficult to be decomposed.1 substance has its own Raman spectral
PAHs are considered as carcinogen, pattern called “molecular fingerprint”.
mutagen, and teratogen.2 We can receive However, the intensity of Raman spectrum
them into our body through breathing, scattering is normally low.
eating, and skin contact. There are several
Surface-enhanced raman scattering Perylene, pyrene, triphenylene were
(SERS) is a technique which helps increase purchased from Dr. Ehrenstorfer GmbH. All
the intensity of Raman signal. Metal chemicals were used as received without any
nanoparticles are used as SERS substrates to further purification. DI water was employed
enhance the signal through a phenomenon as a solvent for preparing aqueous solutions.
called “localized surface plasmon resonance 2.2 Synthesis of AgNPs
(LSPR)”. The oscillation of electron clouds Silver nanoparticle colloid was
on metal surfaces is so-called “plasmon”. By synthesized using previously proposed
applying external light with the frequency of synthesis method.5 Briefly, 90 milligrams of
plasmonic oscillation, the plasmon starts to silver nitrate (AgNO3) was dissolved in 500
resonate and create an LSPR phenomenon. mL DI water, and then heated until boiling.
This phenomenon makes the A 10 mL solution of 1% trisodium citrate
electromagnetic field around the metal was suddenly added into the boiling AgNO3
surface enhance greatly. Then, the solution. The mixture was stirred vigorously
molecules that are adsorbed on the metal and kept boiling for 1 hour. The mixture
surface scatter light more effectively. The color changed from yellow to grey. After the
area in which electromagnetic field is more mixture was cooled down to room
intense is called a “hot spot”.3 Metal temperature, the volume of the mixture was
nanoparticles are commonly used in a adjusted back to 500 mL using DI water.
colloid form which is inconvenient to store. 2.3 Fabrication of AgNP film
Nevertheless, it is not much stable compared The glass slide was cut into the size
to a solid form. Fabricating film as solid of 0.5 × 0.5 cm2, then immersed in a piranha
SERS substrates is another method to create solution (H2O2:conc.H2SO4 = 3:7) and boiled at
a stable and convenient substrate for sample 100°C for 30 minutes. After that, the glass slide
detection. was rinsed with DI water and ethanol. The
In this study, we propose the cleaned glass slide was soaked into a 2% of 3-
detection of PAHs using SERS technique. mercaptopropyltrimethoxysilane (3-MPTMS) in
Silver nanoparticles (AgNPs) are chosen to ethanol solution overnight. Then, it was rinsed
be a SERS substrate. We functionalize the off by ethanol to remove excess 3-MPTMS.
surface of AgNPs with thiol to create a The AgNP films were fabricated
suitable non-polar area for PAHs to attach using a LB method. Briefly, 0.5 μL of thiol
on a substrate. Also, we fabricate AgNPs was added into 10 mL of hexane. The
substrate into films by using a Langmuir- solution was gently poured onto 20 mL of
Blodgett (LB) method4 to make them silver nanoparticle colloid. The interface
convenient for using and storing. between hexane and water was then created.
After that, 12 mL of ethanol was slowly
2. Materials and Methods injected into an aqueous phase. Silver mirror
2.1 Chemicals can be observed in this step. Hexane was
Ethanol, hydrogen peroxide (H2O2), then slowly evaporated at ambient
and sulfuric acid (H2SO4) were purchased conditions, which left the AgNP film on the
from Merck. Octanethiol (OT), pentanethiol (PT), surface of aqueous phase. The AgNP film
dodecanethiol (DT), naphthalenethiol (NT), was transferred to the glass slide by dipping
phenylethanethiol (PhT), chrysene, and 3- the modified glass slide into the aqueous
mercaptopropyl trimethoxysilane (3-MPTMS), phase and then lifted vertically to cover the
were purchased from Sigma-Aldrich Co., Ltd. glass slide with AgNP film. The dried film
Silver nitrate (AgNO3), trisodium citrate was soaked in hexane for 15 min to remove
dihydrate, hexane and naphthalene were excess chemicals. Finally, the film was dried
purchased from Aencore Chemical Co., at room temperature and kept in desiccators.
Ltd., Carlo Erba Reagents S.A.S., Fischer
Scientific, and JT Baker, respectively.
2.4 SERS measurement charges of AgNPs surface which improves
The prepared AgNP films were miscibility of AgNPs in non-polar solvent.
separately immersed into each PAHs However, AgNPs are too large. They cannot
solution (PAHs dissolved in ethanol with escape from an aqueous phase and enter into
different concentrations) for 15 minutes, and hexane phase, which makes them stuck at
then rinsed gently with ethanol before the interface. It results in the observed silver
measuring with DXR Raman microscope mirror film. Without thiols, the AgNPs film
(Thermo Scientific) with a 532 nm cannot be fabricated.
excitation laser. Laser power at 10 mW was
used with 50 μm slit aperture. All spectra
were collected using exposure numbers of
16 and exposure time of 2 seconds. Each
sample was measured at 5 different areas on
the film.
3. Results and Discussion

3.1 Characterization of AgNPs
Figure 2. SERS spectra of thiol-modified

AgNP film
To find a suitable substrate for

detecting each PAH, the surfaces of AgNPs
were functionalized by PT, OT, DT, PhT, and
NT. SERS spectra collected from each
modified film are presented in Figure 2.
3.3 Detection of PAHs using different
Figure 1. UV-visible spectrum of silver thiol-modified AgNP films
nanoparticle colloid The functionalization of AgNPs with
thiols creates a hot spot with non-polar
The advantages of AgNPs are that surfaces which allow the detection of PAHs.
they give the highest electric field Figure 3 shows SERS spectra of AgNPs film
enhancement in visible region and can be soaked in different PAHs at 1.0 × 10-4 M.
easily prepared using few chemicals and We cannot observe any peak shift of thiol-
simple procedure.5 From Figure 1, silver modified AgNP films characteristic peaks
nanoparticle colloid shows the dipole after soaked in PAHs solutions indicating
plasmonic band at 430 nm which indicates that there is no strong interaction between
that the size of synthesized silver substrate and PAHs. Thiol can induce PAHs
nanoparticles average is approximately 70 into the nonpolar environment by van der
nm. The obtained diameter size is in the Waals and/or hydrophobic forces. Each
appropriate range for using as SERS PAHs may be detected by different kinds of
substrate. thiol-modified AgNPs film according to the
3.2 Characterization of thiol-modified structure of alkyl/aryl part of thiols as their
AgNP films property (such as chain length, steric effect,
By a LB method, AgNPs are pushed and aromaticity) might be suitable to some
up to the water/hexane interface due to a PAHs.
decrease in polarity of water by an ethanol
addition. Thiols in hexane phase reduce
Figure 3. SERS spectra collected from (a) PT-, (b) OT-, (c) DT-, (d) PhT-, and (e) NT-
modified AgNP films after soaked with solutions of different PAHs at the concentration of
1.0 × 10-4 M
Chrysene can be detected by 3 types stretching; 1381 cm-1 peak corresponding to

of thiol-modified AgNP film. The skeletal ring vibration; and 1238 cm-1 peak
characteristic peak at 1158 cm-1 corresponding to =C ̶ H in plane
corresponding to =C ̶ H in plane deformation deformation vibration
was found in PT-modified AgNP film (Figure Lastly, triphenylene can be detected
3A). The characteristic peak at 1294 cm-1 in PhT- and DT-modified AgNP film
corresponding to =C ̶ H in plane deformation (Figure 3C and 3D) as it shows
was found in OT-modified AgNP film (Figure characteristic peaks at 1568, and 1368 cm-1
3B). The characteristic peak at 1382 cm-1 corresponding to aromatic ring stretching;
corresponding to the vibration of skeletal and 1294 cm-1 peak corresponding to C ̶ H
ring in chrysene was found in DT-modified bending.
AgNP film (Figure 3C). 3.4 Determination of PAH concentrations
Pentacene can be detected by PhT- To determine the sensitivity of thiol-
modified AgNP film as it shows the modified AgNP film, SERS spectra of
characteristic peak at 793 cm-1 corresponding thiols-modified AgNP films soaked in the
to =C H̶ out of plane deformation vibration solutions of PAHs with various
(Figure 3D). concentrations were measured. The
Perylene can be found in all types of concentrations were varied into 7 conditions
thiol-modified AgNP film as it shows of 1.0 × 10-4, 5.0 × 10-5, 1.0 × 10-5, 5.0 ×
characteristic peaks at 1568 and 1370 cm-1 10-6, 1.0 × 10-6, 5.0 × 10-7, and 1.0 × 10-7 M.
corresponding to aromatic ring stretching; The chosen pairs of PAHs and thiol-
and 1299 cm-1 corresponding to C ̶H modified AgNP are shown in Figure 4 and
bending. the limit of detection was obtained from the
Pyrene can be detected in PT and OT calibration curve between the relative
modified AgNP film (Figure 3A and 3B) as intensity of the marker band of PAHs and
it shows characteristic peaks at 1622, 1587, thiol-modified AgNP film and concentration
and 1502 cm-1 corresponding to C=C ring (an example of perylene on OT-modified Ag
Figure 4. SERS spectra of (a) chrysene on PT-modified AgNP film, (b) pentacene on PhT-
modified AgNP film, (c) perylene on OT-modified AgNP film, (d) pyrene on PT-modified
AgNP film, (e) triphenylene on PhT-modified AgNP film
film is shown in Figure 5). The limit of For further analysis, the mixture of
detection of chrysene and pyrene on PT- PAHs might be detected and use
modified AgNP film are 1.1308 × 10-8 and mathematical method such as chemometric
2.1073 × 10-4 M. The limit of detection of to help analyzing the result as some of PAHs
pentacene and triphenylene on PhT- characteristic peaks are overlapped with
modified AgNP film are 5.7562 × 10-6 and each other which makes it difficult to be
5.3018 × 10-4 M. The limit of detection of determined.
perylene on OT-modified AgNP film is
5.2608 × 10-5 M. 4. Conclusion
SERS can detect PAHs i.e.,
chrysene, pentacene, perylene, pyrene, and
triphenylene, at low concentrations. For this
purpose, AgNPs were functionalized with
different kinds of thiols and formed into an
LB film on a glass slide. The compatible
pairs of PAHs and thiol-modified AgNP
film are chrysene with PT-, OT-, and DT-
modified AgNP film; pentacene with PhT-
modified AgNP film; perylene with all types
of thiols-modified AgNP films; pyrene with
PT-, and OT-modified AgNP film; and
triphenylene with DT-, and PhT- modified
AgNP film.
The advantages of SERS technique
Figure 5. Calibration curve of perylene on are no sample pretreatment, a convenient
OT-modified AgNP film
in situ analysis with inexpensive expenses 2. Boström, C.; Gerde, P.; Hanberg, A.;
and sensitive results. Jernström, B.; Johansson, C.; Kyrklund,
T.; Rannug, A.; Törnqvist, M.; Victorin,
Acknowledgements K.; Westerholm, R. Environ. Health
This research was funded by Perspect. 2002, 110, 451-488.
Ratchadapisek Sompoch Endowment Fund 3. Petryayeva, E.; Kru, U. J. Anal. Chim.
(2016), Chulalongkorn University (CU-59- Acta 2011, 706, 8-24.
015-FW). 4. Pienpinijtham, P.; Han, X. X.; Ekgasit,
S.; Ozaki, Y. Phys. Chem. Chem. Phys.
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The synthesis of metal/PDDA/rGO catalyst for detection of nitrite
Aunyarut Paisanpisuttisin, Chaiya Prasittichai, Wilai Siriwatcharapiboon*
Department of Chemistry, Faculty of Science, Kasetsart University,
Chatuchak, Bangkok 10900, Thailand
*E-mail: fsciwls@ku.ac.th
Abstract:
The metal/poly(diallydimethylammonium chloride)/reduced graphene oxide
(metal/PDDA/rGO) modified glassy carbon electrodes (GCEs) were successfully prepared by
a chemical reduction. The electrodes were used to determine various concentrations of nitrite
by cyclic voltammetry. The formation and composition of metal/PDDA/rGO were
characterised by scanning electron microscopy (SEM) and energy dispersive x-ray
spectroscopy (EDS). The electrocatalytic response of nitrite oxidation measured on
Ni/PDDA/rGO modified electrode is higher than that of Fe, Zn and Co, respectively. The
calibration curve is linear over two concentration ranges from 0.8 to 4.0 µM and 4.0 to 20
µM. The linear regressions are ip (μA) = 0.05312C (μM) – 0.04971 and ip (μA) = 0.08225C
(μM) – 0.14747, respectively. The modified GCE was then employed to determine the
concentration of nitrite in real sample.
1. Introduction suffered from poor sensitivity and high

Nitrite is a well-known threat to overpotential. The modification of the
human health. It is potentially carcinogenic electrode surface is an important issue in
agents and a cause of “blue baby electrochemical detection of nitrite. The
syndrome”.1 Nitrite is highly involved in main purpose of electrode modification is to
human life since it is widely used as a facilitate an electron transfer between the
preservative to cure meat in food industry electrode surface and electroactive species.8
and a corrosion inhibitor in industrial water. To achieve the purpose, carbon-based
The Directive of the Scientific Committee nanostructured materials, such as multi-
on Food and the European Food Safety walled carbon nanotubes, graphene and
Authority has concluded that 50-100 mg graphene nanocomposites, have been
nitrites per kilogram meat may be safe to use applied on the electrode surface. The
for many meat products.2 The World Health graphene oxide (GO) and reduced graphene
Organisation limits the contaminant level in oxide (rGO) incorporated with precious
drinking water such that the concentration of metals (Au,9,10 Pt,11,12 Pd13), non-precious
nitrite must not exceed 3 mg/L.3 Therefore, metals ( Ni,14 Co,15,16 Fe17,18), metal oxides
its levels must be carefully monitoring in (Co3O4,19 Fe2O320) and polyelectrolyte
drinking water, groundwater and meat (PDDA18,21) have been employed for the
products. nitrite oxidation.
There are many techniques that have It is highly interested to develop a
been developed in order to determine the use of non-precious metal electrocatalysts
concentration of nitrite, including chromato- towards the nitrite oxidation due to their low
graphy,4 chemiluminescense,5 spectroscopy6 cost, non-toxicity, long-term stability and
and electrochemistry.7 Among these good electrocatalytic acivities. The PDDA is
techniques, the electrochemical technique is a linear cationic polyelectrolyte which its
widely used due to its rapid response, positive surface charge can provide more
simplicity, high sensitivity, selectivity and active sites and can enhance an electrostatic
low detection limit; however, the ability of a attraction for the nitrite adsorption.18,21,22
bare GCE and a conventional electrode is
In the present work, the Ni, Co, Zn with high-purity nitrogen. The glassy carbon
and Fe/PDDA/rGO modified GCEs for the electrode (0.071 cm2) was polished using
electrocatalytic activity on the nitrite slurries of increasingly finer alumina
oxidation are compared. The metal/PDDA/ powder (1.0, 0.3 and 0.05 μm, Buehler),
rGO was prepared by a chemical reduction. followed by sonication in ultrapure water for
The electrochemical measurements were 5 min. 4 µL of freshly sonicated
employed by using cyclic voltammetry nanoparticle suspension were pipetted onto
(CV). The modified electrodes were applied the surface of the GCE and dried under Ar
for the nitrite determination in real sample. atmosphere at room temperature. The
counter and reference electrodes were a Pt
2. Materials and Methods wire and Ag/AgCl electrode, respectively.
2.1 Reagents and apparatus
The chemicals used were: graphene 3. Results and Discussion
oxide dispersion in water 2 mg/mL (GO, 3.1 Morphological and spectra characteri-
Sigma-Aldrich), poly (diallyldimethyl- sation of Ni/PDDA/rGO nanoparticles
Ammonium chloride) (PDDA, Sigma
Aldrich), potassium dihydrogen phosphate
(KH2PO4, Merck), dipotassium hydrogen
phosphate (Na2HPO4 , Merck), potassium
hexacyanoferrate(II)tri-hydrate
(K4[Fe(CN)6]·3H2O,Merck), cobalt(II)
chloride hexahydrate (CoCl2·6H2O, Merck),
nickel(II) acetatetetrahydrate (C4H6NiO4
·4H2O, Merck), and zinc chloride (ZnCl4,
Merck).
All the electrochemical measure-
ments were performed on Potentiostat
Galvanostat (Autolab). A QUANTA 450
scanning electron microscope was employed Figure 1. (a) SEM image of Ni/PDDA/rGO
for SEM images and EDS spectra. (b) SEM/EDS element mapping of
2.2 Synthesis of metal/PDDA/rGO Ni/PDDA/rGO and (c) EDS spectrum of
nanoparticles Ni/PDDA/rGO
The metal/rGO/PDDA nanoparticles
were prepared by the chemical reduction. Figure 1 shows a scanning electron
0.5 mL GO, 0.3 mL ultrapure water and 0.01 microscopy image and EDS of
M metal salt were mixed together and were Ni/PDDA/rGO used in this study. As shown
sonicated for 30 min. 1 mL sodium in Figure 1 (a), there is wrinkle morphology
borohydride was then added to reduce the of rGO nanosheets, indicating that the
mixture. The mixture was further sonicated reduction process does not cause the
for 30 min and left for 2 h. After that the morphology of rGO to be damaged. It is also
solution of PDDA was added into the observed a relative uniform size distribution
mixture and stirred for 30 min. The mixture and high monodispersity of the Ni
was centrifuged twice at 12,000 rpm for 30 nanoparticles deposited onto rGO sheets.
min. The metal/PDDA/rGO nanoparticles The spherical-shaped particles have an
were redispersed in the solution of ethanol: average size of 60-80 nm. The chemical
ultra-pure water 1:1 ratio. composition of the nanocomposites was
2.3 Electrochemical measurements determined by the energy dispersive
All experiments were carried out at spectrum (EDS). The result in Figure (b)
room temperature. Prior the electrochemical shows the distribution of Ni nanoparticles on
measurements, electrolyte was saturated rGO. Figure 1 (c) verifies the presence of
Ni, C, N and O elements (other peaks The plot of peak current versus
originate from the substrate used). nitrite concentration for individual
3.2 Cyclic voltammetry measurements determination demonstrates a linear
To investigate the performance of
correlation, as shown in Figure 4 (a) and (b).
the modified electrodes, the electrochemical
behavior of nitrite ions was first studied on
the electrode surface with different metals.
The potential range utilised cover the
double-region and nitrite oxidation region.
The cyclic voltammograms of nitrite
oxidation at the different electrodes into 20
μM NaNO2 solution (0.1 M PBS; pH 6.0)
are shown in Figure 2.
Figure 3. Cyclic voltammograms of

Ni/PDDA/rGO in N2 saturated 0.1 M PBS
(pH 6.0) with different concentrations of
NaNO2. Scan rate = 0.05 V s-1
Figure 2. Cyclic voltammograms of (a)

GCE, (b) Fe/PDDA/rGO, (c) Zn/PDDA/
rGO, (d) Co/PDDA/rGO and (e) Ni/PDDA/
rGO in N2 saturated 20 μM NaNO2 + 0.1 M
PBS (pH 6.0). Scan rate = 0.05 V s-1
The electrodes show the irreversible

peak of nitrite oxidation at 0.85 V. The onset
potential of Ni/PDDA/rGO is at more
negative potential than that of Co, Zn, GCE
and Fe, respectively. The anodic peak
current of Ni/PDDA/rGO is higher than that
of Co, Fe, Zn and GCE, respectively. The
introduction of metal and nitrogen atoms can
increase the surface area and conductivity of
the rGO sheets, promoting the nitrite
oxidation.
The Ni/rGO/PDDA was used to
Figure 4. Plots of peak current versus
determine the concentration of nitrite in PBS
concentration of (a) 0.8 - 4.0 μM and (b) 4.0
(0.1 M, pH 6.0). The influence depending on
– 20.0 μM of NaNO2, Error bars represent
nitrite reduction on the response of the
the standard deviation for three independent
Ni/PDDA/rGO electrode was observed. The
measurements
nitrite oxidation peak increases gradually
with the concentrations of nitrite, as shown
The peak current is linearly related to
in Figure 3.
nitrite concentration, over a range of 0.8 -
4.0 μM and 4.0 – 20.0 μM with a correlation
coefficients of 0.9886 and 0.9927, GCE has promising application in
respectively. The detection limit was determining nitrite in food samples.
estimated to be 0.7568 μM (S/N = 3). The
calibration curve between the peak current Acknowledgements
(I) and the nitrite concentration (C) can be The authors thank Kasetsart
described as follows: ip (μA) = 0.0497C University Research and development
(μM) – 0.0377 and ip (μA) = 0.0823C (μM) institute (KURDI), Center of Excellence for
– 0.1475. Innovation in Chemistry (PERCH-CIC) and
3.3 Determination of nitrite in real sample Department of Chemistry, Faculty of
Science, Kasetsart University, Bangkok for
Table 1. Determination of nitrite in sausage support and funding of this research.
sample
Sample Added Found Recovery References
(μM) (μM) (%) 1. Manassaram, D. M.; Moll, D. M. 2006,
Sausage - 6.6741 - 114, 320.
2.4 9.1130 100.4 2. European Food Satefy Authority. EFSA
J. 2010, 5 (8),1538-1539.
To evaluate the analytical appli- 3. World Health Organization. Guide
cability of the prepared electrode, the Lines for Drinking-Water Quality:
Ni/PDDA/rGO was tested to determine the Geneva, 2004.
concentration of nitrite in the sausage 4. Niedzielski, P.; Kurzyca, I.; Siepak, J.
sample. The sample was prepared by boiling Anal. Chim. Acta 2006, 577, 220.
in ultrapure water at 70°C for 2 h. The 5. Lagalante, A. F.; Greenbacker, P. W.,
sample was then filtered and centrifuged at Anal. Chim. Acta 2007, 590, 151
8000 rpm for 10 min. The supernatant was 6. Yao, W.; Byrne, R. H.; Waterbury, R.
kept cold for 20 min and then used as D. Environ. Sci. Technol. 1998, 32,
sample solution. A standard addition method 2646.
was used to estimate the accuracy of this 7. Gopalan, A. I.; Lee, K. P.; Komathi, S.
method. The evaluation of the results was Biosens. Bioelectron. 2010, 26, 1638.
shown in Table 1. According to the result, 8. Bagheri, H.; Hajian, A.; Rezaei, M.;
the obtained recovery is acceptable, Shirzadmehr, A. J. Hazard. Mater.
indicating that the proposed method has 2017, 324, 762.
great potential in the practical sample 9. Zhang, Y.; Wen, F.; Tan, J.; Jiang, C.;
analysis. Zhu, M.; Chen, Y.; Wang, H. J.
Electroanal. Chem. 2017, 786, 43.
4. Conclusion 10. Jiang, J.; Fan, W.; Du, X. Biosens.
In this work Ni/PDDA/rGO modified Bioelectron. 2014, 51, 343.
GCE shows the greater electrocatalytic than 11. Yang, B.; Bin, D.; Wang, H.; Zhu, M.;
that of the other electrodes. The Yang, P.; Du, Y. Colloids Surf. A 2015,
Ni/PDDA/rGO has been successfully 481, 43.
employed for the determination of nitrite at 12. Chen, D.; Zhuang, X.; Zhai, J.; Zheng,
different concentrations. The current Y.; Lu, H.; Chen, L. Sens. Actuators, B
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μM (S/N = 3). Finally, the modified Electroanal. Chem. 2017, 796, 9.
electrodes show the satisfactory result in 15. Cui, L.; Pu, T.; Liu, Y.; He, X.
determination of nitrite in sausage sample, Electrochim. Acta 2013, 88, 559.
suggesting that the Ni/PDDA/rGO modified
16. Luo, X.; Pan, J.; Pan, K.; Yu, Y.; 19. Liu, H.; Guo, K.; Lv, J.; Gao, Y.; Duan,
Zhong, A.; Wei, S.; Li, J.; Shi, J.; Li, X. C.; Deng, L.; Zhu, Z. Sens. Actuators B
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X.; Zhang, W.; Zhang, J.; Ye, J. J. K.;Sekar, C.; Wilson, J.; Kim, S. J.
Electroanal. Chem. 2011, 651, 12. Applied Catalysis, B 2014, 148-149, 22.
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Global calibration model for determination of glucose in non-alcoholic
beverages using near infrared spectroscopy combined with chemometrics
Sureerat Makmuang, Nontawat Sricharoen, Prompong Pienpinijtham,
Sanong Ekgasit, Kanet Wongravee*
Sensor Research Unit, Department of Chemistry, Faculty of Science,
Chulalongkorn University, Bangkok 10330, Thailand
*E-mail: kanet.w@chula.ac.th
Abstract:
Near infrared (NIR) spectroscopy combined with chemometrics has been employed to
determine and quantify amount of glucose. By conventional way, a calibration curve is
required to build a statistical model for quantitative analysis of a system. Several calibration
curve might be required for several systems. To avoid these complicated set up, we propose a
new alternative technique called “global model” for quantitative analysis of glucose in
nonalcoholic drink. To build a global model, it involves the extraction and selection of
significant spectra from water and glucose system. Then, a global model was constructed
using the selected spectra. This model was used to calculate concentrations of glucose in non-
alcoholic drinks such as tea and coffee. Partial least-squares regression (PLSR) was used to
build calibration models to calculate concentrations of glucose. The corresponding values for
the root mean square error of calibration (RMSEC), cross validation (RMSECV) and
prediction (RMSEP) were found to be 0.62, 0.67 and 0.59, respectively. These calculations
are in good agreement with the results from HPLC. Therefore, this global model approach
can be used to calculate concentration of glucose in other non-alcoholic drinks without any
requirement of a new calibration model.
1. Introduction Therefore, the structural information

Overloaded sugar leads to the of the complex chemicals such as food and
negative effects on human health such as drinks can be potentially revealed by NIR.
obesity, diabetes and heart disease However, the overtone and combination
Therefore, it is important to quantify correct bands could be normally occurred in NIR
amount of sugar in food and drink because it spectrum. They contain very complex and
benefits in many aspects such as nutritional many overlapping signals. Therefore, the
labeling, detection of adulteration, food distinguished and characteristic peaks of an
quality, and economics. In the last decade, analyte (glucose in this case) are difficult to
the nondestructive techniques have been identify by the conventional band
attractively focused to use for determination assignment methods and spectral analysis
of sugar. Near infrared (NIR) spectroscopy methods. To build a good calibration curve
displays as one of the promising from a single defined peak from NIR spectra
nondestructive techniques.1 This technique is might not sufficient. Chemometrics has most
widely used in many fields, due to its often been used to extract specific feature to
advantages over other analytical techniques specific chemical components in the NIR
such as no requirement for sample spectra for easy interpretation. Multivariate
pretreatment, very fast and easy to data analysis techniques such as principal
implemen.2 The pattern of the overtone component analysis (PCA) and partial least
vibration bands of the functional groups (C- squares (PLS) are used to mathematically
H, N-H and O-H) canbe significantly altered calculate the pure component spectra and
by the surrounding functional groups. pure component concentration profiles from
the set of NIR spectra. Mostly, PLS have were performed with using tea solution as
been frequently used to build the calibration solvents. The accuracy of glucose contents
model from NIR spectrum.3 By conventional in the solutions were verified by high
way, each calibration model is required for performance liquid chromatography (HPLC)
each system. To obtain an appropriate at food research and testing laboratory
quantification, a new calibration model must (FRTL) Chulalongkorn university. These
be constructed for any new system. It is were defined as actual value of glucose
inevitable, in case of many systems need to concentrations.
build a new calibration model all the time. 2.3 Spectral data acquisition and
In this work, we propose a new processing
alternative method for determination of NIR spectrometer with NIR256-2.5
glucose using a global model. This involves detector, LS-1 tungsten halogen light source
the extraction and selection of significant and fiber optic connector (SMA 905, Ocean
components prior to build the calibration Optics) was used to acquire NIR spectra of
model. In any case, the pure NIR spectra of the samples. The samples were incubated in
water and glucose was extracted using PCA. the water bath at 25 oC and the humidity was
Then, the new NIR spectra was constructed kept at a steady level prior to acquire NIR
by using only the pure NIR spectra (water spectra. The NIR spectra of the samples
and glucose). The calibration model to were collected using transmittance (T) mode
predict concentration of glucose was in the range of 900 and 2300 nm using path
calculated using PLSR from these NIR length 0.5 mm, integration time of 1 and 32
spectra. This protocol can be used in any averaged scans with smoothing windows of
system containing water and glucose as 1. NIR spectra were preprocessed using
major components without any requirement multiplicative scattering correction (MSC)
of set up new calibration curve for to compensate for additive and
quantitative analysis. multiplicative effects in spectral data. The
preprocessed NIR spectra was further used
2. Materials and Methods for the multivariate data analysis.
2.1 Chemicals and materials 2.4 Multivariate data analysis
D (+) - glucose of analytical grade Multivariate method is an essential
was purchased from Ajex Finechem. tool for analyzing NIR spectra showing
Raming Oolong tea purchased from Raming complicated overlapping absorption bands.
Tea Co., Ltd. was used in this work and all In this study, PCA was used to extract the
of solution were prepared by using distilled major components underlying in the
deionized water (DI). All glassware was dataset.4 The key idea of PCA is to represent
clean up with detergent and DI water. the original data matrix X by a product of
2.2 Sample preparation two matrices T and P representing as scores
In the study, glucose solutions were and loading matrix, respectively.
prepared using DI water and tea as solvents. X=TP+E
To use DI water as solvent, a calibration set where T, P and E representing as scores,
of samples was prepared with glucose loading and error matrix, respectively and
concentrations at 1-20% w/w. The ranges of notation  defined as transpose operator. To
the glucose concentration were chosen from use only the significant component, the new
the average of total sugar in commercial dataset (Xnew) was calculated using score
non-alcoholic beverages. The other set of and loading matrix of water and glucose.
samples was validation set which was Xnew = TwaterP water+ TglucoseP glucose
prepared with the glucose concentration at After that, Xnew was used to build the
4-17%w/w. This set was used to validate the calibration model using partial least squares
calibration model build from the calibration regression (PLSR). In its simplest form, a
set of samples. Another set of experiments linear model specifies the linear relationship
between response c (glucose concentrations), be observed at the absorbance at 1450 nm
and a set of variables of the Xnew (1st overtone of O-H bond stretching) and
(wavelength) which can be expressed by 1950 nm (O-H combination band). Whereas,
c = Xnew b + e = T q + e the dominant band of the PC2 was located at
where q is PLS loading vector and b is the 1820 nm corresponding to CH2 stretching of
regression coefficient vector, estimated as glucose. The bands of loading PC3 is only
follows slightly shifted comparing to PC1. This
b=Hq might relate to the interaction between O-H
where H is the PLS weight matrix. of water to O-H of glucose. The loading of
An optimal number of PLS PC4 contains the characteristic bands at
components was determined using leave one 1440, 1760 and 2050 nm which relevant to
out cross-validation (LOOCV). The the 1st overtone OH stretching of water, the
performance of calibration model was 1st overtone C-H stretching and the
evaluated in terms of root mean square error combination bands, respectively. This might
which can be expressed: relate to glucose-glucose interactions.
3.2 Chemometrics
RMSE = In our case, chemometrics approach
Where cact and cpred are actual value and is an alternative way to build an appropriate
predicted value of glucose concentration, calibration model to quantify the glucose
respectively.n is the number of samples. In concentration. This approach involves
this study, root mean square error index was multivariate analysis which refer to use
used in 3 criteria RMSEC to evaluate more than one wavelength in order to build
calibration model, RMSECV to find the the calibration model. Table 1 shows the
optimal PLS components and RMSEP to results obtained from multivariate data
validate the calibration model. Lower value analysis. In the calculation, the calculation
of RMSE, higher accuracy of the prediction models were separated into the 6 models.
was occurred. The calculation models (1-3) were
constructed from full spectrum. The other
3. Result and Discussion models (4-6) were constructed from using
3.1 Spectral analysis optimal number of PCs (4 PCs) as
Transmission NIR spectra of glucose mentioned above. The models 1, 2, 4 and 5
solutions after preprocessing with MSC was require the calibration model of each system
showed in Figure 1a. The band assignments (water and tea) to calculate glucose
of the major components (water and sugar) concentrations. For example, to calculate
are briefly summarized in the highlight glucose concentration in water and tea, two
bands. They involve 1st vibrational overtone calibration models were built from set of
of O-H combination band (1100-1200 nm), sample using water and tea as solvent,
CH2 stretching (1720-1820 nm) and respectively. They represent as conventional
combination bands of CH, CH2, OH (1900- model. In case of model 3, it was built from
2200 nm).5 It can be seen that the intensity calibration set of samples using water as
of NIR spectrum was changed depending on solvent to calculate glucose concentration in
glucose concentration. For the 1st overtone, the tea.
CH, CH2 stretching and combination band In case of model 6, the calibration
increase when the glucose concentration model was constructed using only the
increase. This represents the direct variation. optimal number of PCs. It was extracted and
PCA was used to extract the significant selected only significant components prior
components from NIR spectra that can be to build the calibration model, thereby the
express in the loading profiles showed in model 3 and model 6 defined as a global
Figure 1b. The loading of PC1 describes the model.
characteristic patterns for water which can
(a) (b)
Figure 1. (a) NIR spectra of glucose solutions (in water system) after performing baseline
correction using multiplicative scattering correction (MSC). The red and blue line represent
the NIR spectrum of the highest (20% w/w) and the lowest (1% w/w) glucose concentration
(b) the loading profiles (PC1-PC4) of 4 significant components after performing PCA on the
NIR spectra
Table 1. Details of the calculation models using multivariate data analysis approach (PLS) of
calibration sample set, validation sample set. Number of major components extracted by
PCA. Number of PLS components was selected by leave one-out cross validation. Root mean
square error of calibration (RMSE) index, root mean square error of cross validation
(RMSECV) and root mean square error of prediction (RMSEP) were used as index of
prediction error.
Calibration model
1 2 3 4 5 6
(global model)
Calibration set of samples water tea water water tea water
Validation set of samples water tea tea water tea tea
Number of PCs Full spectrum 4 4 4
Number of PLS 13 13 13 4 4 4
components
RMSEC 0.04 0.19 0.04 0.60 0.48 0.62
RMSECV 0.30 0.42 0.30 0.66 0.51 0.67
RMSEP 0.90 0.50 0.34 1.33 0.75 0.59
R2* 0.9877 0.9983 0.9978 0.9949 0.9958 0.9953
R2* was calculated by using actual value of glucose concentration versus predicted value of glucose
concentration
Table 1 RMSE values represent the 1-3. Optimum number of PCs was done by
average prediction error of the model. It can cross-validation which performed in form of
be seen that RMSEC, which represent root RMSECV, it was insignificantly different in
mean square error of calibration samples, model 1-6. These results indicate that the
are quite low especially in model 1-3 (0.04- calibration model using optimal components
0.19), while the model 4-6 (0.48-0.62) were have the capability in order to be used to
slightly higher. This phenomenon can arise determine the glucose concentration of an
from that full spectrum carried noises which unknown solution.
affects the prediction accuracy in the model
(a) (b) (c)
(d) (e) (f)

Figure 2. a-f show relationship between actual value (x-axis) and predicted value (y-axis) of
glucose concentrations of the model 1-6, respectively
Next step, the generated model was In our case, R2 value more than 0.99
used to calculate the glucose concentration was obtained in model 2-6. These suggest
of the validation set. In order to evaluate the glucose concentrations is linearly related
predictive potential of calibration model, the to the intensity patterns of NIR spectra. It
RMSEP was obtained. The results show that can also be expressed this global model was
the RMSEP using full spectrum (model 1-3) the goodness to fit, and therefore it is very
trend to give less RMSEP than the model reliable model to develop for future
4-6 using only significant components. In prediction.
case of the model 6, the calibration model
was constructed from water to be used to 4. Conclusion
calculate glucose concentration in tea. The results obtained in this work
RMSEP is only 0.59 which insignificantly show the potential of NIR spectroscopy
different from the model 3 (0.34). This combined with chemometrics to quantify
shows that the global model gives the glucose concentration in non-alcoholic
acceptable predictive error range. It implies beverages. The protocol of “global model”
that our procedure of “global model” can be was proposed in order to build the global
performed to calculate glucose concentration calibration model and use the model to
in any non-alcoholic beverages without any calculate glucose concentration of other
requirement of building a new calibration systems (tea in this case). The corresponding
model. The actual values of glucose values for RMSEC, RMSECV and RMSEP
concentration were plotted versus the were found to be 0.62, 0.67 and 0.59,
predicted value of glucose concentration that respectively. Moreover, the correlation (R2)
was showed in figure 2. The R2 value was between actual value of glucose
used to access the accuracy of the model. concentrations and predicted value are
>0.99. Therefore, this global model can be
used to calculate concentration of glucose in References
non-alcoholic drinks without the 1. Weihong, H.; Da-Wen, S.; Hongbin, P.;
requirement of building a new calibration Tingtiao, P. Compr. Rev. Food Sci. Food
model and could be used for routine quality Saf. 2016, 15, 1067-1079.
control of beverage products. 2. Braz, J. Chem. Soc. 2003, 14, 198-219.
3. Ozaki, Y. Anal. Sci., 2012, 28, 545-563.
Acknowledgement 4. Brereton, R. G. in Chemometrics for
This research was funded by Grant Pattern Recognition: Multiclass
for International Research Integration: Chula Classifiers; Richard, G. Wiley:
Research scholar, Ratchadaphiseksomphot Chichester, U.K. 2009, 298-303.
Endowment Fund S Makmuang gratefully 5. Golic, M.; Walsh, K.; Lawson, P. Appl.
appreciated the financial support from Spectrosc. 2003, 57, 139-145.
Science Achievement Scholarship of
Thailand (SAST).
Extraction of phenol using poly(styrene-co-divinylbenzene)
monolithic bead
Yaowalak Wimanthong, Orapin Chienthavon*
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science,
Kasetsart University, Chatuchak, Bangkok 10900, Thailand
*E-mail: fsciopc@ku.ac.th
Abstract:
In this work, poly(styrene-co-divinylbenzene) or PS-DVB monolithic adsorbent was
synthesized as a bead to extract phenol from water matrix. Quantitative determination of the
target analyte was performed by gas chromatography with flame ionization detection (GC-
FID). In the synthesis of this adsorbent, styrene and divinylbenzene and dibenzoyl peroxide
were used as the monomer and initiator, respectively, while decanol and tetrahydrofuran were
added to improve porosity during the polymerization. Morphology of synthesised material
showed an interconnected spherical shape of monolith structure of averagely 2 µm diameter.
Owing to the uniform and thoroughly interconnected particles within the macropore network,
the selectivity and permeability for extraction of phenol from 50 mL of water sample was
improved. In this work, several parameters affecting the extraction efficiency were evaluated.
The results showed recovery in the range of 48-113% and linear relationship with R2 ≥ 0.99.
1. Introduction obstacles, several absorbent materials have

Phenols are widely used in drugs, been studied. One of interesting materials is
dyes, paper, pesticides, disinfectant, petrol, poly (styrene-co-divinylbenzene) or PS-
and polymer manufacture. Although they are DVB monolithic adsorbent. Because of its
essential in many industries and plays an non-polar property, high surface area and
important role in daily life, phenolic selectivity, this type of material likely
compounds potentially generate health adsorbs and preconcentrates phenolic
problems in living organisms and increase compounds. Besides the material can be
toxicity of environmental media such as soil, fabricated in various containers, such as
lake and pond river.1 For these issues, US cartridge, capillary and HPLC column.
Environmental Protection Agency (EPA) Since conventional commercial PS-
has declared phenols as priority pollutants. DVB cartridges, suffer from some problems,
Hence, determination of these compounds including time consuming and limited
has become a significant part of amounts of loaded samples,3 in this research,
environment and public health sectors. a newly poly (styrene-co-divinylbnzene)
Nevertheless, trace level of phenolic monolithic adsorbent was synthesised as a
compounds concentration present in bead for extraction and preconcentration of
environment and their losses incur during some selected phenols from water. Various
sample handling, extraction etc. Pre- parameters that affected extraction
concentration process consequently is an efficiency, such as added sodium chloride
important role to support phenols analysis. concentration, stirring speed, and extraction
Although, over the past decade, time, were optimised. Determination of the
several extraction methods have been target analytes were performed by using gas
developed for preconcentration of these chromatography with flame ionization
compounds, some extraction methods, such detection (GC-FID).
as Soxhlet and liquid-liquid extraction
(LLE), required hazardous organic solvent 2. Materials and Methods
and time consuming.2 To overcome these 2.1 Chemicals and equipment
Divinylbenzene, o-cresol and 1,2- The PS-DVB monolithic bead was
dimethyl-3-nitrobenzene were supplied from immersed into methanol for 1 min for
Aldrich (St. Louis, Mo, USA). 2,4- conditioning before extraction. Then it was
dimethylphenol (2,4-DMP) and 2-Chloro introduced into a glass bottle containing 50
phenol (2-CP) were purchased from TCI mL of water sample, which was pre-adjusted
(Tokyo, Japan). Dibenzoyl peroxide, 4-nitro to pH 2 by using NaOH and HCl. After
phenol (4-NP) and decanol were bought stirring the aqueous sample at 500 rpm for
from Acros organics (New Jersey, USA). 20 min, the monolithic bead was taken into a
Phenol (PH) and styrene were obtained from glass vial which was contained with 3 mL of
Merck (Darmstadt, Germany). Tetrahydro- dichloromethane for desorption. The glass
furan was bought from Fisher scientific vial was subsequently subjected to an ultra-
(Leicestershire, UK). Concentrated hydro sonication for 15 min. Leaving the
chloric acid and methanol (HPLC grade) monolithic bead, the aqueous extract was
were obtained from Burdick & Jacson evaporated by nitrogen stream until near
(Ulsan, Korea). Dichlomethane, acetone, dryness. After that, 1,2-dimethyl-3-
and sodium hydroxide were bought from nitrobenzene was added as an internal
RCI Labscan (Bangkok, Thailand). standard and the mixture was adjusted to
PS-DVB monolithic bead were 1.00 mL with dichloromethane. The
characterised by using a scanning electron extracted solution was injected into GC-FID
microscope (Quanta 450, FEI, Hillsboro, for analysis.
Oregon, USA). A 744 pH meter (Metrohm, 2.4 Chromatographic conditions
Switzerland) was used during the extraction Analysis of phenolic compounds was
process. Deionised water consists of 18 M performed by using a gas chromatograph
resistance was obtained from Elga (Maxima, with flame ionization detector (GC 7890A,
Elga, Bucks, England). Agilent technologies, Santa Clara, CA,
2.2 Synthesis of PS-DVB monolithic bead USA). Five representative phenols, namely
adsorbent phenol (PH), 2-chlorophenol (2-CP), o-
The copolymer of styrene and cresol, 2,4 dimethylphenol (2,4-DMP), and
divinyl benzene was synthesised in 1 dram 4-nitrophenol (4-NP), were selected,
vial, 10 mg of dibenzoyl peroxide was according to their different additional
dissolved with 200 μL styrene and 200 μL functionalities. They were separated on a
divinylbenzene. The mixture was added with 30 m  0.25 mm. id HP-5HT column
400 μL of dodecanol and 40 μL of (Phenomenex, California, USA) with
tetrahydrofuran, homogenised by using a 0.25 µm film thickness. A 99.99% oxygen
vortex mixer. The mixture solution was free nitrogen was used as carrier gas. The
carefully poured into a polypropylene tube. GC oven temperature program was set at an
After that, the tube was tightly sealed and initial temperature of 50 °C held for 1 min,
kept at 70 °C for 24 hours. The synthesised raised to 100 °C at 20 °C/min, then
monolith was removed from the tube and increased to 160 °C at 30 °C/min, and then
sequentially cleaned with methanol and raised to 250 °C at 6 °C/min The detector
acetone in an ultrasonic bath, then dried at and injection port temperature were
45 °C for 6 hours. maintained at 290 and 270°C, respectively
2.3 Extraction procedure The injector was set in a splitless mode.
(a) (b)
1.2 cm.
Figure 1. Schematic representation (a) Scanning electron micrographs of PS-DVB

monolithic bead and (b) PS-DVB monolithic bead adsorbent
3. Results and Discussion increasing the stirring speed significantly

3.1 Characterisation of poly(styrene-co- decreased an analyte response represented
divinylbenzene) monolithic bead by its peak area ratio.
In this work, the synthesis method
was adapted from the research of Ramnut 4
who synthesised PS-DVB monolith in a
capillary column. In the polymerization step,
divinylbenzene and styrene were used as
monomer and dibenzoyl peroxide was used
as initiator. In order to control the pore size
and increase permeability, tetrahydrofuran
and decanol were added into the
polymerization mixture.
Morphology of PS-DVB mono-lithic
material was characterised by using a Figure 2. Effect of stirring rate on the
scanning electron microscope, giving SEM extraction efficiency of selected phenols by
micrographs presented in Figure 1. The using PS-DVB monolithic bead adsorbent.
result showed a structure of monolith with Extraction condition; extraction time 40
interconnected spherical shape of about 2 min, desorption time 15 min, no salt
µm diameter. addition
3.2 Optimisation of PS-DVB monolithic
bead extraction conditions This probably occurred from the
To obtain high recovery, sodium breaking of monolithic bead surface during
chloride concentration, stirring rate, and the extraction, particularly when the water
extraction time that affected extraction sample was vigorously stirred at the speed
efficiency were optimised by using a over 500 rpm. Hence, the stirring speed at
standard solution containing five targeted 500 rpm was selected for further
phenols at a concentration of 40 mg/L each. experiment.
Owing to pH-dependent property of the Since the analyte molecules required
sample solution was therefore adjusted to a suitable time to migrate from the sample
pH 2 in order to increase solubility and pi-pi solution into the macro-pore of PS-DVB
interaction between the analytes and the bead and adsorbed on its porous surface. An
adsorbent surface. extraction time between 10-90 min were
To enhance the mass transfer investigated. The result in Fig.3 showed the
velocity and reduce extraction time, the peak area ratio improved as the extraction
stirring speed in the range of 500-1250 rpm time increased from 10 to 20 min. The
were evaluated. From the result in Figure 2, response significantly decreased when the
extraction time was higher than 40 min.
ratio relatively increased particularly for PH,
2-CP and o-cresol. As a result, the salt
concentration of 0.3 g/mL was selected as
optimum.
3.3 Linearity and recovery
Various concentration of phenol
standard ranging from 20–80 mg/L were
analysed by using GC-FID under the
optimised conditions. In this method, 1,2
dimethyl-3-nitrobenzene was used as
internal standard. Good linearity with
correlation coefficients of greater than 0.99
Figure 3. Effect of extraction time on the were obtained for all target analytes.
extraction efficiency of phenols by using The recovery was examined by
PS-DVB monolithic bead adsorbent. spiking each standard analyte to give the
Extraction condition; stirring rate 500 rpm concentration of 40 mg/L each in 50 mL DI
desorption time 15 min, no salt addition water. The spiked sample was extracted, and
the liquid extract was subjected to the
chromatographic determination.
Table 1. Analytical performance for

phenolic compounds obtained by PS-DVB
monolithic bead adsorbent
Phenols r2 Recovery (%)
(n=3)
PH 0.9961 80.7 ± 7.4
2-CP 0.9968 89.6 ± 8.4
o-cresol 0.9917 81.6 ± 4.7
2,4-DMP 0.9927 113.9 ± 4.1
4-NP 0.9957 48.6 ± 7.5
Figure 4. Effect of salt concentration on the
extraction efficiency of phenols by using
PS-DVB monolithic bead adsorbent. In this work, one time extraction of
Extraction condition; Extraction time 20 water sample gave too low recovery to meet
min, desorption time 15 min, stirring rate the criteria requirement of EPA method.
500 rpm Repeated extraction of the water sample was
performed two more times using a new
Nevertheless, the highest responses monolithic bead. The result of recovery for
of 2CP, o-cresol and 2,4-DMP were each phenolic compound was summarised in
obtained for 40 min, while the responses of Table 1. The results showed the recoveries
PH and PCP were lower than the average of selected phenols of between 48-113%,
extraction time of sample of 20 min. except that of 4-NP, which was much lower
Therefore, 20 min was chosen as the than 80%. It could occur from weak
optimum. hydrophobic interaction between 4-NP and
Effect of salt concentration on peak the PS-DVB adsorbent surface
area ratio was observed by adding NaCl to
give concentrations of 0-0.3 g/mL of water 4. Conclusion
sample. As shown in Fig.4, the peak area In this work, PS-DVB monolithic
ratio slightly changed for the NaCl bead could be used for extraction and
concentration from 0-0.2 g/mL. At the salt preconcentration of PH, o-cresol, 2,4-DMP
concentration over 0.2 g/mL, the peak area and 2-CP in water matrix. The highly porous
material was synthesised as an easy-to-use
bead by a simple polymerization. Owing to References
its high permeability, hydrophobic propertie, 1. Michałowicz, J. Pol. J. Environ. Stud.
surface area, as well as eliminating possible 2007, 16, 347-362.
carry-over effect of the purposed method, 2. Mahugo C.; Ferrera, Z.; Esther, P. M.;
the extraction efficiency was satisfactorily Rodriguez, I. J. Molecules 2009, 14 (1),
obtained. 298-320.
3. Rodrı́guez, I.; Llompart, M. P.; Cela, R.
Acknowledgement J. Chromatogr. A 2000, 885 (1–2), 291-
The authors would like to thank the 304.
Center of Excellence for Innovation in 4. Ramnut, N. Investigation of silica
Chemistry (PERCH-CIC), Commission on monolith as a non-polar matrix retainer
Higher Education, Ministry of Education for for nitrosamines determination.
providing a scholarship to Y. Wimanthong. Kasetsart University, Thailand, 2008.
The Department of Chemistry, Faculty of 5. Hu, C.; Chen, B.; He, M.; Hu, B. J
Science, Kasetsart University was also for Chromatogr A 2013, 1300, 165-72.
partially financial and instrumental support. 6. Saraji, M.; Bakhshi, M. J. Chromatogr
A. 2005, 1098 (1-2), 306.
Determination of mercury(II) by using 3-dimentional microfluidic
paper-based analytical device
Hikmanita Lisan Nashukha1,2, Jirayu Sitanurak1,2, Hermin Sulistryarti3,
Duangjai Nacapricha1,2, Kanchana Uraisin1,2*
1
Flow Innovation-Research for Science and Technology Laboratories (FIRST Labs),
Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
2
Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
3
Department of Chemistry, Brawijaya University, Jl. Veteran Malang 65145, Indonesia
*Email: kanchana.ura@mahidol.ac.th
Abstract:
A novel method for analysis of mercury using paper-based device with colorimetric
detection was developed. The method is based on indirect analysis of mercury using a
concept of iodometry. The analysis of mercury involves the reaction between Hg(II) and
excess amounts of iodide (I-) to form [HgI4]2- in the donor layer of the device. The residual I-
is oxidized by iodate (IO3-) in acidic condition producing iodine gas (I2). The generated I2
diffuses through a spacer to react with iodide-starch at the acceptor layer forming purple
complex of I3- - starch. Photographs of colored product is taken at the end of measurement
and analyzed using Image J software. The blue color intensity is inversely proportional to
mercury contents in sample. The effects of photo capturing time and reagent concentrations
were studied. The calibration graph was linear in the range 50–350 mgHg(II)/L with RSD of
2.2% (150 mgHg(II) L-1, n=12). The method was applied for determination of mercury in
water samples.
1. Introduction vapor atomic absorption spectrometry (CV-

Mercury is known as an element AAS),3,4 high performance liquid
which has high level of toxicity. In 1959, chromatography (HPLC) with chemilu
the first accident of mercury toxication was minescence detection,5 resonance scattering,
reported on the contamination of methyl- gas chromatography (GC) coupled with ICP-
mercury (MeHg) from the wastewater MS.6 These kinds of techniques are suitable
channel of chemical plant (Chisso Co. Ltd) for trace level analysis and provide low limit
in south-west region of Kyushu Island which of detection. However, the cost of analysis,
well-known as Minamata City.1 Naturally, laborious operation and maintenance, and
mercury can be found in free forms but the availability in some laboratories have
commonly find as the compounds, such as been also reported as the drawbacks of the
cinnabar or mercury sulphide. It released to methods.
the atmosphere due to the volcanic activity, Introducing in 2007 by Whitesides,
forest fires, biomass burning, or Earth crust microfluidics paper-based analytical devices
weathering processes. Increasing mercury (µPADs) has been popular as the new
production to the environment also occurred analytical device with several benefits such
by anthropogenic processes including coal as disposable, cost effective, low of reagent
combustion factory, cement production, and consumption, easy fabrication, and provides
wastewater.2 Common techniques for rapid analysis. The device was developed for
detection of mercury in environmental simultaneous analysis of glucose and protein
sample have been reported including cold based on colorimetric detection on a
patterned paper.7 Some researchers including
Cai8 reported µPADs for mercury detection soaked with 10% nitric acid and then rinsed
in water samples with helping of with deionized water before use.
nanoparticles to enhance the color product
due to the high extinction coefficient of the
nanoparticles. The detection step could be
done in low levels by with or without
additional instrument such as digital camera,
scanner, or mobile phone camera. But it
provides some drawbacks, such as high cost
of reagent, and some of the reagent need a
synthesis step. As an option for mercury
detection, use of well-known reaction could
be considered instead of use of nanoparticles
on paper device. Hg(II) easily reacts with
some ligands to form strong complex such
Figure 1. The schematic diagram of 3D-
as iodide and thiocyanate to be [HgI4]2- 9 and
µPAD for Hg(II) detection by combining of
[HgSCN4].2-10 Both complex have strong
two layers of paper with double-sided
constant stability of complex but detectable
adhesive tape as the spacer
in UV region. Even we add the additional
instrument, we cannot see the changing of
2.2 Fabrication of 3D-µPAD for Hg(II)
color formation.
detection
In this work, a new concept of
3D-µPAD was made by screen
colorimetric detection for Hg(II) on a paper
printing technique onto Whatman filter
device was developed by using 3-
paper number 4 (Whatman International,
dimentional microfluidic paper-based
Ltd., UK) as white bare paper. Hydrophobic
analytical device (3D-µPAD) that previously
barriers for acceptor and donor layer was
published by Nacapricha, and teams in
screened by using mixture of diglycidyl
2016,11 based on the generating excess of
ether bisphenol A (epoxy resin), hardener
iodide as iodine gas after reaction with
(amine mixture), and yellow fabric ink as
Hg(II) on donor layer and catch by color
the hydrophobic material. Circular shape
forming reagent on acceptor layer that
pattern was screened for acceptor layer,
inversely proportional to the Hg(II) amounts
denoted as “A” and dumbbell-shape pattern
in samples.
was screened for donor layer, denoted as
“D” (Figure 1). Spacer layer was made by
2. Materials and Methods
double-sided adhesive foam tape (Scotch,
2.1 Chemicals
USA). Left side of spacer layer was drilled
All of chemicals were prepared from
with 7 mm i.d of punch die to make a
analytical reagent grade. Mercury chloride
connection between left donor reservoir and
and potassium iodate were purchased from
acceptor layer. Both of acceptor and donor
Ajax Finechem (Australia). Potassium
layer were combined in the opposite way
iodide was purchased from Merck
with spacer layer in the middle as shown in
(Germany). Starch was purchased from
Figure 1. Digital camera (IXUS125 HS,
AnalaR (England). Nitiric acid and Sulfuric
Canon, Japan) was used as the colorimetric
Acid were purchased from RCI, Lab Scan,
detector of the µPAD inside the homemade
Co. Ltd., (Thailand). Deionized Milli-Q
light controlled box. All the images were
water with a resistivity 18M Ω was prepared
analysed by using ImageJ software obtained
from Easypure II system (USA). All
from (U.S. National Institutes of Health,
glasswares and plastic bottles were firstly
Maryland, USA).
Figure 2. Procedure of operation for Hg(II) detection by using 3D-µPAD
2.3 Operation of 3D-µPAD for Hg(II) 2.4 Sample preparation

detection Water samples were used for Hg(II)
Operation of 3D-µPAD for Hg(II) detection by using proposed method. Water
detection was described in Figure 2. The samples was collected from small-scale gold
first step of operation, 1 µL of 0.2 mol L-1 of mining area, Lombok Island, Indonesia; and
KIO3 in 0.2 mol L-1 H2SO4 was dropped on nearby our university (Phayatai campus,
the left donor reservoir and followed by Faculty of Science, Mahidol University,
letting the device dry for 5 minutes. Second Bangkok, Thailand). The water samples
step, the left donor reservoir was covered were filtrated by using Whatman filter paper
with transparent tape at 4 minutes and 30 number 42 and acidified with nitric acid
second. Third step, the device was turned (HNO3) to be pH 2.
over and dropped 2 µL of 1 mol L-1 KI with
1% of starch) on the acceptor reservoir. 3. Results and Discussion
Fourth step, acceptor reservoir was 3.1 Design and principle
immediately covered with transparent tape The main purpose of this work is to
to prevent the unexpected vaporization of develop simple, cost-friendly, instrument-
reagents. Fifth step, turned over again the free and environmental-friendly microfluidic
device (facing up the donor layer) and paper based analytical device for daily
dropped 8 µL of pre-mixed Hg(II) and 10 screening of Hg(II) detection on water
mmol L-1 KI in the right donor reservoir. It samples. Mercury is well-known as a metal
was prepared on the 5-mL volumetric flask that can easily form strong complex with
before the analysis. The generated iodine some ligands. Mercury ions react with
gas was directly diffused through the spacer iodide to form tetraiodomercurate [HgI4]2-
to react with color development reagent (KI complex and detect the absorbance in the
and starch) and generates purple color UV region by spectrophotometer. However,
complex of triiodide-starch reagents. For the this reaction requires large volume of
last step, the acceptor layer was faced up reagents impossible to apply in the paper-
inside the black tight photographic box. The based device based on colorimetric detection
development color complex was exactly because it has no color of complex. One of
captured at 4 minutes after dropping pre- the 3D-µPADs was reported by Nacapricha
mixed solution. Color intensity of complex and teams for determination of volatile and
was analysed by using ImageJ software and non-volatile compounds11. In our works, we
plotted between green intensity from RGB used the combination of iodometric
scale and concentration of Hg(II). reactions for Hg(II) detection and applied
with 3D-µPAD. Hg(II) reacts with an excess
amount of iodide (I-) to produce [HgI4]2-. However, it was found that when the initial
The residual of I- was placed on the right concentration of iodide higher than 10 mmol
donor reservoir and flowed to the left donor L-1, the color complex formation of various
reservoir which was oxidized by iodate concentration of Hg(II) was not significantly
(IO3-) in acidic condition and produced increased and the signal could not be
iodine gas. The iodine gas (I2) was diffused differentiated as confirmed by result of slope
through the spacer layer reacts with iodide- of calibration (sensitivity). It was concluded
starch and formed purple complex of that the amounts of initial iodide were too
triiodide-starch (I3- -starch) on the acceptor excess enough for complexing with Hg(II).
layer. High amounts of mercury, produced The sensitivity was significantly decreased
less amounts of residual I- and results in at higher concentration of iodide. Thus, 10
decreasing of color intensity in the end of mmol L-1 of iodide was selected.
analysis. The color intensity was measured
by using ImageJ software. RGB channel was
used to measure the color intensity and
green intensity was selected for further
analysis because it showed the highest
sensitivity.
3.2 Optimization of photo capturing time
Photo capturing time is started to
measure after dropping the pre-mixed
solution when Hg(II) and I- in the right
donor reservoir until the color of complex
formation is stable on the acceptor reservoir.
It was captured by digital camera every 2
minutes after dropping of pre-mixed
solution for 8 minutes of total time. The
green intensity of color complex was
gradually increased from 2 minutes to 4
minutes and slightly increased up to 8
minutes (Figure 3.A). This may be due to
the iodine gas completely diffused to the
acceptor layer and form a complex with pre- Figure 3. (A) Effect of photo capturing time
deposited reagents. Thus, 4 minutes was with green intensity of 150 mgHg L-1 compared
selected as the convenient time to capture to sensitivity of Hg(II) detection. (B) Effect of
the purple color of triiodide-starch complex iodide concentration with green intensity of 150
mgHg L-1 compared to sensitivity of Hg(II)
due to the efficiency of time analysis.
detection
3.3 Optimization of initial iodide
The effect of iodide concentration as 3.6 Analytical performance
an initial reagent to react with Hg(II) was The analytical features of the
studied. The concentration was varied from proposed method including linearity of
5 up to 14 mmol L-1 of potassium iodide. measurement, LOD, LOQ, and precision
The result as shown in Figure 3.B, with were performed. The linearity of the
increasing of iodide, the green intensity of calibration curve was plotted between
150 mgHg L-1 of mercury was increased. concentration of Hg(II) with green intensity
Related with the use of residual of iodide in the range of 50-350 mgHg L-1 with
after reacted with Hg(II), increasing the coefficient correlation of 0.9968. The LOD
initial amounts of iodide lead to increasing (3σ of blank) and LOQ (5σ of blank) of
the residue of iodide that further convert to Hg(II) detection were 20.0 mgHg L-1 and
iodine gas for each concentration of Hg(II). 33.3 mgHg L-1, respectively. The precision
of 2.2% RSD (150 mgHg L-1, n=12) was under the optimal conditions. The proposed
achieved. method provided good linear range of
50-350 mgHg L-1. This method is applicable
for daily screening method for determination
of mercury in water samples.
Acknowledgement
This work was supported by Mahidol
University under the National Research
Universities Initiative and Centre of
Excellence for Innovation in Chemistry
(PERCH-CIC) and Faculty of Graduate
Studies, Mahidol University.
Figure 4. Calibration curve of 3D-µPAD for References

Hg(II) detection 1. Harada, M. Crit. Rev. Toxicol. 1995, 25
(1), 1-24.
3.8 Apply to sample 2. Pirrone, N.; Mason, R. P., Mercury fate
The developed method was applied and transport in the global atmosphere:
for the determination of Hg(II) in water
emissions, measurements and models.
samples obtained from small-scale gold
mining area, Lombok Island, Indonesia and Springer. 2009, 3-5.
surface water collected from Mahidol 3. Zavvar M. H.; Asghari, A.;
University. The results of recovery studies Shirkhanloo, H. J. Anal. Chem. 2010,
are summarized at Table 1. From the results, 65 (9), 935-939.
Hg(II) from the samples were spiked by 4. Almeida, I. L. S.; Oliveira, M. D. R.;
using standard Hg(II) 150 mgL-1 and Silva, J. B. B.; Coelho, N. M. M.
provided good percentage of recovery in the Microchem. J. 2016, 124, 326-330.
range of 92-103%. As a promising, the 5. Kodamatani, H.; Tomiyasu, T., J.
developed method is applicable for Chromatogr. A 2013, 1288, 155-159.
determination of Hg(II) in environmental 6. Chung, S. W. C.; Chan, B. T. P. J.
samples without any obstruction from Chromatogr. A 2011, 1218 (9), 1260-
sample matrices. 1265.
7. Martinez, A. W.; Phillips, S. T.; Butte,
Table 1. Study of recovery from water
samples M. J.; Whitesides, G. M. Angewandte
Sample Present Added Found % Rec Chemie (International ed. in English)
(mgL-1) (mgL-1) (mgL-1) 2007, 46 (8), 1318-1320.
1 nd 150 151.8±22.8 101.7 8. Cai, L.; Fang, Y.; Mo, Y.; Huang, Y.;
2 nd 150 145.8±41.0 94.6
3 nd 150 136.1±27.3 92.0
Xu, C.; Zhang, Z.; Wang, M., AIP
4 nd 150 154.4±34.8 103.0 Advances 2017, 7 (8), 085214.
nd
is not detectable (nd < LOD) 9. Pappas, A. J.; Powell, H. B. Anal.
Chem. 1967, 39 (6), 579-581.
4. Conclusion 10. Markle, G. E.; Boltz, D. F. Anal Chem.
A simple, cost-friendly, instrument- 1954, 26 (3), 447-449
free and environmental-friendly has been
11. Phansi, P.; Sumantakul, S.;
developed for determination of Hg(II) in
Wongpakdee, T.; Fukana, N.;
water samples by using 3D-µPAD based on
colorimetric detection of iodide-starch Ratanawimarnwong, N.; Sitanurak, J.;
complex. The samples could be directly Nacapricha, D., Anal. Chem. 2016, 88
applied to water sample after filtration step (17), 8749-8756.
Highly sensitive electrochemical capsaicin sensor based on
polyaniline-derived N-doped mesoporous carbon
Pathaporn Temcheon, Wilai Siriwatcharapiboon, Orapin Chienthavorn, Panitat Hasin*
Kasetsart University, Chatuchak, Bangkok 10900, Thailand
*E-mail: fscipths@ku.ac.th
Abstract:
A glassy carbon electrode (GCE) modified with N-doped mesoporous carbon (N-MC)
with high electrocatalytic activity was employed for sensitive determination of capsaicin. The
N-MC/GCE was characterized by Scanning Electron Microscopy (SEM) and Energy
Dispersive X-ray Spectroscopy (EDX) as well as its electrochemical property was also
investigated by Linear Sweep Voltammetry (LSV). The SEM images revealed the linear
array of connected rod-like particles and confirmed the successful synthesis of the
mesoporous carbon. The electrochemical performance study showed that the N-MC/GCE
facilitates the adsorption of capsaicin on the electrode surface and provides fast electron
transfer due to its high surface area and charge polarization. Furthermore, the presence of N-
MC on GCE significantly enhances the oxidation peak current, indicating that the N-MC
showed excellent electrocatalytic activity to the redox reaction of capsaicin. Under optimized
conditions, the proposed electrochemical sensor showed a linear response to capsaicin from
1.0 to 10.0 µM with detection limit of 0.62 µM (S/N=3) and the coefficient of determination
(R2) of 0.9993. Thus, the N-MC/GCE demonstrated the promising electrochemical sensor
that can be further developed as a sensitive portable electrochemical sensor for the
determination of capsaicin in food and pharmaceuticals.
1. Introduction protective effects against cholesterol and

In recent years, a lot of popular hot obesity. In addition, the pepper spray which
and spicy foods around the world are is widely used as a self-defense chemical
produced by adding chili peppers that have agent also consists of capsaicin as an active
capsaicinoids in large amounts. component. With increasing demand by
Capsaicinoids are the source of pungency, consumers for spicy foods and the
flavor and hotness in food. With the increasing use of capsaicin in
capsaicinoids concentrations of about 0.01- pharmaceutical industry and our daily life,
0.3% (w/w), the spice level of chilies is mild the development of a simple and sensitivity
whereas the hot chilies contain 0.3-1 % analytical method to determine the capsaicin
(w/w) of capsaicinoids. Capsaicin (8- has become more indispensable.
Methyl-N-vanillyl-trans-6-nonenamide) is The heat level of chilies, peppers,
the most active ingredient in capsaicinoids and hot sauces has been measured by a
family responsible for pungent flavor and traditional method known as “the Scoville
occupies up to 70%. Not only is capsaicin Organoleptic Test1” created by the Ameri-
the main reason for the pungent taste but can chemist Wilbur Scoville in 1912.
also it provides many medical needs. It However, this method suffers from its
could be used as a new material in impression and subjectivity because it is
pharmaceutical industry because of its depending on the opinions of tasters, all of
advantageous biological properties, such as whom might probably have different sensi-
anti-bacteria, anti-mutagenic, and antioxidant tivity to the level of pungency.
power. Moreover, the capsaicin also has
The electrochemical technique has In this study, glassy carbon electrode
attracted much attention out of various (GCE) modified with N-doped mesoporous
detection methods due to its low cost, ease carbon (N-MC) has been used for the
of use, quick response time, simplicity, determination of the capsaicin. Under the
sensitivity, and the possibility for on-site optimized condition, GCE modified with N-
field analysis. Several different carbon MC demonstrates an excellent analytical
materials such as boron doped diamond2 or performance in terms of sensitivity, linear
graphene3 have been used as the electrode dynamic range, and it can also be applied to
materials in electrochemical method for the the determination of capsaicin in real
detection of capsaicin due to their high samples.
electrical conductivity, chemical inertness,
and low background current. 2. Materials and Methods
Generally, the electrode materials 2.1 Reagents
with uniform pore structured, many edge- Pluronic®123, Tetraethyl orthosili-
plane-like defective sites, and high specific cate (TEOS), N-(2-aminoethyl)-3 aminopro-
surface areas are beneficial to pyltrimethoxysilane, Capsaicin for analytical
electrocatalytic process. Mesoporous carbon standard were purchased from Sigma-
(MC) has additional benefits of uniform Aldrich. Aniline 99% was purchased from
tailored including unique mesoporous Panreac. Hexamethyldisilazane (HMDM)
structure large pore volumes, large acces- was purchased from Fluka.
sible surface area, and superior stability as 2.2 Synthesis of polyaniline-derived
well as extraordinarily broad chemical and nitrogen doped mesoporous carbon
structural diversity. These unique features N-MC was synthesized according to
maximize interfacial area for reaction, the literatures.4 Briefly, The N-MC was
ensure fast charge transport, and shorter prepared from mesoporous silica (SBA-15)
diffusion path for active species making MC used as a template. Firstly, SBA-15 was
materials suitable for applications in synthesized by stirring a mixture of
electrocatalysis. However, the intrinsic poor Pluronic®123: H2O: HCl: TEOS at 45 ºC
electrical conductivities and less active sites for 20 h with the volume composition of 4:
on the basal plane of carbon structure limit 20: 130: 8.5, respectively and keeping in an
severely the practical application of MC oven at 80 °C for 24 h. Then, SBA-15 was
materials. To overcome these obstacles, a filtered, washed copiously with distilled
variety of efforts have been devoted to water, and then dried at 45 °C. After that,
optimize their electronic characteristics and the external surface of SBA-15 was
increase the active sites on the basal plane. modified with SiMe3 groups by stirring 4 g
To prepare N-doped mesoporous of the material in HMDS/toluene (5 mL/60
carbon with high N content, the mL) solution for 18 h.
carbonization of polyaniline (PANI) Afterward, 0.5 g of the obtained
polymerized in the pores of the mesoporous material was stirred in 100 mL of diethyl-
silica has been chosen. Owing to its high ether: ethanol with 1:1 volume ratio in order
N/C atomic ratio and high molecular weight to remove the Pluronic®123 template. The
combined with confining PANI within the inner channel walls of the obtained product,
nanosized cavities of mesoporous silica, SiMe3-SBA-15, was functionalized with
these can prevent the vaporization of small diamine groups by stirring 0.1 g of SiMe3-
CN species at high temperatures. Therefore, SBA-15 in 1 mL of N-(2-aminoethyl)-3-
this method can provide N-doped aminopropyltrimethoxysilane and 100 mL of
mesoporous carbon with not only high toluene for 18 h at 80 °C. After stirring for
surface area but also high N content leading 18 h, the white precipitate was washed
to the outstanding electrocatalytic activity. copiously with ethanol and dried at 45 °C.
The obtained material is denoted as Voltammetry was carried out by using
SBA-15/Diamine. To polymerize the potentiostat Metrohm; using Ag/AgCl as a
polyaniline inside the pores of SBA- reference electrode, Pt as a counter
15/Diamine, 0.1 g of SBA-15/Diamine was electrode, N-MC/GCE as a working
stirred in the mixture of 10 mL of 1 M HCl electrode, and sodium acetate (NaOAc):
and 0.6 g of sodium persulfate for 2 h. acetic acid (AcOH) (pH 3.0) as a supporting
Afterward, the material was washed to electrolyte.
remove the residual persulfate ions by
distilled water. Then, the product was stirred 3. Results and Discussion
in the solution of 150 μL of aniline and 10 3.1 Physical characterization of N-MC
mL of 1 M HCl in an ice bath (0-5 °C) for 4 Figure 1 shows the SEM images of
h. Shortly, the color of the solution turned the surface for SBA-15 silica and N-MC. It
green. After that, the green precipitate was is found that the short rod-like SBA-15
washed copiously with acetone: ethanol in silica links lengthways to generate the
1:1 volume ratio, treated with 10 mL of 1.0 micrometer rod-like particles as shown in
M ammonia solution, washed several times Figure 1 (a). When PANI/SBA-15 is further
with distilled water, and then dried at 45°C. carbonized and etched, it is obvious that the
Next, the pyrolysis step was operated by N-MC with the length of 500-600 nm and
calcining the obtained material under the the diameter of around 400 nm retains the
protection of argon gas at 200 ºC, 300 ºC, rod-like morphology and delivers a rough
800 ºC for 2 h consecutively. Finally, the surface as shown in Figure 1 (b). Therefore,
mesoporous silica template was removed by the fast diffusion of analyte into and out of
stirring 2.0 mg of the as-prepared N-MC the N-MC could be possible through its
into the 200 ml of 1 M NaOH for 2 h at 100 interconnected porous channels which can
°C and keeping in the oven for 4 h at 100 further enhance the electrochemical
°C. Then, the powder was washed with properties of N-MC.
distilled water by high-speed centrifugation
at 12000 rpm until the pH of supernatant a b
became 7.0.
2.3 Preparation of N-doped mesoporous
carbon modified glassy carbon electrode
(N-MC/GCE)
The glassy carbon electrode (GCE)
was polished with 1 µm, 0.3 µm, and 0.05
µm of alumina slurry consecutively for 3
minutes and rinsed with distilled water.
Then, the N-MC solution was prepared by Figure 1. SEM images of (a) SBA-15 silica
mixing 5 mg of N-MC with 1-propanal: and (b) N-doped mesoporous carbon (N-MC)
water in 1:3 volume ratio and sonicating for
25 minutes. Next, dropping 2 µL of N-MC The EDS analysis show that the N-
solution on the GCE surface and drying the MC is constituted of high carbon content
electrode at 50 °C. Before use, the modified (~ 83.5%) with less amount of silicon (~ 1%) as
electrode (N-MC/GCE) was thoroughly shown in Figure 2. indicating that the silica
rinsed with distilled water. is completely removed during the etching
2.4 Characterization process. This suggests strong evidence of
Scanning Electron Microscopy (SEM) high conductivity of N-MC due to the
images were obtained by using a Quanta 450, presence of high carbon content.
FEI, Netherland, equipped with an Energy
Dispersive Spectrometer (EDS) analyzer.
Figure 2. EDX analysis of the N-MC Figure 3. LSV of 4 µM capsaicin in buffer
pH 3 by N-MC/GCE
3.2 Electrochemical behavior of capsaicin
on N-MC/GCE 3.2.1 Effect of scan rate
To investigate the electrochemical Under different scan rates,
performance of the prepared sensors, Linear electrochemical behavior of capsaicin was
Sweep Voltammetry (LSV) was used to also studied and the results were presented
determine capsaicin in 4 µM capsaicin in in Figure 4. Figure 4 illustrates the LSV
NaOAc/AcOH buffer solution (pH 3.0) signals of scan rates from 12.5 to 100 mV s-1
within the potential range from +0.1 to +0.9 in 10 µM capsaicin solution on the N-
vs. Ag/AgCl (3 M NaCl) at a scan of 50 mV MC/GCE. As shown in Figure 4, it is found
s-1 after the accumulation of capsaicin for that the oxidation peaks shift positively and
250 s with stirring under open circuit it is clearly that Figure 4 (inset) the
potential condition. The responses at oxidation peak current of capsaicin is linear
different electrodes were depicted in Figure with the square root of the applied scan rate
3. The LSV of bare GCE exhibits less over the range 12.5 to 100 mV s-1 (ipa (µA)=
obvious oxidation peak, while LSV signal 17.999ν1/2 + 0.4761 (R2= 0.9683)). This
on N-MC modified GCE shows an oxidation indicates totally irreversible electrode
peak at 0.64 V with sharper outline. In process and adsorption control process of N-
addition, when GCE is modified with N- MC/GCE for electrochemical oxidation of
MC, the oxidation peak current of capsaicin capsaicin rather than a surface controlled
(1.56 µA) is much higher than a bare GCE. process.
The increase of oxidation peak current on N-
MC modified GCE manifests the better
electrochemical activity toward capsaicin
oxidation. This indicates that N-MC is more
electroactive than GCE, likely due to the
higher conductivity and active sites resulting
from the presence of N in the carbon
skeletons. Moreover, the above results also
reveal that the carbonization of the resulting
PANI/SBA-15 material, followed by etching
away the SBA-15 hard template provides N-
MC with high surface area for the
accumulation of the capsaicin resulting in Figure 4. LSV of 10 µM capsaicin in pH 3
the enhancement of sensing property. buffer solution at N-MC/GCE. Scan rate
12.5, 25, 50, 75.5, 100 mV s−1. The
relationship between the ipa values and the
square root of scan rate (inset).
3.2.2 Effect of pH current at a potential of +0.64 V (versus
The effect of pH was explored on the Ag/AgCl, 3 M NaCl) as a function of
oxidation current for 4 µM capsaicin in the capsaicin concentration (Figure 6 (inset)). It
solution pH range form 1.0 – 8.0 in was found that the oxidation peak current is
NaOAc/AcOH buffer solution at the N- linearly related to capsaicin concentration
MC/GCE. As shown in Figure 5 with the within the range of 1 – 10 µM. The equation
increase of pH, the oxidation peak currents for linear regression can be expressed as ipa
increase, and the maximum oxidation peak (µA) = 3.178×10-1 c (µM) – 2.50×10-3
current was obtained at pH 3.0. Then, the (R2=0.9993) and the limit of detection
oxidation peak currents gradually decrease (LOD) is estimated to be 0.62 µM (S/N = 3).
with increase of pH higher than 3.0, which The plot of peak currents with Scoville units
could be attributed to the partial was also shown in Figure 6 (inset). Here,
deprotonation of the phenolic moiety of calculated concentration in µM has been
capsaicin in higher pH. This leads to converted to ppm. The concentration was
inefficient accumulation of capsaicin on the also converted to Scoville units assuming
surface of the modified electrode as previous that 1 ppm is approximately equal to 15
reports5. For this reason, a pH 3.0 was Scoville units. The regression equation can
selected as the optimized pH value in order be expressed as ipa (µA) = 0.0694 Scoville
to obtain a sensitive detection of capsaicin unit - 0.0025. Compared with the reported
for the subsequent experiments. modified electrodes, N-MC/GCE exhibits
comparable electrochemical properties in
linear range and LOD in capsaicin
detection.6 Therefore, it can be concluded
that the present N-MC/GCE is an excellent
electrochemical sensor for the sensitive and
rapid determination of capsaicin.
Figure 5. (a) The relationship between pH

and ipa. (inset) The relationship between the
pH and the Epa values in 4 µM capsaicin
solution varying buffer pH. Scan rate, 50
mV s-1
3.2.3 Calibration curve and detection

limit
Linear Sweep Voltammetry (LSV) at
a scan rate of 50 mVs-1 after 250 s
accumulation was performed to examine the
capsaicin quantitatively at the present N-
MC/GCE due to its simplicity, fast
electrochemical response, low background
current and high sensitivity. Figure 6(a) Figure 6. Showing of (a) LSV and (b)
depicts linear sweep voltammograms of linearity of capsaicin in range of 1-10 µM of
successive additions of capsaicin on the N- capsaicin on the N-MC/GCE in pH 3 buffer
MC/GCE in NaOAc/AcOH buffer solutions solution at accumulation time of 250 s and
(pH 3.0) and a calibration curve was scan rate 50 mV s-1. The relationship
constructed by plotting the oxidation peak between concentration (ppm) and (inset b)
Scoville units
4. Conclusion Acknowledgements
In this work, the polyaniline-derived Financial support from Centre of
nitrogen doped mesoporous carbon (N-MC) Excellence for Innovation in Chemistry and
was synthesized and successfully applied in Kasetsant University Research and
the simple, sensitive and reliable Development Institute (KURDI)
electroanalytical sensing of capsaicin. The
prepared N-MC/GCE significantly enhanced Reference
the oxidation peak current owing to its high 1. http://www.chilly.in/scoville_scale.htm.
conductivity, large surface area, and 2. Yardım, Y., Electroanalysis 2011, 23
increased charge polarization. Therefore, the (10), 2491-2497.
present electrochemical sensor showed 3. Kim, D.-H.; Lee, W.-Y. J. Electroanal.
satisfactory analytical performance in terms Chem. 2016, 776 (Supplement C), 74-
of linear dynamic range (1-10 µM), 81.
detection limit (0.62 µM), and sensitivity 4. Silva, R.; Voiry, D.; Chhowalla, M.;
(0.3178) compare to those obtained at other Asefa, T., JACS. 2013, 135 (21), 7823-
modified electrodes. The good analytical 7826.
performance and the simple fabrication 5. Wang, Y.; Huang, B.-B.; Dai, W.-L.;
process of the sensor implied a potential Xu, B.; Wu, T.-L.; Ye, J.-P.; Ye, J.-S.
replacement for the human-subjective Anal. Sci. 2017, 33 (7), 793-799.
Scoville organoleptic test that can also be 6. Ya, Y.; Mo, L.; Wang, T.; Fan, Y.; Liao,
further developed as a sensitive portable J.; Chen, Z.; Manoj, K. S.; Fang, F.; Li,
electrochemical sensor for the instant C.; Liang, J. Colloids Surf. B: 2012, 95
detection of capsaicin in food and (Supplement C), 90-95.
pharmaceuticals.
m
Facile immunoassay for colorimetric detection of parathyroid hormone

based on aggregation of gold nanoparticles
Aurachat Lert-itthiporn1,2*, Tipachai Vatanavicharn2,3, Nathawut Choengchan1,2*
1
2
Applied Analytical Chemistry Research Unit, Department of Chemistry, Faculty of Science,
King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
3
Department of Applied Biology, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang,
Bangkok 10520, Thailand
*E-mail: aurachat.l@gmail.com, nchoengchan@gmail.com
Abstract:
This work presents the immunoassay based on using gold nanoparticles (AuNPs) for
facile colorimetric detection of parathyroid hormone. AuNPs were synthesized accordingly to
Turkevich’s method and were then conjugated with anti-parathyroid hormone antibody by
incubation for 1 hr at 25 °C. Conjugated AuNPs were applied to detection of parathyroid
hormone based on aggregation of AuNPs in the presence of the hormone. Color of the
solution was changed from red wine to purple and the characteristic surface plasmon peak
was shifted from 521 nm to 542 nm. Under the optimized conditions, the level of parathyroid
hormone can be detected in the range of 10 – 1,000 pg mL-1 with good linearity (r2 = 0.997).
This method was applicable for the parathyroid hormone determination with good recovery
(106.3 – 113.6%).
1. Introduction determination. There have been both

Parathyroid hormone (PTH) is a manual1 and automatic methods.9-11
hormone containing 84 amino acids secreted Although these methods provided high
by the parathyroid glands to maintain selectivity but sensor preparation steps were
normal amount of blood calcium.1 An sophisticated.
amount of PTH for normal people is 10 - 65 To date, applications of
pg mL-1.2 Abnormal excretion of PTH can nanoparticles are widely used due to a
cause hypo-parathyroidism and hyper para- variety of potential applications in biomedical,
thyroidism. Hypoparathyroidism occur when optical and electronic fields.12 For examples,
not enough PTH is produced result in low W.D. Dittmer et al.13 presented labeling
blood calcium.3 Hyperparathyroidism is magnetic particles for quantification of the
caused by secretion of PTH is exorbitant amount of PTH. V. Jarrige et al.14 developed
occur with fragile bones and kidney stones.4 a point of care (POC) device based on
Curing of this symptom is parathyroid magnetically controlled movement of
surgery.5 Therefore detection of the blood superparamagnetic nanoparticles for
PTH level is required for close monitoring. measurement of PTH. Amongst the reported
The most widely used analytical nanoparticles mentioned above, AuNPs are
methods for quantitative analysis of blood the most attractive metallic nanoparticles
PTH level are chromatography and immuno- because of their high capability for
assay. For chromatographic method,6-8 high functionalization with biomolecules.15 This
accuracy and precision are achieved. leads to their application for determination
However, it was found that large volume of of various proteins and hormones.16-18
organic solvent was employed. In addition, However, to the best of our literacy, there
the instrument and its operation procedures has not been any work reported on the
are complicated. The immunoassays have analysis of PTH in blood using the AuNPs
been also developed for the PTH so far.
This work, we consequently develop 2.4 Procedure for PTH determination
a simple method for PTH detection based on A 10 μL of each concentration of
immunoassay by aggregation of AuNPs. The standard PTH was added to the conjugated
AuNPs were preliminarily conjugated with AuNPs and incubated for 1 hr at 25 °C. The
anti PTH antibody. In the presence of PTH, surface plasmon bands of the AuNPs were
aggregation of conjugated AuNPs was recorded from 400 – 800 nm using UV – Vis
occurred and color of the solution was spectrophotometer.
changed from red wine to purple. The
corresponding surface plasmon peak was 3. Results and Discussion
also shifted (521 to 542 nm). Possibility of 3.1 Characterization of the prepared
develop assay for applying to rat blood AuNPs
samples are investigated. Results in Figure 1(a) and (b) reveal
that the surface plasmon peak is located at
2. Materials and Methods 521 nm with red wine colored solution.
2.1 Reagents and materials Results by TEM image show that shape of
AuNPs were prepared from HAuCl4 ∙ the nanoparticles are spherical with the
3H2O (Sigma-Aldrich, USA) by using average size of 25.0 ± 11.4 nm. (Figure 1(c)
sodium citrate tribasic dehydrates (Sigma- and (d)).
Aldrich, USA) as reducing agent. Anti-PTH
antibody (ab53040, Abcam, UK), was used
for conjugation of AuNPs. Standard PTH
was prepared in phosphate buffer saline, pH
7.4 (PBS, Sigma-Aldrich, Switzerland).
Working standard solutions were appro-
priate dilution of this stock solution.
Autoclaved deionized-distilled water was
used throughout the experiment.
2.2 Synthesis of AuNPs
Colloidal AuNPs were prepared
accordingly to Y. Wang et. al.19 Briefly, 100
mL of 0.01% w/v HAuCl4 was heated to
boil, followed by adding 2 mL of 1.0% w/v
sodium citrate and simultaneously stirred for
15 min. The solution was kept stand to Figure 1. (a) surface plasmon peak, (b) red
ambient temperature and stored at 4°C. The wine color solution, (c) TEM image and (d)
particles were characterized by a trans- size distribution chart of the as-prepared
mission electron microscope (TEM) a AuNPs (4.84 x 10-3 nM)
TECNAI-10 (Netherlands) and a UV- Vis
spectrophotometer (JASCO V-630). 3.2 Detection principle
2.3 Conjugation of antibody and colloidal Schematic illustration for the
AuNPs detection principle of PTH using the
A 15 μL of 0.1 mg mL-1 antibody conjugated AuNPs is shown as Schematic 1.
was added to 100 μL of the adjusted pH 7.0 The reaction detection for PTH is
AuNPs. After 5 min, 15 µL of 10% w/v based on aggregation of conjugated AuNPs
NaCl was added. The solution was by adding PTH. The surface plasmon peaks
incubated at 25 °C for 1 hr. The conjugated were changed from 521 to 542 nm
AuNPs solution was used without any (Figure 2(a)). This results in changing of
purification. solution color from red wine to purple
(Figure 2(b)). Aggregation of the conjugated
AuNPs was found proportional to the PTH
concentration (Figure 2(c)).
Schematic 1. Schematic illustration for the

detection principle of PTH using the
Figure 3. Absorbance of AuNPs at
conjugated AuNPs
difference pH
Results in Figure 3 demonstrate that

absorbance of the particles were deviated
after conjugation for most of the studied pH
values, excepting pH 7.0. This is due to
inappropriate pH results in both self-
aggregation of the AuNPs after conjugation
and the denatured protein effect. Therefore,
pH 7 was selected for further study.
3.4 Effect of reaction time.
Effect of reaction time was studied
from 30 min to 2 hr. Poor linearity (r2 =
0.554) at 30 min reaction time was
observed. It is due to incomplete reaction. In
contrast to 1 and 2 hr, they both provided
Figure 2. (a) surface plasmon peaks of good linearity (r2 > 0.98) with no significant
AuNPs with different concentrations of PTH difference in sensitivity. It is because the
((a) – (e) are the AuNPs in the presence of 0, reactions reached the equilibrium. Reaction
10, 100 and 1,000 pg mL-1, respectively), (b) time at 1 hr was therefore chosen.
red wine to purple- colored solution of 3.5 Application to blood sample
AuNPs and (c) linear calibration for PTH The aggregation reaction of AuNPs
detection was applied as detection principle of the
method for colorimetric determination of
3.3 Effect of pH of AuNPs on conjugation PTH in blood samples. Rat blood samples
of antibody were received from National Laboratory
pH of AuNPs were adjusted using Animal Center, Thailand. They were
0.05 mM NaOH to the final pH of 5-9 centrifuged at 13,000 rpm for 40 min and
before conjugated with antibody. Results are filtered through a 0.22 µm Nylon membrane
presented in Figure 3. filter prior to analysis. Results as analytical
recovery are summarized in Table 1.
Table 1. Analytical recovery of spiked 6. Marika, K.; Paul, C.; Richard, D. V.;
blood samples Lily, L.; Zengji, L.; Xiaoyan, M.;
Concentration of PTH Steven, F.; Christopher, M. R. J.
(pg mL-1) Recovery Chromatogr. B 2004, 810, 151–155.
No. 7. Shaligram, S. R.; Alkesh, A.; Rustom,
(%)
Original Added Found M.; Padmaja P. J. Pharm. Analysis 2012,
1 16.6 53.7 77.6 113.6 2 (2), 136-142.
2 288.4 89.1 389.0 112.9 8. Erin, E. C.; Mary, E. L.; Jon, B.; Sally,
3 33.88 1023.3 1122.0 106.3 H.; Cristina, L. Q.; Norman, S.;
Kenneth, J. F.; Eileen, C.; Elizabeth, T.
Results in Table 1 reveal that the J. Chromatogr. B 2013, 938, 96-104.
satisfied recovery was achieved. However, 9. Marίa, T. J.; Jose, M. B.; Marίa, L. G.;
the relatively high recoveries are obtained Pau, M.; Antonio, A.; Manel, P. D.;
for some samples. Prior pre-treatment of Anna, L. Am. J. Surg. 2013, 206, 783-
sample is required and is now under 789.
investigation. 10. Esther, P. G. S.; Jesús, M. F.; Aurora, G.
R.; Bruno, M, T.; Fernando, M. P.;
4. Conclusion Carmen, M. C.; Virginia, M. A.; Susana,
A facile immunoassay for S. G. Endocrinol Nutr. 2014, 61 (1), 3-8.
colorimetric detection of PTH was 11. Valerie, C.; Noah, S.; Michael, P. H.;
developed. The developed method provided Martin, J. B.; Michae,l T. Elizabeth, M.;
a good linearity. The very low concentration Xun, Z.; Richard, J. P. Otolaryngol.
of PTH (~ 10 pg mL-1) were detectable. The Head Neck Surg. 2008, 138, 204-208.
method showed the potential for applying to 12. Abhilash, M. Int. J. Pharma Bio. Sci.
determine the blood PTH. However, sample 2010, 1, 1-12.
pretreatment prior to analysis is necessary. 13. Dittmer, W. U.; Kievit, P.; Prins, M. W.
J.; Vissers, J. L. M.; Mersch, M. E. C.;
Acknowledgements Martens, M. F. W. C. J. Immunol.
Applied Analytical Chemistry Methods. 2008, 338, 40-46.
Research Unit, Department of Chemistry, 14. Veronique, J.; Jeroen, H. N.; Jacco, P. H.
Faculty of Science, King Mongkut’s F. V. S.; Mike, F. W. C. M.; Joost, L. M.
Institute of Technology Ladkrabang is V. Langenbecks Arch Surg. 2011, 396,
grateful acknowledged for equipments and 337-343.
financial support. 15. Ralph, A. S.; Pilar, R. G.; Feng, Z.;
Marco, Z.; Wolfgang, J. P. Chem. Soc.
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Label-free gold nanoparticles for spectrophotometric determination of
creatinine in urine
Arjnarong Mathaweesansurn1,2*, Kanisa Tipanantakun2, Parinya Thaothong2,
Pimmada Ooybumrung2, Nathawut Choengchan1,2*
1
2
*E-mail: a.mathawee@live.com, nchoengchan@gmail.com
Abstract:
A simple method for quantitative analysis of creatinine in urine using label-free gold
nanoparticles (AuNPs) is presented. In the presence of creatinine, the AuNPs are aggregated
and this effect results in a color change from wine-red to purple. The surface plasmon
resonance peak is shifted from 522 to 650 nm. Calibration curve between absorbance ratio
and standard creatinine concentration was made with linearity of 0 to 200 mg L-1 (Abs 650 / 522
= 0.004[Creatinine] + 0.103, r2 = 0.995) was obtained. Other analytical characteristics are
listed as the followings: RSD ≤ 5 %, LOD (3SD of blank/slope) = 0.5 mg L-1 and LOQ
(10SD of blank/slope) = 1.6 mg L-1. The method was successfully applied to determination of
creatinine in spiked urine without any complex pretreatment, only simple dilution with water
was performed. The results obtained by this method and by Jaffé reaction method were not
significant difference when paired t-test at 95 % confidence (tstat = -0.13 and tcri = 2.26) was
used. All the results implied that the method gave high accuracy and high precision for the
quantitative analysis of creatinine in urine sample.
1. Introduction Nowadays, due to the unique optical

Creatinine is a metabolite formed properties (surface plasmon resonance,
from creatine and phosphocreatine and it is SPR), gold nanoparticles (AuNPs) have
excreted in urine by the kidney at a been widely exploited as colorimetric
relatively constant rate. The creatinine sensors.10 Several studies have been reported
concentration in urine is an important on the synthesis of AuNPs and their
parameter for kidney diagnosis. The normal applications in colorimetric detection
range of creatinine concentration in urine are various of biological molecules. J. Sittiwong
3.6 to 27 mmol L-1 and 3.3 to 22.5 mmol L-1 et al. proposed a utilization of AuNPs as
for men and women, respectively.1 sensor for determination of creatinine in
Therefore, creatinine level in urine is an urine after an extraction of sample using
important marker for renal disease. sulfonic acid functionalized silica gel.11
The traditional method for creatinine The aim of this work is to develop a
determination is the colorimetric Jaffé simple method for determination of
reaction.2,3 The method is fast and easy. creatinine in urine using label-free AuNPs as
However, this method is non-specific. The chemical sensor without any serious sample
other reported methods are enzymatic pretreatment. Detection principle is based on
method,4,5 capillary electrophoresis (CE),6 the aggregation of the AuNPs in the
high performance liquid chromatography presence of creatinine. This results in a color
(HPLC)7,8 and amperometric biosensor.9 change from wine-red to purple and surface
Although these methods are high specificity, plasmon peak shift from 522 to 650 nm.
they are complicated and expensive. Validation and application of the method to
human urine were also investigated.
2. Materials and Methods mixed through a Gennie Z Vortex mixer
2.1 Chemicals (Scientific Industries, USA) and was kept
All standard and reagents used were standing for incubation (3 min). The surface
of analytical reagent grade. Deionized- plasmon band were monitored by scanning
distilled water purified by ZENEER UP 900 wavelength from 400 to 800 nm via Jasco
(18 MΩ/cm, human corporation, Korea) was V-630 UV–visible spectrophotometer
used throughout. Tetrachloroauric(III) acid, (USA).
sodium citrate tribasic dihydrate and
creatinine were purchased from Sigma- 3. Results and Discussion
Aldrich, USA. Sodium dihydrogen ortho 3.1 Detection principle
phosphate (Analytical UNIVAR reagent, The detection reaction between
New Zealand) and Disodium hydrogen AuNPs and creatinine is presented in Figure
phosphate heptahydrate (Panreac, E.U.) 1A. Firstly, the AuNPs is capped with citrate
were used to prepare phosphate buffer (pH ion because the as-prepared particles are
6.0). synthesized accordingly to the Turkevich’s
2.2 Synthesis of AuNPs method, where sodium citrate is used for
The AuNPs was synthesized reduction of Au(III) to Au0 nanoparticles.
accordingly to Turkevich’s method.12 The citrate ions are regarded as the stabilizer
Briefly, the 100.0 mL of 1.0 mM for preventing the AuNPs from self-
Tetrachloroauric(III) acid (HAuCl4) solution aggregation (Figure 1B). However, the
was heated. At the boiling point, 7.0 mL of interaction between the AuNPs and citrate
38.8 mM sodium citrate was added to this ions is week. In the presence of creatinine,
solution. The mixture was heated under facile exchange between creatinine and
vigorous stirring for 15 min. Finally, the citrate ion is easily occurred. Creatinine can
solution was cooled down to room strongly bind with the AuNPs and this result
temperature and stored at 4°C for further in aggregation of the AuNPs and the
utilization. solution color change from wine-red to blue
2.3 Standard and sample preparation (Figure 1C).
A creatinine stock standard solution Figure 2 shows the surface plasmon
of 1000 mg L−1 was prepared by dissolving band of the synthesized AuNPs with top
0.0500 g of creatinine powder in 50.0 mL of peak at 522 nm and after addition of
DI water. Working standards were creatinine the peak at 522 nm decrease and a
appropriated diluted from stock solution to new surface plasmon band emerged at
provide final concentration range of 1.0 to around 650nm.
200.0 mg L−1. Human urine samples were 3.2 Appropriate time for measurement
spot collected from normal volunteers. All The effect of the mixing time
samples were diluted with water (100-fold) between AuNPs and creatinine was varied
prior to analysis. Any further pretreatment from 1 to 10 min (Figure 3). In the presence
was not necessary. of 50 mg L-1 creatinine, the absorption peak
2.4 Procedure for determination of at 650 nm of AuNPs gradually increased
creatinine with increasing reaction time. A good
Aliquot of 1.0 mL of AuNPs was linearity of the calibration curve was
transferred to a test tube. 5.0 mL of 10.0 obtained when 3 min was used, this time
mmol L-1 phosphate buffer (pH 6.0) and was therefore selected.
0.05 mL of standard creatinine or urine
sample were then added. The solution was
Figure 2. The surface plasmon band of
AuNPs solution the in the absence and
presence of 200.0 mg L-1 creatinine
Figure 1. A: Schematic illustration of the

interaction of creatinine with AuNPs. B:
TEM image of 1.04 nM AuNPs without
creatinine and C: TEM image of 1.04 nM
AuNPs with 200 mg L-1 creatinine. Inset
photos present bottles of solutions of B and Figure 3. The surface plasmon band of
C, respectively. AuNPs solution in the presence of 50.0 mg
L-1 creatinine recorded at 1 to 10 min
3.3 Effect of the AuNPs concentration
Effect of the concentration of the 3.4 Selectivity study
AuNPs was studied from 0.52 nM to 1.04 The selectivity of this method was
nM. Results are shown in Figure 4. The evaluated using other organic small
absorbance ratio at 650 and 522 nm compounds (albumin, uric acid, ascorbic
(A650/A522) was used to assess the degree acid, L-glutathione, L-tryptophen, L-serine,
of aggregation. When the concentrations L-threonine, L-histidine, L-valine, L-
were increased, sensitivities and linear phenylanine and aspartic acid). The
relationship were increased. The optimal interferences concentration was 10-fold
AuNP concentration of 1.04 nM was higher than that of creatinine. Figure 5A
selected for further study as it gave the clearly illustrated that only creatinine cause
highest sensitivity and linearity. a solution color change from wine-red to
blue. The corresponding surface plasmon
bands (Figure 5B) also show that only standard conditions. It is clearly observed
creatinine cause an increase of absorbance at that good linearity is obtained from 0 to 200
650 nm. This suggests that this method is mg L-1 creatinine.
very selective to creatinine. 3.6 Analytical performances
The analytical performances are
summarized in Table 1. It gives a good
analytical performance.
3.7 Determination of creatinine in urine
sample
The developed method was applied
to determine the creatinine content in urine
of normal volunteers. Urine samples were
diluted with water. Any further sample
pretreatment is not required. Results in
Table 2 are not significantly difference
under the statistical paired t-test14 at 95%
confidence level (tstat = -0.13, tcrit = 2.26).
There results imply that the method was
successfully validated.
Figure 4. Effect of AuNPs concentration on Table 1. Analytical performances of the

sensitivity method for determination of uric acid
Parameter Value
-1
Linearity range (mg L ) 0 – 200
Regression coefficients (r2) 0.9947
RSD (%) (n = 5) 2.6 – 4.9
a -1
LOD (mg L ) 0.5
LOQb (mg L-1) 1.6
a
LOD = 3SD of blank / slope
b
LOQ = 10SD of blank / slope
Table 2. Concentrations of creatinine in

urine samples
Concentration ( mg L-1, mean + SD)
Sample
This work The Jaffé method
1 233.6 ± 1.3 243.0 ± 4.0
2 256.9 ± 9.7 256.1 ± 8.8
Figure 5. (A): The photograph of AuNPs
solutions and (B): The surface plasmon 3 150.5 ± 2.0 137.2 ± 0.9
bands of AuNPs solution in the presence of 4 121.8 ± 1.8 128.0 ± 1.3
creatinine (200 mg L−1) or interferences 5 107.4 ± 3.9 104.9 ± 6.0
(2000 mg L−1) 6 228.4 ± 1.8 206.5 ± 4.4
7 171.6 ± 7.8 192.6 ± 10.2
3.5 Colorimetric detection of creatinine 8 226.8 ± 0.1 227.2 ± 7.3
Figure 6 displays the surface 9 197.4 ± 2.3 197.4 ± 9.5
plasmon band of the synthesized AuNPs 10 94.7 ± 3.2 91.3 ± 5.7
with various concentrations of standard
creatinine and the corresponding calibration
plot between the absorbance ratio and the
Acknowledgements
Financial supports from the Faculty
of Science, King Mongkut’s Institute of
Technology Ladkrabang is gratefully
acknowledged. Instrumental supports from
Applied Analytical Chemistry Research
Unit, Department of Chemistry, Faculty of
Science, King Mongkut’s Institute of
Technology Ladkrabang are also
appreciated.
References
1. Debus, B.; Kirsanov, D.; Yaroshenko, I.;
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2. Vasiliades, J. Clin. Chem. 1976, 22,
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3. Falcó, P. C.; Genaro, L. A. T.; Lloret, S.
M.; Gomez, F. B.; Cabeza, A. S.; Legua,
C. M. Talanta 2001, 55, 1079-1089.
4. Moss, G. A.; Bondar, R. J. L.; Buzzelli,
D. M. Clin. Chem. 1975, 21, 1422-1426.
5. Ogawa, J.; Nirdnoy, W.; Tabata, M.;
Yamada, H.; Shimizu, S. Biosci.
Biotech. Biochem. 1995, 59, 2292-2294.
6. Liotta, E.; Gottardo, R.; Bonizzato, L.;
Pascali, J. P.; Bertaso, A.; Tagliaro, F.
Clin. Chem. 2009, 409, 52-55.
7. Ambrose, R. T.; Ketchum, D. F.; Smith,
Figure 6. (A): The surface plasmon bands of J. W. Clin. Chem. 1983, 29, 256-259.
AuNPs solution in the presence of different 8. Yuen, P. S. T.; Dunn, S. R.; Miyaji, T.;
creatinine standard solution, (B): The linear Yasuda, H.; Sharma K.; Star, R. A. Am.
plots between the absorbance ratio and the J. Physiol. Renal. Physiol. 2004, 286,
creatinine concentration F1116- F1119.
9. Nieh, C. H.; Tsujimura, S.; Shirai, O.;
4. Conclusion Kano, K. Anal. Chim. Acta 2013, 767,
In this work, a simple method for 128–133.
quantitative analysis of creatinine in urine 10. Apyari, V. V.; Arkhipova, V. V.;
using label-free gold nanoparticles was Dmitrienko, S. G.; Zolotov, Y. A. J.
accomplishedly developed. Good analytical Anal. Chem. 2014, 69, 4-15.
performance was obtained. As a result, 11. Sittiwong, J.; Unob, F. Spectrochim.
precise and accurate quantification of the Acta Mol. Biomol. Spectrosc. 2015, 138,
creatinine concentration can be obtained. 381-386.
The proposed method was also successfully 12. Turkevich, J.; Stevenson, P. C.; Hillier,
applied for the creatinine determination in J. Discuss. Faraday Soc. 1951, 11, 55-
urine samples without any sample 75.
pretreatment.
13. He, Y.; Yu, X. H. Microchim. Acta 14. Miller, J. N.; Miller, J. C. Statistics and
2015, 182, 2037-2043 Chemometrics for Analytical Chemistry,
5th. ed., Pearson Education: Harlow,
UK, 2005
Paper-based ammonia gas sensor using zinc complex of
Eriochrome Black T and digital image analysis
Supa Sangdang, Pongsak Lowmunkhong, Pakawadee Sutthivaiyakit*
Department of Chemistry and Center of Excellence in Innovation in Chemistry, Faculty of Science,
Kasetsart University, Bangkhen, Bangkok 10900, Thailand
*Email: fscipws@ku.ac.th
Abstract:
Colorimetric paper-based sensor for ammonia gas has been developed by coating an
aqueous mixture of synthesized Zn complex of Eriochrome Black T and TiO2 in the presence
of polyvinyl alcohol (binder) and polyethylene glycol (dispersing agent) on a filter paper. The
paper changes from red to blue upon exposure to ammonia gas. The quantitation of ammonia
gas was performed by digital image analysis using a commercial scanner and the Image J free
software. The RGB values are converted to 2D CIE 1931chromaticity space followed by the
interpolation technique. The calibration graph is in the range of 200-2,000 ppmv with a
detection limit of 24 ppmv. The method was applied for air samples collected from workplace
environment in a small rubber pilot plant. The results obtained from the method developed are
in good agreement with those obtained from the commercial detector tube method.
1. Introduction 2. Materials and Methods

Ammonia gas is colourless and has a 2.1 Materials
pungent odor.1,2 It is widely used in a variety Sodium salt of eriochrome black T,
of industries including food processing zinc sulphate heptahydrate, polyethylene
industry refrigeration system rubber glycol 6000 and polyvinyl alcohol 28-99
industry, fertilizer plants, petrochemical were obtained from Riedel-de Haen AG,
plants. However, exposure to high ammonia Mallinckrodt, Merck (Darmstadt) and Fluka,
concentration is a serious health threat, respectively. TiO2P25 was a gift from
resulting in the requirement of accurate Degussa AG, Frankfurt. Ultra-pure water
ammonia detector. was obtained from water purification system
Paper is a low cost cellulose fiber model Simplicity ( MilliporeTM, France) .
with high surface area which is readily to Other chemicals are of analytical reagent
modified and fabricated and used at the grade.
point of need.3, 4 It has been used as support 2.2 Instrument
in many applications including Electrospray ionization ( ESI) mass
environmental analysis and medical spectral measurements were recorded on
diagnosis. Among paper based method5-7 Bruker Micromass – microTOF Quattro II
optical paper based method has been mass spectrometer Absorbance
growing interest in the point of need as it is measurement were taken on a Perkin
simple to use, rapid, inexpensive and can be Elmer Lambda35 double beam UV-Vis
integrated with image analysis.8, 9 spectrophotometer. Fourier transform
The present work aimed to develop a infrared (FTIR) spectra were recorded using
new paper based ammonia gas sensor based a Perkin Elmer, 2000 FT-IR. Images were
on zinc complex of Eriochrome Black T and captured on HP Scanjet 7400c color scanner.
digital image analysis.
Ammonia samples were collected by Grab 2.5 Preparation of standard gaseous
air sample (SKC, PA USA). ammonia
2.3 Synthesis of zinc complex of EBT Stock standard ammonia gas was
0.311 g of ZnSO4.7H2O was added to obtained by using a gas tight syringe to
the solution containing 1 g EBT in 250 mL withdraw 10 mL of a headspace gas
of water. The mixture was heated at 80 °C ammonia over 28% ammonia solution and
inject to 1 liter Tedlar bag filled with 990
for 30 min and left at room temperature.
mL N2 and the whole bag shook for one
After centrifugation at 6,000 rpm for 10 min. hour on a shaker. The ammonia concen-
The precipitate was washed with cold water tration was then calibrated with a commer-
and recrystallized in water-methanol cial ammonia gas detecting tube (GASTEC,
mixture. The brownish black crystals were Japan).A working concentration of ammonia
washed with cold water, dried in air and gas was appropriately diluted with nitrogen
then stored in a desiccator. gas and shaken for one hour to ensure the
Molar mass of the zinc-EBT homogeneity of dilute gas.
complex [C20H10N3O7SZn]-1 was confirmed 2.6 Procedure for gaseous ammonia
by using electrospray MS (in MeOH, analysis
negative ion mode): Expected: 501.761 A 50 mL round bottom flask with
(100%), Found: 501.9510 (100%). The zinc- two necks was used as a sample cell. One
EBT complex was also confirmed by the neck has a septum seal at the end and the
presence of its distinctive isotope pattern: other is a ground joint. A detecting paper
64
Zn (48.6%), 66Zn (27.9%), 67Zn (4.1%), was inserted into a holder with a ground
68
Zn (18.8%), 70Zn (0.6%). Infrared (KBr, glass joint at the end, which fitted well in a
cm–1): 3451 (OH), 1616 (C=N), 1503 (NO2), ground joint neck of the round bottom flask.
1404 (N=N), 1231 (C-O). Maximum 50 mL of ammonia gas was injected through
absorption wavelength (λmax) in 50% a septum neck of a cell and allowed for 2
aqueous methanol: 344.52 nm and 546.73 min for ammonia reacting with the Zn-EBT
nm. complex coated on the paper. The coated
2.4 Preparation of detecting papers paper was transferred to a scanner to capture
The detecting solution was prepared the image.
by mixing the solution composed of Zn-EBT
4 mg, and TiO2 8 mg in 20 mL of 0. 80%
polyvinyl alcohol containing 120 µL of
Figure 1 shows the absorbance
0. 36% polyethyleneglycol. A filter paper
spectrum of Zn-EBT complex coated on the
( WhatmanTM No.1) used as a support was
cut into a rectangular size of 1.5 × 6 cm and modified filter paper before and after
exposure to ammonia gas. However, it was
dipped into the detecting solution and dried
in air and stored in a desiccator. The coated observed that absorbance from 550 to 780
decrease significantly. It probably is due to
paper was cut into a circle of 13 mm in
diameter and stored in a desiccator. one ammonia molecule coordinating to the
ZnII ion.
Figure 1. Absorbance spectrum of Zn-EBT complex coated on modified filter paper before
(a) and after exposure (b) to ammonia gas
A circular area of 2000 counts was

selected over the digitized images and RGB
values of digitized images were read by
Image J free software as shown in Figure 2.
Figure 3. Color image of paper based sensor
responding to different ammonia
concentration
The image of detecting paper was

captured and its RGB values were then read.
RGB values were converted to xj,yj values
by converting them first to linear RGB
values, then to XYZ tristimulus values and
finally to the 2D ( x, y) CIE chromaticity
space through the following equations:10
Figure 2. Digitized image of detecting paper

after exposure to ammonia gas. The circle
indicates the measured area
The values of Xj, Yj for the
Figure 3 shows color image of the concentration were used to construct the
paper based sensor responding to different external calibration curve. Additionally, the
ammonia concentration in the range 0-2000 concentration of the sample was calculated
ppmv. by an interpolation technique where its
External calibration graph was magnitude depends on the distance between
constructed over 200-2,000 ppmv by the measurement point and the calibration
injecting appropriate concentration of curve.10 The limit of detection was taken as
ammonia gas into the cell. the concentration corresponding to three
times of S/N ratio. The noise was determined
by measuring the standard deviation of Acknowledgements
blank as nitrogen gas passed through the Financial support from the Center of
sample cell. Limit of detection of the Excellence for Innovation in Chemistry
method was found 24 ppmv (n=7). Samples ( PERCH-CIC) , Office of Higher Education
in different locations of the workplace Commission, Ministry of Education and
environment of a processing rubber plant Kasetsart University Research and
were with drawn into a Tedlar bag at a flow Development Institute (KURDI) are
rate of 1 Lit. min-1. Samples were analyzed gratefully acknowledged.
as described in section 2.2 and also detected
by commercial detector tube. As shown in References
Table 1, the results obtained from developed 1. Tavoli, F.; Alizadeh, N. Sens. Actuators
method show good agreement with those B 2013, 176, 761-767.
obtained by detector tube method. However, 2. Timmer, B.; Olthuis, W.; van den Berg,
the detector tube method has lower limit of A. Sens. Actuators B 2005, 107, 666-677.
detection. 3. Santhiago, M.; Henry C. H.; Kubota, L.
T. Electrochim. Acta 2014, 130, 771-777.
Table 1. Comparison of present method
4. Zhao, W.; Ali, M. M.; Aquirre, D.;
with commercial detector tube method
Sample Present Detector Error Brook, M. A.; Li, Y. Anal Chem. 2008,
method(ppmv) tube(ppmv) 80, 8431-8437.
1 559 600 -6.7%
2 332.3 360 -7.7%
5. Santhiago, M.; Wydallis, J. B.; Kubota,
3 < LOD 16 - L. T.; Henry, C. S. Anal. Chem. 2013, 85,
4 < LOD 5 - 5233-5239.
6. Gu, Y.; Huang, J. Colloids Surf. A. 2013,
4. Conclusion
A new device for ammonia gas 433, 166-172.
detection was developed. The material was 7. Lee, J.; Lee, Y.; Ahn, Y. J.; Choi, S.;
composed of Zn-EBT/TiO2 coated on a filter Lee, G. J. Sens. Actuator B 2018, 256,
paper. With the use of image of detecting 828-834.
paper captured by a commercial scanner and 8. Paciornik, S.; Yallouz, A. V.; Campos, R.
image J software, determination of ammonia
C.; Gannerman, D. J. Braz. Chem. Soc.
gas in work place environment was feasible
and in a good agreement with a detector tube 2006, 17, 156-161.
method. 9. Hong, J.; Chang, B. Y. Lab on a Chip
Additionally, the optical detection on 2014, 14, 1725-1732.
paper-based with colorimetric method was 10. Yetisen, A. K.; Martinez-Hurtado, J. L.;
developed, it is rapid, inexpensive and easy Garcia-Melendrez, A.; Vasconcellos, F.;
to use.
Lowe, C. R. Sens. Actuators B 2014, 196,
156-160.
A ‘contact stamping’ microfluidic paper-based analytical device
for determination of creatinine using camera phone and
standard addition approach
Suthathip Thongrod1,2*, Arjnarong Mathaweesansurn1,2, Nathawut Cheongchan1,2*
1
2
*E-mail: suthathip.stt@gmail.com, ncheongchan@gmail.com
Abstract:
This work presents the use of a paper-based analytical device (µPAD) for
determination of creatinine based on the Jaffé reaction. Patterning of the hydrophobic barrier
was made by contact stamping of the indelible ink onto a laboratory filter paper (WhatmanTM
No.1). The contact stamping was selected because of its fast and simple fabrication method.
This µPAD was designed to compose of one sample area surrounded with eight reagent
(alkaline picrate) and eight detection areas. Aliquots of standard creatinine solutions were
dropped onto the spaces connected between sample and reagent areas for quantitative
analysis of creatinine by standard addition approach. The orange-colored products were
developed on the detection areas and were simultaneously photographed through a camera
phone (Samsung Galaxy Tab S 8.4TM). Intensities of the product colors based on RGB model
were investigated using Image JTM. The intensity ratios of red to green (R/G) were then
plotted against standard creatinine concentrations. Advantages of the developed method are
simple, fast and suitable for ‘point-of-care’ clinical testing. Study on application of the
method to urine samples is now under carried out in order to prove its efficiency for real use.
1. Introduction µPADs, including photolithography,16 inkjet

Creatinine is produced as the printing,17 wax printing,18 laminating,19 and
metabolic waste product resulting from the stamping.20-22 Photolitho- graphy and inkjet
conversion of creatine and phosphocreatine and printing require expensive hardware for
is excreted through urine.1 The urinary fabrication while the use of wax for the
creatinine concentration in normal clinical hydrophibisation of paper normally needs an
range is 280-2590 mg/L,2 however it can be extra heating step, for wax diffusion in
more or less depend on age and gender. paper, which ultimately can increase
Excreated urinary creatinine level can be fabrication costs.
regarded as a marker for kidney dysfunction. Here, we report a simple, fast and
There are many publications reported low-cost method for fabrication of µPADs
on various methods for determination of by contact stamping of the ink onto
creatinine, such as chromatographic laboratory filter paper. The contact stamping
methods,3-8 enzymatic methods,9 electro- µPADs was applied to urinary creatinine
chemical method10,11 and spectrophoto determination based on Jaffé reaction using
metric Jaffé methods.12-15 However, the standard addition approach and camera
methods mentioned above exploited bulky phone for taking photo.
instruments.
Recently, microfluidic paper-based 2. Materials and Methods
analytical device (µPADs), have attractive 2.1 Chemicals and paper material
due to their advantages such as simplicity All chemicals used in this work were
and less volume of employed chemicals. analytical reagent grade. Deionized-distilled
There are several methods for fabrications of water was used throughout. Stock standard
creatinine was prepared by dissolving (more than 1.6 mm) it is time-consuming for
0.1000 g. creatinine anhydrous (Sigma, flowing of the solution. Thus, the 1.6 mm
China) in 100 ml deionized water. Stock channel width was chose.
picrate solution was prepared by dissolving
0.2978 g picrate (Merck,USA) in deionized
water and working alkaline picrate solution
was prepared by mixed 4.81 ml of stock
picrate, 4.0 ml of 2.5 M NaOH and fill up to
10 ml with deionized water. Standard
laboratory filter paper WhatmanTM No.1 was
used as paper-based device.
2.2 Design of µPADs for standard
addition approach
The µPADs in Figure 1 is designed Figure 1. Patterning of the contact stamping
like a flower-shape with 3 areas. Central µPADs for the creatinine determination with
area is sample area. Middle areas (a-h) are standard addition approach
reagent areas and outer areas (A-H) are
detection areas. Diameter of each reagent The channel length can affect the
and detection area is 5 mm while diameter moving time of the liquid. At the lengths of
of the sample area is 10 mm. The channel 3 mm and 5 mm were regarded as the most
between sample area and reagent area (inner appropriate lengths for inner channel and
channel) is 3x1.6 mm2 and another channel outer channel, respectively. Because moving
(outer channel) is 5x1.6 mm2. time of the liquid from sample area to
2.3 Colorimetric detection of creatinine detection area is fastest.
An aliquots of 0.6 µL 0.05 M.
picrate in 1.0 M NaOH were dropped onto
each reagent area. A 0.3 µL of each standard
creatinine solutions (50-1000 mg/L) was
dropped into inner channel. The filter paper
was dried using hot drier for the 30 s. Urine
was dropped onto central area. The sample Figure 2. Patterning of the µPADs for the
propelled all standards and reagents into investigation of channel width effect
detection areas. The orange-colored photographs was taken after dropping a 6.0
products were developed and were µL of red dye solution.
simultaneously photographed through a
camera phone (Samsung Galaxy Tab S 3.1.2 Barrier width
8.4TM). Intensities of the product colors The suitable thickness of the
based on RGB model were investigated hydrophobic barriers was investigated to
using Image JTM. prevent fluid leakage. Leaking test were
studied by a ring pattern diameter of 3 mm
3. Results and Discussion with vary barrier width between 0.4 to 2.4
3.1 Optimization for the µPADs patterning mm. Table1 lists the number of barrier width
3.1.1 Channel width and length obtained among 4 repetitions. At the thin
The effect of channel width was barrier width (less than 1.2 mm), there are
studied. Figure 2 shows a stamped pattern some missing part of the ring. At 1.2 and 1.6
with diameter of 6 mm clear circle mm, the patterning are complete without any
connected with fluid channel at various size missing part. The thickness barrier width of
width (0.4-2.4 mm). By the channel width 1.2 mm is chosen as its stamp line width is
less than 1.6 mm, the ink is able to spread not difference from the expected barrier
and block the channel. At the larger width width.
Table 1. Lists the number of barrier width The results in Table 2 show that
fabricated by contact stamping for aqueous sensitivity is increased when concentration
regarded of picrate is increased. As compromising
between the sensitivity and amount of
consumed reagent, the suitable concentration
of picrate at 0.025 mg/L is selected.
Table 2. List of various concentrations of

picrate, which reaction with standard creatinine
concentration of 50 - 1000 mg/L
Equation R2
0.0125 M picrate 1.63x10-4X +
0.989
in 1.0 M NaOH 1.1628
0.0125 M picrate 2.89x10-4X +
0.995
in 1.0 M NaOH 1.2848
3.1.3 Optimization of detection area 0.0125 M picrate 3.33x10-4X +
0.992
The suitable size of detection area in 1.0 M NaOH 1.3605
were studied. Results are shown in Figure 3. 0.0125 M picrate 4.23x10-4X +
0.992
in 1.0 M NaOH 1.4105
For the dimension of 6 mm, the product
color is uneven. This may result in the
measured color value, while the dimension 3.2.2 Concentration of NaOH
of 4 mm is too small to allow the substance The appropriate concentration of
to be trapped inside the container and there NaOH was investigated in the range of 0.2 to
are some over the channel. The 5 mm 2.0 M. Results are shown in Figure 4. At low
diameter is therefore selected as the concentration of NaOH, products complex is
detection area. pale yellow and it is more dense orange
3.2 Optimization for the creatinine when higher concentration of NaOH is
detection exploited. However, at 2.0 M NaOH, some
3.2.1 Concentration of picrate of sediment was observed on the paper. This
Picrate solutions at various can block the liquid passage. The 1.0 M
concentrations (0.0125 to 0.1 mg/L) were NaOH was selected.
prepared in 1.0 M NaOH. After reaction
with creatinine, the color of the product was
developed on paper and was taken a photo.
Figure 4. Color of product between standard

creatinine and picrate in various
concentration of NaOH in the range of 0.2 to
2.0 M
Figure 3. Photographs of the µPADs with
different diameters of the reagent and
detection areas
3.3 Calibration and analytical performances determined creatinine concentration are not
For the quantification of creatinine different by Bland-Alman plot. This means
by using µPADs with Jaffé reaction, the the developed method is successfully
standard addition curve was established as validated.
shown in Figure 5. The precision of this
method in term of relation standard
deviation (RSD) was less than 3%.
Figure 6. Bland-Altman plot for comparison

of the data obtained from our method with the
Figure 5. Example of the standard addition conventional Jaffé’s method
curve
4. Conclusion
Table 3. Comparison of the measured A contact stamping µPADs for
creatinine concentration in urine samples, determination of creainine in urine was
determined by difference method successfully developed. The contact
Urinary creatinine concentration mg/L
Sample
(Mean±SD)
Recovery stamping method is the simple and cost-
Original Added Found effective fabrication method. An advantage
1 211.7±1.9 500 713.7±1.6 100 of the method of standard additions could
2 180±1.7 500 698.5±0.5 104
still be useful for overcoming matrix effects.
3 231.7±0.9 500 735.6±2.1 101
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Biosen. Bioelectron 2016, 75, 347–351
A Low cost device for colorimetric determination of salicylic acid
Anukool Khammona1,2, Veeramol Vailikhit2,3*
1
The Graduate School, Kasetsart University, Thailand
2
Forensic Science Program, Department of Chemistry, Faculty of Liberal Arts and Science,
Kasetsart University, Kamphaeng Saen, Nakhon Pathom, Thailand
3
Sustainable Chemistry Research Unit, Faculty of Liberal Arts and Science, Kasetsart University,
Kamphaeng Saen, Nakhon Pathom, Thailand
*E-mail: veeramol.v@ku.ac.th
Abstract:
An affordable colorimeter was designed and developed for the determination of
salicylic acid. The proposed device employs a white-LEDs light source, Arduino UNO R3
microcontroller and a TCS3200 colour sensor. The effective intensity from green values of
Fe(III)-salicylic acid, purple complex showed the best linearity (r2 > 0.99) in the
concentration range of 10-200 mg/L. Precision and accuracy were found to be comparable to
a standard UV-VIS spectrophotometer in the concentration range 25-100 mg/L. This is an
effective, affordable and portable D.I.Y. colorimeter for forensic use in the field, also for
education and amateur science use.
1. Introduction analytical performance depends on the

Salicylic acid is prohibited to use in properties of the tools (camera, smartphone
food additive according to the Ministry of or scanner) that are used to produce pictures.
Public Health’s Act No.151 (B.E.2536)
because it’s health hazard. Salicylic acid is
still found, especially in preserved foods.
There are several methods for quantitative
determination of salicylic acid such as Gas
chromatography-mass spectroscopy (GC-
MS), high-performance liquids chromato-
graphy-mass spectroscopy (HPLC-MS),
spectrophotometry, spectrofluorimetry and
electrochemistry.1 The most frequently
analysis is the spectrophotometric method
because it’s accurate, quick analysis ability
and easy to use. However, the spectro-
photometer may not always be available Figure 1. Design device connected with
especially in poorer area. For the computer to displayed data from colour
measurement, usually the purple complex is sensor
formed by the reaction between a single
chromogenic reagent (FeCl3) and salicylic This report describes the
acid. construction of a low cost device using
Recently, the analytical method Arduino UNO R3 microcontroller which is
based on the analysis of colour data in RGB an open-source platform used for building
system from digital image has been applied and programing of electronics,7 low cost
for qualitative determination of substance TCS3200 colour sensor from Texas
such as chlorine,2 chromium,3 formal- Advanced Optoelectronic Solutions
dehyde,4 iron,3,5 methamphetamine6 and (TAOS), LED lamp. The RGB values can be
salicylic acid1 in many field. However, the used with Beer-Lambert law to determine
the concentration of salicylic acid compared
to a standard spectrophotometer.

2.1 Apparatus
The designed device is shown in
Figure 1 and 2(A). It consists of four parts: a
Arduino UNO R3 microcontroller, a
TCS3200 colour sensor as detector, LEDs
lamp and a sample cuvette holding, attached
to a box constructed from acrylic sheet to
give internal dimensions of 14 x 14 x 10 cm
(Width x Length x Height). The schematic
diagram of TCS3200 colour sensor with
Arduino is shown in Figure 2(B). The
Arduino was programmed with the Arduino
IDE program. It converts the output
frequency from the TCS3200 sensor to
colour in RGB system. The system diagram
is shown in Figure 3. A colordetect program
was used to display the data on computer.
(A)
Figure 3. System diagram of the designed

device
The colour given from the designed

device are in the form of RGB system which
(B) is a colour system for representing the colors
used on a computer display. Red(R),
Green(G), and Blue(B) can be combined in
various proportions to obtain any color in
the visible spectrum. RGB colors varied
between 0 and 255, the range that a single
8-bit byte can offer. In the RGB colour
system, any colour is represented in the form
of (R, G, B). The (0, 0, 0) refers to black and
the (255, 255, 255) refers to white. This
system can be made 16,777,216 colours.
Absorbance measurements were
made by using T60 UV-VIS Spectrophoto-
meter PG Instruments and our designed
device. The 1-cm cuvette was use for all
Figure 2. Designed device (A) and measurement.
schematic diagram of TCS3200 colour
sensor with Arduino (B)
2.2 Chemical and reagents (where SD is blank standard deviation and
All chemicals used were analytical m is slope of the calibration curve) and LOQ
grade. The standard solution of salicylic acid was 10 SD/m
was prepared by dissolving 0.1xxx g The precision and accuracy of the
salicylic acid, HOC₆H₄COOH. (Merck) into designed device was determined by spiked
a 100 mL graduate flask with 5 ml ethanol sample with the known standard salicylic
and brought it up to the mark with distilled acid solution for 3 concentrations (25, 50,
water to give the stock solution which was 100 mg/L.) The accuracy and precision were
diluted further as required. A solution of 2% investigated from percent recovery and
(w/v) ferric chloride was prepared by percentage of standard deviation (%RSD).
dissolving 2 g of FeCl3.6H2O (QRec) in 100
mL of 1% Hydrochloric acid. 3. Results and Discussion
2.3 Analytical procedures Salicylic acid reacts with ferric
Standard salicylic acid was prepared chloride to give violet colour. The maximum
in the range of 10 – 800 mg/L by dilute from absorption (λmax) of Fe(III)-salicylic acid
the stock and aliquot 2 mL into test tube purple complex by T60 Spectrophotometer
with 1 mL of 2% ferric chloride. After are 525 nm (Figure 3) The colour reaction is
shaking, an absorbance was measured by stable for more than 1 hours.
T60 spectrophotometer and the designed
device at the same time. For T60
spectrophotometer, the absorbance was
measured at the maximum absorption (λmax)
of the complex. The calibration curve was
prepared by plotting absorbance with
standard concentrations to report the
correlation coefficient (r2), regression
equation and the linear range. For the
designed device, the effective intensity
(absorbance) was calculated from the RGB
values determined by measuring the color
intensity from the detector without sample Figure 3. Maximum absorption (λmax) of
(reagent blank) and comparing with the Fe(III)-salicylic acid, purple complex with
color intensity of the sample through Beer- T60 Spectrophotometer
Lambert law as follows:
3.1 Effect of light and calibration curve
T= I0/I1 = ekbC, In order to study the effect of light
AX =-log T = -log(I0/I1) = ɛbC, on the effective intensity, white, red, green
and blue LEDs were used as light source.
AX =-log(I0/I1) The calibration curve was prepared. The
results are shown in Table 1. From that
Where the AX is the effective intensities and result the effective intensity of red (R) and
I0, I1 are the RGB values of sample and blank green (G) values from white light showed
solution respectively. the best linearity (r2>0.99). Thus for
2.4 LOD, LOQ, precision and accuracy providing the sufficient the white-LEDs was
The analytical performance of the selected to be a light source. Figure 4-5
designed device was performed under the shown the calibration curve of red (R), green
optimum condition. Linearity, precisions, (G) and blue (B) values from white light.
accuracy, limit of detection (LOD) and limit The result showed the linearity range of the
of quantitation (LOQ) were evaluated. The calibration curve from R, G and B values are
limit of detection (LOD) based on 3SD/m 10-400, 10-200 and 10-200 mg/L, respectively.
The correlation coefficient (r2) from R and
G values are 0.99 and 0.98 for B values.
The effective intensity from G values
shows the linearity up to 200 mg/L and then
drops to zero but the effective intensity from
R and B values are stable after 400 mg/L
because the green intensity (G) is completed
absorbed by the purple complex after 200
mg/L but the red (R) and blue (B) intensity
are not completed absorbed. The G values
given by the design device is zero. (When Figure 4. Plots of the effective colour
all of the intensity of the RGB color are intensity versus salicylic acid from 10 – 800
completed absorbed, the color of the mg/L with designed device
solution will turn to black.)
The calibration curve from T60
spectrophotometer is linear up to 350 mg/L
with good correlation coefficients
2
(r = 0.9943). It can be seen that the
correlation coefficients (r2) of the designed
device and T60 spectrophotometer are
similar from the range 10 to 400 mg/L.
Table 1. The correlation coefficient (r2),

regression equation and the linear range Figure 5. Calibration curves of the effective
calculated from the RGB values of the intensity of the designed device with white
designed device light
Colour Regression 3.2 Limit of detection (LOD), limit of

Light Range r2
Intensity Equation quantitation (LOQ), precision and
10 - 400 0.0041x +
accuracy
R 0.9912
0.0073 The limit of detection (LOD) and
10 - 200 0.008x - limit of quantitation (LOQ) were calculated
White G 0.9984
0.0166
from 10 measurements of RGB values. The
10 - 200 0.0037x +
B
0.0112
0.9808 values determined are given in Table 2.
Precision and accuracy were also examined
10 - 400 0.0038x +
R 0.96 by the standards salicylic acid 25, 50 and
0.0457
Red G N N N 100 mg/L. It was found that the precision
B N N N and accuracy calculated from the G values
R N N N
are better than R and B values. Thus, G
10 - 100
values was selected as a values for
0.0082x +
Green
G
0.0226
0.9605 calculated.
10 - 50
0.0228x -
B
0.101
0.9645 Table 2. LOD, LOQ and precision of R, G,
10 - 25 0.0382x -
B values and spectrophotometer T60
R 0.8829 comparison
0.1003
10 - 100 0.0085x + LOD LOQ %RSD Relative error
Blue G 0.9864
0.0047 R 4.3 14.4 20.6 10.1
10 - 200 0.0037x + G 5.8 19.5 4.8 3.8
B 0.984
0.0011 B 10.2 34.0 11.5 17.7
T60 1.4 4.6 3.5 2.1
Table 3. Intra-day and inter-day precisions From Table 3 and 4, the %recovery of the
of the designed device (G values) spiked standard salicylic acid were in range
Precision SA Mean ± sd %RSD 96.6-104.1% and %RSDs are less than 5%
(mg/L) which are acceptable according to the
25 25.6 ± 0.8 3.2
AOAC8. The maximum error between our
Intra-daye 50 51.4 ± 1.3 2.4
100 100.4±2.6 2.5 designed device and T60 Spectrophotometer
25 25.6 ± 0.6 2.4 is -5.2%, which is considered to be
Inter-day 50 50.1 ± 1.6 3.2 acceptable.
100 99.0 ± 2.1 2.1
4. Conclusion
Method repeatability was obtained We have successful developed and
by repeating the assay on the same day for optimized a low cost device for colorimetric
intra-day precision, and on different days for determination of salicylic acid. The
inter-day precision. The %RSDs are 2.1- proposed device shows good analytical
3.2% (Table 3). performances, and acceptable results as that
The device was used to measure obtain from T60 spectrophotometer. This is
salicylic acid in fermented food collected a low price with simple design portable
from supermarket. The food samples were D.I.Y. colorimeter that can be applied for
filtered through a filter paper (Whatman use in the field, also for education and
NO1), and 2 mL of sample were aliquot into amateur science use.
test tube with 1 mL of 2% ferric chloride.
After shaking an absorbance was measured. Acknowledgements
The quantity of salicylic acid in food sample The authors gratefully acknowledge
was calculated by standard addition method. support from the Forensic science program
The same sample was measured at the same also Department of chemistry, Faculty of
time using T60 spectrophotometer as a liberal arts and science, Kasetsart
standard laboratory instrument. The %error University. We are grateful to Mr.Wilhelm
of our device compared with T60 spectro- Josef Holzschuh for all his help.
photometer are calculated as:
%error = Our design – T60 *100
T60
Table 4. Test result of the designed device and T60 spectrophotometer

T60 Spectrophotometer Designed device
Sample
(mg/L) Found (mg/L.) % Recovery % RSD % Error
Sample 1 N N - - -
+25 25.0 25.9 ± 1.2 104.0 4.9 3.9
+50 50.9 48.3 ± 2.3 96.6 4.9 -5.2
+100 98.0 101.5 ± 3.8 101.5 3.8 3.5
Sample 2 N N - - -
+25 24.1 24.5 ± 1.0 98.3 4.2 1.8
+50 49.9 51.6 ± 1.7 103.2 3.4 3.3
+100 100.5 100.5 ± 2.1 100.5 2.1 0.03
Water N N - - -
+25 25.0 25.6 ± 0.6 102.4 2.4 2.3
+50 51.0 48.8 ± 1.5 97.6 3.2 -4.4
+100 99.5 99.0 ± 1.7 99.0 1.7 -0.5
* N: not detected
References
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Abbaspour, A. Pharm. Chem. J. 2017, Peamaroon, Instrum Sci. Technol. 2016,
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Intaravanne, Y. Sens. Actuators B 2013, N.; Daeid, N. N.; Kanatharana, P.;
182, 592-597. Wongniramaikul, W. Forensic Sci. Int.
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J. Biosens. Bioelectron 2016, 75, 48-54.
N
Fiber optical sensor based on surface plasmon resonance spectroscopy for
determination of atrazine
Chidchanok Tabtimhin, Pakorn Varanusupakul*
Chemical Approaches for Food Applications Research Group, Department of Chemistry, Faculty of Science,
Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
*E-mail: pakorn.v@chula.ac.th
Abstract:
A sensitive sensor device based on surface plasmon resonance (SPR) spectroscopy
was developed on an optical fiber and made to be selective to atrazine by molecular
imprinted polymer. The uncladed surface of an optical fiber was coated with a thin film of
gold nanoparticles by self-assembly of gold nanoparticles method. A thin and uniform film of
gold was obtained. The SPR spectra were recognized by the dip of the resonance wavelength
at 507 nm. The thickness of gold film was optimized by varying the soaking time of the
optical fiber. The surface was further modified for selective sensing of atrazine with the
molecularly imprinted polymer (MIP). The atrazine MIP template was synthesized via UV-
polymerization path way using methacrylic acid as a monomer, azobisisobutyronitrile as an
initiator, and ethylene glycol dimethacrylate as a cross-linker. The MIP was tested for the
selectivity with the standard atrazine compared with non-imprinted polymer (NIP). The MIP
was then in-situ polymerized by dropping the pre-polymer solution on the surface of gold
film coated optical fiber under UV lamp. The SPR signal was observed by the shift of the
resonance wavelength. The selective sensing of Atrazine was investigated and reported.
1. Introduction (MIP). MIP is the synthesized material

Surface plasmon resonance (SPR) resembling to lock and key model providing
spectroscopy is a type of spectroscopy binding sites that have specific size and
technique that offers highly sensitive, shape with the template analyte.5 In this
accurate and fast analytical tool.1,2 SPR study, the surface plasmon resonance
takes place at the interface between nano technique in optical fiber configuration is
metal-dielectric interface caused by an developed assembling with MIP for
incident light in total internal reflection selective determination of atrazine
condition. The SPR spectrum is recognized herbicide.
by the dip of the resonance wavelength. This
phenomenon is very sensitive to any change 2. Materials and Methods
such as the adsorption of molecules to the 2.1 Materials
surface that causes the shift of the resonance Methacrylic acid was obtained from
wavelength.3 Fluka Chemika (Gallen, Switzerland). 2,2′-
Generally, SPR probe or device is Azobis(2-methylpropionitrile) 0.2 M in
based on Kretschmann’s configuration, toluene, ethylene glycol dimethacrylate
which is a prism configuration.4 This 98%, gold(III) chloride trihydrate, Atrazine
configuration has limited uses. Recently, PESTANAL®, and sodium formate were
SPR probe using optical fiber has been obtained from Sigma Aldrich (St. Louis,
developed instead of prism based on the USA). A bare optical fiber with diameter of
same principle. The advantages of fiber 600 µm and universal bare fiber terminator
optic SPR probe are small and portable. (BFT1) were purchased from Thorlabs, Inc.
However, the selectivity of the SPR sensor (New Jersey, USA). An UV-Vis light source
is not good. This limitation can be improved (AVALIGHT-DHC) was obtained from
by using a molecularly imprinted polymer Avantes (Apeldoon, the Netherlands). A
spectrometer with charge-coupled device 2.4 Removal of atrazine by MIP
(CCD) array detector (USB400) was 1 g of MIP was added into a 30 ppm
obtained from Ocean Optics Inc. (Florida, of atrazine solution. The absorbance of the
USA) atrazine in the solution was measured at 235
nm. The removal of atrazine was determined
2.2 Fabrication of the fiber optic for SPR
by measuring the absorbance of atrazine
sensor
The optical fiber was cut into 12 cm. remaining in the solution at 235 nm with
About 1 cm in the middle of the optical fiber soaking time for 5-60 min.
was uncladed, washed and sonicated in a 2.5 Assembly of MIP on the fiber optical
surfactant solution for 10 min followed by sensor
deionized water for 10 min. The optical fiber The MIP was in situ synthesized on
was coated with thin layer of gold the optical fiber. The pre-polymer mixture
nanoparticles using self-assembly of thin with atrazine template was dropped on the
film gold-nanoparticle method.6 The optical layer of gold nanoparticle on the optical
fiber was immersed into gold(III) chloride fiber and radiated under UV lamp for 1 hr to
solution mixed with 500 mM of sodium form the MIP. The optical fiber was washed
formate at 1:5 ratio. The concentrations of and the template was removed and dried as
gold(III) chloride were varied from 5-20 described in section 2.3. The NIP was
mM and the immersion times were varied assembled on the fiber optical sensor in the
from 5-80 min. The SPR spectrums were same way without addition of atrazine
obtained using the setup described in section template.
2.6. The particle sizes of gold nanoparticles
2.6 Setup of the SPR based fiber optic
were obtained using scanning electron
sensor
microscope (SEM).
The optical fiber was connected to
2.3 Synthesis of MIP template for the UV-Vis light source and the
atrazine spectrometer with universal bare fiber
The MIP was synthesized using terminator (BFT1). The experiment was
methacrylic acid as a monomer, as 2,2′- conducted in a black box to get rid of extra
azobis(2-methylpropionitrile) an initiator, light from ambient light as shown in Figure
ethylene glycol dimethylacrylate (EGDMA) 1. The SPR spectrum was obtained.
as a cross-linker and atrazine as a template.
A 30 mg of atrazine, 2 mL of monomer,
1 mL of cross-linker were mixed under
nitrogen gas for 5 min, then a 0.5 mL of
initiator was dropped under nitrogen gas for
2 min. The mixture was radiated under UV
lamp for 1 hr. The MIP was formed. The
remaining monomer and cross-linker were
removed by ethanol. The atrazine template
was then removed by washing with sodium
hydroxide. The MIP was dried at 60 °C in
the oven for 30 min and grounded into Figure 1. Setup of the SPR based fiber optic
powder prior to uses. The non-molecularly sensor
imprinted polymer (NIP) was prepared the
same way without addition of atrazine
template. The morphology of MIP and NIP
were studied by scanning electron
microscope (SEM).
The concentrations of gold chloride
could affect the shape and the dip of SPR
signals. Using gold(III) chloride
concentration of 5 mM (Figure 2A), the
spectra were slowly decreased and the dips
of SPR signals were not clearly observed.
Probably, the layer of the gold nanoparticle
was too thin. Using gold(III) chloride
concentrations of 10 mM (Figure 2B), the
spectra were nicely dipped and SPR
characteristic were clearly observed at 521
nm. Using gold(III) chloride concentrations
of 20 mM (Figure 2C), the spectra were
sharply decreased and occurred towards
lower wavelength than those of 10 mM.7 At
high concentration of gold chloride, the gold
nanoparticle layer could be developed
relatively fast, forming large particle sizes
and a thick film, consequently. Therefore,
no SPR characteristic was observed.
The surfaces of the optical fibers
using gold chloride concentration of 10 mM
at different immersion time (Figure 3) show
that the particle sizes of the gold
nanoparticles were bigger and denser with
increased immersion time. At immersion
time of 80 min (Figure 3D), the gold particle
layer started to form a film resulting in
decrease in SPR signal because of the
changed in particle size. Therefore, the gold
Figure 2. The SPR signals obtained from chloride concentration of 10 mM at 60 min
optical fibers coated with gold nanoparticles were chosen to prepare the optical fiber for
at various concentrations of gold chloride SPR sensing probe.
and various immersion times (A) 5 mM of at 3.2 Molecularly imprinted polymer for
30-80 min (B) 10 mM at 30-80 min and (C) adsorption of atrazine
is 20 mM at 10-50 min The MIP and NIP were synthesized
using UV polymerization method. The SEM
3. Results and Discussion images (Figure 4) of both MIP and NIP
3.1 The effect of concentration of gold show different morphology of the surface
solution and immersion time of optical and size. The MIP shows rougher surface
fiber with SPR signal and bigger size compared to NIP.8
The concentrations of gold solution The MIP was tested for adsorption of
were varied at 5, 10, and 20 mM for 15, 20, atrazine. Figure 5 shows the amounts
25, 30, 40, 45, 50, 55, 60 and 80 min (represented as absorbance) of remaining
immersion time. The SPR signals obtained atrazine in the solution after treated with
from optical fibers coated with gold MIP and NIP.
nanoparticles at various concentrations of
gold chloride and various immersion times
were summarized in Figure 2.
Figure 5. Amounts of atrazine after treated
with MIP and NIP at various times, using
0.1 g of polymer
Apparently, atrazine could be

significantly adsorbed to the MIP compared
to the NIP. It suggested that the MIP was
successfully synthesized providing binding
sites, shape and size that fit to the target
atrazine. In addition, the absorption of
atrazine increased with increased adsorption
time.
3.3 Fiber optic SPR sensing of atrazine
Figure 6. Fiber optic SPR with MIP for

Figure 3. SEM images of the optical fiber sensing of atrazine (10 min soaking time,
surfaces using gold chloride concentration 5 mL of 30 mM atrazine standard)
of 10 mM at different immersion time; (A)
35 min, (B) 50 min (C) 60 min and (D) 80 MIP was attached to the gold
min nanoparticle layer on the optical fiber
surface for selective sensing of atrazine.
Figure 6 shows the SPR signals obtained
from optical fibers attached with MIP for
sensing of atrazine compared to those with
NIP. The sensing of the SPR probe with
MIP for the atrazine could be observed by
the shift of the SPR wavelenght from 507 to
Figure 4. SEM images of (A) MIP and (B) 558 nm in a 30 mM of atrazine solution for
NIP 10 minute. .
4. Conclusion References
The optical fiber sensor based on 1. Mayer, K.; Hafner, J. Chem. Rev. 2011,
surface plasmon spectroscopy was 111, 3828–3857.
successfully fabricated with gold 2. Gupta, B.; Shrivastav, A.; Usha, S.
nanoparticle and modified for selective Sensor 2016, 16, 1318-1351.
sensing of atrazine with molecularly 3. Tang, Y.; Zeng, X.; Liang, J. J. Chem.
imprinted polymer. The resonance Educ. 2010, 87, 742–746.
wavelength was observed at 507 nm. The 4. Ahmed, S.; Kim, J.; Tran, V.; Suzuki, T.
shift of the SPR wavelenght from 507 to 558 Sci. Rep. 2017, 7, 495-504.
nm was observed for selective sensing of 5. Roland, R.; Bhawani, S. J. Anal.
atrazine. Methods Chem. 2016, 20, Article ID
5671507.
Acknowledgements 6. Suzuki, H.; Sugimoto, M.; Matsui, Y.;
This work was partially supported by Kondoh, J. Sens. Actuator B 2008, 132,
the Asahi Glass Foundation. 26-33.
7. Chen, J.; Bai, L.; Lui, K.; Lui, R.;
Zhang, Y. Int. J. Mol. Sci. 2014, 15, 574-
587.
Perchlorate analysis of vegetables grown in Chiang Rai, Thailand
Sodkaneung Dachpanya1, Phuangpetch Saysanguan1, Prachak Inkaew1,2,
Siwaporn Praman3, Thaneeya Hawiset3*
1
School of Science, Mae Fah Luang University, Chiang Rai, Thailand
2
Center of Chemical Innovation for Sustainability (CIS), Mae Fah Luang University, Chiang Rai, Thailand
3
School of Medicine, Mae Fah Luang University, Chiang Rai, Thailand
*E-mail: thaneeya.haw@mfu.ac.th
Abstract:
Perchlorate is one of the contaminants found naturally in the environment. The
consumption of food containing large amount of perchlorate affects the absorption of iodine
in human body and interrupts hormone production of thyroid gland. In this work, perchlorate
concentrations in five different kinds of vegetables grown in Chiang Rai, Thailand were
analyzed by perchlorate ion selective electrode. 30 samples of vegetables were collected from
the local markets in Chiang Rai in 2016. The analyzed vegetables were cabbage, Chinese
cabbage, bok choy, Chinese kale and yardlong bean. The average perchlorate concentrations
for cabbage, Chinese cabbage, bok choy and Chinese kale were 3.44, 3.77, 10.95, 7.41
mg/kg, respectively. Perchlorate level of yardlong bean, however, was lower than the limit of
detection (0.3 mg/kg).
1. Introduction suggest that perchlorate could be from the

Perchlorate (ClO4-) is an inorganic reaction of chloride, in the form of sodium
anion and strong oxidizing agent. chloride from the ocean or land-based
Perchlorate can be released from dissolving chloride aerosol, and ozone in the
solid salts of ammonium, potassium, atmosphere. Use of perchlorate-containing
magnesium, and sodium perchlorate and products also contributes to the presence of
perchloric acid into water and soil. perchlorate in the environment.3,4
Perchlorate is highly soluble and able to Perchlorate has been used in military
mobilize in aquatic environment. It is purposes such as solid rocket fuels, signal
relatively stable in ambient condition flares, colored and white smoke generators,
because of its tetrahedral arrangement. As a artillery traces, incendiary delays and
result, perchlorate may persist for many railway torpedoes.5 In industries, perchlorate
years under typical surface water and has been used in gas drying agents,
groundwater conditions.1 Most natural lubricating oils, tanning, finished leather,
perchlorate occurs in arid and semiarid electronic tubes, fabric fixes, dyes, cloud
environments.2 In such environments, seeding, electroplating, aluminum refining,
mobilization of perchlorate to groundwater signal and road flares, rubber manufacture,
is from the precipitation. High evaporation paint, enamel production, cattle feeds,
rate in such environments leads to high magnesium batteries and fertilizer for
accumulation of perchlorate compared to longan and other plants.1 The United States
other places in the world. The Atacama Environmental Protection Agency (US-
Desert in Chile is well-known for its nitrate EPA) has placed perchlorate on the
and perchlorate deposits. Perchlorate can be Contaminant List. The presence of
found in precipitation because it can be perchlorate poses a considerable human
formed naturally by atmospheric processes. health risk even at trace levels.3 Potential
The exact mechanism for the production of human health risks from perchlorate in food
perchlorate is unknown. However, some and drinking water are from the observation
that perchlorate can form strong bond with
sodium (Na+)/iodide (I-) symporter, the Chinese cabbage, bok choy, Chinese kale
protein responsible for transporting iodide and Chinese long bean (Figure 1).
into the thyroid gland. Hence, perchlorate
interferes with the thyroid gland’s ability to
produce thyroid hormones.6,7
Perchlorate is considered to be
inorganic contaminant in dust, soils,
groundwaters, surface waters, and irrigation
waters used for crop production in many
countries.3,8,9 Agriculture as well as
livestock products can be contaminated by
perchlorate because of the use of soil and
irrigation water containing perchlorate.10,11
Various concentrations of perchlorate are
found in different food varieties such as Cabbage Chinese Bok Choy Chinese Yardlong
Cabbage Kale Bean
daily products, beverages, meats, fruit and
vegetables. Several countries including the
USA, Canada, China, South Korea and India Figure 1. A photograph of a cabbage,
have found perchlorate from nanogram to Chinese cabbage, bok choy, Chinese kale
milligram level in water, soil, food and and yardlong bean used in this study
environmental dust.8,9,12-14 Many studies also
focus on the pathway of perchlorate 2.2 Sample preparation
decomposition or the methods to clean The vegetables were clean with
perchlorate from the environment.15 Over distilled water and cut into small pieces of
the past decade, the identification and about 2 cm. 100 g of the vegetables were
measurement of perchlorate in various mixed with 25 mL distilled water and
sources and the environment has improved, chopped in a food processor. Each sample
especially in the micromolar range.16,17 was centrifuged in 50 mL polypropylene
Government agencies in several countries centrifuge tube at 3000 RPM for 10 min.
have begun to monitor the level of The supernatant was used for the analysis.
perchlorate in environment and 2.3 Perchlorate determination
produce.3,12,18,19 However, there is no report The amount of perchlorate in each
of perchlorate monitoring or concerns type of vegetable was determined by
amongst Thai people and government. This perchlorate ion selective electrode (Oakton
work has investigated the levels of Instruments, U.S.A). A linear calibration
perchlorate in some types of vegetables plot was established with five different
common to Thai people. levels of perchlorate standard solutions
dissolved in distilled water. Three replicates
2. Materials and Methods were used to determine the concentration
2.1 Materials and standard deviation of perchlorate in each
Perchlorate standard for ion vegetable samples. The linear response of
chromatography (1000 mg/L) was obtained the electrode, as indicated from Oakton
from Sigma-Aldrich and used to prepare Instruments, is between 0.7 and 98000 ppm.
other perchlorate standard concentrations. However, based on our literature review,
Double distillation water was used to perchlorate found in most food and
prepare diluted standard concentration. beverages are in low ppm range. We
Vegetable samples were obtained from local prepared the calibration between 0.7 and
markets in Chiang Rai province, Thailand in 100 ppm. The levels of perchlorate were
2016. 30 samples of five kinds of vegetable reported according to the fresh weight.
were analyzed. The vegetables are cabbage,
3. Results and Discussion negative because perchlorate holds one
3.1 Calibration plot of perchlorate standard negative charge. The electrode gave the
For ion selective electrode, the reading of 317.5 ± 1.5 in distilled water, 1 M
electrode response (mV) follows the KCl, 1 M H2SO4, and solutions of pH 1-10.
equation Hence, the electrode was used to measure
the levels of perchlorate in the samples
E = E˚- S log X (1) without adjusting the pH of the samples.
The limit of detection of the
where E is the measured electrode potential electrode, as determined from the
E˚ is the reference potential intersection of the two extrapolated
(a constant) segments of the calibration plot, was 0.3
S is the electrode slope ppm.
X is the level of perchlorate ions in Percent recovery was used to check
the solution the response of the electrode toward the
matrix and perchlorate. Specific levels of
perchlorate were added to each type of the
350.0
vegetable samples. Percent recoveries
300.0 obtained were in between 96 and 103%.
250.0 3.2 The levels of perchlorate in vegetable
Response (mV)
200.0
samples
150.0
y = -56.94x + 316.46
R² = 0.992
All of the vegetable samples except
yardlong bean have perchlorate above 0.8
100.0
mg/kg which is considered to be high
50.0
according to the value set by US-EPA and
0.0
0 0.5 1 1.5 2 2.5 3
the European Commission. The mean values
log[ClO4-] for bok choy, Chinese kale, Chinese cabbage
and cabbage are 10.95, 7.41, 3.77, 3.44
Figure 2. A representative of a calibration mg/kg, respectively (Figure 3).
plot of perchlorate standard between 1 and
1000 ppm. The values shown on the x-axis 16
are the logarithm values of these
14
concentrations.
12
Perchlorate levels (mg/kg)
10
Figure 2 shows one of the calibration
8
plots of perchlorate standard between 1 and
1000 ppm. The electrode give a good linear 6
response with R2 = 0.992. The concentration 4
of perchlorate from the sample can be 2
calculated from 0
Chinese cabbage cabbage bok choy Chinese kale
E E
concentrat ion  10 S
(2)
Figure 3. A column chart of the levels of
perchlorate found in vegetable samples.
The calibration plot was checked frequently
Results show as mean values of perchlorate
to determine a proper response of the
levels (mg/kg) of 6 samples for each type of
electrode. The response is close to ideal for
vegetable. The error bars are standard
ion selective electrode for monovalent
deviation values of each type of vegetables.
charge (59 mV/decade or 59 mV for an
order of magnitude change in the
concentration). The slope of the plot is
Table 1. Perchlorate levels of vegetable samples (mg of perchlorate in kg of fresh weight
vegetable samples
na mean ± sd median (mg/kg) minimum maximum
Sample
(mg/kg) (mg/kg) (mg/kg)
Chinese cabbage 6/6 3.77 ± 1.92 3.34 2.14 6.85
cabbage 6/6 3.44 ± 1.73 2.81 1.71 6.19
bok choy 6/6 10.95 ± 5.83 10.23 4.89 21.35
Chinese kale 6/6 7.41 ± 4.41 7.45 0.82 13.87
yardlong bean 0/6 ND ND ND ND
a
n: detection frequency (number of detected samples/number of all analyzed samples)
ND: not detected or below the detection limit of the ion selective electrode
Bok choy has the highest level of that the US population is exposed to about
perchlorate compared to other vegetables. 0.053 µg/kg of body weight/day of
The range of perchlorate levels found on the perchlorate from 27 food and beverage. The
vegetable is 0.82-21.35 mg/kg (Table 1). level of perchlorate exposure measured from
Based on the standard deviation, the levels urine in America is 0.066 µg/kg body
of perchlorate are significantly different weight/day.22 US-EPA has suggested a
even in the same type of vegetable. Since, reference dose (RfD) of 0.7 µg/kg of body
the samples were obtained from three weight/day.3 Based on this recommendation
different markets in Chiang Rai, the soil and and the levels of perchlorate found in
water could be the source of these high vegetables grown in Chiang Rai, most
levels of perchlorate present on the people should consume less than 50 g of the
vegetable and should be investigated further. reported vegetables each day. Food that is
Gan and coworkers reported high levels of consumed in large quantities should also be
perchlorate in soil in Northern China investigated for perchlorate level.
(ranging from 0.001 to 216 mg/kg) and
Southern China (ranging from 0.001 to 25.8 4. Conclusion
mg/kg). In door dust collected from Greece, Perchlorate levels were determined
Japan, the USA, Saudi Arabia, Vietnam also by perchlorate ion selective electrode on 30
showed high levels of perchlorate (between samples of vegetables from local markets in
0.02 and 104 mg/kg).20 Lee and coworker Chiang Rai, Thailand. The range of
investigated the levels of perchlorate in perchlorate level found in Chinese cabbage,
various foods and beverages in Korea and cabbage, bok choy and Chinese kale is 0.82-
found low levels of perchlorate in Chinese 21.35 mg/kg which is considered to be high
cabbage and cabbage.21 However, most of compared to the levels found in other
their samples showed some levels of countries. The sources of this high
perchlorate (in µg/kg level). Gan and perchlorate levels should be investigated
coworker also investigated levels of further.
perchlorate in various foods in China and
found even very low level of perchlorate in Acknowledgments
most vegetable samples. The maximum Financial supports from Mae Fah
values of perchlorate found in spinach and Luang University and National Research
cabbage were 417 and 216 µg/kg, Council of Thailand are greatly appreciated.
respectively.11 The United States Food and
Drug Administration (US FDA) has estimated
References 13. Ye, L.; You, H.; Yao, J.; Kang, X.;
1. Karimi, G.; Rezaee, R. Encyclopedia of Tang, L. Chemosphere 2013, 90, 2493-
Toxicology, 3rd; Academic Press: 2498.
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Ronen, Z.; Nativ, R. J. Hydrol. 2009, Environmental Sciences; Elsevier, 2013.
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46, 2307-2315. 16. Gholamian, F.; Sheikh-Mohseni, M. A.;
4. Dasgupta, P. K. Anal. Chim. Acta 2006, Salavati-Niasari, M. Mater. Sci. Eng.
567, 1-3. 2011, 31, 1688-1691.
5. Walsh, M. R.; Walsh, M. E.; Ramsey, C. 17. Yu, L.; Cheng, Q.; Cañas, J.; Valentin-
A.; Brochu, S.; Thiboutot, S.; Blasini, L.; Blount, B. C.; Anderson, T.
Ampleman, G. J. Hazard. Mater. 2013, Anal. Chim. Acta 2006, 567, 66-72.
262, 228-233. 18. Elston, H. J. J. Chem. Health Saf. 2010,
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Environmental Health; Elsevier: 19. Borjan, M.; Marcella, S.; Blount, B.;
Burlington, 2011, 364-370. Greenberg, M.; Zhang, J.; Murphy, E.;
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4-12. Total Environ. 2011, 409, 460-464.
8. Kannan, K.; Praamsma, M. L.; Oldi, J. 20. Wan, Y.; Wu, Q.; Abualnaja, K. O.;
F.; Kunisue, T.; Sinha, R. K. Asimakopoulos, A. G.; Covaci, A.;
Chemosphere 2009, 76, 22-26. Gevao, B.; Johnson-Restrepo, B.;
9. Kumarathilaka, P.; Oze, C.; Indraratne, Kumosani, T. A.; Malarvannan, G.;
S. P.; Vithanage, M. Chemosphere 2016, Moon, H. B.; Nakata, H.; Sinha, R. K.;
150, 667-677. Minh, T. B.; Kannan, K. Environ. Int.
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Sci. Total Environ. 2014, 470-471, 99- Osterloh, J. D.; Mauldin, J. P.; Pirkle, J.
106. L. J. Exposure Sci. Environ. Epidemiol.
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Development of microfluidic device for determination of
total cholesterol in blood
Suphakorn Katib1, Chalermpong Saenjum2, Plaipol Dedvisitsakul1,
Kanchana Watla-iad1,3*
1
School of Science, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand
2
Center of Natural Active Pharmaceutical Ingredient Research, Faculty of Pharmacy, Chiang Mai University,
Chiang Mai 50200, Thailand
3
Center of Chemical Innovation for Sustainability, School of Science, Mae Fah Luang University, Muang,
Chiang Rai 57100, Thailand
*E-mail: kanchana.wat@mfu.ac.th
Abstract:
A simple and low-cost microfluidic method was developed for total cholesterol
determination in blood. The device was operated by mean of measurement of colorimetric
cholesterol reaction based on reaction with iron(III) chloride (FeCl3) in acetic acid and
sulfuric acid (H2SO4) to obtain red-purple complex. The developed device presented the
concept of online separation and detection simultaneously on silica gel coated on aluminum
device (0.5 cm x 7 cm). Color intensity was detected as RGB value by co-operation of device
and mobile phone application. The systematic device was optimized for reproducibility,
accuracy, and linearity. This device would be used as screening device for taking care of
human healthy and patient. And, it may be able to use for warning and progressing of
myocardial infarction, coronary heart disease, and lipid disorders diseases.
1. Introduction Various techniques for determination

Cholesterol (CHL) is steroid alcohol of blood cholesterol are reported such as
containing with 27 carbons in structure. The colorimetric,6-13,25 high-performance liquid
cholesterol is an essential component of chromatographic,2,9 gas chromatographic,7,14
animal membrane. The CHL is also starting enzymatic,1,7,8,16-20 electrochemistric,18,23-25
substance for producing steroids in human and spectrophotometric methods.12,15,23,26,27
body such as progesterone, aldosterone, All of these methods are high sensitive,
estrogens, testosterone, cortisol, vitamin D, precise, accurate, and high performance
and bile acid.1,2 There are two types of method for quantitative analysis. However,
cholesterol which are free and esterified these methods also require advanced
cholesterols. High content and disorder of instruments, complicated conditions, expert
cholesterol in blood could affect to human experience to operate, long analysis time
health such as myocardial infarction, and expensive cost. Nowadays, many points
coronary heart disease, lipid disorders, liver of care devices for determination of
failure and diabetes.3,4 Therefore, the level cholesterol were developed. Two types of
of blood cholesterol is estimated for developed devices used to determine
warming the initiation and progression of cholesterol in blood sample were enzymatic-
diseases. Generally, normal level of total colorimetric and non-enzymatic methods.
cholesterol in blood is less than 200 The enzymatic method is more specific to
mg.dL-1. The level of total cholesterol cholesterol determination than other method.
increasing the risk of heart disease is from However, analysis cost is quite higher than
200 to 239 mg.dL-1. And more than 240 the common chemical reaction method. In
mg.dL-1 can identify as high blood addition, microfluidic device has become
cholesterol.1,5 more developed for cholesterol
determination because of small volume of
solvent, sample, and reagent consumption.28 based-on chromatographic method for
But, such developed method still expensive separation of cholesterol in the blood
cost. sample. The standard cholesterol was
In this work, the colorimetric device dropped on the TLC plate at starting point
was developed for online-separation and (center and 1 cm from lower edge). After
determination of cholesterol. The that, the device was carried out in a chamber
smartphone with free application software for separation of cholesterol until the
was successfully applied for detection of running solution move to the marked final
cholesterol. The cholesterol content in real point. The mixture solution of ethanol and
samples were analyzed by the developed dichloromethane (60:40) was used as
method. The method was validated with that running reagent. Then, the color developing
of a standard visible spectrophotometric reagent was dropped on the top of the device
method. This device could be alternative where is opposite the position of sample
point of care that was suitable for screening, zone.
warning and progressing of diseases.

2.1 Reagent and chemicals
Cholesterol standard used as
standard solution was purchased from
Merck Chemical Company. It was prepared
by dissolving in ethanol with proper
concentration from 0 to 280 mg.dL-1. Color
developing reagent for cholesterol- Figure 1. Example of colorimetric devices of
colorimetric method was mixed from standard cholesterols from 0.0 to 280 mgdL-1
sulfuric acid (H2SO4), acetic acid and ferric
chloride (FeCl3). Ferric chloride (0.25 g)
was dissolved in 25 mL of 100% acetic acid.
Then, ferric chloride solution (1 mL) was
transferred to mix with 25 mL of 98%
sulfuric acid.
All chemicals such as potassium
hydroxide (KOH), dichloromethane
(CH2Cl2), ethylenediaminetetraacetic acid
(EDTA), and ethanol (C2H6O) were
analytical reagent grade.
2.2 Sample preparation
The EDTA (2 mg) was added into Figure 2. The calibration curve plotted
blood sample (1 mL) filled in a test tube. between the color intensity as G value and
Then, the mixed sample was centrifuged at cholesterol cencentration (0 to 280 mg.dL-1).
1350 rpm for 10 min. The 300 µL of
ethanolic KOH (3% w/v) was added into the The red-purple color generated on
obtained serum (100 µL). Finally, the serum the device was based on the Zlatkis
was extracted with dichloromethane (700 reaction.29,30 The cholesterol was reacted
µL) and distilled water (100 µL) for twice. with H2SO4, acetic acid and ferric chloride
2.3 Instrumentation to produce red-purple complex. The
Device was developed by handmade developed color was detected by using co-
cutting of an aluminum thin layer operated of light-controlling box and
chromatographic plate (TLC plate) with the application program on a smart phone as
size of 0.5x7 cm. The TLC was operated
RGB value. The light-controlling box spectrophotometer was used as reference
created from paper board containing with a method for validation of the developed
5W-LED white light. A visible method.
Table 1. Comparison between this proposed method and visible-spectrophotometry of the

cholesterol content in the real blood samples (3 replications)
Sample 1 2 3 T-test
-1
This research (mg.dL ) 182±9.94 71±7.56 188±8.14
Spectrophotometry 0.3789
180±6.69 78±7.95 177±9.39
(mg.dL-1)
T-critical = 4.3026 at 95 % confidence level
3. Results and Discussion obtained from the proposed method was not
The developed device co-operated significant difference from that of the
with light-controlling box and mobile standard method at 95% confidence level.
application on smartphone was worked This developed method is simple for
successfully for determination of cholesterol determination of cholesterol in blood sample
concentration. The device was designed as than other reported colorimetric-chromatographic
simple model and demonstrated color signal. method operating under tedious step, many
It was employed as separator for separation conditions and advanced spectrophotometer.31,32
of cholesterol based on chromatographic
method. The mobile phase of the device was 4. Conclusion
the mixture of dichloromethane and ethanol The device based on chromatographic
in ratio of 60:40. Then, the separated and colorimetric techniques, co-operated with
cholesterol on the plate (Figure 1) was light-controlling box and mobile application
monitored by colorimetric method via the on smartphone was developed for
Zlatkis reaction.29,30 The colorimetric determination of cholesterol content in
method of Zlatkis reaction was shown red- blood samples. Online separation and
purple color under concentration of reagent. detection of cholesterol in blood could be
The color intensity as RGB values on the performed successfully on the proposed
device was read by mobile application in a device. The detecting reagent was the
light-controlling box. The box was used for combination of FeCl3, acetic acid and
protecting outer light and also reducing H2SO4. The device presented good linearity
noise signal. The color intensity of R and B when using G value of RGB system for
value were not good relationship with plotting calibration curve. This simple and
cholesterol concentration. Because, the color cost effective device could be alternative
obtained from reaction was red-purple tone method for determination of cholesterol in
that was related to G value as blood. It could be used for screening,
complementary color. Other, the increasing warning and progressing of diseases.
concentration of cholesterol was lead to
decrease of G value. The developed method Acknowledgements
presents good linearity as r2 of 0.9952, in a We would like to thank the Scientific
range of cholesterol concentration from 0 to and Technological Instruments Center
280 mg.dL-1 (Figure 2). The method also (STIC) of Mae Fah Luang University and
showed good reproducibility with %RSD the National Research Council of Thailand
less than 4.33%. (NRCT2561005: 797735) for supporting this
The proposed method was validated project.
with visible-spectrophotometry. The results
were shown in Table 1. The result shown
that the cholesterol content in blood samples
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An effective enrichment method using catanionic surfactant couple to high
performance liquid chromatographic determination of
polycyclic aromatic hydrocarbons
Sophon Hem, Suthasinee Boonchiangma, Supalax Srijaranai*
Materials Chemistry Research Center, Department of Chemistry and Center of Excellence for Innovation in
Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
*E-mail: supalax@kku.ac.th
Abstract:
This study demonstrates the using of supramolecular solvent (SUPRAS) as the
extraction solvent of polycyclic aromatic hydrocarbons (PAHs) prior to the analysis by high
performance liquid chromatography (HPLC). Four PAHs studied are naphthalene, fluorene,
phenanthrene and fluoranthrene. The SUPRAS was generated from the mixture of anionic
surfactant (sodium dodecyl sulphate, SDS) and cationic surfactant (tetrabutylammonium
bromide, TBABr) at the ratio 1:3 of (50 mmol L-1 SDS and 150 mmol L-1 TBABr) with the
addition of AlCl3. The extraction of PAHs was performed in a centrifuge tube at room
temperature. SUPRAS was dispersed in aqueous solution containing PAHs in the presence of
AlCl3 salt by vortex. The extracted phase was obtained by centrifugation and was dissolved
with acetonitrile before injection to HPLC. The experimental parameters affecting the
enrichment efficiency were optimized. The chromatographic conditions are a ZORBAX
Eclipse XDB–C18 column (150 × 4.6 mm, 5.0 μm particle size), the mobile phase consisted
of acetonitrile and water at 60:40 (v/v), a flow rate of 1 mL min-1 and UV detection at 254
nm. Four PAHs were completely separated within 20 min. The analytical characteristics and
applicability of the proposed are demonstrated.
1. Introduction surfactant, for anionic surfactant,

Current trend for traditional liquid concentrated HCl is recommended, while
liquid extraction (LLE) is focused on green coacervative agent is needed for cationic
concept including miniaturized scale and the surfactant. In this study, mixed cationic and
use of non-toxic extraction solvent. Several ionic surfactants, called catanionic
solvents have been explored as the surfactant, was investigated as extraction
extraction solvent to replace toxic organic solvent for the extraction of polycyclic
solvents such as ionic liquid, deep eutectic aromatic hydrocarbons (PAHs).
solvent and supramolecular solvent PAHs are compounds containing two
(SUPRAS). SUPRAS is a water–immiscible or more aromatic rings. They are generally
liquid, mostly generated from amphiphiles produced from the burning of plants and
through a sequential self–assembly and fuel. PAHs can easily contaminate to
coacervation processes.1-3 SUPRAS have a environment and foods. They are dangerous
unique array of physico–chemical properties to human because of their high
which are suitable to be used as an lipophilicity.6,7 In addition, some of them are
extraction solvent. These properties are good carcinogen and mutagen. Tea is the most
solubilization, easy to prepare and tunable. popular beverage made from tea leaves.
Surfactants are amphiphilic There are several processes to produce tea
compounds which can be used as precursors including smoke fired, flame fired, pan fired
of SUPRAS under various coacervation or streamed.8-10 Therefore it is worthwhile to
conditions.4,5 Such as heating is required for determine PAHs in tea samples. Analysis of
non-ionic surfactants and zwitterionic PAHs in food sample is a challenging work
as PAHs usually present at trace level in 2.2. Instruments
high matrices samples like food. An Agilent 1220 Series A HPLC
The objective of this study is the (Agilent, USA) equipped with UV–vis diode
development of liquid phase microextraction
array detector was employed for the
(LPME) for the preconcentration of PAHs
before their analysis by HPLC. SUPRAS determination of PAHs. The separation of
was generated from the catanionic surfactant the studied PAHs was performed on an
(tetrabutyl ammonium bromide and sodium Eclipe XDB–C18 column (150 mm × 4.6 mm
dodecyl sulfate) was investigated as an i.d., 5 μm particle size) using isocratic
alternative extraction solvent. PAHs studied elution of acetonitrile and water (60:40
are naphthalene (Nap), fluorene (Flu), (v/v)) at flow rate of 1 mL min-1. The
phenanthrene (Phen) and fluoranthrene (Flt)
injection volume was 20 μL and the
as structures shown in Figure 1 the
applicability of the proposed method is detection wavelength was set at 254 nm. A
demonstrated for tea samples. vortex mixer (50 Hz) from Scientific
Industries, INC (USA) was used for mixing
solution. Centrifuge (Hermle Z 206 A,
Hermle Labortechnik GmbH; Germany) was
used for phase separation.
2.3 Extraction of PAHs
2.3.1 Preparation of SUPRAS
Aliquot 1.00 mL of 50 mmol L-1
SDS and 1.50 mL of 150 mmol L-1 of
TBABr were added to a 10 mL centrifuge
tube and 1.5 g AlCl3 was added. The
mixture solution was adjusted to 10 mL with
Figure 1. Structures of PAHs water. The solution was vortexed for 20 s
and then centrifuged at 3500 rpm for 5 min.
2. Materials and Method The SUPRAS was at the top of centrifuge
2.1. Chemicals and reagents tube. It was separated using syringe and was
Four PAHs including naphthalene kept in a brown vial at room temperature.
(Nap), fluorene (Flu), phenanthrene (Phen) and 2.3.2 Liquid phase microextraction using
fluoranthrene (Flt) were obtained from Sigma SUPRAS
(USA). Acetonitrile (ACN) HPLC grade was Standard or sample solution (10.00
purchased from Merck (Germany). Sodium mL) was placed in a 10 mL centrifuge tube.
dodecyl sulfate (SDS), tetrabutylammonium 25 µL of SUPRAS (from 2.3.1) and 0.1 g of
bromide (TBABr) and sodium chloride (NaCl) AlCl3 were added. The mixture solution
were obtained from Ajax Finechem (Australia). was vortexed for 20 s and centrifuged at
Aluminum chloride (AlCl3) was purchased 3500 rpm for 3 min. The SUPRAS was at
from Sigma (USA). All stock solutions of the bottom of centrifuge tube and water
PAHs (1000 mg L-1) were prepared in ACN phase was removed by syringe. The
and kept in brown vials. It was stored at 4oC SUPRAS was dissolved with 10 µL ACN
in a refrigerator. Ultrapure water with the before injecting to HPLC.
resistivity of 18.2 MΩ cm from RiOs™
Type I Simplicity 185 (Millipore, USA) was
used throughout.
2.4 Determination of PAHs in tea samples
Two samples of tea infusion and two
samples of dried tea leaves were purchased
from Supermarket in Khon Kaen, Thailand.
Accurate weight of tea infusion (2 g) and
dried tea leaves (4 g) were brewed in 20 mL
of water at 85–95 oC. The tea solution was
cooled at room temperature and then was
filtered through filter papers. An aliquot of
1.00 mL of tea solution was added to 9 mL Figure 2. Effect of SUPRAS volume for
of ACN in a 10 mL centrifuge tube. NaCl (1 extraction, Extraction conditions: 10 mL of
g) was added to the solution and then standard solution of PAHs containing 0.5
centrifuged at 3500 rpm for 15 min. The mg L-1 for Nap and Flt, 0.25 mg L-1 for Flu
and 0.1 mg L-1 for Phen, vortex time: 20 s at
supernatant (1.00 mL) was transferred to a
1800 rpm, AlCl3 (1% w/v), centrifuge: 5
10 mL centrifuge tube containing 9 mL of
min at 3500 rpm
water before being extracted follow the
procedure described in 2.3.2.
3.1.2 Effect of amount of salt
The salt addition influences on both
3. Results and Discussion the preparation of SUPRAS and the
The parameters affecting extraction extraction processes. It is not only
efficiency were studied and optimized. The increasing the ionic strength, but also
results are evaluated in term of enrichment assistance the breakup of emulsion, thus
factor (EF), which can be calculated from increase the phase separation in the
the following equation.11 extraction procedure.12 AlCl3 was selected in
this study because of it provided high
extraction efficiency compared to the other
salts. The amount of AlCl3 was then studied.
Where Cex and Co are concentrations of
The results (Figure 3) show that the
PAHs in extraction phase (SUPRAS) and
highest EF was at 0.1g (1%w/v), however
the initial concentration of PAHs in aqueous
when further increasing the amount of AlCl3
phase.
the EFs were decreased.
3.1 Optimization of liquid phase
microextraction using SUPRAS
3.1.1 Effect of volume of SUPRAS
The volume of SUPRAS is one the
most important parameter for extraction of
PAHs. In this study, different volumes of
SUPRAS in the range of 25–100 L were
studied. As the results shown in Figure 2,
the EFs decreased when the volume of
SUPRAS increased which may due to the
dilution. The volume of 25 µL was selected Figure 3. Effect of AlCl3 amount; extraction
according to the highest extraction conditions: As described in Figure 2, except: 25
efficiency of PAHs. µL of SUPRAS was used.
3.1.3 Effect of vortex found that 3 min of centrifugation was
Vortex is used to disperse the enough to separation the phase (Figure 5).
SUPRAS, thus the analytes can transfer 3.2 Analytical performance of liquid
easily to the extraction solvent. It is one of phase microextraction using SUPRAS
the most necessary parameter to obtain high Figure 6 shows the chromatograms
extraction efficiency. In this study, vortex of standard PAHs obtained from (a) the
time was studied from 0 to 80 s at the fixed proposed LPME using SUPRAS as the
speed at 1800 rpm. It can be seen (Figure 4) extraction solvent and (b) direct HPLC
that 20 s for vortex gave high EFs and the analysis (without the preconcentration). It
EFs were quite stable with longer vortex can be seen that LPME could enhance the
times. Therefore, vortex time 20 s was sensitivity for the analysis of PAHs.
chosen.
Figure 4. Effect of vortex time; extraction Figure 6. Chromatograms of standard

conditions: As described in Figure 3, except PAHs: (a) obtained from LPME (0.1 mg L-1
0.1 g AlCl3 was used. for Nap and Flt, 0.03 mg L-1 for Flu and
Phen) (b) obtained from direct HPLC
3.1.4 Effect of centrifugation analysis (3 mg L-1 for Nap and Flt, 1 mg L-1
for Flu and 0.7 mg L-1 for Phen)
The linearity, limit of detection

(LOD) and limit of quantification (LOQ) are
summarized in Table1. The LOD and LOQ
are evaluated from a signal to noise ratio
S/N of 3 and 10, respectively. The LODs
were in range of 0.55–5.00 µg L-1 and LOQ
were 2.18–18.00 µg L-1. For intra–day
(repeatability, n=5) and inter–day
Figure 5. Effect of centrifugation time; (repeatability, n=5x3) precisions of the
extraction conditions: as described in Figure LPME method were evaluated, which the
4 RSDs of peak area were less than 4.1% and
4.7% for intra–day and inter–day,
Centrifugation is another important respectively. In addition, the EFs were
parameter to break up the emulsion and between 46 and 91.
assisting the phase separation in an 3.3 Analysis of tea samples
extraction. The centrifugation time was Matrix match calibration curves were
studied from 1 to 5 min at 3500 rpm. It was used for the quantitative analysis of PAHs in
tea samples. Phen and Flt were detected in
some samples (Table2), however they are at dried tea leaves samples (Table3). The RSD
trace concentrations. Figure 7 shows the of the relative recoveries was less than
chromatograms for dried tea leaves (sample 6.0%.
III).
4. Conclusion
The advantages of the proposed
liquid phase microextraction using SUPRAS
as the extraction solvent are simple,
inexpensive, efficient and environmentally
friendly. The studied SUPRAS was easily
prepared by mixing anionic with cationic
together at room temperature.
Acknowledgements
Figure 7. Chromatograms of dried tea
Financial supports from Royal
leaves (sample III): (a) blank sample, (b)
Scholarship under Her Royal Highness
spiked sample (0.1 mg L-1 for Nap and Flt
Princess Maha Chakri Sirindhorn Education
and 0.05 mg L-1 for Flu and Phen)
Project to the Kingdom of Cambodia and
Materials Chemistry Research Center,
In addition, the accuracy of the
Department of Chemistry and Center of
proposed method was studied in term of
Excellence for Innovation in Chemistry,
recovery by spiking PAHs standard at three
Faculty of Science, Khon Kaen University
different concentrations. The recoveries of
are gratefully acknowledged.
analytes for tea infusion samples were in
range of 91–108%, while the recoveries
ranges from 94–108% were obtained for
Table1. The analytical features of PAHs

Analyte Linear Linear equation R2 LOD LOQ EF
range(µg L-1) (µg L-1) (µg L-1)
Naphthale 15-300 y=1.1881x+6.7633 0.9975 5.00 18.00 46
(600-7000)a (y=0.0258x-1.7742) a (0.9993)a (200)a (630)a
Fluorene 5-70 y=8.7786x-7.5249 0.9973 1.54 5.00 74
(200-3000) (y=0.1191x-11.597) (0.9984) (70) (220)
Phenanthrene 2-70 y=32.773x-23.57 0.9974 0.55 2.18 91
(90-3000) (y=0.3609x-46.433) (0.9972) (30) (90)
Fluoranthrene 10-300 y=5.1094x-33.78 0.9988 3.84 11.00 62
(600-7000) (y=0.0826x-47.751) (0.9985) (200) (470)
( ) a value of standard without LPME (direct HPLC analysis)
Table2. Determination of PAHs in tea samples

Type of tea Sample Concentration (µg L-1)
Naphthalene Fluorene Phenanthrene Fluoranthrene
Tea infusion I ND ND 126± 0.6 518± 1.0
Tea infusion II ND ND ND ND
Dried tea leaves III ND ND 95± 5.3 617± 1.6
Dried tea leaves IV ND ND 55± 2.0 ND
Table3. Recoveries for tea samples
Analyte Spiked level Sample (% recoveries ± SD) n=3
(µg L-1) I II III IV
Naphthalene 20 107.8 ± 0.7 101.4± 3.2 106.7± 3.2 108.7± 2.5
50 101.9± 1.9 105.2± 1.1 103.4± 2.2 107.3± 4.4
100 100.5± 1.0 105.7± 0.8 100.7± 0.6 107.3± 3.4
Fluorene 10 101.1± 1.3 108.7± 0.9 100.4± 1.6 102.5± 4.0
30 102.3± 0.4 104.4± 0.5 102.8± 0.6 105.8± 4.7
50 108.0± 1.2 108.5± 0.4 99.8 ± 0.5 105.4± 4.5
Phenanthrene 10 95.4 ± 0.6 100.7± 0.6 95.1 ± 0.3 96.9 ± 2.6
30 105.6± 1.3 100.1± 0.2 98.6 ± 0.6 105.2± 2.1
50 105.6± 0.1 106.5± 0.1 99.0 ± 0.1 103.5± 2.3
Fluoranthrene 20 96.7 ± 0.5 99.4 ± 1.1 94.4 ± 1.6 105.5± 3.4
50 103.1± 0.4 91.9± 0.1 94.4 ± 0.7 101.6± 4.3
100 106.9± 0.2 94.4 ± 0.2 94.8 ± 0.6 97.6 ± 1.6
References 7. Delgado, B.; Pino, V.; Ayala, J.;

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2016, 1027, 74–80. E.; Moris, G. J. Food Chem. 2014, 14,
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5. Xiong, B.; Zhang, Y.; Hou, Y.; Peter H. K.; Tomaniova, M. Talanta 2012, 100,
Arp, H.; J. Reid, B. J. Chemosphere 207–216.
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6. Feizi, N.; Yamini, Y.; Moradi, M.; Santaladchaiyakit, Y.; Srijaranai, S. RSC
Karimi, M.; Salamat, Q.; Amanzadeh, H. Adv. 2017, 7, 50143–50149.
Anal. Chim. Acta 2017, 953, 1–9. 12. Song, S.; Ediage, E.; Wu, A.; Saeger, S.
J. Chromatogr. A 2013, 1292, 111–120.
Extraction and preconcentration method for trace Pb determination by
UV-Visible spectrophotometry
Apinya Navakhun
Department of Chemistry, and Center of Excellence on Environmental Health and Toxicology (ETH),
Faculty of Science, Burapha University, Chonburi, Thailand
E-mail: apinyan@buu.ac.th
Abstract:
In this research, part per billion level of Pb in water sample was extracted and
preconcentrated by dispersive liquid-liquid microextraction method (DLLME). Optimum
conditions for DLLME were studied in order to obtain high extraction efficiency. The
complex of Pb with dithizone was formed and then extracted prior to UV-visible detection at
517 nm. The DLLME conditions were ammonia buffer (pH10), dithizone concentration of 1
x 10-5 M, 5 mL of the mixture of extraction solvent (dichloromethane) and disperser solvent
(ethanol) at the ratio of 1:3 and extraction time of 5 minutes. Under optimum conditions,
calibration graph was linear in the range of 20-140 µg/L with R2 0.9950. The limit of
detection and limit of quantification were 0.58 and 1.93 µg/L, respectively. The precision and
accuracy of the method were also evaluated. The proposed method was applied for
determination of Pb contaminated from industry in water samples.
1. Introduction Techniques as liquid-liquid extraction (LLE)

Pb has been well known as toxic and solid phase extraction (SPE) have been
elements for human and environment. By reported.3-5 However, extraction techniques
accumulation in bodies, Pb may cause brain such as LLE, SPE require the use of
damage, kidney injury, seizures and anemia. significant quantities of organic solvent,
The relative toxicity translates into low which is flammable, volatile, toxic and
maximum acceptable concentrations (MAC) harmful to the environment. Like the SPE,
of Pb for drinking water in comparison to LLE is another classic pretreatment
other metals (10 µg/L (WHO)1 and 15 µg/L technique that has been widely employed in
(USEPA)).2 Therefore, there has been an analytical chemistry. However, the
increasing need for analytical chemists to conventional LLE uses large amounts of
evaluate the environmental and healthy solvent and it is time-consuming to perform,
quality based on accurate determination of which limits its further application. To
Pb in environmental samples. However, a overcome the disadvantages mentioned
certain preconcentration/separation process above, miniaturized and green extraction
is usually imperative for the concentrations methods have been developed.5
of target analytes. In real samples, the The aim of this work is to
concentration of Pb is commonly too low or determinate trace toxic Pb in environmental
the sample matrix is too complicated to be samples by combining dispersive liquid
determined directly by the instrumental liquid microextraction with UV-Visible
techniques. Therefore, the analytical detection method. The parameters
methods for the measurement of Pb that are influencing the extraction efficiency and the
sensitive, selective, versatile and cost- analytical performance were optimized.
effective are required. Different strategies
have been investigated for the extraction and 2. Materials and Methods
pre-concentration of lead in various 2.1 Chemicals and instruments
environmental and biological matrixes.
Lead standard solutions were 3.1 Pb-dithizone complex formation
prepared from 1000 mg/L AAS standard In this study, dithizone (H2Dz) was
solution (Merck). Dithizone stock solution used as sensitive ligand for Pb determination
was prepared by dissolving of the by UV-Visible detection. The absorption
appropriate amount of solid dithizone (Ajax spectra of free ligand (dithizone) and
Finechem) in hexane. Buffer solution of 0.5 complex (PbDz2) at pH 10 are shown in
M NH3/NH4Cl was prepared in ultrapure Figure 1. The maximum absorption for
water. All chemicals used in this research PbDz2 and free dithizone were found at 517
were analytical reagent grade. A UV-visible and 611 nm, respectively. Unfortunately, the
spectrophotometer model UH5300 (Hitachi) free dithizone was also absorbed at 517 nm.
was used for the absorption measurement. The correct absorption values for PbDz2
The micro quartz cuvette with path length complex need to be corrected by equation
10 mm (Hellma Analytics) was used for (1) and (2).
small volume.
2.2 Procedures Acpx corrected,517 = Atotal,517 –ALr,517 (1)
2.2.1 Complex formation and DLLME ALr,517 = Acpx,611 x (AL,517/AL,611) (2)
extraction
In this work, dithizone (diphenylthio- Where Acpx corrected, 517 is the corrected
carbazone: H2Dz) was used as complexing absorbance for PbDz2 complex, Acpx total,517
agent for formation of lead-dithizone and Acpx,611 are the absorbances of PbDz2 at
complex (PbDz2). The optimum pH, 517 and 611, respectively. ALr, 517 is the
concentration of H2Dz was added into the absorbance of residue ligand at 517 nm,
Pb solution. The extraction procedure was AL,517 and AL,611 are the absorbances of free
performed by injection of the optimum ligand at 517 and 611 nm, respectively.
volume of disperser and extraction solvent In order to optimize PbDz2
into the complex solution. Then the solution formation, the pH of solution and
was stirred for 5 minutes. The organic concentration of dithizone were studied. The
solvent phase was separated from aqueous solution pHs of 8.5, 9 and 10 were carried
phase by centrifugation. The organic out. The difference in absorbance of
solvent (500 µL) was transferred by complex and blank (abs) was calculated
microsyringe. The appropriate amount of and compared. The pH of solution affects to
sodium sulfate anhydrous was added into the formation of PbDz2 complex. The
organic phase in order to remove residue precipitation of Pb(OH)2 could be occurred
water. Finally, the absorbance was when pH higher than 11 was used. pH 10
monitored at 517 and 611 nm for absorption gave the highest absorption (Figure 2), and
of complex and residue free ligand, was set as the optimum pH.
respectively. The various parameters which Concentration of dithizone was the
affected the complex formation and next parameter. The dithizone concentration
extraction efficiency were carried out. between 1-100 µM was studied. The
2.2.2 Method validation unstable complex was found when too low
Various parameters including limit concentration of dithizone was used.
of detection, limit of quantification, Whereas at dithizone of 50 and 100 µM, the
calibration curve, precision and recovery decreasing in absorbance of complex was
were evaluated in this study. Finally, the found due to high background absorbance of
proposed method was applied for excess free ligand. The highest absorbance
determination of Pb in water sample of PbDz2 was found when concentration of
collected from Map Ta Phut industrial dithizone as 10 µM (1 x 10-5 M) was utilized
estate, Rayong province. (Figure 3), thus the dithizone of 10 µM was
set as the optimum concentration for next
3. Results and Discussion experiment.
carried fine droplet of extraction solvent into
the aqueous solution results in increasing the
contact area between extraction solvent and
aqueous solution.7 In this study, methanol
ethanol acetonitrile and acetone were tested.
The result was demonstrated in Figure 4.
The methanol and ethanol were commonly
used as the disperser solvent and illustrated
the same extraction efficiency. However,
Figure 1. Dithizone and PbDz2 spectrum when consider the toxicity, ethanol was
chosen as the disperser solvent in this study.
Figure 2. The effect of pH on the abs of

PbDz2
Figure 4. The effect of disperser solvent on
the absorbance of PbDz2
Figure 3. The effect of dithizone

concentration on the absorbance of PbDz2
Figure 5. The effect of ratio of
dichloromethane: ethanol on the absorbance
3.2 Extraction parameters
The extraction efficiency and of PbDz2
selectivity was affected by the polarity of
extraction solvent and target analyte The ratio of dichloromethane
according to like dissolves like principle. (disperser solvent) and ethanol (extraction
Moreover, the high density and high boiling solvent) of 1:1, 1:2, and 1:3 v/v were also
point solvent performs the advantages that studied. In Figure 5, the highest absorbance
easy to separate from aqueous phase and was obtained when the ration of
less loss of solvent during extraction dichloromethane and ethanol of 1:3 v/v was
process.6 Thus, dichloromethane was chosen used. Thus, this ratio was chosen as the
as an extraction solvent in this study. In optimum and used for next study. The
DLLME procedure, type of disperser solvent volume of disperser and extraction solvent
affects the extraction efficiency. Disperser mixture was the next parameter that affects
solvent function as media that dispersed and the extraction efficiency. The mixture
solvent volumes of 2 - 5 mL were studied.
The highest absorbance was found when 2 Finally, the proposed method was
mL of mixture solvent was used as showed applied for determination of trace level of
in Figure 6. However, large deviation of the Pb in environmental sample. The water
absorbance value was also found in 2-3 mL samples were collected from Map Ta Phut
of mixture solvent. It was because when the industrial estate, Rayong province. The
volume of solvent mixture decreased, the amounts of Pb as < LOD were obtained in
volume of the organic solvent decreased and this study.
difficult for collection. Therefore, the 5 mL
of mixture solvent was chosen as the Table 1. Method validation for the proposed
optimum condition in this study. method
parameters value
LOD 0.58 µg/L
LOQ 1.93 µg/L
Calibration graph
Concentration range 20-140 µg/L
Linear equation y=0.0088x +0.1248
R2 0.9950
Precision
Repeatability 1.75% (n=9)
Reproducibility 3.35% (n=9)
% recovery 115.20±1.24
Figure 6. The effect of volume of extraction
4. Conclusion
mixture solvent on solvent on the
A rapid and simple extraction
absorbance of PbDz2
method for preconcentration of Pb in water
samples has been proposed. The µg/L level
3.3 Method validation and water sample
of Pb was extracted by dispersive liquid
determination
liquid microextraction method. The small
The limit of detection (LOD) and
volume of extracted Pb was detected by
limit of quantification (LOQ) were
spectrophotometer with microvolume
calculated as three times and ten times of
sample cell. This method was applied for
standard deviation of blank signals (n=10),
determination Pb in environmental sample.
respectively. Concentration of 0.58 and 1.93
µg/L were obtained for LOD and LOQ,
Acknowledgements
respectively. Calibration graph was
The author acknowledges Department
constructed from Pb solution that
of Chemistry, Faculty of Science, Burapha
concentration of 20-140 µg/L. In this study,
University for research experience. Mr.
the calibration graph of y = 0.0088x +
Narongsak Phasukcharoen phaibun and Miss
0.1248 with R2 =0.9950 was obtained.
Budsaracam Paritava have also been
Precision of this method was evaluated in
thankful for their experimental work. This
repeatability and reproducibility. The %RSD
research was partly supported by funding
of 1.75 and 3.35 were obtained from nine
from Center of Excellence on Environmental
replicated analysis of 40 µg/L Pb solution.
Health and Toxicology (EHT), Ministry of
The resulting precision was significant
Education, Thailand.
better than acceptable value from AOAC
method.8 The recovery was studied by
References
spiked 40 µg/L of Pb standard into water
1. World Health Organization, Background
samples. The recovery of 115.20±1.24%
document for development of WHO
was obtained and below the acceptable value
guidelines for drinking water quality,
from AOAC. The method validation
Geneva, 2011.
parameter and value were listed in Table 1.
2. Environmental Protection Agency, 5. Li, L.; Bin, H.; Linbo, X.; Zucheng, J.
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1559.
Aqueous copper(II) measurement by smart phone photometry
Tharaphan Youkhong1,2, Rungwalee Suklim1,2, Veeramol Vailikhit1,2*
1
Department of Chemistry, Faculty of Liberal Arts and Science, Kasetsart University,
2
Sustanable Chemistry Research Unit, Faculty of Liberal Arts and Science, Kasetsart University,
*E-mail: veeramol.v@ku.ac.th
Abstract:
Aqueous copper(II) as sulfate was measured with an 6S iPhone based photometer
using a white LED light source. The Apple application iDropper detected RGB intensity and
the Beers-Lambert Law gave the concentration of Cu2+ in solution. The results were linear for
red light between 4 – 16 mM. The precision was 2% and the relative error was 0.2%, thus the
device can be used in teaching laboratories when a standard UV-vis spectrometer is not
available.
1. Introduction an instrument that will probably not be used

Photometry and the application of constantly.3
the Beer-Lambert law is often used to In contrast, smart phones with built-
determine the concentration of a substance in cameras are near ubiquitous in most areas
in a solution as it is simple and requires little of the world, and have software applications
more than a few millilitres of the sample available that give the red, green and blue
solution and a few standard solutions of the (RGB) values of pixels within a picture.
substance with known concentration for This essentially allows almost any common
reference.1 It is used in the teaching smart phone to be used as a crude and
laboratory to introduce students to simple spectrophotometer. This report
spectroscopy in general, and to the practice details the use of a self-made
of sample preparation with the need to spectrophotometer based on an Apple
control either solution concentration or the iPhone 6S with the iDropper software
amount of substance at each step. The application to determine the concentration of
calculations involved in determining Cu2+ in aqueous solution and compares the
concentrations and amounts of substance are results with a Spectronic Educator single
also rudimentary, but an essential skill in beam visible-range instrument (Thermo
understanding and determining the amount Electron Corporation) and a T80 (PG
of a substance in a sample.2 Instrument Ltd.) spectrophotometer dual
The main difficulty involved in using beam UV-Vis instrument used for analytical
this technique is the acquisition of a measurements. The smart phone based
spectrophotometer. These instruments cost spectrophotometer will be shown to be a
thousands of US dollars or euro for fully useful and more easily affordable option for
capable UV-Vis instruments. Smaller teaching basic spectroscopy at the secondary
instruments that allow simple scanning in school and undergraduate level.
the visible range or setting of a fixed
wavelength of light are available, are well 2. Materials and Methods
suited for teaching and cost a few hundred 2.1 Equipment
dollars/euro, and even less for used A box, Figures 1 and 2, was
instruments. However, this is often still too constructed to support and contain the
expensive for many schools, especially in components of the spectrophotometer and is
rural areas of developing countries and for based on previous work.4 It was made from
rectangular pieces of hard plastic sheet solutions. These values were converted to
which were glued together with one piece light absorption values using Ax=-log(Ix/Iin),
hinged at the front as a door for loading where Iin is intensity of the light entering the
sample solutions. The dimensions were 35 sample solution, Ix is the intensity of light
cm tall, 24.5 cm wide and 17.5 cm deep from the solution and Ax the absorbance.
from the door, but are not crucial. The The RGB values range from 0 to 255 for
interior is coloured white and, as a light each of the three colours, and the
source, contains several strips of white light spectrophotometer was prepared so that Ix
Epiled SMD5050 LEDs powered by an was 255 with a blank sample. This was then
external 12VDC electrical supply which taken as the Iin for the other samples. The
allowed control of the brightness. On the calculations were performed, the absorption
right side was a simple metal bracket to hold values plotted, and regression lines fitted
a standard 1cm plastic cuvette. On the left using Microsoft Excel 2007. Where the plots
side was a 1 cm diameter hole through were linear, it was taken as following the
which an external iPhone 6S camera could Beer-Lambert law.
observe the cuvette. The iPhone was secured 2.3 Solutions
on a metal slide with a flexible foil bellows The solutions used consisted of AR
between it and the box to block external grade copper sulfate, CuSO4·5H2O (Merck
light. This allowed varying the distance KGaA), dissolved in distilled water. For the
between the camera and the sample for best standard curves, solutions from 1mM to
results. 100mM were prepared with accurately
known concentrations.
2.4 Measurements
A plastic cuvette was rinsed and
filled with the sample solution being
measured. It was then placed in the bracket
inside the constructed box and the camera of
an iPhone attached on the outside was used
to record an image of the cuvette and
solution. Initial trials showed that sliding the
camera so that is was 27 cm from the
cuvette gave a well focus image that allowed
easy determination of the RGB values at
several points in the sample solution portion
of the image. A blank sample of distilled
water only was used to adjust the LED
brightness so that the RGB values were 255
each, as expected for white light. The
Figure 1. Schematic of the self-made various CuSO4 solutions were then placed in
spectrophotometer the box and an image of each recorded.
Later the RGB values were determined
2.2 Software using the iDropper application and the range
The iPhone 6S with iOS 10.3 of concentrations where the Beer-Lambert
operating system was used to record images law is applicable was found. For
of the sample solutions in the cuvette comparison, standard curves were also
mounted in the box. The iDropper 2.0 determined using a Spectronic and a T80
application available from the Apple App UV-vis spectrophotometer, which are
Store5 was used to determine the RGB (red, commonly found in teaching and research
green and blue) values of the images at laboratories respectively.
several points in the images of the sample
door
adjustable
bellows
hole for LED

camera strips
sample
bracket
Figure 2. Labelled photograph of the self-made spectrophotometer
3. Results and Discussion converted to absorbance using A=log10(I0/I)

The Spectronic and T80 were set to give 0.12 – 0.20 in arbitrary units.
the λmax of Cu2+ in solution, which was 815
nm. Characteristics of the linear standard Table 1. Linear range (Abs. vs. mM) of the
curves between 8 mM and 80 mM are given three instruments where the Beer-Lambert
in Table 1. law applies
With the self-made spectrophoto- iPhone Spectronic T80
meter, the colour images consist of just three R value λmax = 815nm
separate colour readings, namely red (R), linear
4 -16mM 8 - 80mM 8 - 80mM
green (G) and blue (B). These act as a crude range
spectrometer, which was seen in the images r2 0.9918 0.9975 0.9988
of the Cu2+ solutions. As the concentration
intensity 194 - 160
increased and the solutions became more
blue in colour, the G and B values changed Abs 0.12 –0.20* 0.13 - 1.12 0.08 - 0.83
only a small amount, staying near 255. *
calculated
However, the R values dropped
significantly, as expected due to 815nm
being in the red region of the spectrum. Most significantly, it can be noted
Thus, only the R values were used with the that the self-made instrument was more
Cu2+ solutions. The standard curve was sensitive to lower Cu2+ concentrations than
linear between 4mM and 16mM and is the laboratory instruments, 4mM compared
summarised in Table 1. The R values, in the to 8 mM. However, it also gave a non-linear
range 0 to 255, are intensities and when response beyond a lower concentration, 16
mM compared to 80 mM. This suggests the
iPhone camera is not as suited to lower light determined by repeated intraday (10 times)
levels as concentration increases and sets a and interday (10 times on each of 3 days)
limit on the capability of the self-made measurements of the known 12mM sample.
instrument. The T80 was significantly more precise as
expected for an expensive research
Table 2. The limit of detection, LOD, and instrument. However, the over two had
the limit of quantification, LOQ, of the three comparable precision.
instruments (mM) are shown. Lower values
are better. Table 3. Accuracy and precision of
iPhone Spectronic T80 measuring a 12 mM sample of known
R value λmax = 815nm concentration
LOD 0.476mM 1.437mM 0.179mM iPhone Spectronic T80
LOQ 2.455mM 6.341mM 0.794mM relative error 0.17% 0.83% 0.33%
intraday RSD 1.88% 2.07% 0.40%
Table 2 compares the limit of interday RSD 1.76% 0.83% 0.68%
detection, LOD, and the limit of
quantification, LOQ, of the three 4. Conclusion
instruments. While the T80 is easily the The self-made spectrophotometer,
superior instrument, the self-made using a smart-phone camera for recording
instrument was comparable to the images of solutions and software
Spectronic, showing that it is suitable for applications for determining the colour
teaching. intensities, gives comparable results to a
It also appears that as the iPhone Spectronic commonly used in teaching
camera is a more general purpose detector laboratories. The use of the RGB values also
used for everyday photography, it responds allows other coloured solutions to be
well with higher light (RGB) levels analysed, with the appropriate R, G or B
compared to laboratory instruments. This colour used for a standard curve.
gives a more linear response in the low Cu2+ While a research instrument such as
concentration, but high R value, range with the T80 is always preferred due to its high
poorer response at lower signal levels. As accuracy and precision, the affordability of a
the LOD and LOQ are calculated from the self-made instrument using a readily
standard curve, lower values of these for the available smart-phone means that practical
iPhone compared to the Spectronic can be basic spectroscopy is possible in teaching
expected as its standard curve covers a laboratories with very limited resources,
lower concentration level. The T80, including high schools.
however, shows that it is extremely sensitive To further extend the capabilities of
and gives very low LOD and LOQ despite a self-made spectrophotometer, future work
its higher concentration linear range. will involve better control of parameters
Table 3 summarises the accuracy when recording images, and evaluation of
(relative error) and precision (relative using this instrument in a teaching
standard deviation, RSD) of the three environment.
instruments. The relative errors were
calculated after determining the Acknowledgements
concentration of a known 12mM sample The authors would like to thank
using the standard curve. Again, this shows Kasetsart University, the Faculty of Liberal
the sensitivity of the self-made instrument as Arts and Science and Department of
it was slightly more accurate than the other Chemistry for financial support of our
two, although all three were within 1% of Sustainable Chemistry Research Unit. We
the expected result. The precision was are grateful to Ajarn Wilhelm Josef
Holzschuh for discussion and help in the 3. Liebhafsky, H. A.; Winslow, E. H. J.
preparation of this manuscript. Chem. Educ. 1950, 27, 61– 62.
4. Karnsomngam, S. Senior Project
References Report, Kasetsart University, 2016.
1. Pringle, D. L.; Chaloupka, K.; 5. https://itunes.apple.com/us/app/
Varankamartin, M. J. Chem. Educ. idropper-the-color-sampling-
1995, 72, 722– 723. tool/id435292730.
2. Kehoe, E.; Penn, R. L. J. Chem. Educ.
2013, 90 (9), 1191-1195.
Modified silica gel for practical preparative chromatographic resolution of
D,L-aspartic acid derivatives
Varaporn Paradamit1*, Chanakan Maungrong1, Kittisak Thammapichai2,
Kantamanee Pantang1, Punlop Kuntiyong2
1
Analytical Research Laboratory, Department of Chemistry, Faculty of Science,
2
Organic Synthesis Research Laboratory, Department of Chemistry, Faculty of Science,
Silpakorn University, Sanamchandra Palace, Nakhon Pathom 73000, Thailand
*E-mail: vparadamit@yahoo.com
Abstract:
Common method for chromatographic resolution of racemic mixtures employs chiral
absorbent and achiral eluent. Alternatively, achiral adsorbent such as silica gel and eluent
containing chiral selector such as -cyclodextrin can bring about separation of the two
enantiomers. Polar compounds such as amino acids have strong affinity to silica gel therefore
the silica surface has to be modified as in C-18 reverse-phase adsorbent. In this study we
examine cheaper method for practical separation of less polar derivatives of D,L-aspartic acid
and glutamic acid obtained from N-benzylation of the amino acids. The silica gel used in the
separation was modified by simpler and cheaper methods using triethoxymethylsilane and
polyethylene glycol (PEG) then coated on glass plate for preparative thin layer
chromatography. Organic solvent systems containing -cyclodextrin as a chiral selector were
used as the mobile phase and resulted in separation of enantiomers. The effectiveness of
separation was observed by comparing the sign (+ or -) and magnitude of the optical rotation of
each fractions of the chromatogram.
1
1. Introduction the chiral selector in mobile phase for the

The simple and inexpensive separation by reversed phase TLC (C18).
technique for the resolution of enantiomers The purpose of modifying silica gel is to
of amino acids and their derivatives is Thin decrease affinity between polar surface of
Layer Chromatography (TLC).1 The silica gel and some polar molecules. Amino
enantiomer separation can be carried out acids and other highly polar organic
either by using chiral derivatization reagents molecules require the stationary phase to be
to form diastereomeric derivatives or modified to a reverse phase system for
directly using chiral selectors which can be optimal separation. The resolutions of dansyl
incorporated in the stationary phase or the derivatives of leucine, valine, methionine,
mobile phase.2 β-cyclodextrin was bonded to serine, glutamic acid, α- amino, n- butyric
silica gel in order to be the chiral stationary acid, aspartic acid, norvaline, threonine and
phase while using water/methanol/ phenylalanine have been achieved since
acetonitrile 1/1/4 (v/v/v) as mobile phase in 1988.5,6 Two recent examples used
2003.3 Other chiral molecules such as the sulfobutyl ether-β-cyclodextrin and hydroxyl
macrocyclic antibiotic vancomycin have -propyl-β-cyclodextrin as a chiral mobile
also been used as a chiral selector embedded phase additive for analytical separation of
on the stationary phase for separation of (+/-)- anti-depression drug citalopram7 and b-
verapamil, an anti-hypertensive drug.4 On the substituted 2-phenylpropanoic acid,8
other hand, β-cyclodextrin has been used as
respectively, using high performance liquid obtained using a Bruker Avance-300
chromatography (RP-HPLC). spectrometer.
In our organic synthesis laboratory, 2.2 Modification of silica gel
amino acid derivatives such as Type I: Modification with triethoxy-
dibenzylamino-L-aspartic acid are used in methyl-silane
alkaloid syntheses.9 Derivatives of non- Silica gel was prepared in three steps.
natural D-amino acids are also useful for (i) 10.0 g of silica gel was activated with
substrate-control asymmetric synthesis. “piranha solution” (98% H2SO4 / 30% H2O2
However they are generally much more 70:30 v/v) at 95 °C for 1 hour. Filter the
expensive in enantiomerically pure form activated silica gel and rinse with distil
from commercial sources than the naturally water. Heat the activated silica gel at 120 ° C,
abundant L-amino acids. Preparative
24 hours. (ii) Add 25 ml of toluene and 10 ml
separation of cheaper racemic materials thus
of triethoxymethylsilane to the activated
becomes an interesting and more practical
silica gel (from i). Heat at 50 °C and stir
option. With these less polar amino acid
slowly under the Argon gas atmosphere for
derivatives, simpler modification of the
silica stationary phase may be adequate for 14 hours. Filter and rinse the product with 20
practical separation of the racemic mixture ml of dry toluene 2 times and 20 mL of
of these derivatives. CH2Cl2 2 times. Dry at 50-55 °C by Rotary
For this purpose, we attempted evaporator before coating the TLC plate.
chromatographic resolution of D,L-N,N- Type II: Modify by polyethylene glycol
dibenzylaspartic acid by TLC using (PEG) 6000
modified silica gel from 2 methods of Heat the activated silica gel at 110°
modification, namely, modification with C, 1 hour before pouring the silica gel into
triethoxymethyl-silane and modification 20 ml of 10% PEG 6000. After stir 1 hour,
with polyethylene glycol (PEG) 6000. Chiral filter the white solid and dry before coating
the TLC plate
selector β-cyclodextrin was added into the
2.3 Spectroscopic measurement
mixed mobile phase.

2.1 General
Chemicals and silica gel 60 PF254
containing gypsum with 90% 55 µm particle
size were obtained from commercial sources
and were used without further purification.
Solvents were purified by distillation from
the appropriate drying reagents immediately
prior to use. Reaction flasks and glassware
were oven-dried at 105oC overnight. Optical
rotations were measured with a Jasco optical
rotation polarimeter model P-1010 at
ambient temperature using a 0.9998 dm cell Figure 1. IR spectrum from the activated
silica before modifying
with 1 mL capacity. Infrared (IR) spectra
were recorded on a Nicolet 5DXB FT-IR
spectrometer. Proton and carbon nuclear
magnetic resonance (NMR) spectra were
To a solution of D,L-aspartic acid
(5.00 g, 33.3 mmol) in MeOH and H2O (1:1)
100 mL was added NaOH (3.33 g, 83.3
mmol), K2CO3 (11.50 g, 83.8 mmol) and
BnCl (15.46 mL, 133 mmol). The mixture
was refluxed overnight and acidified with
1 M HCl. N,N-dibenzyl aspartic acid
precipitated from the solution and was
filtered under reduced pressure to give
dibenzylaspartic acid (4.33g, 53%) as a
Figure 2. IR spectrum from the Type I
modified silica gel (Modified with white powder; 1H NMR (300 MHz, DMSO)
triethoxymethylsilane)  7.43 – 7.13 (m, 10), 3.70 (d, J = 13.4 Hz,
2H), 3.68 – 3.53 (m, 3H), 2.75 (dd, J = 16.3,
6.4 Hz, 1H), 2.55 (dd, J = 16.3, 8.2 Hz, 1H).
13
C NMR (75 MHz, DMSO) δ 174.85,
172.32, 135.38, 129.59, 128.86, 128.33,
59.81, 54.76, 33.43; max (film) 3192, 2923,
2589, 1996, 1495, 1455, 1364, 1284, 1182,
886 cm-1.
2.5 Separation of N,N-dibenzyl-D,L-aspartic
acid
Separations of N,N-dibenzyl aspartic
acid were performed on the thin layer
chromatography (TLC) plates which were
coated by different types of modified silica
gel. (7.0 g of modified silica gel with small
amount of distil water for the 8x8 inches
Figure 3. IR spectrum from the Type II glass plate). The compound was dissolved in
modified silica gel (Modify by polyethylene
CH2Cl2 0.0075% (w/v) and the solution was
glycol (PEG 6000))
applied onto the preparative thin layer
The IR spectra show modification of chromatographic plates. The loaded Prep-
silica gel in Type I at 1384 cm-1 indicating TLC plate was then placed into a developing
incorporation of triethoxymethyl silane chamber containing the mobile phase. The
(Figure 2). Type II modification (Figure 3) mixed mobile phase system were all the
same (CH3OH: 1% cyclodextrin: acetonitrile
can be observed at 1708 cm-1 which derived
at a ratio of 1:1:4 (v/v/v)). After the
from PEG content and not present in
separation, the stationary phase was divided
unmodified silica’s spectrum (Figure 1).
equally into 6 horizontal strips and the
2.4 Preparation of N,N-dibenzyl-D,L- adsorbent was removed from the plate and
aspartic acid suspended each strip in CH3OH separately.
O O
After 15 minutes the mixture was filtered
BnCl, NaOH, K2CO3
HO
OH
H2O/MeOH, reflux HO
OH
and the filtrate concentrated under reduced
O NH2 53% O NBn2 pressure. The optical rotation of the
D,L-aspartic acid N,N-dibenzyl-D,L-aspartic acid
compound in each fraction was measured by
the Jasco optical rotation polarimeter model
P-1010.
3. Result and Discussion
Table 1. Optical rotation value , c = (1.00 / MeOH) of each fraction separated by two
types of modified silica gel (two duplicates)
Fraction no. Type I (#1) % ee Type I (#2) % ee Type II (#1) % ee Type II (#2) % ee
1 -24.2404 22(L) -21.7050 20(L) -13.41591 12(L) -8.55042 8(L)
2 -18.0004 16(L) -15.6552 14(L) +2.58170. 2(D) +5.01485 4(D)
3 -3.24643 3(L) -2.88571 2(L) +9.88230 9(D) +12.4136 11(D)
4
+0.21943 - +1.09783 - +16.8033 15(D) +15.5313 14(D)
5
6 +7.40024 7(D) +9.32480 8(D) +24.1415 22(D) +22.3203 20(D)
+10.1000 9(D) +12.2654 11(D) +45.7451 41 (D) +44.1744 40(D)
Empirical determination of enantiomeric excess of N,N-dibenzyl derivative of D- and L-
aspartic acid.
% ee = ( measured / of enantiomerically pure compound) x 100
The optical rotation value of enantiomeircally enriched fractions can, in

enantiomerically pure N,N-dibenzyl-L aspartic theory, be further optically purified by
acid is -111.1000 (c = 1.00, MeOH). recrystallization. The optimization of this
The sample obtained from each strip technique in combination with recrystallization
of absorbent removed from the TLC plates attempts are being carried out in our
showed low to moderate enantiomeric laboratories to achieve highly optically pure
purity. The optical roations of the fraction at derivatives of D- and L-amino acids.
both ends of the plates had opposite signs
(+ or -) indicating opposite enantiomers Acknowledgements
predominating at opposite ends. Type I silica Financial support for this research is
gave the highest enantiomeric purity of provided by the Faculty of Science,
L-aspartic acid derivative at 22% in fraction Silpakorn University.
1 whereas Type II silica gave the highest
enantiomeric purity of D-aspartic acid References
derivative in fraction no. 6 at 41%. 1. Gübitz, G.; Martin, G. Chiral
Separations Methods and Protocals
4. Conclusion 2004, Humana Press, USA.
Our modified silica-coated prepara- 2. Ward, T. J. Anal. Chem. 2000, 72, 4521-
tive TLC plate could separate the racemic 4528.
dibenzylamino derivative of D,L-aspartic 3. Hug, R.; Mercier, L.; Kooyman, P. J.
acid when a mobile phase containing
Chem. Mater. 2001, 13, 4512-4519.
β-cyclodextrin. The separation gave enantio-
4. Bhushan, R.; Gupta, D. Biomed.
enriched fractions of dibenzyl-D- or L-
aspartic acid. Optical rotation value from the Chromatogr. 2005, 19, 474–478.
separation by triethoxymethyl-silane- 5. Armstrong, D. W.; He, F.; Han, S. M. J.
modified silica gel and PEG-modified silica Chromatogr. 1998, 448, 345-354.
gel showed partial separation where
6. Gunther, K.; Richer, P.; Moller, K.
triethoxymethyl silane-modified silica gave
Methods in Molecular Biology: Chiral
mixtures enriched with derivative of D-
aspartic acid and PEG-modified silica gave separation: Methods and Protocals 2003,
derivative of L-aspartic acid in a higher 243, 29-59.
enantiomeric excess. Although the 7. Peng, Y.; He, Q. S.; Cai, J. Int. J. Anal.
enantiomeric excess achieved from these Chem. 2016.
experiments were not high, these
8. Tong, S.; Zhang, H.; Yan, J. J. C.; Thongteerapab, S.; Khemthong, P.
Chromatogr. Sci., 2016, 54, 593–597. Tetrahedron 2017, 73, 4426–4432.
9. Kuntiyong, P.; Bunrod, P.; Namborisut,
D.; Inprung, N.; SathongJin, J.; Sae-guay,
Analysis of volatile components in Arabica coffee roasted at different
temperatures by using solid phase microextraction- gas chromatography-
mass spectrometry
Suchawadee Insawang1, Patcharee Pripdeevech1,2*
1
School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
2
Center of Chemical Innovation for Sustainability (CIS), Mae Fah Luang University,
*E-mail: patcharee.pri@mfu.ac.th
Abstract:
Volatile components of Arabica coffee beans from Chiang Rai province were
extracted by using solid phase microextraction (SPME) prior analysis by gas
chromatography-mass spectrometry (GC-MS). Three SPME fibers including
polydimethylsiloxane (PDMS), carboxen/polydimethylsiloxane (CAR/PDMS) and
divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) were selected to extract
volatile components. Fourteen compounds were detected with PDMS fiber. The key volatile
components were sotolone, decanal and cis-pinocarvyl acetate, while eighteen intermediate
polar components including indole, 2-furanmethanol and sotolone were detected when using
CAR/PDMS fiber. Twenty-eight compounds were detected with major volatile components
of 2-furancarboxaldehyde and 2-furanmethanol for DVB/CAR/PDMS fiber which can be
extracted with board range of compounds. According to the highest number of extracted
volatile components, DVB/CAR/ PDMS fiber was further used to extract volatile components
of Arabica coffee beans roasted at different temperatures. It was found that light roasted
coffee at 180 ºC provided vanillin, methyl vanillin, 2-furancarboxaldehyde and acetic acid as
major components. Two-furancarboxaldehyde, vanillin, methyl vanillin and 2-furanmethanol
were detected as major components in medium roasted coffee at 210 ºC while 2-
furancarboxaldehyde, 2-furanmethanol, 2-acetoxymethylfuran and 2-methyl-3-oxo-
tetrahydrofuran were detected as main compounds in dark roasted coffee at 225 ºC. SPME-
GC-MS is efficient method to extract the the key odor compounds in roasted Arabica coffee
beans.
1. Introduction contribute to coffee aroma. Differences in

Coffee is a major beverage in social coffee aroma profile has been detected from
and economic market. It is produced by many factors including coffee plant cultivars
steeping and draining through a flannel or (Robusta, Arabica), intrinsic bag and
muslin bag or using coffee brewing seasonal variation, geographical locations,
equipment with filter paper. The identified processing (wet or dry) and ageing before
odor components in coffee has been roasting process.2 Moreover, roasting
increased continuously every year. At least temperature and type are also play a role in
800 volatile components are detected in differentiating aroma profile of coffees.3 In
roasted coffee. They were classified in a general, roasting temperature is categorized
group of aldehydes, ketones, alcohols, in four types including light, medium,
pyrroles, pyrazines, furans, thiazoles, medium-dark and dark roasting3. Light
thiophenes, esters, oxazoles, lactones, roasted coffee generally reach an internal
sulphur compounds, pyridines, alkanes, temperature of 180 °C to 205 °C while
alkenes, phenolic compounds, benzenic medium roasted coffee reach internal
compounds, acids, pyranones and terpenes temperatures between 210 °C and 220 °C.
and derivatives.1 Only 80 volatiles actually The light and medium roasted coffees are
light and medium brown in color, machine. The obtained samples were packed
respectively. Both types have no oil on the in sealed aluminium foil bag and kept in
bean surfaces. Medium-dark roasted coffee dark at 4 °C until analysis.
have a richer, darker color with some oil on 2.2 Extraction of volatile components by
the bean surface which are roasted from using SPME
temperature between 225 °C and 230 °C. Three SPME fibers including; 100
Dark roasted coffees are dark brown or μm PDMS, 75 μm CAR/PDMS and 50/30
sometimes almost black in color and it has μm DVB/CAR/PDMS were used to extract
sheen of oil on surface due to roasting to an volatile components of roasted Arabica
internal temperature of 240 °C to 250 °C. coffee bean samples. The SPME holder and
Quantification of coffee volatile components all SPME fibers were purchased from
is challenging due to the wide range of Supelco, Bellefonte, PA, USA. Before using
concentrations, high volatilities, wide range of SPME fiber, it was preconditioned at 280
of physicochemical properties such as °C in injection port of GC for 2 hr for
polarity, pK, charge and their potential to eliminating any contaminates. For each
polymerize and bind to other coffee extraction, 10 g of ground roasted Arabica
components. Volatile components in coffee coffee was added to 250 mL-headspace vial.
is successfully analyzed by using solid- The vial was closed with septum and plastic
phase micro extraction (SPME) coupled to cap, respectively. The sample vial was
gas chromatography-mass spectrometry heated at 90 °C for 30 min prior insertion of
(GC-MS) which offer discriminative SPME fiber through the septum. The fiber
sensitivity to different classes of volatile was exposed to the headspace for extraction
compounds, extracted peak areas or spectra of volatile constituents for 30 min before
are then analyzed by standard data analysis desorption of volatile compounds in the
techniques or multivariate approaches.4 The injection port of GC-MS for 5 min.5
evaluation of the different analytical 2.3 Analysis of volatile components by
approaches to understand and to quantify the GC-MS
relative presence of volatile aroma The volatile components of ground
compounds in coffee will be undertaken to dark roasted Arabica coffee beans extracted
evaluate the relative merits of each from all SPME fibers were investigated by
analytical approach and their relative using a Hewlett Packard model HP6890 gas
discriminatory ability. The aim of this study chromatograph (Agilent Technologies, Palo
was to evaluate the discriminatory ability of Alto, CA, USA). It was equipped with 5%
a range of analytical approaches for phenylpolymethylsiloxane (HP-5MS)
determining key volatile components of capillary column (30 m × 0.25 mm i.d., film
Arabica coffee samples obtained from thickness 0.25 μm; Agilent Technologies,
different roasting temperature. The main USA) interfaced to an HP model 5973 mass-
objective of our research was to analyze the selective detector. The oven temperature
volatile compounds using SPME-GC-MS. was initially held at 60 °C and then
increased by 2 °C/min to 220 °C. The
2. Materials and Methods temperatures of injector and detector were
2.1 Arabica coffee beans 250 and 280 °C, respectively. Purified
Arabica coffee beans were obtained helium was used as the carrier gas at a flow
from Chiang Rai province, Thailand (N: rate of 1 mL/min. Electron impact (EI) mass
19.4332◦, E: 100.1516◦). All samples were spectra were collected at 70 eV ionization
collected in November 2017. Arabica coffee voltages over the range of m/z 29-300. The
beans were roasted at three different electron multiplier voltage was 1150 V. The
temperatures including 180 °C (light roast), ion source and quadrupole temperatures
210 °C (medium roast) and 225 °C (dark were set at 230 °C and 150 °C, respectively.
roast) for 10 min by using coffee roasting Identification of volatile components was
performed by comparison of their Kovát furanmethanol and acetic acid. For
retention indices, relative to C6-C19 n- DVB/CAR/PDMS fiber, twenty-eight
alkanes, and comparison of the mass spectra compounds were investigated. The major
of individual components with the reference volatiles were 2-furancarboxaldehyde, 2-
mass spectra in the Wiley7N and NIST05 furanmethanol, sotolone and larixinic acid.
databases. Normally, volatile components were
extracted based on the partition of analytes
3. Results and Discussion between the coating composite, sample
Volatile constituents of dark roasted headspace and sample in SPME technique.
Arabica coffee beans were extracted by The extraction is proceeded when the
using three different SPME fibers including concentration of component closed to
PDMS, CAR/PDMS and DVB/CAR/PDMS equilibrium between three phases.6 Best
prior analysis their volatile components by trapping of components is the most
using GC-MS. All selected fibers were used important factor for selecting of fiber which
comparatively to extract components in can be applied for all samples. In addition,
order to investigate the best fiber for chemical properties of the target
extracting of compounds in dark roasted components may be affected on the
Arabica coffee samples. The potential of extraction. Different numbers and intensity
SPME fiber in extracting of volatile of volatile components of roasted Arabica
compounds is depended on the concept “like coffee samples could be related to fiber
dissolves like” and the fiber thickness. composites. Intermediate and high polar
Basically, non-polar fiber is expected to be volatile components were extracted mostly
effective in extracting non polar compounds by CAR/PDMS and DVB/CAR/PDMS
whereas polar fibers are suitable for fibers, respectively, compared to those
extracting of polar component. The intensity extracted by PDMS fiber demonstrating
of extracted components can differ higher number of non-polar volatile
significantly based on the response of compounds. Higher number of volatile
components to each SPME fiber. It is found components was obtained in fiber containing
that highest number of volatile components at least one composite such as CAR/PDMS
was detected by using DVB/CAR/PDMS and DVB/CAR/PDMS fibers. Therefore,
fiber compared to those found in DVB/CAR/PDMS was appropriate for
PDMS/DVB and PDMS fibers, respectively, extracting volatile components of roasted
under the same conditions. However, Arabica coffee samples obtained from
although PDMS/DVB fiber provided lower different roasting temperatures. The
number of volatiles than DVB/CAR/PDMS identified volatile compounds of all coffee
fiber, data obtained by this fiber may be samples obtained from DVB/CAR/PDMS
used to confirm identification of volatile fiber are listed in Table 1. A total of twenty-
components in all samples. Therefore, the eight volatile components were detected
DVB/CAR/PDMS fiber was chosen to among these roasted Arabica coffee
extract volatile components of all samples. samples. It was found that vanillin
Notable differences in the volatile (14.69%), methyl vanillin (12.04%),
components among these samples using the 2-furancarboxaldehyde (11.24%) and acetic
DVB/CAR/PDMS fiber were detected. acid (5.31%) was considered as key volatile
Fourteen compounds were detected with compounds in light roasted Arabica coffee.
PDMS fiber. The key odor volatiles were Volatile components detected in medium
sotolone, decanal and cis-pinocarvyl acetate. roasted Arabica coffee were 2-furancarbox-
Eighteen intermediate polar volatile aldehyde (15.35%), vanillin (12.11%),
constituents were found when using methyl vanillin (10.26%) and 2-furan
CAR/PDMS fiber with the major methanol (6.58%) while 2-furancarbox-
compounds of indole, sotolone, 2- aldehyde (18.52%), 2-furan-methanol
(10.24%), 2-acetoxy-methylfuran (6.35%) components provided sweet odor such as
and 2-methyl-3-oxo-tetrahydrofuran vanillin, methyl vanillin and sour odor such
(5.36%) were also detected as key volatile as acetic acid could be decreased at higher
components in dark roasted Arabica coffee. roasting temperature.
It was found that content of volatile
Table 1. Volatile compounds of Arabica coffee roasted at different temperatures by using

DVB/CAR/PDMS fiber
% relative peak area
No. Compound RI Light Medium
Dark roast
roast roast
(225 ºC)
(180 ºC) (210 ºC)
1 acetic acid 620 5.31 3.24 0.56
2 2-methyl-3-oxo-tetrahydrofuran 776 1.35 2.85 5.36
3 2-furanmethanol 873 4.58 6.58 10.24
4 2-acetoxymethylfuran 926 3.15 3.87 6.35
5 2-furancarboxaldehyde 974 11.24 15.35 18.52
6 δ-limonene 1045 3.54 2.58 1.25
7 2-ethyl-3,5-dimethylpyrazine 1067 1.25 2.41 3.87
8 larixinic acid 1097 1.32 3.25 1.78
9 sotolone 1118 3.58 3.28 1.25
10 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone 1145 1.63 2.57 4.52
11 3-isobutyl-2-methoxypyrazine 1163 1.08 1.14 2.96
12 2,3-diethyl-5-methylpyrazine 1177 0.47 0.58 2.94
13 decanal 1189 2.14 2.53 0.85
14 2-vinyl-3-ethyl-5-methylpyrazine 1226 1.14 1.25 2.96
15 4-ethylguaiacol 1263 0.69 0.94 1.22
16 indole 1278 0.54 0.73 4.22
17 cis-pinocarvyl acetate 1307 2.56 2.54 3.65
18 4-vinylguaiacol 1318 1.11 1.13 4.12
19 cis-p-mentha-8-thiol-3-one 1355 3.25 3.62 3.61
20 eugenol 1372 2.87 2.58 3.58
21 E-β-damascenone 1389 3.52 2.04 1.11
22 vanillin 1403 14.69 12.11 2.05
23 cis-threo-davanafuran 1417 2.04 2.12 5.04
24 E-cinnamyl acetate 1445 3.11 2.56 1.14
25 methyl vanillin 1479 12.04 10.26 2.38
26 methyl isovalerate 1516 1.89 1.78 0.83
27 dodecanoic acid 1531 2.11 0.52 0.18
28 caffeine 1834 4.58 3.24 0.87
Total relative peak area percentage 96.78 97.65 97.41
RI, kovats index on DB-5MS.
Moreover, caffeine, a central nervous hydrofuran were detected with greater content
system stimulant of methyl- xanthine, was in dark roasted coffee. Major volatile
also detected with low content at high components played an important role as key
roasting temperature whereas volatile compounds in coffee in all samples are
components with woody odor including depicted in Figure 1.
2-furancarbox-aldehyde, 2-furanmethanol, 2-
acetoxy-methylfuran and 2-methyl-3-oxotetra
proteins, peptide, and amino acids generated
ketones, pyrrols, and pyrazines.10,11 Lipids
were responsible for only small amounts of
aldehydes and ketones given their resistance
to changes during the roasting process while
chlorogenic acids produced phenolic volatile
compounds.12 In addition, trigonelline
produced pyrroles, pyridines, and pyrazines.
Almost all thiophenes, oxazoles, and
thiazoles were formed during roasting only
and they are not usually detected in green
coffee beans. The roasting degree and
roasting parameters affect the volatile
composition of coffee.13 The effect of
roasting degree is readily apparent because
the aroma of a light roasted coffee.
Figure 1. Area percentage of key volatile
components including 2-furanmethanol(I), 4. Conclusion
2-acetoxymethylfuran(II), 2-furancar- The study illustrated how aroma
boxaldehyde(III), vanillin(IV), methyl profiles were impacted when three common
vanillin(V) and caffeine(VI) in coffee roasting temperature was applied during
obtained from different roasting coffee roasting. The three specific roast
temperatures coffee (light, medium and dark) were shown
to have elevated contents of six components
Differentiation of volatile components including 2-furanmethanol, 2-
of coffee during roasting may be related to acetoxymethylfuran, 2-furancarboxaldehy-
the formation of complex aroma of coffee de, vanillin, methyl vanillin and caffeine,
by various reactions including pyrolysis, respectively. The association of specific
Strecker degradation, and Maillard changes in aroma profiles for different roast
7,8
reaction. The concentrations of volatile temperature has not been shown previously
components in roasted coffee may be varied in Chiang Rai and could be incorporated
on the nonvolatile compounds in the raw into screening tools to enable the coffee
seeds and on roasting temperature. industry quickly identify if roasting
Therefore, these factors influence the final temperature is used during production.
composition of the volatile components of Although it should be noted that the specific
roasted coffee. This study was similar to marker compounds may change with
study of Baggenstoss et al.9 who reported variations in coffee bean chemistry due to
furans and pyrans, pyrazines, pyrroles, species and production techniques. Sensory
ketones and phenols, hydrocarbons, studies have been carried out to confirm if
alcohols, aldehydes, acids and anhydrides, those chemical changes are perceivable, and
esters, lactones, thiophenes, oxazoles, the results will be published in a future
thiazoles, pyridines, amines, and various publication.
sulfur and nitrogen compounds as major
classes of volatile compounds in roasted Acknowledgements
coffee. However, it is impossible to The authors thank Mae Fah Luang
determine all reactions producing these University for supporting of financial and
volatile compounds according to more than GC-MS.
one route was detected. It was found that
soluble polysaccharides produced furans,
aldehydes, ketones, and phenols whereas
References 7. Schieberle, P. Ann. N. Y. Acad. Sci.
1. Chu, Y. F. in Coffee: Emerging Health 2005, 1043, 236-248.
Effects and Disease Prevention, Aptara® 8. Muller, C.; Hofmann, T. J. Agric. Food
Inc., New Delhi, India, 2012. Chem. 2005, 53, 2623-2629.
2. World Resource Institute (WRI). 9. Baggenstoss, J.; Poisson, L.; Luethi, R.;
Countries by coffee consumption per Perren, R.; Escher, F. J. Agric. Food
capita, 2010. http://earthtrendswri.org/ Chem. 2007, 55, 6685-6691.
(accessed November 2017). 10. Geiger, R.; Perren, R.; Kuenzli, R.;
3. Baggenstoss, J.; Poisson, L.; Kaegi, R.; Escher, F. J. Food Sci. 2005, 70, E124-
Perren, R.; Escher, F. J. Agric. Food E130.
Chem. 2008, 56, 5836–5846. 11. Ortola, M. D.; Londono, L.; Gutierrez,
4. Schenker, S.; Handschin, S.; Frey, B.; C. L.; Chiralt, A. Food Sci. Technol. Int.
Perren, R.; Escher, F. J. Food Sci. 2000, 1998, 4, 59.
65, 452-457. 12. Holscher, W.; Vitzthum, O. G.;
5. Pripdeevech, P. in PACCON proceeding Steinhart, H. J. Agric. Food Chem. 1992,
2017, 33-38. 40, 655-658.
6. Pripdeevech, P.; Machan, T. Food 13. Yeretzian, C.; Jordan, A.; Badoud, R.;
Chem. 2011, 125, 797-802. Lindinger, W. Eur. Food Res. Technol.
2002, 214, 92-104.
Non-enzymatic electrochemical detection of glucose based on copper foam
modified on copper wire
Suntisak Khumngern1,2, Ratchaneekorn Jirakunakorn1,2,3, Ira Musarafa5,
Jittima Choosang1,2,3, Panote Thavarungkul1,3,4, Proespichaya Kanatharana1,2,3,
Apon Numnuam1,2,3*
1
Trace Analysis and Biosensor Research Center, Prince of Songkla University,
Hat Yai, Songkhla 90112, Thailand
2
Department of Chemistry, Faculty of Science, Prince of Songkla University,
3
Center of Excellence for Innovation in Chemistry (PERCH-CIC), Faculty of Science,
Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
4
Department of Physics, Faculty of Science, Prince of Songkla University,
5
Faculty of Mathematics and Natural Science, Yogyakarta State University,
Karangmalang, Yogyakart 55281, Indonesia
*E-mail: apon.n@psu.ac.th
Abstract:
A copper foam modified copper wire electrode was fabricated for the non-enzymatic
detection of glucose. Cu foam was electrodeposited by applying a potential of -5.0 V in 0.30
M CuSO4 and 0.50 M H2SO4. The amperometric detection of glucose was carried out at an
applied potential of +0.45 V (versus Ag/AgCl) in a 0.10 M NaOH supporting electrolyte.
Under the optimal conditions, a linear range of 0.010 to 10.0 mol L-1 was obtained with a
detection limit of 9.0 µM (3Sa/b). The method exhibited short response time (3 s), good
reproducibility (RSD = 3%), excellent operation stability (260 analyses) and negligible
interferences of ascorbic acid and uric acid. Its application in the detection of glucose in
serum samples is promising.
1. Introduction non-enzymatic glucose sensors.5 Among

Diabetes is a worldwide chronic these, transition metal nanomaterials such as
disease resulting from insulin deficiency and Cu,6 Ni,7 and their oxides8 have attracted
hyperglycemia which is reflected by the more attention due to their low cost, plenty
exceeding blood glucose concentration from of morphologies, high specific surface area,
its normal range (80-120 mg dL-1 or 4.4-6.6 good electrocatalytic activity and the
mM).1-2 The diagnosis and management of possibility of promoting electron transfer
this disease thus requires a tight monitoring reactions at a lower overpotential. Among
of blood glucose. Electrochemical enzyme these, Cu nanomaterial have been
biosensors for glucose detection have gain a extensively studied because of their
lot of attention because of its high selectivity numerous applications in catalysis, field-
and sensitivity.3 However, some of their effect transistors and biosensors.9,10 Cu
limitation include high cost, instability and nanomaterials-based amperometric sensors
complex fabrication caused by the inherent for glucose have shown high sensitivity and
nature of the enzymes.4 Therefore, there is a good stability because of their unique
need to develop inexpensive and robust non- nanostructure and extraordinary properties
enzymatic sensors that can accurately detect (e.g. excellent electronic transport property
glucose in blood. and high electrocatalytic activity).11
Many catalysts, such as carbon In this work, a porous Cu foam has
composites, noble metals, transition metals, been electrodeposited onto a Cu wire
transition metal oxides and hydroxides, electrode. This process of fabricating Cu
metal alloys, and bimetals, were explored as foam is simple without the use of complicated
and expensive equipment. The surface consisting of the Cu foam/Cu wire working
morphology of the Cu foam was electrode, an Ag/AgCl reference electrode,
characterized using scanning electron and a platinum wire auxiliary electrode. A
microscopy. The electrochemical characterization 0.10 mol L-1 NaOH (optimal value) was
of the Cu foam/Cu wire electrode was used as the supporting electrolyte.
investigated by cyclic voltammetry (CV)
and chronoamperometry, and the use of the
Cu foam/Cu wire electrode as a glucose
sensor was demonstrated.

2.1 Chemicals and reagents
Acetone and CuSO4.5H2O were Scheme 1. Illustration of the formation
obtained from Scharlau Chemie S.A., process of the porous Cu foams
(Sentmenat, Spain). Glucose and NaOH
were from Merck (Darmstadt, Germany). 3. Results and Discussion
Ascorbic acid and uric acid were from 3.1 Characterization of modified electrode
Sigma-aldrich (Darmstadt, Germany). Figure 1 shows surface morphology
HNO3, H2SO4 were obtained from of electrodes. The bare Cu wire has a
RCi Labscan (Bangkok, Thailand). All other smooth surface (Figure 1A). In Figure 1B, it
chemicals were of analytical grade. All is clear that Cu foam has been
solutions were freshly prepared with electrodeposited on the surface of the Cu
deionized (DI) water. wire, with pores up to 75 µm in diameter,
2.2 Fabrication of Cu foam on Cu wire linking to each other in all direction. At a
Cu wire with a diameter of 0.50 mm higher magnification (Figure 1C), one can
was used as the electrode core. 5.0 mm of see that the pore walls compose of numerous
the wire was first cleaned by immersing in corncob-like deposits with some nano-
0.50 M HNO3 solution for 30 s. After particles. This would supply more active
rinsing with DI water, it was immersed in surface area for catalysis and enhance its
acetone for 30 s. Porous Cu foam was then ability for electron transfer.
electrodeposited on the Cu wire surface in
10.0 mL of the mixture solution of
0.30 mol L-1 CuSO4 and 0.50 mol L-1 H2SO4
at -5.0 V (vs. Ag/AgCl) in 10 s.12 During
this deposition process, bubbles originated
from hydrogen evolution act as templates for
Cu2+ ion electro-reduction to form porous
copper foam (Scheme 1). The surface
morphology was then observed by scanning
electron microscopy (SEM, Quanta 400,
FEI, The Netherlands).
2.3 Electrochemical measurement
Electrochemical measurements were
conducted with a -Autolab type III
Figure 1. SEM images of the electrodes
potentiostat–galvanostat (Metrohm Autolab,
surfaces (A) bare Cu wire electrode, at ×200
Utrecht, The Netherlands), with GPES 4.9
and Cu foam/Cu wire electrode, at ×200 (B)
software for computer control. Cyclic
and ×1,500 (C)
voltammetric and amperometric glucose
detection were operated in a batch cell under
stirring with a three-electrode system
3.2 Electrocatalysis of glucose reaction of this non-enzymatic detection of
The electrocatalytic performance of glucose was assisted by the Cu(II)/Cu(III)
Cu foam electrode toward glucose oxidation redox couple. Thus, an enhanced response
in alkaline medium of 0.10 mol L-1 NaOH appeared on the Cu foam modified electrode
was investigated by cyclic voltammetry in the presence of glucose solution and was
from -0.10 to 0.70 V (vs. Ag/AgCl). As directly correlated to the concentration.
shown in Figure 2, the bare Cu wire
electrode does not show any oxidation or
reduction current in the absence of glucose
(Figure 2a) and its electrochemical response
to glucose is low (Figure 2b). For the Cu
foam/Cu wire electrode, a single broad
reduction peak with a peak potential from
+0.20 to +0.50 V can be observed in the
Scheme 2. Illustration of the non-enzymatic
absence of glucose (Figure 2C) which
electro-oxidation process of glucose to
should correspond to a Cu(II)/Cu(III) redox
gluconolactone and gluconic acid on the
couple. With the addition of 5.0 mM
fabricated Cu foam/Cu wire electrode in
glucose, a notable enhanced catalytic
alkaline media
oxidation current is observed in the range of
+ 0.25 V to + 0.50 V. Compared to the bare
Cu wire, the structured Cu foam/Cu wire
electrode can enhance electrocatalytic
activity toward glucose oxidation, which is
attributed to the larger surface area of the
three-dimensional porous structure of Cu
foam.
Figure 3. Amperometric response of a Cu

wire and a Cu foam/Cu wire electrode for
glucose detection (0.20-2.0 mmol L-1) at an
applied potential of 0.35 V (from Figure 2d)
and 0.45 V (from Figure 2b) for Cu foam/Cu
wire and bare Cu wire, respectively
Figure 2. Cyclic voltammograms of bare Cu
wire (a and b) and Cu foam/Cu wire (c and Figure 3 shows the amperometric
d) electrodes in 0.10 mol L-1 NaOH without response of Cu wire compared to the Cu
(a and c) and with (b and d) of 5.0 mM foam/Cu wire electrode. The sensitivity
glucose at 100 mV s−1 (slope of the calibration plot) of glucose
detection using a Cu foam/Cu wire is
Scheme 2 shows the glucose sensing 3.40 times higher than that of a Cu wire
mechanism of Cu foam/Cu wire electrode. electrode.
During the cyclic voltammetric measurement, 3.3 Optimization
Cu(II) was oxidized to Cu(III), that could In order to improve the sensor
catalyze glucose oxidation to generate performance, various factors affecting its
gluconolactone which was further oxidized response were investigated. The concentration
to gluconic acid.13 That is, the catalytic of NaOH and the applied potential were
optimized by amperometrically detecting a
series of glucose solution at a concentration increased linearly with glucose concentration
range of 0.20-2.0 mol L-1. NaOH was tested between 0.010 and 10 mmol L-1 (Figure 5)
at 0.05, 0.075, 0.10 and 0.125 mol L-1. As with a detection limit of 9.0 µmol L-1(3Sa/b).
shown in Figure 4A, the sensitivity of
glucose detection at 0.35 V (vs. Ag/AgCl)
increased with the concentration of NaOH
until 0.10 mol L-1 (Figure 4A) because glucose
is more easily oxidized and the electrocatalytic
activity of CuO is greatly enhanced at high
OH-. The decrease at higher NaOH
concentration may be because too much OH-
can block further electro-adsorption of glucose
anion, resulting in a decrease in current
signal.14
Figure 5. Calibration curve of the fabricated
glucose electrochemical sensor and an
example of the current response from
various concentrations of glucose in the
batch injection analysis (inset)
3.4.2 Operation stability and repeatability
Figure 4. Effect of the concentration of

NaOH (A) and applied potential (B) on the
sensitivity of glucose detection (0.20-2.0
mmol L-1)
The applied potential was tested at

0.20, 0.35, 0.40, 0.45 and 0.50 V (covering
the oxidation peak in Figure 2d) by detecting
glucose in 0.10 mol L-1 NaOH. As shown in Figure 6. Operational stability of Cu
Figure 4B, the maximum sensitivity was foam/Cu wire sensor for glucose detection
obtained at 0.45 V (vs. Ag/AgCl). Therefore, (A) and repeatability of the response of Cu
0.10 mol L-1 NaOH and an applied potential of foam/Cu wire from six electrode preparations.
0.45 V were used for subsequent experiments.
3.4 Analytical performances The operational stability of one
3.4.1 Linear range and limit of detection electrode was studied by comparing the
Under the optimal conditions, the sensitivity of the current response of ten
change of the oxidation current of glucose
glucose concentrations (0.20 to 2.00 mmol 4. Conclusion
L-1). As shown in Figure 6A, the relative The Cu foam modified Cu wire
sensitivities of the fabricated sensor after 26 electrode prepared by electrodeposition
measurement cycles (a total of 260 injections) provided a uniform structure. High surface
were greater than 90% and dropped below area of the porous Cu foam contributed to an
90% thereafter. At the end of the study the effective catalytic redox reaction of glucose
sensing surface appeared to have lost parts thus provided an enhanced sensitivity. The
of the Cu foam. Thus, the rapid decrease of glucose sensor provided a wide linear range,
the response after 26 cycles was most likely a low limit of detection, good stability,
the result of this loss. The preparation reproducibility and selectivity. This Cu
repeatability of the Cu foam/Cu wire was foam/Cu wire electrode shows potential in
tested by preparing six electrodes to measure applying for the detection glucose in real
a series of concentrations of glucose (0.4, serum samples.
0.8, 1.2, 1.6 and 2.0 mmol L-1). The relative
standard deviations of the currents from the Acknowledgements
five concentrations of glucose were 2.5, 1.6, We would like to thank Science
2.5, 2.0 and 2.1% (Figure 6B), indicated that Achievement Scholarship of Thailand
the electrode preparation has good repeatability. (SAST), the Faculty of Science Research
3.4.3 Selectivity Fund, Prince of Songkla University, Contact
The interference from electroactive no. 1-2559-02-011, Trace Analysis and
compounds normally coexisting with Biosensor Research Center (TAB-RC) and
glucose in real samples (e.g. human blood) Department of Chemistry, Faculty of
such as ascorbic acid and uric acid were Science, Prince of Songkla University,
tested. Thailand.
References
1. Hossain, P.; Kawar, B.; El Nahas, M.
New England J. Med. 2007, 356, 213-
215.
2. Wang, J. Chem. Rev. 2008, 108 (2), 814-
825.
3. Wang, G.; He, X.; Wang, L.; Gu, A.;
Huang, Y.; Fang, B.; Geng, B.; Zhang,
X. Microchim. Acta 2013, 180 , 161-
Figure 7. Interference test of Cu foam/Cu 186.
wire in 0.1 M NaOH at +0.45 V with 5.0 4. Yang, J.; Lin, Q.; Yin, W.; Jiang, T.;
mmol L-1 glucose in the presence of 0.2 mmol Zhao, D.; Jiang, L. Sens. and Actuators
L-1 ascorbic acid and 0.4 mmol L-1 uric acid B Chem. 2017, 253, 1087-1095.
5. Zhang, L.; Ye, C.; Li, X.; Ding, Y.;
There was no influence from Liang, H.; Zhao, G.; Wang, Y. Nano-
0.2 mmol L-1 ascorbic acid and 0.4 mmol L-1 Micro Lett. 2018, 10 (2), 28
uric acid (concentrations at the upper level of 6. Kang, X.; Mai, Z.; Zou, X.; Cai, P.; Mo,
the normal physiological range) on the Zhuang, Z.; Su, X.; Yuan, H.; Sun, Q.;
glucose current response (Figure 7). Thus, Xiao, D.; Choi, M. M. F. Analyst 2008,
the Cu foam/Cu wire presented good 133 (1), 126-132.
selectivity toward glucose detection. 7. Niu, X.; Li, Y.; Tang, J.; Hu, Y.; Zhao,
H.; Lan, M. Biosens Bioelectron 2014,
51, 22-8.
8. Luo, L.; Zhu, L.; Wang, Z. Bio 13. Reitz, E.; Jia, W.; Gentile, M.; Wang,
Electrochem. 2012, 88, 156-163. Y.; Lei, Y. Electroanalysis 2008, 20
9. Yang, J.; Lin, Q.; Yin, W.; Jiang, T.; (22), 2482-2486.
Zhao, D.; Jiang, L. Sens. and Actuators 14. Chen, J.; Deng, S. Z.; Xu, N. S.; Zhang,
B: Chem. 2017, 253, 1087-1095. W.; Wen, X.; Yang, S. Appl. Phys. Lett.
10. Kang, X.; Mai, Z.; Zou, X.; Cai, P.; Mo, 2003, 83 (4), 746-748.
J. Anal. Biochem. 2007, 363 (1), 143- 15. Umar, A.; Rahman, M. M.; Al-Hajry,
150. A.; Hahn, Y. B. Electrochem. Commun.
11. J. Anal. Biochem. 2007, 363 (1), 143- 2009, 11 (2), 278-281.
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12. Ojani, R.; Raoof, J. B.; Fathi, S.
Electroanalysis 2008, 20 (16), 1825-
1830.
A polyaniline/pencil lead solid phase microextractor
for trace phthalate esters
Nichapat Chunin1,3, Morakot Kaewpet1,3, Panwadee Wattanasin1,3, Proespichaya
Kanatharana1,3, Panote Thavarungkul2,3, Chongdee Thammakhet-Buranachai1,3*
1
2
Department of Physics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
3
Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai,
Songkhla 90112, Thailand
3
*E-mail: chongdee.t@psu.ac.th
Abstract:
A solid-phase microextraction (SPME) fiber was fabricated by electrodeposition of
silver-incorporated polyaniline film on a pencil lead core. This fiber was used to extract trace
phthalate esters and later thermally desorbed at an injection port of a gas chromatograph
coupled with an electron capture detector (GC-ECD). Under the optimum conditions,
linearity of two phthalate esters, benzyl butyl phthalate (BBP) and bis(2-ethylhexyl) phthalate
(DEHP) were in the range of 0.05-5.00 mg L-1 and 0.25-5.00 mg L-1 with a coefficient of
determination (R2) greater than 0.99. Limits of detection of 0.030 and 0.24 mg L-1 and limits
of quantification of 0.10 and 0.79 mg L-1 were obtained for BBP and DEHP, respectively.
The developed method was utilized for the simultaneous extraction of spiked BBP and DEHP
in deionized water (1.0 mg L-1). A high preconcentration factor and satisfactory recoveries
were achieved. This study indicated that the developed polyaniline/pencil lead SPME fiber
can be used to extract BBP and DEHP. Further optimization of the extraction and thermal
desorption conditions would make this sorbent applicable for the determination of phthalate
esters contaminated in food and environmental samples.
1. Introduction electron capture detector (GC-ECD) has

Phthalate esters (PAEs) are been recommended by the United States
plasticizers, a class of compounds that can Environmental Protection Agency as a
increase the flexibility of plastics through standard method.5,6 However, the amount of
weak secondary molecular interactions with PAEs contaminated in the samples is in
polymer chains. Since there is no covalent trace level and the direct injection of the
bond between PAEs and plastics, they can sample is not possible due to the complex
easily leach from the plastic containers into sample matrix. Hence, sample preparation
the food that come into contact with.1,2 steps are needed to preconcentrate and
When exposed to PAEs in high level, it reduce the effect of interference in the
poses an effect to human body such as sample.7
vertigo, nausea and vomit. It might also An interesting sample preparation
cause cancer or affect the human technique is solid phase microextraction
reproductive system.3,4 Therefore, the (SPME) due to its simplicity and solvent-
determination of PAEs contamination would free extraction process.8 However,
be beneficial for human health. commercially available SPME devices are
High performance liquid chromato- relatively expensive, fragile, and have low
graphy (HPLC) and gas chromatography sample capacity. This work investigated the
(GC) are the usual analytical techniques for use of pencil lead based SPME fiber to
the analysis of PAEs. GC coupled with an overcome the problems. Pencil lead has
good mechanical rigidity and is very low (18.2 MΏ.cm ELGA, England). All chemicals
cost.9 Surface modification with appropriate used were of analytical grade.
sorbent can be easily performed using an 2.2 Instruments
electrochemical technique. All analyses were performed using a
Conducting polymers are often used gas chromatograph (Shimadzu GC-14B)
as sorbent in many sample preparation equipped with an electron capture detector
techniques, out of which polyaniline (PANI) (ECD) (Kyoto, Japan) and a packed column
is one of the most popular.10 It can be of 3% OV-3 on Supelcoport, 100/120 mesh,
prepared using either chemical 2.1 m × 3.2 mm i.d. (Bellefonte, USA). Data
polymerization or direct electrodeposition processing was carried out with a N2000
onto the surface of the substrate. In addition, Chromatography Data System (Surwit
PANI is particularly suited for the Technology Inc., Hangzhou, China).
adsorption of PAEs because it can form 2.3 Optimization of GC-ECD
interaction and hydrogen bonding Operating conditions of the GC-ECD
with the target analytes.11 However, the system for the analysis of BBP and DEHP
sample loading capacity of PANI is still were optimized, i.e., the carrier gas flow
limited through its film formation and the rate, column, injector and detector
stability of a PANI thin film is somewhat temperatures. The optimization was carried
poor. Nevertheless some recent reports have out by varying one parameter at a time while
mentioned that Ag, MnO2, RuO2 can keeping the others constant. Five
enhance the porosity of PANI film.12,13 This replications of the analysis of 1.0 µL of 1.0
may help increase the surface area and thus, mg L-1 BBP and DEHP working standard
the extraction efficiency. solution were performed for each tested
Here in, we report the development value. The optimum conditions were
of a solid phase microextractor by obtained by considering the best resolution,
electrodepositing silver-incorporated poly- the highest response and the shortest
aniline film onto the surface of a pencil lead analysis time.
for trace phthalate esters detection by GC- 2.4 Preparation of pencil lead SPME fiber
ECD. A pencil lead was immersed in a
mixed solution of 30 mL of conc. H2SO4 and
2. Experiment 3.0 g of KMnO4. It was then washed in
2.1 Materials sequence with 10 mL of H2O2 (30%) (to
Pencil leads (type 2B, diameter reduce Mn species), 10 mL of HCl (to
0.7 mm, length 60 mm) were from A.W. remove Mn(II) ions) and 10 mL of water (to
Faber-Castell (Germany) Ltd. (Thuringia, remove HCl and other ions) and dried at
Germany). Aniline monomer (reagent grade, room temperature.
99.0% purity) was from Sigma-Aldrich (St. The Ag/PANI sorbent was electro-
Louis, Missouri, USA). It was distilled prior deposited onto the pencil lead from a
to use. H2SO4 were from Lab-Scan solution containing 0.2 M aniline and
(Bangkok, Thailand). H2O2 (30%), KMnO4, 0.5 mM AgNO3 in 0.2 M H2SO4. The three-
acetonitrile (HPLC grade) and ethyl acetate electrode system consisted of a pencil lead a
were from Merck (Darmstadt, Germany). working electrode, a platinum wire counter
Di-2-ethylhexyl phthalate (DEHP) (97% electrode, and a Ag/AgCl reference
purity) was from Fluka (Buchs, electrode coupled to a potentiostat (ML 160,
Switzerland). Benzyl butyl phthalate (BBP) AD Instruments, Australia) connected to a
(99% purity) was from Sigma-Aldrich (St. computer. The electrodeposition was
Louis, Missouri, USA). Acetone, ethanol performed with the potential intervals of
and methanol were from Lab-Scan -0.2 to 0.8 V at a scan rate of 10 mV s-1. The
(Bangkok, Thailand). Ultrapure water was morphology of the as-prepared materials
from a maximum ultrapure water system was characterized by scanning electron
microscopy (SEM) (JSM-5800LV, JEOL, Table 1. Optimization of GC-ECD
Massachusetts, USA). The Ag/PANI pencil conditions, investigated and optimum values
lead fiber was conditioned at the GC Parameter Investigated Optimum
values condition
injection port at 180 ºC under a carrier gas Carrier gas flow rate (mL min-1) 20, 30, 40, 50 30
stream for 2 h to remove unreacted residue Column temperature
 Initial temperature (ºC) 210, 220, 230, 240 230
on the fiber before use.  Initial holding time (min) 3, 4, 5, 6 3
2.5 Adsorption and desorption  Ramp rate (ºC min-1) 5, 10, 15, 20 10
procedures  Final temperature (ºC) 270, 280, 290, 300 290
 Final time (min) 2, 3, 4, 5, 6 2
The adsorption procedure was Injector temperature (ºC) 280, 290, 300 300
performed by immersing the pencil lead Detector temperature (ºC) 280, 290, 300 290
2 mL of 1.0 mg L-1 of mixed standard

solution of BBP and DEHP in a 4 mL amber Table 2. Analytical performances of GC-
vial. The mixture was stirred at 2000 rpm ECD for the analysis of BBP and DEHP
Analyte Linear range LOD LOQ
for 30 min at room temperature, during this (mg L-1) (mg L-1) (mg L-1)
time the analytes were adsorbed onto the
BBP 0.05 – 5.0 0.03 0.10
surface of the modified pencil lead fiber.
After that, the analytes were thermally DEHP 0.25 – 5.0 0.24 0.79
desorped at the GC injection port at 300 ºC
under a carrier gas stream for 30 s (Figure 1). 3.2 Characterization of Ag/Polyaniline
film on pencil lead fiber
Morphology
The SEM image of a bare pencil lead
showed a smooth surface (Figure 2a). The
surface became rather rough after it was
immersed in acid solution, just like aged tree
barks (Figure 2b). This suggests that the
oxidation from the mix solution exerts a
severe impact to the pencil lead. When it
Figure 1. Schematic presentation of the was modified with Ag/PANI, a good distributed
extraction and desorption procedures of the porous structure film was observed along the
Ag/PANI pencil lead SPME fiber for BBP pencil lead (Figures 2c and 2d)
and DEHP determination

3.1 Optimization of the GC-ECD operating
conditions
The optimum conditions for the
determination of phthalate esters are
summarized in Table 1. The linear range,
limit of detection (LOD) and limit of
quantification (LOQ) were evaluated by the
IUPAC recommended method, as 3-fold and
10-fold multiples, respectively, of the
standard deviation of the ordinate intercept Figure 2. SEM images: surface morphologies
divided by the slope of regression line of a bare pencil lead at magnification of
(Sa/b). Under these conditions are 100×(a); an etching pencil lead at magnification
summarized in Table 2. of 100×(b); a Ag/PANI deposited on pencil
lead at magnifications of 100×(c) and of
5,000×(d)
Thermal resistance
Thermal desorption at GC injection
port was used in this study, thus, the
thermogravimetric analysis (TGA) was first
performed to investigate the thermal
stability of the prepared fiber. The TGA
result indicated no decomposition of the
deposited Ag/PANI up to 500 ºC thus, this
fiber is suitable for the desorption at high
temperature of the GC injection port.
3.3 Optimization of extraction conditions Figure 4. Effect of desorption time on peak
Adsorption time area of 1.0 mg L-1 BBP and DEHP;
The adsorption time is an important adsorption time of 30 minute
parameter since it would certainly affect the
extraction efficiency of the developed the extraction of DEHP was achieved
SPME fiber. Figure 3 shows the increase of (8-folds) while no enrichment for BBP was
peak area with the adsorption time from 10 observed (Figure 5). This may be because
to 30 minutes. The decrease at a longer time BBP is more polar than DEHP, thus, can
is possibly due to the back stripping of form a stronger interaction with a
analyte from the SPME fiber into the Ag/PANI SPME fiber making it harder to
extracting solution. Thus, 30 minutes was desorb. To improve the enrichment factor
selected for subsequent experiments. for BBP extraction, optimization of other
affecting parameters e.g. stirring rate, salt
addition, would be useful.
Figure 5. Chromatograms of (a) 1.0 mg L-1

Figure 3. Effect of adsorption time on peak standard of BBP and DEHP (b) 1.0 mg L-1
area of 1.0 mg L-1 BBP and DEHP; standard of BBP and DEHP extracted with
desorption time of 40 second the developed SPME fiber and thermally
desorbed at the GC injection port
Desorption time
The effect of desorption time is 3.4 Reusability
shown in Figure 4. Initially the peak area A single fiber was used for the repeat
increased with time, then became stable extraction of 1.0 mg L-1 mixed standard
after 30 seconds. That is, all adsorbed solution of the two phthalate esters. After
analytes were completely desorbed from the the first extraction, the pencil lead SPME
fiber at 30 seconds. fiber was washed in acetonitrile and DI
Under the optimum conditions, the water, at a stirring rate of 900 rpm for 10
prepared Ag/PANI pencil lead SPME fiber min before being reused. Relative peak area
was used for the extraction the BBP and of subsequent extraction cycles were
DEHP from the spiked standard solution in compared to the first one which was set as
deionized water. High enrichment factor for 100%.
Figure 6 shows that one fiber can be Acknowledgements
used up to 12 times) with average% This work was supported by Prince
responses of 97.8 ± 3.0 (RSD = 3.1%) and of Songkla University granting number of
98.6 ± 2.3 (RSD = 2.3%) for BBP and SCI610440S, Scholarship for Outstanding
DEHP, respectively. These values were GPA, The Center of Excellent for
much better than the accepted RSD Innovation in Chemistry (PERCH–CIC),
recommended by AOAC (11% at analyte Trace Analysis and Biosensor Research
concentration level of 1,000 ng mL-1).14 Center (TAB-RC), Department of
Beyond these 12 extraction cycles, the Chemistry, Faculty of Science and Graduate
response decreased to less than 90%. This School, Prince of Songkla University, Hat
may be due to structural deformation of the Yai, Thailand.
PANI/Ag and the leaching out of some part
of it from the fiber and it can be observed by References
naked eyes. 1. Jen, J. F.; Liu, T. C. J. Chromatogr. A.
2006, 1130, 28–33.
2. Balafas, D.; Shaw, K. J.; Whitfield, F. B.
Food Chem. 1999, 65, 279–287.
3. Le Noir, M.; Plieva, F.; Hey, T.;
Guieysse, B. J. Chromatogr. A. 2007,
1154, 158-164
4. Guo, Y.; Kannan, K. Anal. Bioanal.
Chem. 2012, 404, 2539–2554.
5. http://www.epa.gov/oppt/existingchemic
als/pubs/actionplans/phthalates_actionpl
an_revised_2012-03-14.pdf.
6. http://www3.epa.gov/epawaste/hazard/te
stmethods/sw846/pdfs/8061a.pdf.
7. Kueseng, P.; Thavarungkul, P.;
Kanatharana, P. J. Environ. Sci. Health
B. 2007, 42, 569–576.
8. Sungkaew, S.; Thammakhet, C.;
Thavarungkul, P.; Kanatharana P. Anal.
Figure 6. Reusability of Ag/PANI pencil Chim. Acta 2010, 664, 49–55.
lead fiber 9. Heydari, A and Gharibi, H. J. Power
Sources 2016, 325, 808–815.
Conclusions 10. Bhadra, S.; Khastgir, D.; Singha, N. K.;
This study shows the possibility and Lee, J. H., Prog. Polym. Sci. 2009, 34,
capability of the prepared Ag/PANI pencil 783-810.
lead SPME fiber for the extraction of BBP 11. Prasad, B.; Srivastava, A.; Pandey, I.;
and DEHP. This cost effective and easy to Tiwari, M. J. Chromatogr. 2013, 912,
prepare Ag/PANI pencil lead SPME fiber 65-74.
was successfully applied for the extraction 12. Kim, J.; Ahn, K.; WonKang, S.;
of phthalate esters. In addition, the prepared SungRoh, J.; HunKwon, S.; YongKim,
fiber can be reused. Further investigation of J. Curr. Appl. Phys. 2012, 12, 160-163.
some extraction parameters would lead to 13. Sawangphruk, M.; Kaewsongpol, T.
further improvement that would make it Materials Letters 2012, 87, 142-145.
more applicable for real sample analysis. 14. http://www.eoma.aoac.org/appf.pdf
Synthesis of carbon nanotubes as a sorbent for solid-phase extraction of
formaldehyde in water sample
Patthama Yenanong, Netnapit Kaewchuay*
Department of Chemistry, Faculty of Science and Technology, Rajamangala University of Technology
Thanyaburi, Thanyaburi, Pathumthani 12110, Thailand
*E-mail: netnapit_k@rmutt.ac.th
Abstract:
In this work, to study on synthesis of carbon nanotubes as a sorbent for solid-phase
extraction of formaldehyde in water sample with UV-Vis spectrophotometer. The important
parameters influencing the extraction efficiency such as type and volume of eluent, flow rates
of sample loading and eluent, volume of water samples were optimized. Under the optimum
conditions, good linearity was obtained for formaldehyde (r2 > 0.999). The relative standard
deviation of the repeatability and reproducibility were 2.31 and 3.57% (n=9), respectively.
The limits of detection and quantification were 0.027 mg L-1 and 0.089 mg L-1, respectively.
The proposed method was successfully applied to the analysis of market wastewater samples,
and the recoveries for formaldehyde were in the range of 69-87%.
1. Introduction is the extraction time and demanding in

Formaldehyde is used as a terms of the volume of solvent. Solid-phase
disinfectant and prevent spoilage. The microextraction (SPME) is expensive and
medical used formaldehyde make the body need to be imported from foreign. Solid-
not rot, kill germs, and clean the patient phase extraction (SPE) is the most frequent
room. Due to properties anti-spoil and the method used for rapid and selective sample
prier, so there are entrepreneur used in the preparation because rapid phase separation,
prevention of spoilage of fresh food and low consumption of organic solvent and low
there is the dumped of formaldehyde into cost.3
the contamination environment. However, In SPE, C18 is one of the most
the study of the effects of exposure to frequently used sorbents. At the present,
formaldehyde in the body shows that this carbon nanotube (CNTs) are novel and
compound sensor pathological changes in interesting nanomaterials to develop solid-
various organs, especially the respiratory, phase extraction because their large surface
immune, skin, gastrointestinal, nervous, and area to volume ratios and high affinity,
reproductive system. Formaldehyde very CNTs can be considered as excellent
toxic to human health and life.1 Standards sorbents for SPE. These properties can
for industrial wastewater discharge of application for the detection and remediation
Thailand controlled concentration of different contamination and water
formaldehyde is less than 1 mgL-1 in treatment. The use of CNTs as absorbent in
wastewater.2 The analytical for SPE in aqueous environments.4-6 It is also
formaldehyde in wastewater have a problem show that MWCNTs for SPE determination
at the low concentration and the carbamate insecticide from environmental
complexities of their matrices are important. waters.7 To develop determination of 16
Thus, it must have sample preparation PAHs in surface water by a novel flow
technique for clean-up and pre-concentration injection solid-phase extraction using a
before analysis. Many of sample preparation MWCNTs packed micro column.8 Using
technique such as liquid-liquid extraction CNT-packed cartridge for SPE determination
(LLE), solid-phase extraction (SPE), solid- of atrazine and simazine in water sample by
phase microextraction (SPME). While LLE HPLC analysis.9 Determination a group of
seven pesticides in mineral water by of CVD was set at temperature of 800oC,
electrophoresis with UV detection was synthesis time was 60 min and flow rate of
combined with SPE using MWCNTs10 and liquefied petroleum gas (LPG) source was
salicylic acid from river water.11 45 mL min-1. Synthesized CNTs was
In this work, synthesis of multi- characteristic by SEM (Scanning Electron
walled carbon nanotube on steel wire were Microscope).
used as a sorbent in SPE cartridge for 2.4 SPE procedure
extraction and preconcentration of CNTs packed cartridge was prepared
formaldehyde. To the best of knowledge, the by placing 30 mg of CNTs in a
application of multi-walled carbon nanotube polypropylene tube block by two
as SPE sorbent and analysis with UV-vis polypropylene frits. Prior to extraction, the
spectrophotometer has not been reported. column was conditioned with 5 mL of
The SPE conditions (such as type and hexane, ethyl acetate, and methanol,
volume of eluent, flow rates of sample respectively. In adsorption step, loading 10
loading and eluent, volume of water mL of standard formaldehyde (1 mg L-1)
samples) were studied and optimized for the was flow through the column at a flow rate
analysis formaldehyde from water samples. of 1 mL min-1 with vacuum pump. In the
elution step, was used 5 mL of methanol.
2. Materials and Methods Finally, the eluate solution was added
2.1 Chemicals and reagents Nash’reagent, and then analyzed by UV-vis
Ethyl acetate, hexane were spectrophotometer (The wavelenght at 413
purchased from Labscan Limited (Thailand). nm) with external standard method. The
Buthanol, ethanol AR. grade were purchased parameter were studied such as flow rate of
from Ajax Finechem Pty Limited (Australia). sample and eluent, type and volume of
Ammonium acetate, acetic acid, acetyl eluent, volume of sample and amount of
acetone AR. grade from Merk. The water CNTs.
used in the wok was deionized water. The
stock standard solution (1000 mg L-1) of 3. Results and Discussion
formaldehyde was prepared by appropriate 3.1 Scanning electron microscopy (SEM)
dilution of a 35-40% formaldehyde from Image
RCI Labscan Limited (Thailand) and stored As show in the SEM image of CNTs
in the refrigerator at 4 oC. Before analysis, synthesized (Figure1). It presents multiwall
formaldehyde is derivatives in situ with 1.5 carbon nanotube (MWCNTs) with the
mM pentafluorophenyl hydrazine (from diameter ranged from 200-500 nm was
Aldrich). After SPE procedure added Nash’s studied.
reagent in formaldehyde solution before
analysis using UV-vis spectrophotometer.
2.2 Apparatus
The spectrophotometer was carried
out with a Lambada 35 UV-vis spectro-
photometer (Perkin Elmer, USA). The
following instrumental parameters were
chosen, analytical wavelength 413 nm. The
vacuum pump and a polypropylene tube
were used in the SPE process.
2.3 Synthesis carbon nanotube (CNTs)
Synthesis carbon nanotube (CNTs) by
Chemical Vapor Deposition (CVD) method12
directly on steel wire substrate and Fe2O3 Figure 1. The SEM image of CNTs
were used as catalyst. Method conditioned (diameter between 200-500 nm)
3.2 Optimization of enrichment SPE 3.2.3 Type of eluent
condition Different type of eluent for the
Studied six factors, amount of CNTs, desorption standard formaldehyde (1 mg L-1).
flow rate of sample loading and eluent, type Methanol, ethanol, and the mixture of
and volume of eluent, and volume of water methanol: H2O (50:50) were determined. As
samples. The SPE condition was realized shown in Figure 3, the efficiency of each
following a step-by-step approach, since solvent in the elution of the formaldehyde.
SPE procedure was found for the extraction Among the three of solvent tested, methanol
of formaldehyde with CNTs as sorbent provided the highest desorption values
materials. (100%). The mixture of methanol:H2O
3.2.1 Amount of CNTs (50:50) fraction showed the experimental
The effect of CNTs amounts were result at 80%, it cannot completely elution.
optimized. For this purpose, 20, 30, 40, 50 For ethanol; it cannot be elution of
mg of CNTs were packed in each SPE formaldehyde.
cartridge. After loading the standard
formaldehyde (1 mg.L-1) 10 ml pass through
SPE cartridge and the eluate fraction were
kept analysis with UV-vis spectrophoto-
meter. At the results, 30 mg of CNTs was
chosen because it was obtained with the
good recovery (100%).
3.2.2 Flow rate of sample
The sample flow rate during the
loading step is crucial factor that affects the
performance of SPE cartridge. The effect of
the sample flow rate was studied at 1 and 2
mLmin-1, as show in Figure 2. It was found
that a sample flow rate at 1 mL.min-1 was Figure 3. Effect of type of eluent;
efficiency adsorption 100% of standard concentration of standard formaldehyde 1
formaldehyde (1 mg.L-1). It is possible that mg L-1, each eluent volume: 10 mL
the flow rate at 2 mL min-1 is faster, so
formaldehyde cannot be absorbed on CNTs 3.2.4 Volume of eluent
sorbent completely. In the effect the volume of eluent
were designed based on applying different
eluent volumes of 3, 4, 5 and 6 mL. As show
in Figure 4, in order to achieve complete
elution of standard formaldehyde (1 mg L-1)
and preconcentration. It was found that 5
mL of methanol was the optimum volume.
3.2.5 Flow rate of eluent
The effect of eluent flow rate on
desorption of standard formaldehyde
(1 mg L-1) from the CNTs sorbent was
studied 1 and 2 mL min-1. As the results
shown in Figure 5, the flow rate of eluent 1
mL min-1 efficiency desorption of standard
Figure 2. Effect of flow rate of sample; formaldehyde (1 mg L-1). Thus was choose
concentration of standard formaldehyde 1 flow rate of eluent at 1 mL min-1.
mg L-1, 10 mL, eluent volume: 10 mL of
methanol
linearity, a correlation coefficient (R2) was
0.999. The LOD was 0.027 mg L-1 and LOQ
was 0.089 mg L-1. The developed method
was applied to the wastewater of squid and
salted fish from the market in Angthong
Province. The recoveries of formaldehyde in
wastewater samples with the spiked
concentration of 0.5 and 1.0 mg L-1
formaldehyde were in the accepted in the
range of 69 - 86%.
Figure 4. Effect of eluent volume;

concentration of standard formaldehyde 1
mg L-1; eluent volume: methanol
Figure 6. Effect sample volume of sample;

concentration of standard formaldehyde 1
mg L-1; eluent volume: 5 mL of methanol
4. Conclusion
In this work, MWCNTs synthesized
Figure 5. Effect of flow rate of eluent. from steel wire showed excellent adsorption
Concentration of standard formaldehyde 1 properties as SPE adsorbents for the
mgL-1; eluent volume: 5 mL of methanol preconcentration of formaldehyde in water
samples. The obtained results showed that
3.2.6 Volume of sample this method is a sensitive, especially it is
For sample volume of 50, 100, 200 cheap. This method could be future use for
mL containing the same amount of standard determination of organic pollutants in
formaldehyde (1 mg L-1) were passed to the environmental water.
SPE procedure. The experimental results
showed in Figure 6, in order to breakthrough Acknowledgements
volumes and shorten analysis time, a sample I would like to thank Asst. Prof.
volume of 50 mL was adopted throughout Netnapit Kaewchuay, Ph. D. Department of
this work. Chemistry, Faculty of Science and
3.2.7 Analytical performance of the Technology, Rajamangala University of
method Technology.Thanyaburi.
Precision, linearity, limit of detection, and
limit of were investigated under the References
quantification chosen experimental condition. 1. International Agency for Research on
The relative standard deviation (%RSD, Cancer (IRAC) 2006, 39-325.
n=9) of the intraday and interday were 2. php.diw.go.th/safety/wpcontent/uploads/
2.31% and 3.57%, respectively. The good 2015/01/formaldehyde.pdf
3. Soylak, M.; Ercan, O.; Haz, J. Mat. 9. DeMartino, A.; Lorio, M.; Xing, B.;
2011, 1689, 1527-1530. Carpasso, R. RSC Adv. 2012, 2, 5693-
4. Salam, M. J. Environ. Sci. Technol. 5700.
2013, 10, 677-688. 10. Ramos, M. A.; Borges, J. H.; Perez-
5. Salch, T. A. App. Surf. Sci. 2011, 257, Delgado, M. A. Electrophoresis 2008,
7746-7751. 29, 4412-4421.
6. Saleh, T. A.; Gupta, V. K. J. Colloid 11. Caballero-Diaz, E.; Valcarcel, M. J. Sep.
Intrface. Sci. 2012, 211, 93-101. Sci. 2014, 37, 434-39.
7. Song, X. Y.; Shi, Chen Y. P. Food 12. Sub-udom, P. Master Degree Thesis,
Chem. 2013, 139, 246-252. Rajamangala University of Technology
8. Hong, W.; Xuecui, W.; Bing, L.; Jie, L.; Thanyaburi, 2014.
Baaixiang, D.; Luxin, Z.; Jingjing, J.;
Quaoyu, Y.; Baoping, H. J. Chromatogr.
2010, 1217, 2911-2917.
Hydrophilic interaction liquid chromatography (HILIC) with diode array
detection for determination of phenylalanine and tyrosine
in dietary supplements
Woraphot Wanichalanant1,2, Warawut Tiyapongpattana1,2,3*
1
Innovative Green Chemistry Research Centre, Faculty of Science and Technology, Thammasat University
Rangsit Centre, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand
2
Department of Chemistry, Faculty of Science and Technology, Thammasat University Rangsit Centre,
Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand
3
Flow Innovation Research for Science and Technology Laboratories (FIRST Labs)
*E-mail: twarawut@tu.ac.th, twarawut@gmail.com
Abstract:
A hydrophilic interaction liquid chromatography (HILIC) with photodiode array
detection was developed for the analysis of phenylalanine and tyrosine contents in dietary
supplements. The chromatographic conditions were optimized using HILIC column (3.0 mm
x 150 mm x 3 µm) with detection wavelength at 210 and 225 nm for determining
phenylalanine and tyrosine, respectively. The mobile phase consisted of 50 mM ammonium
formate and acetonitrile with isocratic elution at a flow rate of 0.8 mL min-1. Under the
optimal condition, the calibration curves of the method ranged from 2 to 500 mg L-1 and 1 to
500 mg L-1 with the determination coefficient (r2) of 0.9998 and 0.9996 for phenylalanine and
tyrosine, respectively. Limit of detection (LOD) and limit of quantitation (LOQ) were
sufficient for quantitative analysis of the analytes in dietary supplement samples. The
proposed method gave satisfactory precision and accuracy with the short analysis time within
3.5 minutes. This method was successfully applied to determine phenylalanine and tyrosine
content in the multi-nutrients dietary supplements.
1. Introduction Analysis of Phe, Tyr and/or other

Amino acids (AAs) are important amino acids in food and supplement are
biomolecules which participate in a number usually measured by HPLC methods. Ion-
of processes such as neurotransmitter exchange chromatography (IEC)1,2 and
transport and biosynthesis, being precursors reversed phase HPLC methods varying of
of proteins. Phenylalanine (Phe) is an both stationary phase and mobile phase as
essential amino acid and the precursor of well as the use of an alternate detector such
tyrosine (Tyr). Their structures are quite as a mass spectrometer (MS) have been
similar (the presence of a phenolic attempted.3 The use of ion pairing reagents4
hydroxide in tyrosine structure). In human, and pre-column derivatization5,6 were also
both amino acids play important roles in its reported for some methods. However, those
transformations lead to various bio- methods have required multiple steps and
molecules: dopamine (a neuro-transmitter), expensive equipment.
norepinephrine and epinephrine (hormones), New alternative method for selective
melanin (a pigment responsible for color separation of the polar underivatized
skin, eyes and hair) and thyroid hormones.1 analytes such as amino acids can be
Because they have many biological accomplished by using hydrophilic interaction
significances, Phe and Tyr as well as other liquid chromatography (HILIC). Using low
amino acids are important in nutrition and UV cutoff solvent (acetonitrile rich-mobile
commonly used in nutritional supplements phase) is an advantage of HILIC method
and food technology. which allows for the spectrophotometric
detection of free amino acids at low Multi-nutrient (Phe, Tyr with other
wavelength.7 nutrients such as vitamins and plant extracts)
The objective of this work is to dietary supplements were purchased from
develop and validate a HILIC method for NOW FOODS (Illinois, USA). The samples
the simultaneous rapid determination of Phe were kept at room temperature and dry
and Tyr without derivatization in multi- place.
nutrient dietary supplements. Twenty capsules of the dietary
supplement were unwrapped and mixed the
2. Materials and Methods sample powder together. 0.0500 g of the
2.1 Chemicals and reagents powder was accurately weighed and
L-phenylalanine, L-tyrosine and extracted in 25.00 mL of 0.1 M HCl. A
ammonium formate were purchased from solution was vortexed for three minutes and
Sigma-Aldich Company (St. Louis, MO, diluted five times with acetonitrile. Then,
USA). Acetonitrile HPLC grade and the sample solution was filtrated with 0.2
hydrochloric acid (HCl) 37% were obtained µm nylon membrane filter and injected into
from Lab-Scan (Bangkok, Thailand). Formic the chromatographic system for analysis.
acid from Fisher Scientific UK
(Loughborough, UK). All the others 3. Results and Discussion
chemicals were analytical grade. 3.1 Sample preparation
Phenylalanine and tyrosine standard The optimal conditions for the
solutions were prepared in 0.1 M hydro sample preparation were carried out by
chloric acid and stored at 4 °C until used. investigating peak area of the analytes at the
2.2 Instrumentation detection wavelength of 210 nm and 225 nm
Chromatographic analysis was for Phe and Tyr, respectively. A parameter
performed with an ultra-high performance was varied while keeping the others fixed in
liquid chromatograph (Shimadzu, Japan) order to achieve high sensitivity. Various
model Nexera, LC-30AD solvent delivery parameters affecting to extraction efficiency
unit, SIL-30AC autosampler, CTO-20AC such as extraction solvent, agitation method
column oven and SPD-M20A diode array and time were studied.
detector (DAD). LabSolution software 3.1.1 Extraction solvent
(Version 5.81 SP1) was used for instrument The effectiveness of various
control, data acquisition and data extraction solvents was tested. 25.00 mL of
processing. different extraction solvents, as following
2.3 Chromatographic method 0.1 M HCl, DI water, 50 mM ammonium
Chromatographic separation of Phe formate pH 3.0 and the mobile phase were
and Tyr was carried out by isocratic elution added to extract 0.1000 g of the sample
using GL Sciences (Tokyo, Japan) Inertsil powder and then sonicated for 30 minutes.
HILIC column (3.0 mm x 150 mm x 3 µm) Figure 1 showed the comparison of
with HILIC guard column (3.0 mm x 10 mm extraction solvent efficiency of Phe and Tyr
x 3 µm) at 30 ºC. The mobile phase in the sample. The extracted amount of Phe
consisted of 80:20 (v/v) acetonitrile / 50 mM obtaining from all extraction solvents was
ammonium formate pH 3.0. The flow rate not significant difference. However, 0.1 M
was 0.8 mL min-1. Injection volume was 2 HCl gave the highest extraction efficiency of
µL. Under these conditions, Phe and Tyr Tyr because Tyr can be soluble in lower
were monitored by DAD at the wavelength (less than pH 3) and higher pH (more than
of 210 nm and 225 nm, respectively. pH 8) aqueous solution.8 Therefore, 0.1 M
2.4 Sample preparation of multi-nutrient HCl was chosen as an appropriate extraction
dietary supplements solvent.
Figure 1. Effect of different extraction
solvents on Phe and Tyr extraction
efficiency from multi-nutrient dietary
supplement sample (three aliquots and three
replicates of injection for each solvent)
3.1.2 Agitation method and time

Agitation method and time also
affected to extraction efficiency. If agitation
method was inefficient, the analytes would
not be completely extracted to the solvent.
The sample was extracted by 0.1 M HCl and
varied the agitation method and time as
follows: ultrasonication at 5, 15 and 30
minutes, shaker at 1, 3, 5, 15 and 30 minutes
and vortex at 0.5, 1, 2, 3, 4, 5 minutes.
As shown in Figure 2, three different
agitation methods gave the similar trend of
the Phe results because of its high solubility
in acid solution. The selection was therefore
focused on the extraction of Tyr. Peak area
of Tyr increased along with extraction time
and then kept constant at the same level. In
order to achieve a fast analysis method, the
vortex at three minutes was used in this
work. Figure 2. Effect of different agitation
3.2 Method validation method with varying of extraction time on
The analytical performance was Phe and Tyr extraction efficiency from
presented with the optimal extraction multi-nutrient dietary supplement sample.
condition in order to determine its (A) ultrasonication, (B) shaker and (C)
reliability. Linearity, sensitivity, precision vortex at various times, All of the methods
and accuracy were determined. The results were carried out with three aliquots and
were summarized in Table 1. three replicates of injection.
Table 1. Analytical performance of HILIC method for determination of Phe and Tyr
Linear Determination Intermediate
Linear LOD LOQ Repeatabilitya
Analyte range coefficient precisionb
equation (mg L-1) (mg L-1) (% RSD)
(mg L-1) (r2) (% RSD)
Phe 2.0 – 500.0 y = 3032.3x - 6354.4 0.9998 1.0 2.0 1.18 1.47
Tyr 1.0 – 500.0 y = 3267.3x - 10103 0.9996 0.5 1.0 1.48 2.00
a
10 replications, b10 aliquots (3 replicates of injections)
Figure 3. HILIC chromatograms of (A) Mixture of Phe and Tyr standard at concentration 50
mg L-1, (B) Multi-nutrient dietary supplement sample, (C) Dietary supplement sample
fortified with 50 mg L-1 of Phe and Tyr
The linearity of calibration curve The analytical performance showed

was in the range of 2 to 500 mg L-1 and 1 to that the proposed method was highly precise
500 mg L-1 with the determination and accurate measurement. Moreover,
coefficient (r2) of 0.9998 and 0.9996 for Phe simultaneous analysis of Phe and Tyr was
and Tyr, respectively. The limit of detection achieved within 3.5 minutes.
(LOD) defined as three times signal-to-noise 3.3 Application to sample
(S/N) ratio was 1.0 mg L-1 and 0.5 mg L-1 The developed method was applied
for Phe and Tyr, respectively. Limit of to analyze Phe and Tyr in multi-nutrient
quantification (LOQ) defined as ten times dietary supplements with the difference in
signal-to-noise (S/N) ratio for Phe and Tyr lot number. The HILIC chromatograms of
was 2 mg L-1 and 1 mg L-1, respectively. Phe and Tyr standard mixture, multi-
Repeatability and intermediate nutrients dietary supplement sample and the
precision were used to evaluate the precision fortified sample were shown in Figure 3.
of the method. The assessed %RSD of Phe The results of Phe and Tyr contents in multi-
and Tyr in samples were less than 2.0%. nutrient dietary supplement samples were
The accuracy of the method was shown in Table 2. The comparison of the
evaluated by the recovery of the sample Phe and Tyr contents between the results
(Table 2). Multi-nutrient dietary supplement obtaining from the proposed method and the
sample was fortified with 50 mg L-1 of Phe nutrition facts labeled amount (150 mg of
and Tyr standard solution. The recoveries of Phe and 400 mg of Tyr per one capsule) was
Phe and Tyr in the sample were in the range achieved by t Test. There is no difference in
of 93.96 and 103.05%. the statistical significance at 95%
confidence interval.
Table 2. Concentration of Phe and Tyr in dietary supplement samples and % recovery of
sample spiked standard
Phe contenta Tyr contentb % Recovery of Phe % Recovery of Tyr
Lot number
(Mean ± SD, mg L-1) (Mean ± SD, mg L-1) (%) (%)
A 152.8 ± 3.9 397.9 ± 1.5 100.38 99.92
B 152.5 ± 2.0 403.7 ± 3.6 101.97 93.96
C 151.38 ± 1.8 406.7 ± 4.2 103.05 97.43
a,b
n=3
This method provides a very simple 1. Moreira, M. J. A.; Gando-Ferreira, L. M.
(without derivatization) and rapid extraction Biochem Eng. J. 2012, 67, 231-240.
of simultaneous HILIC separation of Phe 2. Allard, P.; Cowell, L. D.; Zytkovicz, T.
and Tyr. The use of DAD was not only H.; Korson, M. S.; Ampola, M. G. Clin.
confirmed the presence of the analytes for Biochem 2004, 37 (10), 857-862.
qualification analysis, but the detection with 3. Socia, A.; Foley, J. P. J. Chrom. A 2016,
dual wavelength was also achieved a high 1446, 41-49.
sensitivity for quantification analysis. 4. Zoppa, M.; Gallo, L.; Zacchello, F.;
Validation studies demonstrated the satisfied Giordano, G. J. Chrom. B 2006, 831 (1),
precision and accuracy of the method. 267-273.
Consequently, the HILIC method was 5. Fekkes, D.; van Dalen, A.; Edelman, M.;
successfully developed for determination of Voskuilen, A. J. Chrom. B 1995, 669 (2),
Phe and Tyr contents in dietary 177-186.
supplements. 6. Dai, Z.; Wu, Z.; Jia, S.; Wu, G. J. Chrom.
B 2014, 964, 116-127.
Acknowledgements 7. Themelis, T.; Gotti, R.; Gatti, R. J.
The authors gratefully acknowledge Pharm. Biomed. Anal. 2017, 145, 751-
the financial support provided by 757.
Thammasat University under the 8. Hitchcock, D. I. J. Gen. Physiol. 1924, 6
Government budget 2018, Contract No. (6), 747-757.
007/2561 and the partial support provided
by Central Scientific Instrument Center
(CSIC) and Department of Chemistry,
Faculty of Science and Technology,
Thammasat University.
A polyaniline-chitosan bead sorbent for parabens extraction
Titiwan Changsan1,3, Proespichaya Kanatharana1,3, Panote Thavarungkul2,3,
Chongdee Thammakhet-Buranachai1,3*
1
2
Department of Physics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
3
HatYai, Songkhla 90112, Thailand
*E-mail: chongdee.t@psu.ac.th
Abstract:
Polyaniline-chitosan beads were synthesized by incorporating polyaniline powder into
chitosan solution. The mixture was added dropwise into a sodium hydroxide solution to form
beads. These non-toxic and low cost sorbent beads were applied to extract parabens
(methylparaben, ethylparaben, propylparaben and butylparaben) under stirring. The
extractants were analyzed by high performance liquid chromatography. The system provided
good linearity for the four parabens in the range of 0.050 mg L-1 to 60 mg L-1. The limits of
detection and limits of quantitation were 0.025 mg L-1 and 0.075 mg L-1 for methylparaben,
ethylparaben and propylparaben and 0.050 mg L-1 and 0.25 mg L-1 for butylparaben,
respectively. The extraction recoveries were in the range of 68-99% (n=4). The results
indicated that the developed polyaniline-chitosan beads can potentially be useful for the
extraction of parabens in real samples such as cosmetics, food, drinks and drugs.
1. Introduction by the high price and fragile fiber with long

Parabens, a group of alkyl esters of sampling/adsorption times.8,9 An alternative
p-hydroxybenzoic acid, are commonly used interesting approach is micro-solid phase
as preservatives in daily products such as extraction (µ-SPE). It is a relatively simple,
cosmetics, foods, drinks, and drugs.1-3 They fast sampling technique that requires
were found to influence endocrine system minimal amount of solvents, sample, and
and development of human breast cancer.4 sorbent.10, 11
The European Economic Community (EEC) This work aims to develop a µ-SPE
law thus regulated parabens at a maximum technique by incorporating polyaniline
concentration of 0.4% (w/w) each and a total powder into chitosan solution before the
maximum concentration of 0.8% (w/w) in mixture was used to form polymer
cosmetic products.5 microbeads. The interactions between the
In real samples, the matrix is sorbent and parabens are π-π interaction via
complex. Thus, sample preparation the benzene rings of parabens and
procedures are generally required to reduce polyaniline and hydrogen bonding between
interferences and to preconcentrate the the hydroxyl group of parabens and amine
analyte. The commonly used sample group of polyaniline.12 These microbeads
preparation techniques are liquid liquid were applied to extract parabens under
extraction (LLE), solid phase extraction stirring with a vortex mixer. Optimization of
(SPE) and solid phase microextraction the analytical performances and validation
(SPME). However, they have some of the method were carried out before
drawbacks, for example, LLE and SPE are applying for the extraction of the target
time-consuming extraction techniques and parabens. Methyl paraben (MP), ethyl
require large amount of sample and toxic paraben (EP), propyl paraben (PP) and butyl
organic solvent6,7 whereas SPME is limited paraben (BP) were detected since they are
the most widely used and found in real dissolved in 100 mL of ultrapure water was
samples.13-15 added dropwise into 2.0 mL of aniline in
100 mL of 1.0 M HCl under vigorous
2. Materials and Methods stirring. The solution was continuously
2.1 Chemicals and reagents stirred for 2 h and then kept in a refrigerator
MP, EP, PP, BP were from Sigma- for 4 h for the complete polymerization of
Aldrich (St. Louis, Missouri, USA). Stock aniline. The obtained dark green precipitate
parabens standard solutions were prepared was treated with 20 mL of 0.50 M of
in acetonitrile (HPLC grade, Sigma-Aldrich, ammonia for 4 h and cleaned with water
St. Louis, Missouri, USA). Series of before it was dried at 60°C in an oven.
working standard solutions were diluted Chitosan, 2.0 g, was dissolved in 60
from stocks with ultrapure water and stored mL of 5% (v/v) acetic acid aqueous solution
at 4 °C. and stirred at 600 rpm.17 Then, 2.0 g of
Chitosan from shrimp shell (low polyaniline powder was added and stirred at
viscosity, < 200 mPas), aniline monomer 600 rpm to obtain the homogeneous
(reagent grade, ≥99.5% purity) and mixture. This polyaniline-chitosan mixture
ammonium perchlorate were from Sigma- was added dropwise into 300 mL of 0.50 M
Aldrich (St. Louis, Missouri, USA). NaOH solution to form polyaniline-chitosan
Hydrochloric acid, acetic acid, ethyl acetate beads (Figure 1).
and sodium hydroxide were from Merck
(Darmstadt, Germany). Ammonia, acetone
and methanol were from Lab-Scan
(Bangkok, Thailand).
2.2 Instruments
Chromatographic studies were
performed using a high performance liquid
chromatography with an ultraviolet detector
(HPLC-UV). Separation of the four
parabens was carried out using a VerticalTM Figure 1. Photos of the prepared poly-
UPS C18 column (5 μm particle size, 150 aniline-chitosan beads with scale for size
mm × 4.6 mm I.D.) (Vertical Chromato- indication
graphy Co., Ltd, Bangkok, Thailand). Data
acquisition and evaluations were performed 2.5 Adsorption and desorption procedure
using eDAQ data acquisition system with Twenty polyaniline-chitosan beads
PowerChrom software (eDAQ Pty, Australia). were added in 2.0 mL of 0.10 mg L−1 mixed
2.3 Optimization of HPLC system MP, EP, PP and BP standard solution with
Parameters of the HPLC-UV for the in a 5.0 mL glass vial. The extraction was
analysis of MP, EP, PP and BP were performed using a vortex mixer for 30 min
optimized, i.e., detection wavelength, ratio to allow the adsorption of target analytes by
and flow rate of mobile phase. The the polyaniline-chitosan beads. After the
optimization was carried out by varying one solution was rinsed out, 2.0 mL of
parameter at a time while keeping the others acetonitrile was added as the elution solvent.
constant. The optimum conditions were Vortex mixing was then applied for 30 min.
obtained by considering the best resolution, Beads were removed before the extractant
the highest response and the shortest was evaporated to dryness using a rotary
analysis time. evaporator (Buchi, Rotavapor, R-200, Flawi,
2.4 Synthesis of polyaniline-chitosan bead Switzerland) and re-dissolved with 1.0 mL
Polyaniline was first synthesized by of acetonitrile before being analyzed with
a previously reported oxidative polymerization the HPLC-UV system.
method16, i.e., 4.0 g of ammonium persulphate
3. Results and Discussion (S/N ≥ 3) of 0.025 mg L-1 for MP, EP and
3.1 Optimization of the HPLC-UV system PP and 0.050 mg L-1 for BP with the LOQs
The detection wavelength for (S/N ≥ 10) of 0.075 mg L-1 for MP, EP and
parabens determination was varied between PP and 0.25 mg L-1 were obtained. These
250 and 260 nm with a 2 nm interval. The values were lower than the allowable
maximum peak areas of the four compounds maximum concentration in cosmetic
were obtained at 256 nm and this was fixed products.5 Therefore, the developed method
for subsequent investigation. has a great potential to be a useful tool for
The ratio of the mobile phase was the determination of these parabens in real
studied by varying the volume of acetonitrile samples.
from 40% to 80% (v/v in water). 60% was 3.2 Amount of sorbent
found to be the best ratio since it provided The effect of the amount sorbent on
the acceptable peak resolution with the the adsorption efficiency toward target
shortest analysis time. analytes was investigated by varying the
The flow rate of the mobile phase number of the polyaniline-chitosan beads
was another main factor affecting the from 10 to 40 beads. The extraction
column efficiency. It was investigated from recovery only increased slightly (7%) from
0.20 to 1.0 mL min-1. The optimum flow 10 to 20 beads. Further increase number of
rate was achieved by considering the lowest beads did not have significantly effect on the
height equivalent to a theoretical plate extraction efficiency (Figure 3).
(HETP) of a van Deemter plot (the plot Consequently, 20 beads of polyaniline-
between mobile phase flow rate and the chitosan were then used.
HETP). 0.6 mL min-1 was found to be the
optimized value since it provided the lowest
HETP, good peak resolution for the
separation of four parabens (Rs > 1.5) and
the total analysis time was only 10 min. The
chromatogram under the optimum
conditions is shown in Figure 2.
Figure 3. The effect of the number of

polyaniline-chitosan beads on the extraction
efficiency
3.3 Adsorption time and desorption time

The effect of adsorption time was
studied at an interval of 10 min from 10 to
Figure 2. Chromatogram of standard 50 min. When the adsorption time was
mixture of MP, EP, PP and BP at the longer than 20 min, there were no
concentration of 0.50 mg L-1 each under the significantly change of the recovery for EP
optimum conditions and BP and only a small change for PP
(3%). However, there was an obviously
A good linearity in the concentration increase of the MP recovery between 20 and
range of 0.050 mg L-1 to 60 mg L-1 was 30 min (Figure 4). Therefore, 30 min was
obtained for all four parabens. The LODs selected for subsequent experiments.
Figure 4. The effect of adsorption time on
the extraction efficiency of four parabens Figure 6. The effect of desorption solvent
on the extraction efficiency of four parabens
When the desorption time was varied
from 10 to 50 min, the recoveries of the four The recoveries under optimal
parabens increased with the desorption time conditions are shown in Table 1. These
till 30 min. There was no significant change extraction recoveries can still be further
thereafter (Figure 5), therefore, 30 min was improved by investigating additional
chosen. affecting parameters such as the speed of
vortex mixer and the volume of desorption
solvent.
Table 1. Extraction recoveries of four

parabens under the optimal conditions
Analytes %Recovery
MP 98.8 ± 3.8
EP 68.0 ± 7.7
PP 82.9 ± 7.1
BP 90.7 ± 4.8
Figure 5. The effect of desorption time on 4. Conclusion

the extraction efficiency of four parabens Non-toxic and low cost polyaniline-
chitosan sorbent beads were simply
3.4 Type of desorption solvent synthesized. When applied for the extraction
Desorption of the four parabens from of parabens (MP, EP, PP and BP) under
the sorbent beads were studied with stirring by a vortex mixer, relatively good
acetonitrile, ethyl acetate, methanol and recoveries were obtained. Further improvement
acetone, chosen because of their different through additional optimization of affecting
polarities. The results are shown in Figure 6. parameters is highly possible. This study
The highest recoveries were obtained from indicates that the use of these beads as
acetonitrile (polarity index = 5.8) while extracting sorbent is promising.
methanol and acetone (polarity index = 5.1)
provided lower recoveries. The lowest Acknowledgments
recoveries were obtained from ethyl acetate This work was supported by the
(polarity index = 4.4). This might be Faculty of Science Research Fund, Prince of
because polar analytes could not be easily Songkla University, Contract no. 1-2559-02-
desorbed using slightly polar desorption 008. The Center of Excellence for Innovation
solvents. Therefore, acetonitrile was chosen in Chemistry (PERCH–CIC), Trace Analysis
for this experiment. and Biosensor Research Center (TAB-RC);
Department of Chemistry, Faculty of Science Moreda-Pineiro, A. Anal. Chim. Acta.
and Graduate School, Prince of Songkla 2015, 898, 50-9.
University, Hat Yai, Songkhla, Thailand. 8. Sajid, M.; Basheer, C.; Mansha, M. J.
Chromatogr. A. 2016, 1475, 110-115.
References 9. Li, Z.; Wang, X.; Li, L.; Zhang, M.;
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Duran, L. M.; Fernandez, M. F.; Olea, 10. Lashgari, M.; Basheer, C.; Kee Lee, H.
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Cantero, A. M. Sci. Total Environ. 2017, 11. Kanimozhi, S.; Basheer, C.;
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2. Shanmugam, G.; Ramaswamy, B. R.;
Chim. Acta. 2011, 687 (1), 56-60.
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J. 2010, 96 (2), 391-396. A.; Rouhi, F. J. Sep. Sci. 2012, 35 (17),
3. Gao, W.; Gray, N.; Heaton, J.; Smith, 2256-65.
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Chromatogr. A. 2012, 1228, 324-8. Weng, J. R.; Feng, C. H. Food Chem.
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Frankowski, R.; Grzeskowiak, T. Z.; Chen, G.; Li, G.; You, J.
J. Environ. Manage. 2017, 204 (Pt 1), Microchem. J. 2017, 130, 420-427.
50-60. 15. He, S.; Zhao, Y.; Zhu, Z.; Liu, H.; Li,
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J.; Mohammadi, J. Microchem. J. 2017, Song, S.; Nie, D.; Yang, X.; Hou, J.;
130, 64-70. Wu, A. Food Control. 2015, 57, 362-
7. Sanchez-Gonzalez, J.; Tabernero, M. J.; 369.
Bermejo, A. M.; Bermejo-Barrera, P.;
Colorimetric sensor for hydrogen sulfide
Supitcha Thammajinno1,2,3, Chongdee Thammakhet-Buranachai 1,2,3,
Proespichaya Kanatharana1,2,3, Panote Thavarungkul2,3,4*
1
Department of Chemistry, Faculty of Science, Prince of Songkla University,
2
Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University,
3
4
Department of Physics, Faculty of Science, Prince of Songkla University,
*E-mail: panote.t@psu.ac.th
Abstract:
A 96-well plate high throughput quantitative colorimetric test was developed for the
determination of hydrogen sulfide ( H2S) . The color change resulted from the formation of
methylene blue from a reaction between the detection reagent and H2S was detected from a
scanned digital image. The real-time Red-Green-Blue ( RGB) basic color data was obtained
by the Image J program. The relationship between the RGB value and concentration of H2S
provided a wide linear range of 3.0 to 60.0 mg L-1 and a low detection limit of 0.84 mg L-1.
This developed method required only 70 µL of sample and 96 samples can be simultaneously
analyzed. The results showed the potential of the method to be applied as a sensor for H2S.
1. Introduction i.e., electrochemical,11 inductively coupled

Hydrogen sulfide (H2S) is a plasma-atomic emission spectrometry (ICP-
poisonous colorless gas with a smell of AES),12 gas chromatography,13,14 UV-visible
rotten eggs. If a high concentration of H2S absorption spectrometry,15 and fluorescence
was discharged beyond pollution limits, the spectrometry.16 Although gas chromato-
quality of natural water may be affected. graphy and ICP-AES show good resolution
This would have an impact on the ecological and sensitivity, they are costly and often
environment and human health. Pollution of requires complicated procedures. In
groundwater is also possible.1-6 comparison, electrochemical and UV–Vis
According to the Agency for Toxic methods are simpler and less costly,
Substances and Disease Registry, hydrogen however, they suffer from interferences of
sulfide concentrations in surface water are coexisting substances and lower sensitivity.
usually very low because it readily An interesting alternative is colorimetric
evaporates from water. It can also be present technique that can provide fast response and
in groundwater that is generally less than 1 naked eyes detection. In order to reduce time
mg L-1.7 and increase the sample throughput, reaction
In mammals, the normal levels of in a 96-well plate combined with the
H2S is about 0.68-5.44 mg L-1.8 Its abnormal detection by a scanner can be a useful tool
levels are associated with a series of for the colorimetric detection of H2S.
diseases, including diabetes, hypertension, A colorimetric reaction with the
Alzheimer's disease and Down's formation of methylene blue has been
syndrome.9,10 Therefore, the detection of widely used for the determination of
H2S is of great importance. Analytical dissolved sulfide in water since its
methods for hydrogen sulfide determination introduction by Fischer in 1883.17 This
are based on various detection principles, method involves the reaction between H2S
and N,N-dimethyl-p-phenylenediamine in 2.2 RGB capturing system and procedure
the presence of the oxidizing agent, Fe(III) A black box scanner cover (54 cm ×
in hydrochloric acid (Figure 1). The reaction 40 cm × 22 cm) was used to eliminate any
between N,N-dimethyl-p-phenylenediamine effects from environmental light (Figure 2).
and hydrogen sulfide involves a 2:1 The intensity of the color product from the
stoichiometry of reagent to sulfide. Thus, the scanned 96-well plate digital image was
concentration of H2S is directly proportional processed using the Image J program. The
to the formation of methylene blue. This Red, Green, and Blue (RGB) intensities
reaction also shows a high selectivity representing the total the photons in each
towards H2S.4 region of the spectrum were used for
quantification. The analytical RGB data
ranged from 0 to 255 for each channel. The
R, G, B values of 0, 0, 0 correspond to a
black image and 255, 255, 255 a white.20
Figure 1. Reaction between hydrogen

sulfide, N,N-dimethyl-p-phenylenedi-amine
and Fe(III) to form methylene blue18
This work shows the potential of Figure 2 . A 96-well plate high throughput
using a scanned digital image based analysis quantitative colorimetric test using Image J
for a fast and direct quantitative program
determinations of H2S. The color change
caused by the formation of methylene blue 2.3 Measurement protocol
from the aforementioned reaction was Two reagents were required for the
detected through the red-green-blue (RGB) methylene blue method, a solution of 0.025
basic color data obtained by the Image J M of FeCl3 and 0.028 M of N,N-dimethyl-p-
program.19 Results are obtained as phenylenediamine dissolved in 10 mL of
individual RGB values. These values can be 6.0 M HCl. N,N-dimethyl-p-phenylenedi-
exploited to produce a data set using amine and FeCl3 were mixed with H2S (3 to
standards of known concentration. 200 mg L-1) in a 96-well plate for a total
volume of 70 µL with a 1:1:5 (v/v) ratio.
2. Materials and Methods The mixture was incubated for 10 minutes
2.1 Chemicals before the color product was scanned and
All chemicals used in this study were analyzed. The color picker tool of the Image
of analytical grade. Ultrapure water was J was used to evaluate the intensity of the
from a maximum ultrapure water system pictures. The RGB color intensities of the
(18.2 MΏ.cm ELGA, England), it was used cropped area (a 0.50 cm diameter circle)
for the preparation and dilution of all were obtained using the ‘histogram’ tool.
solution. N,N-Dimethyl-p-phenylenediamine The data were transferred to an Excel
was from Sigma-Aldrich (Buchs, (version 2010) spreadsheet for subsequent
Switzerland). Iron(III) chloride hexa-hydrate analysis. The color intensity was converted
were from Sigma-Aldrich (Darmstadt, to the absorbance with an equation reported
Germany). Hydrochloric acid was from by Zareh and coworkers.21
Labscan (Bangkok, Thailand). Sodium
sulfide x-hydrate was from AppliChem
Panreac (Darmstadt, Germany).
3. Results and Discussion 0.60
RED
3.1 The RGB responses 0.55 RED: y =(0.0043±0.0000)x +(0.2986±0.0011) R² = 0.9997
GREEN: y = (0.001±0.0000)x + (0.3205±0.0013) R² = 0.9930
The RGB intensities (IR, IG, and IB) 0.50 BLUE: y = (0.0003±0.0000)x + (0.3254±0.0021) R² = 0.7700
Absorbance
from the image are related to the 0.45
concentrations of hydrogen sulfide as shown 0.40

GREEN
in Figure 3. The RGB intensities decreased 0.35
BLUE
with the increasing hydrogen sulfide 0.30
concentrations indicated the possibility of 0 10 20 30 40
Concentration of H2S (mg L-1)
50 60 70
the application of this method as a H2S Figure 4. Relationship between absorbance

sensor. The red intensity showed the highest of each color and hydrogen sulfide
sensitivity (slope of the calibration curve). concentration
130 Repeatability
0.45
120
IBLUE 0.40
110
IGREEN 0.35
Sensitivity x100
100
0.30
(L mg-1)
Intensity
90
0.25
80 0.20
IBLUE : y = -(0.0691±0.020)x + (120.57±0.60)
70 R² = 0.7608 IRED 0.15
IGREEN : y = -(0.2622±0.012)x + (121.78±0.37)
60
R² = 0.9916
0.10
50 IRED : y = -(0.9466±0.035)x + (125.94±1.08) 0.05
R² = 0.9945 0.00
40
0 10 20 30 40 50 60 70 1 2 3 4 5 6
Figure 3. Relationship between intensity of Figure 5. Repeatability of sensor (n=6) in

each color and hydrogen sulfide red channel
concentration
3.3 Linearity and LOD
The linearity was evaluated from the
The molecular absorption of the
red channel to be between 3.0 and 60.0 mg
products was calculated using the equation
L-1 (Figure 6) with the limit of detection
AX = - log[(Ix-I(x,b)/(I(x,w)-I(x,b)]
(LOD) of 0.84 mg L-1 (calculated as 3.3
where AX = absorbance, IX = intensity, IX,b =
S.D./sensitivity, where S.D. is the standard
0, IX,w = 255.21
deviation of intersect). This LOD is within
As shown in Figure 4, the absorption
the normal range of H2S concentration in
increase with the H2S concentration. The
human serum sample (0.68 and 5.44 mg L-
blue products from the reaction 1 8
). Thus, it is possible to apply this method
demonstrated a greater absorption of red
for the determination of hydrogen sulfide in
light than green and blue. The spectral
human serum. To be able to detect H2S in
responsivity of the red channel was typically
ground water sample (less than 1 mg L-1),7
in the range of 580 to 700 nm.22,23 These
the LOD need to be improved. Further
results agreed well with previous studies that
optimization may induce the mole ratio
reported the 𝞴max of methylene blue product
between N,N-dimethyl-p-phenylenediamine
at 670 nm.24
and Fe(III) and incubation time.
3.2 Repeatability
The repeatability of this sensor was 0.60
investigated by preparing six replications of 0.55
calibration curve of the red channel between 0.50
3 and 60 mg L-1 (Figure 5). The sensitivities

Absorbance
0.45
of the H2S were between 0.35±0.01 and 0.40 RED: y =(0.0043±0.0000)x +(0.2986±0.0011)
0.38±0.01 L mg-1, indicated a good 0.35
R² = 0.9997
repeatability. 0.30
0 10 20 30 40 50 60 70
Figure 6. Calibration plot showing the

linearity of H2S between 3.0 and 60 mg L-1
4. Conclusion B. Am. J. Med. Genet. Part A 2003,
This study showed the possibility of 116A, 310–311.
using a scanner captured image from the 11. Savizi, I.; Kariminia, H.; Ghadiri, M.;
reaction in a 96-well plate to detect H2S. Azad, R. Biosens. Bioelectron. 2012,
The proposed sensor offers the advantages 35,297–301.
of simplicity, rapidity, low cost, portability 12. Colon, M.; Iglesias, M.; Hidalgo, M.;
and high sample throughput. Further studies Todoli, J. J. Anal. At. Spectrom. 2008,
of some affecting parameters would 23, 416–418.
certainly improve the sensitivity and limit of 13. Hyspler, R.; Ticha, A.; Indrova, H.;
detection. Zadek, Z.; Hysplerova, L.; Gasparic, J.;
Churacek, J. J. Chromatogr. B 2002,
Acknowledgements 770, 255-2002.
This work was supported by the 14. Faccenda, A.; Wang, J.; Mutus, B. Anal.
Faculty of Science Research Fund, Prince of Chem. 2012, 84, 5243–5249.  
Songkla University, Contract no. 1-2559-02- 15. Jimenez, D.; Martínez-Manez, R.;
010. Partial support from the Trace Analysis Sancenon, F.; Ros-Lis, J.; Benito, A.;
and Biosensor Research Center (TAB-RC), Soto, J. J. Am. Chem. Soc. 2003, 125,
the Center of Excellence for Innovation in 9000–9001.  
Chemistry (PERCH-CIC), Department of 16. Sasakura, K.; Hanaoka, K.; Shibuya, N.;
Chemistry, Faculty of Science and Graduate Mikami, Y.; Kimura, Y.; Komatsu, T.;
School, Prince of Songkla University, Hat Ueno, T.; Terai, T.; Kimura, H.; Nagano,
Yai, Songkhla, Thailand. T. J. Am. Chem. Soc. 2011, 133, 18003–
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10. Kamoun, P.; Belardinelli, M.; Chabli,
A.; Lallouchi, K.; Chadefaux-Vekemans,
Development of latent fingerprints with Lawsone/PVP on thermal paper
Benjawan Timdee, Gedsirin Eksinitkun*
Forensic Science Program, Department of Chemistry, Faculty of Liberal Arts and Science,
Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom, Thailand
*E-mail: faasgre@ku.ac.th
Abstract:
Lawsone is a naphthoquinone natural product from the leaves of Lawsonia inermis, a
group of compounds that are well known for their reactions with amino acids of keratin.
Also, the lawsone solution can react with latent fingerprint deposits on paper surfaces.
Detection of latent fingerprints on thermal paper is still a problem present in forensic science,
because thermal paper shows background black staining on the heat-sensitive front side;
when its treated with conventional techniques such as ninhydrin in petroleum ether. In this
study, we have reported the first utilization of lawsone with PVP (Polyvinylpyrrolidone) for
the development of latent fingerprints on thermal paper. Compared with the conventional
lawsone solution, the mixed solution of lawsone/PVP in HFE-7100, ethanol, ethyl acetate as
a solvent reduces black background staining on latent fingerprint’s area about 20-30%. The
results of the evidence were clear, and detectable minutiae can be used for visualization and
identification of latent prints; by using an Automated Fingerprint Identification System
(AFIS) more than 30 points of minutiae. In conclusion, this study could help forensic
scientists and individuals in the criminalistics field; to be able to develop latent fingerprints
with non-hazardous materials in a quicker fashion.
1. Introduction detail. The best solution is 0.1g lawsone, 8.5

Various methods have been reported mL ethanol, 0.7 mL ethyl acetate, 0.8 mL
for the development of latent fingerprint. acetic acid in 90 mL HFE 7100.2-5
The Optimal method depends on time and Thermal paper is a special paper that
the surface of a fingerprint object. For has a thermal reactive layer in the paper
example, such as a paper, is a porous surface layers found in everyday life, such as paper
when a skin ridge deposits left at the receipts from department stores or shops,
surface. The sweat compound is quickly paper from the ATM Credit card swiping
absorbed into the surface of the paper. The machine, paper from the press machine,
detection of fingerprints on this type of paper queue, fax, etc.6-9
evidence are usually used chemical solution Detection of latent fingerprints on
such as Ninhydrin solution (2,2- thermal paper is an increasing problem in
dihydroxyindane-1,3-dione), 1,2-Indane- criminalistics. The thermal reactive layer of
dione solution, 1,8-diazafluoren-9-one thermal paper treated with conventional
solution (DFO).1-4 techniques like ninhydrin in petroleum ether
Lawsone is a naphthoquinone natural or DFO solution to instantly turn black. The
product from the leaves of Lawsonia inermis hidden fingerprints are also obscured These
has been used as a hair dye, because methods are not applied to thermal paper.8-10
lawsone reacts with amino acids of keratin In 2013, Ya-Ping Luo reported using a 1,8-
in skin and hair. In 2004, Almog and Diazafluoren-9-one solution (DFO)/
colleagues studied the detection of latent Polyvinylpyrrolidone (PVP) solution to
fingerprints from lawsone extracts. Lawsone detect latent fingerprints on thermal paper. It
solution can also react with the amino acids was found that PVP could reduce black on
of latent fingerprints on the filter paper to the background of the paper and made
show purple/brown impressions of ridge appearance of the fingerprint more clear.10
We are interested in the lawsone solution, 2.2 (but no ethanol) mixed with PVP
which is a natural product containing PVP. solution in ethanol 0.85 mL with different
The optimal ratio of lawsone solution that ratio of PVP: ethanol 50:1, 100:1, 150:1 and
was used, had detected latent fingerprints on 200:1, respectively. To determine the ratio
thermal paper. of lawsone/PVP in ethanol, the results were
The following characteristics were scanned with the HP Deskjet Ink Advantage
found: ratio of lawsone solution was PVP in 1515 All-in-One Printer, and using Dell
ethanol, with the increase in the appearance Latitude AFIS for detectable minutiae.
of latent fingerprints, to reduce the
blackness of the paper background and to 3. Results and Discussion
make sure non-toxic materials were used in 3.1 Study of lawsone solution on thermal
a laboratory setting and the environment.6,8,9 paper
From the Figure 1 show the results
2. Materials and Methods of the preparation of the two lawsone
2.1 Fingerprints preparation solutions, it was found that formula 1
The researcher imprinted the right (without acetic acid) can see the pattern of
thumb on the thermal paper. The pressure is ridge detail fingerprints more clearly than
about 300-400 grams without cleansing, and formulation 2 (with acetic acid) that gives
contact the body with many sebaceous the black surface, which sees lots of lines.
glands. There are three brands of Tesco Compared to the Nihon and One Home &
Lotus, Nihon and One Home & Office, and Office brands that didn’t show any visible
then tested within 24 hours. fingerprints, the Tesco Lotus brand showed
2.2 Study of the condition of lawsone the best results; in terms of visible
solution with thermal paper fingerprints. According to Ya-Ping Luo's
In this study, the lawsone solution 2 research, the acidification of the test solution
formulas was prepared as shown in Table 1. reacted with thermal reactive layer of
Then apply the three brands of thermal thermal paper substrates, as a result the
labels with the fingerprints dipped into each paper turned black.
solution. Let it dry for about 60 seconds. 3.2. Ratios of lawsone / PVP
Repeat 3 times. Observation and Table 2 shows the test results of
comparation with the naked eye for Nihon, Tesco Lotus and One Home &
detectable the pattern of ridge detail Office thermal paper of lawsone working
fingerprints and colour of the background. solution with 0.85 mL of PVP in ethanol at
different ratios of 50:1, 100:1, 150:1 and
Table 1. shows the lawsone solution 2 200:1. From the naked eye observation,
formulas there is a change in the background color of
chemical substance the thermal paper in the area where the
fingerprints are deposited. The PVP
formula 1 formula 2
increased so that fingerprints were not
Lawsone 10 mg. 10 mg.
Ethyl acetate 0.07 ml. 0.07 ml. visible at a ratio of 200:1 PVP in ethanol
Ethanol 0.85 ml. 0.85 ml. solution in Nihon and Tesco Lotus thermal
Acetic acid - 0.08 ml. paper. One Home & Office did not produce
HFE7100 9.08 ml. 9 ml. clear lines. Only the boundary of the
fingerprint is visible. In a 100:1 and 150:1
2.3 Ratio of lawsone / PVP solution ratio, PVP can be seen in almost the same
PVP is well soluble in polar solvents way.
such as ethanol and less soluble in non-polar
solvents. Therefore, the researchers applied
the appropriate lawsone solution from study
(a) (b) (c)
Figure 1. A comparison of latent fingerprints, and the background of Nihon brand (a), Tesco
Lotus brand (b) and One Home & Office brand thermal paper after dipping in. lawsone
working solution without acetic acid (formula 1) (left) and acetic acid (formula 2) (right)
Table 2. Thermal background color of the thermal paper with latent fingerprints, and the
fingerprint of thermal paper 3 brands when tested between lawsone working solution mixed
with PVP solution in different ratios
Brand of Ratio of solution PVP ethanol
thermal paper
50 : 1 100 : 1 150 : 1 200 : 1
Nihon
Tesco lotus
One Home&Office
(a) (b) (c)
Figure 2. The number of minutiaes of Nihon(a), Tesco lotus(b) and One Home and Office(c)
thermal paper of lawsone working solution with 0.85 mL of PVP in ethanol at a ratio of 150:1
Table 3. The number of minutiaes with lawsone/PVP at different ratios with Automated
Fingerprint Identification System (AFIS)
Brand of Ratio of solution PVP ethanol
Thermal paper
50 : 1 100 : 1 150 : 1 200 : 1
Nihon 22.66 46.00 53.66 14.33
Tesco lotus 11.33 33.00 58.33 14.00
One Home&Office 5.33 7.66 7.66 5.0

The best = (52.81 – 66.00) Good = (39.61 – 52.80) Medium = (26.41 – 39.60) Poor = (13.21 – 26.40)
The poorest (00.00 – 13.20)
3.3 The number of minutiaes of lawsone 4. Conclusion

with PVP in ethanol at different ratios Detection of fingerprints on thermal
The development latent fingerprints paper with lawsone blends with 100:1 and
on 3 brands of thermal papers using a 150:1 acetic acid-free PVP in ethanol yields
scanner HP Deskjet Ink Advantage 1515 passive fingerprints that can be individually
All-in-One Printer, and Dell Latitude AFIS verified. And as an alternative to reducing
for detectable minutiae. From the average the toxicity to humans and the environment,
numbers of minutiae, the great number reduce costs and easy to find.
means the good result. Figure 2 showed the
best results of the number of minutiaes of Acknowledgements
Nihon, Tesco Lotus and One Home & This work was supported by an
Office showed poorer results for PVP in advisor (Gedsirin Eksinitkun) and Central
ethanol at a ratio of 150:1. Table 3 showed Institute of Forensic Science-CIFS. The
the results of the number of minutiaes on author (Benjawan Timdee) gratefully
Nihon, Tesco Lotus and One Home & acknowledges the Kasetsart University,
Office thermal paper of lawsone working Thailand for providing the fellowship to
solution with 0.85 mL of PVP in ethanol at carry out the research work in the Faculty of
different ratios of 50:1, 100:1, 150:1 and Arts and Science Department of Forensic
200:1. The numbers of minutiae used to Science, Kasetsart University, Thailand.
detect fingerprints, The best = (52.81-
66.00), Good = (39.61-52.80), Medium = References
(26.41-39.60), Poor = (13.21-26.40) and the 1. Jasuja, O. P.; Toofany, M. A.; Singh, G.;
poorest (00.00-13.20). Sodhi, G. S. Sci. Justice 2009, 49, 8-11.
2. Jelly, R.; Lewis, S. W.; Lennard, C.; 6. Drochioiu, G.; Sandu, I.; Olteanu, G. I.;
Lim, K. F.; Almog J. Talanta 2010, 82, Mangalagiu, I. IJCJS 1, 37-51.
1717-1723. 7. Jasuja, O. P.; Singh, G. Forensic Sci. Int.
3. Jelly, R.; Patton, L. T. P.; Lennard, C.; 2009, 192, e11, e12
Lewis, S. W.; Lim, K.F. Anal. Chim. 8. Schwarz, L.; Nat, P.; Klenke, I. J.
Acta 2009, 652, 129-140. Forensic Sci. 2010, 55, 1076, 1077-
4. Thomas, P.; Farrugia, K. Sci. Justice 1079.
2013, 53, 315-317. 9. Zhao, Y.; Feng, Y.; Luo, Y. JFSC. 2013,
5. Jelly, R.; Lewis, S. W.; Lennard, C.; 1, 1-3.
Lim, K. F.; Almog, J. RSC Adv. 2008, 10. Luo, Y. P.; Zhao, Y. B.; Liu, S. Forensic
3513, 3514. Sci. Int. 2013, 229, 75-79.
Novel charged iridium(iii) complexes for colorimetric butylamine sensor
Panida Seetawan1, Witsanu Sombat1, Wimonsiri Amornchai2, Kittiya Wongkhan1*
1
Department of Chemistry, Faculty of Science, Ubon Ratchathani University,
Ubon Ratchathani 34190, Thailand
2
Ubon Ratchathani Forensic Police, Ubon Ratchathani 34000, Thailand
*E-mail: kitt_w_2000@yahoo.ie
Abstract:
We reported three charged iridium() complexes (SY-11), (WS-02) and (NU-02) for
butylamine colorimetric sensor. The complexes were synthesized and characterized by 1H
NMR, 13C NMR, MS and IR techniques. Upon the optimized condition, SY-11 and WS-02
changed the solution colors with butylamine detected by the naked eye within 30 minutes and
30 seconds, respectively. The results showed that the reaction times depended on the reactivity
of the electrophilic ligands. We monitored the absorption spectra which the blue-shifted was
observed from amide intermediate complex (KM-10). The preliminary test with amphetamine
(Yaba′) of WS-02 showed a changing color. This result can be applied for amine drugs in
forensic investigation.
1. Introduction
The organic amine bases such as
amphetamine, methamphetamine and heroin
in Figure 1 have increasingly abused and
caused serious health and social problems.
The convenient for the detection of these
amine drugs is chemical color reaction
(CCR).1 However, this method limited with
the less accuracy compared to the other
methods.2,3 A few examples have been
reported as an amine CCR technique.
Recently, we reported the chemical reaction
of charged iridium(III) complex (NU-02) for
the amine sensor.4
It is well known that the
photophysical properties of iridium(III)
complexes are dependent on the chemical
Figure 2. The structures of our charged
data of ancillary ligands.5-9 With this study
iridium(III) complexes
in hand, SY-11 and WS-02 in Figure 2 were
designed and studied for amine sensor.
2.1 General
All reactants and solvents were
purchased from commercial sources and
used without further purification unless
otherwise noted. The synthesis of iridium
Figure 1. The structures of amphetamine, complexes were used with similarly report.10
methamphetamine and heroin 1
H and 13C NMR spectra were recorded on a
Bruker ARX-300 spectrometer. Molecular
weight was obtained using a Bruker J = 7.8 Hz, 1H), 7.37 – 7.25 (m, 1H), 7.06
MicrOTOF-Q II. Infrared spectra were (dd, J = 12.1, 5.0 Hz, 1H), 7.00 (t, J= 7.5
measured on a Perkin Elmer spectrum RXI Hz, 1H), 6.94 – 6.75 (m, 2H), 6.23 (d, J =
spectrometer. UV-visible absorption was 7.5 Hz, 1H), 3.34 (dd, J = 13.3, 6.6 Hz, 2H),
studied with Perkin Elmer spectro- 1.58 (dt, J = 15.0, 6.6 Hz, 2H), 1.46 – 1.19
photometer (model LS50). The chemical (m, 5H), 0.89 (dd, J = 15.6, 8.2 Hz, 4H); 13C
reaction was investigated by iridium(III) NMR (75 MHz, CDCl3) δ166.8, 164.9,
complexes with 3 eq. buthylamine in the 154.8, 150.4, 149.6, 147.8, 143.4, 138.1,
excess HCl condition. 137.8, 131.8, 130.7, 127.2, 124.6, 123.5,
2.2 Molecular characterization 122.7, 119.4, 40.5, 31.0, 29.6, 20.1, 13.0;
[(Dimethyl 2,2'-bipyridine 3,3'di- ATR-FTIR (neat) 3428, 3063, 2923, 1661,
carboxylate)-bis-(phenyl-4-trifluorome- 1607, 1529, 1478 and 1312 cm-1; MS
thylpyridine)iridium(III)] hexafluoro- (ESI ) m/z calcd for C42H42IrN6O2 (M+-PF6)
+
phosphate (SY-11) was obtained as a light 855.2998, found 855.2889.

orange solid (72%); 1H NMR (300 MHz, [(Dimethyl 2,2’-bipyridine-3,3’-
acetone-D6) δ 8.57 (d, J= 14.3 Hz, 6H), 8.29 dicarboxylate)-bis-(2-(phenyl)-pyridine-
(t, J= 5.2 Hz, 2H), 8.15 (d, J= 8.0 Hz, 2H), C2’,N)-iridium(III)] hexafluorophosphate
7.83 (s, 2H), 7.50 (d, J= 6.0 Hz, 2H), 7.12 (t, (NU-02) was obtained as an orange solid (90
J= 7.3 Hz, 2H), 7.00 (t, J= 7.4 Hz, 2H), 6.37 %); 1H NMR (300 MHz, CDCl3) δ 8.37 (s,
(d, J= 7.5 Hz, 2H), 3.88 (s, 6H); MS (ESI+, 1H), 8.11 (s, 2H), 7.89 (d, J = 7.5 Hz, 1H),
m/z) calcd for C42H42IrN6O2 (M+-PF6) 7.77 (t, J = 7.7 Hz, 1H), 7.66 (d, J = 7.5 Hz,
909.1487, found 909.1556. 1H), 7.49 (s, 1H), 7.19 – 6.97 (m, 2H), 6.90
[(1,10- Phenanthroline-5,6-dione)- (t, J = 7.3 Hz, 1H), 6.22 (d, J = 7.3 Hz, 1H),
bis-(phenyl-4-trifluoromethyl pyridine)- 3.85 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
iridium(III)] hexafluorophosphate (WS- 165.0, 155.9, 151.6, 149.1, 143.3, 139.4,
02) was obtained as a dark brown solid 138.5, 133.1, 131.8, 131.0, 127.8, 124.8,
(60%); 1H NMR (300 MHz, acetone-D6) δ 123.7, 123.0, 119.6, 113.9, 53.6; MS (ESI+,
8.78 (d, J= 7.9 Hz, 1H), 8.61 (s, 2H), 8.33 m/z) calcd for C36H28IrN4O4 773.1740 (M+-
(d, J= 5.1 Hz, 2H), 8.24 (d, J= 6.0 Hz, 2H), PF6), found 773.1668.
8.17 (d, J= 7.7 Hz, 2H), 8.00 (dd, J= 7.9, 5.4
Hz, 2H), 7.43 (d, J= 5.9 Hz, 2H), 7.13 (t, J= 3. Results and Discussion
7.5 Hz, 2H), 7.02 (t, J= 7.3 Hz, 2H), 6.45 (d, 3.1 Synthesis
J= 7.4 Hz, 2H); MS (ESI+, m/z) calcd for The charged iridium(III) complexes
C42H42IrN6O2 (M+-PF6) 847.1120, found were successfully synthesized and
847.1212. characterized as reported in experiment
(N3,N3'-Dibutyl-[2,2'-bipyridine]- section. SY-11, WS-02 and NU-02 were
3,3'-dicarboxamide)-bis-(2-(phenylpyri- obtained in 72% 60% and 90% yields,
dine-C2',N)iridium(III)] hexafluoro- respectively. The molecular are shown in
phosphate (KM-10) was was obtained as a Figure 2.
red-orange solid (86 %); 1H NMR (300 3.2 Chemical reaction study
MHz, CDCl3) δ 8.09 (d, J = 7.8 Hz, 1H), For study the reaction of complexes
7.98 (d, J = 5.3 Hz, 1H), 7.86 (d, J = 7.9 Hz, with butylamine, we monitored the solution
1H), 7.72 (dd, J = 15.9, 7.9 Hz, 1H), 7.64 (d, colors which are shown in Table 1.
Table 1. The pictures of the chemical reaction from NU-02, SY-11 and WS-02 with
buthylamine
Reaction time NU-02 SY-11 WS-02
0 sec
30 sec
600 sec (10 minute)
1800 sec (30 minute)
The solution color of NU-02 showed The results are displayed in Figure 4 which
a slightly changed after 30 minute. The the color solution can be observed by naked
reactive trifluoromethyl groups were eye in 30 seconds.
introduced (SY-11). The result showed that
the orange color was observed within 30
minute. Then, we modified to WS-02 with
very reactive diketone and trifluoromethyl
group. The impressive result was obtained
with buthylamine within 30 seconds which
the solution color changed from colorless to Figure 4. The pictures of WS-02 with
green. The changed photophysical property amphetamine (Yaba') in dichloromethane at
suggested a formation of new chromophore. room temperature; (a) 0 sec (b) 30 sec
The amide formation could be a responsible
from the ester and butylamine. 4. Conclusion
The chemical sensor of n-butylamine
with the iridium(III) complexes was
developed. The reaction color can be
monitored with naked eye within 30 seconds
from WS-02. This study could be a benefit
system to detect various types of narcotic
drugs in the future.
Acknowledgements
The authors would like to
acknowledge the financial support from the
Ubon Ratchathani University under research
Figure 3. Visible absorption spectra of NU- grant NRCT-165264.
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10. Rothe, C.; Chiang, C.; Jankus, V.;
H.; Yang, W. Ca, Y. Synt. Met. 2014, Abdullah, K.; Zeng, X.; Jitchati, R.;
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S.; Xu, Y. Inorg. Chim. Acta 2014, 409,
372-378.
Construction of sensor network on pontoon for monitoring of water quality
in coastal area of Samut Sakorn Province
Paithoon Prasertying1,2*, Khongpan Rungprateepthaworn3, Sommai Chokrung3,
Prachumpong Dangsakul3, Thanika Duangtanoo3, Kamol Kaemarungsri3,
Rachaporn Kienprasit3, Phoonthawee Saetear1,2, Prawpan Inpota1,2,
Thitirat Mantim1,4, Warawut Tiyapongpattana1,5, Nathawut Choengchan1,6,
Kanchana Uraisin1,2, Nuanlaor Ratanawimarnwong1,4, Duangjai Nacapricha1,2
1
Flow Innovation-Research for Science and Technology Laboratories (FIRST Labs.), Thailand
2
Mahidol University, Bangkok 10400, Thailand
3
Embedded System Technology Laboratory, National Electronics and Computer Technology Center (NECTEC),
Prathumthani 10120, Thailand
4
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand
5
Department of Chemistry, Faculty of Science and Technology, Thammasat University,
Pathumthani 10120, Thailand
6
Department of Chemistry and Applied Analytical Chemistry Research Unit, Faculty of Science,
King Mongkut’s Institute of Technology Ladkrabung, Bangkok 10520, Thailand
*E-mail: paithoon.13437@gmail.com
Abstract:
This work presents a development of sensor network on pontoon for monitoring of
water quality in coastal area of Samut Sakorn province. The sensor network consists of 3
units; (1) sensing unit, (2) processing and storage unit, and (3) communication unit. First,
sensing unit established with four commercialized probes (electrical conductivity (EC),
dissolved oxygen (DO), pH and temperature) was located on the floating cube-shaped
pontoon, made of high density polyethylene (HDPE) with anti-UV property. Second,
processing and storage unit collects and storages data obtained from the probes, prior to
transmission of signal to the host receiver. Third, communication unit is wireless
transmission system connected to the host receiver. We designed and installed eight stations
regarding the fisheries area for real-time monitoring the EC, DO, pH and temperature. The
monitored data related to the environmental condition. Regarding the data, it was found that
EC is less than 20 mS/cm with alkaline range (7.5-9.0 pH). DO of 4 ppm and temperature
change in range of 25-32.5 oC were also observed. Maintenance of the probes will also be
discussed. The sensor network would be useful to farmers in term of awareness of quality of
water for coastal fisheries in Samut Sakorn province.
1. Introduction processed food or ready meals. Since Samut

Fishery is a major farming Sakhon is one of the sources of exporting
occupation in Thailand. It is important fishery products monitoring of water quality
economic value. According to a report by is thus needed for maintaining the
the Office of Agricultural Economics, the productivity of the products and the
fishery exportation in 2009 has been economics of the local community.
191,734 million baht, or 19 percent of total Fishery farming in Samut Sakhon
agricultural exportation. Samut Sakhon relies on natural water sources from coastal
province is the area where there have been area such as sea water and canal water
various types of fisheries. Major fishery located nearby the sea. Farming is normally
products of Samut Sakhon province have carried out by digging up to crate ponds.
been shrimp, shellfish and fish exported as Some of the ponds are used for farming
chilled processed food, canned food, frozen mixture of fishery products including fish,
crab, shrimps and shellfish. Some of the Fisheries Research and Development Center
farms focus on only one particular species. regularly go out for field trip to collect water
Each fishery product requires samples and bring back to analyze in
different levels of salinities. Salinity affect laboratory. In order to serve the needs of
the growth of aquatic animals and specify real-time measurement of water quality, we
each species such as the intrabody fluid propose use of pontoon equipped with
when shrimp molt and absorb minerals back sensors immersing in water resources. These
to the body, especially calcium for shrimp sensor signals are collected in wireless
shell. In considerably low level of salinity, format. The data can be stored on the
concentrations of minerals are also low and website reachable by some users such as
are not sufficient for shrimp to utilize the farmers in the local area as well as
mineral to construct its shell.1 pH is an governmental officers work for Samut
indicator of the concentration of hydrogen Sakhon Coastal Fisheries Research and
ions (H+) in water. Changes in the pH of Development Center.
water during the day will affect the growth In this work development of sensor
of aquatic animals. Suitable pH for the network on pontoon for monitoring of water
growth of shrimp is 6-9. If the pH is too quality in coastal area of Samut Sakorn
high or too low, shrimp growth rate will be province is presented. The sensor network
slow and the survival of shrimp is poor.2 consists of 3 units; (1) sensing unit, (2)
Temperature also affects aquatic animals. processing and storage unit, and (3)
Increasing in 1 oC temperature will directly communication unit. First, the sensing was
affect the aquatic animals since the equipped with four commercialized probes
metabolism within the animal body including electrical conductivity (EC),
increases 10 times, resulting in the increases dissolved oxygen (DO), pH and temperature
in food and oxygen demand. Furthermore, sensors. The base of pontoons were made of
temperature affects the activity of organic high density polyethylene (HDPE) floating
degradation of microorganisms and cubes. Secondly, processing and storage unit
dissolved oxygen in water. The appropriate are equipped on the pontoon and securely
water temperature for the growth of aquatic locked in metallic cage. Inside the cage,
animals is 28-33 oC. Some of the sensors is there are electronic circuitry for data
temperature dependent. Thus changes in the collection, storage and wireless
temperature will affect the reading of these communication system for data
sensors. Therefore temperature measurement transmission. In this work, we tested
is necessary for water quality monitoring.3 monitoring of the four parameters for water
Oxygen is important for vital life because all quality as well as investigation of the
kinds of animals need oxygen for growth. At maximum period required for cleaning the
30 ppt salinity and 30 oC of sea water, sensing probes.
dissolved oxygen in water is about 6.39
mg/L. If the water temperature or the 2. Materials and Methods
salinity increases, the amount of dissolved 2.1 Pontoon and commercial probe
oxygen in water decreases. As shown in Figure 1 the circuitry
Since water quality and condition for board are first made and installed into a
aquaculture are strictly important and must plastic box prior fixing inside metallic cage.
be monitored closely, there is needs of real- Above the cage, there is a roof made for
time monitoring particularly in the coastal fixing two solar cell panels for production of
area in which water is pumped from natural the electricity to supply to the circuitry
resources into private fishery farms. For board and the sensors. This housing cage
real-time monitoring in river, canal and in (0.6 m wide × 0.6 m long × 0.5 m tall) has
the sea, wireless sensor network may be a been designed with two side doors that can
good choice. Samut Sakhon Coastal be opened on both sides for convenience in
the maintenance. As shown in Figure 2 each Table 1. Stations of pontoon sensor network
pontoon station is equipped with four launched in Samut Sakorn province
commercialized probes including (1) Site no. Place Coordinate
electrical conductivity (EC), (2) dissolved 1 Pantainorasingh non- N13.48810
hunting area wildlife E100.33025
oxygen (DO), (3) pH sensor and (4)
conservation division
temperature sensor. These sensors are fixed 2 Song canal N13.49878
with a pre-cut circular foam. The foam fits E100.31718
well inside the a polyvinyl chloride tube 3 Pittayalongkorn canal N13.51307
bound on the side of the HDPE floating cube E100.33733
4 Cook Moo restaurant N13.51391
(1 m wide ×1 m long × 0.4 m tall). These
E100.30277
floating cubes have anti-UV property. 5 Samut Sakhon Coastal N13.49529
Fisheries Research and E100.30811
Development Center
6 A house N13.49910
E100.33534
7 Gulf of Thailand N13.46922
E100.33163
8 Pramong district N13.49855
E100.38637
Figure 1. Photograph shows a pontoon

equipped with sensor probes, circuitry box,
housing cage and roof with two solar cell
panels for generation and supply of
electricity.
Figure 3. Map showing locations of eight

stations of sensor network on pontoons
launched in canals and sea near the coast of
Samut Sakorn province.

3.1 Response signal of four commercial
probes
Figure 4 shows example of the
responses from the four sensing probes
Figure 2. Four commercial probes used for
including temperature, pH, electrical
each pontoon including dissolved oxygen
conductivity and dissolved oxygen. These
(DO), pH, electrical conductivity (EC) and
data were collected from site no. 3 for one
temperature from left to right.
month from 13 October to 12 November
2017. During the data collection, these four
2.2 Locations of pontoon station sensing probes were cleaned every week to
Table 1 shows eight locations of the clean off the dirt and some biological
stations of sensor network on pontoons. growth on the sensing area preventing
These eights locations are near the sea of interferences that may lead to error reading
Samut Sakorn province. Figure 3 shows the and wrong analysis of the water condition.
map of these eight locations.
high. Nonetheless, the data from site no. 7
was collected only 5 days due to the damage
of sensors found later after signal missing.
Site no. 7 is located in the sea at
approximately three kilometers away from
the coast. Damage of the sensors was caused
by the wave. This pontoon on site no. 7 was
later brought back to the shore for
maintenance. The rest of other pontoon
sensor networks have been working properly
during the test of one month period. No
damage on sensors were observed indicating
that the design of the pontoon and the
housing of sensor holder (PVC pipe) is
suitable for all canal sites.
3.2 Investigation of requirement for
cleaning frequency
Since the aim of this work was
mainly for remote monitoring of four major
parameters of water quality needed for
fishery farming, we therefore investigate the
minimum time required for sensor
Figure 4. Example of recorded sensors’ maintenance including cleaning of the
signal from temperature, pH, conductivity sensors. We investigated all of the four
and dissolved oxygen probes collected from sensors including DO, EC, pH and
site no.3 from13 October 2560 to 12 temperature probes. These probes were
November 2560. investigated by employing the plan for
prolonging the cleaning period of the probes
According to the results in Figure 4 at the sites for (i) every one week, (ii) every
temperature and pH of water from site no. 3 two weeks and (iii) every three weeks. Z-
did not change much over the period of one score was employed as statistical tool for
month. Nonetheless, there seem to be this evaluation. The average values within
fluctuation in temperature and pH in cycle three days obtained after those three prolong
format. Temperature varied from 25 to 32.5 cleaning periods were compared with the
◦C. Fluctuation of temperature is the cause
average values obtained from the first three
of exposure to sun light during the day and
days after launching the sensors immersing
temperature of air during the night which
into water at the site.
affects the heat transfer. However the Z-score results are shown in Figure
fluctuation of temperature is within the 5. It is observed from the Z-scores that the
appropriate temperature for sea life along values of EC and temperature sensors for the
the coast. pH of water also fluctuate in cycle
period of cleaning every two weeks are
format. pH was in alkaline region from 7.5- outside ±2 region which is the statically
8.5. Conductivity of water fluctuated from acceptable region. After three weeks only
10 to 20 mS/cm. These salinities are still DO sensor gave the response that are still in
suitable for fishery farming in the area the acceptable ±2 region. When cleaning the
depending on the type of animal farming. sensors at the end of two weeks and three
Dissolve oxygen did cycle and fluctuated. weeks, it was observed that there were
Patterning of the fluctuations of these four remarkable accumulation of growth of
parameters are similar with other sites. biofilm and sea scallop on all sensors. From
However the values of conductivity and
these results in Figure 5, it seems that the
dissolved oxygen of site no. 7 were pretty
sensors may tolerate films and sea scallop remove the sea scallop. Thus it is therefore
development within one week without recommended that these sensors should be
cleaning. However at the period of three cleaned every one week in this type of
week, growth of the sea scallop was quite environment.
dominant and took a long time to clean to
Figure 5. Z-score of recorded signal from dissolved oxygen, pH, electrical conductivity and
temperature probes. Collected during 12 November 2560 to 23 December 2017.
4. Conclusion least every week to prevent instability of the

Eight pontoons equipped with four sensing probes.
sensors including DO, pH, EC and
temperature probes were launched into Acknowledgements
canals and sea near the coastal area of Samut Agricultural Research Development
Sakorn province. Firstly, testing of data Agency (Public Organization) is gratefully
collection and transmission via the GPRS acknowledged for providing the generous
system was carried out over a period of one funding to this project. Seeding money from
month. Secondly, the frequency needed for Center of Excellence for Innovation in
cleaning the sensing probes were Chemistry (PERCH-CIC), Commission on
investigated. According to the Z-scores, the Higher Education, Ministry of Education is
probe should be cleaned every week for acknowledged. We would like to
maintaining their performance in these acknowledge kind suggestion of this project
brackish and saline natural water sources. It right at the beginning from Coastal
was clearly observed for all the probes that Aquaculture Research and Development
there was heavily build-up of sea scallop Regional Center 2 (Samut Sakhon). Mr.
after two weeks. Thus for this work, it was Vorapol Dounglomjan and Mr. Narin
decided that the sensors should be cleaned at Bounroum of the Learning Center and
Laboratory Mahachai Rehabilitation Center References
for Natural Resources and Environment, 1. Mevemkamp, L.; Ong, E. Z.; Colen, C.
Samut Sakhon are gratefully acknowledged V.; Vanreusel, A. Mar Environ Res
for contacting and acknowledging local 2018, 133, 32−44.
people of this pontoon sensor network 2. Zlatanovic, L.; Hoek, J. P.; Vreeburg, J.
project. H. G. Water Res 2017, 123, 761–772.
3. Zhang, M.; Sun, Y.; Liu, Y.; Qiao, F.;
Chen, L.; Liu, W. T.; Du, Z.; Li, E.;
Aquaculture 2016, 454, 72–80.
Downscaling ion-association solvent extraction for tetracycline
determination with RGB color detection system
Saiphon Chanpaka1*, Orapan Intharaksa1, Nopbhasinthu Patdhanagul1, Thitikul Boonsri1,
Khotchaporn Chankaew1, Kate Grudpan2,3
1
Department of General Science, Faculty of Science and Engineering, Kasetsart University Chalermphrakiat
Sakon Nakhon Province Campus, Sakhon Nakhon 47000, Thailand
2
Center of Excellence for Innovation in Analytical Science and Technology, Chiang Mai University,
3
Department of Chemistry, Faculty of Science, Chiang Mai University,
*E-mail: saiphon.c@ku.th
Abstract:
Downscaling liquid-liquid micro solvent extraction of tetracyclines based on ion-
association was studied. It is based on the formation of a yellow color ion associate of a
protonated cation of tetracycline with anionic form of organic dye which could be extracted
into dichloromethane. It is performed in microliter scale using RGB color system for direct
detection after phase separation in small PCR tube by compact instrument which could be
connected with the application in mobile phone. The parameters which affected on extraction
efficiency were studied. The method was then applied for determination of tetracyclines in
commercial pharmaceutical samples. It is an alternative way for downscaling of chemical
analysis, cost effective determination method and reducing of solvent consumption and
operating time.
1. Introduction The use of digital images represents

Tetracyclines were discovered in the a current opportunity to develop fast and
1940s and exhibited activity against a wide direct quantitative determinations for
range of microorganisms including gram- analytical chemistry.16-20 In particular,
positive and gram-negative bacteria combined with colorimetric–chromogenic
chlamydiae, mycoplasmas, rickettsiae, and methods that can use RGB data (Red Green
protozoan parasites.1 There are numerous Blue basic color). The values of colour
methodologies have been developed for correspond to the change in colour intensity.
determination of tetracyclines.2-9 Extraction The special current analytical interest are
of tetracycline using anionic dyes for ion those defined as ready-to-use and field
association liquid-liquid extraction has been methods. The availability of these high
reported.10 However, conventional specification cameras at reasonable price
extraction methods have drawback of high makes low cost imaging of colorimetric
consumption of toxic organic solvent. analytical test devices.
Conventional batch wise spectrophotometric This research aims to extraction of
operation, normally involves a relatively tetracyclines based on ion-association
large scale of chemicals used and time reaction in microliter scale, quantitative
consuming. In recent years, the fields of determination by imaging digital analysis
miniaturization in chemical analysis and based on RGB data for direct detection after
cost-effective analysis have gained phase separation by compact instrument
increasing interest. There was a review on which could be connected with the
variety methods for liquid-liquid micro application in mobile phone. Determination
extraction.11-15 of tetracyclines in commercial pharma-
ceutical samples was applied.
2. Materials and Methods 2.3 Extraction procedure
2.1 Chemicals and reagents 100 ppm of tetracycline was pipetted
Tetraclyclines (tetracyclinehydro in 0 to 50 L for each small vial, 40 L 1 x
chloride, oxcytetracycline and 10-2 M was then added and making volume
chlortetracycline, Sigma-Aldrich) (1000 to 100 L by buffer solution and mixed it
ppm) was prepared by 0.1 g of tetraclyclines well. The solution was then pipetted into
dissolved by DI water and adjusted volume PCR tube for 50 L, using of
to 100 mL. dichloromethane as an extraction solvent
Bromophenol blue solution (1 x 10-3 with the ratio between aqueous phase
M) was prepared by dissolving of 0.6908 g solution an organic phase 1:1. Shaking for 1
of bromophenol blue (acid form) in DI minute and waiting for until the solution in
water, 3 mL of 0.1 M NaOH was added and two phases were completely separated. The
then the solution was adjusted to volume of solution was then detected by RGB color
100 mL. absorbance sensor directly without removing
Sodium acetate and phosphate of aqueous phase from organic phase.
solutions were prepared from by dissolving 2.4 Procedure for tablets
proper amounts of (CH3COONa.3H2O, Twenty tablets of 500 and 1000 mg
Carlo Erba, Italy) and (Na2HPO4, BDH, commercial samples of tetracyclines were
Poole, England) by DI water. weighted accurately. The average weight of
Acetate buffers (0.1 M, pHs of 3-6) table was calculated. Crush the tables well in
ware prepared by mixing 0.1 M sodium fine powder. 300 mg portion of powder was
acetate with 0.1 M acetic acid (98.2, 84.7, dissolved in deionized water. The solution
35.7 and 5.22 mL) with 0.1 M sodium was filtered and adjusted to volume of 100
acetate solution (1.77, 15.3, 64.3 and 94.8 mL. The clear solution was prepared for
mL of 0.1 M acetic acid) solution for to pHs extraction in the same procedure as
3, 4, 5 and 6, respectively before making to tetracycline standard solutions.
a final volume of 100 mL.
Phosphate buffers (0.1 M, pH 7-11) 3. Results and Discussion
were prepared by mixing 0.1 M phosphate Ion association or ion-pair is
(75.6, 95.5 and 95.0 mL) with 0.1 M HCl a chemical reaction whereby ions of
(24.4, 4.49 and 4.5 mL) before being opposite electrical charge come together
adjusted to pH 7, 8 and 9, respectively. in solution to form a distinct chemical
Phosphate buffer of pHs 10-11 were entity. There are also classified according to
prepared by mixing 0.1 M phosphate (96.6 the nature of the interaction as contact,
and 96.5 mL) with 0.1 M NaOH (3.4 and 3.5 solvent-shared or solvent-separated.
mL) respectively. All the solutions were
measured for accurate pH values using a pH TCLN+(aq) + Org. dye-(aq) TCLN+(aq) - Org. dye-(aq)
meter. TCLN+ - Org. dye-(aq) TCLN+ - Org. dye-(org)
Deionized water was used for the Figure 1. Reaction diagram of TC and
preparation of all reagents. organic dye
2.2 Instrumentation
PiCOEXPLORER, Photo absorbance The schematic reaction of ion-
sensor USHIO scientific, Japan with PAS- association reaction between protonated
110 software was used for RGB absorption form of tetracycline and anionic form of
detection. 300 L PCR tubing (Eppendoft) anionic dye is presented in Figure 1.
was used as a cuvette for measurement. pH Whereas, TCLN+ is the protonated primary,
measurement were done with pH meter secondary or ternary amine cation, Org. dye-
CONSORT c830 (Belgium). is an anionic form of organic dye and
TCLN+ - Org. dye- is neutral ion-association
compound.
After ion-association compound was Moreover, effect of pH was also
formed the polarity of the compound was studied by varying of pH solution, the
changed. It then could be extracted into the extraction solutions in organic phase were
organic solvent which the polarity is lower measured, graphs plotted between RGB
than an aqueous phase. color for and pHs was shown in Figure 3.
In this experiment, three types of
tetracyclines; tetracycline hydrochloride,
oxytetracycline and chlortetracycline were
investigated for the proposed method.
3.1 Effect of pH
Extraction of tetracycline based on
ion-association reaction using of
bromocresol green as the counter ion has
been reported by author by using sequential
injection lab-at-valve micro extraction
system.21 The association constants of (a)
tetracyclines are approximately of 3, 7 and 9
which was studied by potentiometric
titration.22 Whereas, pKa of bromocresol
green is 4.7.23 The speciation diagram of
tetracycline hydrochloride as a function of
pH is shown in Figure 2 The pH condition
expected for tetracyclines should be highly
protonated. Therefore, this experiment,
bromophenol blue with pKa of 4.0, 3.85,
3.624 was selected for another organic dye
which is expected for more highly (b)
association form between protonated form
of tetracyclines and anionic form of
bromophenol blue than with an anionic form
of bromocresol green as reported in
literature.21 It could be also found that the
sensitivity of extracted solution when using
of bromophenol blue as an organic dye is
three times higher than when using of
bromocresol green from batch experiment.
Therefore, bromophenol blue was selected
for this experiment.
(c)
Figure 3. Effect of pH on extraction
efficiency of tetracyclines (a) tetracycline
hydrochloride (b) oxytetracycline (c)
chlortetracycline
From the results, pH 3.0 was selected

for optimum pH which provides highly
extraction efficiency of association compound
Figure 2. Speciation diagram of tetracycline in extracted organic phase for all tetracyclines
hydrochloride as a function of pH22 studied.
3.2 Effect of concentration of organic dye
Concentration of organic dye has a
significant effect on extraction.
Concentration of bromophenol blue from
2 x 10-3 M to 8 x 10-3 M were studied for
final concentration in aqueous phase before
extraction. 4 x 10-3 M final concentration
was selected which provides high extraction
efficiency after extraction for all
tetracyclines into organic phase.
Figure 4. The RGB color calibration graph
3.3 Analytical characteristics
of tetracycline hydrochloride
Study on the association reaction of
tetracyclines including of tetracycline
3.4 Application to tables
hydrochloride, oxcytetracycline, chlortetra-
Under the proposed selected
cycline and bromophenol blue extracted in
conditions, the procedure was then applied
micro-liter level were investigated. The
for determination of tetracycline
yellow compounds were formed between
hydrochloride in commercial pharmaceutical
negative charge of bromophenol blue and
samples from various companies, the results
positive charge of tetracyclines in buffer pH
are listed in Table 1. The determined amount
3.0 at the final concentration of bromo
of tetracycline hydrochloride were compared
phenol blue in aqueous phase of 4 x 10-3 M.
with the label amount listed from the
The solution could be extracted into
company. The % label is estimated by the
dichloromethane phase in micro-liter level.
(amount found) x 100 / (label amount).
The system was measured by measuring of
RGB color with portable meter connected to
Table 1. Tetracyclines in pharmaceutical
the application-based measurement system
preparation samples in some companies
on a mobile phone. Under the optimum
conditions, all studied tetracyclines could be Sample Label Amount a
% Label
extracted into organic solvent. The RGB amount found (mg)
(mg)
color system was used to generate
Sample 1 500 510 ± 38.2 102 ± 5.34
calibration graph for quantitative analysis. Sample 2 500 500 ± 43.6 100 ± 8.72
Which color should be selected for a
% Label is estimated by (amount found) x 100/(Label
quantitatively of tetracyclines is depend on amount)
which color provided a good calibration
with appropriate slope and correlation 4. Conclusion
coefficient. The linearity of standard The proposed extraction procedure,
solutions at the concentration of 0 to 50 ppm ion-association liquid-liquid extraction in
of tetracyclines were y = 0.001x + 0.0193, y downscaling volume with RGB color
= 0.006x + 0.0448 and y = 0.0039x + 0.0145 detection system is a novel technique which
with correlation coefficients of 0.9915, simple and convenience detection with
0.9992 and 0.9992 (R color) for tetracycline portable and could be connected with mobile
hydrochloride, oxytetracycline and chlor- phone application. The extracted sample can
tetracycline, respectively. The limit of be measured in directly without removing of
detection (LOD) is 7.85 ppm. The limit of aqueous from organic phase before
quantitation (LOQ) is 35.86 ppm. The detection. It is an alternative way for cost
relative standard deviations of 0.48 to 5.53% effective determination method. It could be
were achieved. The example of calibration used for replaced in the places where the
graph of tetracycline hydrochloride which expensive instruments are not available.
the colour correspond to the change in Solvent consumption and operating time are
colour intensity shown in Figure 4. reduced. It should be gained the advantage
of the extraction tetracycline in some 11. Jeannot, M. A.; Cantwell, F. F. Anal.
samples which are limited amount. Chem. 1996, 68, 2236.
12. Jeannot, M. A.; Cantwell, F. F.; Anal.
Acknowledgements Chem. 1997, 69, 235.
The authors would like to thank 13. Pedersen-Bjergaard, S.; Rasmussen, K.
Faculty of science and engineering, E. Anal. Chem. 1999, 71, 2650.
Kasetsart University Chalermphrakiat Sakon 14. Rezaee, M.; Assadi Y.; Hosseini, M. R.
Nakhon Province Campus for research M.; Aghaee, E.; Ahmadi, F; Berijani, S.
facilities. Center of Excellence for J. Chromatogr. A 2006, 1, 1116.
Innovation in Analytical Science and 15. Berijani, S.; Assadi, Y.; Anbia, M.;
Technology (I-ANALY-S-T) and the Milani Hosseini, M. R.; Aghae, E. J.
Science and Technology Service Center, Chromatogr. A 2006, 1, 1123.
Chiang Mai University in cooperation with 16. Lopez-Molinero, A.; Lian, D.; Sipiera,
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867. 34, 2874.
9. Ueno, R.; Uno, K.; Kubora, S. S.; 23. Harvey, D. “Modern Analysis
Horiguchi, Y. Nippon Suisan Gakk. Chemistry” McGraw Hill, USA. 2000.
1989, 55, 1273. 24. Sabnis, R.W. Hand book of Acid-Base
10. Mishra, D. D.; Islam, I.; Sharma, J. P. Indicators CRC Press, USA. 2007.
Microchim. Acta 1985, 3, 97.
Solid phase extraction of uranium and thorium from various multi-element
standard solution and geological sample
Kalaya Changkrueng*, Saowaluck Thong-in, Harinate Mungpayaban
Office of Atoms for Peace, Ministry of Science and Technology, Bangkok 10900, Thailand
*E-mail: kalaya.c@oap.go.th
Abstract:
Matrix effect is one of major problem of Inductively Coupled Plasma-Mass
Spectrometry (ICP-MS) technique. It will make inaccurate results. In some case the ratio of
uranium isotope is quite sensitive necessity such as 235U/238U ratio. Solid phase extraction by
using UTEVA resin is to separate uranium (U), thorium (Th) and other elements before using
ICP-MS measurement. This work tests the extraction process of 2 multi-element standard
solution and 2 geological samples. The experiment focus on percent recovery of U, Th, resin
recycling, and matrix effect of extracted-process. The results show that UTEVA resin is
highly selectivity for U/Th with other elements, then matrix effect is reduced. The other
benefit of this extraction process provides increase U and Th concentration in sample that can
be measured by quadrupole mass spectrometer (or ppb detection limit) and to improved
confidential result of unknown samples for nuclear forensics and safeguards aspect.
1. Introduction determined the solution by ICP-MS.2 The

Nuclear forensics laboratory which interferences with internal standard were
was established in 2013 at Office of Atoms studied. The determination of uranium by
for Peace (OAP). One of the purposes of using spectroanalytical techniques such as
laboratory was to determine illicit nuclear flame atomic absorption spectrometry
materials by using high performance (FAAS), ICP-OES, ICP-MS was reviewed.
instruments i.e. ICP-MS, Gamma Spectro- The comparison of the matrix effects for
metry and Scanning Electron Microscope trace elements among ICP-MS and
with Energy Dispersive X-Ray Spectroscopy radiochemical neutron activation analysis
(SEM/EDS). 235U, 238U, thorium and rare (RNAA) from meteorites was done.3
earth were also originated in the monazite, Uranium and thorium isotopes were
bastnazite and xenotime ores. ICP-MS was measured by magnetic sector inductively
widely used to determine trace elements and coupled plasma mass spectrometry in terms
isotopic measured. The hard work was of precision, accuracy and other statistic
occurred when we need to analyze by using values.4 Uranium isotope in sandstone
mass spectrometry technique because of deposits by using ICP-MS determination
matrix effect among elements and how to technique was studied.5 The determination
purify the mixture into individual element. of quantization of uranium(VI) and
For the uranium-thorium separation, thorium(VI) was reviewed in several
it should be used solid phase extraction or analytical techniques (such as liquid-liquid
column chromatography. The determination extraction, ion exchange, extraction
of elemental impurities in uranium chromatography etc.).6 The matrix effect of
compounds by using several spectrometric trace elements included uranium, thorium
techniques such as ICP-OES, ICP-MS.1 and plutonium by using ICP-OES was
TRU resin cartridges was used to separate studied.7 A new solid phase extractant to
uranium and uranium isotope (235U and purify uranium(VI), thorium(IV) and
238
U) from fresh water and used three lanthanide elements form acidic matrices
alkaline reagents to elute uranium and was developed.8
UTEVA, TRU and TEVA resin are 2.2 Samples preparation
being used for actinide separation from This study tested a geological
various mixtures. The condition for samples: REE-2 contents 737  14 g/g of
uranium-thorium separation in high-purity Th and 3.73  0.12 g/g of U and IAEA-
Lead samples with UTEVA was proposed,9 RGU-1, Uranium ore sample which contents
while other scientists used for geological about 396 – 402 mg/kg of U and less than 1
samples.10 and the condition of extractants mg/kg for Th.
for solution sample (nitric acid form) has For geological sample, it is in solid
been changed.11,12 TRU resin is used to form that it is prepared to be a solution by
separation process of natural water with high using K1 Prime fluxer (Katanax, inc.) which
matrix concentration.13 These resins shows added some lithium borate fluxes.
high recovery yields for uranium and 2.3 Spectroscopic measurement
thorium. A 7700x ICP-MS was used to
This experiment is to study the determine the elements and isotope from
uranium-thorium separation process and every mixture and solution. The tuning
recovery yield of uranium and thorium by solution for ICP-MS was from Agilent
using ICP-MS measurement. The samples Technologies: 7500cs and the concentration
are 4 types which 2 samples are from was 1 g/L of Ce, Co, Li, Mg, Tl and Y in
standard solution, 2 samples are geological 2wt% HNO3 matrix.
sample and the other is unknown sample. Calibration curve for ICP-MS
determination are plotted from CGU1-5 or
2. Materials and Methods uranium isotopes (235U and 238U) from
2.1 General Inorganic Venture : CGU1-5 in 2% HNO3
The sample solution was thorium (v/v), ENV, 2A, thorium standard solution
from AccuStandard in 2% HNO3 Multi- and CCS-1 standard solution from Inorganic
element calibration standard 2A (2A) Venture which contents rare earth elements
contains 9.96 g/ml of uranium and (Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, Sc,
Environment calibration standard (Env) has Sm, Tb, Th, Tm, U, Y and Yb) in 7.14%
9.97 g/ml of uranium and 9.98 g/ml of HNO3 (v/v) has U and Th 100  0.7 g/mL,
thorium in 5% HNO3 from Agilent equally. The step of operating condition is
Technologies. Geological samples: certified shown in Table 1.
reference material for a Carbonatile with
Rare Earth Elements (REE-2) from Table 1. ICP-MS operating conditions
CanmetMINING, Ontario, Canada and Parameter condition
IAEA-RGU-1, Uranium ore (RGU-1) from RF power 1550 W
IAEA: reference products for environment Carrier gas flow rate 1.06 L/min
Make-up gas flow rate 0 L/min
and trade. Nebulizer Micromist
UTEVA resin sized 100-150 m Nebulizer Pump 0.10 rps
(Eichrom Technologies, Lisie, IL) was used Spray chamber 2C
for extraction process. Mass resolution 1u
The mixture of feed, standard Sample depth 8.0 mm
reference material for calibration curves, and
solution were prepared by Type I water 2.4 Methods
(ThermoScientific: Model Barnstead The step of uranium-thorium
SMART2PURE). purification from mixture9 is: packed
All acids, oxalic acid and hydrogen UTEVA resin into the column
peroxide solution were analytical grade. 1) regenerated resin with 0.02 M HCl, 0.5
M oxalic acid, DI water and 3 M HNO3
respectively,
2) poured uranium-thorium with multi- 3. Results and Discussion
element solutions (or feed) into the 3.1 Matrix effects
column, For measurement test about matrix
3) eluted U by using 3 M HNO3, 9 M HCl effect of 5 calibration standard solutions, it
and 0.02 M HCl, respectively then collect is found that it cannot determine uranium or
the uranium-elution, thorium concentration in mixture precisely,
4) 0.5 M oxalic acid was used for eluting of if the sample is unknown. While if it is only
Th, then collect the thorium-elution, one element (of uranium or thorium) in
5) evaporated solution of elution from No.4 solution, it can be predicted concentration in
and No.5 until nearly dryness accuracy.
6) add mixture of conc. HNO3 and H2O2 3.2 Percent recovery
(ratio 1:1) This experiment has not only
The schematic of solid phase uranium and/or thorium in samples but also
separation was shown in Figure 1. have other elements such as rare earth,
however it was not to report about them.
Because they are found rare earth and other
elements concentration which passing
through the column (step 3) were almost the
same amount from feed stock.
Table 2 shows the results of uranium
concentration which is compared with
reference standard. Amount of uranium in
samples from 1 to 400 ppm are tested in
separation columns. The results show the
good recovery of uranium contents.
Tables 3 shows the quantity of
thorium concentration which can be
compared with certified values. The
outcomes of thorium in elution are same as
the uranium amount. Though the elution of
geological samples will be prepared by
adding lithium borates flux, they can be
concluded that is no effect for the results.
Nevertheless, the effect of quantity of flux
Figure 1. The schematic of separation will be verified afterward.
Table 2. The uranium concentration results

Measurement
Sample name Sample type Certified value (ppm) % Recovery
value (ppm)
2A Standard solution 1.04 1 ± 0.05 104  5
ENV Standard solution 104.74 100 ± 5 105  5
RGU-1 Geological 401.17 400 ± 2 101  5
REE-2 Geological 3.85 3.73 ± 0.12 103  5
Table 3. The thorium concentration results

Measurement
Sample name Sample type Certified value (ppm) % Recovery
value (ppm)
ENV Standard solution 104.53 100 ± 5 105  5
REE-2 Geological 750.11 737 ± 14 102  5
Even though, the high recovery References
percentage were shown. It has to be studied 1. Pires, M. A. F.; Luiz de S. A; Cotrim,
about the condition for geological sample M. E. B. Microchem J. 2013, 106, 194-
preparation by using fluxer, in terms of 201.
quantity of fluxes and samples, type of 2. Tagami, K.; Uchida, S. Anal. Chim. Acta
samples). Because of an excess of quantity 2007, 592, 101-105.
of uranium and thorium in elute solution. 3. Ebihara, M.; Shinotsuka, K. Anal. Chim.
Since, fusion technique is a simple, short Acta 1997, 338, 237-246.
preparation time and properly for high Silica 4. Shen, C. C.; Lawrence, E. R.; Cheng, H.;
composition samples, that could not be Dorale, J. A.; Thomas, R. B.; Bradley,
digested by strong acid and microwave M. S.; Weinstein, S. E.; Edmonds, H. N.
digestion technique. Chem Geol. 2002, 185,165-178.
This work is to be adopted for 5. Placzek, C. J.; Heikoop, J. M.; House,
geological sample preparation process by B.; Linhoff, B. S.; Pelizza, M. Chem.
using solid phase extraction. Although, the Geol. 2016, 437, 44-55.
results show a good value when compared to 6. Prasada, R. T.; Metilda, P.; Gladis, M.
the reference standard. It has to be verified Talanta 2006, 68, 1047-1064.
for the more suitable condition of uranium 7. Ramanujam, A.; Gopalkrishnan, M.;
thorium separation in order to enhance Radhakrishnan, K.; Dhami, P. S.;
accuracy and precision value for Kulkarni, V. T.; Joshi, M. V.;
determination of isotopic ratio. Patwardhan, A. B.; Mathur, J. N.
Talanta 1997, 44, 169-176.
4. Conclusion 8. Siva, K. R. Ch.; Subramanian, M. S.
The results of U and Th are agreed Separ. Sci. Technol. 2007, 55, 16-22.
well with the certified values. The fusion 9. Grinberg, P.; Willie, S.; Sturgeon, R. E.
process including with UTEVA resin solid Anal. Chem. 2005, 77, 2432-2436.
phase extraction for geological sample 10. Carter, H. E.; Warwick, P.; Cobb, J.;
preparation is an important technique which Longworth, G. Analyst 1999, 124, 271-
can be arranged the mobile phase for 274.
extraction column. Then, this can be adapted 11. Rozmaric, M.; Ivsic, A. G.; Grahek, Z.
for uranium and thorium determination of Talanta 2009, 80, 352-362.
geological sample and improved 12. Rozmaric, M.; Ivsic, A. G.; Grahek, Z.
confidential result of unknown samples for Talanta 2010, 81, 1884.
nuclear forensics and safeguards aspect. 13. Unsworth, E. R.; Cook, J. M.; Hill, S. J.
Anal. Chim. Acta 2001, 442, 141-146.
Acknowledgements
I would like to thanks Technical Support
Division, Office of Atoms for Peace,
Ministry of Science and Technology;
Department of Mineral Resources, Ministry
of Natural Resources and Environment.
Development of an online flow analysis system for determination of
phosphate
Dechen Pelden1,2, Duangjai Nacapricha1,2, Shoji Motomizu3, Kanchana Uraisin1,2*
1
Flow Innovation Research for Science and Technology Laboratories (FIRST labs)
2
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol
University, Rama VI Rd, Bangkok 10400, Thailand
3
Graduate School of Natural Science and Technology, Okayama University,
Tsushimanaka 1-1-1, Kitaku, Okayama 700-8530, Japan
*E-mail: kanchana.ura@mahidol.ac.th
Abstract:
This work presents a flow-based technique so-called Simultaneous Injection Effective
Mixing Analysis (SIEMA) system for phosphate analysis. The detection principle is based on
molybdenum blue method. The SIEMA system involves the simultaneous aspiration of small
plugs of sample, molybdate solution and stannous chloride in acidic medium through the
individual 3-way solenoid valves. The zones were first rested inside their holding coils via
the control of a syringe pump. The aspirated zones were simultaneous pushed forward to the
detector for colorimetric detection of phosphomolybdenum blue complex at 690 nm. By
using this system, the mixing occurs at a confluence point resulting in efficient turbulent
mixing. Optimization of physical and chemical parameters was carried out. The calibration
graph was linear in the range of 0.2 – 8 mgP L-1. The relative standard deviation (RSD) at 2
mgP L-1 was found to be 1.9% (n = 11). The influence of major interfering ion of silicate was
also studied. The method was applied to analysis of phosphate in natural water samples
1. Introduction the disadvantages of being expensive and

Phosphate is an essential needing skilled personnel for operation
macronutrient present in low levels in while electrochemistry2 suffers from signal
aquatic systems but it can be a potential drift, instability of reagents and requirement
pollutant when present in higher of frequent calibration. Consequently, flow
concentrations. In the aquatic system analytical techniques have been extensively
phosphate can be released from used for phosphate determination due to its
mineralization of algae and phosphate advantages of high sample throughput,
minerals present in anthropogenic sources possibility of automation, facility and
like sewage, industrial effluents and also reproducibility.3
diffuse inputs from grazing and agricultural Flow injection analysis (FIA)4-8 and
lands. The increase in the level of phosphate sequential injection analysis (SIA)5-8 are two
in water due to the aforementioned sources of the well-established and widely used flow
lead to uncontrollable growth of algae/algal techniques for phosphate determination. FIA
bloom and results in depletion of dissolved has the advantages of automation and rapid
oxygen, killing fishes and other aquatic analysis of large number of samples but has
plants and animals. These phenomena can the drawback of using large amount of
ultimately affect the local ecosystem and reagent and carrier solutions owing to its
quality of drinking water supplies. continuous monitoring process. Additionally,
To this end, analytical techniques the flexible tubes of the peristaltic pump
like chromatography and electrochemistry used in FIA makes it less robust. SIA
have been employed for the determination of technique has the advantages of being
phosphate. However, chromatography1 has robust, versatile and using lesser reagent and
carrier compared to FIA. However, the solutions throughout. A stock solution of
stacked zones in SIA results in low mixing 100 mgP L-1 phosphate was prepared by
efficiency and lower sample throughput. dissolving accurate weight 0.22 g of
So there is a need of an analytical potassium dihydrogen phosphate (Merck,
technique that uses less reagent but give Germany) in 500.0 mL of DI water. Reagent
effective mixing at the same time and such a 1 (R1) was prepared by dissolving 2.4 g of
technique called Simultaneous Injection ammonium molybdate tetrahydrate (Qurec,
Effective Mixing Analysis (SIEMA) was New Zealand) in 1.2 mol L-1 sulphuric acid
pioneered by Motomizu in the year 2006. (RCI Labscan, Thailand) making the volume
The SIEMA technique had its first to 100.0 mL. Reagent 2 (R2) was prepared
publication in the year 2010 for the analysis freshly using 0.03 g of stannous chloride
of palladium.9 This technique is said to be a (BDH, England) and 0.2 g of hydrazinium
hybrid between FIA and SIA since it has the sulphate (APS, Australia) in 100.0 mL of 1.2
best features of both techniques incorporated mol L-1 sulphuric acid. A stock solution of
into it. Some of the features include faster 100 mgSi L-1 was prepared by dissolving
analysis than SIA, efficient mixing 0.04 g of sodium metasilicate anhydrous
comparable to FIA, robustness similar to (Fluka, Switzerland) in DI water to make
SIA, lower reagent consumption and simpler 250 mL. Natural water samples were
manifold than FIA.10 prepared by filtration using Whatman filter
This paper presents a SIEMA system paper (Ashless No. 42) and 40 mL of each
developed aiming for the spectrophotometric sample was spiked with standard phosphate
determination of phosphate in natural water 4 mgP L-1 for recovery study.
system using the molybdenum blue 2.2 Apparatus
chemistry. The molybdenum blue chemistry The SIEMA system developed is
is the basis for phosphate analysis in batch shown in Figure 1. It consists of a
wise as well as most automated systems. It bidirectional syringe pump (Hamilton co.,
involves the reaction between phosphate and USA), three 3-way solenoid valves (SV1 –
molybdate heteropolyacids which are SV3) and a 2-way solenoid valve (SV4)
subsequently reduced to a deep blue colored which are controlled by homemade software
compound and determined using a on a laptop computer. A Perkin Elmer
spectrophotometer. spectrophotometer (Lambda 25 UV/Vis
spectrometer) fitted with a flow through cell
2. Materials and Methods (10 mm path length) was used for the
2.1 Chemicals and materials absorbance measurement. The detector
All chemicals used were of analytical output was acquired on a PC using Lambda
reagent grade and deionized water purified 25 software. The flow lines were built with
by an Easypure II system (Barnstead International, 0.75 mm i.d. PTFE tubes except for the
USA) was used for the preparation of auxiliary coil with i.d. 1.5 mm.
Hol di ng coil SV1 (R1)
D Waste
Auxi l i ary SV4 Mi xi ng coil
Hol di ng coil SV2(S)
coi l
H2 O
Syri nge
pump
Hol di ng coil SV3(R2)
Figure 1. Schematic diagram of the SIEMA system. SV1, SV2 and SV3, 3-way solenoid
valves; SV4, 2-way solenoid valve; R1, (NH4)2Mo7O24 prepared in 1.2 mol L-1 H2SO4; S,
Standard/sample; R2, SnCl2 + N2H6SO4 prepared in 1.2 mol L-1 H2SO4; D, Spectrophotometer
2.3 Procedure The flow rate for propelling of all

The phosphate standard (S), R1 and aspirated zones through the detector was
R2 were introduced following the operation varied in the range of 1 – 10 mL min-1. The
protocol given in Table 1. Prior to the result showed that the signal increased by
injection of the standard and reagents the increasing the flow rate as can be seen in
flow lines were filled with water as carrier. Figure 2 (B). the increase in the signal
In the first step, R1, S and R2 were maybe caused by the enhancing of mixing in
simultaneously aspirated into each holding the confluence point when high flowrate was
coil via SV1, SV2 and SV3 using syringe used. Due to high signal and shorter analysis
pump. The total volume of the three time given by the flow rate 8 mL min-1, it
solutions was 600 µL, and the volume of was chosen for further studies.
each solution was approximately 200 µL. In The effect of mixing coil was studied
the second step, carrier water was aspirated in the range of 100 – 400 cm as shown in
into the syringe. Finally, all the solutions in Figure 2 (C). The signal is lower for the
the three holding coils were simultaneously longer mixing coil and the decrease in signal
dispensed and they merge at a confluence maybe caused by more dilution and
point just before entering the mixing coil dispersion compared to shorter length of
and then pushed towards the detector. The mixing coils. The mixing coil of length 200
colored product was measured at 690 nm by cm was selected as it provided satisfactory
the spectrophotometric detector. signal and analysis time is shorter.
3.2 Optimization of chemical parameters
3. Results and Discussion The optimization of chemical
3.1 Optimization of physical parameters parameters in this work was carried out
In this work, optimization of all using 8 mgP L-1. The effect of molybdate
physical parameters were carried out using 2 concentration was studied in the range of
mgP L-1 phosphate. 1.2 – 7.2 g L-1. It was observed that the
The volumes of reagents and signal for phosphate increased gradually till
standard were increased in the range of 100 3.6 g L-1 of molybdate and the signal didn’t
– 600 µL in order to increase the signal as increase significantly after this concentration
shown in Figure 2 (A). It was observed that while the blank signal kept increasing
along with the increase in volume the signal exponentially. The increase in the blank
increased as more volumes of reagents and signal maybe caused due to the difference in
standard were available for reaction. The concentration of the molybdate reagent
volume of 200 µL was selected for further prepared in 1.2 mol L-1 H2SO4 and the
studies since it gave enough sensitivity for carrier DI water resulting in Schlieren effect.
phosphate detection and also provided small
signal of blank.
Table 1. Operation protocol for phosphate determination
Syringe pump (SIA) Solenoid valve Function
Syringe Syringe Flow rate
Step SV1 SV2 SV3 SV4
valve position (µL) (µL/s)
1 Delay 3 s OFF OFF OFF OFF
2 100
ON 600
Simultaneous aspiration of
Delay 25 s standard and reagents
3 OFF 5000 300 Aspiration of carrier water

into syringe
4 Delay 5 s ON ON ON ON
5
Simultaneous dispensing of
ON 0 133 Delay 35 s
the
aspirated zones to detector
The molybdate concentration of 2.4 3.3 Analytical features

g L-1 was selected for further studies as it Under the optimized conditions
gave satisfactory sensitivity and the blank described under the previous section, a
signal was lower. series of phosphate solutions were
Effect of SnCl2 concentration was introduced into the system. A linear
studied in the range of 0.005 – 0.12 g L-1. It calibration graph in the range of 0.2 – 8 mgP
was observed that the phosphate signal L-1 (y= (14.1±0.003) x 10-2x + (12.6±0.012)
increased till the concentration 0.03 g L-1 x 10-2, r2 = 0.997) was obtained with a
after which the signal plateaued while the sample throughput of 60 h-1. A limit of
blank signal gradually increased. The reason detection (LOD) 0f 0.02 mgP L-1 was
for the increment is as described under the achieved (calculated from 3 times standard
study of molybdate concentration. The deviation of blank divided by the slope of
stannous chloride concentration of 0.03 g L-1 the calibration graph). Relative standard
was selected for further studies because it deviation of 11 replicates of 2 mgP L-1 was
gave high sensitivity. 1.9%.
Acid concentration was varied in the 3.4 Interference study
range of 0.2 – 1.4 mol L-1 and the effect was Silicate is known to be a major
studied. As shown in Figure 3 the signal of interference in the analysis of phosphate
phosphate as well as blank signal decreased using molybdenum blue chemistry because
with the increase in the concentration of it can also form heteroployacid which on
H2SO4. The concentration of 0.2 mol L-1 reduction gives molybdate blue species.
gave the highest signal but the blank signal However, previous studies have reported
for the same concentration is very high. The that the signal of silicate can be suppressed
high blank signal is due to the self-reduction when sufficiently high concentrations of
of molybdate reagent at lower acid acid was used.11 In order to determine the
concentration where the molybdate forms tolerance limit of the SIEMA system for
the blue colored complex without the silicate, various concentrations of standard
presence of phosphate. For further studies of silicate were spiked with 1 mgP L-1
1.2 mol L-1 H2SO4 was selected because it phosphate standard solution.
gave enough sensitivity for phosphate
analysis and also the blank signal was low.
A B C
0.40 0.40 0.40 400 0.40 80
Analysis time (sec)
Analysis time (sec)

Abs. at 690 nm
Abs. at 690 nm
0.35 0.35 0.35 350 0.35
Abs. at 690 nm
79
Blank signal
0.30 0.30 0.30 300 0.30
78
0.25 0.25 0.25 250 0.25
0.20 0.20 0.20 200 0.20 77
0.15 0.15 0.15 150 0.15
76
0.10 0.10 0.10 100 0.10
75
0.05 0.05 0.05 50 0.05
0.00 0.00 0.00 0 0.00 74
0 200 400 600 800 0 2 4 6 8 10 12 0 100 200 300 400 500
Volume of standard and reagent (µL) Flow rate (mL min-1 ) Mixing coil length (cm )
Figure 2. Effect of (A) volume of standard and reagent, (B) flow rate, (C) mixing coil length
on the absorbance, blank signal and analysis time of phosphate.
The tolerance limit for silicate is Table 2. Percentage recovery of phosphate
defined as ± 3 standard deviation of signal in natural water sample
of 1 mgP L-1phosphate standard solution. It Sample Founda
Found after
%
was found that the SIEMA system can spikinga,b (mgP
no. (mgP L-1) Recovery
L-1)
tolerate up to 8 mgSi L-1 of silicate.
1 n.d 3.43±0.02 77.6 %
2.5 2.0 2 n.d 3.92±0.01 77.5%
1.8 3 0.29±0.01 4.22±0.01 82.0%
2.0 1.6 4 0.29±0.01 4.89±0.01 99.1%
Abs. at 690 nm
Blank signal
1.4 5 n.d 3.65±0.01 77.5%

1.5 1.2
6 0.23±0.01 3.65±0.03 77.1%
1.0 a
1.0 0.8
mean of triplicate results
b
0.6
spiked concentration of 4 mgP L-1
n.d. Not detected
0.5 0.4
0.2
0.0 0.0 4. Conclusion
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
A SIEMA system was successfully
H2SO4 concentration (mol L-1)
developed for the analysis of phosphate in
Figure 3. Effect of H2SO4 concentration on natural waters. The system developed uses
the absorbance of phosphate and blank lower sample volume and has a fast sample
signal throughput. Application of the developed
system to water sample has been
3.5 Application to water sample
demonstrated with acceptable percentage
Analytical recovery
recovery values. This system can be a
In this work, natural water samples
beneficial alternative to other flow analytical
were collected from Pitthaya Longkon Canal
techniques for the analysis of phosphate.
in the Samut Sakhon Province, Thailand.
Study of % recovery was carried out by
Acknowledgements
using spiked standard phosphate 4 mgP L-1 We would like to extend our thanks
into filtered natural water sample. As shown to Thailand International Corporation
in Table 2, recoveries were found to be in Agency (TICA) and the office of higher
the range of 77.1 – 99.1% which is education commission and Mahidol
acceptable recovery values according to university under the national research
AOAC requirement for single laboratory universities initiative for the financial
validation of chemical methods.12 support rendered.
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Methods 2012, 4 (5), 1452-1457. 11. Horwitz, W. AOAC requirement for
6. Mas-Torres, F.; Estela, J. M.; Miró, M.; single laboratory validation of chemical
Cladera, A.; Cerdà, V. c., Anal. Chim. methods 2002.
Simultaneous determination of p-coumaric acid and naringenin
in honey using dispersive liquid-liquid microextraction and high
performance liquid chromatography
Korbkarn Khukitirat1, Pongsak Lowmunkhong1, Somyote Sutthivaiyakit2,
Pakawadee Sutthivaiyakit1*
1
Kasetsart University, Bangkhen, Bangkok 10900, Thailand
2
Department of Chemistry and Center of Excellence for Innovation in Chemistry,
Faculty of Science,Ramkhamhaeng University, Bangkok 10240, Thailand
*E-mail: fscipws@ku.ac.th
Abstract:
p-Coumaric acid and naringenin have anti-inflammatory and antioxidant benefits.
However, they have not been determined in honey from Thailand. A simple, rapid and
sensitive dispersive liquid-liquid microextraction (DLLME) followed by HPLC with diode
array detection was developed for simultaneous determination of the two compounds. Some
important parameters for DLLME were optimized. A mixture of acetonitrile disperser
solvent) and dichloromethane (extraction solvent) was rapidly injected to a sample solution in
0.01 M ammonium acetate adjusted to pH 3 by a syringe, thereby forming a cloudy solution.
After centrifugation at 4 °C, the enriched analytes in the sedimented phase were filtered prior
to injection. The separation was performed on a Pursuit® XRS Diphenyl column (150x3.0
mm, 3 µm) at 40 °C with 0.1% formic acid – methanol gradient as the mobile phase. The
detection wavelengths were 291 nm and 311 nm for naringenin and p-coumaric acid,
respectively. The flow rate was 0.3 mL min-1. Due to the lack of matrix blank, standard
addition was performed in the method development. The concentrations of p-coumaric acid
and naringenin in lychee are 0.5±0.01µg.g-1 and 0.38 ± 0.03µg.g-1, respectively..
1. Introduction anti-inflammatory.6 As honey is a complex

Honey is a natural product that is natural product, the sample preparation to
rich in both nutritional and medicinal preconcentrate these two compounds and
values.1,2 Phenolic acids and flavonoids are clean up the matrix are needed. Dispersive
among the minor constituents showing liquid-liquid microextraction (DLLME) was
medicinal properties. Thai honey has been selected due to its simplicity, low cost and
reported to possess medicinal properties.3,4 rapidity compared to solid phase extraction
However, there is a lack of both the content method which was previously used.7-9
and type of phenolic acids and flavonoids
reported in Thai honey. The present study 2. Materials and Methods
aims to develop simultaneous determination 2.1 Materials
of p-coumaric acid and naringenin in Thai p-coumaric acid (98%) and
honey using dispersive liquid-liquid microextraction naringenin (˃98%) were purchased from
(DLLME) and high performance l i qui d Sigma Aldrich and TCI, respectively. Ultra-
chromatography. p-Coumaric acid was chosen pure water was obtained from water
due to the fact that it possesses anti-arthritis purification system model SimplicityTM
activities and mitigatory effects against (Millipore, France). Other chemicals are of
diabetes and obesity5 Naringenin was analytical reagent grade.
selected as it has bioeffect on human health
as antioxidant, free radical scavenger and
2.2 Equipment 3. Results and Discussion
Samples were chromatographed on a 3.1 Optimization of DLLME
Pursuit® XRS Diphenyl column (150x3.0 There are several parameters affect
mm, 3 µm) at 40 °C with 0.1% formic acid – the efficiency of DLLME process used in
methanol gradient as the mobile phase. The this study to preconcentrate and clean up
mobile phase was delivered by a gradient honey samples. They are extraction solvent,
system from Agilent 1100 quarternary pump dispersive solvent, ratio of extraction solvent
(Agilent Technology, Waldbronn, Germany) to dispersive solvent, pH of sample solution,
and composed of 0.1% formic acid adjusted sample volume and ionic strength.
to pH 3(eluent A) and methanol (eluent B). Six solvents were selected for the
The gradient elution started from 30% study of the effect of extraction solvent.
methanol and increased linearly to 40% over They are dichloromethane, chloroform,
0.5 min and to 75% over 14.5 min, at a flow carbontetrachloride, dichloroethane, tetra-
rate of 0.3 mL.min-1. The column was chloroethane. These solvents have low
equilibrated for 7 min prior to the next solubility in water and high extraction
injection. The rheodyne valve with 10 µL capability of interested compounds, and
injection loop was used throughout the once were injected into aqueous system,
experiments. The detection wavelengths able to form a stable two-phase system in
were 291 nm and 311 nm for naringenin and the presence of dispersive solvent. In this
p-coumaric acid, respectively. experiment, two grams of honey were use
2.3 Methods and experiment was performed as described
The stock standard solutions were in 2.4 except the type of solvent was
prepared at a concentration of 300 µg. mL-1 changed. As shown in Figure 1, among six
in methanol and stored at 4 °C. Working solvents, dichloromethane is the best solvent
solutions were appropriately diluted in for p-coumaric acid and second best for
methanol as required. naringenin while tetrachloroethane extracted
2.4 Sample preparation very little of p-coumaric acid.
Two grams of honey sample was
added with 6 mL of 0. 01 M ammonium
acetate adjusted to pH 3 and the mixture was
thoroughly stirred. Then, 0. 6 g of sodium
chloride was added and the mixture was
vortexed for 1 min. One mL of 1: 1
acetonitrile- dichloromethane was rapidly
injected twice into the mixture. The cloudy
solution was formed. After centrifuging to
disrupt the dispersion of the organic droplets
at 6,000 rpm for 10 min, the sedimented
phase was separated and collected into a
new tube. Sedimented phases were ruptured
with a glass rod and the solution was filtered
prior to injection.
For the standard addition method Figure 1. Effect of different extraction
appropriate aliquot of the standard mixtures solvents on extraction of p-coumaric acid
was spiked in the honey sample and the and naringenin obtained from DLLME.
mixture was left to room temperature for at Aqueous phase volume, 6 mL; organic
least an hour before analysis was performed. solvent volume 0.5 mL and dispersive
solvent volume 0.5 mL
Thus, dichloromethane was chosen dichloromethane gave the best extraction and
for further experiments. Next, three this ratio was used for further experiments.
dispersive solvents, namely acetonitrile, Next, the effect of pH of sample solution
acetone and methanol, were investigated as was investigated in the range 2 to 7. The
these solvents are partly miscible with experiment was performed as described in
aqueous phase. The experiment was 2.4 except that pH of the buffer solution was
performed as described in 2.4 except that the varied. A pH 3, p-coumaric acid was
type of dispersive solvents was changed. Of extracted most while at pH 5 naringenin was
the three solvents, acetonitrile was the best extracted most. Thus, simultaneous
dispersive solvent for both compounds as determination of both compounds pH 3 was
shown in Figure 2. depicted as it yields best extraction and
second best extraction for p-coumaric acid
and naringenin, respectively.Effect of
sample volume in the range 2 to 10 mL for
two grams of honey sample was studied.
The experiment was performed as described
in 2.4 except that sample volume was varied
in the range of 2 to 10 mL. Six milliliters of
sample volume yield the highest value and
was chosen for next experiments.
The effect of ionic strength in the
range 0.3 to 0.8 gram of sodium chloride for
2 g of honey sample and with other
experimental conditions kept constant on the
extraction efficiency of DLLME was
Figure 2. Effect of different dispersive evaluated.
solvents on extraction of p-coumaric acid The extraction efficiency increases
and naringenin obtained from DLLME. with addition of sodium chloride up to 0.6 g
Experiment was performed as described in and slightly decreased with further addition
2.4 of sodium chloride. This may be owing to
the salting effect plays a significant role.10
Subsequently, the effect of ratio of However, at high ionic strength (0.7 – 0.8 g),
extraction solvent (dichloromethane) to the extraction efficiency for p-coumaric acid
dispersive solvent was investigated. was the same while that for naringenin was
Experiment was done according to 2.4 slightly decreased. Additionally, the density
except that ratio of acetonitrile: of aqueous mixture was so high that after the
dichloromethane was varied while the total centrifugation, the extract was in the upper
volume of the solvent was kept at 1.00 mL. layer resulting in inconvenience for phase
Three ratios of extraction solvent to separation. Thus 0.6 g of sodium chloride was
dispersive solvent i.e. 1:4, 1:1 and 4:1 chosen for further experiments.
acetonitrile to dichloromethane were
studied. The ratio of 1:1 of acetonitrile to
Figure 3. Typical chromatogram of honey sample spiked with 0.5 µg.g-1 detection wavelength
at 311 nm obtained from DLLME. Peak identification (1) p-coumaric acid and (2) naringenin
3.2 Sample analysis The standard addition calibration

Under optimum conditions, the two curves show good linearity in the range up to
peaks are baseline separated from each other 2. 5 µg. g-1 with correlation coefficient better
and from unknown peaks as shown in Figure than 0. 99 for both compounds. Limit of
3. The good performance of column may be detection for each compound was determined
due to the aromatic structure which can as three folds standard deviation of 10
interact with the phenolic compounds via π- replicates of honey sample spiked with 0. 25
π interaction.11 As no blank matrix of honey µg of standard mixture to 1 g of honey
sample is available, standard addition method sample. Their values are shown in Table1.
was performed. Two grams of honey was Furthermore, as is known that no
spiked with different concentrations of reference materials for honey sample is
standard mixtures in the range 0 – 2.5 µg.g-1 commercially available, the accuracy of the
and left for at least one hour before method was validated using the recovery data
performing the extraction. by spiking honey samples at two levels in
triplicate as shown in Table1.
Table 1. Recoveries of p-coumaric acid and naringenin(n=3)

DLLME spike %Recovery (RSD%) LOD (µg.g-1)
Compound
level (µg.g-1) Longan Lychee Longan Lychee
0.5 109.88% (3.77%) 102.41% 7.85%)
Naringenin 0.02 0.09
1.25 93.20% (6.85%) 99.74% (2.50%)
0.5 117.55% (6.41%) 83.41% (5.11%)

p-Coumaric acid 0.04 0.10
1.25 96.78% (2.45%) 97.37% (7.45%)
Table 2. Analysis results of different honey samples
Content (µg.g-1)
Compound
Longan Lychee
Naringenin 0.18 ± 0.03 0.38 ± 0.03
p-Coumaric acid 0.12 ± 0.01 0.50 ± 0.01
The recovery values ranged between References

93.20 and 109.88% with relative standard 1. Lucci, P.; Saurina, J.; Núňez, O. Trends
deviation in the range of 2.50 and 7.85% for Anal. Chem. 2017, 88, 1-24.
naringenin while 83.41 and 117.5% with 2. Mattonai, M.; Parri, E.; Querci, D.;
relative standard deviation in the range Degano, I.; Ribechini, E. Microchem.
2.45 – 7.45% for p- coumaric acid. Addition 2016, 126, 220-229.
nally, the content of each compound found 3. Jantakee, K.; Tragoolpua,Y. Biological
in different honey samples are shown in Res. 2015, 48, 4-18.
Table 2. The variations in concentration of 4. Pattamayutanon, P..; Angeli, S.; Thakeow,
phenolic compounds investigated in Thai P.; Abraham, J.; Disayathanoowat, T.;
honey may be due to the different floral Chantawannakul, P. Food Sci. 2015, 80,
source (longan, lychee). M2228-40.
5. Pei, K.; Ou, J.; Huang, J..; Ou, S. J. Sci.
4. Conclusion Food Agric. 2016, 96, 2952-2962.
The DLLME coupled with HPLC- 6. Alam, M. A.; Subhan, N.; Rahman, M.
DAD gives reproducible and accurate results M.; Uddin, S. J.; Reza, H. M.; Sarker, S.
for determination of p-coumaric acid and D. Adv. Nutr. 2014, 5, 404-417.
naringenin in honey samples. It is the first 7. Campillo, N.; Viňas, P.; Fȅrez-
time that these two compounds were Melgarejo, G.; Hernanȁndez-Coˊrdoba,
analyzed in Thai honey samples. The M.; Talanta 2015, 131, 185-191.
advantages of the method developed are 8. Zhao, J.; Du, X.; Cheng, N.; Chen, L.;
miniaturize preconcentration methodology, Xue, X.; Zhao, J.; Wu, L.; Cao, W. Food
rapid and cost effective. DLLME developed Chemistry 2016, 194, 167-174.
in this method can be applied to determine 9. Chen, H.; Chen, H.; Liao, L.; Ying, J.;
other phenolic compounds in honey. Huang, J. J. Chromatogr. Sci. 2010, 48,
450-455.
Acknowledgement 10. Daneshfar, A.; TAbaraki, R.;
Financial support from the Center of Khodakarami, R.; Khezeli, T. Anal.
Excellence for Innovation in Chemistry Bioanal. Chem. Res. 2016, 3, 41-51.
(PERCH-CIC), Office of Higher Education 11. Pyrzynska, K.: Biesaga, M. Trends in
Commission, Ministry of Education is Anal. Chem. 2009, 28, 893-902.
gratefully acknowledged.
Optimization of oleaginous yeast production by central composite design
Supatsara Rujanant*, Sarun Keansaior, Sasithorn Kongruang*
Bioprocess Engineering and Biotechnology Center, Department of Biotechnology, Faculty of Applied Science,
King Mongkut’s University of Technology North Bangkok, 1518 Pibulsongkram Rd.,
Bangsue, Bangkok 10800, Thailand
*E-mail: s5904071820023@email.kmutnb.ac.th, sasithorn.k@sci.kmutnb.ac.th
Abstract:
Rhodosporidium toruloides is one of the high potential oleaginous yeasts for the
application in food additives, pharmaceuticals and feed ingredients. The effect of growth
parameters to biomass and lipid production of R.toruloides TISTR 5149 in molasses media
was studied and optimized by response surface methodology via Central Composite Design.
Fermentation parameters affecting the lipid production using 40, 50 and 60 g/L molasses as
carbon source, pH 5, 6 and 7, temperatures at 28, 30 and 32 °C and shaking speeds at 200,
250 and 300 rpm were investigated for biomass and lipid production. Results found that
biomass and lipid production can be explained using the quadratic models. Results also
revealed that biomass yielded 15.47 g/L under 50 g/L molasses, 200 rpm , 32 °C, pH 5.0
while pH and shaking speed were significantly effect to lipid production (p = 0.0015 and
0.0006) where the highest lipid production was 11.88 g/L. Models were validated and showed
a good agreement to predict biomass but not lipid production. This mathematical relationship
of biomass production can be applied to scale up in pilot scale.
1. Introduction these can be used as vitamin-A precursors,

Oleaginous yeasts is well known as natural dyes, antioxidants, and possible
microbe that can accumulate high amounts tumor-inhibiting activity, proving their
of intracellular lipids by utilizing various potential for the pharmaceutical, chemical,
types of organic carbon in a short duration food, and feed industries.5 However,
which currently attracted many biodiesel stringent culture conditions are required by
refinery industry regardless of seasonal oleaginous yeasts to induce lipogenesis, with
variation. This is because microbial lipids C/N ratio skewed excessively toward
produced by oleaginous microorganisms carbon, creating nitrogen limitation in the
including microalgae, bacteria, fungi and culture medium.6 To date, most studies on
yeasts have been consider as promising lipid production by oleaginous
potential feedstock for biodiesel production microorganisms have been carried out with
due to the majority of fatty acid and formed glucose as a carbon source,7,8 but it is still
from a long-chain similar to vegetable oils.1 problematic given the high price of raw
Rhodosporidium toruloides has materials. Considerable efforts have led to
conveniently been grown in bioreactors on minimize the production cost and find new
various media based on waste-water, waste alternative carbon sources such as volatile
juices, molasses, crude glycerol for the fatty acids, molasses.9
production of microbial lipids.2,3 Apart from The conventional technique to
lipids, this yeast is also produced carotene optimize factors of process is based on one
that can be used as a therapeutic agent.4 The parameter variable at a time and keeping
accumulation of lipids is up to ∼50% of its other remaining parameters constant. This
total cell weight, during the log phase and technique also takes more time to
amasses significant a mounts of carotenoids experiment and has poor efficiency for
during the stationary phase of growth. As optimizing a lot of factor.10 The approach
known that carotenoids are important as lacks the absoluteness to predict response
© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) EE1
variable under un-experimented trials of was analyzed in triplicate as tabulated in
independent variables. In addition, the Table 1.
interaction amongst the variables affecting
growth and lipid production is not Table 1. Level of the factor tested in CCD
investigated, it does not represent the Factors Symbol Level of factors
complete effects of the parameters on the code -1 0 +1
Molasses (g/L) X1 40 50 60
process and might lead to incorrect pH X2 5 6 7
conclusion. In order to deal with this Temperature (°C) X3 28 30 32
problem, optimization studies can be carried shaking speed (rpm) X4 200 250 300
out using response surface methodology
(RSM). In this work it is aimed to suggest a 2.2 Optimization by response surface
cheaper raw material for the application in methodology
food additives pharmaceuticals and feed Central Composite Design (CCD)
ingredients by using R. toruloides cell. For was operated to optimize the four important
this work, molasses was used as a carbon. In factors (molasses as carbon source, pH,
order to optimize the production of lipid by temperatures and shaking speeds) for
R.toruloides TISTR 5149, the effects of enhancing efficiency of lipid production.
growth parameters of this strain in batch The four independent factors were studied at
fermentation were studied and optimized three levels (-1, 0, 1) (Table 2). A total of 30
through a multivariate approach for lipid experiments were conducted. All factors
production by using response surface were taken at a central code value, which
methodology via Central Composite Design. was considered as zero. The minimum and
maximum ranges of the factors were used.
2. Materials and Methods The biomass concentration, lipid
2.1 Oleaginous yeast inoculums concentration and percent of lipid content
preparation and morphological study were noted as response values (y1, y2, y3),
R. toruloides TISTR5149 was and each trial was the average of the
obtained from Culture Collection of triplicate.
Thailand Institute of Scientific and Second-order polynomial model was
Technology Research (Bangkok, Thailand). fitted to the response data obtained from the
Yeast cultures were maintained on yeast design. The polynomial equation is in the
extract-malt extract (YM) 0.75 g of malt following form:
extract, 1.25 g of peptone and 1.00 L of
distilled water; Medium I (26.46mL of Y = β0 + ΣβiXi + ΣβiXiXj + ΣβiiXi2 …..[1]
molasses, 0.75 g of yeast extract, 0.75 g of
malt extract, 1.25 g of peptone and 1.00 L of where X1, X2 and X3, represent the coded
distilled water), Medium II (33.07mL of levels of the independent variables as
molasses, 0.75 g of yeast extract, 0.75 g of described in Table 1 and β0, βi, and βj (i, j =
malt extract, 1.25 g of peptone and 1.00 L of 1, 2, 3, 4) are the coefficient estimates,
distilled water) and Medium III (39.69 mL where, β0 is the interception term, βi is the
of molasses, 0.75 g of yeast extract, 0.75 g linear term, βii is the quadric term, and βii is
of malt extract, 1.25 g of peptone and 1.00 L the interaction term. For the predicted
of distilled water). The final pH of all these responses, y1 stands for lipid content
mediums were adjusted to 7. These whereas y2 stands for biomass concentration.
mediums contributed to with the levels of The accuracy and general ability of the
coded values of -1, 0 and 1, respectively. above polynomial model could be evaluated
Cultures were then incubated at three by the coefficient of determination (R2). The
temperatures: 28, 30, and 32 °C in an orbital experimental data was analyzed using the
shaking incubator with three shaking speeds: statistical software, Design-Expert software
200, 250 and 300 rpm for 48 h. Each sample version 10 (STAT- EASE Inc., Minneapolis,
Table 2. Central composite design matrix vortexed again for 1 min. The mixture was
Run X1 X2 X3 X4 Responses centrifuged at 3000 rpm for 15min to
Y1 Y2 Y3 separate the aqueous and organic phase. The
1 -1 0 0 0 9.9 2.7 27.3 organic phase was transferred to weight then
2 1 1 -1 1 12.0 10.8 90.0
3 0 0 0 1 2.0 1.8 90.0
calculated and reported as lipid content
4 1 -1 1 -1 14.9 0.3 2.6 (g/L).
5 0 0 0 0 4.0 1.0 27.2
6 -1 1 -1 1 10.0 5 50.0 3. Results and Discussion
7 -1 -1 -1 1 8.0 2 25.0 3.1 Morphological observations of R.
8 -1 -1 1 1 17.9 3.5 19.8 toruloides TISTR5149
9 1 1 -1 -1 10.0 7.5 75.0
Carotenogenic basidiomycete yeast,
10 0 0 -1 0 4.0 1.1 27.2
11 -1 1 -1 -1 6.0 4.5 0.0 R. toruloides TISTR 5149 belongs to the
12 0 0 0 0 4.0 1.1 27.2 species of the ballistoconidia-forming
13 1 1 1 1 17.7 3.5 19.5 genera. This oleaginous strain was isolated
14 -1 1 1 1 12.6 3.9 31.2 from a decaying fruit. It grew on YM agar
15 0 0 0 0 4.0 1.1 27.2 after 48 h of 30°C incubation and showed
16 0 1 0 0 8.0 5.6 69.7 orange, smooth, glistening, convex and the
17 0 0 0 0 4.0 1.1 27.2
18 0 0 -1 0 16.0 4.5 28.1
entries edge (Figure 1a). This oleaginous red
19 1 -1 1 1 25.9 4.8 18.9 yeast accumulate both lipid and carotenoid
20 1 0 -1 0 6.0 3.5 59.5 as observed in Figure 1a. Cells were
21 1 -1 -1 -1 6.0 4.5 0.0 observed in Figure 1b, which cells were the
22 -1 -1 -1 -1 0.0 0.0 0.0 reproduction stage by polar budding, single
23 1 1 1 -1 10.5 3.7 34.7 or in pairs and contain oil droplets.
24 0 0 0 0 4.0 1.1 27.2
3.2 Mathematical relationship
25 0 -1 -1 0 8.1 5.7 69.7
26 0 0 0 0 4.0 1.1 27.2
The second-order polynomial of
27 1 -1 -1 1 0.0 0.0 0.0 equation (2-4). The values of correlation co
28 -1 1 1 -1 9.1 2.2 24.1 efficient of R2, 0.767, 0.523 and 0.617 for
29 0 0 0 -1 4.3 1.2 27.2 biomass concentration, lipid concentration
30 -1 -1 1 -1 13.5 4.7 29.6 and lipid percentage were associated with
equations (4). The R2 of predicted biomass
MN, USA), for regression analysis to fit the production represented the good agreement
equations developed and also for the of the empirical models as represent the
evaluation of the statistical significance of correlation between the experiment results
the equations. and theoretical values predicted by the
2.3 Determination of biomass content model equation.
The fermented broth was centrifuged In case of biomass production had
at 4,000 rpm for 10 min. Precipitated only two fermentation parameters affected
biomass was cleaned with water and to the yield which are temperature and
centrifuged twice. Then washed cells were shaking speed with P<0.0001 and 0.0243 as
dried at 80°C until constant weight. The shown in Table2.
biomass was determined gravimetrically. DCW = +22.13+14.54*pH-1.59
2.4 Determination of lipid content *shaking sp.+4.94*C:N-0.06*pH*shaking
Total lipids within the cell were sp.+4.04E-003* shaking sp.2-0.05*C:N2 ;
extracted by the modified method according R2 = 0.767 …………………...…….…[2]
to Floch et al., 1957.11 20ml of fermentation Lipid = +1.78+1.17 * pH-1.34*
broth were harvested by centrifugation after pH*shaking sp.+2.24* pH2; R2 =0.523..[3]
that washed and then dried to obtain Lipid(%) = +10.23*Temperature-
constant weight. Pellet was resuspended in 3 10.17* shaking speed+13.48* C:N-13.16*
mL of chloroform/methanol (2:1) vortexed Temperature* shaking speed-11.41* shaking
for 15 min, added 1 mL of chloroform and speed* C:N+26.78* pH2-31.03* shaking
(a) (b)
Figure 1. Morphology of R. toruloides TISTR 5149 (a) growth on YM agar. (b)

under a light microscope (1000x)
speed2; R2=0.617 …………….[4] Table 3. Analysis of variance of

3.3 Optimization of biomass experimental results of the reduce quadratic
concentration for biomass production
The biomass optimization was Source Sum of df Mean F- P-
reevaluated through a stepwise process to Squares square ratio value
Model 777.50 6 129.58 12.84 <0.0001
eliminate the insignificant fermentation Linear
parameters. Table 3 presents the significance X2 130.67 1 130.67 12.95 0.0015
X3 158.80 1 158.80 15.74 0.0006
of each coefficient through P-values by an X4 62.98 1 62.98 6.24 0.0243
analysis of variance. The results indicated Quadratic
X32 269.26 1 269.26 26.68 <0.0001
that the differences in the model, lack of fit, X42 58.63 1 58.63 5.81 0.0243
Interaction
as well as three linear term (X1, X2 and X3),
X2 X3 133.69 1 133.69 13.25 0.0014
two quadratic terms (X32 and X42 ) and one Residual 232.09 23 10.09
Lack of fit
interaction terms (X2X3) were all significant Pure error
232.09 17 13.65
0.000 6 0.000
(P < 0.05), while the other terms were Total (corr.) 1009.59 29
insignificant (P > 0.05). The lower the value
of the coefficient of variation, the higher the However, the maximum lipid
reliability of the experiment. Under the concentration could be obtained at 7.37g/L
quadratic equation, the optimum biomass of with 62% lipid when R. toruloides
this model was obtained as 23.05 g/L once TISTR5149 was fermented in 40 g/g C:N at
the fermentation broth contained 50g/L 32°C, pH 7.0 with 200 rpm shaking speed.
molasses, pH=5 which shaking speed at 300 This situation would lead to yield 8.26 g/L
rpm at 32°C. Under this condition, the lipid biomass.
concentration was 4.19g/L resulting in
11.88g/L of lipid percentage. 4. Conclusion
3.4 Response surface plot This work showed the fermentation
The illustration of response surface conditions for biomass and lipid production
methodology yielded the contour plots as the in submerged cultures using industrial
relationships between the biomass waste. The conditions established for
concentration (a-f), lipid percentage (g and biomass production by when R. toruloides
h) and the test variables. The response TISTR5149 using molasses as carbon source
surface plot (Figure 2b) showed curvature and yeast extract as carbon source can be
that the optimum 23.05g/L biomass yielded achieved under the adjustment of significant
at 50.86g/L C/N, pH5, 32°C over 72h fermentation parameters. The potential
fermentation time with 300 rpm shaking biomass cultivation in this investigation is a
speed. For this condition, the maximum lipid great source for food and feed application.
concentration was 4.19g/L resulting in 20.15
lipid percentage.
(a) (b)
(d)
(c)
(e) (f)
(g) (h)
Figure 2. The contour plots for biomass production at varying (a) temperature and shaking
speed, (b) C/N source ratio and pH, (c) C/N source ratio and shaking speed, (d) C/N source
ratio and temperature, (e) pH and shaking speed, (f) pH and temperature, (g)
temperature and shaking speed and for %lipid (fat), (h) C/N source ratio and shaking speed
and for %lipid (fat)
Acknowledgements 5. Natalia, M.; Isabel, C. F. R. F. Trends
This research project has been partly Food Sci. Tech. 2017, 62, 33–48.
supported by National Research Council of 6. Mesut, T.; Serkan, O.; Mehmet, N. A.;
Thailand (NRCT) (Grant no.KMUTNB- Nazli, P. A. Renewable Energy 2016, 99,
GOV-57-22). 198–204.
7. Areesirisuk, A.; Yen, T. B.; Chiu, C. H.;
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Effect of the Pd-Mg/SiO2 on catalytic behavior for partial hydrogenation of
soybean oil biodiesel
Jakkrapong Jitjamnong1*, Chachchaya Thunyaratchatanon2
1
Department of Petroleum Technology, Faculty of Industrial Education and Technology,
Rajamangala University of Technology Srivijaya, Muang, Songkhla 90000, Thailand
2
The Petroleum and Petrochemical College, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
*E-mail: alekjakkrapong@gmail.com
Abstract:
The catalytic behavior of Pd-Mg/SiO2 for the partial hydrogenation of diunsaturated
FAMEs (C18:2) to improve the oxidative stability of soybean oil-derived biodiesel was
studied. The hydrogenation activity and cis-monounsaturated FAME (cis-C18:1) selectivity
of the catalyst were compared with that of the pure silica support (SiO2) at various C18:2
conversion levels, i.e., 12% and 36%. The SiO2 and Pd-Mg/SiO2 catalyst were characterized
using X-ray diffraction (XRD), transmission electron microscopy (TEM), N2 adsorption-
desorption, X-ray photoelectron spectroscopy (XPS), and CO pulse chemisorption. The
FAME composition, oxidative stability and cold flow properties of biodiesel and partially
hydrogenated biodiesel were determined using gas chromatography (GC-FID), Rancimat
method, and cold flow testing. The contents of FAME composition in partially hydrogenated
biodiesel using SiO2 was hardly altered during 4-h reaction owing to the lack of palladium
(Pd) active sites. At 12% C18:2 conversion, the hydrogenation rapidly performed due to an
abundance of the vacant Pd sites, leading to the high turnover frequency (TOF; 24228 h-1);
however, it was then decreased at 36% C18:2 conversion (16863 h-1). The amount of cis-
C18:1 and trans-C18:1 was increased with increasing the hydrogenation of C18:2 content.
The oxidative stability of partially hydrogenated biodiesel using Pd-Mg/SiO2 was noticeably
improved.
1. Introduction monounsaturated FAME-rich fuel (cis-

Biodiesel or fatty acid methyl ester C18:1) is being more preferable
(FAMEs) is the promising alternative energy composition. Partial hydrogenation is
which generally produced from vegetable employed to transform polyunsaturated
oils and animal fats via transesterification FAMEs to monounsaturated FAME.3-4
with a methanol in the presence catalyst. It The aim of this work is focus on the
is non-toxic, biodegradable, and low catalytic performance and cis/trans-C18:1
emission profiles compared to petro-diesel.1 selectivity using Pd-Mg/SiO2 in the partial
Properties of biodiesel strongly depend on hydrogenation of soybean oil-derived
the fatty acid profile in the feedstock, which biodiesel. In addition, oxidative stability and
is identical to that of the resulting biodiesel. cold flow properties, i.e., could point and
Polyunsaturated fatty esters cause low pour point, were also investigated.
oxidation resistance, leading to life-storage
shortening as well as low cetane numbers. 2. Materials and Methods
Conversely, the high level of fully saturated 2.1 Materials
fatty esters in biodiesel exhibits poor cold Soybean oil, purchased from Thai
flow properties.2 Apart from the level of vegetable oil Public Company Limited, was
double bond, the cis-trans isomer is also applied as feedstock for transesterification
important. Owing to the higher melting due to the high level of linoleic (C18:2).
point of trans-isomer compared to that of Tetraamminepalladium (II) chloride
the corresponding cis-isomer, the cis- (Pd(NH3)4Cl2.xH2O), received from N.E.
Chemcat Corparation, Japan, was used as a and desorption isotherms of nitrogen using a
Pd precursor. Magnesium nitrate Quantachrome Autosorb-1MP. Prior to
hexahydrate (Mg(NO3)2.6H2O), bought from analysis, the volatile species adsorbed on the
Sigma-Aldrich Pte Limited, was applied as a catalyst surface were eliminated by
Mg modifier. A commercial mesoporous degassing the catalyst in vacuum
silica (SiO2-Q30), used as a support atmosphere at 250 °C overnight. The
material, was obtained from Fuji Silysia principle of the Brunauer-Emmett-Teller
Chemical Company Limited. (BET) was applied to analyze the specific
2.2 Catalyst preparation surface area (SBET) and average pore
The Pd-Mg/SiO2 was prepared by an diameter (DAvg). The total pre volume
incipient wetness impregnation. Firstly, SiO2 (VTotal) was obtained at a relative pressure of
support was dried in an oven overnight to P/P0 = 0.995.
remove moisture. The SiO2 was then The metal dispersion on the catalyst
impregnated with an aqueous solution was analyzed by CO pulse chemisorption
containing Pd(NH3)4Cl2.xH2O under technique using temperature-programmed
vacuum for 24 h. The total amount of Pd desorption/reduction/oxidation (TPD/R/O),
loading was 1 wt.%. The catalyst was dried Ohkura R6015 model. Firstly, the prepared
in a vacuum oven at 110 °C for 6 h. Next, catalyst was reduced in a flow of hydrogen
the catalyst was calcined under O2 at 300 °C for 4 h, and then helium was
atmosphere at 300 °C with a heating rate of purged for cooling. Pd dispersion was
0.5°C/min and flow rate of 500 mL/min for calculated by assuming a stoichiometry of
3 h. After that, the Mg precursor (4 wt.%) CO:Pd = 1:1.
was doped on the calcined catalyst using the The surface element and composition
same previous method. The Pd-Mg/SiO2 was investigated using X-ray photoelectron
catalyst was then calcined at 400 °C for 3 h spectroscopy (XPS). The spectra were
and reduced under H2 gas flow of 100 recorded on a Kratos Axis Ultra DLD which
mL/min with a heating rate of 5°C/min at carried out by a monochromatic Al Kα
400 °C for 2 h. (1486.6 eV) source at 15 kV. The XPS
2.3 Catalyst characterization spectra were calibrated using the binding
The composition of the catalysts was energy band of C 1s at 284.6 eV.
characterized using X-ray diffraction 2.4 Transesterification of soybean oil
(XRD). The diffraction patterns were The transesterification was
obtained from a Rigaku DMAX 2200 HV, performed in three-neck round-bottom flask.
which performed using CuKα radiation (λ = First, soybean oil was heated at 60 °C and
1.5405 Å) at 40 kV and 30 mA. The then the mixture of methanol (molar ratio of
catalysts were analyzed in a continuous methanol to oil was 9:1) and KOH (1 wt.%
mode, scan step rate of 1º/s, and 2-theta compared to oil) was added and stirred
range of 10-90º. together at 300 rpm for 1 h. Upon completed
The micro-structure of the catalysts reaction, the solution was poured into a
was investigated using Transmission separation funnel and formed two phases
electron microscopy (TEM), a JEOL JEM- completely within overnight. The upper
2100 model. The catalyst was dispersed in phase, which was mainly composed of
ethanol and treated by an ultrasonic biodiesel, was washed with distilled water at
sonicator for 30 min. The prepared solution 60 °C several times to remove residual
was dropped on the carbon film coated on methanol and KOH. Soybean oil-derived
copper grid and then dried under room biodiesel was stored in the glass bottom
temperature overnight. contained sodium sulfate anhydrous
The specific surface area and the (Na2SO4) to remove remaining washed
porosity were determined by the adsorption water.
2.5 Partial hydrogenation of soybean oil ASTM D2500 and ASTM D97,
biodiesel respectively.
Partially hydrogenated FAME of
soybean oil-derived biodiesel was carried 3. Results and Discussion
out in stainless steel semi-batch reactor at 80 3.1 Catalyst characterization
°C and hydrogen pressure of 0.4 MPa. The The X-ray diffraction patterns of
1.304 g of reduced catalyst was placed into SiO2 and reduced catalyst are illustrated in
the reactor containing 130.4 g of biodiesel (1 Figure 1. The diffraction pattern of SiO2
wt.% of catalyst compared to biodiesel). The exhibited a single broad peak that is indexed
reactor was purged with N2 for air removal. to the amorphous silica support, whereas
Next, H2 was flowed into the reactor with that of Pd-Mg/SiO2 presented at least four
the flow rate of 150 mL/min and maintained distinct peaks corresponding to metallic
at 0.4 MPa. The stirrer was applied at 500 palladium (Pd0), one weak peak of
rpm to perfectly mix the biodiesel, solid magnesium (Mg0) as well as the other two
catalyst, and H2 gas. The temperature was peaks of magnesium oxide (MgO).
gradually increased to 80 °C and the first
product was collected. The subsequent
products were kept every 30 min during 4-h
reaction. The compositions of biodiesel and
partially hydrogenated biodiesels were
analyzed by a Hewlett Packard gas
chromatograph 5890 Series II equipped with
a flame ionization detector (GC-FID). The
conditions were applied as carrier gas with a
flow rate of 70 mL/min, the injector
temperature was set at 200 °C with a split
ratio of 75:1, and detector temperature was
230 °C. A 0.2 µL of sample was injected at
an oven temperature of 155 °C after an
isothermal period of 20 min. Then, the GC
oven was heated up to 220 °C with a heating
rate of 2 °C/min and held for 10 min with a Figure 1. XRD patterns of SiO2 and reduced
one cycle running time of 62.5 min. FAME Pd-Mg/SiO2
compositions were specified by reference to
the retention time. The TEM image of the Pd-Mg/SiO2
2.6 Biodiesel analysis catalyst is shown in Figure 2. Although the
The oxidative stability of feed and presence of dark region could be supposed
partially hydrogenation biodiesels were that the metal impregnation was successful
determined using the Metrohm 743 (Figure 2A), the identification of metal type
Rancimat according to EN 14214 standard was rather difficult. Thus, EDS technique
test method. The samples were heated to 110 was used to evaluate the existence of Pd and
°C under air stream with a flow rate of 10 Mg particles in the prepared catalyst, as
L/h. The volatile compounds were shown in Figure 2B. The TEM result was
transferred to conductivity cell containing well conform to the XRD analysis.
DI water. The inflection point was presented The metal dispersion and metal size
as the induction period (IP, h). The cold is listed in Table 1, the metal dispersion and
flow properties, which are cloud point (CP) metal size were 1.93% and 25.2 nm,
and pour point (PP), of biodiesel samples respectively. The SBET, VTotal, and DAvg of
were investigated using a CPP 5Gs SiO2 support were 106 m2/g, 1.3 mL/g, and
automated CP and PP analyzer according to 47.5 nm, respectively (Table 1). For reduced
Pd-Mg/SiO2 catalyst, VTotal and DAvg were particles were successfully impregnated on
slightly decreased compared to that of pure the support.
SiO2, which was elucidated that metal
Figure 2. TEM photograph of (A) Pd-Mg/SiO2 and (B) TEM-EDS of Pd-Mg/SiO2
Figure 3. XPS spectra of (A) Pd 3d of reduced Pd-Mg/SiO2, (B) O 1s of reduced Pd-

Mg/SiO2, and (C) Mg 2p of reduced Pd-Mg/SiO2
Table 1. Characteristics of catalysts and fuel properties of both hydrogenated biodiesel

Catalyst SBET VTotal DAvg Dispersion Metal Fuel properties of partially
(m2/g)a (mL/g)b (nm)a (%)c size hydrogenated biodiesel
(nm)c Oxidative CP PP
stability (h)d (°C)e (°C)f
Feed SO-
– – – – – 2.2 4.0 −2
FAMEs
SiO2
106 1.3 47.5 – – 2.2 4.3 −2
support
Pd-Mg/SiO2 107 1.2 43.6 1.93 25.2 7.3 4.5 −1
a
Determined by the BET method.
b
Determined by N2 adsorption at a relative pressure (P/P 0) = 0.995.
c
Measured by CO pulse chemisorption.
d
Determined by the Rancimat method according to EN 14214.
e
Evaluated according to ASTM D2500.
f
Measured according to ASTM D97.
Table 2. FAMEs composition and turnover frequency (TOF) at different C18:2
conversion levels of partially hydrogenated biodiesel using Pd-Mg/SiO2 catalysts
Feed SO-
SiO2a Pd-Mg/SiO2b Pd-Mg/SiO2c
FAMEs
FAME composition (%)a
C18:3 5.54 4.98 4.25 1.49
C18:2 52.58 49.09 46.47 33.67
C18:1 23.84 27.09 31.23 46.21
C18:0 3.73 3.79 3.93 4.23
Ratio of cis-C18:1/total C18:1 1 1 0.95 0.84
TOF (h−1) – – 24228 16863
a
When 4-h reaction was completed.
b
When C18:2 conversion was approximately 12%.
c
When C18:2 conversion was approximately 36%.
To identify the surface composition adsorption to the metal surface, leading to

of catalyst, the XPS spectra were applied as the high turnover frequency. The ratio of
displayed in Figure 3. According to the XPS cis-C18:1 to total C18:1 was displayed in
spectra, the binding energy of Pd 3d was Table 2. At 12% C18:2 conversion, the cis-
mainly found around 336.8 eV and 340.4 C18:1 selectivity was close to 1 due to the
eV, indicating to the presence of Pd0 (Figure low amount of trans-C18:1; however, it was
3A). In addition, the O 1s spectra exhibited decreased at high C18:2 conversion. To
the two predominant peaks of SiO2 and PdO more clarify, the amount of both cis-C18:1
(Figure 3B). The binding energy of Mg 2p and trans-C18:1 at low and high C18:1
spectra could be corresponded to the conversion level are shown in Figure 4.
magnesium phase in the form of According to this figure, it could be
MgO/Mg(OH)2 at 50.4 eV (Figure 3C). It noticeable that the level of trans-C18:1 was
could be implied that the deposition of unavoidably increased along with cis-C18:1
metallic palladium (Pd0) and magnesium formation during the reaction proceeding. It
(Mg0) was formed. was not mainly formed owing to the cis- to
3.2 Catalytic activity trans-isomerization.
The catalytic activity, represented in
terms of TOF, and the cis-C18:1 selectivity
using Pd-Mg/SiO2 was evaluated by the
different conversion levels in the 4-h partial
hydrogenation reaction (Table 2).5 The
catalytic performance was compared under
two C18:2 conversions, i.e., 12% and 36%.
The highest catalytic performance with a
TOF of 24228 h-1 was obtained at the C18:2
conversion is around 12% within 1-h
reaction time in the partial hydrogenation,
due to the presence of greater numbers of
vacant Pd active sites and well dispersion.
However, it was decreased at 36% of C18:2
conversion at around 4-h reaction time
(16863 h−1) because the catalyst is slightly
deactivated during the partial hydrogenation Figure 4. FAMEs Compositions of both
process. It evidently showed that an percentage of cis-C18:1 and trans-C18:1 in
abundance of active sites and well hydrogenated biodiesel after C18:2
dispersion might enhance the hydrogen conversions were approximately 12% and
36%.
3.3 Biodiesel properties levels were studied. At 12% conversion, the
The oxidative stability, cloud point catalyst exhibited the high TOF due to the
(CP), and pour point (PP) of synthesized abundance of active sites; however, it was
feed biodiesel and partially hydrogenated then decreased with the increasing of the
biodiesel are summarized in Table 1. These reaction time. Besides, the trans-C18:1 was
properties of biodiesel directly depended on inevitably formed along with the production
the degree of unsaturated fatty acid methyl of preferable cis-C18:1. The oxidative
esters. The oxidative stability of feed stability of biodiesel was clearly improved
biodiesel was merely 2.2 h due to the high using partial hydrogenation.
polyunsaturated FAMEs, especially C18:2
(around 52%). After 4-h reaction, the Acknowledgements
partially hydrogenated biodiesel using SiO2 This research was financially
as a catalyst revealed the same oxidative supported by Rajamangala University of
stability (2.2 h) as that of feed biodiesel Technology Srivijaya and The Petroleum
which resulted from the low catalytic and Petrochemical College, Chulalongkorn
performance of pure SiO2. For Pd-Mg/SiO2, University. We also gratefully National
it exhibited the excellent oxidative stability Institute of Advanced Industrial Science and
due to the high ability to diminish the Technology, Japan and National Metal and
amount of unsaturated compounds. For this Materials Technology Center (MTEC) for
result, it apparently showed that the lower providing material and instrumental
composition of C18:3 and C18:2 provided analysis.
the better fuel performance with the higher
oxidative stability.6 Meanwhile, cloud point References
(CP) and pour point (PP) of the partially 1. Hadis, B.; Nastaran, P. J. Mol. Liq.
hydrogenated biodiesel using Pd-Mg/SiO2 2016, 223, 10–15.
catalyst was rather higher than that of feed 2. Hee, Y. S.; Jae, H. R.; Seong, Y. B.;
biodiesel and partially hydrogenated Young, C. K. J. Supercrit. Fluids 2013,
biodiesel using SiO2 owing to the increasing 82, 251–255.
the saturated fatty acid level. Apart from the 3. Plaifa, H.; Apanee, L.; Nuwong, C.;
level of saturated compounds, the existence Yuji, Y. Energy Fuels 2017, 31, 8202–
of the higher level of trans-isomer might be 8209.
the crucial factor for worsening the cold 4. Natthida, N.; Apanee, L.; Nuwong, C.;
flow properties of partially hydrogenated Yuji, Y. Appl. Catal., A. 2012, 441–442,
biodiesel. 72–78.
5. Shin, Y. C.; Lalita, A.; Takehisa, M.;
4. Conclusion Yohko, A.; Makoto, T.; Yuji, Y.;
The Pd-Mg/SiO2 presented the more Chiraphat, K.; Phunthinee, S.; Supranee,
catalytic ability than that using the pure SiO2 L. C. R. Chim. 2016, 19, 1166–1173.
support owing to the presence of Pd active 6. Chachchaya, T.; Jakkapong, J.; Apanee,
species. To consider the performance of Pd- L.; Natthida, N.; Nuwong, C.; Yuji, Y.
Mg/SiO2, the various C18:2 conversion Appl. Catal., A. 2016, 520, 170–177.
Adsorption of methylene blue dye from water using Cymbopogon citratus
Vanida Chairgulprasert*, Sakeeyah Masamae
Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani Campus,
Mueang, Pattani 94000, Thailand
*E-mail: vanida.c@psu.ac.th
Abstract:
The adsorption of methylene blue dye onto a low cost adsorbent of Cymbopogon
citratus has been studied. Removal efficiency was optimized with respect to initial dye
concentration (5-25 ppm), contact time (0-140 min), adsorbent dosage (0.2-1.0 g), pH (4-12)
and temperature (30-60C). The maximum efficiency (87.7%) was achieved by using 0.2 g
Cymbopogon citratus in the methylene blue dye concentration of 15 ppm at neutral
condition and 30C, over a period of time 120 min. The adsorption isotherm has been
analyzed using the Langmuir and Freundlich models. It was found that the data were best
fitted to Freundlich model (R2 = 0.926). The adsorption kinetics followed pseudo-second
order. The thermodynamic parameters have also been evaluated. The enthalpy change was -
13.357 kJ/mol indicating of an exothermic adsorption and the entropy change was -0.034
kJ/mol•K showing that the process was in well arrangement by adsorbing the dye molecule
onto the adsorbent surface. The computed Gibbs free energies were -3.055, -2.715, -2.375
and -2.035 kJ/mol at 30, 40, 50, and 60C respectively showing feasible and spontaneous
processes.
1. Introduction The leaves of lemongrass were

Synthetic dye is a dangerous collected from a local market in Pattani
pollutant in waste water. It is toxic, Province, Thailand. They were washed with
carcinogenic and mutagenic to living distilled water, and then oven drying at 60
creatures.1 The effluents from various C. The dry materials were ground into
industries including paper, textile and powders and kept in air tight plastic
cosmetics using synthetic dyes could cause container prior to the experiments.
water pollution. Even, their effluents have 2.2 Batch adsorption studies
been treated, they could still harm being The batch adsorption experiment
health. Adsorption is one of the most was carried out with MB (100 mL 5-25
effective technique to eliminate dyes from mg/L) with the pH adjusted as desired (4-
water.2 Therefore, this paper reveals the use 12). Next, the adsorbent (0.2-1.0 g) was
of Cymbopogon citratus, known as added and the mixture was shaken at 250
lemongrass, leaf powders to adsorb rpm for the required time (5-140 min).
methylene blue dye from water. After shaking, the suspension was
The adsorption capacity was centrifuged and filtered. Residual MB in the
optimized by varying the parameters of pH, filtrate was analyzed using a UV-Vis
contact time, initial dye concentration, spectrophotometer at a wavelength of 665
adsorbent dose and temperature. Adsorption nm. The adsorption capacity (qt, mg/g) and
isotherms, kinetics and thermodynamics the percentage uptake (% sorption) were
were also studies to assess the experimental calculated from equation 1 and 2.
data. qt = (Ci-Ct)V/W (1)
Sorption (%) = [(Ci-Ct)/Ci]*100 (2)
2. Materials and Methods Where Ci and Ct (mg/L) are the initial and
2.1 Preparation of adsorbent material final MB concentrations, respectively. V
(mL) is the volume of the solution and W time, respectively. The K1 and K2 are the
(g) is the weight of the adsorbent. first and second order rate constants.

3.1 Effect of pH
As shown in Figure 1, a little change
of MB uptake was observed from pH 4 to
12 (6.44-6.58 mg/g, 85.7-87.7%). However,
the maximum efficiency of MB adsorption
by Cymbopogon citratus powders occurred
at the pH 7.
Figure 2. Effect of contact time and

concentration (5-25 mg/L) on MB
adsorption by Cymbopogon citratus [dose
0.2 g, pH = 7.6, 30 C]
It was revealed that all studied MB

adsorptions with lemongrass powders failed
to fit the pseudo-first-order due to the low
regression coefficient (R2). Instead, the
adsorption data were well described for the
Figure 1. Effect of pH (4-12) on MB pseudo-second-order plot giving high R2 at
adsorption by Cymbopogon citratus [initial all initial dye concentrations used (Figure
MB 15 mg/L, dose 0.2 , contact time 120 3).
min, pH = 7, 30 C]
3.2 Effect of contact time and initial

concentration
At the first 5 min, the adsorption
occurred rapidly and then gently slowed
until equilibrium was obtained at 80 min for
all adsorptions as shown in Figure 2. In
addition, the removal capacity of MB onto
lemongrass powders depended on initial dye
concentration. The adsorption capacity Figure 3. Pseudo-second-order plot on MB
increased with the initial MB concentration. removal by Cymbopogon citrates
This is explained from the high driving
force for mass transfer of high initial Table 1. Pseudo-second-order parameters
concentration that promotes adsorption.3 of MB adsorption by Cymbopogon citratus
3.3 Adsorption kinetics Conc.
Two kinetics models of pseudo-first- Pseudo-second-order parameters:
(mg/mL)
order and pseudo-second-order were used to qe(exp) qe(cal) K2 R2
analyze the data obtained as shown in 5 1.86 1.87 0.23 0.9991
Equation 3 and 4.4,5 10 4.12 4.15 0.01 0.9993
log (qe-qt) = log qe - K1. t/2.30 (3) 15 6.15 6.21 0.06 0.9994
20 8.10 8.20 0.04 0.9991
t/qt = 1/K2qe2 + t/qe (4)
25 9.94 10.10 0.02 0.9992
Where qt and qe corresponded to the amount
of MB adsorbed onto the lemongrass
powders at time t (min) and equilibrium
The K2 constants and calculated qe, that 0.2 g of adsorbent contains sufficient
obtained from slopes and intercepts were binding sites for maximum MB adsorption.
reported in Table 1. All calculated qe values
agreed with the experimental values,
confirming that the adsorption data are well
described by this model. It was also noticed
that the K2 constant values decreased with
increasing of initial MB concentration
implying that the removal rate of MB
depend on initial MB concentration.
3.4 Adsorption isotherms
Langmuir6 and Freundlich7
isotherms were applied to model the MB
adsorption. The linear equations of both Figure 4. Freundlich plot on MB removal
isotherms were represented in Equations 5 by Cymbopogon citratus [dose 0.2 g, pH =
and 6. 7.6, 30 C]
Ce/qe = 1/qmKL + Ce/qm (5)
log qe = log KF + 1/n log Ce (6)
Where qe (mg/g) is the mass of MB
adsorbed per unit mass of ash, Ce (mg/L) is
the MB equilibrium concentration in
solution, qm (mg/g) is the monolayer
adsorption capacity and KL is the Langmuir
constant. KF (L/g) is the Freundlich constant
associated with the adsorption capacity and
n is an empirical parameter related to the
adsorption intensity and surface
heterogeneity.
The MB removal data could not be Figure 5. Effect of adsorbent dose (0.1-1.0
modelled to the Langmuir plot due to a g) on MB adsorption by Cymbopogon
lower R2. On the other hand, it agreed well citratus [initial MB 15 mg/L, contact time
to the Freundlich model with R2 = 0.9260 120 min, pH = 7, 30 C]
(Figure 4) attributing that the adsorbate
surfaces are heterogeneous with a lot of 3.7 Effect of temperature and
different effective function groups to bind adsorptionthermodynamics
MB cations resulting in a better adsorption It was found that increasing
at higher concentration. The plot exhibited temperature of the MB solution led to
1/n = 1.14 suggesting that the adsorption is decreasing of adsorption capacity. The
cooperative process. thermodynamic parameters of H and S
3.6 Effect of adsorption dose were determined from the slope and
The different dose of adsorbent (0.2- intercept of the plot between log (qe/Ce) and
1.0 g) was applied to evaluate the MB 1/T using equations 7 (Figure 6).9 The value
removal. From Figure 5, the result showed of G was subsequently obtained from
that using 0.2 g of Cymbopogon citratus equation 8.
was the most effective dose to remove MB log qe/Ce = S/2.303R - H/2.303RT (7)
at initial concentration of 15 mg/L from G = H-TS (8)
water at pH 7, 30 C. Increasing adsorbent Where R is the gas constant (8.314 J/mol.K)
dose over 0.2 g resulted in the decrease of and T is the Kelvin temperature (k).
MB removal capacity. 8 It was suggested The MB adsorption by Cymbopogon
citratus powders is a favorable process with
negative values of G (Table 2). In model. In addition, the MB was adsorbed
addition, the change in enthalpy is a small spontaneously with an exothermic process
negative value corresponding to the onto Cymbopogon citratus.
exothermic process and physical
adsorption.10 Nevertheless, the negative Acknowledgements
entropy change showed a decrease in We would like to thank Division of
disorder at the solid-liquid interface during Chemistry, Department of Science, Faculty
the adsorption.11 of Science and Technology, Prince of
Songkla University, Pattani Campus,
Thailand, for support with chemicals and
equipment.
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citratus 1906, 57, 385–471.
G(kJ/mol), T (C) H S 8. Hanif, M. A.; Nadeem, R. H.; Bhatti,
(kJ/mol) (kJ/mol.K) N.; Ahmad, N. R.; Ansari, T. M. J.
30 40 50 60 Hazard. Mater. 2007, 139, 345–355.
-3.06 -2.72 -2.38 -2.04 -13.36 -0.034 9. Smith, J. M.; Van Ness, H. C.
Introduction to Chemical Engineering
4. Conclusion Thermodynamics, 4th Ed, McGraw-
Cymbopogon citratus could be used Hill, Singapore, 1987.
as a low cost adsorbent to remove MB dye. 10. Djordjevic, D.; Stojkovic, D.;
The adsorption capacity depends on pH, Djordjevic, N.; Smelcerovic, M. Ecol.
initial MB concentration, adsorbent dose Chem. Eng. 2011, 18 (4), 527–536.
and temperature. The adsorption was also 11. Özcan, A.; Öncü, E. M.; Özcan, A. S.
satisfactorily described by the second order Colloids Surf. A. 2006, 277, 90–97.
mechanism and followed the Freundlich
Simulation of in situ removal of ethanol from culture broth using
microbubbles
Rungrote Kokoo1*, Matthana Khangkhamano2
Department of Chemical Engineering, Faculty of Engineering, King Mongkut’s University of Technology
1
North Bangkok, Bangsue, Bangkok 10800, Thailand

2
Department of Mining and Materials Engineering, Faculty of Engineering, Prince of Songkla University,
*E-mail: rungrote.k@eng.kmutnb.ac.th
Abstract:
Ethanol is a renewable and clean biofuel mostly produced via fermentation process. The
productivity of ethanol can be increased when the ethanol was removed from the culture
broth to diminish the inhibition effect on growth rate of microorganisms. In this study, the
conventional processes and in situ removal processes were modeled based on Polymath and
Aspen Plus. During the process of in situ removal, hot air microbubbles (>78 oC) was
generated and fed to ethanol-water mixture. Ethanol, which is lower boiling point than water,
was then evaporated from the mixture into microbubbles. The ethanol in microbubbles was
removed when they floated over the mixture. The rate equations and kinetic parameters of the
formation of by-products i.e., glycerol and acetaldehyde were formulated using Polymath.
Polymath was also used to estimate the kinetic parameters with the rate unit of mol/L·hr.
These parameters were then used in Aspen Plus to estimate the increase of productivity and
energy consumption. In comparison with the conventional processes, the simulation results
showed that the productivity of ethanol from the in situ removal processes increased by 1.5%.
The energy removal at the fermenter section increased by 38.83%. However, the overall
energy consumption including fermentation and separation sections of the in situ removal
processes decreased by 9.73%.
1. Introduction Ethanol is currently produced via

Due to the increasing of the world fermentation process based on the growth of
energy demand, limited fossil resources and microorganisms on biomass materials in an
environmental concerns, scientists are anaerobic environment. Several techniques
seeking some environmental technologies to are employed for conducting the ethanol
produce energy from the renewable sources. fermentation processes i.e., non-continuous
Ethanol is an alternative renewable energy and continuous.1 The non-continuous
that can be produced via fermentation process processes offer lower productivity in
from many biomass materials.1 For comparison with the continuous processes
environmental issue, the use of ethanol does as a result of the idle time. While continuous
not contribute to global warming because processes present a series of advantages
carbon dioxide emitted from its combustion including lower of construction cost of the
can be recycled by plants. The SOx, NOx, fermenter, lower requirements of
and CO emissions are quite low with no dust maintenance and operation, better control of
release. In addition, the octane value of the process.3
ethanol is very high that can be blended with To increase the productivity, the
gasoline in various ratios to produce continuous ethanol removal during the
gasohol, which are currently used in many fermentation can be utilized. This is an
countries, especially Brazil, the United attractive alternative for the intensification of
States and Thailand.2 alcoholic fermentation processes. It can
diminish the inhibition effect on growth rate
of microorganisms when ethanol is removed ethanol-water mixtures by hot microbubble
from the culture broth. Some examples of injection was described by Abdulrazzaq et
the reaction-separation integration processes al.7 Their modeling also shown that the non-
are ethanol removal by vacuum, ethanol equilibrium driving force is kinetically much
removal by gas stripping, ethanol removal more rapid at vaporising ethanol in all liquid
by membranes, and ethanol removal by proportions.8 Moreover, Al-yaqoobi et al.
liquid extraction.4 Gas stripping offers concluded that using microbubble
advantages in terms of its effectiveness and distillation for ethanol removal could
ease of operation.5 Although, these contribute to improving commercial
processes can increase the productivity, viability of ethanol production and other co-
most of them are currently employed in the products of bio-refinery processing.9
laboratory due to the difficulty in industrial The main aim of this research is to
scale. increase the productivity and reducing the
Recently, a rapid evaporation cost of ethanol fermentation using
methodology with hot and dry microbubbles microbubble distillation technology for the
was proposed by Zimmerman et al.6 They recovery of ethanol from fermentation
stated that introducing dry fine bubbles, not broths. A conventional technique and an
even microbubbles, would strip the water alternative fermentation technique using
from the reacting mixture. Microbubbles microbubbles for ethanol removal as shown
also vaporises water dramatically faster than in Figure 1a and 1b, respectively, have been
hot, dry fine bubbles. Conceptually, modeled using Polymath 6.10 (the Fogler
removing of ethanol would drive the Chemical Reaction Engineering) and Aspen
reaction to competition according to Le Plus V8.8. The productivity and energy used
Chatelier's principle for equilibrium of such models have been investigated and
reactions. The strongly non-equilibrium compared.
preference for ethanol vaporisation in
Conc. EtOH + Air
To distillation
Feed Feed Broth Bubble

Heat Column
exchanger
Heat
exchanger Hot air
Fermenter Fermenter
To distillation To distillation
a b
Figure 1. Process of ethanol production (a) conventional process (b) in situ removal process
using microbubbles
2. Model Definition time before feeding to the bubble column.

Both Polymath and Aspen Plus were The results obtained from Polymath were
employed in this study. Polymath was used then utilized to model the fermentation
to estimate the kinetic parameters. It was processes in order to find the productivity
also used to find the optimum fermentation and energy consumption using Aspen Plus.
2.1 Polymath from glucose, respectively; x, s, p and g are
The kinetic model of alcoholic the concentrations of yeast, glucose, ethanol,
fermentation taken from Leksawasdi et al.10 and glycerol, respectively. The values of the
was added into Polymath. This model kinetic parameters obtained from
describes the co-fermentation of two main Leksawasdi et al.10 and Polymath with the
substrates i.e., glucose and xylose using
Zymomonas mobilis strain as different rate units were listed in Table 1.
microorganisms. Pure glucose was, 2.2 Aspen Plus
however, used as substrate in this study. The In this section, Aspen Plus was used
rate equations can be classified into three to model the fermentation process including
groups including the growth rate of the conventional process and the in situ
Zymomonas mobilis, the uptake of glucose, removal process. To obtained the reaction
and the production of ethanol as represented rate with the unit of mol/L·hr, the kinetic
in Eq. 1 to Eq. 6. Moreover, during the parameters with the unit of molarity (mol/L)
fermentation, by-product like acetaldehyde from Polymath must be used. The equations
and glycerol are normally formed. In this of the reaction rates have to be then written
study, the formation of by-products was in FORTRAN statements in the Calculation
assumed that the loss of glucose just block. The reactions used in the fermenter
converts to both acetaldehyde and glycerol. (CSTR) were listed in Eq. 9 to Eq.11. The
The rate equations of acetaldehyde and yeast cells are assumed to have an empirical
glycerol formation have been added for formula (CH1.8O0.5N0.2) derived from the
mass balance as shown in Eq. 7 and Eq. 8.3 elemental analysis of cell biomass.3
Growth rate of Z. mobilis: C6 H 12 O6  1.14 NH 3 
(9)
5.71CH 1.8O0.5 N 0.2  2.57 H 2 O  0.29CO2
 rx x
dx
(1)
dt C6 H 12O6  2C2 H 5OH  2CO2 (10)

rx   max 
s  
  1  p  Pix   K ix


 (2) C6 H 12O6  C3 H 5 OH 3  C 2 H 4 O  2CO2 (11)
K s   Pmx  Pix  K s
  ix 
 sx   This model was assumed that
Glucose uptake: temperature has no effect on the growth rate
of Z. mobilis. In addition, as supported by
 rs x
ds
(3) both CFD and experimental results,2-9 the
dt bubble column in the in situ removal
 s   p  Pis   K is  processes can be replaced by the Separator
rs  q s ,max    1    (4) unit in Aspen Plus. Ethanol with purity of
   K s 
 K ss  s   Pms  Pis   is  92.8 % by volume was achieved with a
Ethanol formation: slight increase in the temperature (4oC) of
the liquid mixture.
dp
dt
 
 rp x (5) A feed stream contains ammonia
(0.17 g/L), glucose, yeast, ethanol, and
 s   p  Pip   K ip  water with their compositions represented in
rp  q p,max    1    (6) Table 1. Total flow rate was set at 1 L/min.
 K sp  s   Pmp  Pip   K ip  s 
      The temperature and pressure of such stream
Glycerol formation: and the reactor were also set at 37oC and 1
atm, respectively. The fermenter volume
dg
 
 rg x (7) was equally set at 421.5 L for both
dt processes. NRTL-RK with Henry’s Law was
used as the property method. The recycle
   p  
n
 s stream of the in situ removal process mainly
rg  q g ,max   1   (8)
 K sg  s    pmg   contains both glucose and water.
     
Where rx are the cell growth rate from 3. Simulation Results and Discussion
glucose; rs is the glucose consumption rate; 3.1 Polymath
rp is the ethanol production rate from Although the kinetic data obtained
glucose, rg is the glycerol production rate from Leksawasdi et al10 can be used to plot
the concentration of substrates in the
fermenter, such data did not provide the 3.2 Aspen Plus
kinetic parameters for glycerol formation. The simulation results obtained from
Based on mass balance, it might be assumed the conventional process show that
that the formation of glycerol results from fermentation process is exothermic reaction.
the loss of glucose. For isothermal condition, heat must be
Table 1. Initial values for the fermentation removed from the fermenter of 291 W.
of Zymomonas mobilis on various glucose Molar flow rates of ethanol, yeast, and
media with x = 0.028 g/L, s = 25.08 g/L, p glycerol before feeding to distillation
= 1.41 g/L, and g = 0 g/L column are 0.2745, 0.0307, 0.0057 mol/min,
respectively. The volumetric flow rate of
Kinetic Rate unit Rate unit product stream is 1.024 L/min. The
parameters (g/L·hr)10 (mol/L·hr)* conversion of glucose at the outlet stream of
µmax 0.31 0.050 the fermenter is 0.92.
For the in situ removal process, the
Ksx 1.45 0.009
fermenter was set of 421.5 L which equals
Pmx 57.2 1.500 to the conventional process. Ethanol can be
Kix 200 0.932 removed from the culture broth through gas
stripping that makes possible the increase in
Pix 28.9 1.380 the concentration of glucose in the
qs,max 10.9 2.908 fermenter. The volumetric flow rate of
Kss 6.32 0.007
CONETOH stream as shown in Figure 3 is
0.0045 L/min which mostly contains ethanol
Pms 75.4 40.977 and water. Mass fraction of ethanol and
Kis 186 0.382 water is 0.928 and 0.072, respectively. The
volumetric flow rate of RECYCLE stream is
Pis 24.6 14.980
0.4425 L/min which mainly contains water
qp,max 5.12 5.310 and glucose. The volumetric flow rate of
Ksp 6.32 0.007 product stream is 1.035 L/min. The
conversion of glucose is identical to the
Pmp 75.4 40.977 conventional process.
Kip 186 0.382 The advantage of using the in situ
Pip 42.6 14.980 removal process is that the volumetric flow
rate of FEED stream must be increased by
qg,max 0.66* 0.177 1.5% to maintain the same conversion with
Ksg 16.97* 0.094 the conventional process. This is because
small amount of ethanol was separated at the
Pmg 12.84* 0.279
bubble column. Another advantage is the
n 17.40* 17.405 reduction of utility cost for separation of
* obtained from Polymath mixtures after the fermentation. This is due
to the lower concentration of ethanol in the
PRODUCT stream.
Therefore, the concentration of As mentioned, during the separation
glycerol can be plotted as shown in Figure using bubble column, the temperature of the
2a. Polymath was then used to solve the liquid stream increases by 4oC. Heat
kinetic parameters as represented in Eq. 8 removed from the in situ removal process of
and the result was shown in Table 1 (R2 = 404 W. Although more heat has to be
0.99). Before adding these kinetic removed from the fermenter when the in situ
parameters into Aspen Plus with the rate removal process was operated, this can be
unit of mol/L·hr, the concentration profile compensated in the separation processes.
(mol/L) of substrates in the fermenter have Overall, energy consumption of both
to be plotted as illustrated in Figure 2b. fermentation and separation sections of the
Polymath was also employed to estimate the in situ removal process decreases by 9.73%.
kinetic parameters and the result was
presented in Table 1.
a
Figure 2. Simulation of substrates in the fermenter (a) g/L (b) mol/L
40
0
CONETOH
Temperature (C )
Volume Flow Rate (l/hr) 40

117 BUBBLE
Duty (W att)
GAS
40
27
40 R1
40
HEAT1 27
P3 27
SEP
R2
37
RECYCLE 26
37
37 40
88
37 89 89
HEAT 40
61
MIX 62
P1 P2
FEED
PRODUCT
SPLIT
285
UMIX CSTR
H1
Figure 3. The in situ removal processes of ethanol production
4. Conclusion C. Eds. Elsevier: San Diego, 2015; pp
In this research, Polymath was 311–340.
employed to estimate the rate equations and 2. Pandey, A. in Concise encyclopedia of
kinetic parameters of the formation of by- bioresource technology. Food Products
product with the rate unit of mol/L·hr. Press: New York : London, 2004.
Aspen Plus was used to investigate the 3. Cardona, C. A.; Sanchez, O. J.; Gutierrez,
productivity and energy consumption of L. F. in Process synthesis for fuel ethanol
both the conventional processes and in situ production. Taylor & Francis Group:
removal processes. The productivity of Florida, 2010.
ethanol increases by 1.5%, when the in situ 4. Cardona, C. A.; Sanchez, O. J. Bioresour.
removal processes were operated, while the Technol. 2007, 98 (12), 2415-2457.
energy consumption at the fermenter section 5. Gong, C. S.; Cao, N. J.; Du, J.; Tsao, G.
increases by 38.83%. Conversely, the T. Adv. Biochem. Eng. Biot. 1999, 65,
overall energy consumption including 207–241.
fermentation and separation sections 6. Zimmerman, W. B.; Al-Mashhadani, M.
decreases by 9.73%. K. H.; Bandulasena, H. C. H. Chem. Eng.
Sci. 2013, 101, 865–877.
Acknowledgements 7. Abdulrazzaq, N.; Al-Sabbagh, B.; Rees,
We would like to thank the J. M.; Zimmerman, W. B. AIChE J. 2016,
Department of Chemical Engineering, King 62 (4), 1192–1199.
Mongkut’s University of Technology North 8. Abdulrazzaq, N. N.; Al-Sabbagh, B. H.;
Bangkok for supporting of software and a Rees, J. M.; Zimmerman, W. B. Ind. Eng.
workstation computer. We also thank Chem. Res. 2016, 55 (50), 12909–12918.
Siraphat Temboonsak and Thanarat 9. Al-Yaqoobi, A.; Hogg, D.; Zimmerman,
Sukkaew for helpful discussions. W. B. Int. J. Chem. Eng. 2016, 2016, 1–
10.
References 10. Leksawasdi, N.; Joachimsthal, E. L.;
1. Vasconcelos, J. N. D. in Sugarcane: Rogers, P. L. Biotechnol. Lett. 2001, 23
agricultural production, bioenergy and (13), 1087–1093.
ethanol; Santos, F.; Borém, A.; Caldas,
Effects of the processing on properties of biochars from rice husk and rice
straw pyrolyzed between 500-1800 C
Pranuda Jivaganont*, Phontip Tammawat, Kritkaew Somton, Pimpa Limthongkul
National Metal and Materials Technology Center, Klong Luang, Pathumthani 12120, Thailand
*E-mail: pranudj@mtec.or.th
Abstract:
This study introduces the processing of rice husk and rice straw by-product waste
from rice fields in Nakorn Ratchasrima, Thailand into biochar. Despite varieties of biochar
applications, this study particularly focuses on its usage toward energy applications. The
current state of the study only concerns the physical and structural aspect. Physical properties
of the as-received state and the ground state of the precursors are compared and found that
there is no distinguished difference in the final structures via X-ray diffraction technique.
Both as-received rice husk and rice straw were pyrolyzed at 500 C, 1360 C and 1800 C.
Graphene-layer-like structure, expectedly benefiting the energy applications, can be observed
at pyrolysis temperatures of 1360 C and 1800 C under N2 via Raman spectroscopy
technique. The pre-treatment with NaOH and HCl helps eliminate Si and metal element. This
precursor purification step offers the possibility to obtain pure carbon phase toward the final
products.
1. Introduction between 300-700 C.4,5 At low pyrolysis

With the increasing energy demand temperature, biochar is known to possess
and the concern of by-product waste pure amorphous structure. The graphene-
management, the conversion of those layer-like structure considered to possess
biomass wastes into biochar has been higher crystallinity, are likely to occur at a
considered value-added. Abundant by- higher pyrolysis temperature. Therefore the
product such as rice husk and rice straw are knowledges of the effects of the higher
easily found in many tropical countries, pyrolysis temperatures, which are rarely
especially in south and Southeast Asia. A documented, are expected to benefit our
growing interest in biochar is due to it being interested applications in both phase and
sustainable to improve soil fertility, nutrient structural aspects.
use efficiency and to increase crop growth In this study, we introduce the
and yield.1,2 The processing for these relatively simple processing and fabrication
agricultural applications are however quite of rice husk and rice straw in order to make
simple and cost effective. Other possible biochar, whose structures show potential
biochar applications produced via a more usages in energy applications. Structural
complex processing are well known for and phase evolution of rice husk and rice
energy applications whose major straw subjected to pyrolysis temperatures
functionality is to aid ion transports. The are investigated. Effects of the pre-treatment
preferred structure for this purpose is so- and effects of the pyrolysis under N2 and Ar
called hard carbon, where graphene-layer- atmospheric, in order to get rid of Si and
likes are embedded in amorphous carbon metal elements are also studied.
matrix.
The common pyrolysis temperatures 2. Materials and Methods
to produce biochar were found to be in the 2.1 Material processing
range of 200-1000 C. 3 and often found Raw rice husk and raw rice straw
were obtained from paddy fields in Nakhorn
under N2 atmosphere. Effects of
atmospheres were studied at the pyrolysis
temperature of 1360C. The pyrolysis under
N2 (Bangkok Industrial Gas) was performed
As-received Raw Rice Husk Ground Raw Rice Husk
and compared with Ar gas (Bangkok
Industrial Gas). The clean precursors in their
as-received state were soaked in NaOH
(Alfa Aesar) solution in order to remove Si
and further soaked in HCl (Sigma Aldrich)
solution to remove metal elements. The
As-received Raw Rice Straw Ground Raw Rice Straw pyrolysis temperatures were varied from
500 C to 1800 C. The biochar products
Figure 1. Morphologies of raw rice husk from rice husk without the pre-treatment
and rice straw at its as-received state and with NaOH and HCl were denoted as RH
ground state and TRH for treated rice husk respectively.
Products from rice straw were named in
similar manner as RS for untreated specimen
and TRS for treated rice straw.
2.2 Characterizations
Loss on ignition (LOI) analysis was
As-received RH 500C Ground RH 500C performed in order to investigate the
percentage of moisture and organic matter
content in rice husk and rice straw. Raw
samples were dried and heated up to
1025 C and hold for 4 hrs. Basic physical
properties of the samples such as filled
As-received RS 500C Ground RS 500C density, tap density were characterized.
Figure 2. Morphologies of rice husk and Morphologies were investigated via Field
rice straw at its as-received state and ground Emission Scanning Electron Microscope
state pyrolyzed at 500 C under N2 (FE-SEM Hitachi SU-8030) while the
structures were explored by X-Ray
Ratchasrima province, the north eastern part Diffractrometry (XRD, PANalytical) and
of Thailand. The raw precursors were Raman Spectroscopy (ND-MDT, Ntegra
washed with tap water for 5 times in order to Spectra).
remove the visible contamination such as
dust, soil etc. The washed precursors were 3. Results and Discussion
ultrasonicated in DI water for 15 minutes for 3.1 Physical properties
another 4 times, when the water appeared Basic properties characterization was
clear without visible color. The clean performed for all firing batches. Significant
precursors were dried in the oven at 110 C amount of white ash residues are present for
for 16 hrs. both rice straw and rice husk after LOI tests.
The dried and clean precursors were As LOI percentage indicates the loss of
further processed under their as-received organic matter, generally carbon, hydrogen
state and their ground state via disc grinding and oxygen which are contained as parts of
mill. The grinding process was added in organic molecules, the results reveal that
order to study the effects of the status of the rice straw consists of slightly higher amount
starting materials. The biomass precursors of organic matter than rice husk (LOI 87%
were pyrolyzed at different temperatures VS 84%). This is possible as the
composition of the rice husk is known to
varying from 500 C to 1800 C for 3 hrs
contain the highest amount of ash compared
with the other materials due to the highest (untreated) RH and raw (untreated) RS show
content of silica (up to 90-98%) based similar X-ray diffraction patterns (Figure 4
material in the rice husk.6 and 5 bottom graphs). Three major peaks at
Physical characteristics of the as- 2θ of 35°, 22° and 18° are observed. The
received and ground states are investigated. highest intensity peak at 22° could be
Cross sectional microstructures of the as- determined as the diffraction from
received raw RH in Figure 1 show a grain crystalline cellulose structure, while the
revealing inner cellular compartment as tube smaller and broader peak at 18° represents a
bundles under the ridges. Outer epidermis of less organized amorphous cellulose
the as-received raw RS exhibits more linear structure.7 The small peak near 35° observed
ridge patterns. Subjected to disc grinding, in raw rice husk could possibly result from
the ground raw materials are found to retain cristobalite (SiO2).
most of their original microstructures. Upon firing under different pyrolysis
In Figure 2, bulky piece with rice temperatures between 500-1800 °C, various
husk grain shape up to 50 micron in size is phases were obtained. At low firing
still found at the pyrolysis of the as-received temperatures (500 °C), both RH and RS
rice husk at 500 C. The bulk part is porous, samples show broad peaks around 23°
while other rectangular bulk shape shows corresponding to the carbon (002) planes
less pores. The ground RH pyrolyzed at with a small bump around 45° corresponding
500 C contains the broken pieces of 4-10 to (100) plane.
m in size. The as-received and the ground Upon firing at higher temperatures
of RS pyrolyzed at the same temperature up to 1360 °C, the carbon of rice husk show
shows similar morphologies. more evidences of ordering of graphite in
Phase formations are investigated via which the (100) peak appears to be sharper.
X-ray diffractrometry. Effects of the starting A small spike on top of (002) diffraction
materials are illustrated in Figure 3. peak carbon for RH1360 might result from
Diffraction patterns of the as-received and the existence of SiO2.
the ground states for both untreated RH and
RS pyrolyzed at 500 C are compared.
Typical amorphous carbon structure is RS500 ground
achieved at the pyrolysis temperature of 500
Intensity / arb. unit
C. It is found that the obtained structure of RS500 as received

the pre-processed samples after the pyrolysis
almost resembles that of the as-received
RH500 ground
ones. This result confirms that there is no
distinguishable difference between the
RH500 as received
samples that were ground and unground. In
order to reduce the processing time as well
10 30 50 70
as cost, the as-received state of both RH and
RS were used to make biochars in the rest of 2q / degree
this study.
Figure 3. XRD patterns of rice husk and
3.2 Effects of pyrolysis temperatures
rice straw from the as-received and ground
Phase evolution of rice husk and rice
straw upon increasing carbonization states pyrolyzed at 500 C under N2
temperatures are shown in Figure 4 and 5,
respectively. Natural rice husk (RH) and rice Untreated RS pyrolyzed at 1360 C
straw (RS) are generally consisted of lignin, under N2 is found to form a completely
cellulose and hemicellulose, which are different phase. Peaks corresponded to
complex polymer consisting of crystalline SiNiO2 (JCPDS#47-1627) with large bumps
and amorphous structure. Unfired raw resulting from the diffractions of (002) and
At 1800 C, both RH and RS
pyrolyzed under N2 show additional phases
RH1800 besides carbon. The filled circle and square
symbols in Figure 4 and 5 corresponds to the
(002) (100) RH1360 diffractions peaks of silicon carbide (SiC)

with 3C and 2C structure. For RH1800,
weak signal of carbon diffraction is
RH500
observed, however the small peak is likely
(200)
(004)
to be (002) diffraction of graphite. The
(110) Raw RH graphite signal for RS1800 is stronger, as
indicated by filled triangle symbol in Figure
10 30 50 70
2q / degree
5. Graphite is generally found to form at
T>1900 C,8 but in this case however, the
Figure 4. XRD patterns of rice husk and ordering of carbon structure to form graphite
untreated rice husk pyrolyzed at various is found to form earlier than expected at the
temperatures under N2. Filled circle temperature as low as 1800 °C.
indicates formation of SiC Mossainite-3C The higher order structure is revealed
and filled square indicated SiC Mossainite- as graphene structure via Raman features in
2C. Figure 6 and 7 for RH and RS respectively.
The spectrum shows the typical D (1355
cm-1) and G (1575 cm-1) lines for higher
RS1800 ordered and less ordered carbon structure,
respectively. The small peaks near 2700
cm-1, typically called 2D or G* band

RS1360
correspond to the formation of graphene
(002)
structure. The information given here also
RS500 implies that upon increasing pyrolysis
(200)
(004)
temperature, the carbon structure has
(110) raw RS evolved from being completely disordered
(amorphous) into a higher ordered structure.
10 30 50 70
2q / degree G D
2D
RH1800
Figure 5. XRD of raw rice straw and
untreated rice straw pyrolyzed at various

temperatures under N2 reveals SiC as seen in
RH1360
raw rice husk. Filled triangle indicates
formation of SiO2. RH500
(100) graphite peaks were still found.

Although, both rice husk and rice straw
3000 2500 2000 1500 1000
contain high amount of silica, it is evident
Raman Shift / cm-1
from these results that the silica contained in
rice husk and rice straw are of different Figure 6. Raman spectrum of RH pyrolyzed
nature resulting in different phases when at various temperatures
pyrolyzed under the same temperature under
N2 and filled square indicated SiC
Mossainite-2C. Filled triangle indicated
graphite.
3.3 Effects of acid and base pretreatments
G D
and firing atmosphere
2D Effects of various treatment
conditions for rice husk on the resulting
RS1800
structures are compared. Figure 8 shows the
XRD results of the untreated rice husk fired
RS1360 in N2 (RH1360) vs untreated samples fired
RS500 in Ar (RH1360Ar) vs acid and base treated
sample fired in N2 (TRH1360) XRD pattern
of the untreated rice husk fired in N2 shows
typical (002) and (100) diffractions peaks of
3000 2500 2000 1500 1000
Raman Shift / cm-1
medium ordered carbon structure along with
small peaks near 21° and 36 corresponding
Figure 7. Raman spectrum of RS pyrolyzed to that of crystobalite (SiO2 JCPDS#
at various temperatures 893435) and SiC (JCPDS# 74-2307),
respectively. Some evidences of the Si-N-O
phases could also be observed as small
(002) peaks on top of (002) carbon peaks although
not as prominent as in the rice straw
(100) samples. When the untreated rice husk was
TRH1360
SiO2 fired in Ar atmosphere, peaks corresponded
with SiO2 and SiC could be observed more
SiC RH1360Ar clearly along with large bumps around 27°
which corresponding to the starting of the
formation of graphite phase.
RH1360 When the rice husk was treated to
remove the Si and other metal impurities
10 30 50 70 and was pyrolyzed at 1360 °C under N2, it
was found that only peaks corresponding to
2q / degree
carbon ordering in the form of graphite
Figure 8. XRD pattern of rice husk ((002), and (100) planes) started to form.
pyrolyzed at 1360 C at various conditions Crystalline structures of rice straw
are investigated in a similar manner.
Structures of the untreated rice straw fired
(002) under N2 (RS1360) untreated RS fired under
Ar (RS1360Ar) and treated RS fired under
(100) N2 (TRS1360) are compared (Figure 9).
SiO2
TRS1360
Rice straw carbonized at 1360 C under both
N2 and Ar do not reveal typical amorphous
SiC
RS1360Ar
carbon pattern. The peaks with various
intensities corresponded to Si2Ni2O
Si2NiO2
composite according to JCPDS# 47-1627 for
RS1360 the carbonization in N2. The existence of Si
is confirmed with Si-composite formation.
10 30 50 70
This evidence aids the identification of the
ash purity. Despite typical amorphous
2q / degree
carbon seen in the pyrolysis under Ar
Figure 9. XRD patterns of rice straw atmosphere, impurities such as crystobalite
pyrolyzed at 1360 C at various conditions SiO2 (JCPDS# 89-3435) and SiC (JCPDS#
74-2307) are found to form along with the
desired carbon structure. The pure carbon Acknowledgments
structure could only be obtained in the National Metal and Materials
treated conditions, i.e. TRH1360 and Technology Center, National Science and
TRS1360. Technology Development (Project#
P1450850).
4. Conclusion
A simple process is used to produce References
biochar from rice husk and rice straw and 1. Asai, H.; Samson, B. K.; Stephan, H. M.;
the final products have potential usage for Songyikhangsuthor, K.; Homma, K.;
energy applications. Despite the varieties in Kiyono, Y.; Inoue, Y.; Shiraiwa, T.;
morphologies as observed via SEM images, Horie, T. Field Crop. Res. 2009, 111, 81–
the resulting structural characteristics and 84.
the uniformity appear to be the same 2. Zhang, A.; Cui, L.; Pan, G.; Li, L.;
regardless of preparation process. Hussain, Q.; Zhang, X.; Zheng, J.;
Different phases and structures Crowley, D. Agr. Ecosyst. Environ.
could be obtained by varying the pyrolysis 2010, 139, 469–475.
temperatures. The higher pyrolysis 3. Haoran, Y.; Tao, L.; Hongyu, H.; Dandan
temperature offers the possibilities of Zhao.; Noriyuki K.; Yong C. J. Anal.
producing graphene-layer-like structure Appl. Pyrolysis 2015, 112, 284–289.
embedded in amorphous carbon matrix. The 4. Uchimiya, M.; Wartelle L. H.; Klasson
atmosphere rather has impact on extra phase K. T.; Fortier C. A.; Lima I. M. J. Agric.
formation such as Si2NiO2 composite for Food Chem. 2011, 59 (6), 2501–2510.
rice straw pyrolyzed at 1360 C under N2 5. Verheijen, F.; Jeffery S.; Bastos, A.
and SiC in other cases. However the extra C.; Van der Velde, M.; Diafas, I. JRC
phases would not form under the pyrolysis Scientific and Technical Reports-A
of the high purity precursors, i.e. raw Critical Scientific Review of Effects on
material containing less Si and other metal Soil Properties, Processes and Functions,
elements. The pre-treatment has then Luxembourg, 2010.
influenced the improvement in properties. 6. Goto, M.; Yokoe, Y. Anim. Feed Sci.
Pure carbon without the disturbance of extra Tech. 1996, 58, 239–247.
phases, which will be used in the future 7. Socha, A. M.; Plummer, S. P.; Stavila,
work, could be obtained with the pre- V.; Simmons, B. A.; Singh, S.
treatment with NaOH and HCl. Biotechnol. Biofuels 2013, 6 (1), 61-70.
At the current state, we have only 8. Guentert, O. J. J. Chem. Phys. 1962, 37,
achieved the condition to obtain hard carbon 884–890.
structure. To able to use the products in
energy applications requires further
investigations toward functionalization of
the biochar materials as well as testing other
key properties such as surface area.
Surface and cross-sectional comparative study of natural indigo dyed
cotton fabrics using Na2S2O4 and monosaccharides as reducing agents in
different alkaline solutions
Laksanawadee Saikhao1, Jantip Setthayanond2, Thitinun Karpkird1, Potjanart Suwanruji1*
1
Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
2
Department of Textile Science, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand
*E-mail: fscipjs@ku.ac.th
Abstract:
Sodium dithionite (Na2S2O4) is a conventional reducing agent for indigo dyeing with
cotton in an alkaline solution in order to reduce an indigo pigment to a water soluble leuco
form. In this study, three monosaccharides (glucose, fructose and galactose) were studied as
eco-friendly reducing agents to compare with Na2S2O4. Two alkaline media: NaOH and
Ca(OH)2 were applied in the reducing system. The optimum condition for reduction of
natural indigo was found at 50oC and 30 min. The results showed that surface and cross-
section of the dyed cotton fabrics using Na2S2O4 and monosaccharides as reducing agents in
NaOH solution could give darker blue shade than in Ca(OH)2 solution. In addition, color
strengths (K/S) of fabrics in NaOH medium from glucose were higher than the ones from
other reducing sugars and Na2S2O4. On the other hand, Na2S2O4 gave higher color strength
fabrics than that from monosaccharides in Ca(OH)2 solution. From the build-up study, shade
depths of cotton fabrics increased as indigo concentrations in the dyebath were raised.
Therefore, monosaccharides could effectively use as green reducing agents in NaOH solution
for natural indigo dyeing on cotton fabrics.
1. Introduction
Indigo is a famous vat blue dye that
has been used more than 5,000 years.1,2 The
dye is obtained from the plants, Indigofera
tinctoria which are normally found in
tropical countries, Africa, Asia and
Southeast Asia. Indican from indigo leaves
is hydrolyzed by enzyme to indoxyl and
glucose.3,4 Then indoxyl is oxidized by
oxygen in air to indigo (Figure 1).
In Thailand, natural indigo dyes are
mainly produced in the northeastern part
such as Surin, Udon Thani, Kalasin and
Sakon Nakhon.5,6 For indigo dyeing process,
insoluble indigo dye can be converted to
soluble form (leuco form) by reducing
agents in an alkaline solution prior to
applying onto cotton fabrics. The absorbed
indigo inside the cellulosic fiber is
reoxidized to the insoluble form by air as Figure 1. Natural indigo production from
shown in Figure 2. indigo plants
reducing agents were investigated in two
different alkaline (NaOH and Ca(OH)2)
solutions.
Figure 2. Oxidation-reduction of indigo 2. Materials and Methods

Natural indigo dye was provided
Sodium dithionite (Na2S2O4) is a from Ban Tam Tao, Sakon Nakhon,
traditional reducing agent for indigo dyeing Thailand. Cotton fabrics were supplied by
but it is considered environmentally P.S.Jevasy. Co. Ltd, Thailand. Reducing
unfavorable and it has corrosive effect on sugars (glucose, fructose and galactose),
wastewater line due to by-products such as Na2S2O4 and bases (NaOH and Ca(OH)2)
sulfites (SO32-) and sulfates (SO42-).7,8 are AR grade.
This work focused on using 2.1 Preparation of cotton fabrics
monosaccharides i.e. glucose, fructose and Cotton fabrics were scoured in a bath
galactose as green reducing agents instead of containing 4 g/L NaOH and 1 g/L wetting
Na2S2O4. Aldehyde and ketone functional agent at 90-95 oC for 45 min. After scouring,
groups in monosaccharides can reduce the cotton fabrics were rinsed with water
indigo to leuco dyes.7,9,10 Surface and cross- and then air-dried.
section of natural indigo dyed cotton fabrics 2.2 Preparation of natural indigo dye
using Na2S2O4 and monosaccharides as
Table 1. Microscopic surface and cross-section views of indigo dyed cotton fabrics from
different reducing agents and bases
Base
Reducing agent NaOH Ca(OH)2
Surface Cross-section Surface Cross-section
Na2S2O4
Glucose
Fructose
Galactose
Natural indigo paste was vacuum Table 2. Color strength (K/S) of indigo
filtered and then oven-dried at 60oC for 24 dyed samples using Na2S2O4 and reducing
hrs. The dried indigo powder was milled and sugars in different alkaline solutions
then stored at room temperature. Samples L* a* b* K/S
2.3 Indigo reduction and dyeing NaOH
Cotton fabrics (2 g) were applied
with natural indigo dye using a liquor ratio Na2S2O4 42.51 -2.97 -22.99 7.34
of 50:1, 12 g/L NaOH, 15 g/L Ca(OH)2, 10 Glucose 41.45 -2.69 -23.83 8.04
g/L Na2S2O4 or 20 g/L reducing sugars. The Fructose 42.43 -2.55 -24.07 7.41
reduction temperature (TR) and reduction Galactose 41.69 -2.72 -23.01 7.53
time (tR) were set at 50oC and 30 min,
Ca(OH)2
respectively. Dyeing temperature (TD) was
set at 30oC and dyeing time (tD) was 30 min. Na2S2O4 33.61 -0.66 -23.26 11.41
The schematic diagram of natural indigo Glucose 49.85 -3.55 -24.98 4.97
dyeing process is shown in Figure 3.
Fructose 52.35 -3.89 -24.05 4.06
TR=50oC Galactose 54.53 -3.38 -24.36 3.40

tR= 30
2oC/min min 2oC/min
min min Table 3. Microscopic view of indigo dyed
tD= 30 cotton fabrics from various indigo
30 oC TD=30 oC concentrations
min
Indigo
Natural indigo dye Cotton fabrics concentration Na2S2O4 Glucose
Reducing agents (g/L)
Alkali
Figure 3. Schematic diagram of natural 0.2

indigo dyeing process
2.4 Characterization of surface and cross-

section of indigo dyed cotton fabrics 0.4
Surface and cross-section of indigo
dyed cotton were characterized by Olympus
SZX16 and CX41 research
stereomicroscope optical microscope. 0.6
2.5 Color measurement
The color strength (K/S) values of
indigo dyed cotton fabrics were evaluated
using a Macbeth ColorEye 7000
0.8
Spectrophotometer (Illuminant D65 at 10o
observer) and calculated by Kubelka-Munk
Equation.
K/S = (1-R)2/2R 1.0
Where; R is the reflection of all indigo dyed

cotton fabrics samples.
3.1 Surface and cross-section of dyed
cotton
3. Results and Discussion After dyeing, surface and cross-
section of the dyed cotton fabrics were
investigated by a light microscope at 10X
magnification. The results in Table 1 Na2S2O4
revealed that types of reducing agents and Glucose
12
using bases affect the color depth of indigo
on cotton. In general, the samples treated 10
with NaOH gave darker blue than the ones

8
treated with Ca(OH)2 as we can visually
K/S
observe from the images of surface and 6
cross-section. In the case of NaOH solution,

4
glucose seemed to give the darkest blue
among all samples. According to the 2
0.2 0.4 0.6 0.8 1.0
Ca(OH)2 solution, cotton dyed from Indigo concentration (g/L)
Na2S2O4 was the darkest one. The
microscope results were in a good Figure 4. Color build-up of indigo dye using
agreement with the color strength (K/S) as Na2S2O4 and glucose as reducing agent
reported in Table 2. Indigo dyeing with
NaOH provided K/S higher than with and monosaccharides (glucose, fructose and
Ca(OH)2 about 2 times for all samples using galactose) as reducing agents in NaOH and
monosaccharides as a reducing agent. In Ca(OH)2 mediums were compared. The
contrast, Na2S2O4 with Ca(OH)2 gave higher results of surface and cross-section of
K/S than with NaOH. The optimum pH of natural indigo dyed fabrics used NaOH
dyebath from base used can lead to greater solution gave bluer shade than Ca(OH)2. In
penetration of indigo dye into the cellulosic addition, glucose gave the highest color
fiber. For the colorimetric data, b* was strength when compared with other reducing
about the same for all samples. Except for agents in NaOH solution. However, the dyed
Na2S2O4 samples, L* from Ca(OH)2 fabric with Na2S2O4 in Ca(OH)2 solution
expressed lighter color than that from NaOH gave the darkest blue color because of the
while a* from Ca(OH)2 showed greener optimum pH of dyebath for Na2S2O4. The
shade than that from NaOH. The results K/S values of the dyed samples that used
suggested that glucose-NaOH system has a monosaccharides as a reducing agent in
potential to be utilized in indigo dyeing on NaOH solution were higher than that in
cotton because its color strengths were Ca(OH)2 about 2 times for all samples. The
comparable. colorimetric data from Ca(OH)2 samples
3.2 Build-up results displayed lighter color and greener shade
As the indigo concentration than from NaOH samples. The results
increased, the color strength of the dyed indicated that glucose could effectively
cotton also increased for both Na2S2O4 and reduce natural indigo dye in NaOH system.
glucose with NaOH, indicating a good The build-up results showed that K/S
build-up property of indigo dyes (Figure 4). increased with increasing dyed
Glucose gave mostly higher color strength concentration, which is corresponding to
than Na2S2O4. The microscopic results of microscopic results. Therefore,
fabric surface in Table 3 also confirmed the monosaccharides could be an eco-friendly
build-up ability of indigo on cotton in the reducing agent to replace Na2S2O4 in NaOH
same manner. The greater amount of indigo medium in natural indigo dyeing process.
in the dyebath could diffuse and fix inside
the fiber in higher extent, yielding higher Acknowledgements
K/S. The authors gratefully acknowledge
Kasetsart University Research and
Development Institute, Bangkhen Campus,
4. Conclusion
Faculty of Science, The Graduate School
Surface and cross-section of natural
and International Affairs Division, Kasetsart
indigo dyed cotton fabrics using Na 2 S 2 O4
University, Thailand, for the financial 6. Duangbubpa, C.; Pratumnet, S. C. N.
support of this research. A great APJMR 2015, 3 (3), 6–10.
acknowledgement is also given to 7. Saikhao, L.; Setthayanond, J.; Karpkird,
Department of Chemistry, Faculty of T.; Suwanruji, P. In Comparison of
Science and Department of Textile Science, sodium dithionite and glucose as a
Faculty of Agro-Industry, Kasetsart reducing agent for natural indigo dyeing
University, Thailand. on cotton fabrics, MATEC Web of
Conferences, EDP Sciences: 2017; p
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Mhenni, M. Int. J. Environ. Res. 2013, 7 Sci. technol. 2004, 38 (14), 4034–4039.
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Cheney: 1993.
Effect of molybdenum disulfide (MoS2) on carbon support from sawdust
via hydrothermal carbonization for jatropha oil residue upgrading
Peerawith Sumtong1, Vituruch Goodwin2, Nuwong Chollacoop2, Apiluck Eiad-ua1*
1
College of Nanotechnology, King Mongkut’s Institute of Technology Ladkrabang, Chalongkrung Road,
Ladkrabang, Bangkok 10520, Thailand
2
National Metal and Materials Technology Center (MTEC) 114 Thailand Science Park (TSP),
Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathumthani 12120, Thailand
*E-mail: apiluck.ei@kmitl.ac.th
Abstract:
The oxygenated compounds in jatropha oil residue has been improved upgraded by
using molybdenum disulfide (MoS2) catalyst on carbon support from sawdust obtained via
hydrothermal carbonization and pyrolysis processes. The lignocellulosic biomass composed
of cellulose, hemicellulose and lignin from sawdust were hydrothermally carbonized between
160–200 oC for 24 hr. The MoS2 catalyst was prepared with mixing between thiourea and
ammonium molybdate tetrahydrate and impregnated on carbon supported materials. The
lignocellulosic structure were decomposed at temperature higher than 180 oC from
hydrothermal carbonization and pyrolysis. These processes decrease cellulose, hemicellulose
and lignin in carbon support with increasing of temperature and time of hydrothermal
carbonization. Jatropha oil residue was upgraded via catalytic deoxygenation to decrease
oxygenated compounds using MoS2 on carbon support carbonized at 200 oC. The upgrading
process was performed using pyrolyzer gas chromatography mass spectroscopy (Py-GC/MS).
It showed that the MoS2 on carbon support carbonized at 200 oC can deoxygenate oxygenated
compounds in jatropha oil residue more than carbon support carbonized at other conditions.
1. Introduction resistance to acid, abundant source, low cost

At present, the problem of energy and no coking. Sawdust is a by-product of
shortages from fossil results in demand for sawing which is a fine wood powder.
renewable energy such as bio-oil. Jatropha Sawdust contains mostly organic
bio-oil is generated from jatropha oil residue compounds including cellulose,
by fast pyrolysis process at high temperature hemicellulose and lignin that contains
rapidly and give bio-oil yield more than polyphenols. For example, sawdust from
75%.1 However, the chemical properties of pines and fir trees reacted with sodium
jatropha bio-oil is less stable than petroleum hydroxide and sodium carbonate was used
fuel because it has high oxygen content, to absorb copper and zinc. Sawdust from
viscosity and acidity.2 Hydrodeoxygenation coconut trees was converted by
(HDO)3 is the process of removal oxygen hydrothermal carbonization and reacted with
atoms in a form of water molecules which sulfuric acid to absorb nickel and mercury.5
improves chemical stability of jatropha bio- Hydrothermal carbonization (HTC) is a
oil. The molybdenum disulfide (MoS2) thermochemical conversion process with
catalyst was used in hydrodeoxygenation temperature, pressure and duration time
process due to its activity and cost range from 160 to 250 oC in close system.
effectiveness, most often supported on Hydrothermal carbonization converts
-Al2O3 but this support material is highly lignocellulosic biomass including cellulose,
acid and accelerates coking reaction.4 The hemicellulose and lignin to producing
carbon support from natural materials are carbon support materials by hydrolysis and
good canditates as catalyst support due to dehydration reaction.6,7 The lignocellulosic
their diverse porous structure, good structure in biomass was removed and gave
porosity on carbon materials. In this Figure 1 shows the functional group
research, sawdust from wood furniture was from FT-IR analysis of sawdust under
synthesized into carbon support materials by hydrothermal carbonization. At 3350 cm-1
hydrothermal carbonization at 160-200 oC. indicates OH stretch in cellulose,
The MoS2 catalyst for jatropha oil residue hemicellulose and lignin. At 2922 cm-1
upgrading was prepared by loading MoS2 on indicates CH stretch in cellulose. At 1634-
carbon support materials via impregnation 1328 cm-1 indicates C=C aromatic and CH2
and pyrolysis technique at 300-500 oC in N2 bending of lignin. At 1200-950 cm-1
atmosphere. indicates C-O stretch in hemicellulose. At
900-700 cm-1 indicates CH bending. The
2. Materials and Methods C-O peak has been decreased and
2.1 Sawdust pre-treatment disappeared in pyrolysis under N2 air by
Sawdust from wood furniture was using high temperature. Decreasing of OH
milled and dried at 90 oC for 24 hr. Mixing and C-O peak show that hemicellulose in
30 g of sawdust with 120 ml of deionized lignocellulosic has been decomposed with
water (water to biomass ratios of 4:1) and hydrolysis and dehydration reaction in HTC
loaded into reactor for HTC. The reaction over than 180 oC. At C=C aromatic
temperature for sawdust treatment was set at and CH2 bending peaks has been changed
200 oC for 24 hr. The reactor was quenching slightly because both peaks are lignin
in water to stop the reaction immediately structures and high stability.
then the products were dried at 90 oC for
24 hr.
2.2 Catalyst preparation
Molybdenum disulfide catalyst was
prepared by mixing between 40 mmol of
thiourea (CH4N2S) and 1 mmol in deionized
water of ammonium molybdate tetrahydrate
((NH4)6Mo7O24.4H2O). The precursor
catalyst was loaded on carbon support
materials by impregnation process at 80 oC
and dried at 90 oC for 24 hr. MoS2 was
formed with pyrolysis at 300-500 oC for 2 hr
under N2 atmosphere.
2.3 Measurement
The morphology of carbon support
by hydrothermal carbonization (HTC) and Figure 1. FTIR analysis of sawdust after
pyrolysis were characterized by scanning hydrothermal carbonization at 160, 180 and
electron microscope (SEM) and the 200 oC for 24 hr
functional group in lignocellulosic under
HTC treatment were investigated by fourier Figure 2 shows the functional group
transform infrared spectroscopy (FT-IR). from FT-IR analysis of sawdust under
Finally, jatropha oil residue upgrading were pyrolysis process. At 3350 cm-1 indicates
analyzed oxygenated, fatty acid and OH stretch in cellulose, hemicellulose and
hydrocarbon compounds by pyrolyzer gas lignin. At 2922 cm-1 indicates CH stretch in
chromatography mass spectroscopy (Py- cellulose. At 1634-1328 cm-1 indicates C=C
GC/MS). aromatic and CH2 bending of lignin. At
1200-950 cm-1 indicates C-O stretch in
3. Results and Discussion hemicellulose. At 900-700 cm-1 indicates
3.1 Hydrothermal carbonization and CH bending. The C-O, C=C, CH2 bending,
pyrolysis of carbon support material CH stretch and OH stretch peak has been
decreased dramatically in pyrolysis under N2 The MoS2 particle has been appeared
air by using high temperature. Decreasing of on the surface of carbon support materials.
all peaks show that increasing of carbon SEM micrograph reveals that the average
content and porosity on sawdust from particle size measure by ImageJ software are
decompose of lignocellulosic structure in 2.73, 2.77, 2.79, 2.22 and 1.28 µm in A, B,
pyrolysis process. C, D and E image, respectively. The MoS2
particle size was decreased due the
increasing of temperature in pyrolysis
reaction in Table 1.
Table 1. Percentage of molybdenum (Mo)

and sulfur (S) in MoS2 on carbon support
materials
Pyrolysis Mo (%wt) S (%wt)
300 oC 3.04 2.45
350 oC 5.77 1.67
400 oC 7.54 2.64
450 oC 7.52 2.77
500 oC 8.22 3.89
Table 1 indicate that the increasing

percentage of molybdenum and sulfur in
Figure 2. FTIR analysis of sawdust after MoS2 at high temperature in pyrolysis
hydrothermal carbonization at 200 oC for 24 reaction. Increasing of pyrolysis temperature
hr and pyrolysis at different temperature for results in more dispersion of the particles on
2 hr the surface because the carbon support is
high porosity from decomposing of
Figure 3 shows the morphology of lignocellulosic in pyrolysis reaction.
catalyst from hydrothermal carbonization
and pyrolysis process. The lignocellulosic 3.2 Jatropha oil residue upgrading
structure was decomposed by increasing of Figure 4 shows the organic
temperature in HTC and pyrolysis reaction.
Figure 3. SEM image (1000X) of catalyst on sawdust from hydrothermal carbonization at

200 oC for 24 hr and pyrolysis under N2 atmosphere for 2 hr (A) 300 oC (B) 350 oC
(C) 400 oC (D) 450 oC and (E) 500 oC
Figure 4. The total compound of jatropha oil residue by using MoS2 catalyst (loaded on
carbon support) from pyrolysis under N2 atmosphere for 2 hr
compounds in jatropha oil residue after hydrothermal carbonization and pyrolysis

catalytic pyrolysis using MoS2 (loaded on reaction. Fatty acid compounds in jatropha
carbon support) by pyrolyzer gas oil residue were transformed to
chromatography mass spectroscopy (Py- hydrocarbons via catalytic pyrolysis using
GC/MS). The total oxygenated compound MoS2 catalyst supported on carbon.
including fatty acid, ketone, alcohol, Approximately 8% of fatty acid compounds
aldehyde, and nitrogen oxygenated were reduced after catalytic pyrolysis
compounds in jatropha oil residue upgrading process compare to 59.25% of fatty acid
were decreased by using MoS2 loaded on compounds in the original jatropha oil
carbon support. In contrast, the total residue.
hydrocarbon compounds including
aliphatics, olefins and aromatics were Acknowledgements
increased. As a result, sawdust pyrolyzed at This work was financially supported
400 oC can reduce fatty acid compounds by the Research and Researcher for Industry
about 8%. Decreasing of the total (RRi), The Thailand Research Fund (TRF)
oxygenated compound indicate that the and thankful to Nanoporous Research
hydrodeoxygenation reaction between MoS2 Laboratory at College of Nanotechnology,
catalyst. Molybdenum disulfide remove King Mongkut’s Institute of Technology
oxygen molecule in total oxygenated Ladkrabang for their supporting.
compound with hydrogenation and
hydrogenolysis in hydrodeoxygenation References
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Shaukat, A.; Brahim, S. Renew. Sust.
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The MoS2 catalysts have been 3. Yaseen, E.; Charles, A. M.; Anna, L.;
successfully synthesized and loaded on Akwasi, A.B. Fuel Process. Technol.
carbon support materials from sawdust via 2014, 123, 11–18.
4. Vorranutch, I.; Chalita, R.; Masaki, O.; 6. Zhengang, L.; Augustine, Q.; Kent
Kajornsak, F. Catal. Commun. 2015, 68, Hoekman, S.; Balasubramanian, R. Fuel
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Bioresource Technol. 2008, 99, 3935– Robert, M.; Gleb, Y. Adv. Energy Mater.
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Lewis base poly(ethylene glycol) immobilized solid amine sorbent
for carbon dioxide capture
Pathompong Janetaisong*, Buppa Somchoam, Somsak Supasitmongkol
National Metal and Materials Technology Center (MTEC),
National Science and Technology Development Agency (NSTDA)
114 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathumthani 12120, Thailand
*E-mail: pathompong.jan@mtec.or.th
Abstract:
Carbon dioxide (CO2) is one the main greenhouse gases associated with global
climate change. CO2 removal from high concentration streams like flue gases and natural gas
wells is therefore becoming increasingly important. This work studied the performance of
CO2 removal in silica-supported polyethylenimine (PEI-based sorbent) and modifiers
incorporated into PEI-based sorbent from a simulated gas stream (80% vol.% CO2 balanced
N2). Poly(ethylene glycol) of low molecular weights (PEG, MW ca. 200 and 400) were used
as Lewis basic modifiers. Many factors affecting the behavior of gas adsorption such as PEI
loadings, adsorption temperature, modifier types/loadings and textural characteristics were
considered. It was found that the utilization ratio of amine group in sorbents increased with
increasing PEI loadings up to 40 wt.%, relating to the exposing more active surfaces for CO2
uptake. Enhancing temperatures resulted in diminished diffusion resistance, leading to an
increased CO2 uptake in the PEI-based sorbents. With the same active PEI sorbent, the PEG
modifiers improved CO2 uptake capacity and desorption rates of the PEI-based sorbents. The
regeneration of spent PEG-modified sorbents can be readily achieved using a small increase
in temperature. Thus the modified PEI-based sorbent with PEG represents an attractive
candidate for potential CO2 separation systems associated with a decrease of the capture
costs.
1. Introduction much attention with many advantages:

As a result of climate change and possibility of upgrading in industrial scale,
global warming issues, reducing greenhouse enhancing CO2 capture compared to
gases emission into atmosphere has been aqueous amine solution, and the simplicity
urgently concerned in industries for several of regeneration.1,2
years. Carbon dioxide (CO2) produced by Studies have shown that immobilizing
combustion of flue gases, biological gas and amine on solid support becomes attractive
petroleum distillates such as natural gas for for CO2 capture. With the numerous CO2
power generation is one of these major sorption sites of amine group,
pollutant problems. CO2 removal technology polyethylenimine (PEI) impregnated on the
from industrial gas streams is therefore ordered mesoporous silica (MCM-41,
exhibiting increasingly important. Many MCM-48 and SBA-15) has widely been
technologies such as liquid phase amine used as the adsorbent for gas purification.3,4
scrubbing and permeation through However, these supporting materials suffer
membrane have been reported for capturing problems in small pore volume restricting
CO2 from high concentration streams. the PEI loading and associated CO2 uptake.5
Unfortunately, liquid phase amine is limited PEI modified fumed silica (FS) was found to
by drawback as equipment corrosion and the be an inexpensive, easy-to-prepare and
cost of membrane separation method is still apparently higher pore volume. PEI/FS
high in material replacement. Gas adsorptive showed the best performance among other
method by using a solid sorbent becomes mesoporous silica nanoparticles for the CO2
capture directly from air.6 Interestingly, the 2.3 Characterization
adsorbed CO2 can be enhanced from the The textural characteristics of sorbents
PEI-based sorbent under a moist gas stream were conducted by using N2 adsorption
due to the interface of free base H2O isotherms at 77K in a Quantachrome
molecule in the adsorption mechanism. The Autosorb automated gas sorption and the
incorporation of poly(ethylene glycol) specific surface area was calculated by BET
(PEG) into PEI-based sorbent could change equation.
the reaction stoichiometry between amine Temperature-programmed desorption
group and CO2, resulting from the presence of CO2 (TPD-CO2) was used to evaluate
of terminal hydroxyl groups in PEG CO2 desorption behavior as a function
structure.7,8 of temperatures using a BELCAT
This study aims to examine the effects chemisorption analyzer. The given sample
of a Lewis acid-base interaction on CO2 was pretreated at 90 C under helium (50
capture/release properties using PEI-based mL/min) for 3 hours, and followed by
sorbent with and without incorporation of thermal equilibration at 55 C under 100
different Lewis basic PEG modifiers (MW vol.% CO2 (30 mL/min) for 2 hours.
ca. 200 and 400). Effect of parameters on Estimates of CO2 sorption capacity
adsorption characteristics were also (mmol/g) were obtained by heating up to
considered in this study. 90C under helium (50 mL/min) for 1 hour.
2.4 CO2 adsorption process
2. Materials and Methods Adsorption/regeneration process was
2.1 Materials performed in a fixed-bed flow absorber.
All chemicals were used as purchased Adsorbent (5 grams) was packed into the
from the supplier. These included: fumed quartz tube (ID 2 cm) with 5 cm of bed
silica (FS, commercial grade, Warker height and then pretreated at 90 C for 3
Chemicals), branched polyethylenimine hours under N2 (45 mL/min). After cooling
(PEI, MW ca. 800, Aldrich), methanol (AR down to the desired adsorption temperature,
grade, Carlo Erba), poly(ethylene glycol) a simulated gas stream passed through the
(PEG, MW ca. 200 and 400, Sigma Aldrich), bed columns with certain flow rate until the
nitrogen (N2, 99.999 vol.%, Praxair) and sorption capacity reached the saturation.
simulated CO2 mixtures (10 vol.% and 80 Desorption process immediately started by
vol.% balanced in N2, Linde). purging N2 and heating up to 90 C. The
2.2 Preparation of sorbents changed outlet gas from the absorber was
PEI-immobilized mesoporous silica analyzed online using a gas chromatograph
with and without incorporation of PEG (7890A, Agilent) equipped with a thermal
modifiers were prepared by wet conductivity detector (GC-TCD). The
impregnation method. A desired amount of obtained breakthrough curves were
PEI and PEG were separately dissolved in calculated as breakthrough CO2 capacity and
methanol before mixing together. A solution amine efficiency by Equations 1 and 2.
containing PEI and PEG was gradually
added to a solution of FS dispersed in (1)
methanol. The resulting solution was stirred
at ambient temperature until the absence of
(2)
immiscible solution. The mixture was
evaporated at 60C and then the residual
where Cap(B) is breakthrough capacity
volatile compounds in the product were
(mmol/g); tB is the breakthrough time (min),
further removed using a drying oven at 60C while the outlet gas concentration is zero;
overnight. The product was obtained as FR is the total flow rate (ml/min); Cin is the
white powder after drying the hardened CO2 concentration of inlet gas stream; Vmol
mass was finely ground. is the molar volume (22.4 ml/mol at STP);
W is the sorbent weight (grams); MW is Table 1. Textural characteristics of sorbents
molecular weight of amines, generally 800 Sorbents Pore volume Surface area
for this study; N is the amount of nitrogen (cm3/g) (m2/g)
atom in PEI; NL is the percentage of FS 1.86 202.8
PEI-20/FS 1.83 91.31
nitrogen loading onto supports (wt.%). PEI-30/FS 1.51 81.18
PEI-40/FS 0.82 43.51
3. Results and Discussion PEI-50/FS 0.37 20.33
3.1 PEI loadings PEI-60/FS 0.08 4.23
The behavior of both breakthrough
CO2 capacity and amine efficiency of by PEI molecules. Up to a PEI content of 40
different PEI loadings in PEI-based sorbent wt.%, CO2 molecules may easily diffuse into
was evaluated at 80 C under 10 vol.% CO2 the pore channels of sorbents, making more
balanced in N2 (5 ml/min) as shown in available amine sites for CO2 adsorption.
Figure 1. However, it is difficult to diffuse into the
bulky and sticky PEI phase, when the FS
pores are impregnated by excess PEI
molecules. The PEI-40/FS sorbent is more
flexible and readily accessible for CO2
uptake.
3.2 Adsorption isotherms
To examine the effect of adsorption
temperature on the CO2 adsorption potential,
PEI-40/FS sorbent packed in the fixed-bed
flow sorption system was performed under
10 vol.% CO2 balanced in N2 (5 ml/min) at
different isotherms (Figure 2).
Figure 1. Effect of PEI loadings on the

breakthrough CO2 uptake
The effect of PEI loadings on CO2

uptake is directly related to the amine
efficiency of the PEI-based sorbent.
Enhancing PEI loadings from 20 wt.% to
40 wt.% increased breakthrough CO2
capacity (Figure 1). Higher PEI contents
provide the amount of amine sites to
chemically react with acidic CO2. However,
the CO2 uptake was decreased with PEI
loadings above 40 wt.%. This behavior can Figure 2. Breakthrough curves of PEI-based
be explained by the textural characteristics sorbent at different temperatures
of the sorbents as presented in Table 1.
The pore volume and surface area of Both breakthrough time and CO2
PEI-x/FS sorbents decreased continuously uptake increased with increasing
with increasing PEI contents. This trend temperatures up to 80 C (Figures 2 and 3),
indicates that the FS pores are impregnated although the CO2-amine interaction is
typically exothermic process. This is Table 2. TPD-CO2 results of PEI-based
because the polymer becomes more flexible sorbent incorporated with PEG
and readily for CO2 diffusion to contact Sorbent
Weight ratio CO2 Capacity
more amine groups in PEI at enhanced (g PEG/g PEI) (mmol/g)
temperature, as has been proposed in no PEG 0 2.18
PEG200 0.05 2.34
literature.9 As amine efficiency can be 0.20 2.56
obtained from CO2 adsorbed amount based PEG400 0.05 2.52
on per mole of amine, the theoretical 0.20 2.86
maximum CO2 uptake is limited 0.25 2.41
stochiometrically with an amine efficiency 0.38 2.27
of 0.5 under dry conditions. The highest
utilization of amine group in the PEI-based in PEG may affect the chemical adsorption
sorbent can be achieved at 0.23 (Figure 3), mechanism, in that the OH group promotes
as a result of being relatively site-isolated the formation of carbamate type zwitterions
amines from one another in lower PEI allowing for more CO2 to be captured per
loadings or CO2 diffusional limitations in amine. This behavior is similar to the CO2
highly loaded materials. uptake mechanism of the amine under
humid condition.10 Another reason is that a
lower viscosity of the PEG-modified sorbent
favors increased mass transfer and
correspondingly enhanced CO2 uptake. At
the same PEG/PEI ratio, the incorporation of
PEG400 promoted better CO2 uptake than of
PEG200 due to the amount of OH groups.
The overloading of PEG molecules
may physically shield PEI chains, resulting
in barrier formation and associated CO2
diffusion limitations into the PEI phase. The
CO2 uptake on PEG-modified sorbent
decreased dramatically at the PEG400/PEI
ratios above 0.20.
Once the samples were saturated with
adsorbed CO2, the CO2 desorption behavior
was monitored online using a BELCAT
Figure 3. Effect of PEI-based sorbent on the chemisorption analyzer equipped with a
CO2 uptake at different temperatures thermal conductivity detector. The CO2
desorption profile of PEG modifier can
3.3 PEG modifiers reach to a steady state in a shorter time (30
With the same 40 wt.% PEI, the PEI- min) compared to the PEG-based sorbent
based sorbent with incorporation of Lewis (40 min) as shown in Figure 4. It implies
basic PEG modifiers (MW ca. 200 and 400) that the PEG modifier exhibited remarkably
was evaluated the CO2 uptake using TPD- faster CO2 desorption over the PEI-based
CO2 technique described in Table 2. sorbent at equilibrium.
All the PEI-based sorbents with 3.4 Regeneration process
adding the PEG modifiers exhibited higher Regenerability was tested for sorbent
CO2 uptake compared to the absence of PEG carrying out 3 cycles in the fixed bed
addition case (no PEG) in Table 2. The reactor. The adsorption cycles were carried
equilibrium CO2 uptake in both PEG200 and out at 55 ºC exposing the sorbent to 80
PEG400 increased as the PEG/PEI weight vol.% CO2/N2 (5 ml/min) for 3 hours.
ratio increased. The presence of OH groups Desorption was carried out at 90 ºC for at
least 3 hours under 45 mL/min sweep gas of cycles, indicating a good regenerability and
N2. The breakthrough CO2 uptake versus relative high stability of both sorbents. The
test cycle number is plotted in Figure 5. CO2 adsorbed on the PEI-based sorbent in
the 3rd cycle decreased around 33% and 17%
for the PEG-modified sorbent, when
compared with the original CO2 uptake. This
was agreement with the findings in Table 2
and Figure 4, where the carbamate type
zwitterions formed in the existence of
hydroxyl group guest (PEG) may lower
thermal stability which facilitates the
regeneration of adsorbent.
4. Conclusion
PEI-impregnated sorbents have been
successfully developed. The amount of
loaded PEI (40 wt.%) presented the higher
dispersion of amine active sites and
correspondingly higher utilization ratio of
amine group in sorbents. With the same
Figure 4. TPD-CO2 profile of PEI-based active PEI sorbent, adding PEG into the
sorbent with and without PEG modifiers PEI-based sorbent can increase not only the
CO2 uptake, but also the CO2 desorption
rate. The PEG-modified sorbent represents a
serious candidate for use in CO2 removal
technology as a commercial absorber.
Acknowledgements
Supports, facilities and fundamental
research funds of this study provides from
National Metal and Materials Technology
Center (MTEC) in a part of National
Science and Technology Development
Agency (NSTDA).
References
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they were readily regenerated by heating to Sharma, P.; Nelson, T. O.; Lali, M.;
90C. The regenerated sorbent was Coleman, L. J.I.; Abu-Zahra, M. R. M.
repeatedly used in further adsorption Powder Technol. 2016, 301, 449−462.
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The methanol synthesis from glycerol in a one-step
over basic oxide catalysts
Rujira Jitrwung1, Jiraporn Chalorngtham1*, Mukrawee Maneewuthiworasakul2,
Kamonrat Leeheng1, Kuntima Krekkeitsakul1, Anantachai Wannajampa1
1
Expert Centre of Innovative Clean Energy and Environment, Thailand Institute of Scientific and Technological
Research, Khlong Luang, Pathum Thani 12120, Thailand
2
Department of Chemical Engineering, Faculty of Engineering and Industrial Technology,
Silapakorn University, Nakhon Pathom 73000, Thailand
*E-mail: jiraporn_c@tistr.or.th
Abstract:
Crude glycerol, obtained as a by-product from biodiesel production, is recently over-
supplied due to limited world demand. As a result, utilization of the glycerol can be realized
by converting glycerol to value-added chemicals such as methanol, hydrogen, and propylene.
In this work, glycerol was catalytically transformed to methanol in a one-step using 16 mm
diameter of the fixed-bed reactor with basic oxide catalysts; CaO and MgO. Reliance of
methanol yield on operating parameters and types of catalyst were investigated. The results
showed that both (CaO and MgO) provided catalytic activities in converting glycerol mainly
to methanol. By-products, observed from the operation, included ethanol, propanol, ethanal,
2,3-butanedione, acetol, ethylene glycol, CO, and CO2. The MgO catalyst provided the
highest methanol selectivity and methanol yield of 44.8% and 43.5%, respectively. In
addition, glycerol conversion was increased 1.4 fold of using 1wt%Ce doping on MgO
catalyst comparing with MgO catalyst.
1. Introduction glycerol is also generated hugely leading to

The world energy demand increases oversupply of glycerol in the world market
continuously in spite of the limited amount along with falling of glycerol prices.
of fossil fuels. Therefore, the shortage of Moreover, it is forecasted that the global
fossil fuels has been concerned seriously production of glycerol will reach to 36
over the decades led to exploring renewable billion liters by 2018 and increase
energy source and developing alternative continucally.3,7 To manage with surplus
fuels.1,2 Biodiesel is attracted much attention generating of glycerol, it is interested in
as promising candidate of alternative fuels conversion of glycerol to high valuable
that it could efficiently substitute the global chemicals such as hydrogen,8 synthetic gas,9
dependence on the fossil fuels.3 methanol,4,9,10 propylene,11 and acrolein.12
It is well known that biodiesel is The glycerol to methanol is an
generally produced via transesterification of interesting research topic and is extensively
animal fats or vegetable oils with methanol studied because methanol can be returned to
in the present of an alkali or acid catalyst. At the biodiesel process as well as raw material.
the same time, glycerol is generated as a Furthermore, it is one of major feedstock
by-product about 10 wt% of biodiesel of various chemical production in the
product as well.3,4 petrochemical industry such as acetic acid,
The biodiesel industry is growing up methyl tert-butyl ether, formaldehyde, and
rapidly over all the world. It is reported that chloromethane13 and it can be used as a fuel
the biodiesel production was 33.2 billion additive or clean fuel.14 Typically, the
liters in 2016 and increased by 9.6 percent transformation of glycerol to methanol can
per year on average during 2010 – 2016 produce via two-step processes, which
period.5,6 Consequently, the amount of consist of glycerol reforming for synthetic
gas (H2/CO) and following by methanol 2.3 Catalytic activity test
synthesis process.9 However, the methanol Prior to test catalyst performance,
production from glycerol through two-step 4 g of catalyst was packed in stainless-steel
processes required high temperature and fixed bed tubular reactor (16 mm inner
high pressure, which their factors effect to diameter) and held in place between plugs of
high cost. Accordingly, the direct glycerol glass wool. After that, the 10 wt% glycerol
conversion to methanol under mild in water solution was injected by using a
condition has been investigated intensely to HPLC pump with feed flow rate of 0.1
solve the production cost. ml/min into the pre-heater (230 °C) to
In this work, the catalytic evaporate the feed completely in stream of
performances of basic oxide catalysts (CaO N2 (60 ml/min) and then the vaporized feed
and MgO) and operating parameter for was introduced into reactor at atmospheric
methanol synthesis from glycerol in a one- pressure. Catalytic activity was investigated
step were investigated. In addition, a study at 250-380 °C. The effluent products
of the influence of CeO2 loading as were condensed through a cooler to
promoter on methanol synthesis over basic separate liquid and gaseous products.
oxide catalyst was also discussed. The compositions of gaseous products
were analyzed online by using gas
2. Materials and Methods chromatography (GC, Agilent Technologies
2.1 Catalyst preparation 7890B) equipped with thermal conductivity
The CaO catalyst was purchased detector (TCD) and Q-HT Molsieve 13X
from Ajax Finechem Pty Limited, Australia. column. Meanwhile, the compositions of
Meanwhile, the MgO catalyst was obtained liquid products were determined on
from ITW Reagents Division, USA. Before an Agilent 7890B gas chromatography
testing catalytic performance, both of the equipped with flame ionization detector
catalysts (CaO and MgO) were calcined in (FID) and DB 1701 column.
air at 650 °C for 6 h. Glycerol conversion, methanol
CeO2/MgO catalyst was prepared selectivity and methanol yield were defined
by incipient wetness impregnation. as follows:
Initially, cerium (III) nitrate hexa-hydrate
(Ce(NO3)3·6H2O) used as precursor was Glycerol conversion (%) (1)
dissolved in deionized water and then
dropped into the CaO or MgO catalysts. Wglycerol ,in − Wglycerol ,out
After that, the mixture were dried at 80 °C = × 100
Wglycerol ,in
and 110 °C for 12 h, respectively. The
obtained catalysts were calcined in air at 600 Methanol selectivity (%) (2)
°C for 6 h. The amount of Ce loaded on
based-catalyst was about 1 wt%. CeO2 Wmethanol
supported on MgO catalysts were denoted as = × 100
WT
1Ce/MgO.
2.2 Catalyst characterization Methanol yield (%) (3)
The BET specific surface area, total
pore volume and average pore diameter of Wmethanol
the basic catalysts were measured by using a = × 100
Quantachrome Autosorp IQ instrument. Wglycerol ,in
Nitrogen adsorption/desorption isotherms of
samples at -196 °C were obtained after Where WT is total weight of products.
outgassing the samples under vacuum at 300
°C overnight.
3. Results and Discussion 3.2 Effect of basic oxide types
3.1 Catalyst characterization The catalytic performance of various
The physical properties including basic oxide catalysts (CaO and MgO) is
BET surface area, total pore volume, and shown in Figure 1 in terms of product
average pore diameter of basic catalysts are distribution (a), glycerol conversion and
summarized in table 1. Comparing CaO with methanol selectivity (b). The result
MgO, total pore volume and average pore (Figure 1(a)) demonstrated that methanol
diameter of the MgO catalyst are larger than was formed as major product over CaO and
theirs of the CaO catalyst, whereas their MgO catalyst. Other products formed in low
BET surface areas are not quite different. selectivity were found such as ethanal,
ethanol, propanol, 2,3-butanedione, acetol,
Table 1. Physical properties of basic ethylene glycol, CO, and CO2. It is related to
catalyst a research of Haider et al.,10 which proposed
Sample SBET a VPa DAPa that the mainly products can be generated
2 3
(m /g) (cm /g) (nm) via dehydration pathway over basic catalyst
CaO 18 0.08 17
and indicated that ethylene glycol was a key
MgO 29 0.38 53
a
SBET, Vp and DAP represented the BET surface area, intermediate for the formation of methanol,
total pore volume, and average pore diameter, while water played a role as a source of the
respectively. hydrogen.
In case of MgO, it was observed that
100% (a) the MgO catalyst provided the highest
90% methanol selectivity and methanol yield of
Ethylene glycol
80% 44.8% and 43.5%, respectively. Meanwhile,
Acetol
70% the formation of methanol over CaO catalyst
2,3-Butanedione
Selectivity (%)
60% was lower, even though the conversion of

propanol
50% glycerol achieved to 80%. This observation
Ethanol
40% indicated that the types of the basic catalyst
Methanol
30% Ethanal
significantly influenced on the methanol
20% CO
synthesis from glycerol.
10% CO2
3.3 Effect of the reaction temperature
0%
The effect of the reaction
CaO MgO temperature on methanol synthesis from
glycerol over MgO catalyst is shown in
(b) Figure 2. The results demonstrated that
100
glycerol conversion monotonically increased
90
when increasing the reaction temperature
Glycerol conversion/ CH3OH Yield (%)
80 Glycerol conversion (%)

and achieving almost complete conversion
70
Methanol yield (%) at 380 °C, which is in good agreement with
60 the result of Kim et al.15 At the same
50 condition, the methanol yield dramatically
40 increased from 21.4% to 43.5% when the
30 reaction temperature in range of 250 °C to
20 300 °C, and then decreased from 43.5% to
10 20.7% when increasing the reaction
0 temperature above 300 °C. The methanol
CaO MgO selectivity was similar trend to the methanol
Figure 1. Catalytic activity of basic oxide yield, which it was 44.8% at reaction
catalysts at 300 °C (a) product distribution temperature of 300 °C. This phenomenon
(b) glycerol conversion and methanol yield indicated that the reaction temperature is
suitable for methanol synthesis from
Methanol yield
glycerol over MgO catalyst at 300 °C. Methanol Selectivity
Glycerol conversion
1Ce/MgO
100 50
80 40
Conversion/Selectivity (%)
Methanol Yield (%)

60 30
MgO
40 20
Glycerol conversion
20 10 0 20 40 60 80 100
Methanol selectivity (%)
methanol yield
0 0
Figure 3. Effect of CeO2 loading on
240 260 280 300 320 340 360 380 400 catalytic activity of MgO catalyst for
Temperature (°C) methanol synthesis from glycerol at 300 °C
Figure 2. Effect of temperature on catalytic
activity of MgO catalyst for methanol Acknowledgements
synthesis from glycerol The authors would like to
acknowledge the National Research Council
3.4 Effect of CeO2 loading of Thailand for approval and financial
Figure 3 displays the effect of CeO2 support in this research.
loading on catalytic performance of MgO
for methanol synthesis from glycerol at 300 References
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Preparation of activated carbon from Salak seeds for dye removal
Supattra Raksaphort*, Nuntaporn Moonrungsee, Wiparat Srimueng, Jatupong Pampaisong
Department of Chemistry, Faculty of Science and Technology,
Rambhai Barni Rajabhat University, Muang, Chanthaburi 22000, Thailand
*E-mail: supattra.r@rbru.ac.th
Abstract:
The one of environmental pollutions has centered on pigment-containing wastewater
from various industrial sources. The potential treatment technique for dye removal is
adsorption using activated carbon produced by local agricultural wastes. In this study, the
activated carbon from Salak (cultivar: Sumalee) seed was prepared from the pyrolysis (400
°C, 1 h), activated with 50% v/v H3PO4 at the impregnation ratio is 1:3, and activated
temperature of 500 °C for 1 h. The activated carbon had the high iodine number of
843.46±49.01 mg/g, while the methylene blue index (12.40±0.04 mg/g) was very low
because the activated carbon was microporous material. The effects of contact time, initial
concentration, and adsorbent dosage were investigated in the batch testing. It can be
concluded that the activated carbon and dye solution ratio of 30:1 (mg/ml) has the highest
dye removal capacity for all initial concentration. Thus, this study demonstrated the potential
of using Salak (cultivar: Sumalee) seed as cheap and efficient raw material to produce
activated carbon for dye removal from wastewater.
1. Introduction sludge-free operation and recovery of the

Dyes are widely used in various sorbate.5
industries such as textiles, rubber, plastics, Activated carbon is the most widely
printing, cosmetics, etc., to color their used adsorbent for dye removal because of
products. As a result, they generate a its extended surface area, micro-pore
considerable amount of colored waste water. structures, high adsorption capacity and high
Since most dyes used today are of synthetic degree of surface reactivity. However, the
origin, it poses a problem to the environment commercially available activated carbon is
if it is not treated properly. Amongst the very expensive and has high regeneration
problems associated with dye contamination cost while being exhausted. Many research
includes visible pollution, limiting the reported the developing more suitable,
penetration of light into the water1,2 as well efficient and cheap, and easily available
as the potential mutagenic and carcinogenic types of adsorbents. Agricultural wastes are
effects.3 Conventional techniques are popular to use for preparing the activated
available for dye removal such as Fenton- carbon due to their accessibility and
oxidation, irradiation, photochemical abundantly availability.6-9 Salak (Salacca
decomposition, adsorption and membrane Zalacca) is a species of palm tree. It is an
filtration4 which are efficient and attractive tropical fruit, is widely planted in
economically feasible but some techniques Indonesia, Malaysia, and Thailand in a large
create secondary pollution notably spent scale. Salak fruit consists of pulp and hard-
catalyst and fouled membrane that require coated seeds. The pulp taste is sweet and
further disposal. Among treatment acidic, while the seed is inedible and
technologies, adsorption is rapidly gaining disposed to the sanitary landfills. In this
prominence as a method of treating effluent. study, Salak seed was chosen as raw
Some of the advantages of adsorption materials for the production of activated
process are possible regeneration at lowcost, carbon. Because it is abundance, similar
availability of known process equipment, lignocellulosic properties as other plant
biomass. Moreover, the very limited studies 2.3 Dye removal of the activated carbon
reported on the use of Salak seed as One type of the activated carbon
precursor for the production of activated prepared from Salak seeds was selected and
carbon, hence adding to the novelty feature further evaluated for its dye removal
of this study as well as at reducing the cost capacity through batch equilibrium
of waste management. The adsorption of adsorption studies. Stock dye solution, 1000
iodine and methylene blue was also mg/L was prepared by dissolving 1 g of
determined. Finally, the activated carbon dyestuff in 1 L of distilled water. The stock
prepared in this study was used in solution was then kept in a dark surrounding
preliminary test for dye removal from at room temperature prior to use. The effect
wastewater. of initial dye concentration, adsorbent
dosage and contact time on dye removal by
2. Materials and Methods activated carbon was investigated. Batch
2.1 Adsorbent preparation experiments of adsorption were performed
Raw material for the preparation of under agitation (150 rpm), 0.4 g of the
activated carbon is Salak (cultivar: Sumalee) prepared activated carbon was added into a
seeds which were collected from series of 100 mL Erlenmeyer flasks
Chanthaburi province. Salak seeds were cut containing 20 mL solutions of 25, 50 and
into smaller pieces, then washed with tap 100 mg/L of dye solution prepared from
water to eliminate any impurities and dried serial dilution of the dye stock solution. The
at 105 °C until a moisture content of 5-10% mixtures were then stirred for 2-80 min on a
was achieved. The Salak seeds were then stirrer hot plate. For the adsorbent dosage
pyrolysed at 400 °C for 1 h. The activated study, 0.4 and 0.6 g of activated carbon
carbons were prepared through the chemical (activated carbon:dye solution ratio of 20:1
activation method using 50%v/v H3PO4 with and 30:1, respectively) were added into a
a ratio of 1:3 (Salak seeds:H3PO4) and left to series of 100 mL Erlenmeyer flasks
immersed for 12 h at 105 °C. It was then containing 20 mL of 100 mg/L of dye
carbonized at 500 °C for 1 h (Carbolite, solution. The mixtures were then stirred for
CWF 1300). After cooling to room 2-80 min on the stirrer hot plate followed by
temperature, the carbonized sample was determination of residual dye concentration
washed with distilled water to remove using UV-Vis spectrophotometer (Thermo
residual H3PO4 and dried at 105 °C for 12 h Scientific, Genesys 10S UV-VIS, USA) at
prior to further use. 600 nm. The extent of dye removal by
2.2 Characterization of the activated activated carbon at equilibrium condition
carbon was calculated using typical mass balance
The surface area of the activated correlation as shown in the following
carbon was determined using a Surface Area equation:
Analyzer (QuantaChrome Corporation, (C  C e )V
NovaWin) through nitrogen adsorption qe = 0
W
desorption isotherm at 77 K. The Brunauer where Co and Ce (mg/L) sre the
Emmet Teller (BET) model was applied to concentrations of dye solutions at initial and
determine the surface area, SBET (m2/g), total equilibrium. V (L) is the total volume of the
pore volume and average pore size radius. solution and W(g) is the mass of activated
The moisture content, iodine value and carbon used.
methylene blue index were determined
according to the methods by American 3. Results and Discussion
Society for Testing Material (ASTM) 3.1 Salak seeds activated carbon
namely ASTM 2867-70, ASTM 4607-94, The yield of the Salak seed based
and Japanese Industrial Standard (JIS) K activated carbon prepared by the chemical
1474:1991, respectively. activation method with H3PO4 was
21.26±0.89%. For that purpose, it was radius of activated carbon are 1196 m2/g,
suspected that a pyrolysis step (above 400 0.56 cm3/g, and 0.94 nm, respectively. The
°C) can reduce significantly the burn-off proximate composition of Salak seed based
values.10 The pyrolysis of lignocellulosic activated carbon is 8.06±0.73 wt% moisture,
materials gives rise to the three phases: char, which makes it suitable precursor for
oils (tars) and gases. A rudimentary porosity obtaining activated carbon.14
is obtained on the char fraction as a
consequence of the release of non-carbon
elements, mainly hydrogen, oxygen and
nitrogen in the form of gases and tars,
leaving a rigid carbon skeleton formed by
aromatic structures.11,12
Figure 2. Removal of 100 mg/L of dye

solution by various dosage of activated
carbon within 80 min of contact time
3.3 Effect of initial concentration and

contact time
Figure 1. Removal of 25-100 mg/L of dye The ability of Salak seed based
solution by 20:1 mg/mL of activated activated carbon to remove dye at various
carbon:dye solution ratio within 80 min of initial concentrations (25-100 mg/L) is
contact time shown in Figure1. The removal of dye using
the activated carbon was rapid at the initial
3.2 Characteristics of activated carbon period of contact time which became slow
Iodine number and methylene blue and constant with the increase in contact
index are widely used parameters for time. The Salak seeds based activated
activated carbon testing because of its carbon showed the ability to completely
simplicity and a rapid assessment of remove 25 mg/L of dye solution within 10
adsorbent quality. It gives an estimate of its min with maximum removal of 0.67 mg/g.
surface area and porosity. The iodine However, higher contact time was required
adsorption of Salak seed based activated to remove more concentrated dye solution.
carbon is 843.46±49.01 mg/g. The typical The removal was recorded as 2.90 mg/g
range is 500-1200 mg/g, which is equivalent (100 mg/L) after 40 min of contact time.
to surface area of carbon between 900 and The Salak seeds based activated carbon
1100 m2/g.13 It is mainly related to the shows a high capability to reach saturation
surface characteristics using a Surface Area of dye removal for the first 40 min. After 40
Analyzer. The methylene blue adsorption min, no significant difference in the amount
capacity of Salak seed based activated of dye uptake was observed indicating the
carbon is very low (12.40±0.04 mg/g). This equilibrium has been attained. This
revealed the fact that the pore size of phenomenon can be attributed to the driving
activated carbon with H3PO4 activation is force to overcome the mass transfer
mainly in the micro-pore regime. These resistance on the adsorbent phase through
results are in accordance with the results of the use of high concentration of dye
BET, where the average BET surface area, solution. After a period of time, it is
total pore volume, and average pore size assumed that no further adsorption takes
place. Generally, with the increasing contact
time the rate of adsorption (slopes) becomes 2. Ong, S. T.; Keng, P. S.; Lee, C. K. Am.
slower with gradual removal until a plateau J. Appl. Sci. 2010, 74, 447–452.
is reached indicating the equilibrium 3. Ratna; Padhi, B. S. Int. J. Environ. Sci.
adsorption of dye on the surface of the 2012, 3, 940–955.
activated carbon. 4. Robinson, E. H.; Li, M. H.; Manning, B.
3.4 Effect of adsorbent dosage B. J. World Aquac. Soc. 2001, 32, 68–
The effect of different adsorbent 71.
dosage (0.40-0.60 g of activated carbon or 5. Kapdan, I. K.; Kargi, F. Enzyme Microb.
20:1-30:1 activated carbon:dye solution Technol. 2002, 30, 195–199.
ratio) was investigated on the removal of 6. Mohammed, N. M.; Muhammad, A. A.
100 mg/L of dye solution. Figure 2 showed Z.; Zainul, A. Z. Int. Biodeterior
that the use of 0.60 g of the activated carbon Biodegradation 2015, 102, 274–280.
was able to remove the highest amount of 7. Sait, Y.; Naile, V.; Hakan, D.
dye (3.23 mg/g) at the shortest contact time Microporous Mesoporous Mater. 2009,
of 40 min. At the same contact time, 0.40 g 122, 189–194.
of the activated carbon was only able to 8. Marielen, C. R.; Matthew, A. A.; Lizie,
remove 2.90 mg/g of dye. Similar pattern D. T. P.; Eder, C. L.; Renato, C.; Liliana,
was also reported by other researchers using A. F.; Puchana-Rosero, M. J.; Fernando,
different types of adsorbents and dyes.15-18 M. M.; Flávio, A. P.; Tatiana, C. Chem.
Higher dye removal from the use of higher Eng. J. 2014, 248, 315–326.
dosages of the activated carbon can be 9. Suzimara, R.; Monique, T. C. Sidnei, L.
associated with higher availability of active P.; Èder, C. L. Renato, C.; Andreia, N.
binding sites present on the surface of the F. J. Hazard. Mater. 2014, 271, 311–
activated carbon. 320.
10. Laksaci, H.; Khelifi, A.; Trari, M.;
4. Conclusion Addoun, A. J. Clean. Prod. 2017, 147,
The high surface area Salak seed 254–262.
based activated carbon, was produced by 11. Mohamed, A. R.; Mohammadi, M.;
H3PO4 activation, showed the potential as Darzi, G. N. Renew. Sustain. Energy
sustainable adsorbent. It is an alternative Rev. 2010, 14, 1591–1599.
adsorbent for the removal of dye from 12. Yakout, S. M.; Sharaf El-Deen, G. Arab
wastewater based on its high removal J. Chem. 2016, 9, 1155–1162.
capacity of dye. The adsorption capacities 13. Saka, C. J. Anal. Appl. Pyrol. 2012, 95,
are significantly influenced by the initial dye 21–24.
concentration, contact time, and adsorbent 14. Gil, R. R.; Ruiz, B.; Lozano, M. S.;
dosage. Fuente, E. J. Anal. Appl. Pyrol. 2014,
110, 194–204.
Acknowledgements 15. Prahas, D.; Kartika, Y.; Indraswati, N.;
The authors would like to Ismadji, S. J. Environ. Prot. Sci. 2008, 2,
acknowledge the financial support from 1–10.
Faculty of Science and Technology, 16. Ahmed, M. J.; Dhedan, S. K. Fluid
Rambhai Barni Rajabhat University, Phase Equilib. 2012, 317, 9–14.
Thailand, and the partially support from the 17. Akkaya, G.; Güzel, F. Chem. Eng.
Department of Chemistry, Rambhai Barni Commun. 2013, 201, 557–578.
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112–118.
Extraction and characterization of humic substances derived from
Thailand’s leonardite
Buntita Jomhataikool1, Sanchai kuboon2, Wasawat Kraithong3, Apiluck Eiad-ua4*
1
College of Nanotechnology, King Mongkut's Institute of Technology Ladkrabang (KMITL),
Ladkrabang, Bangkok 10520, Thailand
2
National Nanotechnology Center, National Science and Technology Development Agency (NSTDA),
111 Pathumthani, Thailand
Abstract:
The major product of lignite mine in Lampang province, Thailand is lignite that uses
as source of energy for electricity and steam engine industry. Leonardite is a byproduct of
lignite mine. It is natural oxidation product of lignite that associated with near surface
mining. It was founded as a rich source of humic substance (HS). Humic substance consists
of 3 fractions of humin (HU), humic acid (HA) and fulvic acid (FA) that can separate by
different pH of base-acid treatment process. This research studied the effect of base
concentration in base treatment process and effect of time during base-acid treatment process.
The method consists of mixing of leonardite with base solution and stirring during condition
time and base concentration. Then, the soluble was separated from insoluble fraction (humin)
by centrifugation. The solution was adjusted to pH 2.0 by adding concentrate HCl. The
precipitates formed at lower pH that conventionally known as humic acid (HA) was separated
from solution by centrifuge. Humic substance was studied for morphology, chemical
structure and element by SEM, FT-IR, UV-Vis and CHNO, respectively. Base concentration
and reaction time have effect to solid yield and chemical structure. Solid yield and percentage
of carbon of HA increase with increasing time and base concentration. Conversely, the
oxidize molecule has decrease. This research provides relative of base treatment condition on
humic substance extraction.
1. Introduction Humic acids are insoluble at low pH, that

Thailand is a significant producer of they can precipitated by adding strong acid
lignite, which is used almost exclusively for or adjust the solution to pH 1. Fulvic acid is
power generation or used as source of the materials that can soluble at all pH and
energy of cement and steam engine can precipitate by adjust pH to 4-5.4 Humic
industrial. Total national lignite production substance can be extracted by 2
is around 21 million tons per years.1 conventional method that are NAGOYA
Leonardite is a byproduct of lignite mine. It method and IHSS method. NAGOYA
is natural oxidation product of lignite with method, in base extraction process must be
brown and coal-like appearance, associated standed in N2 atmosphere and HA solutions
with near surface mining.2 It is a rich source was diluted with 50 times the volume of 3%
of humic substance.3 Humic substance (HSs) NaCl solution. In IHSS method, soil sample
can be divided into three mainfractions by must be acidified to pH 1-2 before extracted.
solubility in varios pH: Humin, Humic acid The extraction process must be stand in N2
(HA) and Fulvic acid (FA). Humin is an atmosphere.5 Humic acid is the principle
organic matter that is insoluble in water at component of humic substance extraction
all pH. Their structures are often vaguely cuase it can used in various application.
described. Humic acid and Fulvic acid Humic acid are known to significantly affect
solution can extracted form soil and other the behavior of pollutants in natural
solid phase using a strong base at high pH. environments, such as trace metal speciation
and toxicity,6 used as a natural feedstuff, it selection of particle size between 180 to 500
has positive effects on the health of various μm.
animal species. Humic acids are thought to 2.2 Method of extraction
be complex aromatic macromolecules with The Humic substance extraction
amino acids, amino sugars, peptides, from leonardite had been carried out
aliphatic compounds involved in linkages following the method by suggest by D.
between the aromatic groups. The gracia,8 using base-acid treatment. Soil
hypothetical structure for humic acid, shown extraction experimental consists of mixing
in Figure 1, contains free and bound 40 g of dry leonardite powder with 400 ml
phenolic OH groups, quinone structures, of KOH solutions of concentrations of 0.05,
nitrogen and oxygen as bridge units and 0.1, 0.25 or 0.5 M solution during 0.5 1, 2,
COOH groups variously placed on aromatic 3, 6, 12 and 24 hours at room temperature.
rings.7 Then, the soluble containing HA+FA were
separated from insoluble fraction containing
humin by centrifuge at 5000 rpm for 15
minutes. The humin fraction was dried at
90ºC for 24 hours. The HA+FA solution
were carefully removed and adjust to pH 2.0
by adding concentrate HCl. The precipitates
formed at pH 2.0 that conventionally known
as humic acid (HA) were separated from
solution by centrifuge at 5000 rpm for 15
minutes and dried at 90ºC for 24 hours.
2.3 Spectroscopic measurement
Figure 1. Hypothetical molecular structure The humic substances were
of humic acid, showing important functional characterizing morphology, element
groups7 composition and chemical sructure by
Scanning electron microscope (SEM), CHN
The research is study extraction of analyzer, Fourier transform infrared
humic substance from leonardite of lignite spectroscope (FT-IR) and UV-Vis
Mae Mho Mine from Thailand by using spectroscope (UV-Vis) respectively. In FT-
base-acid treatment and centrifugation. This IR spectroscopy, one milligram of dried
work studied the effect of time and and base humic fraction was mixed, ground with 100
concentrationin base treatment process and mg of potassium bromide (KBr) and then
the effect of base type. The objective of this mechanically pressed to form a pellet. For
work is to find the obtimize condition for UV-Vis spectroscopy, Humic acid were
extraction humic acid from Thailand’s dissovled with 0.05 NaHCO3 (4 mg/10 mL).
leonardite soil with easy, safety and low A 0.05 M NaHCO3 solution was used as the
energy process. blank and humic acid was detected at a
wavelength of 280 nm and 360 nm for
2. Materials and Methods E2/E3 and 465 nm and 665 nm for E4/E6
2.1 Materials ratio.
Leonardite soil sample was collected
from lignite mine at Mae moh, Lamphang
province, Northern Thailand since August
3.1 Yield of humic substance
2016. The sample was dried at 80ºC for 24
The solid yield of humic acid was
hours to remove water and moisture
displayed on Figure 2. In Figure 2(a), humic
contents. After that, the soil piece was
acid with different of base concentration
milled into powder by mortar and sieved for
were showed. The result indicates that solid
yield of humic acid was increased with
increasing base concentration to 0.1M but Figure 3(b) and (c) are showing the
solid yield of humic acids was decreased microstructure morphology of humic acid
with increasing base concentration to 0.5M. and fulvic acid, respectively. The HA and
Because of with high concentration, the FA fraction morphology are composed of
potassium species in base solution was particles with various shapes and sizes
formed into metal halide salt (KCl).8 In distribution.
different reaction time on base extraction as
shown in Figure 2(b), the humic acid yield a
was increased with increasing reaction time.
However, the solid yield of humic acid not
significant change and reach a stable state
with increase reaction time from 3 to 24
hours.
a
2 μm
2 μm
b
2 μm
Figure 2. Solid yeld of humic acid extracted Figure 3. SEM image of (a) Humin, (b)
from leonardite with various (a) base Humic acid and (c) Fulvic acid that
concentrations and (b) times extracted from leonardite soil with 0.01M
KOH and particle size at 180 μm for 3h at
3.2 Morphology and structural of humic room temperature
substance
SEM images in Figure 2 indicated 3.3 Element analysis of humic acids
the three fractions of humic substances with Element compositions study of
different microstructures morphology. In humic substances has been review by
Figure 3(a), the morphology of humin is steelink (1985).9 Because humic substances
shown which is nonconductive bulk material are non-stoichiometric materials they must
with few porous structure. Furthermore, in be characterized in terms of their average
properties. Element compositions (C, H, N, 3.4 Structural and spectroscopic
O plus S, atomic H/C, O/C and N/C ration) characteristics of the humic substances
of humic acid was shown in Table 1. Humic UV-Vis spectroscopy
acid extracted from leonardite with The value of E2/E3 and E4/E6 ratios
increasing base concentration from 0.05M to is known to be inversely related to
0.1M showed increased percentages of C. molecular weight and aromaticity, but
However, percentage of C at higher proportional to O, C, and COOH content
concentration was decreased that according and total acidity.12 A low ratio (<5) indicates
with solid yield result. In different reaction a high degree of condensation of aromatic
time, average percentage of C was increased humic components, high molecular weight
with increasing reaction time up to 3 hours and low acidity. High ratio (>5) indicates a
but decreased with increasing time from 3 to greater presence of aliphatic compounds,
24 hours. Percentage of C is highest at 3 low molecular weight and high acidity.13
hours reaction time due to the reaction is The E2/E3 and E4/E6 values of humic acid
reach a stable state at higher reaction time. extracted from leonardite was shown in
The percentage of H and N was not Table 1. From absorbance ratio result
significant change in different condition. indicate that the E2/E3 and E4/E6 values of
The H/C ratio has also been used to humic acid extracted with different reaction
indicate the degree of aromaticity or time lower than 6 hours was not significant
unsaturation (a small value) or aliphaticity (a change but lowest at 3 hours. At higher
large value) of materials.10 H/C ratio is a reaction time, absorbance ratio was
function of hydrogen and carbon present in increased. In different base concentration,
the functional group; primary carboxyl and the E2/E3 and E4/E6 values of humic acid
carbonyl groups.11 The O/C ratio appears to was decreased with increasing concentration
be qualitatively parameter for comparing the up to 0.1M. However, the absorbance ratio
oxygen content of humic substance. As was increased in higher base concentration
displays in Table 1, the H/C ratio showed and lowest 0.1M. This means that the
similar ratio with increasing reaction time up aromaticity, molecular weight and acidity of
to 3 hours. However, H/C ratio was humic acid extracted with higher reaction
increased at higher reaction time. The time and higher base concentration was
oxygen content from O/C ratio was lowest at relatively lower than lower reaction time and
reaction time at 3 hours. In various base lower base concentration, which accords
concentration, the H/C was increased with with H/C and O/C results and report from
increasing concentration up to 0.1M but Kang et al.14
decreased with higher concentration (0.1M
to 0.5M). The oxygen content from O/C
ratio was lowest at 0.01M.
Table 1. Element composition and absorbance ratio of humic acid extracted with various
time and base concentration
Time (hours) Base concentration (Molar)
Parameters
0.5 1 2 3 6 12 24 0.05 0.1 0.25 0.5
C/% 24.19 26.73 27.07 31.78 26.42 25.93 21.67 10.28 31.78 26.06 25.35
H/% 2.15 2.30 2.39 2.99 2.74 2.73 2.49 1.83 2.99 2.22 2.08
N/% 1.20 1.28 1.31 1.35 1.05 1.15 0.98 0.56 1.35 1.17 1.12
O plus S/% a 72.46 69.68 69.23 63.88 69.79 70.19 74.85 87.33 63.88 70.55 71.46
H/C b 1.07 1.03 1.06 1.13 1.24 1.26 1.38 2.13 1.13 1.02 0.99
N/C b 0.04 0.04 0.04 0.04 0.03 0.04 0.04 0.05 0.04 0.04 0.04
O/C b 2.25 1.96 1.92 1.51 1.98 2.03 2.59 6.37 1.51 2.03 2.11
E2/E3 1.96 1.90 1.86 1.84 1.97 2.01 2.05 1.89 1.84 1.97 2.06
E4/E6 5.18 5.07 4.9 4.63 4.88 6.88 7.33 4.73 4.63 5.04 5.27
a
by different b atomic ratio x 100
deformation and C–OH aliphatic was
increased but C=O, C=C and H–bond was
similar. For different base concentration, –
a
COOH deformation and C–OH aliphatic was
slightly decreased with increasing base
concentration. However, C=O, C=C and H
bond was increased but decreased at higher
0.5M that according with Element
composition and absorbance ratio result.
Acidity of humic acid can be predict by
using the peak area of FTIR spectra (–COOH
and C–OH group). The acidity of humic acid
was decreased with increased base
concentration. However, acidity of humic
acid was higher at higher reaction time (more
than 3 hours).
b
4. Conclusion
Humic substance can be extracted
from Thailand’s leonardite soil by base-acid
treatment and centrifugation. Humic acid
was major product of extraction. Solid yield
result indicated that yield of humic acid was
increased with increasing reaction. But, yield
of humic acid was reach to stable state at
higher reaction. In different base
Figure 4. FT-IR spectra of humic acid
concentration, solid yield of humic acid was
derived from leonardite with varios (a) base
highest at 0.1M and was decreased at higher
concentration and (b) reaction time
concentration. The H/C, O/C, E2/E3, E4/E6
and peak area of functional group can be
IR spectra spectrometry
used to indicate three degrees of aromaticity,
The FTIR spectra of humic acid in
oxygen content and molecular weight of
different conditions recorded in the 4000-
humic acid. Acidity of humic acid can be
400 cm-1 range are presented in Figure 4.
predict by using the peak area of acidic
The HA spectra bands were similar in
functional group. From solid yield,
various condition. In addition, the spectra
percentage of C, atomic H/C and O/C ratio,
bands around 3400-3300 cm-1 (O–H and N–
absorbance ratio and peak area of functional
H stretch), 1680-1490 cm-1 (aromatic C=C,
group result can be report that humic acid
C=O, COO–), 1120-920 cm-1 (C–OH
extracted at reaction time 3 hours and used
stretch of aliphatic alcohol) and 720-550
base concentration at 0.1M was the optimum
cm-1 (deformation of −COOH). These
condition for extract purity humic acid from
results accord with those reported by Naidja
Thailand’s leonardite.
A. et al.15 The results indicate humic acid
that was extracted from Thailand’s
Acknowledgements
leonardite is purity product with similar
The authors are grateful to the
spectra with commercial HA.13 Table 2.
Research and Researcher for Industry (RRi),
showed the peak area of OH group, C=C
The Thailand Reseach Fund (TRF) and
group, C−OH group and COOH group from
Suntitranon Co., Ltd,. The authors are also
FTIR spectra of humic acid product. With
thankful to the National Nanotechnology
increasing reaction time, –COOH slightly
Table 2. The peak area of functional group of humic acid acid extracted with various time
and base concentration
Time (hours) Base concentration (Molar)
Parameters
0.5 1 2 3 6 12 24 0.05 0.1 0.25 0.5
-COOH deform 0.32 0.33 0.32 0.38 0.43 0.44 0.46 0.38 0.38 0.22 0.22
-C-OH aliphatic 3.21 3.6 3.4 3.34 4.79 4.11 4.64 2.82 3.34 2.89 2.3
C=C, C=O aromatic 0.6 0.62 0.53 0.68 0.7 0.65 0.56 0.37 0.68 0.56 0.36
H bond in O-H 0.26 0.22 0.28 0.28 0.21 0.25 0.23 0.15 0.28 0.22 0.05
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Primary Industries and Mines Office Environ. Sci. Technol. 1994, 28 (11),
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1993, 27 (5), 846−856. W. Appl. Spectrosc. 2002, 56 (3),
318−320.
Modification of electrodes by diamond-like carbon for electrochemical
advanced oxidation of diuron
Nimit Kantiyawong1, Boonchoat Paosawatyanyong2, Varong Pavarajarn1*
1
Center of Excellent in Particle Technology, Department of Chemical Engineering,
Faculty of Engineering, Chulalongkorn University, Thailand
2
Department of Physics, Faculty of Science, Chulalongkorn University, Thailand
*E-mail: varong.P@chula.ac.th
Abstract:
Among pesticides used in agriculture, herbicides are used in the highest amount. In
Thailand, diuron is the top herbicides used for eliminating weed. It is a toxic substance (LD50
1017 mg/kg). Since diuron has excellent chemical stability and half-life over a year in nature,
therefore diuron can reside and penetrate through the soil, causing contamination in water.
The diuron contamination can spread widely. An electrochemical advanced oxidation process
(EAOP), in which highly active hydroxyl radicals are generated at the anode to decompose
organic contaminant, can be used to degrade diuron. In this work, the EAOP was applied in a
microchannel so that diffusion distance can be reduced and high degradation efficiency can
be achieved. According to the past works, graphite was used as an anode for the EAOP,
resulting in up to 90% removal of diuron within 100 s of residence time. However, corrosion
of the graphite occurred under high applied current. In this research, diamond-like carbon
was deposited onto the surface of stainless steel plate via DC magnetron sputtering under
different operating pressure. The coated plate was then used as an anode for the EAOP in a
microreactor. Its effects on the degradation of diuron are investigated and reported.
1. Introduction AOPs include photocatalytic process,

Nowadays, herbicides are widely Fenton’s process, and electrochemical
used in agriculture activity for elimination advanced oxidation process (EAOP).5 At
and control unwanted weeds. Diuron is one present, the AOPs, especially EAOP have
of the top ten herbicides used in Thailand. It begun to be used for removal of organic
is classified as a phenylurea compound that compounds contaminating in water because
can inhibit photosynthesis. Diuron is a toxic these processes have high degradation
substance having the oral LD50 in rats of efficiency.6 In the EAOP, when electrical
1057 mg/kg.1 Because diuron has excellent current is applied to the electrodes, hydroxyl
chemical stability, it has half-life over a year radicals are generated at the surface of the
in nature that can reside in soil and in anode from dissociation of water molecules.
natural water leading to contamination The hydroxyl radicals generated can react
problems. Moreover, diuron is a priority and decompose organic compounds.
toxic compound in the European Union Consequently, this highly active technique
Waste Framework Directive.2 can be used for degradation of diuron.7
Removal of contaminating diuron in Usually, boron-doped diamond (BDD) is
water has been proposed using many used as an anode in the EAOP.8 However,
techniques, such as biodegradation,3 the synthesis of BDD is complicated and
adsorption,4 and advanced oxidation very expensive. Recently, graphite has been
processes (AOPs). The AOPs are developed used in place of BDD for EAOP operating
for a treatment of many types of organic under low electric current. Since the
pollutants in the water. These processes efficiency of the EAOP is commonly limited
generate highly active hydroxyl radicals by issues such as mass diffusion of organic
(OH˚) by different methods. Examples of compounds to the surface of the electrodes,
the EAOP is effectively utilized in a ppm. Potassium iodide (KI), potassium
microchannel reactor. Because of the small hydrogen phthalate (C8H5KO4), ammonium
distance between electrodes, the diffusion molybdate ((NH4)6Mo7O24), and sodium
distance of organic compound is reduced, hydroxide (NaOH) were purchased from
resulting in high degradation efficiency. The Ajax Finechem and used as reagents for
reduced gap size of the microchannel also detection of hydrogen peroxide formed
requires low applied current, which is within the reactor. All of the chemicals used
suitable for graphite to be used as electrodes. were in analytical grade. The carbon target
The degradation of diuron can achieve up to with 99.999% purity was purchased from
90% in a microchannel reactor with Kurt J. Lesker. It was used as a target for the
residence time of 100s.9 However, corrosion sputtering. Argon gas with 99.999% purity
of the graphite electrode might take place was purchased from Praxair and used for the
during the operation. sputtering process. The stainless steel 304
Diamond-like carbon (DLC) is grade was used as the substrate in the
amorphous carbon having combination of sputtering.
sp2 (graphite-like bond) and sp3 (diamond- 2.2 Diamond-like carbon coating via dc
like bond) structure. It has been widely used magnetron sputtering
for many applications such as corrosion The DLC thin film was deposited via
protection, and in electronics.10 DLC has DC magnetron sputtering using solid carbon
excellent properties for the application as an target (99.999%) on 304 stainless steel
electrochemical electrode, such as, high substrates (SS). Before deposition of the
hardness, high chemical inertness, corrosion DLC thin film, the surface of stainless steel
resistance, and electrically conductive. The substrates was coated with Ti as an
techniques for DLC coatings can be divided interlayer between DLC and stainless steel.
into two types: chemical vapor deposition In the deposition process, the chamber was
(CVD) and physical vapor deposition evacuated to a base pressure of 310-5 torr.
(PVD). For PVD, which is much simpler Then, argon gas was supplied to the
than CVD, it is consisted of many sputtering chamber through a mass flow
techniques such as arc, sputtering, and laser controller at flow rate of 2 sccm. The
vapor deposition methods.11 The DC distance between the target to the substrate
magnetron sputtering is one of the PVD holder was set at 10 cm. The DLC thin film
methods for DLC coating. It is the most was deposited on Ti/SS substrates at room
uncomplicated and inexpensive type of temperature. The operating pressure for the
sputtering process. The advantages of DC sputtering process was varied in the range of
magnetron sputtering are control simplicity 2510-3 torr to 510-3 Torr. The sputtering
and low cost of construction and operation.12 voltage and time were fixed at 550 V and
This research aims to study the 1.5 h, respectively. The film was
surface modification of stainless steel characterized using X-ray photoelectron
electrode with DLC via DC magnetron spectroscopy (XPS).
sputtering process under different operating 2.3 Degradation of diuron via EAOP
pressure. Their effects of the effectiveness in within microchannel reactor
the degradation of diuron via EAOP within In the experiment, 10 ppm diuron
microchannel reactor are reported. solution in deionized water was prepared.
The diuron solution was supplied through
2. Materials and Methods the microchannel reactor (thickness of 250
2.1 Chemicals and materials µm) at controlled velocity by a syringe
Diuron or 3-(3,4-dichlorophenyl)- pump. The flow rate was fixed at 1.5 mL/h,
1,1-dimethyl urea, 99.5% purity, was which corresponds to fixed mean residence
purchased from Sigma Aldrich. It was time of 100 s. Before applying the current to
dissolved in water at concentration of 10 the electrodes, diuron solution was flowed
through the microchannel for 1 hour to
ensure complete adsorption of diuron onto
the surface of the electrodes. Subsequently,
DC power supply was used to apply a direct
current of 1 mA across the gap between the
electrodes. The solution coming out from
the microchannel reactor was sampled and
analyzed. A high performance liquid
chromatography (HPLC) and total organic
carbon analyzer (TOC analyzer) were used
to identify concentration of diuron and total
organic carbon content in water respectively.

The DLC film was successfully
deposited on the surface of substrates via
Figure 1. The relation between the relative
DC magnetron sputtering method. The
concentration of diuron at the outlet of the
substrate was titanium coated stainless steel
microchannel reactor and operating time,
(SPT). The operating pressures investigated
comparing the different type of electrodes
were 5 mTorr (SPTC5), 15 mTorr
(SPTC15), 20 mTorr (SPTC20), and 25
Concentrations of hydrogen peroxide
mTorr (SPTC25). For degradation of diuron
detected when graphite and DLC covered
via EAOP in a microchannel reactor under
stainless steel (SPTC5, SPTC15, SPTC20,
an applied current of 1 mA, the results are
and SPTC25) are used as the anode are
shown in Figure 1. It is indicated that the
different, indicating that amount of
degradation of diuron takes place, but the
hydrogen peroxide generated on different
extent of the degradation depends on the
electrodes are not the same. It is
electrode. The steady-state concentration of
hypothesized that the activity of the anode is
diuron at the outlet of the microchannel
relating to molecular structure of the
reactor is lower than the concentration at the
electrode. Graphite is pure sp2 structure,
inlet. However, the bare stainless steel (SS)
while DLC is amorphous carbon with
and titanium coated stainless steel (SPT)
combination of sp2 and sp3 structure.13 The
electrode are not suitable as an electrode for sp2 structure can transfer electron easily.7
EAOP because the diuron degradation
Therefore the graphite electrode can
achievable is small since both electrodes are
generate hydroxyl radicals more than DLC
reactive surface. In order to generate stable
electrodes. When diuron is present in the
hydroxyl radicals, the electrodes must be solution, the concentration of hydrogen
inert. In this results, the behavior of diuron
peroxide is decreased, which indicates that
degradation using DLC electrodes could be
hydroxyl radicals interact with the
fitted with a pseudo-first-order kinetics,
molecules of diuron and induces the diuron
comparable with when graphite electrode is
degradation.
used.9
The change in either the material or
Hydrogen peroxide (H2O2) is
nanostructure of carbon (i.e., sp3-to-sp2
detected at the outlet stream of the
ratio) of the anode affects degradation of
microchannel reactor during the EAOP. The
diuron because of the change in amount of
results using different type of electrodes are
hydroxyl radicals generated as shown in
shown in Figure 2.
Figure 3. The relationship between percent
The formation of hydrogen peroxide
of diuron degradation and hydrogen
has been suggested as the result of
peroxide generated during the process is
recombination of reactive hydroxyl radicals.7
Figure 2. The effects of electrodes on Figure 3. The correlation between the
hydrogen peroxide detected at the outlet of concentration of hydrogen peroxide
the microchannel reactor, comparing generated in pure DI water and the percent
between pure DI water and 10 ppm of degradation of diuron contaminants
diuron contaminant
correlated. It should be noted that the

concentration of hydrogen peroxide is a
direct representation of the hydroxyl radicals
generated under the same condition of
EAOP. Although the efficiency of diuron
degradation is affected by structure of
carbon or sp3/sp2 ratio within the DLC film,
these properties do not affect the order of
degradation reaction. The reaction is still a
pseudo-first-order reaction.
The influence of operating pressure
for DLC deposition process on the
degradation of diuron was studied by
varying pressure of Ar in the chamber, in the
range of 5 mTorr to 25 mTorr, as shown in Figure 4. The effects of operating pressure
Figure 4. The concentration of hydrogen for DLC sputtering on the concentration of
peroxide and percent of degradation are hydrogen peroxide generated in pure DI
different when the operating pressure is
water and percent of diuron degradation
varied.
In the DC magnetron sputtering The corrosion of DLC electrodes
process, the increasing operating pressure
during EAOP was investigated with the total
increases the sp3/sp2 ratio of the DLC film.10 organic compound contamination in DI
Although the sp2 structure results in more water at the outlet stream of the
hydroxyl radical then the sp3 structure, the microchannel reactor. These results are
sp3 structure has higher hardness than the shown in Figure 5. It is clear that the
sp2 structure. Therefore, DLC with high sp3 corrosion of the DLC film deposited at high
content should have higher resistance to operating pressure is less than the DLC
corrosion. electrode deposited at low operating
pressure. These results confirm the previous can form titanium carbine with carbon
discussion. atoms, leading to better adhesion to the
The comparison of corrosion during substrate.
EAOP using different electrodes is shown in
Figure 6. The graphite electrode shows the 4. Conclusion
highest corrosion because it is pure sp2 DLC film can be used to increase
structure. Considering the DLC film that resistant to corrosion of the electrodes for
was deposited on Ti/SS substrate, it shows EAOP process in a microchannel reactor.
less extent of corrosion than the DLC film Nevertheless, the nanostructure of DLC (i.e.,
deposited on SS substrate because titanium sp3-to-sp2 ratio) affects amount of hydroxyl
radicals generated at the surface of anode
such that smaller amount of hydroxyl
radicals are generated if sp3 content in the
DLC film is high. The Ti interlayer
enhances adhesion between DLC films and
substrate, reducing the corrosion.
Acknowledgements
This research was supported by
Research Program in Hazardous Substance
Management in Agricultural Industry,
Center of Excellence on Hazardous
Substance Management (HSM), Bangkok,
Thailand.
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Carbon dioxide capture on solid amine sorbent: breakthrough behavior
and kinetic analysis
Buppa Shomchoam*, Pathompong Janetaisong, Somsak Supasitmongkol
National Metal and Materials Technology Center (MTEC), National Science and Technology Development
Agency (NSTDA), 114 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Nueng,
Amphoe Khlong Luang, Pathumthani 12120, Thailand
*E-mail: buppap@mtec.or.th
Abstract:
Polyethylenimine (PEI)-extruded pellet and commercial zeolite pellet (molecular
sieve, MS 13X) were experimentally investigated to determine the technical feasibility for
using PEI-extruded pellet in carbon dioxide (CO2) removal from a simulated hydrocarbon gas
streams (15% CH4, 3% C2H6, 2% C3H8, 110 ppm H2S balance CO2). It was found that the
higher temperature favors the diffusion of CO2 molecules from the surface into the bulk of
PEI-extruded pellet. At the same temperature, the volume-based CO2 uptake of PEI-extruded
pellet measured in the fixed-bed flow system was slightly lower than that of MS 13X. The
amine group in PEI structure dominates the higher CO2/C3H8 selectivity relative to the MS
13X sorbent. The constricted pores of MS 13X play a role on controlling CO2 diffusion,
resulting in a slower CO2 sorption rate. Pseudo-first order, Pseudo-second order and Avrami
models were applied to fit experimental data. Among all the kinetic models examined,
Avrami model was found to be most suitable for simulation of the breakthrough curve of CO2
uptake on PEI-extruded pellet in fixed bed column, with average relative errors of less than
6% over the temperature range of 30-80 oC and the nonlinear coefficient of determination
(R2) ≥ 0.99. The PEI-extruded pellet could be a potential sorbent for efficient removal of CO2
in fixed-bed sorption column.
1. Introduction in natural gas we have developed a high

Carbon dioxide (CO2) is the sorption capacity sorbent by immobilizing
greenhouse gas that makes the largest amine functional groups, such as those
contribution from human activities. It is found in polyethylenimine (PEI) on high
released into the atmosphere by: the surface area supports such as fume silicas.
combustion of fossil fuels such as coal, oil However, the using of powders as sorbent in
or natural gas, and renewable fuels like fixed bed sorption system give rise to some
biomass; by the burning of, for example, serious problems like pressure drop and
forests during land clearance; and from retarded mass transfer rate. Therefore, these
certain industrial and resource extraction powdered sorbents are invariably needed to
processes.1 The conventional technology for be shaped into larger particles such as
CO2 removal is amine sorption. However, extrudes, granules, and sphere prior to their
the process has a number of drawbacks such use as sorbent.
as high energy consumption for solvent The literature provides a large
regeneration, equipment corrosion and low number of expressions describing the
contact area between gas and liquid.2 kinetics of sorption on solid surfaces for
CO2 is a weak Lewis acid that can liquid-solid system; but much less
interact with electron donors; therefore, the information is available to describe the gas
presence of basic nitrogen-containing sorption kinetic on functionalized
surface group is throught to provide sorbents mesoporous materials. In this study, the
capable of interacting strongly with CO2.3-5 experimental kinetics data of CO2 sorption
Therefore, for the purpose of reducing CO2 at different temperatures were fitted to three
kinetic models (Pseudo-first order, Pseudo- conductivity detector (GC-TCD) and
second order and Avrami models) to columns (Hayesep Q and molecular sieve
describe the sorbent-sorbate interactions and 5A). The dynamic capacity of CO2 capture
sorption rate behavior. onto the sorbent was calculated using
Equations (1) and (2):
2.1 Sorbent preparation (1)
All chemicals were used as
purchased from the supplier. These
included: Cab-O-Sil, fumed silica (2)
(commercial grade, Concrete Composite
Co., Ltd), methanol (AR, Carlo Erba), where q is the adsorbed CO2 content (mmol
polyethylenimine (Mw 800, Mn 600, CO2/g sorbent), F is the total flow rate
Aldrich), polyvinyl alcohol binder (PVA- (ml/min), tq is the stoichiometric time (min),
18Y, Japan Vam & Poval), N2 (99.999%, W is the mass of sorbent (g), Vmol is the molar
Praxair), a hydrocarbon gas mixture (15% volume (22.4 ml/mmol), Co and CA are the
CH4, 3% C2H6, 2% C3H8, 110 ppm H2S inlet and effluent CO2 concentrations
balance CO2, Praxair). Polyethylenimine- (%vol.) respectively.
immobilized fumed silica (PEI/FS) and PEI-
extruded pellets were prepared as reported in
our research.6
40 wt% PEI loading in fumed silica
(PEI-40/FS) was gradually added to a binder
solution in distilled water and stirred at 60
o
C for 1 hrs. The resulting solution was
evaporated the solvent completely at 80 oC
until the mixture became a sticky enough for Figure 1. Schematic diagram of the
pelletizing adsorbent via a plunger extruder. experimental apparatus
Cylindrical extrudate with small diameter
(ca. 2 mm) and 4 mm in length was obtained 2.3 Kinetic of sorption
after dried overnight at ambient temperature The experimental CO2 sorption
and cut to small piece. The size of pellets kinetics data is examined by fitting it to
agreed well with the length range (4-6 mm) three models i.e. pseudo-first order, pseudo-
of a commercial zeolite type MS 13X (1.6 second order and Avrami kinetic models as
mm pellets, Sigma-Aldrich). given in Table 1.
2.2 CO2 sorption process
The CO2 sorption from the model Table 1. Kinetics sorption models7
gases was performed in a fixed-bed Kinetic Equation Differential form
model
adsorption system (Figure 1). Sorbent pellet Pseudo-
(5 g) was loaded in a quartz column with first order
inner diameter of 2 cm and 4 cm of packed Pseudo-

second
height. Before each run, the sorbent was order
pretreated at 90 oC under N2 (45 ml/min) for Avrami
3 hrs. After cooling down to desired sorption
temperature, the gas flow was switched to a
hydrocarbon gas mixture at a constant flow where qe and qt (mmol/g) are sorption
rate (5 ml/min) to investigate CO2 sorption. capacities at equilibrium and at time
The treated gas out of the adsorber was respectively, kf, ks and kA are the pseudo-
analyzed online using a gas chromatography first order, pseudo-second order and Avrami
(7890A, Agilent) equipped with a thermal rate constant, n is the Avrami's kinetic order.
The best fit of sorption kinetic model became faster than gas sorption, the
was evaluated by Equation 3 and coefficient equilibrium was achieved and the CO2
of determination (R2). uptake decreased as expected (Figure 3).
3.2 CO2 selectivity of sorbents
Competition between CO2 and other
components in the sorption process on PEI-
(3) based pellet and zeolite 13X was evaluated
where Err(%) is the error function, qt(exp) and at 55 oC under 15% CH4, 3% C2H6, 2%
qt(mod) are the experimental and calculated C3H8, 110 ppm H2S balanced in CO2 (5
adsorption capacities at a given time, N is ml/min) as shown in Figure 4.
the total number of experimental points.
a)
Figure 2. Breakthrough curves of CO2
uptake in PEI-extruded pellets at different
isotherms
b)
Figure 4. Breakthrough curves of gas
uptake in a) PEI-extruded pellet and b)
zeolite 13X sorbent
Figure 3. Breakthrough CO2 capacity as a
function of sorption isotherms The breakthrough curves of
hydrocarbons (CH4, C2H6 and C3H8) had
3. Results and Discussion C/C0 values higher than 1 and came back to
3.1 Sorption isotherms steady state at C/C0=1 (Figure 4a). This
The breakthrough curves and CO2 behavior may be explained that some
sorption of PEI-extruded pellet are shown in hydrocarbon molecules can be weakly
Figures 2 and 3. Both breakthrough time and adsorbed in PEI-based sorbent and then
CO2 uptake of PEI-based pellet were occurred roll-up effects. The more strongly
increased significantly with the temperature adsorbed components (CO2 and H2S)
enhanced from 30 oC to 55 oC. This is displace some hydrocarbon from the
probably due to the nature of highly flexible sorbent. This behavior was also found in the
polymer for easier CO2 diffusion from the breakthrough curves of zeolite 13X, except
surface into the bulk of PEI-based pellet by for the C3H8 case (Figure 4b). This implies
overcoming the kinetic barrier at enhanced that the amine group in PEI structure
temperature, although the actual sorption is dominates the higher CO2/C3H8 selectivity
exothermic. Beyond 80 oC, gas diffusion
relative to the zeolite 13X at the same
condition studied.
For a large scale CO2 capture
process, higher volume-based CO2 sorption
capacity is very important because it will
reduce the sorbent bed size, thus reducing
the infrastructure investment for mass CO2
capture. Based on packing density of PEI-
extruded pellet (0.398 g/ml) and zeolite 13X a)
(0.385 g/ml), the volume-based CO2 uptake
of PEI-extruded pellet measured in the
fixed-bed flow system was slightly lower
than that of zeolite 13X (Table 2).
Table 2. Comparison between PEI-extruded

pellet and zeolite 13X
Sorbents Surface Pore size Pore Volume-
area, distribution volume based
BET (Ao) (cm3/g capacity
(m2/g) [STP]) (mg
CO2/ml b)
sorbent)
PEI Figure 5. Experimental CO2 uptake at 55 oC
extruded 32.30 350 0.28 26.02 and corresponding fit to kinetic models on a)
pellet
MS 13X 700.40 6.27 0.49 27.19 PEI-extruded pellet and b) zeolite 13X
sorbent
3.3 Sorption kinetics of CO2
Among the properties expected in a extruded pellet and zeolite 13X over the
good sorbent, fast sorption kinetics is one of range of temperatures considered (Table 3).
the most important, since the efficiency of a The kinetic rate constants (kA) were
sorbent to withstand large sorbate flows are calculated from the best fit parameters to the
related with its rate of sorption. experimental data using Avrami’s model, in
Figure 5 shows the CO2 uptake which the correlation coefficient (R2) was
versus time at 55 oC, and the corresponding greater than 0.98 for PEI-extruded pellet and
profiles as predicted by the different kinetic 0.96 for zeolite 13X. The values of q
models. For all sorbents, it was observed reported in Table 3 reflected closely the
that the pseudo-first and pseudo-second experimental sorption capacity.
order kinetic models presented some The calculated Avrami’s kinetic
limitations. The pseudo-first order model order (n) was a fractional value (ca. 1.3-1.4)
overestimated the CO2 uptake until the with respect to the sorption temperature
process approached equilibrium. The within the range studied. The fractional
pseudo-second order model underestimated order of this model has been suggested as a
the CO2 uptake beyond ca. 40 min and then change in sorption mechanisms.8 This is
overestimated after ca. 100 min. The best fit consistent with the assumption that a
to a kinetic model was obtained using physical state change of PEI occurs at higher
Avrami’s equation. This is clearly consistent temperature as mentioned previously.
with the calculated error (q(%)) and Enhancing temperature facilitates the mass
coefficient of determination (R2) in Table 3. transfer of the physisorbed CO2 molecules
It was observed that both the pseudo- from the surface into the deeper multilayers
first and pseudo-second order kinetic models of PEI, when loaded on mesoporous sorbent
presented some limitations with respect to pellet. This leads to a significant
the prediction of CO2 uptake on PEI- enhancement of the equilibrium CO2 uptake
capacity with temperatures. The PEI- resulting in a slower CO2 uptake rate
extruded pellet exhibited a higher compared to the PEI-based sorbent in pellet
equilibrium capacity for CO2 at 80 oC than form.
at 55 oC and 30 oC, respectively (not
shown). This result was in contrast to the 4. Conclusion
result for the kinetic rate constants (kA) in The CO2 uptake on
Table 3. Overall, the rate of CO2 uptake polyethylenimine-extruded pellet sorbents
decreased as the temperature increased. This was evaluated using fixed bed. The
may be attributed to the interactions between proposed Avrami adsorption model seems to
the polyamine and the adsorbed CO2 be a good alternative to the prediction of
through forming a strong pervasive CO2 uptake over the entire temperature
hydrogen bonded network, thus limiting range studied. Besides the faster CO2 uptake
translational and rotational dynamics. Such rate, the PEI-extruded pellet presented a
network can be thought to contribute to higher CO2/C3H8 selectivity and a slightly
mass transfer barriers of CO2 between the lower volume-based CO2 uptake relative to
CO2 and solid phases. the zeolite 13X under the same conditions.
In contrast with the PEI-extruded The PEI-extruded pellet represents an
pellet, the increased temperature increased attractive candidate for use in commercial
the rate of CO2 uptake (kA) on zeolite 13X adsorbers.
(Table 3). Enhancing temperature simply
provides more internal energy to gaseous Acknowledgements
molecules, resulting in the faster migration The author gratefully acknowledges
of CO2 molecules inside the pores. At the the support of National Metal and Materials
same temperature, the highest CO2 uptake Technology Center (MTEC), National
rate on the PEI-extruded pellet compared Science and Technology Development
with the zeolite 13X. This may correspond Agency (NSTDA).
with hindered diffusion in the constricted
pores of commercial zeolite 13X (Table 2),
Table 3. Values of kinetic model parameters for CO2 sorption on sorbents at different
temperatures
Sorbents Sorption temperature (oC)
30 55 80
Pseudo-first order KF(S-1) 0.0578 0.0534 0.042
R2 0.9794 0.9858 0.9800
q(%) 25.45 51.83 43.91
PEI extruded pellet Pseudo-second order KS(mol Kg-1S-1) 0.0167 0.0056 0.0046
R2 0.9379 0.8934 0.8930
q(%) 13.38 13.53 13.63
Avrami KA(S-1) 0.0420 0.0302 0.0263
n 1.275 1.4415 1.3776
R2 0.9805 0.9886 0.9855
q(%) 4.46 5.43 5.16
Pseudo-first order KF(S-1) 0.0526 0.0576 0.0737
R2 0.8633 0.8679 0.8844
q(%) 85.6613 73.0600 59.1752
MS 13X Pseudo-second order KS(mol Kg-1S-1) 0.0003 0.0008 0.0022
R2 0.6883 0.7634 0.8227
q(%) 7.8219 14.0467 22.7232
Avrami KA(S-1) 0.0196 0.0215 0.0266
n 1.1646 1.6182 1.6483
R2 0.9568 0.9614 0.9686
q(%) 14.3060 12.9060 11.4593
References IPCC Special Report on Carbon Dioxide

1. Adegbulugbe, A.; Christophersen, Q.; Capture and Storage 2005, 54.
Ishitani, H.; Moomaw, W.; Moreira, J. 2. Gorji, A.; Sayari, A. Chem. Eng. J. 2011,
173, 72–79. 6. Suwannakij, K.; Lailuck, V.;
3. Plaza, M. G.; Garcia, S.; Rubiera, F.; Pis, Supasitmongkol, S. KMUTNB Int. J.
J. J.; Pevida, C. Sep. Purif. Technol. Appl. Sci. Technol. 2017.
2011, 80, 96–104. 7. Rodrigo, S. G.; Abdelhamid, S. Chem.
4. Zhang, Z.; Xu, M.; Wang, H.; Li, Z. Eng. J. 2010, 161, 182–190.
Chem. Eng. J. 2010, 160, 571–577. 8. Cestari, A. R.; Vieira, E. F. S.; Vieria, G.
5. Houshmand, A.; Daud, W. M. A. W.; S.; Almeida, L. E. J. Hazard. Mater. B.
Shafeeyan, M. S. Sep. Sci. Technol. 2011, 2006, 138, 133–141.
46, 1098–1112.
Performance comparison of different membrane micro-channel reactors
for methanol production from biogas and hydrogen
Khunnawat Ountaksinkul1, Tara Jiwanuruk2, Nattapong Kasempremchit1,
Paravee Vas-Umnuay3*, Palang Bumroongsakulsawat1, Sompong Putivisutisak4,
Suttichai Assabumrungrat1
1
Center of Excellence in Catalysis and Catalytic Reaction Engineering, Department of Chemical Engineering,
Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand
2
National Metal and Materials Technology Center, Thailand Science Park, Pathumthani 12120, Thailand
3
Center of Excellence in Particle Technology, Department of Chemical Engineering, Faculty of Engineering,
4
Advanced Computational Fluid Dynamics Research Unit, Faculty of Engineering,
Chulalongkorn University, Phayathai, Bangkok 10330, Thailand
*E-mail: paravee.v@chula.ac.th
Abstract:
The models of different membrane microchannel reactors (MMR) for methanol
synthesis from biogas and hydrogen (H2) were developed. MMRs combining reaction and
separation by membrane in a single unit were simulated based on 2D pseudo homogeneous
models using COMSOL Multiphysics. Biogas was fed into the MMRs where CO2 in biogas
was separated through a membrane to react with H2 in a reaction channel (RC) to form
methanol. Three configurations of MMR were investigated including 1) addition of CO2 into
1 side of RC, 2) addition of CO2 into both sides of RC and 3) combined addition of CO2 into
RC with simultaneous removal of water from the reaction zone. In case 3, Argon acts as a
sweep gas for permeation side. The yields of methanol achieved from each configuration
under conventional condition were 14.11%, 15.48% and 20.92%, respectively. The
configuration in case 3 outperformed the others because the removal of water in the system
can shift the equilibrium of the reactions to the forward side. The effects of operating
parameters including inlet temperature, pressure and flow rate utilized in case 3 were
investigated by sensitivity analysis. The optimal condition was at 533 K, 40 bar, WHSV 50.7
h-1, BC:RC ratio 2:1 and SC:RC ratio 5:1.
1. Introduction vertical shell & tube heat exchanger

Currently, the abatement of fossil reactor.3 Yet, several investigations have
resources and the growth of energy demand been widely studied about micro-structured
lead to development of renewable energies. and membrane reactor due to high heat and
Biogas is one of these which can be used as mass transfer, providing simultaneous
a fuel for transport vehicle engines. reaction and separation steps and achieving
However, CO2 in biogas is the cause of the conversion over equilibrium limitation.3-11
reduction of engine power output. Thus, In this work, three different types of
CO2 needs to be removed from biogas and planar membrane wall-coated microchannel
the upgraded biogas should have methane reactor (MMRs) for methanol production
content approximately 95 %.1 Many years from biogas and H2 were studied using 2D
ago, there were various researches studying computational fluid dynamics (CFD) model
about CO2 utilization. One of those is to to compare their effects on the performance
produce hydrocarbon fuel like methanol of the reactor. Moreover, effect of operating
which is less polluting and also has high H2 parameters were investigated by sensitivity
to carbon ratio and energy density.2 analysis to find the optimal condition. The
Generally, most of the reactors used for the operating parameters including inlet
commercial methanol synthesis plants are a temperature, pressure, weight hourly space
a. c. d.
Reaction
Sweep gas channel
channel
Biogas channel
Reaction channel
b.
Biogas channel Biogas channel
Reaction CuO/ZnO/Al2O3 Silica H-SOD
channel membrane membrane
Biogas channel Catalyst
Figure 1. The proposed 2D configulation each case a.) 1 biogas channels, b.) 2 biogas channels
c.) water removal d.) 3D configulation
of MMR
velocity (WHSV) of reaction channel and Table 1. Operating condition for base case
flow ratio of reaction channel to biogas Parameters Value (unit)
Inlet temperature 533 (K)
channel were studied. Pressure in RC, BC and SC 30, 31 and 1 (bar)
WHSV in RC 50.7 (h-1)
2. Model Description Mass flow ratio of BC:RC 3:2 (-)
2.1 Reactor geometry Mass flow ratio of SC:RC 2:1 (-)
In this research, the membrane Catalyst loading 0.142 (g)
Density of catalyst 1775 (kg/m3)
microchannel reactor can be divided into Mole fraction of biogas
three cases; 1) addition of CO2 into 1 side of CO2 0.5
RC (Figure 1a), 2) addition of CO2 into both CH4 0.5
sides of RC (Figure 1b) and 3) combined
addition of CO2 into RC with simultaneous The kinetic models of Van-dal &
removal of water from RC (Figure 1c). The Chakib Bouallou12 were applied in this
characteristic of the planar membrane research. The reactions for methanol
micro-structured reactor in all cases is synthesis involve three main equilibrium
performed in Figure 1d. Each channel is 20 reactions: CO hydrogenation (1), CO2
mm long with 0.7 mm x 20 mm (cross- hydrogenation (2) and reverse water gas
sectional area) and thickness of walls (high shift (3). All reactions can be written as
strength metal alloy) are 0.3 mm.2 The follows:
porous silica membrane is deposited on the CO + 2H2 ↔ CH3OH (1)
wall of biogas channel (BC) as a CO2 CO2 + 3H2 ↔ CH3OH + H2O (2)
selective membrane and CuO/ZnO/Al2O3 CO2 + H2 ↔ CO + H2O (3)
catalyst is coated on the walls of reaction The corresponding reaction rates for
channel (RC) for methanol synthesis in all reactions (2, 3) are expressed in the
cases. CO2 in biogas is separated from BC to following equations (4-5):
react with H2 in RC. For case 3 where water k1PCO2 PH 2  k6 PH 2O PCH 3OH PH2
is being removed, a thin defect free hydroxy rCH 3OH  2 (4)
 
3
sodalite (H-SOD) membrane is coated at the 1  k2 PH 2O PH1  k3 PH0.5  k4 PH 2O
2 2
inner wall as a water selective membrane
k5 PCO2  k7 PH 2O PCO PH1
and catalyst is applied over this membrane rRWGS  2
(5)
layer shown in Figure 1c. The water 1  k2 PH 2O PH1  k3 PH0.5  k4 PH 2O
2 2
occurred from methanol synthesis is where pressure of each species (Pi) is given
removed from RC into the sweep-gas in Pa, temperature (T) in K and reaction rate
channel (SC). Argon is fed into SC as a (r) in kmol kgcat-1s-1. the k parameters in
carrier gas. reaction rate equations mentioned previously
2.2 Chemical kinetics can be calculated from the below equation
and Ai, Bi are presented in the research of mixture, Q is heat source term due to heat of
Van-dal & Chakib Bouallou.12 reactions which occur only the wall surfaces
B for this study. For porous support, the
Inki  Ai  i (6)
T effective thermal conductivity is given by:
2.3 Assumptions & governing equations
2.3.1 Model assumptions

keff   p k p  1   p k (10)
In this study, the fluid flow inside the where θp is volume fraction or 1-porosity
membrane microchannel reactor (MMR) and kp is thermal conductivity of porous
was considered to be in a steady state media. The equation of conservation law of
condition, a compressible fluid and a chemical species under steady state
laminar flow. For non-ideal gas system, condition for this study can be written below
 
r uur
Soave Redlich Kwong (SRK) model was  u   i    Ji  Ri  Ni (11)
used as thermodynamic model for predicting
where ωi is the local mass fraction of each
gas behavior in this investigation. The
species. Ji and Ri are the mass diffusion flux
pressure drop in MMR could be neglected
which can be calculated from Fick’s law and
due to no obstruction in the channels. The
the net of rate of reaction on wall surfaces,
transport phenomena in z direction was
respectively. For permeation flux (Ni), the
neglected because of no significant changes
water and carbon dioxide flux can be
in this direction. Moreover, fluid flow in the
adopted from Chen et al.1
catalyst and membrane layers were also
 p 
neglected due to very thin thickness. N i  i   (15)
2.3.2 Governing equations  l 
The equation for conservation of Where Δp is partial pressure difference of
mass or continuity equation at steady state component i between retentate and
condition is shown in Equation 7 permeate. l is thickness of membrane and i
 
r is permeability coefficient of component i.
  u  0 (7)
Water permeation flux was applied from
where  is density of gas mixture and u is Rohde et al.,13 whereas CO2 permeation flux
velocity of fluid. For a laminar flow of was adopted from Masashi & Shin.14
compressible fluid under steady state Finally, the methanol yield can calculated
condition, the equation of conservation of from mole flow rate of methanol (FCH3OH)
momentum or Newton’s second law is and inlet CO2 (FCO2, inlet) by Equation 16.
simplified as follows: FCH 3OH
(16)
yield  100%
 r 
     
r r  r r T  2 FCO2 ,inlet
 u   u      p    u  u     u  
   3 
(8)
where p is the static pressure, µ is the
molecular viscosity which can be
determined based on molar average of
species in the mixture and  is the tensor
unit. For flow in porous media, the velocity
profile can be calculated from Darcy’s law.
The equation of conservation law of energy
for fluid flow under steady state condition Figure 2. Boundary condition
can be written in Equation 9
ur
d z  C p u  T     d z k T   d z Q (9) 2.4 Simulation method
For solving the numerical solution,
where Cp and dz are the specific heat COMSOL Multiphysics version 5.3a was
capacity and the thickness of channel, used by finite element method. Free quad
respectively. k is thermal conductivity of the meshes were assumed at 54400, 86400 and
86400 domain elements for cases 1-3, mole fraction at the surface wall of RC was
respectively. For boundary condition higher than the centre-line of RC in all cases
defined in case 3, it could be summarized in (Figure 3a-c) due to catalyst coated at
Figure 2. For case 1 and case 2, it used the surface wall. At the outlet, the methanol
same boundary condition with biogas mole fractions were 1.17x10-2%, 1.19x10-2%
channel and reaction channel in Figure 2. and 1.80x10-2% for cases 1-3, respectively.
a. The case 3 (Figure 3c) exhibited the
) highest mole fraction of methanol due to
b. increasing net rate of reaction term in
) Equation 11 from disturbance of chemical
equilibrium by using H-SOD membrane.
c.
According to Le chatelier’s principle, the
)
water removal out of reaction channel can
0 0 0 0 0 1 1 1 1 1 shift the reaction equilibrium (Equations 2,
3) to forward side. The yields of methanol in
Figure. 3. . Mole
. fraction
. . of. methanol
. . . along
each case were compared in Figure 4. The
with reaction channel of a.) case 1,6 b.)8case 2
2 4 6 8 0 2 4
methanol yields were 14.11%, 15.48% and
and c.) case 3 20.92% for cases 1-3, respectively. Hence,
the case 3 provided the highest performance
which corresponding with the results from
Figure 3. For methane content in BC, the
results are expressed in Figure 5. The red
line represents the inlet methane mole
fraction.
The methane contents at outlet were
88.77%, 91.42% and 89.00% for cases 1-3,
Figure 4. The yield of methanol in RC of respectively. The methane contents were
case 1, case 2 and case 3 improved to be over 38.77%, 41.42% and
39.00%. It could be concluded that the
methane contents in each case do not
significantly change. Yet, the case 2
provided the highest methane content
because the high membrane area led to
enhancing of permeation flux in Equation
15. From all results, the water removal case
is suitability due to providing high yield of
Figure 5. The methane content in BC of methanol and methane content. Hence, the
case 1, case 2 and case 3 operating parameters of case 3 were studied.
3.2 Operating parameters study
3. Results and Discussion In Figure 6, the effect of temperature
3.1 Base case study on the yield of methanol at predetermined
In this section, cases 1-3 were range (493 K, 533 K and 573 K) was studied
simulated by using operating parameters along the centre line of the channel.
shown in Table 1 and governing equations According to the results, it was found that
according to section 2.3. The mole fractions the yield of methanol at the outlet were
of methanol obtained from each case are 22.60%, 30.98% and 19.64% for 493 K, 533
performed in Figure 3. The blue colour K and 573 K, respectively. Increasing
represents to the low methanol mole fraction temperature from 493 K (solid line) to 533
area and the red colour represents to the high K (dot line) increases reaction rate of CO2
methanol mole fraction area. The methanol hydrogenation (Equation 2) because the
temperature affects the kinetic constants in According to Le Chatelier’s
Equation 6. Moreover, the high reaction rate principle, the increased gaseous pressure
also promotes the CO2 and water flux due to causes CO2 hydrogenation (Equation 2)
improving driving force or partial pressure reaction to move forward. However,
difference in Equation 15. Hence, the yield increasing pressure causes the system to be
of methanol was enhanced to be over 8.38%. more complicated and uses more energy
consumption. The effect of WHSV on the
yield of methanol at fixed catalyst loading
0.142 g is expressed in Figure 8. The yields
of methanol at 20 mm were 30.8%, 31.21%,
30.78% and 29.98% for 50.70, 76.06,
101.41 and 126.76 h-1, respectively.
Figure 6. Effect of temperature on the yield

of methanol along with RC
Figure 8. Effect of WHSV in RC on the

yield of methanol along with RC
Figure 7. Effect of pressure on the yield of

methanol along with RC
Yet, increasing temperature to 573 K

(dash line) reduces the yield of methanol
about 11.34% because the excessive
enhancement of the temperature increases Figure 9. Effect of mass flow BC:RC ratio
thermodynamics equilibrium of endothermic on the yield of methanol along with RC
reaction. Therefore, the reverse water gas
shift (Equation 3) is promoted more than
CO2 hydrogenation (Equation 2). From this
results, the temperature about 533 K is
considered to be the suitable parameter for
this system.
In Figure 7, the effect of pressure on
the yield of methanol along the center line of
RC was studied at 20, 30 and 40 bar. At the
outlet, the yields of methanol were 14.65%, Figure 10. Effect of mass flow SC:RC ratio
20.92% and 26.27% for 20, 30 and 40 bar, on the yield of methanol along with RC
respectively. Hence, increasing pressure
leads to improvement of methanol yield Based on these results, enhancement
because the higher pressure directly affects of WHSV reduces the performance of MMR
Yet, increasing temperature to 573 K (dash slightly. Increasing WHSV increases the gas
line) reduces the yield of methanol about flow rate and gas velocity but decreases
11.34% because the excessive enhancement contact time of reactants. Hence, the
the rate of reaction (Equations 4, 5). methanol yield is reduced. Furthermore,
enhancement of flow rate improves the Acknowledgement
convection term and dilutes water in RC. This work was financially supported
Thus, the water permeation flux is weakened by the Ratchadapisek Sompoch Endowment
due to low driving force. In addition, the Fund (2016), Chulalongkorn University
effect of mass flow ratio of BC:RC was also (CU-59-003-IC).
investigated, as shown in Figure 9.
According to the results, yields of methanol References
were 24.70%, 17.48%, 13.46% and 10.94% 1. Chen, X. Y.; Vinh-Thang, H.; Ramirez,
for ratio 2, 3, 4 and 5 times, respectively. A. A.; Rodrigue, D.; Kaliaguine, S. RSC
Increasing BC:RC ratio reduces yield of Advances 2015, 5, 24399–24448.
methanol because increasing BC:RC ratio or 2. Tadbir, M. A.; Akbari, M. H. Int. J. of
gas flow rate in biogas channel reduce Hydrogen Energy 2011, 36, 12822–
driving force for CO2 permeation flux. Thus, 12832.
both rates of reaction (Equations 4, 5) and 3. Farsi, M.; Jahanmiri, A. J. Natural Gas
methane content in BC are dropped due to Chem. 2012, 21, 407–414.
the lower amount of CO2 diffusing through 4. Tonkovich, A.; Jarosch, K.; Arora, R.;
the membrane. For effect of mass flow ratio Silva, L.; Perry, S.; McDaniel, J.; Daly,
of SC:RC, the results are expressed in F.; Litt, B. Chem. Eng. J. 2008, 135,
Figure 10. The yields of methanol were S2–S8.
30.98%, 31.33%, 31.53% and 31.66% for 5. Bakhtiary-Davijany, H.; Hayer, F.;
ratio 2, 3, 4 and 5 times, respectively. Thus, Phan, X. K.; Myrstad, R.; Venvik, H. J.;
it could be summarized that the effect of Pfeifer, P.; Holmen, A. Chem. Eng. J.
mass flow rate ratios of SC:RC on 2011, 167, 496–503.
performance of MMR were not significant 6. Bakhtiary-Davijany, H.; Dadgar, F.;
change. Hayer, F.; Phan, X. K.; Myrstad, R.;
Venvik, H. J.; Pfeifer, P.; Holmen, A.
4. Conclusion Ind. Eng. Chem. Res. 2012, 51, 13574–
The three designs of membrane 13579.
microchannel reactors for methanol 7. Hayer, F.; Bakhtiary-Davijany, H.;
synthesis and biogas upgrading were Myrstad, R.; Holmen, A.; Pfeifer, P.;
investigated by COMSOL Multiphysics 5.3a Venvik, H. J. Chem. Eng. J. 2011, 167,
in order to find the suitable case. The results 610–615.
show that case 3 which is the water removal 8. Xuyen, K.; Phan, H. D. B.; Myrstad, R.;
provided a high yield of methanol and Thormann, J.; Pfeifer, P.; Venvik, H. J.;
methane content due to the reaction Holmen, A. Ind. Eng. Chem. Res. 2010,
equilibrium shifting forward. Thus, both rate 49, 10934–10941.
of reaction and permeation flux were 9. Struis, R. P. W. J.; Stucki, S., Appl.
improved. The effect of operating Catal. A: General 2001, 216, 117–129.
parameters of water removal case were also 10. Struis, R. P. W. J.; Stucki, S.; Wiedorn,
studied. Based on these results, the M., J. Membr. Sci. 1996, 113, 93-100.
increasing WHSV and BC:RC ratio reduced 11. Tonkovich, F. D. A. L. Stud. Sur. Sci.
the performance of MMR, while the Catal. 2004, 147, 415–420.
increment of pressure and temperature could 12. Van-Dal, É. S.; Bouallou, C. J. Cleaner
improve the performance of MMR. Yet, Production 2013, 57, 38–45.
excessive increment of temperature also 13. Rohde, M. P.; Schaub, G.; Khajavi, S.;
decreased the performance of MMR. Jansen, J. C.; Kapteijn, F. Microporous
Finally, the optimal condition was at 533 K, Mesoporous Mater. 2008, 115, 123–
40 bar, WHSV of 50.7 h-1, BC:RC of ratio 136.
2:1 and SC:RC of ratio 5:1. 14. Masashi Asaeda, S. Y. Sep. Purif.
Technol. 2001, 25, 151–159.
Catalytic dehydration of ethanol to diethyl ether over ZSM-5
catalysts with different Si/Al molar ratios
Nattawat Nampipat1, Peangpit Glinrun2, Thongchai Glinrun2, Nattaya Comsup2,
Bunjerd Jongsomjit1*
1
Center of Excellence on Catalysis and Catalytic Reaction Engineering Department of Chemical Engineering,
Faculty of Engineering Chulalongkorn University, Bangkok 10330, Thailand
2
Department of Petrochemicals and Environmental Management, Faculty of Engineering,
Pathumwan Institute of Technology, Panhuman, Bangkok 10330, Thailand
*E-mail: bunjerd.J@chula.ac.th
Abstract:
Dehydration of ethanol to diethyl ether was studied over ZSM-5 catalysts over the
temperature range of 200−400 °C. The ZSM-5 catalysts were synthesized by the
hydrothermal process with the desired Si/Al molar ratios of 20, 40, and 60. These catalysts
were characterized using N2 physisorption, X-ray diffraction and temperature-programmed
desorption of NH3. The results showed that the various Si/Al molar ratios led to effect on the
amount of acidity on the surface of catalysts. The ZSM-5(20) catalyst exhibited the highest
ethanol conversion giving the DEE yield of 100% at 200 oC. This can be attributed to the
largest amount of acid sites present in ZSM-5(20) catalyst.
1. Introduction of applications in fuel chemical industry.3,4

The dehydration of ethanol has long Diethyl ether (DEE) has high octane and
been of interest to produce ethylene and cetane number, thus, it is used as an ignition
diethyl ether from non-petroleum feedstock additive for gasoline and diesel engines.
because it can reduce greenhouse gas Although diethyl ether is used in many
emissions and depletion of fossil fuel application areas, the dehydration of ethanol
reserves. In addition, ethanol can be easily to diethyl ether has received less attention.
produced from biomass materials such as It is well known that many solid acid
cassava, sugarcane, corn, rice and others catalysts such as zeolites, silica-alumina and
through fermentation process.1 alumina4,5 have been used for ethanol
Generally, there are two competitive dehydration to produce diethyl ether. ZSM-5
pathways during catalytic dehydration of is one type of zeolite catalysts that is widely
ethanol. Ethanol can be dehydrated to used for catalytic dehydration of ethanol to
produce ethylene at high temperature diethyl ether because it has a good
(endothermic reaction) as the first reaction, performance at lower reaction temperature
while diethyl ether was formed at lower with a higher product yield.6,7 ZSM-5 zeolite
temperature (exothermic reaction) as the can be easily synthesized via hydrothermal
second one. process. The molar ratio of silicon to
aluminium of ZSM-5 can be controlled by
changing the amount of sodium silicate
solution.8,9
The aim of this research is to
investigate the effect of different molar
Ethylene is an essential chemical in ratios of silicon to aluminium in ZSM-5
the petrochemical and polymer industries,2 catalysts synthesized by hydrothermal
while diethyl ether has been also used as a process. The catalytic activity of ZSM-5
solvent in the chemical, fragrance, and catalysts was studied in ethanol dehydration.
pharmaceutical industries and has a number The reaction was preformed in a
microreactor and all products were analyzed pore volume was estimated from the total
by gas chromatography (GC) with FID amount of adsorbed nitrogen by using the
detectors. ZSM-5 catalysts were BJH method.
characterized by various techniques such as X-ray diffraction (XRD): The
N2-physisorption (BET & BJH), X-ray crystalline phases were identified using a
diffraction (XRD) and ammonia SIEMENS D-5000 X-ray diffractometer
temperature-programmed desorption (NH3- with Cu Kα(λ = 1.54439Å). The patterns
TPD). were recorded over the 2θ between 5º and 60º.
Temperature programmed desorption
2. Materials and Methods of ammonia (NH3-TPD): NH3-TPD was
2.1 Materials performed using Micromeritics chemisorp
In this work, Aluminium nitrate 2750 pulse chemisorption system to measure
(Sigma-Aldrich Company Ltd.), sodium the acid properties. In the measurement, 0.1
silicate solution (Merck Company Ltd.), g of catalyst was packed in a U-tube glass
tetrapropylammonium bromide (98% with 0.03 g of quartz wool and pretreated at
Sigma-Aldrich Company Ltd.) and 1 M 500 ºC under helium flow for 1 h. Then, the
hydrochloric solution (HCl, 37% Qrёc Ltd.) sample was saturated with 15% of NH3/He
were used to synthesize the ZSM-5. Ethanol and the physisorbed ammonia was desorbed
(99.99% Merck Company Ltd.) and under helium gas flow after saturation. The
ultrahigh purity nitrogen gas (99.99% Linde sample was heated from 40 ºC to 500 ºC at
Thailand Public Company Ltd.) were used heating rate of 10 ºC/min to desorb the
for the reaction study. chemisorbed NH3.
2.2 Catalyst preparation 2.4 Reaction test
Tetrapropylammonium bromide was The catalytic dehydration of ethanol
dissloved in de-ionized water and mixed was carried out in a fixed-bed continuous
with sodium silicate and aluminium nitrate flow microreactor made from a borosilicate
that was dissollved in de-ionized water. 1 M glass with an inside diameter of 0.7 cm and
HCl was added to the mixture solution for length of 33 cm. The catalyst (0.05 g) was
adjusting the final pH = 10.5. The mixture packed with quartz wool into the middle of
solution of aluminium, silicon and microreactor. The impurity was eliminated
tetrapropylammonium bromide was stirred from surface of catalyst prior to reaction by
until it was a homogenous solution on the pretreating with nitrogen with 60 mL/min at
hot plate stirrer. The homogenous solution 200 ºC for 1 h. Then, ethanol was vaporized,
was brought into stainless-steel autoclave fed and controlled by a single syringe pump
for synthesis at 210 ºC for 24 h. After the at total flow rate of 1.45 ml/h (WHSV =
synthesis is completed, the solution was 22.9 h-1). Finally, all products were collected
centrifuged at 2000 rpm with de-ionized and analyzed at temperatures of 200 ºC,
water for 20 minutes at 5 cycles in order to 250 ºC, 300 ºC, 350 ºC and 400 ºC by a GC-
adjust pH to 7. Then, the precipitate was FID equipped with a DB-5 capillary column.
dried at 110 ºC overnight in oven. Finally,
the dried solid catalysts were calcined in air
at 550 ºC for 4.5 h.
3.1 Characteristics of catalysts
2.3 Catalyst characterization
The XRD patterns of ZSM-5
N2 physisorption: The adsorption-
catalysts are recorded in Figure 1. It can be
desorption isotherms of nitrogen at -196 ºC
observed that all catalysts exhibited the
was obtained from adsorptiometer
characteristic sharp peaks of ZSM-5
(Micromeritics ASAP 2010). The specific
presenting at 2θ = 7.8º, 8.7º, 22.94º, 23.6º
surface areas were determined from
and 24.26º.10 It appeared that the intensity of
adsorption values for relative pressure
characteristic peaks (2θ = 21º and 27º)11 of
(P/P0) by using the BET method. The total
SiO2 increase with increasing Si/Al ratio. ZSM-5 decreases with increasing Si/Al
The BET surface area and total pore volume molar ratio.9
of all catalysts determined by N2
physisorption (BET&BJH) were reported in
Table 1. It shows that the surface area and
the pore volume decrease with increasing
Si/Al ratio.
Figure 2. NH3-TPD profiles of ZSM-5 with

different molar ratios of Si/Al (a) ZSM-
5(20), (b) ZSM-5(40) and (c) ZSM-5(60)
Table 2. Total acidity from NH3-TPD

Figure 1. XRD patterns of ZSM-5 with
different molar ratios of Si/Al (a) ZSM- NH3 desorption (µmol Total
NH3/gcat) acidity
5(20), (b) ZSM-5(40) and (c) ZSM-5(60) Catalysts (µmol
Weak Medium NH3/gcat)
Table 1. BET surface area and pore volume to Strong
BET surface ZSM-5(20) 472 203 675
Pore volume
Catalysts area ZSM-5(40) 335 179 514
(cm3/g)
(m2/g) ZSM-5(60) 303 69 372
ZSM-5(20) 207 0.10
ZSM-5(40) 172 0.10
3.2 Ethanol dehydration reaction
ZSM-5(60) 177 0.09
The catalytic ethanol dehydration to
DEE over ZSM-5 catalysts having different
The acidities of ZSM-5 catalysts Si/Al molar ratios was tested at temperature
with different Si/Al molar ratios determined of 200 ºC to 400 ºC. The conversion and
by NH3 -TPD were shown in Figure 2. The selectivity of ZSM-5 zeolite catalysts were
typical acid site distributions of these graphs reported in Table 3. It shows that the ethanol
were weak acid site (below 250 ºC), medium conversion increases with increased reaction
acid site (250 ºC – 400 ºC) and strong acid temperature for all catalysts. The ZSM-5(20)
site (above 400 ºC)12 from NH3-TPD in all catalyst exhibited the highest activity. It can
of ZSM-5 ratios. Moreover, the intensity of be observed that at 400 oC, the conversions
TCD signal decreases when the ratio of 70.3, 38.2, and 3.5% were obtained for
ZSM-5 increases. It can be said that the catalysts having the Si/Al molar ratios of 20,
acidity decreased as the ratio of silica to 40 and 60, respectively.
aluminum increases due to the amount of The selectivity to ethylene decreases
sodium silicate solution with high pH was with increasing Si/Al ratio. This is attributed
added and cannot be removed completely. to the decrease in the acidity. For DEE
Amount of total acidity is shown in Table 2. selectivity, it was found that the selectivity of
This table shows that the total acid sites of
Table 3. Conversion and product selectivity of all catalysts
Temperature Ethanol Conversion Product Selectivity (%)
Catalysts
(ºC) (%) Ethylene Acetaldehyde Diethyl ether
200 7.1 0.0 0.0 100.0
250 12.8 4.9 0.8 94.4
ZSM-5(20) 300 16.4 32.4 1.1 66.5
350 43.1 80.0 1.6 18.4
400 70.3 95.6 2.0 2.5
200 2.8 0.0 0 100.0

250 9.4 3.1 11.2 85.7
ZSM-5(40) 300 12.0 11.0 4.5 84.5
350 20.5 43.3 5.2 51.5
400 38.2 83.6 3.8 12.7
200 0.0 0.0 0.0 0.0

250 0.0 0.0 0.0 0.0
ZSM-5(60) 300 1.2 0.0 100.0 0.0
350 2.1 4.2 95.3 0.6
400 3.5 10.3 86.0 3.7
DEE was high at low temperatures and For ethylene yield, it also gives 70.3% of
decreased when temperature is increased ethanol conversion and 95.6% of ethylene
because of the ethanol dehydration to diethyl selectivity at a temperature of 400 ºC. The
ether favours exothermic reaction and high activity obtained from ZSM-5 having
undergoes diethyl ether cracking reaction Si/Al of 20 can be attributed to high surface
that turn into ethylene and ethanol.4 This area and high total acidity.
results in the observation of high ethylene
selectivity at high temperature. From the Acknowledgements
results, it can be concluded that the ratio of The authors thank the Grant for
Si/Al in ZSM-5 essentially affects the International Research Integration: Chula
catalytic ethanol dehydration to diethyl ether Research Scholar, Ratchadaphiseksomphot
and ethylene due to surface area and acidity. Endowment Fund for financial support of
These factors were the important keys to this project.
obtain the highest activity in ethanol
dehydration. References
1. Thatoi, H.; Dash, P. K.; Mohapatra,
4. Conclusion S.; Swain, M. R. Int. J. Sustain. Energ.
The ZSM-5 catalysts with different 2016, 35, 443–468.
Si/Al molar ratios were tested in ethanol 2. Soh, J. C.; Chong, S. L.; Hossain, S. S.;
dehydration to diethyl ether. In order to Cheng, C. K. Fuel Process. Technol.
maintain high yield of diethyl ether, this 2017, 158, 85–95.
reaction must be operated at low 3. Kamsuwan, T.; Praserthdam, P.;
temperature. The ZSM-5 having Si/Al of 20 Jongsomjit, B. J. Oleo Sci. 2017, 66,
is the best catalyst among the other catalysts 199–207.
(ratios of 40 and 60) and produces the 4. Phung, T. K.; Busca, G. Chem. Eng. J.
highest yield of diethyl ether at 250 ºC 2015, 272, 92–101.
(conversion 12.8% and selectivity 94.4%).
5. Phung, T. K.; Hernández, L. P.; R. Cryst. Res. Technol. 2008, 43, 1300–
Lagazzo, A., Busca, G. Appl. Catal. A. 1306.
2015, 493, 77–89. 10. Zhang, X.; Lin, L.; Zhang, T.; Liu, H.;
6. Wu, C. Y.; Wu, H. S. ACS Omega 2017, Zhang, X. Chem. Eng. J. 2016, 284,
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9. Shirazi, L.; Jamshidi, E.; Ghasemi, M.
Decarboxylation of free fatty acid over Ni-MCM-41
Orhathai Jirarattanapochai1, Duangkamon Jiraroj1, Joseph Samec2*,
Duangamol N. Tungasmita1*
1
Center of Excellence in Catalysis for Bioenergy and Renewable Chemicals (CBRC), Department of Chemistry,
Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
2
Department of Organic Chemistry, Stockholm University, Stockholm SE-10691, Sweden
*E-mail: joseph.samec@su.se, duangamol.n@chula.ac.th
Abstract:
The decarboxylation of free fatty acid over metal catalysts supported on porous
materials can produce alkane and alkene that contain carbon 16-18 atoms. These products can
be used in lubricant and fuel industries especially diesel oil and jet oil. In this work, 10 wt%
Ni-MCM-41 was used as catalyst for decarboxylation of oleic acid. Ni-MCM-41 was
prepared via hydrothermal method and impregnated nickel by incipient wetness impregnation
method. The catalyst was characterized by XRD, SEM and N2 adsorption desorption
technique. From the characterization, XRD pattern demonstrated the characteristic peaks of
mesoporous compound and long wavelength XRD (2Ɵ=10-80o) exhibited characteristic
peaks of NiO. Additional, the N2 adsorption-desorption isotherm indicated mesoporous
behaviour and BET surface area was 804 m2/g for Ni-MCM-41. The reaction was carried out
in batch reactor at 400 oC for 30 min under N2 atmosphere. The composition of the products
was analyzed by gas chromatography. It was showed that Ni-MCM-41 can promote
decarboxylation of oleic acid that the selectivity for heptadecane (C17H36), heptadecene
(C17H34) and conversion of oleic acid was 18.1%, 36.6% and 92.0%, respectively.
1. Introduction Nevertheless, these metals are rare

The increasing energy demand led to and expensive. Subsequently, hydrotalcites
quickly decline fossil fuels and enlarge (MgO–Al2O3) showed the activity of
environmental problems such as air decarboxylation. However, the selectivity to
pollution and global warming. Many heptadecene, a product by direct
researches interested in the production of decarboxylation of oleic acid, was very low
renewable energy. Fatty acid methyl ester, due to cracking reaction.2 In this work, the
provided from esterification of fatty acid and catalyst MCM-41 was chosen as a catalytic
transesterification of triglycerides has been support due to high surface area and pore
widely studied due to its high lubricity, diameters ranged from about 2 to 10 nm that
renewable and non-toxicity. However, fatty is close to the molecule size of the
acid methyl ester has many disadvantages hydrocarbon jet fuels. However, the pure
such as high oxygen content, high viscosity silica MCM-41 displays poor ion-exchange
and low stability.1 As an alternative, ability, weak acid intensity and low
hydrodeoxygenation process has been hydrothermal stability due to the absence of
developed to eliminate the oxygenated active sites. For this reason, introducing Al
compounds. However, this process into the framework could be increased the
consumes excess hydrogen. Thus, acidity and catalytic activity. Moreover, the
decarboxylation of lipids can be determined incorporation of the metal, could be
because it does not require hydrogen and improved the catalytic activity further. In
produce water, which causes catalyst this work, Al-MCM-41, Ni-MCM-41 and
deactivation.2 The earliest studied in the Ni-Al-MCM-41 were synthesized. The aim
decarboxylation reaction used precious of this work was to investigate the potential
metals such as Pt and Pd as catalysts.3 to heptadecane and heptadecene production
from the decarboxylation of oleic acid over 2.1.3 Synthesis of nickel mesoporous
these synthesized catalysts. The catalyst materials
properties have been characterized by XRD, Nickel mesoporous materials were
SEM and N2 adsorption technique related to prepared by impregnation method from
the activity results in decarboxylation. nickel(II) nitrate hexahydrate that contain
nickel 10%wt. The solution was added drop
2. Materials and Methods wise into mesoporous materials. Then,
Cetyl trimethylammonium bromide stirred and dried at 80 oC. After that, the
(C19H42BrN, 97%, Merck), ammonia sample was calcined at 550 oC for 3 h.
solution (NH3, 25%, Merck), tetraethyl 2.2 Catalysts characterization
orthosilicate (C6H20O4Si, 99%, Merck), DI The X-ray diffraction patterns were
water, ethanol (Merck), aluminium determined by a Rigaku, Dmax
+
triisoproproxide ([(CH3)2CHO]3Al, 98%, 2200/Ultima diffractometer equipped with
Aldrich), toluene (99.8%, Carlo Eeba), Cu Kα radiation. The X-ray tube was
nickel(II) nitrate hexahydrate operated at 40 kV and 30 mA. For the
(Ni(NO3)2.6H2O, 99%, Carlo Eeba), N,O- analysis of crystalline structure, the range of
bis(trimethyl)trifloroacetamide, (BSTFA, 2Ɵ was 1.5-6.0 degree with a scan speed of
95%, TCI), oleic acid (C18H34O2, Fluka, 5 min-1. The metallic phase on the supports
99.9%), pyridine (Merck), tetrahydrofuran was approved over 2Ɵ range of 10-80
(THF, 99.8%, RCI Labscan), naphthalene degree with a scan speed of 1 min-1. The
(98%, Fluka) particles size and morphology of
2.1 Catalysts preparation mesoporous compounds were analyzed by
2.1.1 Synthesis of MCM-41 JEOL JSM-6480LV scanning electron
MCM-41 was prepared by microscopy (SEM) with 15 kV of
hydrothermal method that reported by T. acceleration voltage. All samples were
Klamrassamee et al.4 In the typical coated with sputtering gold under vacuum
synthesis, 7.2 g of Cetyltrimethylammonium for conductivity. N2 adsorption desorption
bromide as a structure directing agent was isotherms were carried out using a BEL
dissolved in 360 g of deionized with stirred Japan, BELSORP-mini nitrogen
at room temperature. After finishing, 30.6 g adsorptometer at 77 K. The surface area was
of 25% ammonia solution, as base was determined by the Brunauer-Emmett-Teller
added and stirred for 10 min. Then, 30 g of (BET) equation. The pore volume and
tetraethyl orthosilicate as a Si source was average pore diameters were determined by
added drop wise and the solution was stirred the Barrett-Joyner-Halenda (BJH) method.
at room temperature for 18 h. After that, the Before the measurement, the samples were
milky solution was filtered, washed with weighed approximately 40 mg and
deionized water: ethanol = 4:1 v/v. Dried at pretreated at 200 oC for 3 h. Acidity of
60°C overnight and calcined at 550 °C for 5 materials were examined by titration using
h. NaOH as a standard solution and
2.1.2 Synthesis of Al-MCM-41 phenolphthalein as an indicator.
Al-MCM-41 was synthesized by the 2.3 Catalytic decarboxylation of oleic acid
post synthesis that reported by Kamlesh N. The decarboxylation reaction was
Tayade et al.5 with a mole ratio of carried out in a reactor assembled from 3/8-
Si/Al=14.1. In the procedure, 0.23 g of inch stainless steel Swagelok parts, sealed
aluminium triisoproproxide and 1 g of with a cap on each end to give a reactor
MCM-41 were dissolved in 100 mL of volume of 1.52 mL6 operating in a batch
toluene with stirred at room temperature for mode. The temperature was measured with a
12 h. After finishing, filtered and dried at K-type thermocouple. In a typical batch
100 oC overnight and then calcined the experiment, 2.0 g of oleic acid and 0.2 g of
product at 550 oC for 5 h. catalyst were placed in the reactor. After
oleic acid and catalyst were loaded in the X-ray diffractograms of Al-MCM-41, Ni-
reactor, the reactor was flushed with MCM-41 and Ni-Al-MCM-41 were similar
nitrogen to remove the remaining oxygen. to MCM-41 but the intensity decreased
Then, the reactor was heated to 300 or 400 significantly. This indicated that aluminium
o
C for 30 min by using fluidize sand bath and nickel were loaded into the porous
(Omega FSB-4). After the reaction, the structure of MCM-41. XRD patterns are
reactor was subsequently cooled down to shown in Figure 1.
room temperature and washed with THF to
remove any residual materials.
2.4 Product analysis
The liquid product withdrawn from
the reactor was analyzed with a gas
chromatograph (Varian CP 3800) equipped
with a 30 m in length of CP-sikl-8 capillary
column equivalent to HP-5 with 0.25 mm
outer diameter and 0.25 µm film thickness.
It was used for analyzing reaction from the
decarboxylation of oleic acid. The liquid Figure 1. XRD patterns of (a) MCM-41, (b)
sample 500 µL was added pyridine 150 µL Al-MCM-41, (c) 10 wt% Ni-MCM-41 and
and silylated with BSTFA 150 µl. Then, (d) 10 wt% Ni-Al-MCM-41 at small angle
mixture was kept in an oven at 60 °C for 30
min. The internal standard naphthalene was The XRD at wide angle
added for quantitative calculations. The demonstrated the characteristic peaks of
sample 1 µL was injected to into the GC NiO, which are assigned at 2Ɵ=37.5o, 43.4o,
with a split ratio 20:1 and the carrier gas was 63.0o, 75.5o and 79.6o were observed clearly
nitrogen with the flow rate 1 mL/min. Flame indicated that Ni particles existed as NiO7,
ionization detector was used as a detector. as shown in Figure 2b and 2d. The peak
The conversion (X) of oleic acid was intensity of Ni-MCM-41 was similar to Ni-
calculated by Equation 1. Al-MCM-41. This referred that the amount
of nickel resemble dispersed in the porous
% conversion, X= (m1-m2)/m1 x 100 (1) structure of MCM-41.
The selectivity (S) of products (p)
was calculated using Equation 2.
% selectivity, Sp= mp/(m1-m2) x 100 (2)
Where mp, m1 and m2 are mole of product,

initial oleic acid and unconverted oleic acid,
respectively.
3. Results and Discussion Figure 2. XRD patterns of (a) MCM-41, (b)

3.1 Catalysts characterization 10 wt% Ni-MCM-41, (c) Al-MCM-41, and
The XRD at low angle shows three (d) 10 wt% Ni-Al-MCM-41 at wide angle
well resolved peaks of MCM-41
corresponding to the (100), (110) and (200) The scanning electron images for
reflections of the hexagonal MCM-41 each catalyst were shown in Figure 3.
lattice. The d100 reflection was very strong Morphology of Al-MCM-41 was similar to
and sharp, indicating a good crystallinity of MCM-41 which was round shape but Al-
mesoporous structure. When aluminium, and MCM-41 was aggregate more than MCM-
nickel were introduced into MCM-41, the 41. Morphology of Ni-MCM-41 and Ni-Al-
MCM-41 were round shape with rough pore volume, which reason might also
surface. The bulk NiO particles were mainly relate to the type of metal employed.
obviously observed on the surface of Ni- The acidity of mesoporous silica was
MCM-41 and Ni-Al-MCM-41, indicating measured quantitatively by acid-base
that NiO particles on MCM-41 were titration using sodium chloride as ion-
accumulated due to the sintering during the exchange agent. As a result, the acidity was
thermal treatment when too much NiO was increased from 1.11 to 1.40 mmol when
loaded on MCM-41.8 modified MCM-41 introducing Al into the
framework. The acidity of Al-MCM-41 and
Ni-Al-MCM-41 were no significant
difference.
Figure 3. The SEM of (a) MCM-41, (b) Al- Figure 4. N2 adsorption/desorption of (a)
MCM-41, (c) 10 wt% Ni-MCM-41 and (d) MCM-41, (b) Al-MCM-41, (c) 10 wt% Ni-
10 wt% Ni-Al-MCM-41 MCM-41 and (d) 10 wt% Ni-Al-MCM-41
N2 adsorption desorption isotherms 3.2 Reaction results

and pore size distributions of catalysts are The decarboxylation reactions of
shown in Figure 4. All samples exhibited oleic acid over the MCM-41 catalysts were
IUPAC type IV isotherms with type-H1 performed in a batch reactor. The results are
hysteresis loop corresponding to the typical shown in Table 2. All of the reaction
mesoporous structure. The adsorption of N2 provided saturated C17 and unsaturated C17
due to capillary condensation that was compounds. During the decarboxylation of
observed at relative pressure (p/p0) ranges of oleic acid, the carboxylic acid gives protons
0.3–0.4 showed a sharp capillary by the heterolytic cleavage of the O-H bond,
condensation step in the adsorption generating a carboxylate and hydrogen ions.
isotherm, suggesting that the MCM-41 Heptadecene (unsaturated C17) is produced
structure still remained even Ni particles due to the removal of CO2. The in situ
were grafted.7 The textural properties of generated hydrogen, as a result of
catalysts are summarized in Table 1. The heterolytic cleavage of the O-H bond in
surface areas of catalysts were successively oleic acid is consumed by the hydrogenation
decreased from 1004 to 838 and 804 m2/g of unsaturated C17 to form heptadecane
with the loading of Al and Ni, respectively. (saturated C17).6 Moreover, it obtained
The pore volume of Al-MCM-41 was higher saturated C15 and C13 compounds due to
than Ni-MCM-41 due to partially dispersion the cracking reaction of oleic acid. At the
of NiO in the porous structure of MCM-41. reaction temperature of 400oC exhibited
The significant decrease of specific surface 13% higher oleic acid conversion and
area and pore volume with the loading of Ni selectivity of saturated C17 and unsaturated
content indicated that the NiO particle was C17 compounds than at the reaction
dispersed in the porous structure of MCM- temperature of 300oC. This indicates that the
41. The tendency of change in total surface reaction preferred at this temperature. In
area of samples was similar to that in total case of, Al-MCM-41, Ni-MCM-41 and Ni-
Al-MCM-41 demonstrated similar compounds, which are desirable products in
conversion of oleic acid (91.0–93.8%) decarboxylation of oleic acid.
which can be attributed to the absence of
rich H2 environment. However, Al-MCM-41 4. Conclusion
provided lower selectivity of C17 Catalytic decarboxylation of oleic
unsaturated compound than MCM-41 due to acid is dominant at the reaction temperature
acidity of Al-MCM-41. Ni-Al-MCM-41 of 400 oC. The modified MCM-41 showed
exhibited selectivity of C17 saturated and high oleic acid conversion. Incorporate of Al
C17 unsaturated compound that were no to the framework of MCM-41 exhibited
significant difference to the Al-MCM-41. lower selectivity of C17 unsaturated
The reaction over Ni-MCM-41 provided the compound. The Ni-MCM-41 exhibited
highest selectivity of C17 saturated and C17 higher decarboxylation activity than other
unsaturated compound 18.1% and 36.3%, catalysts and showed the highest selectivity
respectively. Metallic Ni species are to C17 compounds (18.1% and 36.6%) due
expected to be active sites to produce C17 to Ni species, which have advantageous
effect on decarboxylation of oleic acid.
Table 1. BET surface area, total pore volume average pore diameter and acidity of the
catalysts
a b b c c
Catalyst BET Internal External Total pore Average pore Acidity
surface area surface area surface area volume diameter (nm) (mmol)
(m2 g-1) (m2 g-1) (m2 g-1) (cm3 g-1)
MCM-41 1004 967 35 0.80 2.43 1.11
Al-MCM-41 838 800 32 0.68 2.43 1.40
Ni-MCM-41 804 784 16 0.59 2.43 1.20
Ni-Al-MCM-41 706 676 25 0.55 2.43 1.42
a
Calculate from the BET method
b
Calculate from the t-plot method
c
Calculate from the BJH method
Table 2. Decarboxylation of oleic acida

Reaction % Selectivity % Conversion
C17 sat C17 unsat C15 sat C13 sat
Thermal (300oC) 0.4 0.9 0.2 0.2 59.1
o
Thermal (400 C) 0.7 3.3 0.4 0.4 72.5
MCM-41 4.8 23.4 3.8 2.3 81.0
Al-MCM-41 5.7 8.7 4.0 1.9 93.8
Ni-MCM-41 18.1 36.6 8.3 2.5 92.0
Ni-Al-MCM-41 7.3 9.4 5.6 3.2 91.0
a
Reaction condition: batch reactor, oleic acid 2.0 g, catalyst 0.2 g, time 30 min, N2 atmosphere
Acknowledgements Chulalongkorn University for the fluidize

This work was financially supported sand bath.
by Center of Excellence in Petrochemical
and Materials Technology (PETROMAT), References
the 90th Anniversary of Chulalongkorn 1. Liu, Y.; Yao, L.; Xin, H.; Wang, G.; Li,
University (Ratchadaphiseksomphot D.; Hu, C. Appl. Catal. B-Environ. 2015,
Endowment Fund) and International 174-175, 504–514.
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Scholar, Ratchadaphiseksomphot B. E.; Na, J.G.; Ko, C. H. Catal. Today
Endowment Fund. Besides, the authors are 2011, 164, 457−460.
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Technology, Faculty of Science, P.; Eränen, K.; Muzin, D. Y. Ind. Eng.
Chem. Res. 2016, 45, 5708-5715. 7. Lu, B.; Ju, Y.; Abe, T.; Kawamoto, K.
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Systematic investigation of biofilm formation of acetogens and
methanogens derived from agro-industrial wastewater using microfluidic
devices and fabricated biointerface
Arslan Siddique1, Benjaphon Suraraksa2, Sukunya Oaew2, Sarawut Cheunkar1*
1
Biotechnology Division, School of Bioresources and Technology, King Mongkut University of Technology
Thonburi, Bang Khun Tian, Bangkok 10150, Thailand
2
Biochemical Engineering and Pilot Plant Research and Development Laboratory, National Center for Genetic
Engineering and Biotechnology, National Science and Technology Development Agency, King Mongkut's
University of Technology Thonburi, Bang Khun Tian, Bangkok 10150, Thailand
*E-mail: sarawut.che@mail.kmutt.ac.th
Abstract:
In the anaerobic digestion of agro-industrial wastewater for biomethane production,
microbial consortia are naturally aggregated as biofilms. Nowadays, the initiation mechanism
of such bacterial biofilm is a subject undergoing intense exploration. Instead of a
conventional wastewater bioreactor, we employ a microfluidic-integrated device to simulate
biofilm formation, which can be microscopically studied thereon. We set out to operate this
device for 2 and 24 hr to ascertain the initial attachment and progressive phase, respectively.
Interestingly, our results revealed that surface coverage by biofilm on a positively-charged
surface after 2 hr in the presence of 4 g/l propionic acid is about 10 times higher as compared
to an unmodified surface. This could be because of electrostatic attraction between a
positively-charged surface and negatively-charged bacterial membranes. Furthermore,
fluorescence in-situ hybridization (FISH) analysis of this surface attached biofilm exhibited
rapid attachment of methanogens as compared to other bacteria after 2 hr indicating the
survival strategy of the methanogens under acidic conditions. This suggests that such
positively-charged surface could significantly promote the retention of active bacteria in the
reactor even under highly-acidic conditions. Indeed, this ideal platform is manifested as a
potential tool for exploration of anaerobic bacterial biofilm on different supporting media.
1. Introduction products from acetogenic phase into carbon

Biogas production is one of the dioxide and methane. Under environmental
renewable, sustainable, and eco-friendly stresses, such as VFAs and H2 accumulation,
sources of producing energy as it is derived methanogens’ activities are inhibited
from the organic waste materials, such as yielding lower or no methane production. In
wastewater. Biogas is primarily generated in order to avoid disturbances, bacteria arrange
the absence of oxygen in the process called themselves in the form of granular biofilm
anaerobic digestion. This process takes place where bacteria organize in layers with
in an air-tight biogas reactor. In there, methanogens in the middle followed by
bacteria are ascertained as key players in layers of acetogens, acidogens and
biodegradation in four distinct phases hydrolytic bacteria. Especially, acetogenic
including hydrolysis, acidogenesis, and methanogenic bacteria remain in close
acetogenesis, and methanogenesis.1 Usually, proximity to ensure the efficient substrate
the biogas productivity relies on the utilization while coping up with the
association of bacteria in the last two phases. environmental stresses. This association of
By functions, acetogenic bacteria convert the bacteria is called syntrophism where
volatile fatty acids (VFAs) into the acetic both bacterial groups work in
acid, carbon dioxide, and hydrogen (H2) synchronicity.2
while methanogenic bacteria convert the
Apart from that, retention of active this device an ideal platform for simulation
bacterial biomass in the wastewater of surface attached wastewater microbial
treatment plant has been a process challenge biofilm.
because of short wastewater residence time
and long generation time of some key 2. Material and Methods
bacteria. In order to mitigate this issue, 2.1 Materials
synthetic surfaces are introduced into the 4’,6-diamidino-2-phenylindole (DAPI)
reactors with the purpose of harboring and and poly-l-lysine coated slides were
retaining active bacteria.3 The surface purchased from sigma aldrich (USA).
attachment of the bacteria is enormously Paraformaldehyde and ethanol were
reported, as it is believed that control gained obtained from Merck (schuchardt,
over the initiation of biofilm formation leads Germany). Formamide was bought from the
to whether enhance or reduce biofilm. Bio-Rad (CA, USA). Peristaltic pump was
Hence, the region of contact between purchased from Watson Marlow (USA).
bacteria and surface, so-called bio-interface, Polydimethylsiloxane (PDMS) sylgard 184
plays a significant role in determining the and a curing agent were procured from Dow
bacterial initial adhesion and biofilm Corning (USA). Phosphate buffered saline
formation.4 To the best of our knowledge, (PBS, 1X, pH=7.4) and Tris-HCl (20 mM,
research on promoting initial biofilm pH=7.2), 0.9 M NaCl and 0.01% sodium
formation in wastewater by altering surface dodecyl sulphate (SDS) were prepared for
chemistry is scare.5 This may be because making hybridization buffer. Commercially
conventional techniques, such as lab-scale available oligonucleotide probes (EUB 338
bioreactors, flow cells etc., available for the and ARC 915) were used for FISH analysis.
insightful study of surface-attached biofilm 2.2 Methods
have many limitations. 2.2.1 Designing of microfluidic mold
With the advancement of As shown in Figure1, the mold for
microfluidics technology, study of biofilm at the microfluidic device was made using the
micro-scale in dynamic flows is highly acrylic sheet. One basal piece (45x35x4
encouraged. Recently, some microfluidic mm) was laser cut and four smaller bits
devices have emerged as a potential tool for (35x2.5x3.0 mm) were gently glued onto it
simulating factors affecting biofilm separated from each other by 2.5 mm. The
formation.6 Microfluidic devices provide an mold was then placed into a receptacle made
ample chance to mimic the natural setting of from aluminum foil.
the bioreactors while providing high 2.2.2 Fabrication of microfluidic channels
throughput data on surface grown biofilm. Polydimethylsiloxane (PDMS) and
To address all the aforementioned curing agent (10:1 ratio) were thoroughly
challenges, we have developed a state-of- mixed in a plastic cup. The bubbles
the-art detachable microfluidic device for produced during this step were removed by
investigation of the anaerobic microbial putting the mixture in a vacuum degassing
biofilm on positively-charged bio-interface. chamber for about 20 min. Later, the
We hypothesize that provision of positive mixture was poured onto the acrylic mold.
charges on the surface will lead to The thickness of the PDMS was
immediate and enhanced surface coverage approximately 1 cm. The PDMS was cured
by biofilm in presence of higher nutrient on the hotplate for 2.30 hr at 60 C. Next,
concentration. Similarly, we also focused on PDMS channel was peeled off from the
simultaneously investigating initial biofilm mold and was carefully cut in appropriate
formation under different growth conditions dimensions. Before experimenting, the
with subsequent staining and examination PDMS channels were punched at both ends
under fluorescence microscope. We believe of the channels as inlets and outlets.
that this experimental miniaturization makes
PBS and stained with nucleic acid staining
dye, DAPI for 10 min in dark. It was then
washed and mounted with glycerol followed
by covering with coverslip and examined
under Epi-fluorescence microscope. All
images were captured using fluorescence
microscope (Olympus BX60 microscope)
with oil immersion lens (100X). Moreover,
FISH was performed to differentiate archaea
(methanogens) from eubacteria (acetogens)
by preparing hybridization buffer and probes
Figure 1. Illustration of steps for fabrication with 8:2 ratios. In this study, ARC915 and
of microfluidic device and staining EUB 338 probes were used to visualize
these bacteria.
It was then bound to the microscope 2.2.5 Image analysis
slides with two sheets of acrylic clamps as Surface coverage by biofilm was
shown in figure 1 to prevent liquid leakage. measured and quantified using ImageJ
Then, the device was connected to the software. Data analysis was accomplished
peristaltic pump with silicone tubes. The through MS Excel 2013.
device setup is shown in Figure 2.
3.1 Operability of microfluidic device
The detachable microfluidic device
was tested for its proper functioning. Four
different solutions were taken under
investigations. There were four channels in
the device and one of the channels was
supplied with PBS as a control. The second
channel was injected with bacterial solution
without any nutrient. The third and fourth
Figure 2. Schematic illustration of channel was provided with bacterial solution
operational microfluidic setup containing 2g/L and 4g/L propionic acid
(PA) respectively, to probe biofilm
2.2.3 Preparation of bacterial co-culture formation under different nutrient conditions
In this work, synthetic wastewater in the same platform. It was operated at
was prepared and micro-organisms injected room temperature in continuous flow
in it were derived from Tapioca starch condition for 2 and 24 hr. at flow rate (~5
industry (courtesy of Ecowaste Lab, PDTI, mL/min) to gain laminar flow considering
KMUTT). In brief, the bacterial inoculum channel’s dimensions. Careful examination
was enriched with syntroph specific of this device under stereomicroscope
substrate, propionic acid (PA) to promote revealed no leakage and blockage in the
the growth of acetogenic and methanogenic channels confirming the successful
bacteria. Similarly, PA as nutrient media operability of the device.
was also provided with bacterial suspension 3.2 Effect of positively charged surface on
while investigating biofilm formation on the biofilm
positively-charged substrate. It is well known that biofilm
2.2.4 Staining and fluorescence in-situ formation starts with initial bacterial contact
hybridization (FISH) with the surface and promotion of this step
The glass slide was taken out by may lead to the enhanced bacterial adhesion.
detaching microfluidic device, washed with We hypothesized that provision of positive
charges on the surface may lead to enhanced higher nutrient concentration at ~5ml/min
initial bacterial biofilm. This is because of corresponding to the velocity and shear rate
the negatively-charged nature of the bacteria of 1.69 cm/s and 4.2 s-1 respectively. This
due to presence of teichoic acid in their cell finding further proves our hypothesis that
walls and peptidoglycan moieties. In a quest surface modification with higher nutrient
to explore the initial biofilm formation (2 provision escalates immediate biofilm
hr.) by bacterial consortia, we employed formation. It is expected that this positively-
poly l-lysine coated glass slide as an charged surface with increased attachment
attachment surface. of active acetogens will help mitigate the
After staining with DAPI as shown issue of PA accumulation in the biogas
in Figure 3, quantitative image analysis reactors by its rapid degradation.
(Figures 4, 5) represent the higher
percentage of surface coverage/µm2 (11.1%)
on modified surface in comparison to 1.85%
on unmodified surface after 2 hr, in presence
of 4g/l PA in bacterial suspension.
Furthermore, the progression of biofilm was
also investigated after 24 hr, which gave the
values of 24% surface coverage on
unmodified surface and 26.8% on modified
surface indicating the continuous biofilm
formation.
Figure 4. Percentage of surface covered by

biofilm on unmodified surface
3.4 Differentiation of acetogens and

Figure 3. Surface attached biofilm stained methanogens
with DAPI on unmodified (a) and modified Syntrophically associated acetogens
(b) surface at 4 g/L PA and methanogens are significantly crucial in
biogas production and their biofilm forming
3.3 Nutrient variation affects biofilm behavior determines their activity.3a,8 Most
development of the methanogens belong to Achaea and
Nutrient provision plays an essential acetogens to Eubacteria. In addition to DAPI
role in biofilm by determining its various
features. We simulated biofilm formation
under different nutrient concentrations.
Propionic acid (PA) having 2 g/L and 4 g/L
conc. was provided along with bacterial
suspension and biofilm formation after 2 and
24 hr was examined. Moreover, it is
reported that increased flow rate surmounts
biofilm thickening by erosion and sloughing,
and keeps the degradation rate of organic
substrate at higher level due to existence of
highly active surface attached bacteria.7 Figure 5. Percentage of surface covered by
Similarly, it is evident from Figures biofilm on modified (positively charged)
4 and 5 that biofilm formation on positively- surface
charged surface is further strengthened by
staining, we also performed fluorescence in- juxtaposed with flow cells to make it
situ hybridization (FISH) technique to economical and favorable while furnishing
envisage their biofilm development after 2 high throughput biofilm data. In summary,
and 24 hr. As shown in Figure 6, it was the findings from this study portray the
observed that methanogens rapidly adhere to higher initial biofilm formation on positively
the surface before the acetogens do based on charged bio-interface, which was further
the color intensity variation measured by reinforced at higher concentration (4g/l) of
ImageJ (data not shown). This may be propionic acid (PA) after 2 hr.
because methanogens are highly sensitive to Furthermore, differentiation of
the acidic conditions. In order to methanogens and acetogens residing in the
acclimatize, they initially form biofilm biofilm also manifested accelerated initial
followed by recruitment of acetogens from growth on the modified surface. Such
the bulk phase, which provides optimum positively charged surfaces could potentially
substrate transfer rate to thrive. Moreover, in be applicable in wastewater treatment plants
the microfluidic channel, concentration where the residence time of the wastewater
gradient is higher along the walls of the is low and retention of active biomass is
channels while velocity gradient is lower challenging. These surfaces will also
and certainly biofilm growth was higher in certainly help to reduce hydraulic retention
that regime.9 Figure 6 (a-d), illustrates the time (HRT) while promoting immediate
differentiation of initial biofilm formed by biogas production.
methanogens and acetogens on unmodified
and modified surfaces after 2 hr specifying Acknowledgement
the enhanced adhesion on positively-charged We cordially thank School of
substrate (c,d). Bioresources and Technology (SBT) for
conference support and Petchra Pra Jom
Klao scholarship, KMUTT for financial
support.
References
1. Ali Shah, F.; Mahmood, Q.; Maroof
Shah, M.; Pervez, A.; Ahmad Asad, S.
The Scientific World Journal 2014,
183752.
2. Hudayah, N.; Suraraksa, B.; Chaiprasert,
P. Environ. Technol. 2016, 37 (21),
Figure 6. Fluorescent images of Archaea 2713–2722.
and Eubacteria stained with ARC915 and 3. a) Langer, S.; Schropp, D.; Bengelsdorf,
EUB338 probes. (a-b and c-d), F. R.; Othman, M.; Kazda, M. Anaerobe
methanogenic and acetogenic biofilm on 2014, 29, 44-51; b) Schmidt, T.;
unmodified and modified surfaces after 2hr. Ziganshin, A. M.; Nikolausz, M.;
respectively at 4g/L PA Scholwin, F.; Nelles, M.; Kleinsteuber,
S.; Pröter, J. Biomass Bioenergy 2014,
4. Conclusion 69, 241–248.
We successfully employed 4. Tuson, H. H.; Weibel, D. B. Soft Matter
detachable microfluidic device for insightful 2013, 9 (17), 4368–4380.
investigation of anaerobic microbial biofilm. 5. Nguyen, V.; Karunakaran, E.; Collins,
Moreover, this microfluidic platform G.; Biggs, C. A. Colloids Surf. B:
requires minute amount of sample thereby Biointerfaces 2016, 143, 518–525.
reducing waste as compared to lab-scale bio- 6. Yawata, Y.; Nguyen, J.; Stocker, R.;
reactors. The functioning of this device is Rusconi, R. J. Bacteriol. 2016, 198 (19),
2589–2595. 2007, 41 (13), 2885–2892.
7. Zhang, C.; Chen, R.; Wang, Y.; Wang, 9. Guermonprez, C.; Michelin, S.; Baroud,
Y.; Zhang, Q. Energy Procedia 2014, 61, C. N. Biomicrofluidics 2015, 9 (5),
1455–1459. 054119.
8. Rochex, A.; Lebeault, J.-M. Water res.
The improvement in the properties of fatty acid methyl ester using partial
hydrogenation reaction in a continuous fixed-bed reactor
Atichat Khongngam1, Chaiya Roopsung1, Thanita Sonthisawate2, Yoothana Thanmongkhon2,
Piyanan Sreesiri2, Weerawat Patthaveekongka1*
1
Silpakorn University, Sanam Chandra Palace, Nakhon Pathom 73000, Thailand
2
Research, Khlong Luang, Pathum Thani 12120, Thailand
*E-mail: patthaveekongka_w@silpakorn.edu
Abstract:
This research studied the improvement in the properties of fatty acid methyl ester
(FAME), produced from palm biodiesel via partial hydrogenation reaction in a continuous
fixed-bed reactor. The palm biodiesel was mixed with hydrogen before the liquid-gas mixture
was fed into the reactor. The partial hydrogenation reaction was catalyzed using packed
commercial Pd/Al2O3 as the catalyst. Several parameters for the improvement in the
properties of FAME were studied such as reaction temperatures (80, 100 and 120 °C),
catalyst weights (2, 2.5 and 3 g) and the flow rate of palm biodiesel (0.38 mL/min). After the
reaction, the properties of palm biodiesel were measured using gas chromatography (GC), in
order to study the composition and the changes in the structure of fatty acid and glyceride.
The result showed that the partial hydrogenation reaction was able to improve the properties
of palm biodiesel. The suitable condition for partial hydrogenation reaction in the continuous
fixed-bed reactor was carried out using reaction temperature of 80 °C, catalyst weight of
2.5 g and flow rate of palm biodiesel of 0.38 mL/min. This condition resulted in the decrease
of polyunsaturated FAME contents more than 85%, while saturated monoglyceride contents
increased more than 80%.
1. Introduction biodiesel is targeted at 14 million liters per

At present, there is an increase in the day at the end of 2036.2 Therefore, the
energy consumption due to large amount of quality of biodiesel is the important key
population and the rapid expansion of factor to increase the use of biodiesel more
industry and transportation. Therefore, the than 7% in the mixture of fuel. However,
renewable energy becomes more important there is a limitation of biodiesel usage if
for energy security because the amount of biodiesel contains high content of
diesel from fossil source is limited. In contaminants, i.e. glycerides especially
addition, the renewable energy is sustainable monoglyceride, unsaturated components.
and environmentally friendly. In Thailand, Those components react with oxygen in the
the transportation sector has consumed the air, resulting in acid formation or
great amount of diesel and the consumption polymerization formed solid precipitation.
of diesel will continually increase every The nozzle or tube in the engine might be
year. As a result, the Department of Energy clogged by the precipitate.3 As a result, the
Business, Thailand, launched the Alternative improvement of biodiesel properties is
Energy and Alternative Energy crucially necessary.
Development plan 2015-2036, in order to Partial hydrogenation process is one
increase the use of biodiesel. In addition, of potential process to improve the
diesel composition was formulated to properties of biodiesel. Double bond in the
contain 7% biodiesel by volume since molecules are added with hydrogen
2014.1 Furthermore, the consumption of becoming single bond, known as
hydrogenation reaction. This process ester or FAME compositions (Agilent
changes residue monoglyceride to be more 6890N) and glycerides (EN14105:2011,
saturated, resulting in higher itself melting Shimadzu 2010).
point which will agglomerate, precipitate
and be removed before using as a final
product. This biodiesel could prevent the
transportation and engine problems, and it
could reduce the emission of soot.4-6 This
work studied the effect of reaction
temperature and the amount of catalyst
(Pd/Al2O3) in partial hydrogenation reaction
using a fixed-bed reactor with continuous
operation mode, in order to improve the Figure 1. Partial hydrogenation process in a
properties of palm biodiesel. continuous fixed-bed reactor
2. Materials and Methods 3. Results and Discussion

2.1 Materials The properties of feed palm biodiesel
Palm biodiesel used in this work used in the experiment are shown in the
contains properties in Table 1. Hydrogen Table 1. The effect of reaction temperature
(99.99%) is used for partial hydrogenation of partial hydrogenation was presented in
reaction using Pd/Al2O3 as the catalyst Figure 2.
(commercial grade). The catalyst is in the
ball shape with approximate diameter of Table 1. Properties of feed palm biodiesel
Feed biodiesel Limitation Result
3 mm and was added in stainless steel tube
Monoglyceride (%wt) < 0.70 0.40
reactor (37cm in length and 1.27cm in outer Diglyceride (%wt) < 0.20 0.09
diameter). Triglyceride (%wt) < 0.20 0.02
2.2 Experimental Free Glycerine (%wt) < 0.02 0.009
The partial hydrogenation reaction Total Glycerine (%wt) < 0.25 0.139
took place in the fixed-bed reactor with Cloud Point (°C) < 16.0 14
Acid Value (mg KOH/g) < 0.50 0.38
continuous operation mode, as seen in the Cetane Number > 51.0 51
Figure 1. Feed palm biodiesel was fed at
0.38 mL/min and then mixed with hydrogen
gas of 200 SCCM (standard cubic
centimeters per minutes) before flowing into
the reactor. The pressure was controlled at
5 bars. The effect of reaction temperature
used in partial hydrogenation reaction was
studied. Reaction temperatures were varied
at 80, 100 and 120 °C, using Pd/Al2O3
catalyst of 2 g. In addition, the effect of the Figure 2. FAME composition of palm
amount of catalyst was investigated, using 2, biodiesel after partial hydrogenation, using 2
2.5 and 3 g with reaction temperature of g of catalyst with different temperature of
80 °C. The product was collected at 2 h for 80, 100 and 120 °C
the characterization.
2.3 Characterization It was found that higher reaction
The properties of feed palm biodiesel temperature increased the content of
and the product after partial hydrogenation saturated FAME because monounsaturated
reaction were analyzed, using gas FAME and poly unsaturated FAME changes
chromatography (GC) to determine the into saturated FAME.7 At high temperature,
composition contents as free acid methyl the solubility of hydrogen increases.
Therefore, hydrogen can effectively transfer catalyst (Figure 5). This might be from the
to the surface of catalyst, in order to unsteady condition in the reactor. In fact,
promote the addition of hydrogen at double using 3 g catalyst should give more content
bonds in the molecules.8 of saturated monoglyceride than that of
In addition, there is a decrease in the using 2 g catalyst. This should be further
content of unsaturated monoglyceride from investigated.
47.7% into 24.0%, 19.8% and 16.6%, using
reaction temperature of 80, 100 and 120 °C,
respectively, as seen in Figure 3. This means
that the partial hydrogenation reaction was
carried out in the reactor. However, if the
content of saturated FAME is over than
60%, it increases the cloud point and pour
point of palm biodiesel. As a result, the
viscosity of palm biodiesel is high at low
temperature or there is a precipitation of Figure 4. FAME composition of palm
palm biodiesel at high temperature. This biodiesel after partial hydrogenation, under
might be the problem in the transportation or 80 °C reaction temperature with different
in the engine. amount of catalyst of 2, 2.5 and 3 g
Figure 3. The content of monoglyceride Figure 5. The content of monoglyceride

after partial hydrogenation, using 2 g of after partial hydrogenation, under 80 °C
catalyst with different temperature of 80, reaction temperature with different amount
100 and 120 °C, at 2 h time on stream of catalyst of 2, 2.5 and 3 g, at 2 h time on
stream
Figure 4 shows that the content of
saturated FAME increased when more The hydrogenation of palm biodiesel
amount of catalyst was used. More amount was investigated for a longer period of time,
of catalyst offered higher surface area to i.e. 7 hours, to observe the continuous mode
catalyze partial hydrogenation reaction. In reaction. It was found that the content of
addition, the resident time of reaction polyunsaturated FAME (i.e. C18:3 and
increased as higher amount of catalyst was C18:2) decreased which contradicted to the
used. Pd/Al2O3 balls, which were packed in increasing of monounsaturated FAME (i.e.
stainless steel reactor, increased the reaction C18:1) and saturated FAME (i.e. C18:0) as
length inside of the reactor. Therefore, the shown in Figure 6. Moreover, after 2 hours,
reactants took a longer period for partial monounsaturated FAME started to slightly
hydrogenation reaction, promoting the increase with the decreasing of saturated
decrease of the contents of unsaturated FAME. This might be conveyed that the
monoglyceride. However, the content of reaction pathway of polyunsaturated FAME
saturated monoglyceride using 3 g catalyst is tends to convert to monounsaturated FAME
slightly lower than that of using 2.5 g rather than saturated FAME for this
continuous fixed-bed reactor. This temperature or the precipitation at high
selectivity probably useful to the partial temperature. In this work, the suitable
hydrogenation that aiming to minimize the reaction temperature and amount of catalyst
polyunsaturated compounds by parallel is 80 °C and 2.5 g, respectively. This
maximizing the monounsaturated condition changed more than 80%
compounds without further converting to the unsaturated monoglyceride into saturated
saturated ones. However, the experiment monoglyceride.
with longer operation time is recommended
for observing the steady state and confirm Acknowledgements
the behavior with this continuous process. This research was supported by
Thailand Institute of Scientific and
Technological Research (TISTR). The
authors deeply appreciate in the excellent
collaboration with all experts from National
Institute of Advanced Industrial Science and
Technology (AIST) in Tsukuba, Japan.
References
1. http;//www.weben.dede.go.th/webmax/c
ontent/government-targets-start-selling-
b7-January-1-2014.
2. http://www.dede.go.th/download/files/A
Figure 6. FAME composition of palm EDP2015_Final_version.pdf.
biodiesel after partial hydrogenation, using 3. http://www.jama.or.jp/eco/wwfc/pdf/JA
2.5 g of catalyst with reaction temperature MA_FQ_PositionStatement_FAME.pdf.
80 °C and pressure 5 bars 4. Haas, W.; Mittelbach, M. Ind. Crops
Prod. 2000, 12, 111–118.
4. Conclusion 5. Van, G. J. H.; Hammond, E. H.; Yu, L.;
Partial hydrogenation reaction can be Monyem, A. Soc. Automotive Engineers
used to improve the properties of palm Technical Paper Series 1997, paper
biodiesel using continuous fixed-bed No.971685.
reactor. It concludes that higher temperature 6. Tongroon, M.; Suebwong, A.; Kananont,
and more amount of catalyst (Pd/Al2O3) M.; Aunchaisri, J.; Chollacoop, N.
used can decrease the content of Renew. Energ. 2017, 113, 660–668.
polyunsaturated FAME and mono 7. Numwong, N.; Luengnaruemitchai, A.;
unsaturated FAME. In addition, it could Chollacoop, N.; Yoshimura, Y. Chem.
further convert unsaturated monoglyceride Eng. J. 2012, 210, 173–181.
into saturated monoglyceride. However, this 8. Alsobaai, A. M.; Al Shaibani, A. M.;
might affect the cloud point and pour point Moustafa, T.; Derhem, A.; JKSUES.
of palm biodiesel, leading to the increase in 2012, 24, 45–51.
viscosity of palm biodiesel at low
Production processes and characterization of high α-cellulose pulp from
sugar cane bagasse and oil palm empty fruit bunch for development
in cellulose fiber
Sappasit Winthachai1, Wallop Arirob2, Vittaya Punsuvon1,3*
1
Center of Excellence-oil palm, Kasetsart University, Bangkok 10900, Thailand
2
Department of Botany, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
3
*E-mail: fscivit@ku.ac.th
Abstract:
High α-cellulose pulp is used as raw material for the production of rayon fiber. In this
study, sugar cane bagasse (SCB) and oil palm empty fruit bunch (OPEFB) were used as raw
material for high α-cellulose pulp. The pulp from SCB and OPEFB was produced through 3
processes, steam explosion, pulping with 20%wt of NaOH cooperated with 0.1%wt of
anthraquinone (AQ) and bleaching with NaClO2 and followed with H2O2. The results showed
that the amounts of holocellulose, α-cellulose, pentosan, ash and lignin in SCB and OPEFB
were 66.55%, 43.79%, 18.38%, 4.72% and 21.76%, and 69.87%, 45.21%, 26.46%, 1.85%
and 21.26%, respectively. Hemicellulose in SCB and OPEFB was reduced by steam
explosion. Pulping and bleaching removed lignin and also reduced ash content including to
increasing of pulp brightness. After these processes, the produced pulp from SCB had
90.04% of α-cellulose while α-cellulose of the OPEFB pulp had increased to 98.80%. In
summary, both SCB and OPEFB have the potential to be used as raw materials in high α-
cellulose pulp production for rayon fiber.
1. Introduction palm oil extraction, respectively. Since

Regenerated cellulose is produced many tons of SCB and OPEFB are produced
from dissolving pulp (High α-cellulose pulp) annually.3,4 Most of the wastes are not
with a high content of α-cellulose and utilized and most of OPEFB are left in the
relatively low hemicellulose and lignin plantation site without further processing.
content. It can be used as raw material in However, SCB and OPEFB have potential
biocomposite, biomedical, textile and food as a raw material for the production of
application. The most widely-used of cellulose-based products such as high α-
regenerated cellulose are fabricated via cellulose pulp because of containing of high
viscose procedure especially, viscose rayon.1 cellulose content and low hemicellulose and
In recent year, there has been an increasing lignin content.
interesting in agro-industrial wastes for their The aim of this research is to
bioconversion into value-added products of produce high α-cellulose pulp from SCB and
commercial scale because there are many OPEFB via three processes. They are
tons of wastes every year which have exploded with steam for hemicellulose
potential as low-cost sources including removing prior to soda-anthraquinone (AQ)
environmentally friendly and biodegradable pulping and the two steps of bleaching with
materials.2 NaClO2 and H2O2. Finally, the high α-
In Thailand, sugar canes and oil cellulose pulps were characterized theirs
palms are two of the important industrial chemical composition and physical
crops. They can be cultivated in all area. A properties and compared with commercial
number of sugar canes and oil palms are rayon fiber.
produced each year. SCB and OPEFB are a
waste left after the sugar cane juice and
2. Materials and Methods exploded at 205 °C, 15 bar of pressure and 7
2.1 Raw materials min of retention time. After the explosion,
SCB was obtained from the pulp slurry was collected and the pulps
Environment Pulp and Paper Co. Ltd. were separated from the water-soluble
(EPPCO), Thailand. It consisted of 66.55% fraction by filtration, washed with water
holocellulose, 43.79% α-cellulose, 18.38% until the clean pulps, air-dried at room
pentosan, 3.12% ethanol/benzene temperature and the pulp yields were
extractives, 21.76% lignin and 4.72% ash. determined after hot-air drying at 105 °C for
OPEFB was provided by Kasetsart 24 h.
Agricultural and Agro-Industrial Product
Improvement Institute (KAPI). The amounts
of holocellulose, α-cellulose, pentosan,
extractives, lignin and ash in the OPEFB
were 69.87%, 45.21%, 26.46%, 10.08%,
21.26% and 1.85%, respectively. The pith
fraction and residues were separated. The
SCB and OPEFB samples were shown in
Figure 1.
Figure 2. Working plan for production

processes of high α-cellulose pulp from SCB
(OPEFB)
Figure 1. SCB (left) and OPEFB (right)
samples 2.4 Pulping process
The soda-anthraquinone (AQ)
2.2 Working plan pulping of the produced pulps from previous
Figure 2 showed all processes in the process was carried out in pulping unit
production of high α-cellulose pulp from which is located in Kasetsart Agricultural
SCB and OPEFB. The first process, 1.5 kg and Agro-Industrial Product Improvement
of the depithed raw materials was loaded Institute (KAPI), Bangkok. 1.2 kg of pulps
into steam explosion apparatus. After that, was loaded to pulping at 160 °C for 120 min
the dried pulps were soaked in soda- with 20%wt of NaOH and 0.1%wt
anthraquinone (AQ) solution. Final process, anthraquinone (AQ) and the pulp to water
the resulting pulps were bleached with two ratio was 1:10 w/v. After pulping complete,
steps of NaClO2 followed with H2O2. The the pulps were collected from black liquor
remaining pulps of each process were by filtration, washed with water several
characterized the chemical composition, times until the clean pulps, air-dried and the
viscosity and brightness. In addition, theirs yield was determined by hot-air drying at
chemical compositions were compared with 105 °C for 24 h.
commercial rayon pulp. 2.5 Bleaching process
2.3 Steam explosion process The two pulps from pulping process
1.5 Kg raw materials of each batch were bleached in two steps. In the first step,
were loaded into 200 L steam explosion the pulps (30 g) reacted with 50%wt of
apparatus. The raw materials were steam- NaClO2 and adjusted pH with 5 mL of acetic
acid. The volume of water, bleaching hydrolyzed to soluble sugars.6,7 However,
temperature and time were 1600 mL, 70-80 the content of lignin was increased because
°C and 1 h, respectively. After NaClO2 soluble components from lignin were re-
bleaching, the pulps were separated from condensed back into the pulps.
yellow liquor by stainless steel screen box,
washed with water until the clean pulps and Table 1. Chemical composition in SCB and
dried by hot-air oven at 50 °C for 24 h. After OPEFB pulps obtained after steam
that, the pulps were bleached again. The explosion
second step, the pulps after NaClO2 Chemical
bleaching were treated with 4%wt of H2O2, composition SCB pulp OPEFB pulp
(%wt)
3%wt of NaOH and 0.5%wt of MaSO4. The
Holocellulose 68.40 54.44
pulp to water ratio, bleaching temperature α-cellulose 58.88 47.53
and time were 1:10 w/v, 80 °C and 3 h, Pentosan 3.76 4.20
respectively. Lignin 30.06 40.04
2.6 Characterization of pulps Ash 2.32 1.57
The raw materials and the products
of each process were characterized for their 3.2 Soda-anthraquinone (AQ) pulping
chemical composition following a standard The chemical composition of SCB
method of Technical Association of Pulp and OPEFB pulps after delignification were
and Paper Industry (TAPPI). The contents of shown in Table 2. The yields of soda-AQ
holocellulose, α-cellulose, pentosan, lignin pulping were 43.62% and 41.06% for the
and ash were determined by the acid chlorite SCB and OPEFB pulps, respectively. The
method according to Browning,5 TAPPI contents of holocellulose and α-cellulose
T203 om-88, TAPPI T223 cm-84, TAPPI increase while the contents of pentosan,
T222 om-98 and TAPPI T211 om-93, lignin and ash decrease. The lignin content
respectively. Viscosity (TAPPI T230 om- reduced from 30.06% and 40.04% of steam
94) and brightness (ISO 2470-1) were exploded pulps to 1.71% and 2.01% of the
measured for characterization of the high α- SCB and OPEFB pulps, respectively. The
cellulose pulps. prehydrolysis with water such as steam
explosion weakened the bond between
3. Results and Discussion lignin and cellulose. Thus, it can be inferred
3.1 Steam explosion that the steam explosion can lead to a more
The steam exploded pulp from SCB effective removal of lignin during the soda-
and OPEFB showed 58.98% and 54% of AQ process.8
pulp yield and the chemical compositions of
SCB and OPEFB pulps were shown in Table Table 2. Chemical composition of SCB and
1. Hemicellulose is undesirable components OPEFB pulps obtained after soda-AQ
in dissolving pulp because it causes problem pulping
in viscose pulp process. The results showed Chemical
composition SCB pulp OPEFB pulp
that the steam explosion is the result of a (%wt)
significant decreased in the hemicellulose Holocellulose 96.54 96.52
with a consequent proportional increased in α-cellulose 82.01 93.91
the holocellulose and α-cellulose contents. Pentosan 2.52 1.31
The hemicellulose which was expressed in Lignin 1.71 2.01
Ash 2.17 0.53
pentosan content reduced from 18.38% and
26.46% of raw materials to 3.76% and
4.20% of the SCB and OPEFB pulps, 3.3 Bleaching
The two-step bleaching with NaClO2
respectively. In the steam explosion process,
and H2O2 significantly improved the quality
the hemicellulose-lignin bonds are cleaved
of SCB and OPEFB pulps. As shown in
and the hemicelluloses are further
Table 3, The SCB pulp contained 97.22%
holocellulose and 90.04% α-cellulose after dissolving pulp. Although, viscosity of
bleaching that yield 91.51% and the OPEFB OPEFB pulp is lower, it is necessary to
pulp contained 99.69% holocellulose and decrease pulp viscosity during viscose rayon
98.80% α-cellulose after bleaching that yield process in the result of the reduction of
90.22%. In addition, the contents of viscosity enhanced the reactivity of
pentosan, lignin and ash were reduced less cellulose.10
than 2% after bleaching. Although,
brightness of SCB and OPEFB pulps was Table 3. Chemical compositions and
higher after the H2O2 bleaching, but the properties of SCB and OPEFB pulps after
viscosities were reduced because the NaClO2 and H2O2 bleaching
decomposition of H2O2 generates free Chemical SCB pulp OPEFB pulp
radicals which attack the cellulose chain.9 composition
(%wt) NaClO2 H2O2 NaClO2 H2O2
For the SCB pulp, it was found that the
Holo
content of α-cellulose was reduced from cellulose
98.30 97.22 98.18 99.69
90.58% to 90.04%. Generally, the SCB α-cellulose 90.58 90.04 96.49 98.80
contained many short fibers and cellulose Pentosan 2.21 2.04 1.61 1.50
chains will be hydrolyzed to soluble sugar Lignin 0.81 0.65 1.07 0.10
Ash 1.98 1.19 0.33 0.20
after many processes.
Brightness
3.4 Properties of high α-cellulose pulps (%ISO)
66.68 73.64 82.54 86.04
The properties of high α-cellulose Viscosity
5.57 4.69 7.90 5.28
pulps from SCB and OPEFB were shown in (cP)
Figure 3. The properties of the pulps were
compared with commercial rayon pulp that
the results were shown in the Table 4. From
the result, holocellulose of SCB and OPEFB
pulps were higher than the commercial
rayon pulp but α-cellulose content,
brightness and viscosity of SCB pulp were
lower than commercial rayon pulp.
Moreover, it showed the undesirable
impurities such as pentosan, lignin and ash
are higher. For the high α-cellulose pulp
from OPEFB, its chemical compositions Figure 3. High α-cellulose pulp from SCB
were improved better than the commercial and OPEFB
rayon pulp. In addition, brightness and
viscosity are within acceptable range for
Table 4. Comparison of properties between two high α-cellulose pulps with commercial
rayon pulp
Chemical composition/ High α-cellulose pulp High α-cellulose pulp
Commercial rayon pulp
physical properties from SCB from OPEFB
Holocellulose (%wt) 97.22 99.69 97.14
α-cellulose (%wt) 90.04 98.80 94.72
Pentosan (%wt) 2.04 1.50 1.79
Lignin (%wt) 0.65 0.10 0.20
Ash (%wt) 1.19 0.20 0.09
Brightness (% ISO) 73.64 86.04 86.52
Viscosity (cP) 4.69 5.28 7.03
4. Conclusion 2. Chandel, A. K.; da Silva, S. S.;
The production of high α-cellulose Carvalho, W.; Singh, O. V. J. Chem.
pulp from SCB and OPEFB was achieved Technol. Biotechnol. 2012, 87, 11–20.
via three processes, steam explosion, soda- 3. Sangyoka, S.; Reungsang, A.; Lin, C. Y.
anthraquinone (AQ) pulping and NaClO2 Sustainable Environment Research
followed with H2O2 bleaching. The 2016, 26, 235−242.
properties of high α-cellulose pulp obtained 4. Sharma, A. K.; Anupam, K.; Swaroop,
from SCB and OPEFB are nearly to the V.; Lal, P. S.; Bist, V. J. Clean. Prod.
properties of commercial rayon pulp that 2015, 106, 422–429.
indicated that both SCB and OPEFB can be 5. Browning, B. in Method of Wood
used as raw materials for highα-cellulose Chemistry. John Wiley and Sons: New
pulp production in regenerated cellulose York, 1967.
industry. 6. Chen, X. W.; Lawoko, M.; Van
Heiningen, A. Bioresour. Technol. 2010,
Acknowledgements 101 (20), 7812–7819.
The authors acknowledge the 7. Martino, D. C.; Colodette, J. L.;
research fund from Ministry of Industry and Chandra, R.; Saddler, J. Wood Sci.
Thailand Textile Institute (THTI). We also Technol. 2017, 51 (3), 557–569.
would like to thanks Department of 8. Harsono, H.; Putra, A. S.; Maryana, R.;
Chemistry, Center Excellence-oil palm and Rizaluddin, A. T.; H’ng, Y. Y.;
Kasetsart Agricultural and Agro-Industrial Nakagawa-izumi, A.; Ohi, H. J. Wood.
Product Improvement Institute (KAPI), Sci. 2016, 62, 65–73.
Kasetsart University for partial support in 9. Andrade, M. F.; Colodette, J. L. Ind.
this research. Crops Prod. 2014, 52, 58–64.
10. Ibarra, D.; Kopcke, V.; Ek, M. Enzym.
References Microb. Technol. 2010, 47, 355–362.
1. Wang, S.; Lu, A.; Zhang, L. Progress in
Polymer Science 2016, 53, 169–206.
The effect of contaminated gases in syngas on biomethanol production
Rujira Jitrwung, Kuntima Krekkeitsakul*, Kamonrat Leeheng, Jiraporn Chalorngtham,
Anantachai Wannajampa
Expert Center of Innovative Clean Energy and Environment, Thailand Institute of Scientific and Technological
Research (TISTR), KhlongLuang, Pathumthani 12120, Thailand
*E-mail: kuntima@tistr.or.th
Abstract:
A biomethanol was produced via hydrogenation of synthetic gas (syngas, a mixture of
hydrogen (H2) and carbon monoxide (CO)) under the optimum using of a Cu/ZnO/Al2O3
catalyst in fixed bed reactor with 16 mm diameter at temperature of 170 °C and pressure of
40 bars. The syngas was surrounded by contaminated gases, including methane (CH4), carbon
dioxide (CO2), and oxygen (O2). The syngas used for biomethanol production is obtained
from reforming of biogas (55 vol.% of CH4 and 45 vol.% of CO2) using catalytic reforming
over Ni/Al2O3 catalyst at 750 °C and atmospheric pressure. The reforming process generated
mixed gas of desired gas (CO and H2) and undesired gas (CO2, CH4, and O2). The purpose of
this study was to investigate the effects of contaminated gases (CO2, CH4, and O2) on
biomethanol production and catalyst deactivation. The levels of the contaminants were
introduced into the feed of H2/CO, keeping ratio of 2/1 by ranging of vol.% 5-25 CO2, 5-25
CH4 and 5-25 O2. The experiment result showed that only the presence 25 vol.% of O2
impacted significantly to both methanol production and reduced catalyst performance.
It can be indicated that Cu/ZnO/Al2O3 catalyst was effective for the production of methanol
from biogas and resistant to contaminated atmospheres.
1. Introduction supply, a promising way is transforming

Nowadays, alternative fuels and biogas to synthesis gas by reforming
renewable energies are continually received process, and then converting syngas to value
attention due to the issues of global warming chemicals such as methanol.
and energy security. Biogas is an alternative The conversion of syngas into
energy source which is produced from methanol has received much attention in
anaerobic digestion of organic matter. The industrial field, because it can used as a
constituents of biogas including methane, solvent, a gasoline blending, and a chemical
CH4 (60% to 80%), carbon dioxide, CO2 feedstock for biodiesel and other chemicals.
(20% to 40%), and trace amounts of Most of the commercial methanol is
hydrogen sulfide, nitrogen, and other produced from fossil fuels and coke-oven
impurities. The biogas composition varies gas which it is not a green technology.
depending on the chemical properties of the Nevertheless, in this work, a new approach
feedstock and the process used. Major of methanol synthesis by using syngas
sources of biogas derived from municipal obtained from reforming biogas was known
wastewater treatment plants, industrial waste as biomethanol.
treatment facilities, landfills, and Methanol is produced from syngas
agricultural sources such as manure and via CO hydrogenation (Equation 1), CO2
energy crops.1,2 hydrogenation (Equation 2) and water-gas
In Thailand, the surplus of biogas is shift reaction (Equation 3) can be written as
produced over the demand of its using as follow:3
biomethane and electricity. Therefore this CO + 2H2 ↔ CH3OH
over supply was burned and emitted to ΔH298 K = - 90.85 kJ/mol. (1)
atmosphere. To manage with the surplus
CO2+ 3H2 ↔ CH3OH + H2O Gas Co., Ltd.) was used for reduction
ΔH298 K = - 49.82 kJ/mol. (2) reaction of metal catalyst. Syngas composed
CO + H2O ↔ CO2+ H2 of 63 vol.% H2, 32 vol.% CO and 5 vol.%
ΔH298 K = - 41.03 kJ/mol (3) CO2 (mixed by Thai Special Gas Co., Ltd.)
was used as a reactant gas for methanol
Both of hydrogenation reactions are synthesis reaction. Purity 99.99 vol.% of O2,
exothermic under low temperatures (200- CH4, CO2 (Thai Special Gas Co., Ltd.) was
250 °C) and high pressures (over 60 bar). To used to mix with syngas in a reactant stream
obtain methanol, the appropriate ratio of for methanol synthesis. All chemicals were
CO/H2 and CO2/H2 for syngas is required. If used as purchased without further
the syngas is composed of high purification.
concentration of CO2, the methanol reaction 2.2 Characterization of Cu/ZnO/Al2O3
is followed by Equation 2 and obtaining catalyst
methanol and water. In case of the syngas is The surface area and pore volume of
composed of pure CO and H2, the methanol samples were measured by N2 adsorption at-
reaction agrees with Equation 1 and the 196 °C using the BET method
product is only methanol. (Quantachrome Autosorb 1). A Scanning
Therefore, the syngas is needed to Electron Microscope (SEM) was conducted
control for receiving the optimal content of (JEOL model JEM 6340J) to examine
H2, CO, and CO2.4,6 The optimal of CO2 topology of the surface of catalyst.
concentration is ranged between 2% and 8% 2.3 Methanol synthesis reaction
as well as the (H2-CO2)/(CO + CO2) ratio in The experiments were performed in
the range of 2-3.7,8 Normally, the reforming fixed-bed continuous flow system as shown
of biogas generated syngas (a mixture of CO in Figure 1. The reactor was made of the 304
and H2 in range of 1.1-2.3) and remaining stainless steel with the length, inside
some CH4, CO2, and O2. diameter and outside diameter of 16 cm, 1.6
For these reasons, this work was cm. and 2.0 cm, respectively. The catalyst
investigated the effect of contaminated gases bed was heated by external electrical
(CO2, CH4, and O2) in reactive gases for the furnace. A K-type thermocouple was placed
synthesis of biomethanol which the in the catalyst bed to measure the
contaminated gases can be affected temperature. The flow of mixed gas was
biomethanol production and catalyst controlled by mass-flow controller (Brooks,
deactivation. Model SLA5850).
The catalyst with mass of 8 g was
2. Materials and Methods reduced at 230 °C and 3 bar with 5 vol.% H2
2.1 Chemicals and gases in N2 flow of 20 ml/min for 10 h. Methanol
Commercial CuO/ZnO/Al2O3 synthesis reaction of synthesis gas (63% H2,
catalyst with ratio of 38 wt.% CuO, 38 wt.% 32% CO, 5% CO2, by volume) mixed with
ZnO and 24 wt.% Al2O3 (from Xi’an contaminated gas either or CO2, CH4, O2
Sunward Aeromat Co., Ltd., China.) was over commercial Cu/ZnO/Al2O3 catalyst
used as a catalyst for methanol synthesis. with the composition of 38/38/24 wt. %
Hydranol®-Composite 5 (Fluka, were operated at 170 °C, 40 bar and 100
lot#SZBE1680V) and Hydranol®-Methanol ml/min of total flow rate. The contaminated
dry (Fluka, lot#SZBE2460V) were used as gases were adjusted by varying from 5% to
titrant and medium substance in analysis by 25% of CO2, CH4, and O2 by volume and
Karl Fischer method. Standard mixed gas 5 keeping total flow rate of 100 ml/min before
vol.% of H2 in N2 (mixed by Thai Special introducing to synthesis gas.
Figure 1. The fixed-bed continuous flow system of biomethanol synthesis
The outlet product was sampled for

analysis every 3 hours. The effluent gas was
passed through a cold trap, followed by back
pressure regulator and cyclone before A
analyzing by Gas Chromatography (GC,
Agilent Technologies 7890B). The gaseous
products (H2, CO, CO2, CH4, O2, N2 and
others) were investigated by GC equipped
with thermal conductivity detector (TCD)
and flame ionization detector (FID) using B
Mol Sieve 13X, Porapak Q and Pora PLOT,
Q-HT columns. The condensed liquid
products were analyzed by GC-FID using a
DB-170 column.

3.1 Cu/ZnO/Al2O3 catalyst C
characterization
Figure 2 shows the morphology of
commercial Cu/ZnO/Al2O3 catalyst (A) and
the effect of contaminant (CO2, CH4, and
O2) in the feed syngas (B, C and D) which
they were analyzed by SEM. The surface of
Cu/ZnO/Al2O3 catalyst was changed from D
smooth surface (Figure 2A) to random sizes
(large and small) as see in Figure 2 (B, C
and D).This phenomena was happened after Figure 2. SEM images of Cu/ZnO/Al2O3
the catalyst surface contacted with excess catalyst (A) a commercial Cu/ZnO/Al2O3
O2. catalyst without any effect (B) the effect of
The large agglomeration particles CO2 on Cu/ZnO/Al2O3catalyst (C) the effect
influenced to the possibility of decreasing on of CH4 on Cu/ZnO/Al2O3 catalyst (D) the
surface area and losing in active site. effect of O2 on Cu/ZnO/Al2O3 catalyst
Table 1. The textural properties of on the catalyst surface and resulting in
Cu/ZnO/Al2O3 catalysts (CZA) and its catalyst deactivation.
effects 2CO (g) ↔ CO2 (g) + C(s)
Samples Surface Pore Average ΔH298 K = - 172 kJ/mol (4)
area volume Pore However, the excess amount of CO2
(m2/g) (ml/g) Diameter
(°A)
in feed stream has restricted CO
Commercial CZA 118.2 0.395 134.0 hydrogenation by its competing adsorption
Effect of CO2 67.2 0.460 274.4 with CO on the active sites of catalyst, and
Effect of CH4 78.0 0.510 261.0 resulting in coke formation.9
Effect of O2 54.1 0.203 150.2 In case of large amount of CO2 it can
generate more water which will destroy
The characteristic of surface area and catalyst surface. In support of this, Fazeli et
pore volume of commercial Cu/ZnO/Al2O3 al. suggested that the presence of water in
catalyst both before and after reaction by the gas phase as a product of synthesis from
vary vol.% of 5-25 CO2, CH4 and O2 CO2/H2 promotes Cu crystallite growth
contaminants are summarized in Table 1. which would result in catalyst
The surface area of commercial deactivation.10,11
2
Cu/ZnO/Al2O3 (118.2 m /g) is reduced when 3.2.2 Catalytic activity in the presence of
applying the contaminant gases CO2, CH4 CH4 containing contaminant in reactant.
and O2, (67.2, 78.0, 54.1 m2/g respectively).
O2 is the most effect to reduce the surface
area about 54.23% and also affected to
decrease pore volume 48.61%. These
phenomena led to catalyst deactivation.
3.2 Methanol synthesis
3.2.1 Catalytic activity in the presence of
CO2 containing contaminant in reactant
Figure 4. The influence of methane (CH4)

contaminated in reactant on CO conversion
and methanol yield
The effect of CH4 in feed syngas on

CO conversion and methanol yield can be
shown in Figure 4. This result indicated that
methane did not effect on CO conversion
Figure 3. The influence of carbon dioxide and methanol yield.
(CO2) contaminated in reactant on CO 3.2.3 Catalytic activity in the presence of
conversion and methanol yield O2 containing contaminant in reactant
The effects of 5-25 vol.% CO2 were
investigated as shown in Figure 3. The
results were found that increasing CO2 in
reactive gas led to decreasing of CO
conversion and slightly changing of
methanol yield.
The present of CO2 in syngas stream
can prevent the coke formation by following Figure 5. The influence of oxygen (O2)
Boudouard reaction (Equation 4). Without contaminated in reactant on CO conversion
CO2 in the feed gas, carbon easily to deposit and methanol yield
The contamination of oxygen (O2) except 25 vol.% of O2 in reactive gas. On
in reactant on CO conversion and methanol the other hand, gaseous product is
yield are shown in Figure 5. The level of 25 depending on the compositions in the feed
vol.% O2 was found that it effected to lower reactant. In case of highly O2 in feed
methanol yield comparing with 5 and 15 affected to CO2 formation which it can
vol.% of O2 according to bring about promote methanol combustion reaction as
catalyst deactivation. Jame T. report that O2 shown in Equation 8.13
determined of CO and CO2 selectivity in
case of presentation of O2, The O2(g) is CH3OH+O2 → CO2+2H2O+Heat (8)
dissociated to O(a) and This O(a) will react
with CO then generating CO2 as shown in 4. Conclusion
Equation 5. The large amount of CO2 can The experimental results exhibited
effect to methanol synthesis was described that commercial Cu/ZnO/Al2O3 catalyst with
in 3.2.1.12 38%CuO/38%ZnO and 24%Al2O3 is high
CO2 (g) ↔ CO (g) + O (a) (5) performance to endore the contaminated gas
It is caused that copper can be in feedstock. These results provided that the
oxidized with O2 to form CuO by following concerning of excess oxygen in is
copper oxidation equation (Equation 6, 7) considered for catalyst deactivation.
which is an unactive form of Cu.12
Cu+0.5O2 → CuO Acknowledgements
ΔH0 = -155.2 KJ/mol (6) The authors would like to
2Cu + 0.5O2 →Cu2O acknowledge the National Research Council
ΔH0 = -166.7 KJ/mol (7) of Thailand (NRCT) for the approval and
financial support in this research.
3.2.4 Product distribution over
Cu/ZnO/Al2O3 catalyst for methanol References
The composition of methanol 1. http://www.powermag.com/biogas-an-
synthesis's product obtained from biogas alternative-energy-source
reforming process was shown in Figure 6. 2. Bozzano, G.; Pino, L.; Manenti, F.
The product is divided into 2 groups; Renew. Energ. 2018, 118, 673–684.
gas phase and liquid phase. The amount of 3. Peter, M; Fichtl, M. B; Ruland, H.;
liquid phase of each condition was similar Chem. Eng. J. 2012, 203, 480-491.
Figure 6. The outlet composition of reaction with difference contaminant

in level of 5-25 % by volume Kaluza, S.; Muhler, M.; Hinrichsen, O.
4. Yoo, C. J.; Lee, D. W.; Kim, M. S.; 9. Wang, L.; Fang, D.; Huang, X.; Zhang,
Moon, D. J.; Lee, K. Y. J. Mol. Catal. A S.; Qi, Y.; Liu, Z. J. Nat. Gas Chem.
Chem. 2013, 378, 255–262. 2006, 15, 38–44.
5. Park, N.; Park, M. J.; Ha, K. S.; Lee, Y. 10. Zhang, Y.; Sun, Q.; Deng, J.; Wu, D.;
J.; Jun, K. W. Fuel 2014, 129, 163–172. Chen, S. Appl. Catal. A: Gen. 1997, 158
6. Cai, W.; Ramirezdela, P. P.; Toyir, J.; (1-2), 105–120.
Homs, N. Catal. Today 2015, 242, 193– 11. Fazeli, A; Khodadadi, A. A.; Mortazavi,
199. Y. I. J. Chem. Eng. 2013, 32, 45–59.
7. Bozzano, G.; Manenti, F. Prog. Energy 12. James, T. S.; Ian, S. M.; Mortaza, S. Ind.
Combust. Sci. 2016, 56, 71–105. Eng. Chem. Res. 1999, 38, 3868–3872.
8. Marechal, F. ; Heyen, G.; Kalitventzeff 13. http://www.iun.edu/~cpanhd/C101webn
B. Comput. Chem. Eng. 1997, 21, 511– webn/chemical%20reactions/combustion
516. .html
Halogen-free catalysts for the cycloaddition reaction of CO2 to aziridines
Prapussorn Yingcharoen, Sunatda Arayachukiat, Valerio D’Elia*
Department of Materials Science and Engineering, School of Molecular Science and Engineering,
Vidyasirimedhi institute, Payupnai, Wangchan, Rayong 21210, Thailand
*E-mail: valerio.delia@vistec.ac.th
Abstract:
CO2 is considered as an abundant and readily available carbon source. The
cycloaddition reaction of CO2 to aziridines is a relevant pathway for the conversion of CO2 to
value-added chemicals such as oxazolidinones that have found a wide range of applications
such as chiral auxiliaries in organic synthesis, agrochemicals, pharmaceutical products and
versatile intermediates. The combination of early transition metal coordination compounds as
Lewis acids and nucleophilic cocatalysts developed in this work was found to form highly
efficient dual catalytic system for the synthesis variously substituted oxazolidinones from
CO2 and aziridines. DFT calculations carried out in collaboration with the Poater group in
Girona shows that the rate determining step of the reaction is CO2 insertion in the ring-
opened 3-membered ring heterocycles. Moreover, the reaction catalyzed by our halogen-free
system was found to be highly regioselective towards the formation of 3,5-disubstituted
oxazolidinones as the major product.
1. Introduction oxazolidinones with excellent

Carbon dioxide (CO2) is naturally regioselectivity under ambient conditions.7
present in the atmosphere as a part of the in addition, many metal compounds were
Earth's carbon cycle, but human activities investigated for this coupling reaction. For
are altering the carbon cycle as carbon example, zinc-glutamate MOF/ TBAB was
dioxide is one greenhouse gas emitted from found of interest because of it could be
fossil fuel combustion. It is important to find catalyze under room temperature.8 besides,
ways to reduce CO2 emission. Scientist zirconyl chloride was not only an efficient
perceive CO2 as the most abundant carbon catalyst but also cheap, resistant to moisture
source, so they investigate ways to convert stable and reusable.9 Many studies about
CO21-3 to produce material of commercial both organo and metal catalysts were carried
interest. One interesting reaction is between out to improve catalytic performance. In this
CO2 and aziridines for the synthesis of work, various early transition metal catalysts
oxazolidinones4 which are important will be employed to explore their catalytic
heterocyclic compounds. Oxazolidinone has potential toward the cycloaddition of CO2 to
a wide range of applications5 as chiral aziridines. Moreover, the halogen-free early
auxiliaries in organic synthesis, transition metal catalysts will be
agrochemicals, pharmaceutical products and investigated in this cycloaddition reaction to
versatile intermediates. Several catalysts are study the conversion efficiency and
available to synthesize oxazolidinones. regioselectivity of oxazolidinones. The
Bifunctional aluminum complexes were reaction condition will be optimized and the
used in combination with quaternary resulting compounds will be characterized
ammonium salts.6 This catalytic system was using nuclear magnetic resonance (NMR)
found to afford the highly selective ring and high resolution mass spectroscopy.
opening of aziridine before CO2 insertion.
Moreover, Polyethylene glycol 2. Materials and Methods
functionalized phosphonium salt was an 2.1 General
effective recyclable catalyst to produce
For aziridines synthesis section: atmosphere. Finally, CO2 was added.
diethyl chlorophosphate, amine, Reaction was heated to 100 °C for 2 days.
triethylamine, n-butyllithium, epoxide, After this period, the conversion of substrate
dichlomethane (DCM), dimethoxyethane was determined by 1H NMR. The product
(DME), were purchased from commercial was purified by column chromatrography
sources. Nitrogen was purchased from (hexane/ ethyl acetate 7:3) and characterized
Praxair. For cycloaddition section: catalysts by 1H NMR, 13C NMR and mass
consist Yttrium(III) chloride (YCl3), spectroscopy.
Zirconium(IV) chloride (ZrCl4), Zinc 2.4 Spectroscopic measurement
chloride (ZnCl2), Niobium(V)chloride NMR spectra were measured on an
(NbCl5), Scandium(III) chloride, (ScCl3), automated “Bruker” 600 MHz for 1H (151
Titanium(IV) ethoxide (TiEtO4), MHz for 13C). Chemical shifts are reported
Niobium(V) ethoxide (NbEtO5), in ppm (δ, relative to TMS) using CHCl3
Zirconium(IV) ethoxide (ZrEtO4) and residual peak (δ = 7.26 ppm) in CDCl3 as an
nucleophiles co-catalysts consisting of tetra- internal standard. Mass of compounds were
n-butylammonium bromide (TBAB), tetra- measured on LC-Quadrupole-time-of-flight
n-butylammonium iodine (TBAI), 4- Tandem mass spectroscopy “Bruker
(dimethylamino) pyridine (DMAP) were compact QTOF” with APCI mode.
purchased from commercial sources, and
stored in a glovebox (MBraun 3. Results and Discussion
MB200MOD). Carbon dioxide (>99.999%) 3.1 Catalytic screening
was purchased from Praxair. For solvent: The combination of early transition
tetrahydrofuran (THF), dichlomethane metal coordination compounds as Lewis
(DCM), diethyl ether (DEE), acetonitrile acids and nucleophilic cocatalysts was
(CH3CN), Dimethylformamide (DMF) were
developed for cycloaddition reaction of CO2
purchased from commercial sources, and
purified by solvent purification system to aziridines.
(MBraun MB-SPS).
2.2 Synthesis of aziridines
The synthesis of aziridines consists
of 2 steps. Firstly, to synthesize
phosphoramidic acid, diethyl
chlorophosphate was added drop wise to a
cooled solution of amine and then
triethylamine was added under a nitrogen
atmosphere. The reaction was left stirring at
room temperature for 1 day.10 Secondly, the
mixture of aziridine, phosphoramidic acid in
dimethoxyethane (DME) was added n-
butyllithium under nitrogen followed by the
addition of epoxide to the reaction. The
Reaction was heated to 140 °C for 18 h. The
Figure 1. The proposed mechanism
residue was purified by column
11
chromatrography.
2.3 Synthesis of oxazolidinones The reaction mechanism was
In glovebox, catalyst and co-catalyst proposed in Figure 1. These Lewis-acidic
were charged into a stainless steel autoclave. transition metal catalysts activate the
After removal of autoclave from glovebox, electrophilic carbon atoms adjacent to the N-
aziridine was dissolved in solvent, and heteroatom of aziridines thus accelerating
added into a reactor under nitrogen
ring opening by the nucleophile (Nu). After
that CO2 is inserted into the metal free catalysts (TiEtO4, NbEtO5, ZrEtO4)
coordinated anion generated by ring were presented (Table 1, Entries 6-8). The
opening. Finally, the ring closure leads to combination of NbEtO5/ TBAB has more
the final oxazoridinones. The cycloaddition efficiency catalytic system than other metal
of CO2 to 2-ethyl-1-phenylaziridine (1a) to compounds. After that, the condition was
produce oxazoridinones (1b) was chosen as optimized. THF was an outstanding solvent
a model reaction for the catalytic screening because of high solubility (Table 1, Entries
and optimized the reaction conditions in 11-14). Furthermore, increasing of CO2
Table 1. transition metal halides including pressure, time and the amount of catalyst
YCl3, ZrCl4, ZnCl2, NbCl5 and ScCl3 were loading were improved the yield of
combined with TBAB in THF at 100 °C, 20 oxazoridinones (1b) (Table 1, Entries 15-
bar CO2 for 1 day (Table 1, Entries 1-5). 17). In addition, when temperature was
Among the metal halide catalysts, the decreased down from 100 °C to 80 °C, the
combination of YCl3/ TBAB was the best yield of the product was decreased (Table 1,
catalyst for CO2 conversion. Moreover, the Entry 18). And at room temperature 2-ethyl-
results of the catalytic screening in the 1-phenylaziridine (1a) was not able to
presence of early transition metal halogen- convert to the desired final product
Table 1. The cycloaddition reaction of CO2 to aziridines by early transition metal

coordination compounds and nucleophilic cocatalysts
Isolate Regioselecti-
Entries Catalyst Co-catalyst Solvent Temperature Time
yielda (%) vityb 1b: 1c
1 2 mol % YCl3 4 mol % TBAB THF 100 °C 1 day 24 97 : 3
2 2 mol % ZrCl4 4 mol % TBAB THF 100 °C 1 day 23 87 : 13
3 2 mol % ZnCl2 4 mol % TBAB THF 100 °C 1 day 14d
4 2 mol % NbCl5 4 mol % TBAB THF 100 °C 1 day 3 55 : 45
5 2 mol % ScCl3 4 mol % TBAB THF 100 °C 1 day 23 90 : 10
6 2 mol % TiEtO4 4 mol % TBAB THF 100 °C 1 day 18d
7 2 mol % NbEtO5 4 mol % TBAB THF 100 °C 1 day 42 93 : 7
8 2 mol % ZrEtO4 4 mol % TBAB THF 100 °C 1 day 14d
9 2 mol % NbEtO5 4 mol % TBAI THF 100 °C 1 day - -
10 2 mol % NbEtO5 4 mol %DMAP THF 100 °C 1 day - -
11 2 mol % NbEtO5 4 mol % TBAB DCM 100 °C 1 day 40 91 : 9
12 2 mol % NbEtO5 4 mol % TBAB DEE 100 °C 1 day 7 98 : 2
13 2 mol % NbEtO5 4 mol % TBAB CH3CN 100 °C 1 day - -
14 2 mol % NbEtO5 4 mol % TBAB DMF 100 °C 1 day - -
15c 2 mol % NbEtO5 4 mol % TBAB THF 100 °C 1 day 50 93 : 7
19c 4 mol % NbEtO5 8 mol % TBAB THF rt 2 day - -
a
Isolate yield calculated from compound 1b after purify by column chromatography.
b
Regioselectivity calculated from both isomer after purify by column chromatography
c
using 30 bar CO2.
d
%conversion of product determined by 1H NMR.
(Table 1, Entry 19). Finally, the optimized 4. Conclusion
catalytic system was 4 mol% NbEtO5/ 8 The cycloaddition reaction of CO2 to
mol% TBAB in THF at 100 °C, and 30 bar aziridines was carried out using a dual
catalyst based on the combination of less
CO2 for 2 day. This catalytic system could
corrosive halogen-free Lewis acids metal
promote the yield of the final product up to catalyst Nb(OEt)5 and a nucleophilic
68% and provided the high regioselective cocatalyst (TBAB). The catalyst was found
(89:11) of 3,5-disubstituted oxazolidinones to be highly regioselectivity for the
(1b) as the major product (Table 1, Entry formation of 3,5-disubstituted
17). oxazolidinones but the yields were just
3.2 Substrate screening moderate. Several attempts are ongoing in
The dual catalyst consisting of our laboratories to promote this reaction
NbEtO5 as a Lewis acids and TBAB as a using more efficient organocatalysts.
nucleophilic is a highly active combination
compounds for the oxazolidinones Acknowledgement
formation. Various terminal aziridines This work has been supported by the
(Table 2) were further studied in this Department of Molecular Science and
reaction under the optimized condition (100 Engineering, Vidyasirimedhi Institute of
°C, 30 bar CO2, THF, 2 days). The reaction Science and Technology.
catalyzed by our halogen-free system was
found to be highly regioselective towards References
the formation of 3, 5-disubstituted 1. Seo, U. R.; Chung, Y. K. Green Chem.
oxazolidinones (b) as the major product. 2017, 19 (3), 803–808.
2. Tamura, M.; Honda, M.; Noro, K.;
Table 2. The synthesis of oxazolidinones Nakagawa, Y.; Tomishige, K. J. Catal.
from the coupling reaction between CO2 and 2013, 305, 191–203.
various aziridinesa 3. Zhou, H.; Wang, G.-X.; Zhang, W.-Z.;
Lu, X.-B. ACS Catal. 2015, 5 (11),
4 mol% NbEtO5,
8 mol% TBAB, 6773–6779.
4. Yang, Z.-Z.; He, L.-N.; Gao, J.; Liu, A.-
THF, 100 °C, 2 days
H.; Yu, B. Energy Environ. Sci. 2012, 5
Isolate Regioselectivity (5), 6602.
Entries Substrates
yield (%) b: c 5. Nale, D. B.; Rana, S.; Parida, K.;
1 31 71 : 29 Bhanage, B. M. Appl. Catal. A: General
2014, 469, 340–349.
N 6. Ren, W. M.; Liu, Y.; Lu, X. B. J Org
2a Chem. 2014, 79 (20), 9771–9777.
7. Watile, R. A.; Bagal, D. B.; Patil, Y. P.;
2 Cl 48 86 : 14 Bhanage, B. M. Tetrahedron Lett. 2011,
(65)b 52 (48), 6383–6387.
8. Kathalikkattil, A. C.; Roshan, R.;
N
Tharun, J.; Babu, R.; Jeong, G. S.; Kim,
3a D. W.; Cho, S. J.; Park, D. W. Chem
Commun (Camb) 2016, 52 (2), 280–
a
Using aziridines (1 mmol), Nb(OEt)5 (0.04 mmol, 4
283.
mol%), TBAI (0.08 mmol, 8 mol%) in THF (2 mL) 9. Wu, Y.; He, L. N.; Wang, J. Q.; Miao,
at 100°C, 30 bar CO2 for 2 days. C. X.; Li, W. Tetrahedron 2009, 65
b
Using 80°C f(31), 6204–6210.
10. Kumar, G. K.; Saenz, D.; Lokesh, G.; 11. Minicone, F.; Rogers, W. J.; Green, J.
Natarajan, A. Tetrahedron lett. 2006, 47 F.; Khan, M.; Smith, G. M.; Bray, C. D.
(35), 6281–6284. Tetrahedron Lett. 2014, 55 (43), 5890–
5891.
The utilization of biomass waste from the palm oil industry
using hydrothermal carbonization process
Theerapat Nangam1, Chayut Pornprasitpol1, Amornrat Suemanotham2,
Lalita Attanatho2, Weerawat Patthaveekongka1*
1
Department of Chemical Engineering, Faculty of Engineering and Industrial Technology, Silpakorn University,
SanamChandra Palace, Nakhon Pathom 73000, Thailand
2
Research (TISTR), Khlong Luang, Pathum Thani 12120, Thailand
*E-mail: patthaveekongka_w@silpakorn.edu
Abstract:
This research studied the utilization of biomass waste from the palm oil industry using
a hydrothermal carbonization (HTC) process. This process is a thermochemical conversion of
wet biomass into high quality solid fuels called biochar by processing in a hot, pressurized
water environment. Several factors, which affect the efficiency of HTC, were investigated,
such as types of biomass waste (palm kernel shell, PS and palm empty fruit brunch, EFB),
reaction temperature (160, 180 and 200 °C) and reaction time (10, 30 and 60 minutes). The
quality of the biochar was determined by means of the elemental and proximate analysis and
the heating value. The results indicated that the fuel properties of solid products from HTC of
PS and EFB were significant improved such as the increasing of carbon content and higher
heating value in the range of 2-13% and 1-9%, respectively compared to the raw palm oil
industry wastes. The increasing of reaction temperature and time was shown to decrease
biochar yield whereas the crude bio-oil was observed to increase. The HTC is able to improve
the properties of biomass wastes for use as a solid fuel.
1. Introduction convert wet biomass into liquid products

In Thailand, Palm oil tree is one of (bio-oil) together with solid products
the most important crops. The cultivation (biochar) and non-condensable gases. The
area of palm oil is about 5 million acres, hydrothermal process operates at high
which was ranked as the third largest temperature and pressure, mostly near the
cultivation area in the world. There are lots critical point (Pc=22.1 MPa and Tc=374
of biomass wastes generated from palm oil °C).2 It is very promising reaction medium
mill industry. They include palm empty fruit for the conversion of lignocellulosic
bunch, palm shell, palm fiber, palm kernel biomass, since it has ability to break the
cake and waste decanter cake. Ministry of rigid structure of lignocellulosic complexes
Energy launched the Alternative Energy and to decompose it into smaller component
Development Plan (AEDP2015)1 to increase by hydrolysis and further reactions.3 The
the blending ratio of biodiesel and diesel advantages of HT compare to other thermal
from B7 up to B10 and B20. Biodiesel in process are the avoidance of energy-
Thailand uses crude palm oil as a feedstock. extensive drying process, high conversion
Therefore, there are a large amount of efficiency and relative low operation
biomass wastes that are generated from oil temperature. Hydrothermal process can be
palm industry. classified into three categories including
The high amount of oil palm wastes hydrothermal carbonization, hydrothermal
can be utilized and upgraded as a feedstock liquefaction and hydrothermal gasification
for hydrothermal process (HT) to generate using main product as criteria. The main
high value liquid and solid products. The objective of this study focuses on
HT is a thermal decomposition process to hydrothermal carbonization process (HTC)
to convert wet biomass into high quality of
solid fuel (biochar). The applications of
biochar include as a carbon material, a soil
amendment, a substitute for activated carbon
and as an adsorbent as well as a solid fuel.
In this present study, the utilization
of oil palm wastes using hydrothermal
carbonization process was investigated at
different biomass wastes, temperature and
reaction time. The waste biomass used in the
experimental tests were palm shell and palm
empty fruit bunch. The biochar products Figure 1. Flow diagram of the experimental
were all analyzed in terms of elemental set up
composition, proximate analysis and heating
value. The yield of HTC products was 2.3 Characterization of raw materials and
measured and compared for the difference biochar products
conditions. The raw materials were characterized
their lignocellulosic compositions including
2. Materials and Methods hemicellulose, cellulose and lignin contents.
2.1 Materials The elemental composition (ultimate
Palm shell (PS) and palm empty fruit analysis) and proximate analysis (volatile,
bunch (EFB), obtained from an oil palm mill fixed carbon, ash and moisture) of feedstock
in Thailand, were used as raw materials in and solid product were determined with
this study. The PS and EFB were dried in TruSpec CHN analyzer and TGA 701,
the oven at 90 °C for 12 hour and then respectively. The higher heating value
crushed to obtain 2 mm in size of PS and (HHV) of raw material and solid product
EFB. was analyzed by bomb calorimeter. The
2.2 Experimental procedure composition of bio-oil product was analyzed
The hydrothermal carbonization and identified by a gas chromatograph-mass
process was conducted in a batch type spectrometry (GC-MS).
reactor (Parr reactor) equipped with an
electric furnace. The raw material was 3. Results and Discussion
loaded into reaction vessel and mixed with 3.1 Characterization of raw materials
deionized water at biomass to water ratio of Table 1 presented lignocellulosic
1:10. The reactor was closed tightly and components, proximate and ultimate
pure nitrogen gas was purged to remove the analysis, as well as heating value of oil palm
residual gas. Then, the reactor was heated wastes. Both of palm shell and palm empty
with stirring to the desired temperature (160- fruit bunch has high moisture content at
200 ºC) with the autogenous pressure and about 26% of PS and 33% of EFB. The
desired reaction time (10-60 min). After the volatile and fixed carbon of raw material has
end of reaction, solid and liquid products a value of 75-78 wt% and 15-23 wt%. The
were separated by filtration under vacuum. PS has higher lignin content (50 wt%) than
The solid product was dried in an oven at EFB (14 wt%). The higher heating value of
110 ºC overnight to yield the final biochar the raw materials is almost same in the range
product. Liquid phase was extracted with of 19-21 MJ/kg.
dichloromethane. Bio-oil was obtained after 3.2 Effect of reaction temperature
removal of dichloromethane. The The effect of reaction temperature on
experimental flow diagram is shown in the yield of biochar and bio-oil as well as
Figure 1. quality of biochar in HTC was investigated
in the range of 160-200 ºC for 30 min in 6
batch reactor. As shown in Figure 2, the Palm Shell
4.78
5
biochar yield of PS and EFB decreased from
Yield Bio-oil (wt%)

Empty Fruit Bunch
79 to 56 wt% of PS and 68 to 47 wt% of 4

3.31
4.34
EFB with increasing in reaction temperature. 3
PS, which contained higher lignin content
than EFB, produced the highest yield of 2 2.42
biochar. On the other hand, Figure 3 1 0.76

presented the bio-oil yield of PS and EFB 0.36
increased from 0.8 to 4 wt%. for both of 0
150 160 170 180 190 200 210 220
biomass wastes at high temperature. It can Temperature (˚C)
be noticed that at low temperature favors the
maximum solid product. As the temperature Figure 3. Effect of reaction temperature on
rises, the solid product may decrease bio-oil yield (30 min)
whereas liquid and gaseous products will be
increase. 80
Proximate analysis (wt%)

70 76.08 75.17
71.99
Table 1. Characteristic of oil palm wastes 60 Volatile Matter
Properties PS EFB 50 Fixed Carbon
Ash
Proximate analysis (%) dry basis 40
Moisture content 0.0 0.0 30 27.29
23.04 23.05
Volatile matter 74.9 78.5 20
Fixed carbon 22.5 15.3
10 0.89
Ash 2.6 6.2 0.59 0.71
0
Ultimate analysis (%) dry ash basis
150 160 170 180 190 200 210 220
Carbon 52.2 53.8 Temperature (˚C)
Hydrogen 5.9 6.8
Oxygen 41.5 38.4 Figure 4. Effect of reaction temperature on
Nitrogen 0.3 0.8 proximate analysis of biochar from PS (30
Lignocellulosic compositions (%)
Cellulose 28.7 28.9
min)
Hemicellulose 24.5 19.2
Lignin 49.6 14.4 fixed carbon content enhanced from 23 to 27
Extractive 16.3 30.2 wt%, As shown in Figure 4. The results of
Heating value (MJ/kg) biochar from EFB also showed the volatile
Higher heating value (HHV) 21.0 19.8
and fixed carbon content as the same trend.
100
The increase in fixed carbon content was
Palm Shell justified by the release of volatile matter that
79.32
80 Empty Fruit Bunch occurred in the HTC.
60.21 The elemental compositions results
Yield (wt%)
60 67.66 55.96
54.16 showed that the carbon content of biochar

40 47.22
from PS and EFB was observed to increase
20
whereas the hydrogen and oxygen contents
reduced with increasing temperature.
0
150 160 170 180 190 200 210
Another important characteristic of biochar
Temperature (˚C) is their higher heating value (HHV).
Figure 2. Effect of reaction temperature on All experiment test, biochar enhanced
biochar yield (30 min) from 21.0 to 22.8 MJ/kg of biochar from PS
and 19.8 to 20.9 MJ/kg of biochar from EFB
At higher temperature, the proximate with increasing reaction temperature, as
analysis results indicated that volatile shown in Figure 5.
content of biochar from PS slightly
decreased from 76 to 72 wt%. In contrast the
90
3.3 Effect of reaction time
81.65 10 min
The influence of reaction time on 80 79.01 30 min
yield of solid and liquid products and 79.32 72.52 60 min
Yield (wt%)
biochar quality in HTC was studied at 160- 70
64.89
200 ºC and reaction 10-60 min in batch 60 57.22
reactor. The biochar yield from PS and EFB 60.21 57.03
55.96
is presented in Figure 6 and 7. It can be seen 50
that the biochar yield of PS and EFB 40

decreased with longer reaction time at all 150 160 170 180 190 200 210
Temperature (˚C)
temperatures. In contrast the bio-oil yield of
PS and EFB was observed to increase and Figure 6. Effect of reaction time on biochar
the highest bio-oil yield was about 6 wt%, as yield from PS
shown in Figure 8 and 9. According to 90
experiments, with longer reaction time 80
10 min
74.15 30 min
provided higher bio-oil products and lower 71.14 60 min
70 66.34
Yield (wt%)
biochar products. The chemical 67.66
58.11
compositions of bio-oil from PS and EFB 60
51.94
were analyzed by GC-MS. Bio-oil 50 54.16 51.61
comprised of various organic compounds. 40

47.22
Phenolic compounds were the main

30
components whereas minor components 150 160 170 180 190 200 210
consisted of ketones, alcohols and other Temperature (˚C)
heterocyclic compounds. Figure 7. Effect of reaction time on biochar

Figure 10 and 11 implied that the yield from EFB
proximate and ultimate analysis of biochar
8
at 10-60 min and 160-200 ºC were 10 min
7
determined. Carbon content of biochar from 30 min 6.59
Bio-oil Yield (wt%)
6 60 min
PS and EFB had value in the range 50-57 5 4.78
wt% and increased with increasing 4
3.31 3.66
temperature and time of reaction. While 3
oxygen content significant decreased for all 2 1.82
experimental tests with increasing reaction 1 0.83

0.56 1.29
0.36
time. 0
140 150 160 170 180 190 200 210 220
Temperature (˚C)
24
Palm Shell
22.8
Figure 8. Effect of reaction time on bio-oil
23 Empty Fruit Bunch
yield from PS
HHV (MJ/kg)
22
22
21 20.9 8
21 10 min
20.4 7
30 min 6.12
Bio-oil Yield (wt%)
20 19.8 6 60 min
5
19
4 4.34
150 160 170 180 190 200 210 220
2.96
Temperature (˚C) 3
1.94
2 2.42
2.01
Figure 5. Effect of reaction temperature on 1 0.76
HHV (30 min) 0
0.66 0.57
140 150 160 170 180 190 200 210 220

Temperature (˚C)
Figure 9. Effect of reaction time on bio-oil

yield from EFB
60 60
10 min 10 min
Carbon content (wt%)

Carbon content (wt%)
30 min 30 min
60 min 60 min
55 55
50 50
45 45
140 160 180 200 220 140 160 180 200 220
Temperature ( C) Temperature ( C)
Figure 10. Effect of reaction time on carbon Figure 11. Effect of reaction time on carbon
content of biochar from PS content of biochar from EFB
1.8
PS
PS_160_60
1.6
PS_180_60
PS_200_60
Atomic H/C ratio
1.4 EFB
EFB_160_60
EFB_180_60
1.2
EFB_200_60
1.0
0.8
0.40 0.45 0.50 0.55 0.60 0.65
Atomic O/C ratio
Figure 12. Van krevelen diagram; between O/C and H/C atomic ratios of PS, EFB and
biochar from PS and EFB at 60 min
The higher heating value of biochar 4. Conclusion

from PS was improved from 21 to 23 MJ/kg This present study focused on the
whereas biochar from EFB slightly utilization of oil palm wastes to produce
increased from 19 to 21 MJ/kg. The solid product by HTC process. The physical
increasing in HHV is mainly released to and chemical properties of biochar from PS
increase of carbon content and decreased of and EFB have changed by the operating
oxygen content in the HT treated biomass conditions. The results indicated that
due to dehydration and decarboxylation.4-6 increasing reaction temperature and time
The conversion of biomass to carbon rich during HTC led to obtained high carbon
materials is commonly represented using a content and heating value while lower
van krevelen diagram as shown in Figure 12. biochar yield. The heating value indicated
The O/C and H/C atomic ratios of biochar that the biochar was improved and suitable
from PS and EFB were observed to decrease for use as solid fuel. The biochar obtained
compared with raw materials. from HTC had lower oxygen and volatile
contents and higher fixed carbon and heating 2. Prado, J. M.; Follegatti-Romero, L. A.;
value compared with original raw materials, Foster-Carneiro, T.; Rostagno, M. A.;
because of hydrolysis, dehydration and Filho, F. M.; Meireles, M. A. A. J.
decarboxylation reactions during HTC. Supercrit. Fluid 2014, 86, 15-22.
3. He, C.; Giannis, A.; Wang, J. Y. Appl.
Acknowledgements Energ. 2016, 111, 257-266.
The authors would like to express the 4. Parshetti, G. K.; Hoekman, S. K.;
grateful acknowledgement to TISTR and all Balasubramanian, R. Bioresource
staff for supporting and providing all Technol. 2013, 135, 683-689.
instruments and utilities as well as National 5. Wei, H. C.; Song, C. Y.; Herng, K. S.
Research Council of Thailand for the Bioresource Technol. 2012, 118, 195-
financial support in this present study. 203.
6. http://www.dede.go.th
References
1. Xiu, U.; Shahbazi, A. Renew. Sustain.
Energy Rev. 2012, 16, 4406-4414.
Electrochromic properties of polyaniline on sputtered crystalline TiO2 on
fluorine-doped tin oxide substrate prepared by electropolymerization for
solar cell applications
Thanapat Chaipitisiri*, Palang Bumroongsakulsawat
Center of Excellence on Catalysis and Catalytic Reaction Engineering, Department of Chemical Engineering,
Faculty of Engineering, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
*E-mail: t.chaipitisiri@gmail.com
Abstract:
Thin films of electropolymerized polyaniline on n-type titanium oxide (n-TiO2)
nanoparticles were fabricated and studied. PANI/TiO2 has drawn widespread attraction for its
easy facile fabrication and is promising for extraction of solar energy. However, before
commercialization, the problems regarding stability and electrical hysteresis of PANI/TiO2
still need to be tackled. Structural, optical, and electrical properties of the films were studied
by XRD, SEM and UV-VIS spectroscopy. This study finds that PANI/TiO2 composite films
prepared with optimized conditions are potentially useful for low-cost hybrid solar cell and
electrochromic applications.
1. Introduction
Nowadays, the increasing
environmental pollution and growing energy
demand are two critical tasks for
researchers. Among various conjugated
conductive polymers,1-3 polyaniline (PANI)
is a promising material due to its simple
electrochemistry, ease of synthesis,
environmental stability and cheap monomer.
It has three distinguishable oxidation
states,4,5 whose structures are presented in Scheme 1. Different oxidation states of
Scheme 1, where y, (0<=y<=1), corresponds PANI
to the fraction of the reduced unit and (1-y)
corresponds to the fraction of the oxidized mm thick, 10 ohm/sq.) was used as the
unit. The fully reduced form (y=1) is called transparent working electrode, where
leucoemeraldine base (LB). The half electropolymerization of aniline to form
oxidized form (y=0.5) is named emeraldine polyaniline was carried out. Analytical
base (EB), which can also be easily doped to grades of aniline, H2SO4, acetone and
obtain the highly conductive emeraldine salt ethanol were purchased from Sigma-
(ES). Lastly, the fully oxidized form (y=0) is Aldrich. FTO-coated glass was cleaned with
referred to as pernigraniline base (PNB). acetone, ethanol and DI water, respectively,
These unique structures of PANI has been and was finally sonicated for 15 min before
exploited in many applications.6-8 use.
2.2 Deposition of TiO2 layer
2. Experiment Figure 1 shows the illustration of the
2.1 Materials and Methods RF magnetron sputtering apparatus. A TiO2
Sodalime glass coated with fluorine- layer was deposited on FTO glass in Ar/O2
doped tin oxide (FTO) (TCO 30-10/LI, = 80/20 atmosphere at 35 mTorr. The
Solaronix S.A. - Aubonne, Switzerland, 3 background pressure in the process chamber
was 5.0 x 10-5 mbar. The depositions was Electropolymerization of aniline in the
performed on non-heated substrates. RF electrolyte were conducted with cyclic
power between the target and substrate was potential scan in the range of -0.3-1 V at a
varied between 100, 200, 300 and 400 W. scan rate of 10 mV s-1 on FTO glass for 10
The distance between substrate and target cycles. After deposition, the coated
surfaces was 100 mm. The deposition time substrates were washed with distilled water
was maintained at 1 hr. and acetone to remove remaining chemicals
such as monomer and oligomers.
2.4 Electrochemical characterization of
PANI/TiO2 film
Cyclic voltammetry of PANI/TiO2
films between -0.3 - 1 V in 1 M H2SO4 at a
scan rate of 10 mV s-1 was performed.

3.1 Structural characterization
400
A(004) 300
W W
R(110) 200 W
100 W
R(211)
R(220) R(301)
Figure 1. Schematic set-up of the
sputtering system
2.3 Electrochemical synthesis
Figure 3. X-ray diffraction pattern of TiO2

deposited on FTO under the different
condition
The film structure was investigated

by X-ray diffraction (XRD). Fig.3. shows
Figure 2. Schematic set-up of a three- the patterns at different discharge powers
electrode cell for electropolymerization and from 100 to 400 W and at 35 mTorr of
electrochemical characterization Ar/O2 gas pressure. According to Fig.4, the
patterns show anatase structure with peak A
All electrochemical measurements (004) and rutile structure with peak R (110),
were performed with Autolab R (211), R (220) and R (301). It can be seen
PGSTAT128N controlled by NOVA1.10 that the intensity of R (110) increases as the
software in three-electrode cell (Figure 2) RF power was varied from 100 W to 400 W.
using FTO-coated glass (1 × 2.5 cm2) as the 3.2 Electrochemical measurement
working electrode, Ag/AgCl electrode as the Cyclic voltammograms (CV) of
reference electrode and a titanium foil as the PANI/TiO2 films are shown in Figure 3 and
counter electrode. The electrolyte contains all show the traditional characteristics of
0.1 M aniline and 1 M H2SO4. PANI.11-14 The peaks at ~0.3 V corresponds
to leucoemeraldine/emeraldine transition.15
However, there are some differences in the a b
peak height. The voltammograms of PANI ) )
on TiO2 sputtered at RF power of 100 W is
wider than the others due to the smallest
presence of rutile phase of TiO2, as
suggested by XRD patterns in Figure 3.
c d
) )
Figure 5. SEM images of (a) 100 W, (b)

200 W, (c) 300 W and (d) 400 W
Figure 4 shows the surface

morphology of the polyaniline film doped
with Sulfuric acid at 1 V constantly versus
Ag/AgCl and fully stopped at 10 min. It was
found that the surface is rough and consist of
dense layer on the FTO glass. In literature, it
can be found that for deposition time of 10
c) min, emeraldine structure of PANI strongly
shows dense layer and its color intensity on
the FTO glass after electropolymerized
deposition. And if we compare PANI films
electrodeposition for each condition of RF
power (Figures 5(a-d)). We can conclude
that the film has the similar morphology.
4. Conclusion
The Titanium dioxide (TiO2) films
d) under various deposition conditions of RF
power were deposited on Fluorine-tin oxide
(FTO) glass substrate by using RF
magnetron sputtering method. The RF
power condition from 100 to 400 W showed
the increased intensity peak of rutile phase
(110). And electrochromic PANI film was
successfully synthesized on FTO-TiO2
coated glass substrated by potential cycling.
Figure 4. Cyclic voltammogram of According to the cyclic voltammetry, it was
found that potential was varied with the RF
Figure 4. PANI/TiO2 under different power, which was using to deposite TiO2
conditions of RF power. a) 100 W, b) 200 previously, the optimum condition for this
W, c) 300 W and d) 400 W experiment is 100 W.
Acknowledgements 7. He, B.; Tang, Q.; Liang, T.; Li, Q. J.
The authors would like to thank Prof. Mater. Chem. 2014, A 2.9, 3119–3126.
Peter Kelly at Manchester Metropolitan 8. Pilan, L.; Raicopol, M. UPB Sci. Bull.
University for his help on TiO2 coating. 2014, 76, 1454–2331.
9. Francombe, M. H.; Vossen, J. L.
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180.
Thermodynamic analysis of the chemical-looping water splitting and
sorption enhanced reforming process for hydrogen production from biogas
Natthaporn Saithong, Karittha Im-orb, Amornchai Arpornwichanop*
Computational Process Engineering Research Unit, Department of Chemical Engineering,
*E-mail: amornchai.a@chula.ac.th
Abstract:
Hydrogen has been considered as clean energy carrier and an important chemical.
Presently, a steam reforming process is widely used for hydrogen production; however, it
requires high energy demand and subsequent hydrogen-purification units. In this study, the
novel chemical looping process integrating water splitting and sorption enhanced reforming
is proposed. In the proposed process, biogas as a renewable feedstock is partially oxidized
with iron-oxide and CO2 is captured by CaO in the fuel reactor (FR) to produce H2-rich
syngas. The iron-oxide is re-oxidized in the steam reactor (SR) to generate an additional H2
stream and CaO is regenerated in the calcinator. Process modeling using a thermodynamic
approach is performed by Aspen Plus simulator and the Redlich-Kwong-Soave equation of
state with Boston-Mathias modification is used to determine thermodynamic properties. The
effects of key parameters such as SFR-to-methane and Fe3O4-to-methane ratios are studied.
The results show that when the FR is run at 610 oC, H2 product in the syngas and high-purity
H2 streams is obtained at 97.01 % and 99.93 % purity, respectively. The conversions of CH4
and CO2 are 95.61 % and 96.45 % and the total hydrogen yield per one mole of methane in
biogas is 3.8. Increasing the H2OFR-to-methane ratio enhances H2 yield and purity.
1. Introduction Presently, the concept of chemical

Hydrogen (H2) has been regarded as looping has been introduced to H2
an important alternative fuel which has high production process in several ways such as
energy density and heating value. It is chemical looping H2 generation (CLHG)
considered the clean and environmentally process (sometimes called chemical looping
friendly fuel. In addition, H2 can be used as water splitting process (CLWS)) and
feedstock for ammonia synthesis, methanol sorption enhanced chemical looping
production and petrochemical processes as reforming (SE-CLR) process. The CLWS is
well as used in fuel cells for electrical the process developed from the chemical
production.1 looping combustion (CLC) process, to
Conventionally, H2 is produced by produce H2, CO2, and N2 using iron oxide
the steam reforming process using natural (FexOy) as an oxygen carrier. H2 produced
gas as a feedstock.2 In this process, a from this process in the re-oxidized stage of
synthesis gas (syngas), which is mainly iron-oxide is sufficiently pure for utilization
composed of H2 and carbon monoxide (CO), in fuel cell systems and the gas separation
is produced from the steam-methane unit is unnecessary. However, the gaseous
reforming (SMR) using catalyst. This products in the reduction state of Fe3O2 to
process is widely used because it is very FeO by fuel is often contaminated with H2
economical; however, it requires several and CO due to the presence of the partial
downstream units for H2 purification. In oxidation reaction, so the gas separation unit
addition, the catalyst used in the process can is needed. In addition, the H2 obtaine from
be deactivated due to coking and high reacting of FeO with steam by steam-iron
energy demand is needed in the reforming reaction is often contaminated by CO2 and
reactor.3
CO because of carbon deposition on iron simulator. A equilibrium reactor model
oxide in the reduction step.4 (RGibbs module) is used to predict the
The SE-CLR is developed from equilibrium gaseous composition by the
chemical looping reforming (CLR) to minimization of Gibbs free energy. The
improve the steam reforming process using iron-oxide in magnetite state (Fe3O4) and
calcium oxide (CaO) as a CO2 adsorbent and CaO are used as an oxygen carrier and CO2
nickel oxide (NiO), as an oxygen carrier. absorbent, respectively. Therefore, the
The solid mixture of NiO and CaO is fed to possible gaseous components are H2, CH4,
a reactor and the syngas is produced from CO2 and CO, and the solids components are
CH4 (as a fuel) reacting with H2O and NiO CaCO3, CaO, Fe3O4, and Fe0.947O.6-7 The
by SMR and partial oxidation reaction, process is operated at the steady state
respectively. At the same time, CaO adsorbs condition. All reactors are assumed at
CO2 and thus, water-gas shift reaction is isothermal and isobaric conditions. The
improved and the fractions of CO and CO2 thermodynamic properties are determined
in the gaseous product are reduced. After based on the Redlich-Kwong-Soave
that, the produced calcium carbonate equation of state with Boston-Mathias
(CaCO3) and Ni is regenerated. A gas modification (ESRKS).
separation unit is not needed for this process The process flow diagram of SE-
because CO2 from steam reforming is CLRWS process for hydrogen production
captured by CaO. As a result, this process is from biogas is shown in Figure 1. The SE-
less complex when compared with a CLRWS process consists of three reactors: a
conventional steam reforming. However, the fuel reactor (FR), steam reactor (SR), and
purity of H2 stream is still not enough to be calcinator. Biogas (BIOGAS), steam
used in some applications such as low- (STEAM-FR), and Fe3O4 and CaO in the
temperature polymer electrolyte membrane SOLID-RE stream are fed into the FR to
fuel cells (LT-PEMFC).5 produce H2-rich syngas stream. The iron-
Regarding the advantages of the SE- oxide in magnetite state is reduced to
CLR and CLWS processes, the integration wüstite state (Fe0.947O) by the oxidation
of such the two processes is proposed in this reaction. At the same time, the CO2
study. The performance of the integrated produced by the complete oxidation reaction
sorption enhanced chemical looping (Eq. (5)), is adsorbed by CaO according to
reforming and water splitting process (SE- carbonation reaction (Eq. (3)), resulting in
CLRWS) for H2 production is investigated. the equilibrium of the water-gas shift
In the proposed process, iron-oxide, CaO reaction (Eq. (2)) shifted to the product side.
and H2O are used as an oxygen carrier, CO2 The gaseous product and solids from the FR
adsorbent and oxidizing agent, respectively. (FR-CYC) are fed to the first cyclone
Biogas is considered as renewable (CYC1) to separate the gas and solid. The
feedstock. A thermodynamic approach is main product of the FR is H2-rich syngas
used to analyze the effects of operating stream (SYNGAS). The solids stream from
parameters on the SE-CLRWS regarding the the FR (FEOCACO3) is fed to the second
H2 yield, H2 purity and CH4 conversion. The reactor, SR. In the SR, Fe0.947O is re-
key operating parameters considered are the oxidized to Fe3O4 using the steam as an
temperature of FR, steam feed to FR-to- oxidizing agent by steam-iron reaction (Eq.
methane (SFR/CH4) molar ratio, and Fe3O4- (6)) to produce high pure H2 stream (H2).
to-methane (Fe3O4/CH4) molar ratio. The mixed gas and solid product
stream (SR-CYC) is sent to the second
2. Modeling of the SE-CLRWS process cyclone (CYC2). Then, the solid stream
The model of the SE-CLRWS from the SR (CACO3FE3) is fed to the
process is developed based on the calcinatory (CAL) to regenerate the CaCO3
thermodynamic approach using Aspen Plus by calcining (Eq. (8)) the CaCO3 to CaO.
Figure 1. Simulation flow diagram of the combined process of the sorption enhanced
chemical looping reforming and water splitting (SE-CLRWS)
The mixed gas and solid stream from the (TFR), Fe3O4/CH4 molar ratio (Eq. (9)), and
calcinator (CLC-CYC3) is fed to the last SFR/CH4 molar ratio (Eq. (10)). The
cyclone (CYC3) to separate the CO2 gas. performance of the process is explained in
The solid stream from the CYC3 is feedback terms of the H2 yield in FR, H2 yield in SR,
to the FR for completing the cycle of the total H2 yield, H2 purity in FR, H2 purity in
process. SR, and CH4 conversion, which are
The main reactions occurring in the calculated by Eqs. (11) - (16), respectively,
SE-CLRWS process are shown in Eqs. (1)- nFe3O4 ,feed
(8).7-8 Fe3O4 / CH 4  (9)
nCH4 ,feed
Fuel reactor:
CH4  H2O  CO  3H2 (1) nFR,feed to FR
SFR /CH 4  (10)
CO  H2O  CO2  H2 (2) nCH4 ,feed
CaO  CO2  CaCO3 (3) nH2 ,produced from FR
H 2 yield in FR  (11)
1.2Fe3O4  CH 4  nCH4 ,feed
3.8Fe0.947 O  CO  2H 2 (4) nH2 ,produced from SR
H 2 yield in SR  (12)
4.807Fe3O4 +CH 4  nCH4 ,feed
15.228Fe0.947 O+CO2 +2H 2O (5) nH2 ,produced from FR andSR
Total H 2 yield  (13)
Steam reactor: nCH4 ,feed
3.808Fe0.947O+H2O  1.202Fe3O4 +H2 (6)
nH2 ,produced from FR
3.8071Fe0.947 O+CO2  H 2 ,FR-pur
(%)  100 (14)
ntotalfrom FR (dry basis)
1.2018Fe3O4 +CO (7)
nH2 ,produced from SR
Calcinator: H 2 ,SR-pur
(%)  100 (15)
CaCO3  CaO+CO2 (8) ntotalfrom SR (dry basis)
The sensitivity analysis is performed nCH4 ,in  nCH4 ,out
to investigate the effect of operating  CH 4 -conv
(%)  100 (16)
nCH4 ,in
conditions on the process performance. The
key operating parameters in the SE-CLRWS where ni and  i are the molar flow rate and
process include fuel reactor temperature the purity (or conversion) of component i.
Table 1 summarizes the standard reaction (Eq. (2)) move reversely. In
operating conditions of the SE-CLRWS addition, at FR temperatures of 500, 600 and
process used in this study. 700 oC, when the SFR/CH4 molar ratio is
increased in the first stage, the H2 purity in
Table 1. Base case condition FR is increased significantly until the re-
Parameter Value oxidation occurs. Under this condition, the
Feed condition H2 purity in FR is constant even though the
CO2 molar concentration in
H2 yield in FR is increased.
biogas feed stream (%) 40
Temperature (oC) 400
Pressure (atm) 1
Steam feed FR-to-CH4 molar ratio 2.2
Fe3O4-to-CH4 molar ratio 1
CaO-to-CH4 molar ratio 1.66
Steam feed SR-to-CH4 molar ratio 2.87
FR temperature (oC) 610
SR temperature (oC) 500
Calcinator temperature (oC) 860

3.1 Effect of the steam feed FR-to- Figure 2. The H2 yield in FR and SR as a
methane molar ratio function of SFR/CH4 and TFR
Figure 2 shows the H2 yield in FR
and SR as a function of SFR/CH4 molar ratio
at different operating temperatures. In the
FR, the reaction system can be divided by
three phases. The first phase involves the
partial oxidation (Eq. (4)), water-gas shift
(Eq. (2)), and steam-methane reforming (Eq.
(1)), resulting in more H2 product in FR.
When the SFR/CH4 ratio is still increased, the
system goes to the second phase where
Fe0.947O is partially re-oxidized to Fe3O4
with steam in enriching H2O environment
due to the steam-iron reaction (Eq. (6)).9 As Figure 3. The H2 purity as a function of
Fe0.947O decreases, the H2 yield in SR is SFR/CH4 and TFR
gradually reduced. Then, at high SFR/CH4
molar ratio, the main reaction in the final 3.2 Effect of the Fe3O4-to-methane molar
phase is the steam-methane reforming ratio
reaction (Eq. (1) because Fe0.947O is Figures 4 and 5 show the effect of
completely reacted with steam to Fe3O4. the Fe3O4-to-methane molar ratio on the H2
Figure 3 shows a change in H2 purity yield in FR and SR, and H2 purity in FR,
by varying the SFR/CH4 molar ratio at respectively. The results show that at the FR
different temperatures. It is found that when temperature of 500 oC, the increase of
the FR temperature is increased, the Fe3O4/CH4 molar ratio does not affect the
maximum H2 purity in FR is decreased. As process performance. At the FR temperature
the carbonation reaction (Eq. (3)) is favored higher than 600 oC, increasing the
at low temperatures, the high amount of CO2 Fe3O4/CH4 molar ratio increases the H2 yield
remains in the FR when operated at high in SR but decreases the H2 yield in FR as
temperatures. This effect causes the shown in Figure 4. This is because, in the
equilibrium of the steam-methane reforming earliest stage, Fe3O4 can be reacted with
reaction (Eq. (1)) and water gas shift CH4 to form Fe0.947O by the complete
oxidation reaction (Eq. (5)), resulting in an
increase of CH4 conversion, and H2 yield in
SR due to the high amount of reactant,
Fe0.947O, for steam-iron reaction (Eq. (6)).
The decrease of H2 yield in FR is the result
of the steam-methane reforming reaction
(Eq. (1) and the reverse steam-iron reaction
(Eq. (6)). When increasing the Fe3O4/CH4
molar ratio reach to the equilibrium, the
amount of Fe0.947O and the gaseous
composition are constant. Although the
increase of Fe3O4/CH4 molar ratio results in Figure 4. The H2 yield in FR and SR as a
a decrease of H2 yield in FR, however, the function of Fe3O4/CH4 and TFR
total H2 yield is oppositely affected. The
total H2 yield can be increased by increasing
of Fe3O4/CH4 molar ratio due to more
Fe0.947O reacting with steam to produce high
purity H2 in SR. In addition, an increase in
the FR temperature decreases the H2 yield in
FR since the carbonation reaction (Eq. (3))
is favored at low temperatures. Although the
maximum total H2 yield of 3.79 can be
obtained at the FR temperature of 700 oC
and Fe3O4/CH4 molar ratio of 1.75,
however, the H2 purity at the FR
temperature of 700 oC, which is 84.77 %, is Figure 5. The H2 purity as a function of
lower than at the FR temperature of 600 oC, Fe3O4/CH4 and TFR
where the maximum H2 purity is 97.01 %
with the H2 in FR and total H2 yield of 3.04 in this study based on a thermodynamic
and 3.75, respectively, at the Fe3O4/CH4 approach. From the parametric analysis, it
molar ratio of 0.92. was found that the H2 purity in H2-rich
The H2 purity in FR can be syngas and high purity H2 stream of 97.01
increased by increasing the Fe3O4/CH4 and 99.93 %, respectively, with high total H2
molar ratio at FR temperature of 600 and yield of 3.8 can be obtained at TFR of 610
o
700 oC, and can be decreased by increasing C, SFR/CH4 molar ratio of 2.2, and
of the Fe3O4/CH4 molar ratio at FR Fe3O4/CH4 molar ratio of 1. At low FR
temperature of 800 oC, as shown in Figure 5. temperature (less than 800 oC), increasing
This is caused by the carbonation reaction the SFR/CH4 ratio can improve H2 yield in
(Eq. (3)). When the amount of Fe3O4 is FR, total H2 yield, and H2 purity in FR
increased, CH4 can be reacted with excess because of the sorption enhanced reforming.
oxygen carrier to obtain CO and CO2. At The high value of Fe3O4/CH4 molar ratio has
low temperatures, CO2 can be adsorbed by a negative effect on the H2 yield in FR due
CaO and CO can be reacted with steam by to the oxidation of CH4 and H2.
water-gas shift reaction (Eq. (2)), resulting
in a lower amount of CH4, CO, and CO2. Acknowledgements
Support from Chulalongkorn
4. Conclusion Academic Advancement into Its 2nd
The integrated process of sorption Century Project is gratefully acknowledged.
enhanced chemical looping reforming and
water splitting (SE-CLRWS) was investigated
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Effect of the electrolyte imbalance on performance of a vanadium redox
flow battery: dynamic simulation and analysis
Tossaporn Jirabovornwisut1, Yong-Song Chen2, Amornchai Arpornwichanop1*
1
2
Advanced Institute of Manufacturing with High-tech Innovations and Department of Mechanical Engineering,
National Chung Cheng University, Chiayi 62102, Taiwan
Abstract:
Electrolyte imbalance of a vanadium redox flow batteries (VRFB) has a significant
impact on its energy capacity during a long-term operation. The side reactions and vanadium
ions diffusion across the membrane lead to the imbalance problem. In this study, a dynamic
model of the VRFB consisting of conservation equations coupled with an electrochemical
model was developed and used to investigate the electrical characteristics and energy
capacity of the VRFB. To develop the VRFB model, uniform temperature and concentration
distributions and instantaneous self-discharge reaction were assumed. The model prediction
was in good agreement with experimental data with the maximum relative error of 2.14%.
Analysis of key operating parameters affecting the battery performance showed that the
hydrogen and oxygen gassing side reactions caused current losses during a charging process,
which were proportional to the state of charge and voltage losses in both the half-cell of the
VRFB. Also, increases in the electrolyte flow rate and initial vanadium concentration reduced
the overpotential of both the half-cells, expanded the operating time to reach the cut-off
voltage and improved the energy capacity and efficiency of the VRFB.
1. Introduction by the concentration gradient between half-

Electrical energy storage (EES) plays cell. This factor results in the self-discharge
a significant role in the alternative solution side reaction, leading to the capacity loss of
for balancing the demand and supply of
the VRFB. In normal operation of VRFB,
electricity. The intermittent and fluctuant
problems of renewable energy sources when the electrode voltage is higher than
requires the reliable storage technologies to standard potential of the oxygen evolution
stabilize an electrical grid.1 Vanadium redox reaction (OER) and hydrogen evolution
flow batteries (VRFB) have a potential reaction (HER), the HER and OER reactions
technology that can be utilized to store can occur at the electrode surface, causing a
energy. Due to employing the same consumption of applied current for charge
vanadium element in the two half cells, the battery.2-3 Thus, the capacity prediction
cross-contamination problem, which is the plays a significant role for scheduling
electrolyte regeneration to recover the
mechanism that a species crosses over and
capacity loss. To study the capacity fade and
reacts irreversibly with elements in the
opposite half-cell, leading to loss of capacity enable extended cycling of the VRFB, a
zero-dimensional or lumped system model
and degradation of system performance, can
be avoided. Although the membrane used in was developed by reducing dimensionality
while capturing the key physics necessary.4
VRFB is designed for allowing only protons However, the existing lumped model does
and other charge-carrying ions pass through, not include both effects of gassing side
it cannot completely prevent the diffusion of reaction and ions diffusion through the
vanadium ions across the membrane caused
membrane for long-term performance model was simplified by using the following
analysis and capacity prediction. assumptions:
To investigate the performance of 1. Electrode physical properties, electrode
the VRFB during long-term operation, a surface area, pore dimeter and thickness of
dynamic model of the VRFB based on electrode are constant.
conservation equations coupled with an 2. Equilibrium of side reactions in solution
electrochemical model is developed and are instantaneously reached.
validated with experimental data of the 3. The electrolyte is perfectly mixed which
the temperature and concentration in each
laboratory unit cell of VRFB. The effects of
position of the cell are uniform and remain
operating parameters such as vanadium constant at room temperature.
concentrations, electrolyte flow rate and 4. The electrolyte properties, density,
applied current, on the electrical viscosity and volume are constant.
characteristics and energy capacity are
5. The effect of air oxidation of the V 2  ion
analyzed.
at negative electrolyte tank is neglected by
purging an inert gas into the void volume of
2. Modelling of VRFB
electrolyte tank for covering the electrolyte
The schematic and mechanism of
surface.5
redox reactions in VRFB are shown in
2.2 Electrochemical model
Figure 1. The oxidation and reduction
The electrochemical model is used to
reactions at the negative and positive
electrodes are explained by the half-cell determine the actual cell voltage ( U cell ) and
reactions. The forward direction of the half- the current loss by gassing side reaction. The
cell reaction represents charging while the cell voltage can be described by the
backward is discharging process. summation of open circuit voltage ( EOCV )
The positive electrode: and overpotential (  ). The open circuit
VO2+ +H2O  VO2+ +2H+ +e- , E0 =1.00V (1) voltage can be described by the Nernst
The negative electrode: equation which is the voltage of the cell
V3+ +e-  V2+ , E0  0.26V (2) when no current flow. During charging or
discharging, some of voltage is lost for
activating the movement of electrons. The
variation of voltage losses depends on the
cell configuration and the mechanism of
electrochemical reaction.
U cell  EOCV  S  C  Ohm  (3)
RT CV (V )CH2  CV ( II ) 
EOCV  E 0    (4)
nF  C V ( IV ) CV ( III ) 
where s is the activation overpotential, a
driving force is required to force an
electrochemical reaction to take place at the
electrode, C is the concentration
Figure 1. Schematic of the VRFB overpotential which is caused by the
potential different between the bulk of
electrolyte and the electrode surface and
2.1 Model assumptions
A dynamic model for a unit cell of Ohm is the ohmic overpotential due to the
VRFB was conducted by including the resistance of electron flow and ion
effects of ions diffusion through the movement balancing the charge, R is the
membrane and gassing side reactions. The gas constant, T is the cell temperature, n is
the number of electrons transfer, F is the 2.3 Mass balance
Faraday constants and Ci represents the For modelling VRFB, the mass
concentration of each specie. balance in electrode and electrolyte tank is
The ohmic overpotential in VRFB derived based on molar concentration in
system occurs at bipolar plates, membrane liquid electrolyte solution (Eq. (9)).
dc j cj
and electrolyte, as described by Eq. (5): Ve k  Q j  ckt  ckj   redox  Dk k Amem
I
d d d  dt nF d mem
ohm  i  c  m  e  (5) (9)
 c m e   ckj ,op 
   si Dk Amem 
The correlation of the current density  d mem 
( i ) on the activation overpotential can be where Dk is the diffusion coefficient
represented by the Butler-Volmer equation.
through the membrane of species k , d mem
The anodic  a  and cathodic  c  transfer
and Amem are the thickness and surface area
coefficient are assumed to equal 0.5 by the
symmetry of electron transfer. The of the membrane. The current for redox
activation loss of each half-cell in VRFB reaction ( I Redox ) in Eq. (9) is the remainder
can be determined by Eq. (6). of applied current which is consumed by
2 RT  1  i 
 gassing side reaction. The rate of self-
S  sinh   (6) discharge reaction depends on the
nF   2i0   concentration of species k in the opposite of
where io is the exchange current density, half-cell ( Ckj ,op ) and the stoichiometric
which depends on the concentration of coefficient ratio ( si ) of self-discharge
species and rate constant.
reaction as summarized in Table 1.6 The
The surface concentration ( CS ) of
concentration variation in the negative and
the active species is influenced by the positive electrolyte tanks is shown in Eq.
electrolyte flow rate and concentration at (10).
bulk electrolyte ( CB ). Thus, the dct
concentration overpotential can be Vt k  Qt  ckj  ckt  (10)
dt
determined by including the effects of local where Vt represents the volume of
mass transfer coefficient ( km ) of the active
electrolyte tank.
species which is proportional to electrolyte
flow rate and bulk concentration as shown in Table 1. Self-discharge reactions by
Eq. (7). vanadium ions cross-over membrane and
RT  i  their stoichiometric coefficient ratio6
con   ln 1   (7) Self-discharge
F  km nFCB  reaction
( Ck ) ( si )
The current density loss associated At negative half-cell CV ( IV ) CV (V )
with the OER and HER occurring during
charging process is approximated by the V 4   V 2   2V 3 CV ( II ) -1 -2
5 2 3
V  2V  3V CV ( III ) 2 3
Tafel relationship (Eq. (8)).2,3
 F ii 
At positive half-cell CV ( II ) CV ( III )
ji  j0,i exp   (8) V 2
V 5
 3V 4
CV ( IV ) 3 2
 RT  3 5 4
V V  2V CV (V ) -2 -1
where ji represent the current loss from the
OER and HER and  and  are the
overpotential and transfer coefficient of 3.1 Model validation
gassing side reaction, respectively. The presented model was validated
with the experimental data of the VRFB.7
The experiment was conducted by charging Figure 3. The highest value of activation
and discharging the battery with a constant loss is obtained when the state of charge
electric current of 10 A and 20 A. Two (SOC) is close to 0 and 1 while the lowest of
peristaltic pumps were used to circulate the activation loss is obtained at an
electrolyte solution through the cell and the approximately of 0.5 SOC.
pumps were operated at constant electrolyte
flow rate of 130 ml min.1 Figure 2 shows the
comparison of experiment data with the
simulation results and it can be noticed that
the predicted results are in good agreement
with experimental data with the maximum
relative error of 2.14%.
Figure 3. Activation overpotentials in both

electrodes at three different charging
currents
Furthermore, the increasing of

current density contributes to the rise in
Figure 2. Comparisons of simulation results ohmic loss (Eq. (5)). The high overpotential
with experimental data enhances the current loss from the gassing
side reactions when compare at the same
3.2 Effect of current density SOC and the magnitude of current loss is
In this section, the battery voltages proportional to the SOC as indicated in
during charging and discharging under Figure 4.
different current densities which are 140 mA
cm-2, 180 mA cm-2 and 220 mA cm-2 are
investigated. The electrolyte flow rate in
both of half-cells are set to be 2 ml s-1. The
initial condition of vanadium concentration
in cell and electrolyte tank are defined at 1.6
M, whereas the proton concentration is 4 M.
The battery is charged and discharged until
the battery voltage reached the cut-off
voltage. The upper and lower limit of
voltage are set at 1.7 V and 1.1 V,
respectively. The results show that the
higher current density is the cause of faster
increase and decrease voltage which means Figure 4. The current density loss
the battery takes shorter time to be fully associated with the OER and HER during
charge and discharge similar to Figure 2. charging
The variation activation losses of positive
and negative electrode are shown in
As a result of increasing current 3.4 Effect of electrolyte flow rate
density from 140 mA cm-2 to 220 mA cm-2,
the coulombic efficiency is decreased
approximately 10% due to the partial of
applied current is consumed for driving
HER and OER leading to reduction in the
current density for the redox reaction at both
electrodes. It is clear that the capacity loss
during 200 cycles increase with the
magnitude of gassing side reactions occur.
In additional, the initiation of SOC for
oxygen evolution is reduced with the
increase in charging current density due to
the oxygen current density is controlled by
the voltage loss at positive electrode.
3.3 Effect of vanadium concentration Figure 5. The battery voltages curve for
Figure 5 shows the battery voltage different initial vanadium concentration
variation during charging and discharging
due to the different of initial vanadium
concentration of 1.6, 1.8 and 2.0 M. It is
noticed that the time for charging and
discharging battery is increased with the
addition of initial vanadium concentration
which means more energy density can be
stored when increase the vanadium
concentration. The vanadium concentration
affects to the overpotential which are
activation and concentration loss on both
electrode during charging period. The
overpotential on both of electrode is reduced
when increase the vanadium concentration.
The battery energy capacity variation during Figure 6. The energy capacity variation
200 cycles of charging and discharging are during 200 cycles of charging-discharging
shown in Figure 6. It is clear that the for different initial vanadium concentration
increase in initial vanadium concentration,
the battery capacity is increased. However, it The effect of the different electrolyte
is interesting that the rate of capacity loss flow rate of 0.5, 1.5 2.5 ml s-1 on the
per cycle correspondingly due to the effect capacity loss during the charge-discharge
of vanadium ions transfer through the cycles are studied. The low electrolyte flow
membrane and the gassing side reaction rate can be reduced the battery SOC at the
occur as mentioned in previous section is upper limit voltage due to the high of
increased when used the high initial concentration loss and reduci the time to
vanadium concentration. The rates of cut-off voltage in both the electrodes as
capacity drop are 0.031, 0.0332 and 0.0361 shown in Figure 7. The energy efficiency for
Ah per cycle corresponding to vanadium flow rate of 0.5, 1.5 and 2.5 ml s-1 are
concentration 1.6, 1.8 and 2.0 M, 65.301%, 82.75% and 86.36%, respectively.
respectively. It can be contributed to the This can be noticed that increase the
reason that the diffusion rate of vanadium electrolyte flow rate from 1.5 ml s-1 to 2.5
ions across the membrane is enhanced with ml s-1 do not much increase of the energy
the increase in initial vanadium.
efficiency. Figure 8 shows the capacity drop overpotential on the electrode can increase
in 200 cycles. It confirmed that changing the the coulombic efficiency and energy
electrolyte flow rate from 1.5 ml s-1 to 2.5 efficiency including the hindering of current
ml s-1 slightly increase the available capacity loss from gassing side reactions. Operating
at the end of 200 cycles with 8.09% while the battery with the low current density can
changing the electrolyte flow rate from 0.5 decrease the overpotential, but it takes such
ml s-1 to 1.5 ml s-1 significantly increase a long time to be fully charged and
with 46.96%. discharged. The increase in the vanadium
concentration and electrolyte flow rate can
mainly reduce the concentration
overpotential and expand the time to the cut-
off voltage. The rate of capacity loss
enhanced by the increase in the initial
vanadium concentration results from higher
diffusion rate of vanadium ions across the
membrane. The increment of electrolyte
flow rate is considered by a tradeoff
between the increased energy efficiency and
more pump power consumption.
Acknowledgements
Support from Chulalongkorn
Figure 7. The charging curves for different Academic Advancement into Its 2nd
electrolyte flow rates Century Project is gratefully acknowledged.
References
1. Weber, A. Z.; Mench, M. M.; Meyers, J.
P.; Ross, P. N.; Gostick, J. T.; Liu, Q. J.
Appl. Electrochem. 2011, 41 (10), 1137.
2. Al-Fetlawi, H.; Shah, A. A.; Walsh, F.
C. Electrochim. Acta 2010, 55 (9), 3192-
3205.
3. Shah, A. A.; Al-Fetlawi, H.; Walsh, F.
C. Electrochim. Acta 2010, 55 (3), 1125-
1139.
4. Boettcher, P. A.; Agar, E.; Dennison, C.;
Kumbur, E. C. J. Electrochem. Soc.
2016, 163 (1), A5244-A5252.
Figure 8. The energy capacity variation
5. Ngamsai, K.; Arpornwichanop, A. J.
during 200 cycles of charging-discharging
Power Sources 2015, 295, 292-298.
for different electrolyte flow rates
6. Merei, G.; Adler, S.; Magnor, D.; Sauer,
D. U. Electrochim. Acta 2015, 174, 945-
4. Conclusion
954.
The dynamic model of the VRFB
7. Ngamsai, K.; Arpornwichanop, A. J.
was developed for investigating the
Power Sources 2015, 282, 534-543.
electrical characteristics and energy
capacity. Reducing the magnitude of the
Removal of copper in aqueous solution by an alternative adsorbent
modified napier grass stem
Wimonrat Tongpoothorn*, Pitchayapa Kampeaw, Chittinan Kamjung
Department of Chemistry, Faculty of Engineering, Rajamangala University of Technology Issan,
Khon Kaen Campus, Muang, Khon Kaen 40000, Thailand
*E-mail: aammyytt@hotmail.com
Abstract:
This study reports on the potential feasibility of modified napier grass stem (mNGS)
for removal of copper in aqueous solution. The adsorption of copper on mNGS was studied
to determine the effect of initial pH, initial concentration, and adsorbent dosage by batch
experiments. The adsorption capacity of copper was found to be 1.15 mg copper per gram of
adsorbent at 28 C. The results of the study showed that adsorption percentage was found to
be 97.02% with optimal conditions of pH 5, initial concentration 50 mg/L and adsorbent
dosage 1 g. The experimental equilibrium data is well fitted with the Langmuir isotherm
model (R2 = 0.9844, RMSE = 1.2599E-5) than Freundlich isotherm model (R2 = 0.9692,
RMSE = 3.1733E-5), indication of monolayer adsorption. The kinetics of copper sorption on
mNGS followed the pseudo-second order model. This suggests that the adsorption process
was controlled by chemisorptions. Thermodynamic studies revealed that the adsorption
process is non-spontaneous and endothermic in nature (∆G = ∆H - T∆S). Therefore, mNGS
has a potentials for application as adsorbent for the removal of copper in aqueous solution.
1. Introduction to 2.0 mg/L.6 Several methods were applied

Currently, the discharge of heavy to remove copper concentration from
metals into water has led to increase effluents for instance by membrane
pollution problem which may affect the filtration,7 chemical precipitation,8
9
quality of water supply due to their non- electrocoagulation, reverse osmosis,1
degradability and toxicity. Contamination of solvent extraction, biological degradation12
11
numerous metals is known to be toxic even and ion exchange.13 Nevertheless, these
at low concentration. In the cause of tissue methods are high capital or high operational
mineral research, copper (Cu) toxicity is a cost or disposal of the resulting sludge.14
common finding.1 Copper is a widely used Adsorption is one of the most cost
industrial metal for machinery and effective method, but using of activated
metallurgy, plumbing, electrical wiring, and carbon as adsorbent is limited due to its
transportation industry.2 Furthermore, expensive. Thus, cheap and efficient
copper can also be found in food as adsorbents based on renewable resources
contaminant, for example, in mushroom, has proved very promising for removal of
liver, shellfish, chocolate, and nuts.3 Too copper from wastewater. In this work,
much copper in water may damage marine efforts have been made to investigate the use
and freshwater organisms such as fish and of napier grass stem (NGS) as an
molluscs.4 Excess of copper in the human economical and available adsorbent for
body resulted in serious health harms, removing copper from aqueous solutions. As
causing anemia, kidney damage, stomach a first step, characterization of modified
intestinal distress, coma and eventual death.5 NGS was carried out in order to determine
The suggested safe level of copper in the possibility of its use as an adsorbent.
drinking water varies depending on the Later, the conditions of adsorption were
source is recommended in the range of 1.5 optimized by varying parameters including
pH of the solution, initial copper respectively, V is the volume of the copper
concentration and adsorbent dosage to solution (L) and W is the dosage of the
obtain a maximum percentage of removal. adsorbent used (g).
Kinetic, isotherm and thermodynamic 2.4 Kinetic of adsorption process
studies were performed to evaluate the Kinetics study was performed for
experimental data to understand the copper explains the uptake rate of adsorbate. The
removal performance and the feasibility of rate and mechanism of copper adsorption
using NGS as an alternative adsorbent. process onto adsorbent could be clarified
based on kinetic studies. In order to
2. Materials and Methods elucidate the adsorption kinetics, the
2.1 Preparation and characterization of equation of pseudo-first-order (3) and
adsorbent pseudo second-order (4) were applied.
The adsorbent was prepared by (3)
mixing powder of NGS with 1.0 M sodium
hydroxide solution in the bottom and then it (4)
was autoclaved at 121 C, 15 psi for 15 min.
After that it was filtered and washed with 2.5 Adsorption isotherms
distilled water until neutral obtained. The The adsorption isotherm may
residual was dried at 80 C for 24 h. describe the probable mechanism of
Consequently, adsorbent which defined as adsorption. In present studied, the two
modified NGS (mNGS) was obtained. important isotherms of Langmuir and
Surface characteristics of the adsorbent were Freundlich were applied. The Langmuir
studied using scanning electron microscope isotherm is represented by the following
(SEM, Hitachi TM3030, Japan), while linear equation (5):
attenuated total reflectance fourier transform (5)
infrared (ATR-FTIR, Bruker tenser 27,
where qe and qmax are the equilibrium
Germany) analysis was done to demonstrate
adsorption capacity and maximum
the functional groups present on the surface
adsorption capacity per gram dry weight of
of the adsorbent.
adsorbent, respectively. Ce expressed in
2.2 Optimization of adsorption
mg/L, and KL is an equilibrium constant
In this study, the conditions were
(L/g). Adsorbents that follow the Freundlich
optimized by varying pH of the solution (3-
isotherm equation (6) are presented as
7), initial copper concentration (25-100
bellow.
mg/L), and adsorbent dosage (0.5-2.0 g).
The experiments were carried out via a (6)
batch adsorption process. After adsorption, where KF is constant (L/g) and n is a
the solution was filtered and remaining constant related to adsorption intensity.15
copper concentration in the solution was 2.6 Adsorption thermodynamics
analyzed by using a frame atomic absorption Adsorption thermodynamics
spectrophotometer (FAAS, AA-240FS, experiments were performed at the optimum
Varain, USA.) The percentage of removal condition obtained for various temperatures
and adsorption capacity (q, mg/g) were (25, 35, and 45 C). Thermodynamic
calculated by solving the following equation parameters were calculated by the following
(1) and (2), respectively: equations where ΔG (kJ/mol) is the Gibbs
% removal = (C0 − Ce) x 100 (1) free energy change, R (8.314 J/mol.K) is the
C0 universal gas constant, T (K) is the
q = (C0 − Ce) V (2) temperature, ΔHº (kJ/mol) is the enthalpy
W change and ΔSº (kJ/mol.K ) is the entropy
where C0 and Ce are the initial and change following equation (7) and (8).16
equilibrium copper concentrations (mg/L),
ln qe = ∆S - ∆H (7) it. mNGS with these characteristics
generally have high specific surface which
Ce R RT provides it a good adsorption capacity.
∆Gº = ∆Hº - T∆Sº (8)

3.1 The characteristics of adsorbent
ATR-FTIR spectrum of mNGS is
shown in Figure 1. Strong absorption band
appeared at 3340 cm-1 related to the OH-
streching vibration found in lignin units and
cellulose. Band at 2895 cm-1 assign to
stretching vibration of –CH2-, -CH3 in
alkane and aliphatic groups. Band around
the 1645, 1425, and 1367 cm-1 regions are
probable related to vibration of C-O
stretching found in carboxyl group, whereas
vibration within 1645 cm-1 region are linked Figure 2. SEM image of mNGS
to C=O type bonds presence in carbonyl and
carboxyl groups, which party constitute 3.2 The optimum conditions
lingo-cellulose compound.17 Band at 1512 The highest copper removal when
and 1425 cm-1 may be related to C=C type the initial pH of the solution was equal to 5
bonds of aromatic compounds. Band at 1262 as shown in Figure 3(a). For the reason, as
cm-1 assign to –COO vibration of acetyl pH increase, more metal binding sites could
group in hemicelluloses. Dominant peak at be exposed and carried more negative
1027 cm-1 was attributed to C-O type bond charge surface becomes available thus
provided by compounds such as lignin and facilitating grater copper removal. However,
phenol of the carboxyl groups, which are above pH 5, the percentage of removal was
known to make available adsorption sites for decreased because the occurrence of copper
metal ions.18,19 precipitation and interfered with the
accumulation or adsorbent deterioration.20
The result of the effect of initial
1.0
copper concentration on the adsorption
0.9
efficiency is shown in Figure 3(b). First an
increase in percentage of removal with
transmittance (%)
0.8
increase in metal ion concentration was
observed. This may be explained by the
0.7
presence of more copper ions in solution
0.6 available for binding onto the active sites of
the mNGS. Consequently, the adsorption
0.5
4000 3500 3000 2500 2000 1500 1000 500 reached to a saturation value at initial copper
wavenumber (cm-1) concentration of 50 mg/L, this can be due to
Figure 1. ATR-FTIR spectrum of mNGS the fact that the adsorbent has a limited
number of active sites and at higher
The SEM image in Figure 2 reveals concentrations the active sites became
the nature of surface of mNGS. It was saturated.21 The percentage removal of
evident that mNGS has a very rough and a copper increased with the increasing of
porous surface, which can be favorable for adsorbent dosage because the increase in
copper ion to diffuse and penetrate into the adsorbent surface area as shown in Figure
interior part of adsorbent and be trapped on 3(c). The maximum removal occurred at the
dosage of 1 g adsorbent. After this point,
further increase in adsorbent dosage lead to concentration and 1.0 g of adsorbent dosage.
no increase in percentage removal. This may Under these condition, it gave 97.02%
due to the overlapping of adsorption sites as removal with adsorption capacity of 1.15
a result of overcrowding of adsorbent mg/g.
particles or else to the screening effect of the 3.3 Adsorption kinetics
outer layer imposed by the high adsorbent
1.90
dosage, thereby shelding the active sites 1.88
R2 = 0.9378
y = -8.7461x + 1.9267
from copper ions.22 1.86
1.84
1.82
100 1.80
t/qt
1.78
95
1.76
90 1.74
85 1.72
(a)
Removal (%)
1.70
80 0.006 0.009 0.012 0.015 0.018 0.021
t
75
70 180 R2 = 0.9998
Y = 0.9710X - 5.0795
65 (a) 160
60 140
2 3 4 5 6 7 8
pH 120
t/qt
100
100
80
60
98
40
(b)
96 40 60 80 100 120 140 160 180
Removal (%)
94
Figure 4. Pseudo-first-order (a) and pseudo-
92 second-order (b) plots for adsorption of
(b) copper onto mNGS
90
0 25 50 75 100 125
Initial concentration (mg/L)
Table 1. Kinetic parameters for copper onto
mNGS adsorbent
100 Kinetic models Parameters
99 Pseudo-first-order R2 0.9378
98
K1 4.5392
97
qcal 0.5190
96
Pseudo-second-order R2 0.9998
Removal (%)
95
K2 0.1856
94
qcal 1.0299
93
92
qexp 1.1256
91 (c)
90
0.0 0.5 1.0 1.5 2.0 2.5
Based on the high coefficient (R2),
Adsorbent dosage (g) which close to 1. The adsorption kinetics
data were described well using the pseudo-
Figure 3. The effect of pH), (a) initial second-order model. Moreover, the
concentration (b) and adsorbent dosage (c) experimental qe values were very close to
on percentage removal the calculated qcal value for pseudo-second-
order model as shown in Figure 4 and Table
Therefore, based on the results as 2. The results suggest that the adsorption
mention above, the optimum condition of process between copper ion and the active
copper adsorption was obtained at pH 5 of sites of the adsorbent occur by chemical
solution, 50 mg/L of initial copper
adsorption, through valence forces and adsorption depends on the saturation level of
sharing of electrons.23-25 monolayer.3
3.4 Adsorption isotherms 3.5 Adsorption thermodynamics
0.92
R2 = 0.9844
The thermodynamic parameters were
0.91 y = 0.1005x + 0.7947 present in Table 3. The positive values of
0.90 ∆G showed that adsorbent system was non-
0.89
spontaneous in nature. Furthermore, the
0.88
decrease in the values of ∆G with increasing
1/qe
0.87
temperature showed that the adsorption
0.86
trend to spontaneous at higher temperatures.
The positive values of ∆H indicate an
0.85
0.84
0.5 0.6 0.7 0.8 0.9
1/Ce
1.0 1.1 1.2 1.3
endothermic nature of the process. On the
other hand, the solution interface during the
0.18
adsorption process. The positive value of ∆S
R2 = 0.9692
0.17 y = 0.0967x + 0.1008 displayed that the affinity of the adsorbent to
0.16
the copper and the degrees of freedom of the
0.15
0.14
adsorption process at the solid-liquid
interface increased.15,26
ln qe
0.13
0.12
0.11
0.10
Table 3. Thermodynamic parameters of
0.09 mNGS on the removal of copper at
0.08
-0.2 0.0 0.2 0.4 0.6 temperature range from 25 to 45 C
ln Ce
∆H ∆S ∆G
Figure 5. Lanmuir (a) and Freundlich (b) 25ºC 35ºC 45ºC
+28.2037 +0.0883 +3.3803 +1.0073 +0.1243
adsorption isotherms of copper onto mNGS
Table 2. Adsorption isotherm parameters 4. Conclusion

for each isotherm model used in the studies The characteristics of mNGS
Adsorption Isotherm demonstrated that it consist of cellulose
medels parameters hemicelluloses and lignin, which present
Langmuir R2 0.9844 hydroxyl, carbonyl, carbonyl groups and
qm (mg/g) 1.258 phenol that make possible the links with
KL (L/mg) 7.90 copper ion in solution. Moreover, due to the
RL 0.0025
Freundlich R2 0.9692 rough and porous structure of mNGS make
1/n 0.0967 it has high specific surface for adsorption of
KF (L/mg) 1.114 copper ion. The best condition for the
adsorption process are the solution’s pH at
According to the correlation 5.0, 50 mg/L of initial concentration and 1 g
coefficients and parameters which of adsorbent dosage. Under these conditions,
summarized in Figure 5 and Table 2. R2 of the maximum percentage removal of 97.02
Langmuir model was very close to 1 and with adsorption capacity of 1.15 mg/g were
larger than Freundlich model which suggests obtained. Adsorption kinetic indicated that
that the adsorption was a process which adsorption of the active sites of the
occurred in a homogeneous surface. The adsorbent occurs chemically, with the
adsorption based upon three assumptions, pseudo-second order model. Since Langmuir
sorption is limited to monolayer coverage, isotherm model had the best adjustment in
all surface sites are identical and can this study, it illustrated that the prevalence
accommodate only one adsorbed atom, and of chemisorptions by monolayers. The
interaction forces between adsorbed atoms positive value of ∆G and positive value of
are negligible. In addition, the maximum ∆H and ∆S indicated that the copper
adsorption process is non-spontaneous, 12. Patil, Y. B.; Paknikar, K. M.;
endothermic and increase in randomness Biotechnol. Lett. 1999, 21, 913–919.
occurs at solid-solution interface. In 13. Matyjaszewski, K.; Pintauer, T.;
conclusion, overall results showed that Gaynor, S. Macromolecules 2000, 33,
mNGS is an alternative and low-cost 1476–1478.
adsorbent for the removal of copper from 14. Thenmozhi, R.; Santhi, T. Research on
aqueous solution. Chemical Intermediates 2013, DOI
10.1007/s11164-013-1276-z.
Acknowledgements 15. Noha, A.; El, E.; Safa, M. A.; Hassan,
The author is grateful to A. F.; Abdelaziz, H. K.; Mohamed, E.;
Rajamangala University of Technology Isan Hesham, A. H. Ecotox. Environ. Safe.
(RMUTI), Khonkaen Campus, Khon Kaen, 2017, 145, 57–68.
Thailand for their supporting. I am also 16. Herbert, N.; Affonso, C. G. Jr.;
kindly acknowledged the Department of Marcelo, A. C.; Gustavo, F. C.; Daniel
Engineering, RMUTI for their foundation. S.; Marcelo, G. S.; Dionir, L. B. Jr.;
Juliano, Z. Open. Chem. 2016, 14, 103–
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The optimization of esterification reaction for biodiesel production derived
from high FFA non-edible oils
Nichaphat Sitthisuk1*, Charoenporn Lertsatitthanakorn1, Vittaya Punsuvon2,
Kaokanya Sudaprasert1, Rewadee Anuwattana3, Chanakan Puemchalad3
1
Division of Energy Technology, School of Energy, Environment and Materials,
King Mongkut’s University of Technology Thonburi, Bangkok 10140, Thailand
2
3
Energy Technology Department, Thailand Institute of Scientific and Technological Research,
Pathum Thani 12120, Thailand
*E-mail: sofaii@hotmail.com
Abstract:
The improvement of biodiesel production from non-edible vegetable oil is proposed
to deal with the biodiesel feedstock limitations and its qualities for a decade. This method
normally has some practical drawbacks about the quantity of undesired free fatty acid and the
identifying suitable reaction conditions for each difference raw materials. These problems
cause difficulty in obtaining maximum yield and best quality of biodiesel properties that
needs the support method to gain the best conditions. Therefore, Response surface
methodology with central composite design and artifitial neautral network, statistical analysis
technique for empirical modeling and optimization, is applied to solve such problem. In this
study, non-edible feedstocks, namely Jatropha curcas and Pongamia pinnata, is used as a
case study to optimize esterification step in biodiesel production. Three reaction conditions,
catalyst concentration (0.5–1.5 wt.% H2SO4), methanol/oil molar ratio (3:1–15:1) and
reaction time 90–150 min were appraised. Detailed case study shows that the undesired FFA
in both JCO and PPO are converted in esterification with appropriate conditions from RSM-
CCD. These optimum conditions gives the highest FFA conversion more than 93% for the
both biodiesel and the physicochemical properties that fulfill requirements in ASTM D6751
and EN14214 standard.
1. Introduction conversion to yield product is normally

Biodiesel is the popular alternative more than 95%.3,4
energy in this century due to many The effective condition factors that
advantages over petroleum-based diesel fuel affected to %FFA content reduction of TGs
such as renewable ability, environmental in esterification reaction were investigated
friendly and low price sources.1 Biodiesel through statistical model and optimization
can normally produced by transesterification technique of response surface methodology
of triglycerides (TGs). However, it need to (RSM) and artificial neural network
pretreat the free fatty acids (FFAs) of TGs (ANN).5,6
by using acid catalytic esterification process. In This work, RSM and ANN is used
The %FFAs content in this reaction step as the method to optimize three effective
should be less than 2% in order to convert factors (methanol to oil molar ratio, H2SO4
biodiesel by transesterification process. acid catalyst concentration and reaction
The Pongamia pinnata (PPO) and time) of PPO and JCO esterification process
Jatropha curcas (JCO) are one of the toxic which have high %FFA content. In addition,
and high FFAs in non-edible oil types.2 the models were compared by R-squared
Hence, the optimization procedure for and absolute average deviation (AAD).
reducing %FFAs content in PPO and JCO is
needed because the effect of the %FFA 2. Materials and Methods
2.1. Experimental setup Table 1. Comparison results of statistical
Crude PPO and JCO was obtained analysis for each modeling sources
from Ranong (province in Thailand). All Std. R-
Adjusted Predicted
chemicals in the experiments were analytical Source R- R- PRESS
Dev. Squared
Squared Squared
reagent (AR) grade. In this work, the
Linear 7.92 0.57 0.49 0.29 1663
experiment followed to the PPO biodiesel 2FI 8.57 0.59 0.41 -0.04 2432
production.5 Quadratic 6.84 0.80 0.62 -0.51 3545
initial - final Cubic 4.58 0.95 0.83 -10.81 27745
%FFA Conversion = 100 (1)
initial
where the initial and final acid value of relative to other model sources. However,
acidified oil of sample. (mg KOH/g oil). the large predicted R-Square and PRESS
The samples were repeated three times. quantity of cubic equation created much
2.2. Modeling of experiments more error than quadratic equation.
The predicted process using RSM Therefore, the quadratic equation is used as
was simple method as in Panpraneecharoen the model structure.
et al.5 In ANN model, the %FFA conversion The Design expert software is used
is predicted by using feed forward artificial to evaluate the coefficient of RSM model
neural network (FF-ANN) algorithm that with quadratic equation structure through
presented the input layer (3 neurons), hidden the 20 screening experiments (selection
layer (7 neurons for PPO, 4 neurons for based on Central Composite design (CCD))
JCO) and output layer (1 neuron). In this in this work. Equation 3 is the structure of
study, the hidden layer has a logistic quadratic equation which relates the %FFA
sigmoid transfer function and the output conversion to the effective factors in terms
layer has a nonlinear autoregressive with of actual process conditions.
exogenous function. The logistic sigmoid Y = 52.22996 + 37.44232A + 0.86302B -0.29938C
transfer and nonlinear autoregressive with - 0.70833AB + 0.275AC +6.9444x10 -4 BC
exogenous function are as follows:
yi ( xi )  ( xi  ri ) PL
- 24.89113A 2 - 9.888x10-4 B2 - 3.955x10-5C2
(3)
i i  d  ai f i (( xi  ri )Qb
i i  ci )
(2) where Y is %FFA conversion, and A, B and
C are actual values of catalyst concentration,
3. Results and Discussion methanol to oil molar ratio and reaction
3.1. RSM modeling and optimization of time, respectively. The positive sign of the
PPO esterification coefficients (A, B, AC, and BC) present a
As the %FFA conversion analysis of preferable effect on the increase of the
these experiments (variation of 3 effective %FFA conversion while the negative sign of
factors: catalyst concentration, methanol to the coefficients (C, AB, A2, B2 and C2) show
oil molar ratio and reaction time), the an invert effect on the increase of the %FFA
response surface model of RSM is suggested conversion.
in form of quadratic equation. The results of For estimating the values of %FFA
statistical analysis for each modeling conversion by using RSM model, the
sources are compared and given in Table 1. experimental results are found that RSM
The comparison results demonstrate model with the quadratic equation has much
that, in terms of all statistical indexes more mismatch prediction, which are 7.89%
relative to all modeling sources, quadratic of the average %AAD and 45.6% of the
equation are the best model structure for the maximum %AAD, caused by the quadratic
relational model of this study. The values of equation structure is poor performance for
standard deviation, R-Squared and Adjusted capturing the highly nonlinear relation.
R-Squared show that cubic equation and However, the statistical index (R-squared)
quadratic equation perform noticeably better indicate that the model is acceptable (R-
squared value is equal to 0.80).
In this study, the predicted optimum
conditions of the reaction by using RSM
model is observed by the 3D surface plot
and contour plot of %FFA conversion as
shown in Figure 1-3. They are 1.5 %wt. of
the catalyst concentration, 11.41:1 of the
methanol to oil molar ratio, and 150 minutes
of the reaction time.
Table 2. Anova analysis

Source of Sum of F- Significant
df P-value
variation squares value 90%CI
model 1882.2 9 4.5 0.014 Yes
A 659.7 1 14.1 0.004 Yes
B 210.9 1 4.5 0.060 Yes
C 475.5 1 10.2 0.010 Yes
AB 47.3 1 1.0 0.338 No
AC 0.5 1 0.01 0.918 No
BC 1.3 1 0.03 0.871 No
A2 365.4 1 7.8 0.019 Yes
B2 3.7 1 0.08 0.785 No
C2 164.6 1 3.5 0.090 Yes
Residual 467.5 10 - - - Figure 2. The 3D surface plot and contour
Lack of plot of %FFA conversion as the function of
467.5 5 2x105 1x10-4 Yes
fit methanol to oil molar ratio and catalyst
Pure error 0.003 5 - - - concentration at reaction time 150 min
Figure 1. The 3D surface plot and contour Figure 3. The 3D surface plot and contour
plot of %FFA conversion as the function of plot of %FFA conversion as the function of
reaction time and catalyst concentration at methanol to oil molar ratio and reaction time
the methanol to oil molar ratio 11.41:1 at catalyst concentration 1.5 %wt
Based on the plots and Table 2, the valid to use in the prediction (the average
effect of each term on the %FFA conversion %AAD = 0.14).
can be observed. The most significant effect
is the catalyst concentration (highest slope
of curve in Figure 1 and 2 and highest F-
value in Table 2), followed by the methanol
to oil molar ratio (See in Figure 2 and 3) and
had slightly invert effect from reaction time
while the large reaction time (See in Figure
1 and 3). Therefore, the plotting results are
supportive of the interaction term of the
model that A and B terms are preferable
effect on the increase of %FFA conversion
while C term is invert effect on its.
3.2. ANN modeling and optimization of
PPO esterification
In ANN model, the predicted model
is highly sensitive to the experimental data
due to its modeling structure. Therefore, the
experimental data sets are need to set into
two subsets as training (70% of the
experimental data sets) and validation
Figure 4. The 3D surface plot and contour
(30% of the experimental data sets). In the
plot of %FFA conversion as the function of
training data set, these data are presented to
reaction time and catalyst concentration at
the model during training step, and the
the methanol to oil molar ratio 15:1
model is adjusted model parameters
following to its error. In the validating
subset, these data are no effect on training
and used to measure the model mismatch. In
this work, training subset is set to 20
different cases (based on run cases of RSM
model) and validating subset is added 9
experimental data (based on the
experimental data point near optimum
conditions).
As mention in the section 2.2, the
coefficients of ANN model (in Equation 2)
are calculated by using the training data sets.
The predicted results have much more
accuracy in prediction, which are 0.008% of
the average %AAD and 0.06% of the
maximum %AAD, caused by the nonlinear
weighting structure of ANN model is high
performance for capturing the highly
nonlinear relation.
As the sensitivity of ANN model, the
Figure 5. The 3D surface plot and contour
validating data are used to re-test the model
plot of %FFA conversion as the function of
performance. The low %AAD of all the
methanol to oil molar ratio and catalyst
validating data indicate that the model is
concentration at reaction time 127.71 min
In order to find the optimum Table 3. Statistical analysis for each
condition of the reaction by using ANN modeling sources
model, the 3D surface plot and contour plot Std. R-
Adjusted Predicted
of %FFA conversion is observed as shown Source R- R- PRESS
Dev. Squared
Squared Squared
in Figure 4-6. The optimum condition,
Linear 2.73 0.62 0.55 0.30 217
obtained from the ANN model, are 1.05% 2FI 2.78 0.68 0.53 0.10 281
wt. of the catalyst concentration, 15:1 of the Quadratic 1.97 0.88 0.76 0.07 291
methanol to oil molar ratio, and 127.71 Cubic 1.06 0.98 0.93 -2.90 1218
minutes of the reaction time.
Based on the 3D surface plot and The comparison results demonstrate
contour plot, the effect of each term on the that quadratic equation are the best model
%FFA conversion is shown in Figure 4-6 structure for the relational model of this
that are the same trend as in RSM model study. The values of predicted R-Square and
results (See in Figure 1-3). The catalyst PRESS show that cubic equation created
concentration is the most effective factor. much more error in the prediction.
Then, the methanol to oil molar ratio is Therefore, cubic equation should
followed (See in Figure 5 and 6). Reaction aliased. Although, the standard deviation, R-
time has also slightly invert effect while the Squared and Adjusted R-Squared of three
large reaction time (See in Figure 4 and 6). other modeling source are slightly the same
value, the quadratic equation perform
noticeably better relative to other model
sources. So, the quadratic equation is used
as the model structure.
The Design expert software is used
to evaluate the coefficient of RSM model
with quadratic equation structure through
the 20 screening experiments (as the same
method as PPO esterification). Equation 4 is
the structure of quadratic equation which
relates the %FFA conversion to the effective
factors in terms of actual process conditions.
Y = 117.03008 - 33.09132A + 2.44484B -0.89836C
+ 0.46250AB + 0.14917AC +0.016972BC
+ 4.19247A 2 - 0.17621B2 + 2.152x10 -3C2 (4)
where Y is %FFA conversion, and A, B and
C are actual values of catalyst concentration,
methanol to oil molar ratio and reaction
time, respectively.
Figure 6. The 3D surface plot and contour For predicting %FFA conversion by
plot of %FFA conversion as the function of using RSM model with the quadratic
methanol to oil molar ratio and reaction time equation, it created large error in the
at catalyst concentration 1.05 %wt prediction, which are 1.75% of the average
%AAD and 6.85% of the maximum %AAD,
3.3. RSM modeling and optimization of caused by the poor performance of quadratic
JCO esterification equation as mention in previous section of
As the modeling source selection of PPO esterification. However, the statistical
RSM, quadratic equation is selected as the index (R-squared) indicate that the model is
model structure due to the results in Table 3. acceptable (R-squared value up to 0.8).
As the optimization method of RSM performance and trend of optimization than
model, the optimum condition of JCO the RSM (Compare Figure 1-2 with Figure
esterification are 1.75 %wt. of the catalyst 4-5). This may cause by the non-linearity of
concentration, 14.82:1 of the methanol to oil ANN. The evaluated optimum conditions of
molar ratio, and 149.79 minutes of the both model are slightly the same as shown in
reaction time. Table 4.
3.4. ANN modeling and optimization of
JCO esterification 4. Conclusion
The results of ANN model prediction Three operation factors are focused
that has more accuracy than the prediction as the case study to reduce the amount of
of RSM, which can observe from 0.18% of FFA in the PPO and JCO sources. RSM and
the average %AAD and 1.18% of the ANN are compared to find the optimum
maximum %AAD. The performance for condition. The results of RSM and ANN
capturing the highly nonlinear is indicated model give slightly the same optimum
by R-squared that equal to 0.9963. conditions. Although, both models can
As the validation step, the low search in the optimum conditions, the
%AAD of all the validating data indicate optimum condition results of ANN model is
that the model is acceptable to use for more accurate than the RSM model.
estimate %FFA conversion of JCO However, the ANN model has a drawback in
esterification (the average %AAD = 0.11). the modeling formulation that need a lot of
As the optimization method of ANN data for training and validating the model in
model, the optimum condition of JCO order to gain high performance in
esterification are 1.75 %wt. of the catalyst prediction.
concentration, 15:1 of the methanol to oil
molar ratio, and 149.89 minutes of the Acknowledgements
reaction time. This work was supported by The
3.5. Performance evaluation of RSM and Thailand Institute of Scientific and
ANN modeling Technological Research (TISTR) and
In case study of PPO and JCO Department of Chemistry, Faculty of
esterification, the model performance of Science, Kasetsart University.
both models are compared by observed
%FFA conversion and predicted %FFA References
conversion. The average %AAD values for 1. Ong, H. C.; Silitonga, A. S.; Masjuki,
RSM and ANN are [7.89 (RSM-PPO), 1.75 H. H.; Mahlia, T. M. I.; Chong, W. T.;
(RSM-JCO)] and [0.008 (ANN-PPO), 0.18 Boosroh, M. H. E. Conv. M. 2013, 73,
(ANN-JCO)], respectively. These results 245–255.
indicate that the ANN model has a lower 2. Dharmaa, S.; Masjukia, H. H.; Ong, H.
deviation than the RSM and both techniques C.; Sebayanga, A. H.; Silitonga, A. S.;
are suitable for this experiment. However, Kusumoa, F.; Mahliac, T. M. I. E. Conv.
the ANN model presents better predictive M. 2016, 115, 178–190.
3. Patil, D. P.; Deng, S. Fuel 2009, 88,
Table 4. The comparison of the optimum 1302–1306.
condition in each model 4. Verma, P.; Sharma, M. P. Fuel 2016,
Oil A C % 180, 164–174.
Model B FFA
source (%wt) min conversion 5. Panpraneecharoen, S.; Punsuvon, V.;
PPO RSM 1.50 11.41:1 150.00 73.04 Puemchalad, C. Asia. J. Chem. 2015, 27,
ANN 1.05 15.00:1 127.71 79.75
JCO RSM 1.75 14.82:1 149.79 71.98 1023–1027.
ANN 1.75 15.00:1 149.89 71.53 6. Ma, L.; Lv, E.; Du, L.; Ding, J. E. Conv.
M. 2016, 122, 411–418.
Production of biofuels in Fischer-Tropsch synthesis using
mono- and bi-metallic Fe-based catalysts
Napaporn Chancharoenlap, Borisut Chantrawongphaisal*, Patthanant Natpinit,
Phavit Thapnui, Phongsak Hongcharoensri, Phichai Wongharn,
Chotika Kongsomboon, Manoo Boonsae
Expert Centre of Innovation Clean Energy and Environment, Thailand Institute of Scientific and Technological
Research (TISTR), Pathum Thani 12120, Thailand
*E-mail: napaporn_c@tistr.or.th
Abstract:
This investigation researched on the production of biofuels using Fischer–Tropsch
synthesis (FTS). The FTS was conducted in a continuous flow type fixed bed reactor under
pressure 15 bar, temperature 240 C and reaction time 6 h. The 2 types of metal catalyst,
Fe/Al2O3 (mono-metallic Fe-based catalyst) and Fe/Cu/Mn/K with Co/Al2O3 (bi-metallic Fe-
based catalyst) were studied to form liquid hydrocarbon in the same reactor. It was found that
80 g of Fe-based catalyst could convert CO and H2 to CH4 as 74.16% conversion. In addition,
bimetallic catalysts (Co/Al2O3-Fe-based catalyst) could form liquid hydrocarbon compounds
which were as paraffin. The highest of %selectivity was 34.76 in which C11-C14 as
kerosene. Moreover, the liquid hydrocarbon contained 53.56% of carbon, hydrogen 11.33%,
and the heating value was 9,944.54 kcal/kg.
1. Introduction Alcohol reaction:

At the present, the growth of world nCO + 2nH2 → CnH2n + 2O + (n-1)H2O
energy consumption rises continuously. The Water Gas Shift:
liquid fuel from fossil resource such as oil, CO + H2O → CO2 + H2
remained the world’s leading fuel which Boudouard reaction:
effects to natural environment such as global 2CO → C + CO2
climate change and air quality due to CO2 Coke deposition:
gas releasing from combustion of fossil CO + H2→ C + H2O
fuel,1 Therefore, one of the attractive The FTS is an exothermic reaction
renewable energy resources is biomass, it occurring over the surface of the metal
does not increase CO2 to atmosphere.2,3 The catalyst. The product selectivity mainly
using of biomass for production of depends on the catalyst and operating
sustainable liquid fuels is so-called biomass condition such as temperature and pressure.5
to liquid process (BTL). For the BTL For the metal catalyst in FTS process, Fe
process, biomass convert to syngas which and Co are used extensively however, these
composed of carbon monoxide (CO) and catalysts are own advantages. For example,
hydrogen (H2) via gasification technologies, Fe-based catalysts have been shown to be
then syngas can be directly synthesis active for the formation of hydrocarbons at
hydrocarbons as liquefied fuels such as higher temperature reaction but the cost is
biodiesel or biojet through the Fischer- generally lower. While, Co-base catalysts
Tropsch synthesis (FTS). The major FTS are active at lower temperatures than Fe and
reactions occur given below;4 Co tend to produce light hydrocarbons,
Paraffin reaction: which is particularly promoted with
nCO + (2n+1)H2→ CnH2n+2 + nH2O manganese oxide.6-8 Kumabe and
6
Olefin reaction: co-workers researched the production of
nCO+ 2nH2 → CnH2n + nH2O hydrocarbon equivalent to kerosene in FTS
with Fe-based catalysts with other species, The FTS was conducted in a
the selectivity of CO to produce C11-C14 continuous flow fixed bed reactor under
hydrocarbons was found to be the second pressure 15 bar and temperature 240 C,
highest, while the highest selectivity was using the synthesized mono- and bi-metallic
C20+ hydrocarbons. In Addition, an Fe-based as catalysts.
increasing the dispersion and stability of 2.1 Catalyst preparation
metal for better catalytic activity was used a The single metal catalyst, Iron (Fe)
supporter as shown in several researches. metal as an active catalyst on supported
Iqbal and co-workers7 compared between Alumina balls (Fe/Al2O3) was synthesized
carbon-supported CoMnOx catalysts and an by impregnation method. The Fe-based
unsupported CoMnOx catalyst under the catalyst prepared from Fe(NO3)2∙6H2O
same operating conditions. The result was which it was dissolved in H2O. Then Al2O3
shown that the use of supporter increased balls (size 4-8 mm.) were impregnated onto
both the catalyst activity and the selectivity Fe solution at 70 C for 8 h. After that, the
to produce C2+ hydrocarbons while catalyst was dried at 80 C for 6 h, and
lowering the selectivity to CH4 and CO2. calcined at 800 C for 3 h.
Bukur and Sivaraj8 found that alumina- For bi-metallic Fe-based catalysts
supported iron catalyst had higher olefin (Co/Al2O3-Fe-based catalyst), Cobalt (Co)
content than silica-supported iron catalyst. was coated on aluminium support
Zhang and co-workers9 showed the addition (Co/Al2O3) via impregnation method while
of Cu as promoter into Fe-based catalyst Fe-based catalyst was synthesized by co-
could improve the catalyst activation rate precipitation method with Fe, Mn, Cu, and
and shortened the induction period, but no K. Fe, Mn and Cu were mixed in the
apparent influence on the steady-state solution with the ratio 100:10:1 respectively.
activity of the catalyst. Apart from the Then mixed solution was adjusted pH at 8.0
catalyst, the operating condition was an to form gel. After that, filtered the
important role in FTS process. Farias and precipitate washed distilled water. Dried at
co-workers10 studied the FTS liquid 120 C for 12 h. Then impregnated 2% of K
hydrocarbon product distribution on into the catalyst, dried at 120 C for 3 h and
operating pressure and temperature over
calcined at 400 C for 3 h.
three different molar of K-promoted Fe
2.2 Fischer–Tropsch synthesis (FTS)
catalysts. The results showed that high
pressures favored the production of waxes
while greater direct selectivity towards
diesel was favored by low pressure.
Moreover, using a Fe catalyst with high K
content presented a higher number-average
number of carbons. Savost'yanov and co-
workers11 showed the positive effect in
terms of increasing reaction rate and
improving of C5+ selectivity when the
pressure was increased in a gas recycle
mode with using Co-Al2O3/SiO2 as the
catalyst. Therefore, production of biofuels in
Figure 1. The schematic of an experiment
FTS between using mono- and bi-metallic
Fe-based catalysts were studied in this
The schematic of an experiment was
research.
shown in Figure 1. The prepared catalyst
was placed in the continuous flow fixed bed
2. Materials and Methods reactor. The reduction of Fe and metal
species at 300 C for 6 h with H2 gas. The
FTS reaction was carried out using syngas Water content occurred depending on
H2: CO with ratio 1:1 under pressure 15 bar catalyst amount, flow ratio syngas and
and temperature 240 C. The gas sample pressure.15
was collected in the gas bag and analyzed by For the other reason, the chain
gas chromatography (GC, Shimadzu 2014) reaction of 2 steps as followed.16
and the liquid products was collected and CO + H2 → -(CH2)- + H2O (3)
analyzed ultimate analysis by elemental CO + H2O → CO2 + H2 (4)
analysis (TruSpecCHN) and their The generated H2 that occurred in
composition by gas chromatography with Equation (4) was reacted with CO to form
mass spectrometry (GC/MS, Agilent msd hydrocarbon continuously. In this
5975). The heating value was determined experiment, hydrocarbon production was
using a bomb calorimeter (Parr 6300 only CH4 gas. It seemed that mono-catalyst
calorimeter). was less selectivity to form linear product or
-(CH2)- monomers and low activity to react
3. Results and Discussion water gas shift (WGS) process. In order to
3.1 Mono metallic Fe-based catalysts improve the activity of Fe/Al2O3 catalyst,
The reaction based on the flow ratio other transition metal such as Cu and Mn
of H2 and CO at 250:60 ml/min, pressure 15 could be promoted to form co-catalyst with
bar, temperature 300 C and retention time Fe /Al2O3.
6 h. The result for Fe-based catalyst was 3.2 Bi-metallic Fe-based catalysts
shown in Table 1. The metal species could increase the
It showed that 80 g of Fe/Al2O3 catalyst activity and stability of CO
could produce higher CH4 and CO2 than activated and selectivity of hydrocarbon.17
others. Besides, the reaction could generate Fe/Al2O3 catalyst or mono-catalyst had
water also. The highest CO conversion to lower syngas conversion, so K promoter was
CH4 and CO2 of 80 g Fe/Al2O3 catalyst was studied to improve the effective
18
74.16% and the productivity of CH4 and hydrocarbon selectivity. Cu promoted in
CO2 were 6.11% and 1.04% respectively. Fe-based catalyst increased active sites and
The reaction of Fe/Al2O3 with H2 and CO rate reduction of iron.19 Co-based catalyst
occurs in 2 steps as below.12 was favorite to enhance the catalytic activity
3Fe2O3 + H2 → Fe3O4 + H2O (1) and stability to form hydrocarbon.20 Al2O3
Fe3O4 + CO → FexC + CO2 (2) had capability for alcohol dehydration to
In the first step, Fe2O3 was reduced hydrocarbons.21,22 Therefore, Fe-based
with H2 to Fe3O4 rapidly, then slowly catalyst with Cu/Mn/K promoter was used in
reacted with CO to be formed FexC this study with Co/Al2O3 catalyst. The
(carbide).13 After that, FexC released carbon reaction was carried out using H2 and CO at
to be form hydrocarbon compounds. In the 40:40 ml/min, pressure 15 bar, temperature
process, carbon that adsorbed on the surface 240 C, retention time 24 h and placed 20 g
of Fe/Al2O3 reacted with H2 producing CH4 of Fe/Cu/Mn/K with 60 g of Co/Al2O3
gas as the first -(CH2)- monomer.14 catalyst.
Table 1. CO conversion to CH4 and CO2 with Fe/Al2O3 catalyst with different grams of
catalyst
Fe/Al2O3 COin CH4 CO2 %CO
Catalyst conversiona
%V/V %C-CO %V/V %C-CH4 %V/V %C-CO2
40 g 18.31 7.85 3.75 2.83 3.16 0.86 47.09
80 g 17.68 7.58 6.11 4.58 1.04 1.03 74.16
120 g 16.44 7.05 4.11 3.08 3.03 0.83 55.74
a
%CO conversion = (% C-CH4+%C-CO2)pdt/ %C-COin
Table 2. CO conversion to CH4 and CO2 with Co/Al2O3-Fe/Cu/Mn/K catalyst
Co/Al2O3- %CO
COin CH4 CO2
Fe/Cu/Mn/K
conversiona
catalyst %V/V %C-CO %V/V %C-CH4 %V/V %C-CO2
1 63.14 27.06 9.19 6.89 40.97 11.17 66.76
2 59.50 25.50 8.02 6.01 40.73 11.11 67.13
3 62.56 26.81 8.17 6.12 43.61 11.89 67.21
4 59.93 25.68 8.67 6.51 45.86 12.51 74.02
5 60.73 26.03 8.85 6.63 43.58 11.88 71.15
6 57.93 24.83 9.54 7.16 42.38 11.56 75.37
a
%CO conversion =( % C-CH4+%C-CO2)pdt/ %C-COin
From the Table 2, it showed that the

average of CO conversion to CH4 and CO2
of as Co/Al2O3-Fe/Cu/Mn/K catalyst was
70.23% (min : max; 66.76% : 75.37%) and
the productivity of CH4 and CO2 were
8.74% (min : max; 8.02% : 9.54%) and
48.86% (min : max; 40.73% : 45.86%)
respectively. Moreover, the use of
Co/Al2O3-Fe/Cu/Mn/K catalyst could
produce 11 mL of liquid product which was
separated into 3 ml of H2O and 8 mL of
liquid hydrocarbon. The liquid hydrocarbon
composed of carbon 53.56%wt, hydrogen
Figure 2. Hydrocarbon selectivity with
11.33%wt, and oxygen 35.11%wt as well as
carbon no. of Co/Al2O3-Fe-based catalyst
the heating value was 9,944.54 kcal/kg as
shown in Table 3.
Table 4. Hydrocarbon selectivity of
Co/Al2O3 - Fe/Cu/Mn/K catalyst
Table 3. Properties of liquid HC product
%Selectivity
Properties Liquid HC product
Element composition (%wt) Paraffins
C 53.56 C8-C10 20.25
H 11.33 C11-C14 34.76
O 35.11 C15-C20 21.50
Heating Value (kcal/kg) 9,944.54 Total 76.52
Olefins
In addition, the results from GC/MS C8-C10 7.45
C11-C14 5.40
was shown in Table 4 and Figure 2. In this C15-C20 -
experiment, the carbon number in liquid Total 12.85
hydrocarbon was divided to C8-C10, C11- Others
C14 and C15-C20 equivalent as gasoline, C8-C10 8.60
kerosene and diesel, respectively. C11-C14 1.75
It was found that the liquid C15-C20 0.27
Total 10.63
hydrocarbon had the carbon number in range
of C8-C20. It could classify that
In this process, H2 gas reduced the
%selectivity of liquid hydrocarbon were
catalyst which Fe2O3→Fe3O4 (as shown in
paraffin, olefin and the others (cyclic and
Equation (1)) and Co3O4 →Co0 (as shown in
aromatic) as 76.52%, 12.85% and 10.63
Equation (5))
respectively. Moreover, the range of C11-
Co3O4 + 4H2 → 3Co + 3H2O (5)
C14 had the highest of %selectivity so that
However, H2 could reduce Fe species
the liquid hydrocarbon was kerosene.
better than Co species due to strongly
interacting between Co species and References
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which formed -(CH2)- monomer,14 and lead
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7. Iqbala, S.; Daviesb, T. E.; Morgana, D.
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liquid hydrocarbon product from this
Bartleya, J. K.; Taylora, S.H.;
research were containing normal paraffin
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the ratio of carbon monoxide and hydrogen V. G.; Soromotin, V. N.; Mitchenko, S.
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Oxidative dehydrogenation of ethanol to acetaldehyde over
AgLi/SSP catalyst
Narawich Mukda, Jakrapan Janlamool, Bunjerd Jongsomjit*
Center of Excellence on Catalysis and Catalytic Reaction Engineering Department of Chemical Engineering,
*E-mail: bunjerd.J@chula.ac.th
Abstract:
This present study focuses on investigation of the oxidative dehydrogenation of
ethanol over AgLi/SSP catalyst to produce acetaldehyde. First, the spherical silica particle
(SSP) as the support was synthesized using the modified sol-gel method. The SSP support
was impregnated with silver (Ag) and lithium (Li) precursors using the incipient wetness
method in order to have a AgLi loading of 5wt%. The obtained catalyst was calcined at 400
°C for 4 hours. After calcination, the catalyst was characterized using various techniques such
as X-ray powder diffraction (XRD), nitrogen physisorption (BET & BJH methods), scanning
electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX), and UV-
visible spectroscopy. The oxidative dehydrogenation of vaporized ethanol was performed to
test the catalytic activity and product distribution. It was conducted in a fixed-bed continuous
flow microreactor made from a borosilicate glass with an inside diameter of 0.7 cm and
length of 33 cm. The reaction temperatures were ranged from 200 to 400 °C. It was found
that the AgLi/SSP catalyst exhibited high activity and high selectivity of acetaldehyde in
oxidative dehydrogenation reaction of ethanol. The AgLi/SSP catalyst showed the highest
yield of acetaldehyde (47.74%) and conversion of ethanol (89.12%) at 350 oC.
1. Introduction support such as their pore size distribution,

Nowadays, acetaldehyde is mostly thermal stability, and high surface area.
used in petrochemical industry for the In this research, the oxidative
preparation of pyridines, acetic acid, dehydrogenation of ethanol over AgLi/SSP
ethylidene diacetate, vinyl acetate, and catalyst to produce acetaldehyde will be
useful resin. In addition, the demand for investigated. Firstly, the spherical silica
acetaldehyde is in an increasing trend during particle will be synthesized by sol-gel
2017-2022.1 Acetaldehyde can be produced methods. Then the AgLi/SSP catalyst will
from dehydrogenation reaction and be prepared by using incipient wetness
oxidative dehydrogenation reaction. The impregnation technique. Secondly, the
difference between these reactions is the characteristics of the catalyst will be
catalyst that was used for each reaction.2 measure by various techniques such as BET,
The Ag-based catalyst has been used SEM/EDX, and UV-visible spectroscopy.
on various supported and give the high Lastly, oxidative dehydrogenation of ethanol
activity in the dehydrogenation of alcohols.3 will be performed to test the catalytic
Silica-supported catalyst is the most popular activity and product distribution.
to use as the support for the silver catalyst in
the reaction of oxidative dehydrogenation 2. Material and Methods
because its activity that is high even the 2.1 General
content of the metal is low. Spherical silica Tetraethyl orthosilicate,
particle (SSP) is one type of silica that has cetrimonium bromide, and ammonium
the appropriate properties to be a great solution reagent grade from Sigma Aldrich
were used as component of SSP
synthesizing. Silver nitrate and lithium 2.4 Oxidative dehydrogenation of ethanol
nitrate reagent grade from Sigma Aldrich The activity and product distribution
were used as silver and lithium precursors, from the oxidative dehydrogenation reaction
respectively. of vaporized ethanol were tested by
2.2 Catalyst preparation conducting in a fixed-bed continuous flow
The spherical silica particle (SSP) microreactor made from a borosilicate glass
supports were synthesized by using sol-gel with an inside diameter of 0.7 cm and length
method. The TEOS, CTAB, NH3, ethanol, of 33 cm. The reaction temperatures were
and H2O with the molar ratio of 1 TEOS: 0.3 ranged from 200 to 400 °C. In this work, the
CTAB: 11 NH3: 58 Ethanol: 144 H2O were amount of catalyst that was used at a time is
mixed and stirred at the room temperature about 0.05 g. The catalyst was reduced in
for 2 hours. Then, the precipitate were flowing H2 at 300 °C for 1 hour before it
washed with DI water and filtrated. After was used.
that, the precipitate were dried at 110 °C for
12 hours and calcined with air flow at 550 3. Results and Discussion
°C for 6 hours. 3.1 Catalyst characterization
The AgLi/SSP catalyst was prepared The XRD patterns of SSP support
by incipient wetness impregnation method. and AgLi/SSP catalyst are shown in Figure
The SSP supports were impregnated with 1. The measurements were carried out at
the mixture of AgNO3 and LiNO3 solution then diffraction angles (2θ) between 15º and
in the appropriate concentration to obtain 80°.
5wt% of AgLi. Then, the catalysts were
dried at 110 °C overnight and calcined in the
air at 400 °C for 4 hours.
2.3 Characterization techniques
X-ray diffraction (XRD): Bruker D8
Advance X-ray diffractometer with Cu-Kα
radiation (λ = 1.54056 Å) was used to
determine the phase composition of catalysts
The BET surface area, pore volume and pore
size were determined by physisorption of N2
using a BET and BJH methods. UV–visible
absorption spectroscopy: the oxidation state Figure 1. XRD patterns of SSP and
of Ag was determined using UV–visible AgLi/SSP
absorption spectroscopy (Perkin Elmer
Lamda-650, wavelength of 200–800 nm It can be observed that SSP and
with a step size at 1 nm). Scanning electron AgLi/SSP has the same XRD peaks at 24,
microscopy (SEM) and energy dispersive X- 31, 35, and 43°. The results indicate that Ag
ray spectroscopy (EDX): SEM and EDX and Li particles possess a good dispersion
were used to investigate the morphology and on the SSP support with very small size. The
elemental distribution at the surface of the metals particles were too small to be
samples by using Hitashi mode S-3400 N. detected by XRD.
Micrographs were taken at the accelerating The textural properties and nitrogen
voltage of 30 kV and magnification ranging adsorption/desorption isotherms of the
from 1,000 to 10,000 and the resolution of 3 catalysts are summarized in Table 1 and
nm. The SEM was operated by using the Figure 2. According to the IUPAC
secondary scattering electron (SE) mode classification, after silver and lithium were
EDX was performed by using Apollo X loaded into the support, the type of isotherm
Silicon Drift Detector Series by EDAX. was changed into type IV. This means the
Table1. BET surface area, pore volume and pore diameter of samples
sample BET surface area Pore volumea Pore diametera
(m2/g) (cm3/g) (nm)
SSP 1023.05 0.90 20.30
AgLi/SSP 163.78 0.33 56.15
a
Determined from the Barret-Joyner-Halenda (BJH) desorption method
catalyst is mesopores and broad pore size

distribution. The observation is consistent
with the value of surface area, pore volume,
and pore diameter as shown in Table 1, the
surface area of the catalyst decreased from
1023.05 to 163.78 m2/g. Also the pore
volume of SSP and AgLi/SSP that decreased
from 0.9 to 0.33 cm3/g. The decreasing of
surface area of the catalyst was due to the
silver and lithium species blocked the pore
after they were loaded. The UV-visible
spectroscopy was operated for SSP and
AgLi/SSP catalyst as shown in Figure 3. Figure 2. Nitrogen adsorption/desorption
After the AgLi was loaded into the SSP, isotherms of SSP and AgLi/SSP
there are the absorption bands occurred at
210, 310, and 430 nm. Pestryakov and
Davydov4 reported that the bands around
210 is the charge transfer bands of Ag+. The
210 band of AgLi/SSP is the tallest band
indicates that a large amount of Ag+ species
are presented.
Table 2. The amount of each element of

SSP and AgLi/SSP obtained from EDX
Sample Element (Wt%) Figure 3. UV-visible spectra of SSP and
Si O Ag AgLi/SSP
SSP 60.26 39.74 -
AgLi/SSP 50.34 43.58 6.08 The density of Si and O was strongly
observed because silica is the main
The morphology of the SSP and component of AgLi/SSP catalyst and the
AgLi/SSP catalyst were determined by using good distribution of Ag was also observed.
SEM as shown in Figure 4. The morphology This confirmed the XRD result that the Ag
of SSP support was agglomeration of particles are good dispersion on the SSP.
particles and having the average size of Moreover, the quantity of each element can
particles around 5 microns. be detected by EDX as shown in Table 2. It
After AgLi was loaded into SSP can be seen that the Si and O were the main
support, it can be observed that the species (Si = 50.34%, O = 43.58%) of the
morphology does not change. The dispersive catalyst while a few of Ag nanoparticles
X-ray spectroscopy (EDX) is used to were located on the surface of catalyst (Ag =
determine the element distribution. The 6.08%).
location of the specific element such as Ag, 3.2 Ethanol oxidative dehydrogenation
Si, O can be detected by using EDX reaction
mapping mode as shown in Figure 5. In order to measure the catalytic activity
Figure 4. The SEM micrographs of SSP and AgLi/SSP catalyst
Figure 5. EDX mapping of AgLi/SSP catalyst
and product distribution, ethanol oxidative SSP catalyst gives the higher ethanol
dehydrogenation reaction was operated for conversion than the SSP catalyst. It can be
SSP support and AgLi/SSP catalyst at observed that the catalytic properties of
atmospheric pressure and the temperature at AgLi/SSP catalyst promoted conversion of
the range of 200-400 °C. ethanol into acetaldehyde. Besides, the
acetaldehyde selectivity was decreased due
to there are many the side reactions that
produce the by-product such as diethylether
(DEE), ethylene and when the oxygen was
used in the dehydrogenation reaction the CO
Figure 6. Ethanol conversion percentage

and acetaldehyde selectivity percentage at
different temperature of SSP and AgLi/SSP
catalyst
The results of SSP support and

AgLi/SSP catalyst were shown in Figure 6.
The ethanolconversion for both SSP and
AgLi/SSP catalyst was increased with Figure 7. Acetaldehyde yield percentage at
increasing reaction temperature. The AgLi/ different temperature of SSP and AgLi/SSP
catalyst
and CO2 were produced as the by-product at the SSP catalyst. This can be concluded that
high temperature. To confirm that there are the presence of silver-lithium improved the
the catalytic properties to change the ethanol conversion and acetaldehyde yield
reactant into the desired product on the of the SSP catalyst.
silver-lithium as the active metal,
acetaldehyde yield percentage of SSP and Acknowledgements
AgLi/SSP was shown in Figure 7. The The authors thank the Grant for
acetaldehyde yield percentage of AgLi/SSP International Research Integration: Chula
catalyst was surely higher than SSP support Research Scholar, Ratchadaphiseksomphot
and the highest yield percentage was 47.74 Endowment Fund for financial support of
% at the temperature of 350 °C while the this project.
highest acetaldehyde yield percentage when
the SSP is used as the catalyst was 16.75 % References
at the temperature of 400 °C. 1. https://www.mordorintelligence.com/ind
ustry-reports/acetaldehyde-market
4. Conclusion 2. Jumming, S.; Yong, W. ACS Catal.
The oxidative dehydrogenation of 2014, 4, 1078−1090.
ethanol into acetaldehyde in the range 3. Vitaly, L.; Irina, I.; Esben, T.
temperature of 200-400 °C over the SSP and ChemCatChem. 2013, 5, 1−8.
AgLi/SSP catalysts was investigated. The 4. Pestryakov, A. N.; Davydov, A. A. Appl.
AgLi/SSP catalyst exhibited higher Catal., A 1994, 120, 7−15.
ethanolconversion (89.12%) and
acetaldehyde yield (47.74%) at 350 °C than
Conductivity of Nb-, V- doped Sr2FeTiO6- materials for
solid oxide fuel cells
Ausa Potong, Soamwadee Chaianansutcharit*
Department of Chemistry, Faculty of Science, Chulalongkorn University and
Center of Excellence on Petrochemical and Materials Technology,
*E-mail: ssuwanak@yahoo.com
Abstract:
Double perovskite Sr2FeTiO6- (SFT) has been developed by substitution of Nb and V
on Ti site to produce Sr2FeTi1-x(Nb, V)xO6- (x = 0.05 - 0.5) and tested for conductivity. The
materials were synthesized by a conventional solid-state reaction with calcination and
sintering temperatures of 1050 C for 12 hours and 1250 C for 12 hours, respectively. Phase
identification carried out using x-ray powder diffractometer indicated the materials consist of
double-perovskite structure and impurity phase. The intensity of impurity phase was
increased with the increasing of Nb and V content. The electrical conductivity determined by
a 4-point probe DC technique in air indicated among the oxide examined,
Sr2FeTi0.95Nb0.05O6- and Sr2FeTi0.95V0.05O6- show the increase in conductivity with
maximum conductivities of 2.4 S cm-1 and 3.9 S cm-1 at 700 C respectively which is a result
of the electronic compensation of Nb and V at the B-site cation. Moreover, the application of
Sr2FeTi0.95(Nb, V)0.05O6- as electrode material for single - cell SOFC will be investigated.
1. Introduction side. Also, the electrolyte material is the

Electricity is mainly generated from oxide ion conductor and therefore only the
the combustion of fossil fuels (coal, natural oxide ion pass through the anode. Next, the
and oil) that releases carbon dioxide (CO2) migrated oxide ion combines with the
which is a source of greenhouse effect. hydrogen gas and produces water and
Nowadays, worldwide attention has electrons (Eq. 2). After that, the electron
refocused on renewable energy such as solar moves to the cathode side via an external
cell, wind energy, biomass energy, ocean circuit; therefore, the electric power is
energy and fuel cell because of their occurred from the cell. Lastly, the overall
environment friendly. Solid oxide fuel cells reaction of SOFC is summarized in Eq. 3.
(SOFCs) are also captured researchers’ Cathode reduction of oxygen:
interest because of their ability to produce ½O2 + 2e-  O2- (1)
high electricity and low pollution.1 Anode oxidation of hydrogen:
SOFCs are electrochemical devices H2 + O2-  H2O + 2e- (2)
that convert the chemical energy of fuels to Total SOFC reaction:
electricity with very high efficiency via the H2 + ½O2  H2O (3)
chemical reaction. The SOFC consists of With regard to high temperature
anode and cathode connected by electrolyte operation (>800 C), SOFC has plenty of
and external circuit. During the SOFC advantages such as relative high power
operation, the air or oxygen gas is fed into density, fuel flexibility and no gas
cathode and the fuel or hydrogen gas is fed emission.2 However, high operating
into anode. At the cathode, the oxygen gas temperature involves some problems e.g.
reacts with the electron and that becomes an sealing issues, relatively expensive
oxide ion, O2- (Eq. 1). Then, the oxide ion components and materials compatibility. In
migrates through the electrolyte to the anode recent year, many researchers have been
studied about to reduce the operation materials were carried out using a Rigaku,
temperature of SOFC into the intermediate DMAX 2002 Ultima Plus X-Ray powder
range (600-800 C) which is known as diffractometer equipped with a
intermediate temperature solid oxide fuel monochromator and a Cu-target X-ray tube
cell (IT-SOFC).3-4 Unfortunately, the (40 kV, 30 mA). The data were recorded and
reducing operation temperature of SOFC the range of two-theta was from 20 to 80
decreases power density; therefore, the degree at room temperature in air.
searching of new electrode materials for IT- Electrical conductivity of the
SOFC that can run more efficiency have specimens was measured by the DC four-
been developed.5 point probes method. The sintered-pellet
The most interested SOFC electrode sample were cut into a rectangular shape
material is metal oxide, especially double then two Pt wires, the currenent electrode,
perovskite structure with A2BB’O6 were attached to the outer of the rectangular
stoichiometric formula because it is mixed specimen using a Pt paste binder and the
oxide ion and electron conductor, where another two wires which corresponds to the
oxide ion transport occurs via vacancy voltage probes were placed between the
hopping mechanism and electron transport outer probes. The specimen was heated at
through the overlapping of d-orbital of metal 950 C for 10 minutes in order to assure a
and p-orbital of oxygen. In this work, good electronic contact between wires and
significant attention has been focused on the specimen. The difference in voltage between
development of Sr2FeTiO6- (SFT) double the Pt probes was recorded from 300 to 800
perovskite materials because it can be used C with 50 C increments in air using an
as both the anode and cathode and also Autolab PGSTAT302N potentiostat/
exhibit a good conductivity. The addition of galvanostat. The data were calculated using
Nb and V was proposed into Ti-site of SFT the equation 4:
structure, Sr2FeTi1-x (Nb,V)xO6-, in order to
create a charge imbalance in the structure (4)
which leads to the change in electrochemical where;
property of materials. Therefore, the  is the electrical conductivity (S cm-1)
substituted oxide materials were primarily I is the applied DC current (A)
investigated for their conductivities. L is length between two inner probes (cm)
V is the different voltage (V)
2. Materials and Methods W is width of specimen (cm)
2.1 Material synthesis T is the thickness of specimen (cm)
The SFT and Sr2FeTi1-x(Nb,V)xO6-
(x=0.05-0.5) double perovskites were 3. Results and Discussion
synthesized by a conventional solid-state 3.1 Crystal structure
reaction. Firstly, the stoichiometric amounts The XRD pattern shown in Figure 1
of commercial powders SrCO3 (99.9%), indicates that SFT is pure and has double
Fe2O3 (81%), TiO2 (99.8%) and Nb2O5 perovskite structure (JCPDF 84-1004).6 The
(99.99%) or V2O5 (98.5%) were thoroughly XRD patterns of Sr2FeTi1-xNbxO6-
mixed by a mortar and a pestle with ethanol (x=0.05-0.5) at room temperature are
for 1 hour and calcined at 1050 C for 12 illustrated in Figure 2. All samples have a
hours in air. Next, the calcined sample was double perovskite structure with
reground with ethanol for 1 hour and Sr(FeNb)0.5O3 (JCPDF 29-1318) impurity
pressed into pellets. Finally, the obtained phase, except in Sr2FeTi0.95Nb0.05O6-. The
pellets were sintered at 1250 C for 12 hours intensity of impurity peaks increased with
in air. increasing of Nb content. Similarly, the
2.2 Material characterization XRD patterns of Sr2FeTi1-xVxO6- (x=0.05-
The crystal structure of synthesized 0.5) illustrated in Figure 3 show the double
perovskite structures are mainly observed
and the concentration of Sr3(VO4)2 (JCPDF
29-1318) impurity phases increases with the
V doping content.
Figure 3. XRD patterns of Sr2FeTi1-xVxO6-

(x=0.05-0.5) at room temperature
Figure 1. XRD pattern of SFT at room
temperature and the overall (x=0.05-0.5) decreases
obviously with the increase in Nb content, as
shown in Figure 5 However,
Sr2FeTi0.95Nb0.05O6- shows higher electrical
conductivity value than other Nb-dope SFT,
which is approximately 2.44 S cm-1 at 700
C. Figure 6 shows the variation on
electrical conductivity of Sr2FeTi1-xVxO6-
(x=0.05-0.5) samples with temperature in
air. The Sr2FeTi0.95V0.05O6- sample shows
the electrical conductivity value higher than
all V samples, which is around 3.93 Scm-1
at 700 C. Thus, it can be suggested that the
introduction of small Nb and V content,
Sr2FeTi0.95Nb0.05O6- and Sr2FeTi0.95V0.05O6-
Figure 2. XRD patterns of Sr2FeTi1-xNbxO6- , can improve the electrical conductivity of
 (x=0.05-0.5) at room temperature

SFT. The effect of Nb and V substitution
maybe explained by the charge
3.2 Electrical conductivity compensation of Fe in the structure.
The temperature dependence of However, further investigation on these
electrical conductivity of SFT in air is materials using XPS will be employed to
presented in Figure 4. The electrical confirm the explanation.
conductivity increases with temperature, 3.3 Electrochemical impedance
suggesting a semiconducting behavior and a Figure 7 shows the Nyquist plots of
typical metallic behavior above 600 C. The SFT, SFTV05 and SFTNb05 symmetric
electrical conductivity values of SFT are cells with LSGM electrolyte at 800C in air.
2.00-1.75 Scm-1 in the temperature range of The difference between the high-frequency
600-800 C with a maximum value of 2.00 and the low-frequency intercepts on the real
axis corresponds to the area specific
S cm-1 at 600 C. After Nb is added, the
resistance (ASR) and the ohmic resistance
semiconducting behavior is still observed
was set as a zero in the Nyquist plots. It is
electrical conductivity of Sr 2FeTi1-xNbxO6-
clearly seen that the ASR values of SFTV05
and SFTNb05 are significant lower than
SFT. The ASR values of SFT, SFTV05 and
SFTNb05 were 6.42, 1.89 and 1.91  cm2,
respectively. The lower in ASR values of
materials also results in the high
conductivity of the materials.
Figure 6. Electrical conductivity of

Sr2FeTi1-VxO6- (x=0.05-0.5) in air as a
function of temperature
Figure 4. Temparature depandance of the

electrical conductivity for SFT sample in
air
Figure 7. Typical impedance spectra of

SFT, Sr2FeTi0.95V0.05O6- (SFTV05) and
Sr2FeTi0.95Nb0.05O6- (SFTNb05) with
LSGM electrolyte at 800 C
the composition of Sr2Fe Ti0.95Nb0.05O6- and

Sr2FeTi0.95V0.05O6-. The materials were 2.44
and 3.93 S cm-1, respectively, at 700 C
which are higher than the electrical
conductivity of SFT material. From these
preliminary results, the double perovskite
Figure 5. Electrical conductivity of Sr2FeTi0.95(Nb,V)0.05O6- materials has
Sr2FeTi1-xNbxO6- (x=0.05-0.5) in air as a potential to be applied for electrode material
function of temperature in IT-SOFCs.
4. Conclusion Acknowledgements
In summary, double perovskite SFT This work is partially supported by
and Sr2FeTi1-x(Nb, V)xO6- (x = 0.05 - 0.5) Research group on Materials for clean
were successfully synthesized by the solid- energy conversion.
state reaction. The XRD analyses reveal the
double perovskite structure and the impurity References
phase which has been increasing with 1. Kong, X.; Zhou, X.; Tian, Y.; Wu, X.;
increasing of Nb and V content. However, Zhang, J.; Zuo, W. J. Power Sources
the pure phase was successfully obtained 2016, 326, 35–42.
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2. O'Hayre, R.; Cha, S. W.; Colella, W.; 5. Jiang, S.; Sunarso, J.; Zhou, W.; Shen, J.;
Prinz, F. B. Fuel Cell Fundamentals Ran, R.; Shao, Z. J. Power Sources 2015,
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3. Muñoz-García, A. B.; Pavone, M. Chem. 6. Vinaykumar, R.; Mazumder, R.; Bera, J.
Mater. 2016, 28 (2), 490–500. J. Magn. Magn. Mater. 2017, 429, 359–
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4536.
Influence of minerals in crude glycerol from biodiesel production on
products distribution of bio-oil via co-pyrolysis with palm oil residues
Teerawit Laosombut*, Lalita Attanatho, Amornrat Suemanotham, Warunyoo Phondate,
Panida Thepkhun, Yoothana Thanmongkhon
Expert Centre of Innovative Clean Energy and Environment,
Thailand Institute of Scientific and Technological Research (TISTR), Pathum Thani 12120, Thailand
*E-mail: teerawit@tistr.or.th
Abstract:
Palm oil is a major feedstock for biodiesel production in Thailand. According to
AEDP2015, the need of biodiesel will be increased to 14 ML/D in 2036. Generally, 10%wt of
crude glycerol is obtained from transesterification process. Then the large amount of glycerol
by-product could cause waste management problem in the future. Additionally, a large
amount of palm residues have been generated in the palm oil industry. Both crude glycerol
and palm oil residues are interesting feedstocks for producing biofuel or biochemical via fast
pyrolysis. In this study, the effects of mineral remained in crude glycerol pyrolysis, as well
as, the glycerol co-pyrolysis with palm oil residues were investigated by Py-GC-MS. For
glycerol pyrolysis, the volatile products concentration, including alcohol, ketone, aldehyde
and small alcohol, increased with the increasing of mineral content in glycerol. In contrast,
glycerol content in products decreased dramatically at high % of mineral. For glycerol co-
pyrolysis with palm residues, bio-oil products obtained contained a verity of volatile
hydrocarbons. Moreover, amount of phenolic compound was apparently increased.
1. Introduction Na+ are the main minerals in crude glycerol

Crude glycerol is a waste from from biodiesel production process.
biodiesel process which is generated around Moreover, main oil feedstock for
10% in biodiesel product.1 Crude glycerol biodiesel production is palm oil which is
can be purified to pure glycerol and then produced from palm oil industry.5 The palm
used in pharmaceutical, cosmetic, soap, industry generated large amount of palm
toothpaste, etc.2 Crude glycerol purification residues during both plantation and oil
process needs high investment cost. Thus it milling process. Most of palm residues are
is probably not suitable for medium or small trivial used in the market due to low values
scale industry. The alternative utilization of of application. Some of residues were
crude glycerol without purify process should identified as waste due to worthless of
be considered. Crude glycerol contained not application. Thus the new opportunity for
only glycerol but also other impurity. The utilization of palm residues should be
characterization of crude glycerol produced concerned.
from difference producer and used Pyrolysis is well known thermo-
difference oil feedstock were investigated.1,3 chemical conversion process for converting
Settapong and co-worker analyzed crude biomass to liquid chemical product.
glycerol obtained from three biodiesel plants Numerous research groups have been
in Thailand.4 Glycerol obtained from large studied the pyrolysis of crude glycerol and
scale producer had high amount of Na+ and biomass. Xiaohu and co-workers
K+ which was used as commercial catalysts investigated co-pyrolysis process in
and was used during biodiesel neutralization laboratory scale by using greek lignite and
process. These results indicated that K+ and crude glycerol from biodiesel as feedstocks,
at pyrolysis temperature of 300-1000 C.6 It 2.2 Thermogravimetric analysis (TGA) of
was found that blending crude glycerol into palm residues
greek lignite enhanced hydrogen yield. Co- Thermal behavior of palm residues
pyrolysis of crude glycerol and olive kernel was determined using a thermogravimetric
for the purpose of hydrogen production were analyzer (Thermo star, Pfeiffer model).
investigated by Skoulou et al. in laboratory Approximately 0.5 g of sample was heated
scale.7 Crude glycerol which contained from 25 to 1000 C with the heating rate of
methanol and KOH was used in this study. 10 C/min in nitrogen gas.
The obtained results indicated the addition 2.3 Pyrolysis gas chromatography mass
of biomass and potassium ion to crude spectroscopy (PY-GC/MS)
glycerol enhanced the hydrogen yield. H2 Pyrolysis experiments were
concentration increased to 11.6% when conducted using a pyrolyzer (model PY-
using mixture of 25 wt% of crude glycerol 2020ID, Frontier) with direct connection to
and 75 wt% olive kernel. GC/MS (Agilent 5975 and 7820A model) as
With the aim to find an alternative shown in Figure 1. Approximately 0.0010 g
approach for utilizing palm residues, as well of sample was weighed into sample cup and
as, glycerol from biodiesel industry. The then loaded to pyrolyszer. UA5MS-30M-
purpose of this study was to investigate co- 0.25F column was used for chemical
pyrolysis of glycerol and palm residues by composition separation in GC/MS. The oven
using pyrolysis gas chromatography mass temperature was programmed from 40 C
spectroscopy (PY-GC/MS). In addition, the (3 min of hold time) to 200 C at the heating
effect of sodium and potassium, which are rate of 5 C/min then increased to 350 C at
the impurities remaining in crude glycerol the heating rate of 10 C/min with a hold
from biodiesel industry, was studied in time of 10 min. The split ratio 20:1 was set
pyrolysis process. in an inlet under 280 C injector temperature
with He inert gas flow at 1 mL/min.
2.1 Glycerol and palm residues
The effect of mineral in crude
glycerol was studied in this work by using
glycerol model compound. This model
compound, so-called model glycerol, was
made by adding around 0 to 3.0 wt% of
sodium hydroxide (AR grade, Lab-scan
Analytical Science) or potassium hydroxide
(AR grade, CARLO ERBA Reagents) into Figure 1. Schematic diagram of Gas
pure glycerol (99.5%, Ajax Finechem), chromatography (7820A) mass spectroscopy
Sodium and potassium ion were used as (5975) with pyrolysis probe (PY-2020DI) on
representative of inorganic mineral impurity the top of injector
in glycerol because there were mineral from
commercial catalyst in biodiesel 3. Results and Discussion
production. 4 3.1 TGA results
Palm residues including palm shell Thermo gravimetric characteristic of
(PS), palm empty fruit bunch (PEFB) and PS, PEFB and WD was shown in Figure 2.
waste decanter (WD) were obtained from The results indicated that all palm residues
palm oil mill industry in Thailand. Firstly, used in this study decomposed around 200
palm residues was dried and then crushed to 600 C. Firstly, the moisture was released
and sieved to particle size in the range of 75- from palm residues around 90 to 180 C.
150 m. After that cellulose and hemicellulose were
decomposed at 230 to 400 C. Finally, lignin
was slightly degraded above 400 C.8 Our when increasing Na+ content in glycerol
previous work9 showed that the from 0.5-3% as shown in Table 1. The
decomposition of lignocellulose and fatty results revealed that the addition of
acid in palm residues increased when inorganic mineral to glycerol slightly
increasing pyrolysis temperature from 480 enhanced glycerol decomposition during
to 580 C and then the decomposition rate pyrolysis process. However, the difference
remained constant at temperature above between the addition of K+ and Na+ was
580 C. Therefore the pyrolysis temperature observed. K+ gave more significant effect on
using in PY-GC/MS experiment in current glycerol conversion during pyrolysis
work was set at 580 C. compared with Na+. This was probably due
to the catalytic effect of K+ that which
cracking glycerol into small hydrocarbon.7
Figure 2. TGA results of palm empty fruit

bunch (__), palm shell (--) and waste
decanter (….)
3.2 Effect of inorganic mineral in glycerol

pyrolysis process
The PY-GC/MS results indicated
that low glycerol conversion was achieved
when pyrolyzed pure glycerol at 580 C. The
area of glycerol was 98.84% while the sum
area of hydrocarbon products was only
1.16% as shown in Table 1. This result was
in accordance with Castello’s work,10 which
also showed little conversion of pure
Figure 3. GC/MS chromatograms of
glycerol during pyrolysis process at 600 C.1
glycerol and model glycerol with K+ at 0 to
The addition of K+ impurity to pure glycerol
had a major effect on the amount of glycerol 3.0%wt after pyrolysis at 580 C: (a) pure
remaining in pyrolysis product as shown in glycerol, (b, c and d) glycerol with 0.5, 2.0
Figure 3. Hydrocarbon content in product and 3.0 %wt of K+, respectively
increased from 24.05 to 56.36% when
increasing K+ impurity from 0.5 to 3%. 3.3 Pyrolysis of palm residues and co-
Moreover, the small amount of alcohol, pyrolysis with model glycerol
aldehyde and ketone were found in the The PY-GC/MS of PEFB and WD
products. gave the bio-oil products with high content
The Figure 4 showed PY-GC/MS of fatty acid (47.30 and 30.19%,
results of glycerol with Na+ impurity. The respectively) and similar other compounds
slightly increase of hydrocarbons content in distribution as shown in Figure 5. However,
products from 2.86 to 5.07 % was obtained, PS which is lignin rich biomass showed
difference pyrolysis behavior. The bio-oil
with high percentage of phenolic compound decomposition. Therefore, 10% of model
(53.05%) was obtained when pyrolyzed PS glycerol with K+ was added to 90% of palm
at 580 C. residues and then used as feedstock for
investigating the behavior of co-pyrolysis
process. Figure 6 showed the chemical
composition of bio-oil obtained from co-
pyrolysis. When comparing with palm
residue pyrolysis, fatty acid content in
products obtained from co-pyrolysis of
PEFB and WD with model glycerol
decreased from 47.30 and 30.19% to 34.03
and 24.7%. In contrast, phenolic compound
in product obtained from co-pyrolysis of
PEFB and WD increased from 7.83 and
12.99 % to 34.03 and 24.7%. In case of PS
co-pyrolysis, phenolic content reduced from
53.05 to 47.88%. The dramatically increase
of fatty acid (from 10.66 to 33.82%) and
slightly increase of aromatic content (from 0
to 2.70%) in the obtained product from PS
co-pyrolysis were observed. This increasing
of aromatic content was probably due to
phenolic decomposition.
The addition of model glycerol
affected the distribution of pyrolysis product
in difference way. For example, pyrolysis
products from PEFB and WD had high fatty
Figure 4. GC/MS chromatograms of acid concentration. Thus, fatty acid was
glycerol and model glycerol with Na+ at 0 to converted to other compounds during co-
3.0%wt after pyrolysis at 580 C: (a) pure pyrolysis process when adding model
glycerol, (b, c and d) glycerol with 0.5, 1.5 glycerol into PEFB and WD. There results
and 3.0 wt% of Na+, respectively could be explained by following reasons.
(i) The small volatile hydrocarbon
Table 1. Chemical composition of product generated from glycerol probably interacted
from pyrolysis of pure glycerol and model with biomass during devolatilization
glycerol at 580 C process.8
Chemical Composition (ii) During co-pyrolysis, K+ induced
(%Area Normalization) glycerol cracking into small hydrocarbon
Glycerol small HCa product. This small hydrocarbon product
Pure Glycerol 98.84 1.16 was then interacted with biomass to generate
Glycerol+K+ (%wt)
0.5 74.65 24.05 volatile product.
2.0 54.22 45.78 Bio-oil consisted of various types of
3.0 42.48 56.36 chemicals. So, operating condition used in
Glycerol+Na+ (%wt) pyrolysis process depended on the target
0.5 97.14 2.86 product. Some of the obtained chemical can
1.5 95.42 4.57
3.0 94.93 5.07 be used for chemical feedstock such as
a
Small hydrocarbon molecules phenol and some kind of hetero-atoms,
while almost of product can be upgraded to
From the previous section, K+ alternative fuel.7
exhibited a positive effect for glycerol
content of fatty acids and phenolic
compounds. Thus, this bio-oil could be used
as biochemical or biofuel after upgrading
process.
Acknowledgements
acknowledge National Research Council of
Thailand (NRCT) for financial support of
this research.
Figure 5. Chemical compositions of the
obtained product from pyrolysis GC/MS of References
palm shell (PS), palm empty fruit bunch 1. Thompson, J. C.; He, B. B. Appl. Eng.
(PEFB) and waste decanter (WD) at 580 C Agric. 2006, 22, 261–264.
2. Fan, X.; Burton, R.; Zhou, Y. The Open
Fuels & Energy Science Journal 2010,
3, 17–22.
3. Hu, S.; Luo, X.; Wan, C.; Li, Y. J.
Agric. Food Chem. 2012, 60, 5915–
5921.
4. Settapong, A.; Wattanachant, C.; CMU.
J. Nat. Sci. 2012, 11, 157–161.
5. http:// www.krungsri.com/ (Thailand
industrial outlook 2017)
6. Manara, P.; Zabaniotou, A. J. Anal.
Figure 6. Chemicals composition of the Appl. Pyrolysis. 2013, 100, 166–172.
obatained products from co-pyrolysis of 10 7. Skoulou, V. K.; Manara, P.;
% of model glycerol with 90% of palm Zabaniotou, A. A. J. Anal. Appl.
residues at 580 C Pyrolysis 2012, 97, 198–204.
8. Delgado, R.; Rosas, J. G.; Gómez, N.;
4. Conclusion Martínez, O.; Sanchez, M. E. J. Cara.
Co-pyrolysis of palm residues and Fuel 2013, 112, 31–37.
glycerol from biodiesel industry could be an 9. Laosombut, T.; Thanmongkhon, Y.;
alternative approach for utilizing these Attanatho, L.; Suemanotham, A.;
biomass wastes. During glycerol pyrolysis, Thepkhun, P.; Boonsae, M., Co-
the potassium impurity in glycerol exhibited pyrolysis and Palm Empty Fruit Bunch:
more positive effect on glycerol A Preliminary Study via PY-GC/MS,
decomposition into small hydrocarbon, Pure and Applied Chemistry
comparing with sodium impurity. The co- International Conference, Bangkok,
pyrolysis of palm residues and model Thailand, Feb. 2-3, 2017.
glycerol gave the bio-oil product with high 10. Castello, M. L.; Dweck, J. JSBS 2014,
4, 61–67.
Parametric study of CZTS thin film solar cells by
convective deposition method
Thanawat Anantamongkolchai1, Sojiphong Chatraphorn2, Paravee Vas-Umnuay1*
1
Center of Excellence in Particle Technology, Faculty of Engineering, Chulalongkorn University,
Bangkok, Thailand
2
Semiconductor Physics Research Laboratory, Department of Physics, Faculty of Science,
Chulalongkorn University, Bangkok, Thailand
*E-mail: paravee.v@chula.ac.th
Abstract:
Nowadays, most of the energy production is derived from fossil fuel-resources, which
are a major source of greenhouse gases. Therefore, several researchers have been looking for
the alternative energy that is clean and renewable. One of the outstanding alternatives is to
utilize solar energy for conversion into electricity. Recently, copper zinc tin sulphide
(Cu2ZnSnS4, CZTS) thin film solar cells have received numerous interests because of its
decent energy band gap matching with the solar spectrum and its high absorption coefficient.
Nevertheless, the efficiency of CZTS solar cells has been reported lower than other
commercial solar cells. Therefore, the challenge of this work is to fabricate CZTS thin film
solar cells via a solution-based process and investigate process parameters to be able to
control the film structure to achieve appropriate CZTS films for solar cell applications. In this
work, a convective deposition method is used for deposition of CZTS thin films because of
its advantages including low quantity of precursor solution utilization, simplicity, and rapid
and controllable deposition. The parametric study includes the different types of solvents,
different fractions of solvents, and the variation of deposition speed. These variable operating
parameters significantly have effects on film morphology, microstructure, and film thickness,
which directly influence on the efficiency of the cell.
1. Introduction cells, in particular, are composed of many

Energy issues are one of the types of materials based on the absorber
important problems due to the dramatically layer, for example copper indium gallium
increasing energy demand. Fossil fuels are diselenide solar cells (CIGS, CuInxGa(1-
provided more than 80% of the total energy x)Se2), copper zinc tin sulfide solar cells
consumption; nevertheless, fossil energy (CZTS, Cu2ZnSnS4) and perovskite solar
production has become a major source of cells. CZTS has been considered as a
greenhouse gases.1 Moreover, fossil fuels noticeable candidate for photovoltaic
will be gradually depleted in the near future.
applications because of its nontoxic
composition and its low-cost and earth-
Consequently, many researchers have been
abundant elements. Furthermore, CZTS has
looking for the alternative energy that is
a suitable energy band gap of about 1.4-1.6
clean and renewable. One of the outstanding
eV and a high absorption coefficient in the
renewable energy sources is solar energy
visible region with >104 cm-1.2
because it is inexpensive for energy
CZTS solar cells are composed of
conversion production and adequate for
many layers of different materials in thin
energy consumption.
film forms, including substrate, transparent
In terms of practical use, solar cells
conducting oxide (TCO), window layer
are electrical devices that convert sunlight
(p or n-type), absorber layer and metal
energy into direct electrical energy by the
contact layer.3
photovoltaic phenomenon. Thin film solar
In general, CZTS thin films can be
fabricated on either flexible or rigid
substrates by various gas-phase and
solution-based methods, for example
sputtering, thermal evaporation, spray
pyrolysis, electrochemical deposition,
chemical bath deposition and spin
coating.4 However, most of the gas-phase
techniques require expensive and
sophisticated equipment with intense
operating conditions, for example under
vacuum atmosphere. Therefore, the
processes require high cleanliness and high
energy input. On the other hand, most of the
solution-based techniques require a lot of
precursor solution and time for deposition.
For this reason, convective deposition
method can be the alternative approach to
solve aforementioned problems, because this
method is carried out under ambient Figure 1. The illustrative representation of
condition and requires simple equipment. In (a) experimental setup and (b) convective
addition, this method takes less time- deposition technique for fabricating thin
consuming and inexpensive apparatus for films5
fabrication.
Principally, convective deposition to each other and forming into a close
has been used to fabricate a monolayer film packed structure, as a result of the
from colloidal or nanoparticles. The minimization of energy.6 The parameters
equipment of this method shown in Figure such as deposition speed, volume of
1(a) consists of a deposition blade of about precursor solution, concentration of solution,
10 µm above the substrate and the etc., affect the physical processes and
translation stage at the bottom. A microliter properties of thin films.
of precursor solution is injected between the In this research, the parametric study
deposition blade and substrate and then the of deposition speed, types of solvents, and
substrate on the stage is pulled in the fractions of solvents, for the deposition of
horizontal axis with a controllable speed. A CZTS thin films was investigated on a
meniscus of the solution occurs between a contact layer of molybdenum by convective
substrate and a blade at a certain speed deposition method. The obtained optimized
forming a thin film of solution on the condition will be appropriate for fabrication
substrate.5 of high efficiency CZTS thin film solar
During deposition, two physical cells.
processes occur simultaneously. First is the
coffee ring effect, where a pattern is left by a 2. Materials and Methods
puddle of particle-laden liquid. The 2.1 Sputtering deposition of contact layer
convective flow to the edge is generated by (Molybdenum, Mo)
the evaporation losses and left only Mo layer was prepared by DC
aggregated thin film on the substrate, as magnetron sputtering using 99.99% pure Mo
shown in Figure 1(b). target. The Mo layer was deposited on clean
Secondly, the cheerio effect occurs soda lime glass under vacuum system in
which is the capillary attraction between Argon atmosphere at 6x10-3 mbar with the DC
particles on the liquid interface that are drawn sputtering power of 550 Watts for 11 min.
2.2 Synthesis of CZTS films
In a typical experimental procedure,
copper(II) chloride, zinc(II) chloride, tin(II)
chloride, and thiourea in stoichiometric ratio
of 2:1:1:8 were dissolved in 2-
methoxyethanol/deionized water and
triethanolamine (TEA) as a solvent and
stabilizer, respectively. A variation of
solvent fraction of 2-methoxyethanol
/deionized water of 100:0, 70:30, and 30:70
was investigated. Subsequently, the
precursor solution was stirred at 50 °C for
30 min. CZTS films were fabricated on Mo
Figure 2. XRD pattern of the CZTS
substrates at different convective deposition
prepared using different fractions of solvents
speeds in the range of 500 – 1000 μm/s.
(a) 2-methoxyethanol:DIW (100:0) on glass
After deposition, the film was dried on a
slide, (b) 2-methoxyethanol:DIW (100:0) on
hotplate at 200 °C for 5 min to remove
Mo substrate, (c) 2-methoxyethanol:DIW
solvent from the films. The coating and
(70:30) on Mo substrate, and (d) 2-
drying processes were repeated for 6 times.
methoxyethanol:DIW (30:70) on Mo
Finally, all the films were annealed in a
substrate
nitrogen atmosphere at 550 °C for 30 min.
2.3 Characterization
56.117° could be assigned to (002) and
The crystallographic structure of the
(312) of wurtzite structure, respectively.
CZTS layer was identified by X-ray
Accordingly, the peaks at 2Ɵ = 40.212° and
diffraction (XRD) in the range of 20-80°.
The morphology and thickness of absorber 73.541° correspond to the diffraction of
layer were obtained from scanning electron (110) and (211) planes of Mo. It can be
microscopy (SEM). Moreover, energy- observed that CZTS films deposited on Mo
dispersive X-ray spectroscopy (EDS) was substrates, as shown in Figures 2(b-d),
used to estimate the stoichiometric ratio of exhibit slightly higher crystallinity than that
Cu:Zn:Sn:S. deposited on glass substrate, as shown in
Figure 2(a). This could be attributed to a
3. Results and Discussion well lattice matching of CZTS with Mo,
3.1 Phase structure compared to CZTS and glass substrate.
The crystal structure of CZTS films Moreover, CZTS films prepared using
prepared by convective deposition method various fractions of solvents do not show
using different types of solvents was much difference in the intensity.
investigated by XRD and the results are 3.2 Effect of deposition parameters on
shown in Figure 2. The kesterite structure film morphology
of CZTS was detected in these synthesized The morphology and structure of
samples, which were obtained from different CZTS films deposited on Mo substrates was
solvent fractions of 2-methoxyethanol: investigated under various deposition
deionized water (DIW) as follows: 100:0, parameters via SEM, as shown in Figure 3.
70:30, and 30:70. The diffraction peaks Figures 3(a-c) demonstrate the films
prepared from solvent of
occurring at 2Ɵ = 28.449°, 32.970°, 47.314°,
methoxyethanol:DIW 30:70, while Figures
and 56.117° are assigned to (112), (200),
3(d-f) demonstrate the films prepared from
(220), and (200) planes of kesterite structure
solvents of methoxyethanol:DIW 70:30, and
of CZTS (JCPDS 26-0575), respectively. In
Figures 3(g-i) demonstrate the films prepared
addition, other peaks at 2Ɵ = 28.449°, and
(a) 2 µm (b) 2 µm (c) 2 µm
(d) 2 µm (e) 2 µm (f) 2 µm
(g) 2 µm (h) 2 µm (i) 2 µm
Figure 3. SEM micrographs of CZTS films deposited on Mo substrates prepared using

different solvents and fractions of solvents as follows; (a)-(c) 2-methoxyethanol:DIW 30:70
at deposition speeds of 500, 750 and 1000 µm/s, respectively, (d)-(f) 2-
methoxyethanol:DIW 70:30 at deposition speeds of 500, 750, and 1000 µm/s, respectively,
and (g)-(i) 2-methoxyethanol:DIW 100:0 at deposition speeds of 500, 750, and 1000 µm/s,
respectively
from solvents of methoDIW 100:0. Under methoxyethanol tends to evaporate slower

each condition, the deposition speeds were than DIW, leaving a remaining solution
varied from 500, 750, and 1000 µm/s, above the monolayer of CZTS film.
respectively. Moreover, the evaporation rates of different
According to the results, it can been solvents are not equal. As a result, there is
seen that the films prepared from high more room for particles within the
fraction of DIW at any speed are smooth and remaining volume of solution above the
compact, as shown in Figures 3(a-c). monolayer of CZTS to relocate until they
Whereas the films prepared from higher are settled and locked to each other by
fraction of 2-methoxyethanol, shown in capillary force on the surface. Thus, a larger
Figures 3(d-i), consist of voids, presenting volume of remaining solution above the
as porous films. This is significantly monolayer could result in a porous film.
attributed to the type of solvent, where 2- Conversely, the sol-gel precursor that has
methoxyethanol has a higher solvent boiling higher DIW fraction of solvent results in a
point compared to DIW. Therefore during denser film. This is attributed to a lower
the deposition by convective deposition volume (less space) above the monolayer of
method, the as-prepared solution with 2- CZTS, as a consequence of a higher
evaporation rate; therefore, particles tend to (a)
draw to each other more compactly.
In order to determine the thickness of Higher velocity
CZTS films, cross-sectional images were
obtained from SEM to identify the thickness
of CZTS film. The thickness of film depends
on two factors: 1) boiling point of solvent
and 2) deposition speed. The deposition of
the precursor solution with the solvent with
higher boiling point results in a higher
volume of solvent above the monolayer as
result of lower evaporation rate, as (b)
previously described. For this reason, the
films prepared using higher fraction of 2– Lower velocity
methoxyethanol at any deposition speeds
have higher thickness, as present in Table 1.
In terms of the effect of deposition
speed on film thickness, at higher speed,
more particles in the bulk precursor solution
are dragged along the convective line
towards the settled particles due to a higher Figure 4. Comparison of film formation by
drag force. As a result, thicker CZTS layer is convective deposition method at (a) a higher
obtained owing to more dragged particles, as deposition speed (b) a lower deposition
shown in Figure 4(a). speed
Table 1. The thicknesses of samples were fabricated using different solvents and deposition
Solvents Avg. thickness Avg. thickness Avg. thickness
Speed 500 µm/s Speed 750 µm/s Speed 1000 µm/s
2-methox 100: DIW 0 579 nm 710 nm 829 nm
Table 2. Atomic percentages of CZTS thin films synthesized by different solvents

Solvents Cu Zn Sn S total
2-methox 100: DIW 0 24.89 13.42 9.30 52.39 100
2-methox 70: DIW 30 22.46 13.74 14.16 49.64 100
2-methox 30: DIW 70 34.29 20.72 14.69 30.31 100
On the other hand, at lower applications. In this study, the stoichiometric

deposition speed less particles are drawn composition of CZTS film was detected by
towards the settled particles as a result of a EDS. Table 2 shows the atomic percentage
lower drag force. Therefore, the film of Cu:Zn:Sn:S in CZTS films that were
becomes thinner, as illustrated in Figure prepared using different solvents. From the
4(b). Suumarized results of film thickness results, the composition of CZTS using 2-
obtained at different deposition speeds are methoxyethanol 70:DIW 30 as the solvent is
shown in Table 1. the closest to the ideal stoichiometric value
Lately, the chemical composition of of 2:1:1:4.
CZTS film has been intensively studied According to these results, CZTS
because the composition can be adjusted to thin films prepared by convective deposition
be near the ideal stoichiometry for certain method can be controlled in order to obtain
an appropriate structure to be used as an Acknowledgements
absorber layer in thin film solar cell Authors are thankful to the Ph.D.
applications. student and officer of Semiconductor
Physics Research Laboratory for their help
4. Conclusion and support.
CZTS thin films were deposited on
Mo substrates by convective deposition References
method, where evaporation and capillary 1. Pike, W. J. World Oil 2016, 237, 1.
force take control of the film formation. In 2. Jimbo, K.; Kimura, R.; Kamimura, T.;
this work, we investigated the effects of Yamada, S.; Maw, W. S.; Araki, H.;
variable operating parameters such as types Oishi, K.; Katagiri, H. Thin Solid Films
of solvents, fractions of solvents, and 2007, 515 (15), 5997−5999.
deposition speed on CZTS characteristics. 3. Green, M. A., J. Mater. Sci. Mater.
The structure and morphology of the film Electron. 2007, 18 (S1), 15−19.
prepared from the precursor solution using a 4. Chopra, K. L.; Paulson, P. D.; Dutta, V.
higher fraction of DIW (lower boiling point) Prog. Photovoltaics 2004, 12 (23),
exhibits a uniform and compact film, which 69−92.
is suitable to be used as an absorber layer for 5. Chonsut, T.; Rangkasikorn, A.;
thin film solar cells. The results show that Wirunchit, S.; Kaewprajak, A.;
the convective deposition method is an Kumnorkaew, P.; Kayunkid, N.; Nukeaw,
efficient solution-based technique for thin J. Materials Today: Proceedings 2017, 4
film fabrication and the film structure can be (5), 6134−6139.
simply controlled for thin film solar cell 6. Joshi, K.; Muangnapoh, T.; Stever, M.
application. D.; Gilchrist, J. F. Langmuir 2015, 31
(45), 12348−53.
Design and performance evaluation of sustainable process combining
hydrotreating and hydrogen generation for biojet fuel production
from palm oil
Siriporn Boonsuk1, Worapon Kiatkittipong2, Kanokwan Ngaosuwan3,
Doonyapong Wongsawaeng4, Suttichai Assabumrungrat1*
1
2
Department of Chemical Engineering, Faculty of Engineering and Industrial Technology, Silpakorn University,
Nakhon Pathom 73000, Thailand
3
Department of Chemical Engineering, Faculty of Engineering, Rajamangala University of Technology
Krungthep, Bangkok 10120, Thailand
4
Department of Nuclear Engineering, Faculty of Engineering, Chulalongkorn University,
*E-mail: suttichai.A@chula.ac.th
Abstract:
This research work proposed a sustainable hydrotreating process for a production of
biojet fuel, whereas hydrogen (H2) could also be produced simultaneously. Palm oil is one of
the most potential feedstock for renewable fuel production in Thailand and is the target
resource in this study. Combining process was proposed contain with hydrotreating of
triglycerides (TG) and steam reforming (SR) of propane by-product. For the H2 production,
the SR of propane was operated at 525 °C and 8 bar. The obtained H2 were employed for
biojet fuel production which are consisted of several reactions; hydrogenolysis,
hydrogenation, deoxygenation (DO) and hydrocracking. Studies of H2 consumption, H2
generation and energy consumption by DO reaction pathways via hydrodeoxygenation
(HDO) were carried out. The system is considered as it can sustain itself without external
support of H2 and heat. The result found that process heat integration is necessary for
efficient operation of the combined process.
1. Introduction including hydrotreating, deoxygenation

The rapid increasing of energy (DO) and isomerization/hydrocracking.
consumption in the world caused by Palm oil is one of the most potential
increasing growth of civilization and human feedstock for renewable fuel production in
population leads to rapid industrialization Thailand, compared to other vegetable oils,
and urbanization, which has resulted in the palm oil consumes less hydrogen (H2) in
significant depletion of fossil based fuel.1,2 hydroprocessing reaction because it contains
The development of renewable fuel from a lower fraction of unsaturated free fatty
bioresources has been an important key to acid (FFA).4 Using palm fatty acid distillate
the future energy.3 Hence, the requirement (PFAD), by-product from palm oil refinery,
for sustainable and environment friendly as a raw material consumes less H2 than
energy sources have become essential for triglycerides (TG) as a raw material. On the
fulfilling the energy scarcity in the field of other hand, to produce H2 from by-product
industry and all modes of transportation.1 of hydroprocessing for biojet fuel
Biojet fuel is a promising alternative production, using palm oil as a raw material
fuel due to it having similar properties to jet can produce by-product propane via
fuel derived from petroleum and can be hydrogenolysis which used for H2
produced from vegetable oil or animal fat production.
through hydroprocessing technologies, Propane can be converted to H2 via
Figure 1. Block diagram of process combining hydrotreating and hydrogen production
propane steam reforming (PSR) using 2.1 Modeling of hydrotreating

Al2O3-supported nickel catalysts which The hydrotreating process consists of
being the most widely used for large scale the transformation of vegetable oil through
industrial reformers.5 hydrogenolysis, hydrogenation, DO,
In this work, we proposed a hydrocracking and hydroisomerization to
sustainable hydrotreating process to produce generate renewable fuels. Thereby, the
biojet fuel which H2 could be produced in process is integrated by two consecutive
situ simultaneously. Combining process was reactors.6
proposed contain with hydrotreating of TG The vegetable oil as triolein (OOO)
and SR of propane by-product which shown is fed with compressed H2 into the reactor,
in Figure 1. This study aims to investigate which contains the nickel-molybdenum
performance evaluation of H2 consumption, catalysts (NiMo).10
H2 generation and energy consumption. First reaction, hydrogenolysis of TG
converted into FFA and propane by-product.
2. Process Simulation and Methodology Second reaction, hydrogenation of
The sustainable process of biojet fuel unsaturated FFA converted into saturated
production using the hydroprocessing is FFA. Third reaction, DO of FFA via
comprised of three different processes, decarboxylation (DCO2), decarbonylation
including hydrotreating, separation and (DCO) and hydrodeoxygenation (HDO)
reforming as will be described below. converted into alkane hydrocarbon and
Conventional hydrotreating process were water (H2O), carbon dioxide (CO2), carbon
designed and simulated using the Aspen monoxide (CO) are by products. The
Plus simulation software to produce selectivity of the FFA to undergo DO
renewable alkanes.6,7 The reactions and reaction pathway is dictated by catalysts
conversion of TG as the literature review6,8,9 used and process condition. The operating
were modeled in Microsoft Excel which condition of the hydrotreating reactor are
product distribution and H2 demand were temperature of 320 °C and pressure of 80
calculated based upon the conversion bar since Antonio and co-workers6 reported
reaction. Palm oil was used as the feedstock that conversion of vegetable oil to long
material in the process model. Since this chain hydrocarbon products (C15-C18) is
feedstock is predominantly as C16:0 and C18:1 almost complete.
fatty acid. The model assumes C18:1 as a DO processes was assumed to occur
feedstock to investigate a scenario of the via HDO without DCO and DCO2 which is
highest H2 consumption. a scenario of the highest H2 consumption
This assumption is possible in case of using Table 1. Stoichiometric reactions involved
NiMo catalyst.10 The model is the in the hydrotreating and reforming process
stoichiometric reactor based on fractional Stoichiometric reactions
conversion, RStoic (R-101). Due to HDO Eq. (1) Hydrogenolysis
C57H104O6+3H2 → 3C17H33COOH +C3H8
consume H2 more than DCO2.
Eq. (2) Hydrogenation
The flow of vegetable oil feeding the C17H33COOH +H2 → C17H35COOH
reactor is 1000 kg/hr, while the H2 is initial Eq. (3) Hydrodeoxygenation
fed 98.26 kmol/hr which is in a ratio of 5 C17H35COOH+3H2 → C18H38+2H2O
times of theoretical requirement for HDO.3 Eq. (4) Hydrocracking
C18H38+2H2 → C10H22+C5H12+C3H8
Main product of first reactor is C18. H2O as a
Eq. (5) Propane steam reforming
by-product from HDO (RE-H2O-1) which C3H8+H2O → CO+3H2
was adsorbed by molecular sieves (SEP-1) Eq. (6) Water gas shift
and sent to mix with makeup H2O for steam CO+H2O → CO2+H2
reforming process.
In the second reactor, hydrocracking (FSPLIT-2) in order to split light gas for
is carried out. In a process that targets the produce necessary H2 in reforming unit.
production of biojet fuel, diesel fraction is LIQ-2 were sent to second distillation
further cracked into kerosene range column (D-102) to distillated naphtha
hydrocarbons. Additional light hydrocarbons (NAPH) as a top product. The bottom
are inevitably produced as it is difficult to products of D-102 (LIQ-3) were sent to
control the degree of cracking.2 The distillated at D-103 where biojet fuel leaving
operating condition of the hydrocracking at the top and green diesel or bio
reactor are temperature 480 °C and pressure hydrogenated diesel (BHD) leaving at the
80 bar, since Shayegh and co-workers8 bottom of the column. The distillation trains
reported the conversion of reaction were designed with shortcut methodology
maximum was 40% from gas oil to gasoline Winn-Underwood-Gilliland method,
and light Gas (C1-C4). through the DSTWU module in Aspen Plus.
The assumption of the reaction The recoveries of the key components were
pathway of hydrocracking, C18H38 was established in 99.9%, while the
cracked to kerosene, naphtha and light gas. thermodynamic model employed was
Stoichiometric reactions were specified in Dortmund modified UNIFAC (UNIF-
the model for the stoichiometric reactor DMD). Distillation column was simulated
based on fractional conversion, RStoic (R- using RadFrac model in order to consider
102). The overall stoichiometric reactions material and energy balances along with the
are outlined in Table 1. Light hydrocarbons rigorous calculation of phase equilibrium.6
generated as by-product of the process were Table 2 shows the main design parameters
fed to a reformer unit to produce H2 for the of each distillation column.
process. 2.3 Modeling of stream reformer
2.2 Modeling of separation In the third reactor, PSR is carried
The products from the hydrocracking out. The operating conditions of the
process (PROD-2) were sent to a separation reformer are temperature of 525 °C and
unit, where excess H2 gas (RE-H2-1) was pressure of 8 bar. Steam 586 kg/hr (steam to
adsorbed by pressure swing adsorption, PSA carbon molar ratio of 5.0) was fed since
(SEP-1) and was recycled to hydrotreating Rakib and co-workers5 reported the
process, while other products were sent into maximum H2 yield was 9.26 mol of total H2
turbine to produce electricity before cooled per mole of propane fed can be achieved.
down by total condenser. The liquid The PSR and water gas shift reaction was
products (LIQ-1) were sent to first specified in the model for the Rigorous
distillation column (D-101) to distill light equilibrium reactor, REqui (R-103). The
gas (L-GAS) and were sent to stream splitter products from the reforming process
Figure 2. Model of sustainable process combining hydrotreating and hydrogen generation for
biojet fuel production from palm oil
Table 2. Design variables of direct H2O (RE-H2O-2) were sent to makeup H2O
conventional distillation trains for (MIX-H2O) for recycle to reforming
hydrocarbon products separation process. The gaseous products (MIXGAS)
Parameter
Column were sent to separate H2 gas product (RE-
D-101 D-102 D-103 H2-2) in order to makeup H2 in stream
No. of stages 7 6 7 splitter (FSPLIT-1) for recycle to
Feed stage no. 5 4 4
Type of condenser Partial Total Total hydrotreating process, while excess gas
Operating press. 1.01 1.01 1.01 (OFF-GAS) such as CO2 and CO are
(bar) combusted by a flare. The overall process is
Distillation flow 108.86 99.09 193.12 shown in Figure 2.
(kg/hr)
Reboiler duty (kW) 68.29 89.87 68.88
3.1 Process synthesis and result analysis
Table 3. Hydrogen consumption of
Table 3 shows the H2 consumption
complete hydrotreating process and partial
and H2 generation from the process. H2
hydrocracking and necessary hydrogen
consumed in hydrogenolysis, hydrogenation,
generation of triolein
H2 consumption/
DO via HDO and hydrocracking. DCO2
Reaction/pathways generation (kmol/hr) pathway does not require H2 for reaction.
Hydrogenolysis -3.39 Theoretically, the maximum H2
Hydrogenation -3.39 consumption is 19.65 kmol/hr.
Deoxygenation Propane is by-product derived from
via DCO2 0 hydrogenolysis 1.13 kmol/hr and
via DCO -3.39
hydrocracking 1.36 kmol/hr as the
via HDO -10.16
Hydrocracking -2.71 stoichiometry reactions. Propane was
Total H2 consumption -19.65 distillated from first distillation column at
(via HDO) top of the train in light gas stream. 2.2
Reforming 20.28 kmol/hr of light gas (approx. 89% of light
gas production) was split into reforming unit
(PROD-3) were sent to a flash drum (F- to produce H2 required for hydrotreating
101), separation unit, where gaseous process.
products were cooled to obtained excess Naphtha was distillated from second
H2O (RE-H2O-2) and separated from distillation column produce 9.91% wt. of
gaseous products (MIXGAS). The excess feedstock. Biojet yield of 19.31 % can be
Table 4. The overall material balance of the model
Stream OOO H2O PROD-1 PROD-2 PROD-3 OFF-GAS L-GAS NAPH BIOJET BHD
Temp. (°C) 25 25 320 480 525 25 -37 24 163 313
Pressure (bar) 1.01 1.01 80 80 8 1.01 1.01 1.01 1.01 1.01
Molar flow 1.13 5.98 92.61 85.90 47.71 7.61 0.27 1.42 1.37 2.04
rate (kmol/hr)
Mass flow 1000 108 1198 1077 683 284 12 99 193 518
rate (kg/hr)
Mass fraction
Triolein 1 - - - - - - - - -
H2 - - 0.14 0.15 0.06 - - - - -
H2O - 1 0.10 - 0.55 0.06 - - - -
CO2 - - - - 0.34 0.82 - - - -
CO - - - - 0.05 0.12 - - - -
Light gas - - 0.04 0.10 - - 0.99 0.02 - -
Naphtha - - - 0.09 - - 0.01 0.95 0.01 -
Biojet - - - 0.18 - - - 0.02 0.98 -
Green diesel - - 0.72 0.48 - - - - 0.01 0.99
Table 5. The utilitiy requirement of process CO2 is by-product from reforming process
combining hydrotreating and hydrogen and main content of OFF-GAS stream.
generation for biojet fuel production from 3.2 Process energy demand
palm oil The heating and cooling
Utilities Heat Saving requirements for the hydrotreating,
Actual Target
(MJ/hr) integrated (%)
separation and reforming process were
Heating 4,834 0 0 100
Cooling 7,647 2,813 2,965 61.23 calculated using Aspen Plus, and are
Total 12,481 2,813 2,965 76.24 presented in Table 5. Heat integration using
energy analysis was performed in order to
achieved which is consistent with the model a realistic production facility. The
literature reported by Antonio (18.6%)6 with HDO and hydrocracking are exothermic
green diesel yield of 51.8% wt. Table 4 reaction. The reforming is an endothermic
shows the overall material balance of the reaction. Due to hydrotreating process
process system. required to operate at high pressure, H2 was
The theoretical of reforming process, compressed which required high power of
PSR can produce H2 maximum 10 mol per 820 kW. Reforming process required power
propane 1 mol as stoichiometric reactions. for 107 kW and 8 kW for compressor and
The equilibrium reaction depends on pump, respectively. The gas turbine can
operating condition of reaction such as generate power 34 kW, therefore net
temperature, pressure and steam to carbon electricity of 901 kW was required. In
ratio. Reforming process operating at addition, the overall process is an
temperature 525 °C, pressure 8 bar and exothermic, 2,965 MJ/hr of cooling utilities
steam to carbon ratio 5 times produced H2 are required.
20.48 kmol/hr from 2.17 kmol/hr of propane
feed. The obtained H2 yield was 9.43 which 4. Conclusion
corresponding well with the experiment The combining process contain with
from the literature Rakib and co-workers.5 hydrotreating of triglycerides and SR of
The H2 was produced sufficient for propane by-product. The H2 was produced
hydrotreating process that no need H2 from sufficient for hydrotreating process that no
external sources. Due to H2O is by-product need H2 from external sources. The result
from HDO pathway of DO and sent to found that process heat integration is
makeup H2O combined with excess H2O for necessary for efficient operation of the
recycle to reforming process. Initial H2O combined process. However, net electricity
feed can be decreased to 107.70 kg/hr which of 901 kW was required mainly for
steam to carbon ratio remains 5 times. The compression process since both reforming
and hydroprocessing are operating at high 4. S.Kantama, A.; Narataruksa, P.;
pressure. The BHD, jet and naphtha yield of Hunpinyo, P.; Prapainainar, C. Biomass
51.8, 19.3 and 9.9 can be achieved. Bioenergy 2015, 83, 448–459.
5. Rakib, M. A.; Grace, J. R.; Lim, C. J.;
Acknowledgements Elnashaie, S. S.; Ghiasi, B. Int. J.
This work has been financially Hydrogen Energ. 2010, 35 (12), 6276–
supported by the Thailand Research Fund 6290.
(DPG5880003). 6. Gutiérrez-Antonio, C.; Gómez-Castro,
F.; Romero-Izquierdo, A.; Hernández, S.
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Development of lignocellulose sugarcane bagasse
as a cooperative catalyst in cycloaddition reaction with CO2
Kawisa Chaipojjana1, Mutsee Termtanun1, Worapon Kiatkittipong1*
Kunlanan Kiatkittipong2, Sushil Adhikari3, Navadol Laosiripojana4,
Suttichai Assabumrungrat5
1
Silpakorn University, Nakhon Pathom 73000, Thailand
2
Department of Chemical Engineering, Faculty of Engineering,
3
Center for Bioenergy and Bioproducts, Department of Biosystems Engineering,
Auburn University, Auburn, Alabama, 36849, United States
4
The Joint Graduate School of Energy and Environment,
5
*E-mail: kiatkittipong_w@su.ac.th
Abstract:
There have been many attempts among researchers for development of CO2
conversion to be value-added chemicals and fuels. However, the requirement of harsh
conditions is still the drawback. In this research, the utilization of lignocellulose of dewaxed
sugarcane bagasse as a novel cooperative catalyst with potassium iodide (KI) in cycloaddition
of 1,2-butylene oxide with CO2 towards 1,2-butylene carbonates was studied under mild
conditions. According to the results, this catalytic system exhibited excellent cycloaddition
activities since the immense hydroxyl groups in dewaxed sugarcane bagasse are able to
coordinate with oxygen atoms of epoxide and activate ring opening of epoxide through
hydrogen bonding. The suitable operating conditions for cycloaddition reaction to achieve
high 1,2-butylene carbonate yield are 120 °C, and 20 bar CO2 pressure.
1. Introduction A wide range of homogeneous

Carbon dioxide (CO2) is majority catalysts have been used for production of
effluent from various industries and cyclic carbonate including alkali metal
transportation sector that use fossil fuels as compounds,4 and ionic liquid (ILs),5
resource.1 Many attempts have to be However, the separate the catalyst from the
undertaken to reduce these greenhouse gas product is complicated and the
emissions including the production of decomposition of catalyst. To resolve this
chemicals. However, the biggest drawback problem, heterogeneous catalysts have been
associated with CO2 utilization is its widely developed, for example, metal
thermodynamic stability and kinetic oxide,6 molecular sieves,7 and supported
inertness, which challenges the conversion polymer8 but they are limited from low
of CO2 to chemicals and fuels. However, activity, poor stability and reusability. Alkali
the synthesis of cyclic carbonate through metal salts are alternative catalyst for the
cycloaddition of CO2 with epoxides is one cycloaddition because of inexpensive,
of the most interesting processes because abundance and nontoxic, but suffer from
this reaction has 100% atom economy and low activities.9 Therefore, they require
cyclic carbonate products are widely used as addition of co-catalysts or solvent for
polar aprotic solvents, electrolytes, catalytic efficiency improvement10 which, in
precursors of polymeric materials, and fine recent studies found that hydroxyl and
chemical intermediates.2,3 carbonyl group-containing catalytic systems
can coordinate with the oxygen atom of 2.3 Characterization methods of the
epoxides through the hydrogen bond and dewaxed sugarcane bagasse co-catalyst
accelerate the cycloaddition of epoxides 2.3.1 Fourier Transform Infrared
with CO211,12. Therefore, lignocellulosic Spectroscopy (FT-IR)
sugarcane bagasse with the hydroxyl and FT-IR spectra were obtained on an
carbonyl groups as contained in cellulose, FT-IR spectrophotometer using a potassium
hemicellulose and lignin would be a bromide (KBr) disk containing
promising biomass-based catalyst for approximately 1% finely ground samples.
catalytic cycloaddition of epoxide and CO2 The FT-IR spectrometer was scanned over
because it is natural biopolymers and has the frequency at a resolution of 2 cm-1 in
excellent properties such as transmission mode in the range of 400-4000
biocompatibility, biodegradability, non- cm-1.
toxicity, and good adsorption. 2.3.2 Thermogravimetric analysis (TGA)
In this work, lignocellulosic SCB and dewaxed SCB were
component in dewaxed sugarcane bagasse performed thermal analysis using
(SCB) was then applied due to the electron- thermogravimetric analysis (TGA) and
rich feature of hydroxyl groups as a differential scanning calorimetry (DSC).
cooperative catalytic system with the The scans were run from room temperature
presence of potassium iodide (KI) for to 1000 °C at a constant heating rate of 10
cycloaddition reaction of epoxides and CO2 °C/min. The apparatus was continually
to form cyclic carbonates under solvent-free flushed with air flow of 30 mL/min.
conditions. 2.4 Cycloaddition reaction of 1,2-butylene
oxide and CO2
2. Experimental All the cycloaddition reactions were
2.1 Chemicals and material conducted in a 100 mL stainless steel
CO2 gas with 99.5% purity was autoclave equipped with a mechanical stirrer
supplied by Masser Specialty Gas Co., Ltd. and automatic temperature control system.
1,2-Butylene oxide (99%) and 1,2-butylene As a standard condition, 1,2-Butylene oxide
carbonate (98%) were purchased from TCI (300 mmol), dewaxed SCB (1,500 mg), and
chemicals. Potassium iodide (99%) was potassium iodide (7.5 mmol) dissolved in
obtained from Ajax Finechem. Ethanol and water 2 ml were added into the reactor. The
toluene bought from Quality Reagent reactor was purged with CO2 for several
Chemical and Merck. Sugarcane bagasse times after heat up to desired temperature.
was obtained from a local factory. All Later, the reactor was pressurized with CO2
chemicals were reagent grade without to a desired pressure, and was stirred at
further purification. All chemicals were maximum speed of 200 rpm in order to
reagent grade without further purification. avoid mass transfer limitation. After
2.2 Preparation of co-catalyst from completion of the reaction, the autoclave
sugarcane bagasse (SCB) was cooled in a cooling bath, and the excess
Sugarcane bagasse (SCB) was dried CO2 was slowly vented. The reaction
in sunlight and then cut into small pieces (1- mixture was then dissolved in ethanol (20
3 cm.). The cut sugarcane bagasse was mL) and filtered to remove the co-catalyst.
milled and dried in oven for 16 h at 60 °C. The co-catalyst was washed with ethanol
Wax was extracted from the dried powder twice times and dried in oven. The
with toluene/ethanol (2:1, v/v) in Soxhlet conversion of 1,2-butylene oxide and the
extractor for 6 h. After the extraction was selectivity of 1,2-butylene carbonate from
completed, dewaxed SCB was washed and the cycloaddition reaction were determined
dried again in an oven for 16 h at 60 °C. The using gas chromatography flame ionized
solvent mixture was then recovered by a detector (GC-FID).
reduced pressure distillation.
3. Results and Discussion enhanced with increasing pressure in the low
3.1 Effect of the reaction temperature pressure-region (10-20 bar), they both
The effect of the reaction somewhat declined in the high pressure-
temperature in range 80 C to 140 C on the region. This phenomenon can be explained
cycloaddition reaction of 1,2-butylene oxide by the appearance of two phases in an
and carbon dioxide to form 1,2-butylene autoclave system, the top phase was CO2-
carbonate as a product is shown in Figure 1. rich phase and the bottom phase was BO-
The yield of 1,2-butylene carbonate rich phase. The concentration of the CO2 in
increased from 63% to 72% when raised up the bottom phase increased with an
the temperature from 80 to 120 C. increasing pressure, leading to the 1,2-
However, mildly decrease appeared at the butylene carbonate yield increased.
140 °C, leading 70% yield of 1,2-butylene However, in the vicinity of the
carbonate. This might possibly due to the catalyst,16,17 high CO2 pressure generated a
formation of 1,2-butanediols,13-15 a by- low concentration of 1,2-butylene oxide and
product from the hydrolysis of 1,2-butylene could retard the interaction between 1,2-
oxide and water. The presence of 1,2- butylene oxide and the catalyst, thus leading
butanediols was confirmed by GC-MS. to a low yield of 1,2-butylene carbonate.
Moreover, it was found to be nil Therefore, the suitable CO2 pressure for
isomerization products, confirmed by 1H- operating this cycloaddition reaction is equal
NMR and 13C-NMR. On the other hand, the to 20 bar.
conversion of 1,2-butylene oxide increased
with the temperature to some extent, relating
to the dependence of the kinetic rate with the
reaction temperature. Therefore, 120 °C was
the suitable reaction temperature in this
cycloaddition reaction.
Figure 2. The influence of CO2 pressure on

1,2-butylene carbonate yield and 1,2-
butylene oxide conversion. (reaction
temperature of 120 °C, and 6 h of reaction
time)
Figure 1. The influence of reaction
temperature on 1,2-butylene carbonate yield 3.3 Characterization of dewaxed SCB co-
and 1,2-butylene oxide conversion. (CO2 catalyst
initial pressure of 2 MPa, and 6 h of reaction FT-IR spectroscopy analysis
time) In Figure 3, FT-IR results reveal that
there is no structural transformation of the
3.2 Effect of the CO2 pressure SCB after wax extracted using
The effect of CO2 pressure on the toluene/ethanol as solvents when compared
catalytic activity of KI/dewaxed SCB to a non-dewaxed. The absorption bands FT-
system was determined at 120 °C in the IR are spectra of SCB and dewaxed SCB
range of 10-30 bar, shown in Figure 2. shown in Table 1.
Although the yield of 1,2-butylene carbonate Thermal analysis
and conversion of 1,2-butylene oxide Both TGA and DSC curve of SCB
and dewaxed SCB are presented in Figure Table 1. Band component of the FT-IR
4, comprised of three and two mass loss spectra of SCB and dewaxed SCB
steps, respectively. The first one up to 200 Band position (cm-1) Functional group
°C is considered as an organic extractives
e.g. fats, waxes, alkaloids, terpenes, 3334 O-H stretching vibration
glycosides, etc. Hence, the weight loss of of cellulose
dewaxed SCB begins at higher temperature 2920 C-H stretching vibration
than that of SCB. The second step occurring of cellulose
between 200 °C and 350 °C is assigned to
the cellulose and hemicellulose. The third 2361 CO2 vibration
(associated to CO2 in
step appearing from 350 °C to 500 °C is
the air)
attributed to the decomposition of lignin.
1733 C=O stretching
vibration of carbonyl in
ester
1645, 1604, 1455 Aromatic ring stretching

(benzene in lignin)
1511 C=C stretching

vibration of aromatic
(carbonyl, ketone in
lignin)
1369 CH2 bending vibration

Figure 3. FT-IR spectrum of SCB and 1321, 1159 C-C, C-O stretching
dewaxed SCB vibration
1238 C-O-C stretching

vibration of pyranose
skeletal
1035 C-O, C-H stretching

vibration of guaiacyl
unit in lignin
through a hydrogen bond; (2) nucleophilic

attacking on the less hindered carbon atom
Figure 4. TGA and DSC curves of SCB and of the epoxide in the activation of the ring of
dewaxed SCB epoxide by I- anion of KI; (3) CO2 resulting
in the cyclic carbonate formation by
3.5 Reaction mechanism intramolecular cyclic elimination, and the
Based on the previous literatures13,18,19 regenerating of catalyst.
and the results of this research, the possible
mechanism displaying the synergistic effect 4. Conclusion
of KI/dewaxed SCB catalytic system on the This research was successful in the
occurrence of cycloaddition reaction are utilization of lignocellulose component in
shown in Figure 5. Three involving steps in dewaxed SCB as a co-catalyst with
this mechanism are provided in the potassium (KI) on the cycloaddition reaction
following logical order: (1) the formation of to produce 1,2-butylene carbonate from 1,2-
the coordination of the hydroxyl groups in butylene oxide and CO2 as reactants. The
dewaxed SCB with an O atom of epoxide massive hydroxyl groups in sugarcane bagasse
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Financially supported by Silpakorn 14. Xiao, L.; Su, D.; Yue, C.; Wu, W.; J.
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Bhanage, B. M. Catal. Lett. 2006, 112, 17. Song, J.; Zhang, Z.; Han, B.; Hu, S.; Li,
51−55. W.; Xie, Y. Green Chem. 2008, 10,
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47, 77−87. Carbon 2015, 8, 1−11.
4. Sanders, D. P.; Coady, D. J.; Yasumoto,
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The studies of ethane dehydrogenation over supported gallium catalysts
Kittipong Prakobtham, Korawich Trangwachirachai, Kittisak Choojun*, Tawan Sooknoi*
Catalytic Chemistry Research Unit, Department of Chemistry, Faculty of Science,
King Mongkut’s Institute of Technology Ladkrabang, Thailand
*E-mail: kittisak.ch@kmitl.ac.th, kstawan@gmail.com
Abstract:
Ethylene is one of the most demands and valuable olefins used in industries as the
intermediate substrate. Mostly, it is derived from the dehydrogenation of ethane using
supported metal oxide as a catalyst. In this research, the use of 3wt.% Ga/HZSM-5 (28) and
3wt.% Ga/SiO2 prepared by wet-impregnation method for ethane dehydrogenation was
investigated. The effect of reaction temperature from 600 - 700 oC was studied using 3wt.%
Ga/HZSM-5 (28). The total yield of ethane dehydrogenation was enhanced with 9.07, 13.37,
and 19.53% at 600, 650, and 700 oC, respectively. Similarly to total yield, the ethylene
selectivity was increased with 27.27, 47.78, and 78.48%, respectively. Moreover, methane
selectivity obtaining from protolytic cracking was increased with the raise of reaction
temperatures (0.55, 1.04 and 1.71%). Interestingly, BTX was increased with along
temperature profiles then depleted at 700 oC because the aromatization was an exothermic
reaction that is thermodynamic unfavorable at high temperature. In comparison between
HZSM-5 and SiO2 supports, ethane dehydrogenation over 3wt.%Ga/SiO2 at 650 oC provides
lower conversion than 3wt.%Ga/HZSM-5 (28); however, it gives 100% ethylene selectivity.
This could attribute to both the poorer dispersion Ga on SiO2 support and the weak acidic
terminal silanol at the SiO2 surface.
1. Introduction Palladium10 and platinum-based catalysts11

Ethylene is used extensively as a typically provide around 15% conversion.
building block for a wide array of products However, they suffer from the deactivation
including, plastics, rubbers, fuel blending due to the metal sintering. Furthermore,
agents, and chemical intermediates.1-3 The these noble metals favored hydrogenolysis
production of ethylene is mainly via steam leading to high methane selectivity. They
cracking, catalytic cracking and ethane are also relatively expensive. The second
dehydrogenation.4 However, the crude oil class is the metal oxides, for instance,
and natural gas, primary sources to produce chromium oxide, vanadium oxide, and
ethylene, is nonrenewable and the concern gallium oxide. Nakagawa et al.12 showed
of its depletion is raising. While, the that the catalytic activity for ethane
production of light alkanes, especially, dehydrogenation is in the order of β-Ga2O3
ethane is nowadays increased from shale gas (19.6%) > Cr2O3 (12.1%) > V2O5 (9.8%),
extraction process.5 As a result, the cost of where the ethylene selectivity is 95.0%,
these light alkanes has decreased 93.8%, and 97.1%, respectively. This trend
substantially, leading to an opportunity to is also seen in propane dehydrogenation
use ethane as a feedstock for ethylene indicating that gallium oxide is capable to
production. activate C-H bond in light alkanes. In 2017,
Currently, many researchers have Chun-Tao Shao et al.13 shows that gallium
paid attention on the catalytic ethane oxide on HZSM-5 has a higher initial
dehydrogenation (EDH) at temperature in activity for propane dehydrogenation as
the range of 550-700 oC.6-9 The first class of compared to gallium oxide on SiO2, SBA-
catalysts is noble metals over various 15, and Al2O3. However, the gallium oxide
supports, for example, palladium on silica. on HZSM-5 catalyst gives the highest
deactivation rate due to coke formation. In Finally, the H2-TPR analysis was evaluated
order to decrease the deactivation rate of this using 10% H2 in Argon with the total flow
gallium oxide over HZSM-5, Ausavasukhi of 30 mL/min and heating rate of 10 oC/min.
et al.9 reported that under the reduction Water produced from the reduction process
condition (hydrogen atmosphere) the was trapped in a U-shape stainless trap at
deactivation of this catalyst was suppressed. 196 oC (liquid nitrogen) before entering the
This contribute to the readily generation of TCD. The TCD signal was calibrated
gallium hydride (GaH2+) as an active species employing a known mass of CuO standard.
under reducible condition. Though, the 2.3 Catalyst testing
active species and effect of gallium over Gas phase catalytic dehydrogenation
various supports has not been evaluated for of ethane was investigated using fixed-bed
ethane dehydrogenation. In this paper, we continuous flow reactor made with quartz
report the influence of acidity and tube (8.0 mm O.D.) at atmospheric pressure.
confinement of supports towards the Before the catalytic testing, the catalyst was
catalytic performance of gallium-based first activated at 450 oC for 1 h. with the
catalysts for ethane dehydrogenation. flow rate of 30 mL/min of air using the
Moreover, the effect of reaction temperature heating rate of 5 oC/min and 2 oC/min for
for ethane dehydrogenation using supported SiO2 and HZSM-5 catalysts, respectively.
gallium catalyst was investigated. The sample was then cooled down to below
100 oC under N2 atmosphere. It was then
2. Materials and Methods reduced at 550 oC under the stream of H2 (50
2.1 Catalyst preparation mL/min) for 3 h. After that, ethane (99.50%,
Prior to use, Silica (99.00%, Carlo Linde) and helium gas were fed into the
Erba) and NH4-ZSM-5 ratio 28 (Zeolite reactor by mass flow controller (20 mL/min
international) were calcined under zero and 55 mL/min, respectively). The catalytic
grade air in tube furnace by ramping to 550 testing was conducted for the total time on
o
C for 1 h. with heating rate 5 oC/min and 2 stream (TOS) of 6 h. The total products
o
C/min, respectively. The gallium loaded on were analyzed using online-gas
HZSM-5 and SiO2 were prepared by wetness chromatograph equipped with flame
impregnation method using gallium nitrate ionization detector (GC-FID). The heavy
nonahydrate (99.99%, Italmer) as a products were collected in gas sampling
precursor. Then, the catalysts were dried in loop, then injected into the GC column
oven at 80 oC for 24 h. The dried catalysts (Equity-1 column (30m × 0.53mm)). In part
were then calcined using zero grade air for 5 of light products, will be collected in gas
h. at 550 oC with the heating rate of 5 sampling loop, then injected into the GC
o
C/min and 2 oC/min for SiO2 and HZSM-5, column (Rt®-Q-BOND column (30m ×
respectively. 0.53mm)).
2.2 Catalysts characterization
Temperature programmed reduction 3. Result and Discussion
by H2 gas (H2-TPR) was operated using 3.1 Characterization
thermal conductivity detector (TCD). A 100 The surface area and total pore
mg of sample was placed into a quartz tube volume of the catalysts are showed in
reactor that is located inside a temperature- Table1. HZSM-5 and SiO2 possess the
regulated furnace. Before the H2-TPR surface area of 414 and 319 m2/g with a total
operation, each sample was first activated at pore volume of 192 and 1070 µL/g,
450 oC for 1 h. with the flow rate of 30 respectively. The surface area and total pore
mL/min of air using the heating rate of 5 volume after loading Ga is decreased to 351
o
C/min and 2 oC/min for SiO2 and HZSM-5, m2/g (163 µL/g) for 3wt.%Ga/HZSM-5 and
respectively. The sample was then cooled 270 m2/g (1023 µL/g) for 3wt.%Ga/SiO2.
down to below 100 oC under N2 atmosphere. The decrease in surface area and total pore
volume could due to the metal gathering on particle size which requires the higher
the supports and blockage of the pores. reduction temperature.14 Interestingly, the
Eventhough, the surface area of all catalysts deconvolution of reduction peak of
were decreased in the same manner, the 3wt.%[Ga]/HZSM-5 consists of two peaks
decline of total pore volume of similarly to the previous literatures9,15. The
3wt.%Ga/HZSM-5 was more pronounce. It proposed reduction of gallium oxide over
is indicated that the Ga can be well- HZSM-5 was suggested to be GaO+ to Ga+
dispersed in the pore of HZSM-5 as reported and Ga+ to GaH2+ respectively. In silica
by Ausavasukhi et al.9 On the other hand, matrix, the reduction peaks indicates the
bulk particle would be formed in SiO2 nonuniform of gallium compared with
surface as reported by Chun-Tao Shao et HZSM-5 that suggested to be bulk Ga2O3.
al.13
Table 1. The BET surface area and total

pore volume of the catalysts
Catalysts SBETa Total pore volumeb
2
(m /g) (µL/g)
HZSM-5 414 192
3wt.%Ga/HZSM-5 351 163
SiO2 320 1070
3wt.%Ga/SiO2 270 1023
a
BET surface area calculated from the adsorption
branch of the N2 isotherm.
b
.Total pore volumes calculated from the desorption
branch using BJH method.
Figure 1. H2-TPR-profile of a) 3wt.%
The prepared catalysts were
[Ga]/HZSM-5 and b) 3wt.%[Ga]/SiO2
determined the reducibility by using H2-
TPR. The reduction profile of 3wt.%[Ga]
3.2 Catalytic activity
supported on HZSM-5 (28) and SiO2 are
3.2.1 Effect of reaction temperature
illustrated in Figure 1. For
The effect of reaction temperature on
3wt.%[Ga]/HZSM-5 (Fig. 1a) shows the
ethane dehydrogenation was evaluated from
reduction peak at 359-662 oC with the
600 to 700 oC using 3%wt.[Ga]/HZSM-5 as
highest intensity at 491 oC. In sharp contrast,
a catalyst. The results are presented in Table
3wt.%[Ga]/SiO2 shows reduction peaks
2. The total yield is increased from 9.07%,
started from 467 oC and kept raising to 900
o 13.37%, to19.53% upon increasing the
C. This indicates that gallium species over
temperature from 600, 650, to 700 oC,
3wt.%[Ga]/HZSM-5 and 3wt.%[Ga]/SiO2
respectively. Ethylene is a major product at
are not similar. The nature of HZSM-5 is a
this range of temperature study. Similarly to
uniform porous material; while, silica is not.
the total yield, the ethylene selectivity
Therefore, the confinement of a support
enhances with the raise of temperature
could be one of causes of this different
(27.27% (600 oC), 47.78% (650 oC), and
behavior. The dispersion was mainly
78.48% (700 oC). Benzene, toluene, and
corresponded to the different TPR profile.
xylene known as BTX were observed as a
Generally, the small particle size has easily
minor product. It is well known that acidity
reduced more than large particle size. From
in HZSM-5 could convert ethylene to BTX
BET result, it is suggested that Ga is located
via the aromatization reaction. In this case,
inside the pore of HZSM-5 resulting in the
yield of BTX products were increased from
well-dispersed with small particle size
600 to 650 oC but depleted at 700 oC. This
which requires the lower temperature of
could contribute to the exothermic reaction
reduction. Furthermore, Ga over SiO2 tends
of aromatization resulting in the
to have a low dispersion with the larger
Table 2. Ethane dehydrogenation at various temperature over 3 wt.% [Ga]/ HZSM-5 (28)
Yield (%)
Cyclopentene
Total yield
Propylene
Acetylene
Ethylene
Methane
Benzene
Toluene
Temperature
xylene
C4
(oC)
600 9.07 0.05 2.47 0.00 0.07 0.04 0.03 3.35 2.52 0.59
650 13.37 0.14 6.39 0.01 0.11 0.11 0.03 3.75 2.32 0.67
700 19.53 0.35 15.33 0.05 0.15 0.18 0.03 2.23 1.19 0.37
*Condition: 20 mL/min ethane + 55 mL/min Helium, W/F 4 g.h.mol-1, activation in 30 mL/min
zero grade air 450 oC for 1 h and reduction in 50 mL/min hydrogen at 550 oC for 3 h.
thermodynamic unfavorable at temperature xylene are additional products. However, the

700 oC and above. Considering acetylene, it deactivation of the 3wt.%[Ga]/HZSM-5 was
was observed at the temperature above 650 observed consecutively with time on stream.
o
C. Meanwhile, methane yield was increased The total yield of ethane dehydrogenation
from 0.05%, 0.14%, to 0.35% at 600, 650, was decreased from initially 13.37% to
and 700 oC, respectively. Methane is the 6.69% in 6 h. Though, ethylene is still the
cracking product of ethane in which its value majority product. This could contribute to
is lost compared with other products. the BTX formation and coke formation
Therefore, the reaction temperature at 650 remaining in the catalysts which suppress
O
C was selected for further investigation. the catalytic conversion of ethane
dehydrogenation. In sharp contrast, Ga
3.2.2 Effect of supports loaded SiO2 shows the lowest total yield
The comparative study of ethane (0.52%) and gives only ethylene (Figure 2c)
dehydrogenation using gallium oxide on compared with Ga loaded HZSM-5 (28).
different supports was determined. The This clearly shows that gallium species on
supports were categorized in acidity and HZSM-5 and silica are not the same as
confinement; SiO2 (neutral and no mentioned in H2-TPR causing the different
confinement) and HZSM-5 (28) (acidic and catalytic activity. In addition, gallium
channel structure). The ethane dispersion could be another factor. The
dehydrogenation activity over HZM-5 (28) lower gallium dispersion of SiO2 compared
is depicted in Figure 2a. The total yield is with HZSM-5 could retard the ethane
initially at 0.65% and slightly depleted to dehydrogenation. Moreover, the acidity and
0.59% in 6 h. The ethylene is a major confinement of HZSM-5 could more readily
product yielding 0.55%, approximately. stabilize gallium cationic species (GaO+,
Methane, propylene, benzene, and toluene Ga+, and GaH2+) and generate the gallium
are also produced at 0.17, 0.04, 0.04, and active species better than silica.
0.03%, respectively. This indicates that in
condition of HZSM-5 can promoted ethane 4. Conclusion
dehydrogenation better than cracking and Ethane dehydrogenation over 3wt.%
aromatization reaction. The activity and [Ga]/ HZSM-5 (28) at 650 oC gives the
product distribution are the result of only moderate total yield and low amount of
acid sites in this HZSM-5. When Ga was undesirable methane. Ga loaded on different
loaded in this support, total yield (13.37%) support shows the different activity. On
and all products are significantly promoted silica matrix, Ga species are in the form of
as shown in Figure 2b, especially ethylene bulk Ga2O3 which is not completely reduced
yield (6.39%). It indicates that Ga species and inactive for ethane dehydrogenation.
facilitate ethane dehydrogenation. While, in HZSM-5 (28), Ga species are at
Moreover, acetylene, C4, cyclopentene, and exchangeable site in form of GaO+ which
can be reduced to Ga+ and GaH2+. References
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N.; Suzuki, T.; Kobayashi, T. Chem.
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Figure 2. Ethane dehydrogenation over a) 13. Shao, C. T.; Lang, W. Z.; Yan, X.; Guo,
HZSM-5 (28), b) 3wt.%[Ga]/ HZSM-5 (28) Y. J. RSC Adv., 2017, 7, 4710
and c) 3wt.%[Ga]/ SiO2
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This research was supported by SCG 78.
Chemicals Co., Ltd and faculty of science, 15. Trangwachorachai, K.; Numwong, N.;
King Mongkut’s Institute of Technology Sooknoi, T. PACCON. 2017, 1296–
Ladkrabang. 1300.
Nitrogen-enriched activated carbon from shrimp shells as the electrodes
for supercapacitors
Weerawit Luewanichwong, Parinya Inthasuwan, Chakorn Tangsirisatian, Nopachote Sandhu,
Khanin Nueangnoraj*
School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology,
Thammasat University, Khlong Luang, Pathum Thani 12120, Thailand
*E-mail: khanin@siit.tu.ac.th
Abstract:
Supercapacitors (also known as electrochemical capacitors) have transpired as
attractive power sources with high energy densities and long cycling life substituting batteries
in some energy storage fields. Porous carbon materials amalgamated with heteroatoms have
been extensively researched as promising electrode materials for supercapacitors. The
activated carbon is produced from shrimp shells, which naturally contain nitrogen in a form
of chitin and proteins. The raw materials are mixed with lignin to accomplish sufficient
carbon content. Shrimp shells are used to avoid the need to chemically obtain nitrogen from
the doping process. Nitrogen enriched activated carbon is of interest as nitrogen, which can
increase the specific surface area of the original activated carbon by enhancing its surface
functionalities. We have found that by increasing the nitrogen content, specific surface area
will be decreased as a result from the collapse of carbon framework. However, regardless the
low specific surface area, the capacitance is enhanced. This clearly show the effect from N
species present in the carbon framework, which could contribute to pseudocapacitance.
Therefore, the obtained nitrogen enriched activated carbon could potentially be used as
electrode materials for the development of high-performance supercapacitors without the
chemical doping.
1. Introduction capacitor.4 Although metal oxides and their

Energy has become a topic of special composites have shown great potential as
interest over the past few years within electrode materials, carbon is still being
scientific communities due to the rapid used as the leading electrode material for
consumption of natural resources and severe supercapacitors because of its low cost, high
environmental concerns.1 To address and electrical conductivity, excellent chemical
meet the environmental issues and the stability and long cycle life.5 Porous carbon
worldwide energy demand; innovative, materials are strongly recommended for
effective and sustainable solutions are high-performance electrode materials
urgently required. Among the different because these materials have high surface
energy storage technologies, supercapacitors area which can increase the capacity to store
have attracted extensive interest because of electrical charge.
their high power density, fast charge- Recently, the introduction of hetero-
discharge rates, and exceptionally long cycle atoms, such as N, O, P, S or B into the
life.2 Supercapacitors are suitable for high carbon framework is being considered as an
power demanding systems, such as load effective strategy to improve their
levelling and emergency braking. The electrochemical activity, catalytic behaviors
performance of supercapacitors are mainly and adsorption performances. Nitrogen-
dependent on electrode materials.3 enriched activated carbons (NACs) have
Therefore, the fabrication of high attracted significant attention because of
performance electrode materials is essential their excellent conductivity, oxidation
to improve the performance of the super stability and adsorption wettability. In
addition, the presence of nitrogen in the reached the surrounding temperature. The
carbon framework can greatly enhance its as-prepared products were washed with 1 M
surface functionalities. This makes NACs an HCl and finally dried at 105 °C in an oven
attractive area of research for use in overnight.
supercapacitors and as an adsorbent 2.3 Preparation of electrodes for
material. supercapacitor
This work, we aimed to synthesize For the preparation of electrodes, the
the NACs from shrimp shells without using obtained activated carbon is mixed with
chemical doping with higher specific surface carbon black and polytetrafluoroethylene
area than that of commercial AC. (PTFE) with the ratio of 9:0.5:0.5 in a
Additionally, the effect from nitrogen mortar until a thin uniform sheet is obtained.
heteroatom on specific surface area, pore After that, the thin sheet is cut into 1×1 cm2
volume, and capacitive performances will be square shape and placed on the mesh with
investigated. the size of 1.5×10 cm2 and folded. The
folded mesh is then pressed and placed in
2. Materials and Methods the beaker with a 0.5 M of sulfuric acid. The
2.1 Materials electrode is then soaked in the electrolyte
NACs from shrimp shells as the overnight before the measurement. Finally,
electrodes for supercapacitors were prepared the sample is characterized by cyclic
from two types of raw materials, red and voltammetry. Three electrodes configuration
white shrimp shells. The shells were was used with the Ag/AgCl reference.
acquired from Prantalay Marketing Public 2.4 Characterization
Co., Ltd. Lignin, alkali with 3 wt% of sulfur, Activated carbons were characterized
and potassium carbonate (K2CO3) were by CHN analysis to determine the amount of
purchased from Sigma-Aldrich Co., LLC. C, H, and N in the samples. Nitrogen
Hydrochloric acid (HCl) was purchased adsorption-desorption was performed to
from Merck Ltd. characterized the porous textures of the
2.2 Preparation samples. Specific surface area (SBET) was
Each type of shrimp shells was calculated from BET equation. Total pore
prepared separately by the following steps: volume (VTotal) was estimated at relative
first by washing with tap water, then boiling pressure (P/P°) of 0.95. Micropore volume
followed by drying, and finally grinding into (VMicro) was calculated by DR-method.
fine powder. After these steps, the shells Mesopore volume (VMeso) was calculated by
were mixed with lignin and activating agent a difference between total pore volume and
in different compositions as indicated in micropore volume.
Table 1, where WS, RS, L, and K stand for
white shrimp shell, red shrimp shell, lignin, 3. Results and Discussion
and K2CO3 respectively. Each sample was 3.1 Carbon, hydrogen and nitrogen
impregnated with potassium carbonate at content
200 °C. After that, the samples were dried in The CHN results shown in Table 1
the oven at 105 °C overnight. represent the amount of carbon, hydrogen
The impregnated samples were put and nitrogen. The raw materials, white and
into an alumina boat and placed into a red shrimp shells have an amount of
tubular furnace to undergo the carbonization nitrogen approximately 6-7% from chitin
and activation under nitrogen atmosphere. and protein. Therefore, activated carbons
The furnace was heated with the heating rate that prepared from shrimp shells are
of 5 °C per minute to the activation expected to have nitrogen. The ratios of
temperature of 900 °C, held at this shrimp shell are varied from 1:1:2 to 3:1:2
temperature for 1 hour and cooled down as the amount of shrimp shells would
until the temperature of the furnace almost increase the amount of nitrogen in activated
carbon. The results show that increasing the higher than that of the commercial activated
mixing ratio from 1:1:2 to 3:1:2 will carbon (ca. 1,000 m2/g).
increase the nitrogen content from 1.47 to 3.3 Electrochemical behavior
3.98% as expected. Cyclic voltammograms of the
3.2 Porous textures prepared NACs are shown in Figure 1. It can
Specific surface area of activated be seen that all of the samples are mostly
carbon can be characterized by N2-sorption electric double-layer (EDL) capacitance
measurement at 77 K followed by applying since their voltammograms exhibit a
Brunauer-Emmett-teller (BET) equation. rectangular shape. However, a broad peak
The results from N2-sorption measurement around 0.4 V (vs Ag/AgCl) can be observed,
in Table 1 show that increasing the mixing which can attribute to the pseudocapacitance
ratio of shrimp shells tends to increase the from surface functionalities. Specific
surface area to a certain point. After this capacitance was calculated from the
point, the surface area starts to decline. voltammogram and is listed in Table 2. The
Similarly, total pore volume increases to the result show that by increasing the N/C ratio,
certain limit before declining. The mesopore the capacitance will be increased. Generally,
and micropore volume decrease inversely the capacitance of electrode is governed by
proportion to the amount of shrimp shells. two parameters, specific surface area and the
Also, the presence of nitrogen in carbon presence of heteroatoms (nitrogen). In order
framework can increase the specific surface to eliminate the effect from surface area, the
area due to the development of surface capacitance was normalized into farad per
functionalities, but this happens only until a square meter, as shown in Figure 1 and
certain limit. This is because nitrogen atom listed in Table 2. Similarly, the same
has the ability to replace the carbon atom in tendency was observed. By increasing the
the framework. Therefore, adding excess N/C, the normalized capacitance was
amount of nitrogen can cause the framework increased up to 0.13 F m-2, approximately.
of carbon to collapse and yield a lower These results clearly show the effect from
specific surface area. The highest specific nitrogen heteroatom, which improves the
surface area is 2,777 m2/g, which is much electrical storage capacity of the AC.
Table 1. Carbon, hydrogen, nitrogen content (excluding ash) and BET surface area
Sample Mixing C H N SBET VTotal VMeso VMicro
ratio [%w/w] [%w/w] [%w/w] (m2/g)
(cm3/g) (cm3/g) (cm3/g)
White shrimp - 32.161 5.731 6.787 - - - -

shell
Red shrimp - 31.127 5.046 6.653 - - - -
shell
RS:L:K 1:1:2 77.533 3.7561 1.6843 2,777 1.80 0.63 1.17
RS:L:K 2:1:2 75.344 4.2099 2.3389 2,279 1.42 0.40 1.02
RS:L:K 3:1:2 75.763 4.2755 3.4856 1,861 1.28 0.39 0.89
WS:L:K 1:1:2 74.718 3.9185 1.4702 1,830 1.16 0.32 0.84
WS: L:K 2:1:2 79.258 4.0045 3.0961 2,098 1.37 0.39 0.98
WS:L:K 3:1:2 73.839 4.3899 3.9836 1,531 1.01 0.32 0.69
Figure 1. Cyclic Voltammograms of activated carbon with ratio of (a) RS:L:K = 1:1:2, (b)
RS:L:K = 2:1:2, (c) RS:L:K = 3:1:2, (d) WS:L:K = 1:1:2, (e) WS:L:K = 2:1:2, and (f)
WS:L:K = 3:1:2
Table 2. Capacitance of NAC electrode 4. Conclusion

Nitrogen-enriched activated carbon
Sample N/C Capacitance Normalized
(F/g) Capacitance can be successfully prepared without
(F/ m2) chemical doping process. Increasing the
RS:L:K 0.0217 179.7259 0.0647 amount of shrimp shell, which is raw
RS:L:K 0.0310 179.9075 0.0789 material, leads to a higher nitrogen content
RS:L:K 0.0460 200.9881 0.1079 but lower specific surface area in the
WS:L:K 0.0196 179.0526 0.0978
WS:L:K 0.0390 189.3967 0.0902 prepared activated carbon. However,
WS:L:K 0.0539 197.3967 0.1289 regardless the effect from specific surface
area, the normalized capacitance is enhanced
by increasing the amount of nitrogen as a
result of nitrogen functionalities present in References
the carbon framework, which could 1. Abbasi, T.; Abbasi, S. A. Renew.
contribute to pseudocapacitance. Therefore, Sustain. Energy Rev. 2011, 15 (4), 1828–
the obtained nitrogen enriched activated 1834.
carbon could potentially be used as electrode 2. Miller, J. R.; Simon, P. Science 2008,
materials for high-performance 321 (5889), 651–652.
supercapacitors without the chemical 3. Kötz, R.; Carlen, M. Electrochim. Acta
doping. 2000, I (15–16), 2483–2498.
4. Li, C.; Zhang, X.; Wang, K.; Zhang, H.
Acknowledgements T.; Sun, X. Z.; Ma, Y. W.; Xinxing. T.
The authors gratefully acknowledge C. New Carbon Mater. 2015, 30 (3),
the raw materials support from Prantalay 193–206.
Marketing Public Co., Ltd. This work was 5. Liu, W. F.; Yang, Y. Z.; Liu, X. G.; Xu,
supported by Thailand Research Fund (TRF) B. S.; Xinxing, T. C. New Carbon
under a contract number MRG6080153 and Mater. 2016, 31 (6), 594–599.
Thailand Energy Storage System from
National Science and Technology
Development Agency (P-17-50509).
Hydrotreating of pyrolysis oil model compounds with fatty acid and high
water content over molybdenum sulfide catalyst supported on titania
Vituruch Goodwin1*, Paweekasit Sitthanawaitin2,3, Tanakorn Ratana2,3,
Sabaithip Tungkamani2,3
1
National Metal and Materials Technology Center (MTEC), 114 Thailand Science Park, Phahonyothin Road,
Khlong Nueng, Khlong Luang, Pathumthani 12120, Thailand
2
Department of Industrial Chemistry, Faculty of Applied Science,
King Mongkut’s University of Technology North Bangkok, Bang Sue, Bangkok 10800, Thailand
3
Research and Development Center for Chemical Engineering Unit Operation and Catalyst Design (RCC),
King Mongkut’s University of Technology North Bangkok, Bang Sue, Bangkok 10800, Thailand
*E-mail: viturucg@mtec.or.th
Abstract:
The hydrotreating of pyrolysis oil is typically based on hydrodeoxygenation (HDO)
reaction to remove oxygenate compounds and produce hydrocarbons. Pyrolysis oil obtained from
fast pyrolysis of oil containing biomass contained high amount of water and free fatty acid. The
hydrodeoxygenation reaction of pyrolysis oil model compounds with free fatty acid and water
was investigated. The feedstock with fatty acid, guaiacol and up to 10% wt of water was
hydrotreated at 573K for 1 hour. The liquid products were analyzed by GC-MS and GC-FID.
At low water content, 1% wt, catalyst supported on titania showed higher HDO activity and
produced more hydrocarbons than commercial catalyst supported on -Al2O3 due to the
electronic promoting effect of titania. While at high amount of water, 10% wt, catalyst
supported on titania exhibited lower HDO activity compared to commercial catalyst. This
may be due to the active CUS sites of catalyst supported on titania was poisoned by water.
Characterization of the commercial catalyst revealed higher surface area and well dispersion
of CUS active sites than catalyst supported on titania. The deactivation of CUS site was
reversible, and catalyst with more CUS sites could sustain its HDO activity better than
catalyst with lower CUS sites.
1. Introduction to fuel. The catalytic hydrodeoxygenation

Recent interest in development of reaction which reduces oxygenates and
renewable energy and environmentally transforms them to hydrocarbons containing
friendly alternative fuel is increasing compounds is a crucial process to improve
worldwide. The alternative fuel from local pyrolysis fuel properties.3 Molybdenum
biomass resources can be produced to sulfide catalyst is very common and most
promote Thailand’s energy security policy. efficient hydrotreatment catalyst which has
Pyrolysis oil from fast pyrolysis of biomass been studied and showed good catalytic
gives high bio-organic liquid yield that can performance for pyrolysis oil upgrading.4,5
be converted to fuel. The liquid product However, typical pyrolysis oil from fast
from pyrolysis of lignocellulosic materials pyrolysis of biomass contained high amount
contains hundreds of hydrocarbons and of water which cause detrimental effect on
oxygen containing organic compounds such catalyst activity. The molybdenum sulfide
as organic acids, aldehydes, ketones and catalyst is deactivated by water reacting to the
phenolics. These oxygenates in pyrolysis oil catalyst’s active site. In this study, the effect
give poor fuel properties including low of high water content in pyrolysis oil on
heating value and low stability.1,2 The hydrodeoxygenation was investigated using
catalytic hydrotreatment of pyrolysis oil is pyrolysis oil model compounds consisting of
an effective process to upgrade pyrolysis oil guaiacol, free fatty acid, linoleic acid and
water added into the mixtures and real The hydrodeoxygenation reaction,
pyrolysis-oil (10%wt. water) from fast HDO, of pyrolysis oil model compounds
pyrolysis of jatropha cake. Hydrotreatment mixture and real pyrolysis oil was carried
of feedstock with 1%wt. and 10%wt. water out in a 50 mL high pressure microreactor
added was done using CoMo sulfide catalyst (Parr model 4590). Each batch was filled
supported on titania, CMT and commercial with 20 mL of feedstock, which consisted of
CoMo sulfide catalyst supported on -Al2O3, a mixture of 5%wt. guaiacol as pyrolysis oil
CMA-Comm as catalyst to evaluate high model compound, 2.5%wt. linoleic acid as
water content effect on catalytic activity in free fatty acid, water and dodecane as
hydrodeoxygenation reaction. solvent. Up to 10% wt. of water was added
to evaluate the effect of high water content
2. Materials and Methods feedstock. For real pyrolysis oil, only 5%wt.
2.1 Preparation and pretreatment of of pyrolysis oil (contained 10%wt. of water)
catalysts in dodecane was used as feedstock. Then 0.4
All catalysts used in this project are g of sulfide catalyst was added with N2 gas
molybdenum sulfide catalysts. The Mo-TiO2 flushing to avoid any air contamination and
catalyst synthesized in our laboratory was the closed reactor was flowed with N2 gas
prepared via sol-gel synthesis of TiO2 for 10 min before H2 gas was added. All
support with ammonium molybdate as Mo hydrotreating experiments were performed
precursor and titanium (IV) isopropoxide at 573 K for 1 hour with initial hydrogen
(Acros OrganicTM, 98+%) as TiO2 precursor. pressure of 4 MPa. Investigation of catalytic
The synthesized Mo-TiO2 gel was dried at hydrotreatment was performed using two
318K for 48 h and calcined at 823K for 4 h supported catalysts, cobalt molybdenum
then sieved to 250-600 mm range. This sulfide on TiO2 (CMT) and commercial
calcined catalyst was then impregnated with cobalt molybdenum sulfide on -Al2O3
cobalt (II) nitrate aqueous solution then (CMA-Comm). The liquid hydrotreated
dried at 318K for 48 h and calcined at 823K product was collected and analyzed by GC-
for 4 h again to obtain CoMo-TiO2 calcined MS and GC-FID equipped with a non-polar
catalyst, CMT. Commercial CoMo--Al2O3 DB-1, 60m × 250μm × 0.1μm column.
calcined catalyst, CMA-Comm and CMT
catalysts were pretreatment to sulfide form 3. Results and Discussion
with 5%H2S/H2 at 673K for 4 h before 3.1 Catalyst characterization
hydrodeoxygenation reaction. Two calcined catalysts, cobalt
2.2 Catalyst characterization molybdenum supported on TiO2, CMT, and
Both calcined catalysts were commercial cobalt molybdenum supported
analyzed for surface area, pore volume and on -Al2O3, CMA-Comm, were analyzed by
pore diameter using BELsorp-mini N2 adsorption, BET, method to determine
instrument to measure volume of N2 gas surface area and pore properties. The results
adsorbed at 77K, using P/P0 of 0.1, 0.2 and from BET analysis are shown in Table 1.
0.3 according to BET technique. The sulfide
catalysts were characterized for sulfide edge Table 1. BET analysis results of two
active sites, CUS, using NO pulse catalysts, CMT and CMA-Comm (analysis
adsorption method conducted in BELCAT-B in calcine form)
chemisorption equipment. TEM Catalysts Surface Pore Average
Micrographs of sulfide catalysts were taken Area Volume Pore
BET (cm3/g) Diamerter
with Jeol JEM-2010 Transmission Electron (m2/g) (nm)
Microscope. CMT 91.07 0.18 7.89
2.3 Catalytic hydrotreatment of pyrolysis CMA-Comm 219.45 0.41 8.36
oil and model compounds
The BET analysis elucidated that amount of NO which is selectively adsorbed
CMA-Comm catalyst has more surface area on the MoS2 CUS sites. On the other hand,
at 219.45 m2/g compared to CMT catalyst the spent-m-CMT catalyst after being used
which has lower surface area at 91.07 m2/g. in hydrotreatment of pyrolysis oil model
Moreover, CMA-Comm also exhibited more (10%wt. water content) exhibited lower
pore volume than CMT but both catalysts decrease in NO absorption at 28% but the
have similar average pore diameter in the spent-m-CMA-Comm had 44% decrease of
range of 7.89 to 8.36 nm. The large pore CUS sites on its surface. This inferred that
diameter is suitable to accommodate large CMT catalyst is more water tolerate than
molecules in pyrolysis oil models feedstock CMA-Comm. The NO absorbed by spent-o-
such as guaiacol and linoleic acid. In CMT also higher than CMA-Comm. The
commercial catalyst, CMA-Comm support strong interaction of CoMoS to -Al2O3 may
is -Al2O3 in amorphous phase while CMT play an important role in water poisoning on
support is an anatase TiO2 in semi- CMA-Comm CUS sites.5 On the contrary,
crystalline phase which resulted in lower water poisoning on CMT supported on
surface area of TiO2 support catalyst. Both titania was inhibited due to its weak
sulfide catalysts, CMT and CMA-Comm
were characterized before hydrotreatment,
denoted fresh-, and after hydrotreatment
experiment, denoted spent-, to evaluate
sulfide edge active sites, CUS, via NO pulse
chemisorption method. The NO adsorbed is
(a) CMT
calculated and reported in total NO amount
absorbed, NO absorbed per gram of catalyst
and NO absorbed per catalyst surface area as
shown in Table 2.
Table 2. NO chemisorption analysis results

of CMT and CMA-Comm (in sulfide form)
before hydrotreament, fresh-catalyst, and
after hydrotreatment, spent-catalyst
Catalysts NO abs. NO abs.
volume /surface
(cm3/g) area
(cm3/m2)
Fresh-CMT 5.0152 0.0551
Spent-m-CMTa 3.5941 0.0395
Spent-o-CMTb 3.7007 0.0406
Fresh-CMA-Comm 9.7877 0.0446
Spent-m-CMA-Comma 5.4454 0.0248
Spent-o-CMA-Commb 3.4561 0.0157
a
Spent-m- = catalyst after hydrotreatment in model
compounds with 10%wt. water.
b
Spent-o- = catalyst after hydrotreatment in real
pyrolysis oil compounds (~10%wt. water).
For fresh-catalysts, amount of NO

absorbed on CMA-Comm was higher at
9.7877 mol/g catalyst than CMT at at 5.0152 (b) CMA-Comm
mol/g catalyst. These results indicated that Figure 1. TEM micrographs of (a) freshly
CMA-Comm in fresh form contains more sulfided CMT and (b) freshly sulfided
MoS2 active CUS sites according to higher CMA-Comm
catalyst-support interaction from electronic from GC-MS results and product
induced promote effect of titania.6 The TEM distribution was analyzed by GC-FID as
micrographs of fresh sulfide catalysts, CMT shown in Figure 2. The hydrotreatment of
and CMA-Comm, were depicted Figure 1. m-feed with CMA-Comm exhibits higher
TEM micrograph of CMT revealed HDO activity, lower oxygenates in products,
that CoMoS edge active site7 was formed and produces more hydrocarbons than CMT
which can be seen as lines with a long slab catalyst. However, it should be noted that
length between 2-6 nm. Long sulfide edge majority of oxygenates product from m-
was formed due to its weak interaction with feed-CMT is phenol which is actually a
titania. However CMT as seen in Figure 1 product from HDO of Guaiacol. Thus, CMT
had low number of sulfide edge stacking. In is still active for HDO but the reversible of
the other hand, CoMoS edge active site on water poisoning on the active CUS site of
CMA-Comm was formed with a short slab catalyst supported on titania is slower than
length around 1-3 nm and high number of commercial catalyst supported on. -Al2O3.
stacking. This formation pattern of CoMoS Since CMT has lower surface area and
edge confirmed the strong interaction lower CoMoS edge site per gram of catalyst,
between CoMoS with -Al2O3 support. at 10%wt. of water added most of its active
3.2 Catalytic hydrotreatment of pyrolysis site is affected by water. Contrary to CMA-
oil and model compounds Comm that has high surface area and well
The hydrodeoxygenation reaction of dispersion of CUS active site, at 10%wt. of
pyrolysis oil model compounds containing water added, the catalyst still retain its CUS
guaiacol and linoleic acid as free fatty acid active site. For hydrotreatment of real
and 10%wt. of water added, named m-feed, pyrolysis oil, o-feed, CMT exhibits higher
and real pyrolysis oil from fast pyrolysis of HDO activity, this is due to the high free
jatropha cake, named o-feed, was performed fatty acid content in pyrolysis oil from
at 573K under 4 MPa of H2 atmosphere for jatropha cake. The free fatty acid reactant is
1 hour. The hydrotreated liquid products more reactive and goes through HDO
from hydrotreatment using two catalysts, process before others oxygenates groups
CMT supported on titania and CMA-Comm such as phenolics, aldehydes or ketones.
supported on -Al2O3 were analyzed by GC- This is also the effect of electronic
MS and GC-FID. The hydrocarbon, HCs, promotion by titania support of CMT which
and oxygenates compounds were identified accelerate the HDO of free
m-feed-CMA-Comm | m-feed-CMT | o-feed-CMA-Comm | o-feed_CMT
Figure 2. Product distribution from hydrotreatment of m-feed-CMA-Comm, m-feed-CMT, o-

feed-CMA-Comm and o-feed-CMT
fatty acid. While CMA-Comm CUS site is Acknowledgements
poison by free fatty acid which caused steric The authors would like to thank
hindrance effect on CoMoS CUS site of National Metal and Materials Technology
CMA-Comm. Hydrodeoxygenation of real Center (MTEC), NSTDA for financial
pyrolysis oil from jatropha cake with CMA- support. The authors gratefully acknowledge
Comm given mixtures of products because Development Center for Chemical
CoMoS on alumina is less selective Engineering Unit Operation and Catalyst
compared to CMT. Free fatty acid has effect Design (RCC), KMUTNB for the
on CMA-Comm more than CMT. collaboration and support of research
facilities for this project.
4. Conclusion
CMT catalyst supported on titania References
showed higher HDO activity for 1. Czernik, S.; Bridgwater, A. V. Energ.
hydrodeoxygenation of real pyrolysis oil Fuel. 2004, 18 (2), 590−598.
from jatropha cake residue and produced 2. Blin, J.; Volle, G.; Girard, P.;
more hydrocarbons than commercial catalyst Bridgwater, T.; Meier, D. Fuel 2007, 86
supported on -Al2O3 due to the electronic (17-18), 2679−2686.
promoting effect of titania. The effect of 3. Mortensen, P. M.; Grunwaldt, J. D.;
High amount of water content at 10%wt. Jensen, P. A.; Knudsen, K. G.; Jensen,
was investigated. CMT catalyst supported A. D. Appl. Catal., A 2011, 407 (1-2),
on titania exhibited lower HDO activity in 1−19.
hydrodeoxygenation of model compounds 4. Bui, V. N.; Laurenti, D.; Delichère, P.;
compared to commercial catalyst. This may Geantet, C. Appl. Catal. B. 2011, 101 (3-
be due to the active CUS sites of catalyst 4), 246−255.
supported on titania was poisoned by water. 5. Goodwin, V. PhD's Degree Thesis, King
Characterization of the commercial catalyst Mongkut’s University of Technology,
revealed higher surface area and well North Bangkok, 2014.
dispersion of CUS active sites than CMT 6. Walton, A. S.; Laritsen, J. V.; Topsøe,
catalyst on titania. The deactivation of CUS H.; Besenbacher, F. J. Catal. 2013, 308,
site was reversible, and catalyst with more 306−318.
CUS sites could sustain its HDO activity
better than catalyst with lower CUS sites.
The effect of carbon, nitrogen, and CuSO4 on laccase production by
Bacillus sp. strain LA1
Prasert Nimnual*, Sinthuwat Ritthitham
Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University,
Muang, Nakhon Pathom 73000, Thailand
*E-mail: nimnual_p2@su.ac.th
Abstract:
Laccase is widely used in bioremediation and beverage manufacturing. The aim of
this study was to optimize the concentration of carbon and nitrogen sources including the
effect of CuSO4 on growth and laccase production from Bacillus sp. strain LA1. The laccase
activity was maximum at 119.5 units/mL in the mineral medium containing soluble starch
(20.0 g/L), yeast extract (5.0 g/L) and CuSO4 (0.05 mM). The cell growth and laccase
production were completely inhibited with the medium containing with CuSO4 concentration
higher than 0.2 mM.

Laccases (p-benzenediol: oxygen 2.1 Microorganism
oxidoreductase EC 1.10.3.2)1 are the Bacillus sp. strain LA1 was
member of multicopper oxidases catalyzing employed as a laccase producer throughout
the removal of a hydrogen atom from the experiments. The bacterial cells were
phonolic and non-phenolic compounds cultivated at 37 °C in minimum medium
leading to form free radicals and undergoing agar plate containing glucose 10 g/L,
further depolymerization, demethylation or peptone 3.0 g/L, KH2PO4 0.6 g/L, K2HPO4
quinine formation2 Laccases have been used 0.4 g/L, ZnSO4·7H2O 0.001 g/L,
in many industrial processes especially FeSO4·7H2O 0.0005 g/L, MgSO4·7H2O 0.5
industries to increase product quality. In g/L, MnSO4·H2O 0.05 g/L with 0.2% (v/v)
textile industry, laccases have been used to guaiacol.7 After incubation for 4 days, the
remove colors in wastewater treatment. bacteria were stored at 4 °C and the stock
Laccase is widely distributed in were then transferred to fresh medium every
plants and wood-degrading fungi; however, month.
bacteria-producing laccases were also The pre-cultures were incubated at
reported, such as laccase from Bacillus sp. 37 °C for 4 days with agitation at 200 rpm.
WD23,1 Bacillus vallismortis,3 Enterobacter 5 mL of pre-cultures was subsequently
cloacae,4 Bacillus sp. PK45 and from transferred to 45 mL fresh liquid medium in
Bacillus subtilis MTCC 1039.6 Recently, 250 mL flasks following by incubation at
Bacillus sp. strain LA1 producing laccase 37 °C using a speed of 200 rpm.
was isolated from soil samples. The laccase 2.2 Effects of carbon and nitrogen sources
production by Bacillus sp. strain LA1 in on laccase production
minimum medium was relatively low. The General full factorial design was
laccase synthesis and secretion is influenced carried out to verify the effects and
by many factors, including nutrient levels, interactions of carbon sources (glucose,
culture conditions, and addition of inducers fructose, sucrose, lactose and starch) and
to the culture mediums. Therefore, nitrogen sources (yeast extract, peptone and
optimization of medium compositions and ammonium nitrate) on laccase production.
their concentration was the aims of this The Minitab version 16 software was used
study. for regression and graphical analyses of the
data obtained. A set of 15 experiments with
three replicates was performed. The levels water with starch and yeast extract as the
of the variables herein investigated were carbon and nitrogen sources, respectively.
shown in Table 1. The components of the The inoculum (10% V/V) was transferred to
minimum medium were dissolved in 45 mL the flasks and the culture was incubated at
of distilled water, with 10 g/L of carbon 37 °C with a speed of 200 rpm. After
source and 2.5 g/L of nitrogen source.8 The incubation for 96 hours, the cultures were
pH of the medium was adjusted to 6.0 taken for the determination of cell dry
before autoclaving (121 °C /15 min). The weight and laccase activity. Data are means
inoculum (10% V/V) was transferred to the of three replicates and evaluated by Minitab
flasks and the culture was incubated at program.
37 °C on a rotary shaker at 200 rpm. After
incubation for 96 hours, the cell cultures Table 2. Levels of the independent variables
were taken for the determination of cell dry used in the factorial design for carbon and
weight and laccase activity. nitrogen
(A) Carbon (B) Nitrogen
Run
Table 1. Levels of the independent variables concentration concentration
used in the factorial design for carbon and 1 1 1
nitrogen source 2 1 2
(A) Carbon (B) Nitrogen 3 1 3
Run 4 2 1
source source
1 1 1 5 2 2
2 1 2 6 2 3
3 1 3 7 3 1
4 2 1
8 3 2
5 2 2
6 2 3 9 3 3
7 3 1
8 3 2 (A) Carbon (B) Nitrogen
Level
9 3 3 concentration (g/l) concentration (g/l)
10 4 1 1 5 1.25
11 4 2 2 10 2.50
12 4 3 3 20 5.00
13 5 1
14 5 2
15 5 3 2.4 Effect of copper sulfate concentrations
on laccase production
(A) Carbon (B) Nitrogen The optimized medium was
Level
source 10.0 g/l source 2.5 g/l supplemented with different concentrations
1 glucose yeast extract
2 fructose peptone
of copper sulfate varying from 0 to 1.0 mM.9
3 sucrose ammonium nitrate Incubation was carried out at 37 °C, 200
4 lactose rpm. At periodic intervals, 10 mL of
5 starch supernatant was withdrawn and centrifuged
at 12,000 rpm for 10 min. Cell growth was
2.3 Effects of carbon and nitrogen determined gravimetrically after drying cell
concentrations on laccase production pellet at 105 °C and the laccase activity was
The concentration of carbon and measured using 2,6-dimetoxylphenol (DMP)
nitrogen in the production medium was as the substrate. The data were expressed as
further optimized using a General full the means of at least three different cultures.
factorial design by performing a set of 9 2.5. Analytical methods
experiments with three replicates. The levels 2.5.1 Extraction of intracellular enzyme
of the variables investigated were shown in The bacterial cells were centrifuged
Table 2.8 The component of the minimum at 12000 rpm for 10 min at 4 °C. The cells
medium was dissolved in 45 mL of distilled obtained as pellet were washed twice with
25 mM sodium phosphate buffer pH 6.0 and suitable nitrogen source for laccase
resuspended in 2 ml of 25 mM sodium production from Bacillus sp.9
phosphate buffer pH 6.0. The protein
extraction was carried out by sonication for Table 3. Cell dry weight and laccase activity
10 min at 20% amplitude and 10 s / cycle. in the medium with different carbon and
Cell debris was removed by centrifugation at nitrogen sources as described in Table 1
14,000 rpm for 20 min at 4°C and the Run
Cell dry Laccase activity
supernatant obtained was used as the weight (g/L) (U/ml)
1 0.61 ± 0.06 11.44 ± 0.02
intracellular enzyme.
2 0.84 ± 0.05 5.62 ± 0.08
2.5.2 Laccase assay 3 0.62 ± 0.06 0.67 ± 0.00
Laccase activity was estimated by 4 0.72 ± 0.04 10.77 ± 0.06
using DMP as the substrate.5,10 200 µL of 5 0.80 ± 0.02 6.39 ± 0.33
supernatant was added to 800 µL of 25 mM 6 0.73 ± 0.09 3.39 ± 0.59
7 0.91 ± 0.09 5.03 ± 0.75
sodium phosphate buffer pH 6.0 containing
8 0.65 ± 0.02 5.33 ± 0.00
2 mM DMP dissolved in 10% (v/v) absolute 9 0.39 ± 0.05 1.33 ± 0.28
and subsequently incubated at 60 °C for 30 10 1.12 ± 0.03 13.09 ± 0.64
min. One unit of laccase was defined as the 11 1.78 ± 0.07 8.98 ± 0.84
amount of enzyme causing an increase in 12 0.37 ± 0.09 5.90 ± 0.28
13 2.03 ± 0.03 29.48 ± 0.37
0.01 of absorbance at 470 nm in 30 minutes.
14 1.34 ± 0.07 12.36 ± 0.33
2.5.3 Cell dry weight determination 15 1.19 ± 0.09 2.93 ± 0.95
Culture samples were centrifuged at
8000 rpm for 15 min. The cell pellet was 3.2 Effects of carbon and nitrogen
washed and centrifuged and then re- concentration
suspended in distilled water. The dry weight A production medium supplemented
was estimated after dryer at 105 °C.8,11 with 20 g/L starch and 5.0 g/L yeast extract
2.5.4 Total sugar analysis was resulted in the maximum laccase
Total sugar was determined production with 119.59 ± 0.13 U/mL, as
according to the method of Dobois et al 12 shown in Table 4, For Bacillus sp. the
using glucose as the standard. production of laccase achieved the
maximum level of 107.32 U/mL in the
3. Results and Discussion concentrations of 1.0 g/L glucose and 0.1
3.1 Effects of carbon source and nitrogen g/L peptone.9
sources
The different carbon sources Table 4. Cell dry weight and laccase activity
(glucose, fructose, sucrose, lactose and in the medium with different concentration
starch) and nitrogen sources (yeast extract, of starch and yeast extract as described in
peptone and ammonium nitrate) were tested Table 2
at 10.0 g/L and 2.5 g/L; respectively. The Cell dry weight Laccase activity
Run
results of cell growth and laccase activity for (g/L) (U/ml)
all 15 experiments were analyzed by 1 0.49 ± 0.16 42.02 ± 0.14
Minitab version 16. As shown in Table 3, 2 0.80 ± 0.23 31.54 ± 0.61
the maximum cell growth of 2.03 ± 0.03 g/L 3 1.12 ± 0.32 80.68 ± 0.29
and maximum laccase activity of 29.48 ± 4 0.97 ± 0.18 45.01 ± 0.41
0.37 U/mL were obtained in a production 5 1.26 ± 0.42 22.39 ± 0.54
medium containing starch and yeast extract. 6 1.40 ± 0.18 93.20 ± 0.38
7 1.42 ± 0.12 32.34 ± 0.59
As previous reported1, Streptomycetes sp.
8 1.67 ± 0.23 15.72 ± 0.75
could considerably produce laccase in a
9 1.67 ± 0.74 119.59 ± 0.13
medium containing starch as the carbon
source while the yeast extract was the
3.3 Effect of copper sulfate concentrations
on laccase production
The effect of copper sulfate
concentrations on the growth of Bacillus sp.
strain LA1 and laccase production was
investigated.
Figure 2. Time course of cell growth,

laccase production and sugar utilization of
Bacillus sp. strain LA1 in the medium
containing 10 g/L starch and 5 g/L and 0.05
mM CuSO4
0.05 M CuSO4 was presented in Figure 2.

The specific growth rate of 7.78 h-1 was
achieved with the maximum biomass of 56
g/L and laccase activity of 84.40, 0.01 U/mL
after 24 hr of cultivation. The total sugar
was decreased to 4.43 g/L
4. Conclusion
In this study, the compositions of
laccase production medium for Bacillus sp.
strain LA1was optimized by General full
factorial design. Starch (20 g/L) and yeast
Figure 1. The effect of CuSO4 on growth of extract (5 g/L) used as the carbon and
Bacillus sp. strain LA1 and laccase nitrogen sources showed the strongest effect
production in the medium containing 10 g/L on laccase activity among the tested
starch and 5 g/L with different CuSO4 substances. The addition of 0.05 mM copper
concentrations sulfate in the medium was effectively
enhanced the laccase production.
The results shown in Figure 1
indicated that laccase activity in the medium Acknowledgements
with 0.05 mM CuSO4 increased to 28 folds, This work was supported by the
as compared to the medium without CuSO4 Department of Biotechnology, Faculty of
addition. Moreover, it was observed that the Engineering and Industrial Technology,
medium containing CuSO4 higher than 0.2 Silpakorn University.
mM caused the inhibition of cell growth and
laccase production. The highest laccase References
activity of 107.32 U/mL was reported when 1. Wang, C. L.; Zhao, M.; Li, D. B.; Cui,
cultivated Bacillus sp. in the medium D, Z.; Lu, L.; Wei, X. D. Afr. J.
supplemented with 1 mM copper sulfate.9 Biotechnol. 2010, 9, 5496−5502.
Moreover, Bacillus subtilis WD23 required 2. Muthukumarasamy, N. P.; Jackson, B.;
1 mM copper sulfate for maximum laccase Joseph R. A.; Sevanan, M. Biochem.
production (141.4 ± 6.64 U /ml).2 Res. Int. 2015, 2015, 9.
Time course of Bacillus sp. growth 3. Zhang, C.; Diao, H.; Lu, F.; Bie, X.;
and laccase production in liquid medium Wang, Y.; Lu, Z. Bioresour. Technol.
with 20 g/L starch, 5 g/L yeast extract and 2012, 126, 80−86.
4. Demissie, A. G.; Kumar, A. Int. J. Biochem. 2006, 41, 581−588.
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8. Revankar, M. S.; Lele, S. S. Process
Methanol to gasoline conversion over ZSM-5 zeolites: influence of catalyst
property and operating conditions
Lalita Attanatho1*, Wanchana Sisuthog1, Yoothana Thanmongkhon1,
Amornrat Suemanotham1, Teerawit Laosombut1, Siriporn Larpkiattaworn2
1
Research, Pathum Thani 12120, Thailand
2
Expert Centre of Innovative Material, Thailand Institute of Scientific and Technological Research,
*E-mail: lalita@tistr.or.th
Abstract:
The catalytic conversion of methanol to gasoline (MTG) has drawn attention, due to
the possibility to be an alternative pathway for biofuels productions from readily available
carbon sources. The present study focused on the influence of catalyst property and operation
conditions on MTG reaction that performing in milli-fixed bed reactor. The influence of
SiO2/Al2O3 ratios (i.e. 26, 30, 80 and 280) of ZSM-5 zeolite catalysts, reaction temperatures
(250-400 oC) and pressures (1-10 bars) at weigh hourly space velocity of 2 h-1 on methanol
conversion, liquid hydrocarbon yield and product distribution were investigated. Results
revealed that methanol could be converted to gasoline range biofuel over ZSM-5 zeolite
catalysts. At higher SiO2/Al2O3 ratio, the improvement in methanol conversion and
selectivity toward iso-paraffin in liquid hydrocarbons products were achieved, while the C1-
C4 gaseous selectivity decreased. ZSM-5 catalyst with SiO2/Al2O3 ratio of 80 exhibited the
best catalytic performance in terms of highest liquid hydrocarbons yield. The major products
obtained including iso-paraffin, aromatics and naphthenes. Furthermore, yield of liquid
hydrocarbons and selectivity toward aromatics hydrocarbons increased with the increasing of
temperature and pressure.
1. Introduction Numerous studies revealed that the

The catalytic upgrading of methanol, zeolite catalysts, particularly Zeolite Socony
so-called methanol to gasoline (MTG), is Mobile-5 (ZSM-5), have significant effect
one of the promising technologies for on MTG process.2-4 ZSM-5 is a network of
gasoline production from biomass, coal and SiO4 and AlO4 with possess high porosity
natural gas. Methanol, a readily available and interesting pore network. It has been
carbon sources, is normally produced from used as shape selective catalyst, due to the
syngas (mixture of carbon monoxide and presence of micropore in its structure.
hydrogen) through biomass/coal gasification Molecules with a greater critical size than
process or natural gas steam reforming that of durene are difficult to diffuse inside
process.1 ZSM-5 structures which lead to the high
It can be further converted into selectivity in MTG.3
gasoline range hydrocarbons via MTG MTG reaction is highly exothermic
process. The first step of MTG reaction is a and the poor heat and mass transfer for
methanol dehydration to acquire dimethyl conventional fixed bed reactor limit the
ether (DME) which is then converted to further development of this technology.
light olefins. The light olefins further react Several methods have been proposed to
to form products in the gasoline boiling release the reaction heat and preventing hot
range. These products can react with spot creation within the conventional reactor
oxygenates or with light olefins to produce such as lowering the methanol partial
additional gasoline product.2 pressure, addition of water to the feed or
using multi-stage fixed bed reactors with This stream was mixed with nitrogen carrier
inter-stage cooling.4 gas that controlled flow rate at 20 mL min-1
Milli-fixed bed reactor is a reactor by mass flow controller. The mixture was
with the diameter within millimeter scale. then introduced into the reactor which
The reduction of diameter to millimeter controlled temperature by the electric
allows for an intensification of heat transport furnace. A thermocouple was placed at the
in radial direction, avoid unexpected transfer center of catalyst bed for monitoring the
limitation and thus isothermal operation reaction temperature. The system pressure
even of highly exothermic reactions was controlled by back pressure regulator.
becomes feasible. Milli-fixed bed reactor The distributions of gaseous product
has been tested for Fischer-Tropsch reaction were monitored by the on-line GC-FID/TCD
and showed promising results with the high system. The liquid products, i.e. separated
volume specific productivity at acceptable into aqueous phase and oil phase, were
pressure drops.5, 6 Therefore, this reactor is collected from the condenser below the
considered as an interesting concept for reactor. The oil phase was analyzed with
MTG process. The aim of the present study GC-FID with the aided of detailed
was to investigate the influence of catalyst hydrocarbon analysis (DHA) software and
property and operating conditions on MTG aqueous phase was analyzed with GC-FID
process in milli-structured fixed bed reactor. equipped with head space. It should be
Consequently, influence of SiO2/Al2O3 notified that DME is considered to be
ratios of ZSM-5 zeolite catalyst, reaction unconverted reactant and would be summed
temperature and pressure on methanol together with methanol for methanol
conversion, liquid hydrocarbons’ yield and conversion and selectivity calculation in the
product distribution were investigated. present study.
Mass flow controller
2. Materials and Methods N
2.1 Materials T
Methanol (99% purity) was Furnace and

Temperature controller
purchased from RCI Labscan. Commercial
Fixed bed reactor
ZSM-5 zeolites with SiO2/Al2O3 ratio of 26,
30, 80 and 280 (i.e. ZSM5-26, ZSM5-30,
ZSM5-80 and ZSM5-280) were purchased
GC-FID
from ACS Material, Advanced Chemicals Condenser
Supplier and Zeolyst International. Prior to
be used, the ammonium form ZSM-5 zeolite Back pressure
was pretreated under static air at 550 °C for HPLC pump MeOH regulator Gaseous
product
4 h, in order to obtain the catalyst in
protonic form. The catalyst was then Liquid product
compacted by compression, crushed and
sieved to obtain 20-40 mesh size particles. Figure 1. Schematic diagram of MTG
2.2 MTG process process
The MTG experiments were
performed in milli-fixed bed flow reactor 3. Results and Discussion
made of 316 stainless steel tube with 2 mm 3.1 Effect of SiO2/Al2O3 ratios
inner diameter, shown schematically in The MTG reactions over ZSM-5
Figure 1. Typically, 1.0 g of calcined ZSM-5 catalysts with SiO2/Al2O3 ratios ranging
catalyst was loaded into the reactor. from 26 to 280 were performed at 400 °C, 1
Methanol was pumped by high pressure bar and weigh hourly space velocity
liquid pump at a flow rate of 0.042 mL min-1 (WHSV) of 2 h-1. The obtained results
and then passed through pre-heater tubing. indicated that all catalysts performed
efficiently with more than 90% methanol either cracked into smaller molecules or
conversion. As shown in Figure 2, the major trapped and forming coke inside the pores.
products of MTG process were For ZSM-5 catalyst, hydrocarbons higher
hydrocarbons (i.e. gaseous and liquid than C11 are too large to be formed inside
phases) and water that generated from the pores.8 The liquid hydrocarbons products
dehydration reaction. The decreasing of mainly contained C7-C11 hydrocarbons
SiO2/Al2O3 ratio, in particular from 280 to (Figure 3a), which are similar to gasoline
26 resulting for a noticeable improvement in fraction of conventional fossil fuel.
total hydrocarbon yield, i.e. gaseous Therefore, the observed high gasoline
hydrocarbon and liquid hydrocarbon. fraction could be beneficial for producing
ZSM5-26 provided the highest total transportation fuel from methanol. Figure 3b
hydrocarbon yield which probably due to the shows a comparison of liquid hydrocarbons
available numbers of strong acid sites on the products distribution which can be grouped
surface. Generally, the strong acid site into 6 groups: (1) paraffins, (2) iso-paraffins,
density decreased with the increasing of (3) olefins, (4) naphthenes, (5) aromatics,
SiO2/Al2O3 ratio.7 However, the catalyst and (6) oxygenates.
with higher strong acid site density would 40
favor the generation of light hydrocarbons, (a) ZSM5-26
whereas the catalyst with lower strong acid ZSM5-30
30
site density would favor the formation of Selectivity (%) ZSM5-80
higher hydrocarbons. Accordingly, gaseous ZSM5-280
hydrocarbon yield (C1-C4) was found to be 20
increased with the decreasing SiO2/Al2O3
ratio. 10
100
Product distribution (wt%)
0
C9
C5
C6
C7
C8
C15+
C10
C11
C12
C13
C14
80
60 80
products distribution (wt%)
(b) ZSM5-26
70
40
Liquid hydrocarbons
ZSM5-30
60
ZSM5-80
20 50
ZSM5-280
40
0 30
ZSM5-26 ZSM5-30 ZSM5-80 ZSM5-280
20
CO+CO2+H2
CO+CO 2+H2 Unconverted MeOH
Unconverted MeOH 10
Water
Water Gaseous hydrocarbon
Gaseous hydrocarbon 0
Liquid hydrocarbon
Liquid hydrocarbon
Figure 2. Product distribution (400 °C, 1 bar

WHSV=2 h-1)
Figure 3. Carbon distribution (a) and
ZSM-5 catalyst possesses a unique product distribution (b) of liquid
shape enhancing the selectivity property hydrocarbons products, (400 °C, 1 bar,
which related to its pores structure. WHSV=2 h-1)
Particularly, only product species with the
size of molecule less than the pore size For all catalysts, the dominant
could be diffuse out of the catalyst. Product hydrocarbons in liquid hydrocarbons
species with larger molecules would be products were iso-paraffins and aromatics.
It should be notified that emissions from aromatics selectivity. The obtained paraffins
transportation fuel caused by the incomplete content over ZSM5-80 was approximately
combustion of aromatics compound, 28.02%, while the obtained aromatic content
consequently the aromatics compound in was 38.27%.
fuel should be restricted. The desired 3.2 Effect of temperature
products from the MTG process are, Temperature is an important
therefore paraffins and iso-paraffins. parameter that affects the performance of
Additionally, the reduction of aromatics MTG process. Since the best performance of
content in liquid product is by far more MTG process with ZSM5-80 catalyst was
benefit to catalyst stability since aromatics observed, temperatures in the range of 250-
group is renowned to be the coke precursor 400 °C on MTG reaction was investigated
in methanol to hydrocarbons (MTH) with this catalyst.
reaction.9 100 0.5
(a)
Liquid hydrocarbon yield

MeOH conversion (%)
35 80 0.4
in liquid hydrocarbons (wt%)
ZSM5-26
(g/gcatalyst/h)
Selectivity of main aromatics
30
ZSM5-30 60 0.3
25 ZSM5-80
40 0.2
ZSM5-280
20
20 0.1
15
0 0
10 250 C 300 C 400 C
5 MeOH conversion
Liquid hydrocarbon yield (g/gcatalyst/h)
0 100
100 100
80
80 (b)
80
60
60
F 60
40
igure 4. Selectivity of main aromatics in 40
liquid hydrocarbons products (400 °C, 1 bar, 20

20 40
WHSV=2 h-1) 0 0
20
250 C 250 C 300 C 300 C 400 C 400 C
The fractions of aromatics compound 0
were identified to be toluene, xylene,
250 C 300 C 400 C
ethyltoluene, trimethyl benzene and durene
as shown in Figure 4. Durene is undesirable CO+CO2+H2
CO+CO2+H2
CO+CO2 +H2
CO+CO2+H2 Unconverted
Unconverted
Unconverted MeOH
Unconverted MeOH+DME
MeOH+DME
MeOH+DME
due to its high melting point, i.e. 79 °C. Water

Water
Water Water Gaseoushydrocarbon
Gaseous
Gaseous hydrocarbon
Gaseous hydrocarbon
hydrocarbon
Consequently, it might be crystallized and hydrocarbon
Liquid hydrocarbon
Liquidhydrocarbon
Liquid hydrocarbon
caused the engine operation problem at high Figure 5. Catalytic performance (a) product
concentration.10 The decreasing SiO2/Al2O3 distribution (b) MTG reaction over ZSM5-
ratio facilitated aromatization reaction and 80 at various temperatures (WHSV=2 h-1,
the obvious decreasing of aromatics 5 bar)
compounds might be observed via
SiO2/Al2O3 ratio of 80. Overall, The obtained results indicated that
performance of ZSM5-80 in MTG process there was a significant increasing in
was superior to other ZSM-5 catalysts used methanol conversion and liquid
in the present study. It provided higher hydrocarbons yield following the increasing
relative yield of liquid hydrocarbons, fewer of process temperature (Figure 5a). This was
gaseous hydrocarbons yield and fewer probably due to the increasing of movement
rate and collision probability of reactant be attributed to the enhancement of
molecules at higher temperature. The aromatization reaction at higher
selectivity toward total hydrocarbons temperature.
product and liquid hydrocarbons product
were also increased with temperature as 80
(a)
shown in Figure 5b. At 300-400 °C, gaseous 250 ˚C
hydrocarbons fraction was around 7.91- 300 ˚C
Selectivity (wt%)
60
16.04% and liquid hydrocarbons fraction 400 ˚C
was around 16.83-19.96%. It might be seen
40
that the increasing of temperature induced
the decomposition of methanol and produces
other basic components, such as H2, CO and 20
CO2.
At 250 °C, the most abundant 0
C2
C6
C1
C3
C4
C5
C7
C8
C9
C15+
C10
C11
C12
C13
C14
product obtained was C2 (71.0%) followed
by C3 (23.2%) as shown in Figure 6a. On the
contrary, C3 and C8-C12 hydrocarbons were 50
(b)
the major product obtained at 300-400 °C. Liquid hydrocarbon product 300 ˚C
At low temperature, the main reaction was distribution (wt%) 40
the methanol dehydration to become DME 400 ˚C
and light olefin formation. The acquired 30
light olefins could cause the catalyst pore
20
blockage, resulting in the low accessibility
of reactants on active sites of the catalyst 10
causing the low methanol conversion and
low liquid hydrocarbons yield. 0
It should be noted that there was no
liquid product generated at 250 °C. Figure
6b shows hydrocarbons distribution of liquid
products obtained at 300-400 °C. Obviously,
paraffins, iso-parafins and napthenes Figure 6. Carbon distribution of
contents decreased against the increasing of hydrocarbons products (a) liquid
temperature within 300 to 400 °C range. On hydrocarbon product distribution (b) MTG
the contrary, the aromatics content increased reaction over ZSM5-80 at various
along with the rising temperature that might temperatures (WHSV=2 h-1, 5 bar)
Table 1. Methanol conversions and product distributions of MTG reaction over ZSM5-80 at
various pressures
1 bar 5 bar 10 bar
MeOH conversion 90.57 93.44 95.34
Product distribution Total hydrocarcon yield 30.17 36.00 32.84
(wt%) Gaseous hydrocarbon yield 11.87 16.04 12.37
Liquid hydrocarbon yield 18.30 19.96 20.47
Water 54.95 54.44 58.88
Unconverted MeOH 10.82 7.44 4.98
Other (CO+CO2+H2) 4.06 2.12 3.03
Liquid product selectivity Paraffins +iso-paraffins 29.98 23.86 27.01
(wt%) Olefins 4.89 1.52 7.55
Naphthenes 21.25 17.37 13.92
Aromatics 38.27 43.12 40.17
Liquid hydrocarbon (g/gcatalyst/h) 0.286 0.390 0.392
3.3 Effect of pressure Acknowledgements
The effect of pressure in the range of Financial support of the present
1-10 bar on MTG reaction was investigated study by National Research Council of
at 400 °C and the results were exhibited in Thailand is gratefully acknowledged.
Table 1. The increasing pressure facilitated
higher methanol conversion and liquid References
hydrocarbons yield. The total hydrocarbons 1. Rownaghi, A. A.; Hedlund, J. Ind. Eng.
yield and gaseous hydrocarbons yield, Chem. Res. 2011, 50, 11872−11878.
however, were partially increased and 2. Sadeghi, S.; Haghihi, M.; Estifaee, P. J.
reached their maximum value at 5 bar but Nat. Gas Sci. Eng. 2015, 24, 302−310.
declined shortly afterwards. At 10 bar, 3. Yingping, H.; Min, L.; Chengyi, D.;
ZSM5-80 catalyst delivered 0.392 g gcat-1 h-1 Shutao, X.; Yingxu, W.; Zhongmin, L.;
liquid hydrocarbons yield, with most carbon Xinwem, G. Chinese J. Catal. 2013, 34,
distributions in the range of C7-C11. The 1148−1158.
major products obtained including 4. Ghavipour, M.; Behbahani, R. M.;
paraffins+iso-paraffin,aromatics, naphthenes Moradi, G. R.; Soleimanimehr, A. Fuel
and olefins with the amount of 27.01, 40.17, 2013, 113, 310−317.
13.92 and 7.55 wt% respectively. 5. Knochen, J.; Guttel, R.; Knobloch, C.;
Turek, T. Chem. Eng. Process. 2010, 49,
4. Conclusion 958−964.
Conversion of methanol to gasoline 6. Chambrey, S.; Fongarland, P.; Karaca,
range biofuel over ZSM-5 zeolite catalyst in H.; Piche, S.; Griboval-Constant, A.;
milli fixed bed reactor was investigated. Schweich, D.; Luck, F.; Savin, S.;
ZSM-5 catalyst with SiO2/Al2O3 ratio of 80 Khodakov, A. Y. Catal. Today. 2011,
exhibits the best catalytic performance in 171, 201−206.
terms of highest liquid hydrocarbons yield. 7. Al-Dughaither, A. S.; Lasa, H.; Fuel
The major products obtained including iso- 2014, 138, 52−64.
paraffin, aromatics and naphthenes. 8. Lu, Y.; Hu, J.; Han, J.; Yu, F. J. Energy
Furthermore, yield of liquid hydrocarbons Inst. 2016, 89, 782−792.
and selectivity toward aromatics 9. Li, M.; Zhou, Y.; Ju, C.; Fang, Y. Appl.
hydrocarbons increased with the increasing Catal. A. 2016, 512, 1−8.
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SiO2/Al2O3 ratio, the improvement in Zhang, D. Ind. Eng. Chem. Res. 2014,
methanol conversion and selectivity toward 53, 19471−19478.
iso-paraffin in liquid hydrocarbons products
were achieved, while the C1-C4 gaseous
selectivity was decreased.
Effect of operating conditions on synthesis of methanol and dimethyl ether
from syngas over solid catalysts
Amornrat Suemanotham*, Lalita Attanatho, Yoothana Thanmongkhon,
Teerawit Laosombut
*E-mail: amornrat_s@tistr.or.th
Abstract:
Synthesis of methanol (MeOH) and dimethyl ether (DME) from syngas over CuO-
ZnO-Al2O3 and -Al2O3 catalysts in fixed-bed reactor were investigated. This study
conducted on the effect of operating conditions, including reactor size (milli- and centi- fixed
bed), reaction temperature (280 and 300 ºC) and gas hourly space velocity (GHSV) (1.5 and 3
NL/g catalyst/h) on CO conversion, MeOH production yield and selectivity. The results
showed that the milli-fixed bed reactor had higher MeOH space time yield (MeOH STY) and
MeOH selectivity than the conventional centi-fixed bed reactor due to better heat and mass
transfer as well as slow catalyst deactivation. The highest MeOH STY of 186.7 g/kg
catalyst/h with MeOH selectivity of 95.0 % was found at 280 ºC and GHSV of 1.5 NL/g
catalyst/h. For the production of DME, the results indicated that indirect DME synthesis
achieved production yield of 292.5 g/kg catalyst/h and selectivity of 92.3 % at 320 ºC and
weight hourly space velocity of 2 g MeOH/g catalyst/h.
1. Introduction bifunctional catalysts which consist of

Methanol (CH3OH) is one of the methanol synthesis catalysts (composed of
basic alcohol. It is an important intermediate oxides such as CuO, ZnO, Al2O3 and/or
used in the chemical industry to produce Cr2O3)1 and solid acid catalysts for methanol
chemicals such as formaldehyde, dimethyl dehydration (such as -Al2O3, HZSM-5 or
ether, acetic acid, etc. Methanol can be used HY-zeolite).2-4 One of an important problem
directly or blended with petroleum products related to the operations of solid catalysts is
as a clean fuel. Methanol is synthesized the loss of catalyst activity with time on
directly from syngas over solid catalysts. stream. Overall reactions taking place in the
Syngas could be produced from coal, natural syngas to DME process are as follows:
gas, and biomass gasification. Syngas Methanol synthesis:
mainly consists of hydrogen (H2) and carbon CO + 2H2  CH3OH (1)
monoxide (CO).
Dimethyl ether (CH3OCH3) is the Methanol dehydration:
simplest of ether. It has potential as 2CH3OH  CH3OCH3 + H2O (2)
alternative of energy and fuels and a useful
intermediate for many chemical productions. Water-gas shift reaction:
DME can be also used to substitute liquefied CO + H2O  CO2 + H2 (3)
petroleum gas (LPG). There are two
processes for DME production including The net reaction:
indirect and direct processes. Indirect 3CO + 3H2  CH3OCH3 + CO2 (4)
process is composed of two steps involving 2CO + 4H2  CH3OCH3 + H2O (5)
methanol synthesis from syngas in the first
step followed by the methanol dehydration In the present study, the effect of
in the second. Direct process or one step, parameters (temperature, reactor size, space
syngas is converted to DME over
velocity) on the conversion, production N2
yield, and selectivity of methanol and DME
were investigated over solid catalysts in H2 MFC
fixed bed reactor. TC
2. Materials and Methods Syngas
2.1 Material Reactor
The chemicals used in this study

were all analytical grade. The MFC Mass flow controller
Cu(NO3)2.3H2O, Zn(NO3)2.6H2O, Na2CO3, TC
GC
Thermocouple
Gas chromatograph
Al(NO3)3.9H2O, and -Al2O3 were supplied Back pressure
regulator Condenser
by Ajax finechem. Methanol (99% purity)
was purchased from Merck. GC

The CuO-ZnO-Al2O3 (CZA) catalyst Liquid sampling
was prepared by a co-precipitation of an Figure 1. Schematic of the experimental set

aqueous solution containing Cu-Zn-Al up
precursors (weight ratio of CuO:ZnO:Al2O3
=5:4:1) and sodium carbonate as a carried out in the milli- (5 mm) and centi-
precipitating agent. The pH was controlled (1.5 cm) fixed bed reactor at temperature
about 7 during precipitation for 2 h. The range 280-300 ºC and gas hour space
precipitate was washed several times with velocity (GHSV) 1.5-3.0 NL/g catalyst /h
deionized water and dried at 105 ºC for 6 h for 24 h. The conversion, productivity and
and further calcined at 380 ºC. The -Al2O3 products distribution were obtained from the
was prepared by thermal decomposition steady-state average values after 24 h on
under static air at 550 C for 3 h. stream.
The BET surface, pore volume and 2.4 Product analysis
pore diameter of catalyst were measured by The products were analyzed by the
N2 adsorption-desorption isotherm using online gas chromatograph (Agilent, Model
Brunauer, Emmett and Teller surface area 7890B), equipped with a thermal
instrument. conductivity detector (TCD) to analyze CO,
2.3 Catalytic activity test CO2 and CH4 as well as a flame ionization
All of experiments were carried out detector (FID) for the separation of
in fixed-bed reactor made of stainless steel methanol, DME, and hydrocarbon, etc. The
placed in tubular furnace. The syngas flow conversion, products yield, and selectivity
rate and pressure of reactor were controlled were calculated as follows:
by mass flow controller and back pressure CO conversion (%):
regulator. The catalyst bed temperature was
measured by a thermocouple positioned in
the center. Prior the reaction, the catalyst
was reduced in situ with 5% H2/N2 at MeOH productivity or MeOH space time
220 ºC for 10 h under atmospheric pressure. yield (gMeOH/kgcat/h):
After reduction, the reactor was pressurized
up to 4 MPa under syngas. A schematic
diagram of the experimental setup is shown
in Figure 1. The syngas composition used MeOH selectivity (%):
for methanol and direct DME synthesis was
H2:CO ratio of 1:1. The activity tests were
MeOH conversion (%): catalyst activity by inhibiting methanol
synthesis. It can be concluded that the milli-
fixed bed reactor had better heat and mass
transfer than conventional centi-fixed bed
DME selectivity (%): reactor.
95.04
100
81.23
Products distribution (%)

80
3. Results and Discussion 60

40
The BET surface area, pore volume 16.85
and pore diameter of solid catalysts are 20
4.38
0.28 0.54 0.30 1.37
given in Table 1. The BET results of the 0
prepared catalyst (CZA) had surface area, CH4
CH4 CO2
CO2 C2-C6
C2-C6 MeOH
MeOH
Reactor size 5 mm. Reactor size 1.5 cm.
pore volume and pore size equal to 70.5
m2/g, 0.029 cm3/g and 1.78 nm. The Figure 2. Effect of reactor size on products
elemental composition of CZA was distribution for methanol synthesis
analyzed by scanning electron microscope
energy dispersive x-ray spectrometer (SEM- 3.2.2 Effect of temperature
EDX). The CZA consisted of 38.4% of Cu, The effect of temperature on the CO
32.1% of Zn, 21.4% of O and 8.1% of Al conversion, yield and selectivity of methanol
that correlated to Cu/Zn/Al ratio of 5/4/1. was investigated in the range of 280-300 ºC,
P=4 MPa and GHSV=1.5 NL/g catalyst/h
Table 1. Characteristics of solid catalysts over CZA catalyst in milli-fixed bed reactor.
Properties CZA -Al2O3 The CO conversion and yield of methanol
Surface area (m2/g) 70.46 104.20 enhanced with increasing reaction
Pore volume (cm3/g) 0.029 0.041 temperature. The CO conversion increased
Pore diameter (nm) 1.78 1.84
from 22% to 38% and MeOH STY
enhanced from 186.72 to 227.7 g/kg catalyst
3.2 Methanol synthesis
/h. As shown in Figure 3, the selectivity to
3.2.1 Effect of reactor size
The performance of reactor with methanol slightly decreased from 95% to
different size, milli- and centi-fixed bed 88% with increasing reaction temperature.
reactors, was investigated in methanol On the contrary, the selectivity of CO2 and
synthesis at syngas ratio of 1, GHSV 1.5 hydrocarbons (C1-C6) increased from 4.4%
NL/g catalyst/h, 280 ºC and 4 MPa over to 10.6% and 0.3% to 0.9%, respectively.
CZA catalyst. It was found that CO At high temperature, CO conversion and
conversion increased from 22% to 35% with methanol STY enhanced due to fast reaction
increasing reactor size whereas MeOH STY rate whereas the selectivity of methanol
decreased from 186.7 to 104.2 g/kg catalyst decreased due to the undesired side reaction
/h. The influence of size of reactor on the including water gas shift reaction Equation
products distribution including methane, (3) and methanation (CO + 3H2  CH4 +
carbon dioxide, hydrocarbons and methanol H2O). These side reactions were produced
is shown in Figure 2. The MeOH selectivity by-products including CO2, methane and
decreased from 95% to 81% by increasing water. The temperature is a crucial factor
the reactor size. The lower selectivity of that impacts on the selectivity and yield of
methanol in centi-fixed bed reactor caused methanol.
by the hot spot and produced undesired side 3.2.3 Effect of GHSV
reaction as well as by-products particularly This present study, the influence of
CO2. At high CO2 concentration reduced GHSV on the conversion, yield and
selectivity of methanol has been investigated
for GHSV=1.5 and 3.0 NL/g catalyst/h with 4 MPa using mixed of CZA and -Al2O3 as
temperature 280 ºC and 4 MPa over the solid catalysts. Syngas ratio of 1 was used in
CZA catalyst in milli-fixed bed reactor. this study. It was found that the low CO
Increasing GHSV will cause an increase in conversion (28%), low DME yield (9 g/kg
the gas velocity that promotes mass transfer catalyst/h) and low DME selectivity (4.2%)
but leads to a decrease in the contact time of were obtained whereas high MeOH yield
reactant.5 and selectivity (204.4 g/kg catalyst/h and
95.04
84.3% selectivity). The products distribution
100 87.86
for DME synthesis is shown in Figure 5.
80 The low DME selectivity and yield caused

by incomplete methanol dehydration
60
Equation (2) resulted in high amount of
40 methanol remaining. The DME productivity
20 10.63 and selectivity were attributable to the
4.38
0.28 0.63 0.30 0.88 concentration and suitable acidity of solid
0
CH4
CH4 CO2
CO2 C2-C6
C2-C6 MeOH
MeOH
catalysts.6 If acidity of catalyst is too low,
280 °C 300 °C methanol formed cannot be dehydrated with
Figure 3. Effect of reaction temperature on appropriate efficiency. If acidity is too high,
products distribution for methanol synthesis DME produced can be converted further to
hydrocarbons.7
The result showed that both of the 100
84.32
CO conversion decreased from 22% to 9%
80
and MeOH STY significant reduced from
186.7 to 26.5 g/kg catalyst /h. It can be 60
concluded that the conversion and methanol 40

yield decreased at higher GHSV because of 10.73
20
the less residence time. Figure 4 indicated 0.13 0.67 4.16
the products distribution as a function of 0

CH4 CO2
CH4 CO 2 CC2-C6
2-C6
MeOH
MeOH DME
DME
GHSV. The selectivity of methanol slightly
decreased from 95% to 88% as GHSV Figure 5. The products distribution for
increased while produced higher amount of direct DME synthesis (280 ºC, 4 MPa,
CO2. GHSV=1.5 NL/g catalyst/h)
95.04
100 88.48
3.3.2 Indirect process
80 Dehydration of methanol to DME

60 was carried out in milli-fixed bed reactor at
280-320 ºC, weight hourly space velocity
40
(WHSV)=2 g MeOH/g catalyst/h and P=0.1
20
4.38
8.99 MPa using -Al2O3 as a catalyst. The results
0.28 0.96 0.30 0.38
of conversion, yield and selectivity of DME
0
CH4
CH4 CO2
CO2 C2-C6
C2-C6 MeOH
MeOH were given in Table 2. It was found that
GHSV 1.5 NL/g catalyst/h GHSV 3.0 NL/g catalyst/h methanol conversion, DME yield and
Figure 4. Effect of GHSV on the product selectivity enhanced by increasing operating
distribution on methanol synthesis temperature. At 320 ºC provided the highest
conversion, yield and selectivity of DME
3.3 DME synthesis (66.9% conversion, 41.9% of DME yield
3.3.1 Direct process and 92.3% of DME selectivity). This can be
DME production was carried out in attributed to increasing the reaction rate. The
milli-fixed bed reactor (5 mm) at 280 ºC and conversion of methanol to DME produced
water as well. The reversible exothermic selectivity than direct process. This is
reaction, catalyst deactivation and low probably due to incomplete methanol
activity occurred due to the presence of dehydration as well as the concentration and
water.7 performance of solid catalysts used in this
study.
Table 2. Effect of temperature on
conversion, DME yield and selectivity for Acknowledgements
indirect DME synthesis The authors wish to acknowledge the
Temperature MeOH DME DME financial support from National Research
conversion yield selectivity Council of Thailand.
(ºC) (%) (%) (%)
280 12.2 4.6 56.2
300 25.7 13.4 75.3 References
320 66.9 41.9 92.3 1. Yin, X.; Leung, D. Y. C. Energ. Fuel
2005, 19, 305-310.
4. Conclusion 2. Kim., S. D.; Beak, S. C.; Lee, Y. J.; Jun,
There are several factors that affect K. W.; Kim, M. J.; Yoo, I. S. Appl.
to the conversion of syngas to methanol and Catal. A. 2006, 309, 139-143.
syngas to DME over solid catalysts in fixed 3. Garcia-Trenco, A.; Martinez, A. Appl.
bed reactor. Methanol selectivity and Catal. A. 2011, 411-412, 170-179.
productivity were enhanced as the reactor 4. Abu-Dahrieh, J.; Rooney, D.; Goguet,
size was reduced from milli- to centi- owing A.; Saih, Y. Chem. Eng. J. 2012, 203,
to faster heat and mass transfer. The 201-211.
conversion and methanol yield increased by 5. Hosseini, S. Y.; Nikou, M. R. K. J. Am.
increasing reaction temperature because of Sci. 2012, 8, 235-239.
the higher reaction rate. In contrast, yield 6. Azizi, A.; Rezaeimanesh, M.; Tohidian,
and conversion decreased by increasing T.; Rahimpour, M. R. Chem. Eng.
GHSV due to the less residence time and Process 2014, 82, 150-172.
low reaction rate. Indirect DME synthesis 7. Stiefel, M.; Ahmad, R.; Doring, A. M.
can achieve the higher production yield and Fuel Process Technol. 2011, 92, 1466-
1474.
Preparation and properties of carboxylated styrene-butadiene
rubber/cassava starch/cellulose fiber composite
Kanokwan Chantawee, Sa-Ad Riyajan*
*E-mail: saadriyajan@hotmail.com
Abstract:
Many polymers have been used to develop biodegradable film. Cassava starch is a
promising material for this purpose owing to their abundance, renewable, and low cost. In
this study, the effect of cellulose fiber on the physical properties of carboxylated styrene-
butadiene rubber (CSBR)/cassava starch (CS) blend film was investigated. The recycling of
plant wasted materials from sugarcane leaves into useful product represented a green
alternative in order to prevent environmental problems due to the large amount of wasted
sugarcane leaves. The cellulose fiber (CF) was isolated by using acid hydrolysis under
heating. The average particle size of cellulose fiber was 27 microns observed from laser
scattering particle size distribution analyzer. The results showed that the addition of cellulose
exhibited higher in tensile strength compared to unfilled cellulose fiber sample. The films
also showed lower moisture absorption ability and moisture content with increasing
concentration of cellulose fiber because the cellulose fiber improved the interaction strength
between the polymer chains, leading less mobility. This study demonstrated a significant
concentration of cellulose fiber improved the film properties and inexpensive biodegradable
film was expected to be a promising candidate for alternative packaging materials.
1. Introduction promising candidates for future materials.

Cassava (Manihot esculenta C.) is an However, the CS based films are limited in
important economic crop for Thailand their application due to its poor mechanical
agriculture. Cassava starch (CS) is planted strength, high water sensitivity, brittle
in approximately 7 million raise of land in character compared to synthetic materials
48 provinces in Thailand. Production level which are undesirable characteristics.5
has expanded continuously, approximately A solution to this problem, a native
30 million tonnes in 2012-2016. CS has starch was modified using various methods
been sold at a lower price than other cereal to obtain high-performance and desirable
starch crops, approximately 10 baht per properties.6 For example, the addition of
kilogram.1,2 Another, it used as raw material plasticizer and physical modification
in varied industries, such as food (non- techniques.7,8 Chemical modifications have
gluten starch, thickener, animal feed), paper, been also reported, such as graft
adhesive component, bioethanol, plywood, copolymerization,9,10 crosslinking,11 and
etc.3 In addition, CS has been extensively acetylation12 for introducing a new
studied to produce biodegradable films by substituent group to starch molecule, leading
many processing techniques, such as its good required properties.
casting, extrusion, blow molding,4 Due to In addition, there are other methods
the fact that it is able to form transparent by mixing of two or more different polymers
film, good film-forming, excellent with/without any chemical bonding between
thickening character, low gelatinization them to achieve various property
temperature, good gel stability, and semi- combinations of the resulting film, such as
permeable to carbon dioxide-oxygen. It has polymer blend13,14 and polymer composite.15
been stated that CS is one of the most
The recent interest in using amylose was purchased from General Starch
carboxylated styrene-butadiene rubber Ltd. (Bangkok, Thailand). The potassium
(CSBR) blended with the CS has gained persulfate was derived from the RFCL Ltd.
attention to encourage the mechanical (India). 10% Triton™ X-100 used as a non-
strength and hydrophobic character of the ionic surfactant which was obtained from
CS.16 CSBR is a copolymer of styrene, Lucky Four Co. Ltd. Thailand. Glycerol was
butadiene, and small amount unsaturated used as plasticizer (Assay 99.5%, ACS
carboxylic acid which prepared by the reagent).
emulsion polymerization. Furthermore, it 2.2 Methods and preparation
possesses very soft, high molecular weight, 2.2.1 Extraction cellulose fiber from
and widely used in water-based binder.17 wasted sugarcane leaves
Furthermore, cellulose nanocrystal Wasted sugarcane leaves were
has been also used in composite materials, bleached in sodium hydroxide solution,
which can isolate from different sources of washed with deionize water and dried in an
cellulosic fibers or animal sources. It is not oven. Then, acid hydrolysis was performed
only encouraged environmental benefit with by using 50% sulfuric acid at 140 ºC for 7 h
ultimate disposability, but also improved the under vigorous stirring. The sediment was
mechanical strength and electrical property diluted 10-fold with water to stop hydrolysis
of polymer. Moreover, it still has many reaction, centrifuged at 10000 rpm, 15 ºC for
features, such as abundance, environmental 15 min. The suspension was dialyzed
compatibility, lightweight, low cost and against distilled water until reach a pH of 7,
biodegradability.18 Moreover, previous sonicated in cold water for 15 min and
researches reported the starch/lignin stored in a refrigerator.
composite materials which presented higher 2.2.2 Preparation of CSBR/CS blend
tensile strength, increased the thermal Cassava starch (50 g) was dissolved
stability, and to be explored for food in 950 mL of distilled water at 90 ± 3 ºC
packaging purposes.19 under stirring, resulting in the starch paste.
In this work, we attempted to modify Potassium persulfate solution (3 g in
a native CS that is widespread in Thailand aqueous solution) was added to the mixture.
by using small amount CSBR and cellulose The gelatinized starch was homogenized
fiber (CF) from renewable resources for with CSBR latex (22 g) in the presence of
improving its mechanical performance. The Triton™ X-100 (1 g in DI water 10 mL) at
CSBR/CS/CF composite films were 220 rpm, 70 ºC for 1 h. The CSBR/CS blend
prepared by casting technique. Moreover, was carried out and used in further steps.
the effect of fiber on the mechanical 2.2.3 Preparation of CSBR/CS/CF
strength, moisture resistance, physical and composite film
thermal properties of these composite films CSBR/CS blend was mixed with CS
were investigated in this work. gel (CS 50 g in DI water 500 mL) at ratio
1:1 to get a mixture suspension. Then,
2. Materials and Methods glycerol (2 wt%) and cellulose fiber (0 to 6
2.1 Materials wt%) were added to the mixture suspension,
The carboxylated styrene-butadiene sonicated for 15 min to remove the bubble.
latex emulsion (Rovene® 9410) was Finally, the mixture suspension was cast on
obtained from Mallard Creek Polymers glass plate and then left at room temperature
(Charlotte, North Carolina, USA). The for 3-4 days.
styrene/butadiene ratio is 25/75 and the 2.3 Characterizations
nominal particle size of the latex is 100 nm. The effect of cellulose fiber on the
The glass transition temperature and solid physical and mechanical properties of
content are -56 ºC and ~49%, respectively composite film will be investigated as the
with pH ~8.5. The CS containing 32% following:
The particle size and distribution of glucose ring.20 The band at 892 cm-1 was
cellulose fiber were determined using LA- referred to the glycosidic C-H vibration
950 laser scattering particle size distribution which was characteristic of cellulose
analyzer. The chemical structure and structure.
interaction bonds were characterized by IR
spectrometer with a resolution of 4 cm-1 and
16 scans in range of 4000 to 500 cm-1. Water
sensitivity was evaluated by water contact
angle measurement. One drop of water
obtained from a Milli-Q system using
OCA20 software. An apparent contact angle
(θ) was measured in 10 second. Moisture
content was measure the loss weight before
and after drying in oven at 110 ºC for 2 h,
and then was calculated as follow equation.
Moisture content (%) = (Wi-Wf)/Wi×100 Figure 1. Particle size and distribution of
Film solubility was estimated the CF
gain weight. A piece of film was cut and
immersed in water for 3 h. The swelling
ratio was calculated as follow equation.
Swelling ratio (%) = (Wf-Wi)/Wi×100
The moisture absorption was
evaluated the gain weight. A piece of film
was cut and placed middle of beaker
containing water at the corners of a square.
The percentage of water absorbed by the
samples were calculated using the following
formula.
Moisture absorption (%) = (Wf-Wi)/Wi×100 Figure 2. FTIR spectra for CF from
Tensile strength test and elongation sugarcane leaves compared to commercial
at break were evaluated by using a Universal CF powder (control)
Testing Machine with stretched at crosshead
speed of 100 mm/min, in accordance to Swelling ratio was used to verify the
ASTM D638. Wi and Wf are initial and final hydrophobic nature of the polymer material.
weight of sample, respectively. Water molecules diffuse into the polymer
chains, leading the film swelling. The
3. Results and Discussion swelling ratio of the sample without CF was
3.1 Physical properties of composites 109 %. After the addition of the CF at 0.4%
The average particle size of cellulose in the CSBR/CS blend, the swelling
fiber after acid hydrolysis process observing increased from 110 to 140% due to water
from particle size analyzers was 27 microns affinity of reactive OH groups on the
as presented in Figure 1. surface. However, the amount of CF over
Figure 2 shows FTIR spectra of CF 1.6%, the swelling ratio started to deteriorate
isolated from sugarcane leaves compared to as presented in Figure 3 because hydrogen
commercial cellulose powder. The peak near bond formed between the hydrophilic
3300 cm-1 is ascribed to OH stretching in functional groups of CF and starch thereby
cellulose molecules. The FTIR band at 1158 increasing water resistance that related the
cm-1 and 1051 cm-1 are ascribed to C-O moisture content results.21
stretching of the C-O-H group and C-O Moisture content which is shown in
stretching of C-O-C in the anhydrous Figure 4 decreased at higher levels of fiber
loading. Due to the fact that pure starch has
poor moisture resistance, whereas the
addition of CF helped to decrease its
sensitivity to moisture.
Figure 5. Contact angle () of CSBR/CS

blend loading with different CF contents
Figure 3. Swelling ratio of CSBR/CS blend

loading with different CF contents
Figure 6. Moisture absorption of CSBR/CS

blend loading with different CF contents
These results are ascribed to a good

interfacial adhesion between starch molecule
and CF that can form hydrogen bonds and
Figure 4. Moisture content of CSBR/CS dipole-dipole force through hydroxyl groups
blend loading with different CF contents on both components.22
In addition, hydroxyl groups on
Wettability of films was measured cellulose fiber can interact with CSBR
by contact angle measurement which containing carboxyl groups via polar-polar
increase in the contact angle represents an interaction between the interfaces of
increase in film hydrophobicity. Figure 5 components. However, tensile strength value
shows no significantly improved surface deteriorated at 2 wt% CF loading due to the
property (~50º) after the addition of the appearance of the phase separation between
CF. Moisture absorption was studied with the cellulose fiber aggregates and polymer
different CF loading levels as a function of matrix, which reduced chain mobility and
time as presented in Figure 6. The kinetics were more rigidity.23
of water uptake showed the same trend for
all CF loading levels which readily absorbed 4. Conclusion
at the early stage and then decreased after This starch-based film plasticized
2.5 h. with glycerol and reinforced with cellulose
3.2 Mechanical properties of composites fiber was successfully carried out from the
The tensile strength and elongation process (CSBR/CS/CF). The cellulose fiber
at break were demonstrated in Figure 7. The from sugarcane leaves that used as
addition of CF significantly improved reinforcing agent in CSBR/CS matrix
tensile strength from 13.4 to 28 MPa with exhibited a remarkable mechanical
increasing of CF content up to 1.6 wt%. performance in tensile strength at 1.6 wt%
Figure 7. Tensile strength (left), Elongation at break (right) of CSBR/CS blend with
different CF contents
loading level. Starch composite films 2012, 48, 125–130.
reduced swelling ratio and decreased 11. Chung, H. -J.; Woo, K. -S.; Lim, S. -T.
moisture content due to its strong hydrogen Carbohydr. Polym. 2004, 55, 9–15.
bonding between natural fiber and polymer
12. Wang, Y. -J.; Wang, L. Starch/Stärke
matrix.
2002, 54, 25–30.
Acknowledgements 13. Wu, Y. -P.; Ji, M. -Q.; Qi, Q.; Wang, Y.
This research was supported by -Q.; Zhang, L. -Q. Macromol. Rapid
Department of Chemistry and Department of Commun. 2004, 25, 565–57.
Textile, Faculty of Science and Technology, 14. Li, M. -C.; Ge, X.; Cho, U.R. Macromol.
Thammasat University, Thailand. Res. 2013, 21 (7), 793–800.
15. Savadekar, N. R.; Mhaske, S. T.
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Investigation of the operation parameters affected on biofuel production
via Fischer-Tropsch synthesis in milli-tubular reactor
Warunyoo Phondate, Lalita Attanatho, Amornrat Suemanotham, Teerawit Laosombut,
Yoothana Thanmongkhon*
Expert Centre of Innovative Clean Energy and Environment,
Thailand Institute of Scientific and Technological Research (TISTR), Pathum Thani 12120, Thailand
*E-mail: yoothana_t@tistr.or.th
Abstract:
Biomass to liquid fuel has been interestingly promising process. Conversion of
producer gas, generally obtained from biomass gasification, to biofuel is widely studied and
developed due to its plausible pathway. The present research investigated the effects of
operating conditions, i.e., temperature of 453-553 K, pressure of 0.5-2 MPa and mixture gas
(CO and H2) hourly space velocity (GHSV) of 1.10-8.78 h-1, on hydrocarbon conversion via
the Fischer-Tropsch process. An artificial syngas was continuously fed into a downdraft
milli-tubular reactor using Co-based catalyst. The experimental results revealed that carbon
balance was approximately recovered by 97%. The maximum yield of liquid fuel was
optimized at the reaction conditions of 493 K, 2 MPa and 4.39 h-1. Increase of temperature
enhanced CO conversion and, also, CH4 and CO2 selectivity. Furthermore, when the higher
operating pressure was applied, the larger amount of liquid hydrocarbon was generated.
However, CO conversion was diminished from 44% to 15% when increasing of GHSV from
1.10 h-1 to 4.39 h-1. The yield of liquid biofuel, on contrary, was remarkably increased from
0.11 goil/h.gcat to 0.19 goil/h.gcat. Stability of catalytic reaction was reported within 72 hours of
continuous operation and the production of liquid hydrocarbon was moderately decreased due
to the deactivation of catalyst.
1. Introduction could be gasified to syngas (CO+H2).

Increasing environmental and energy Moreover, life cycle assessment of BTL
security concerns are the major driving reports that greenhouse gases emission from
forces that bring about promotion of biofuel. the process is relatively less and more
Trends to improve air quality, climate environmental friendly than that of liquid
change and sustainability have globally fossil fuels.3,4
encouraged many researchers to study on Fischer-Tropsch process involves a
bio-origin fuels instead of petroleum fossil. series of chemical reactions that produce a
Among conversion technologies for biofuel variety of hydrocarbon molecules according
derived from biomass to hydrocarbons, the to the formula (CnH(2n+2)). During this
BTL or biomass-to-liquid process usually process CHx monomers, formed via
consisting of gasification for generating hydrogenation of adsorbed CO on transition
producer gas or syngas, cleaning unit for metals, produce hydrocarbons and
eliminating dust and tars from gaseous oxygenates with a broad range of chain
product, reformer for gas conditioning, gas lengths and functional groups. The major
compressing unit, Fischer-Tropsch (FT) products are linear paraffins and olefins
synthesis, hydro-cracking unit and product being used as alternative fuel, like gasoline
purification or distillation, is particularly and diesel.5
promising.1,2 A wide variety of feedstock
can be utilized as feed such as, coal, (2n + 1)H2 + nCO → CnH(2n+2) + nH2O (1)
biomass, municipal waste, etc., for BTL
rather than other technologies because they
“CnH(2n+2)” represents a product volume specific productivity at acceptable
consisting mainly of paraffinic hydrocarbons pressure drops. Thus, the reactor has been
(HCs) of variable chain length. For considered as a noticeable idea for FT
synthesizing HCs from FT process, Fe-, Co-, process. In the present research, the
and Ru-based catalysts are widely used. The conversion of synthesis gas to biofuel using
Fe and Co catalysts are more efficient and cobalt catalyst is fundamentally optimized in
economic than the other, and can be used for terms of the primary liquid fuel yield and
both low and high temperature (453-523 K carbon balance in order to produce HCs as
and 573-623 K) conversions, depending on alternative fuel. The parameters including
the reactor type.6 The higher temperatures temperature, pressure, gas hourly space
lead to faster reactions and higher velocity and size of reactor that influenced
conversion rates but also tend to favor CH4 on carbon monoxide conversion, methane
production. The temperature, as this reason, and carbon dioxide selectivity, and HCs
is usually kept at low to middle of the range. productivity are fully investigated.
Increasing pressure leads to higher
conversion rates and also favors formation 2. Materials and Methods
of long-chained alkanes which are desirable The Fischer-Tropsch synthesis was
both. Typical pressure range is in the range carried out in a milli-tubular reactor made of
of 0.1-0.6 MPa. Even higher pressures stainless steel tubing with 5 mm inner
would be favorable, but the benefits may not diameter and 350 mm long. Around 0.0625-
justify the costs of high-pressurized 0.5 g of 20%Co.1%Ru/Al2O3 catalyst were
equipment. Normally, the H/C molar ratio loaded as the packed bed of reactor which
for Fe is under 1 but Co could be used for was connected to the apparatus as seen in
the ratio of 1-2. Concerning to the reactor Figure 1. After purging the system with N2
types, separation of the liquid media from (99.99%, Praxair (Thailand) Co., Ltd.), the
the desired product is necessarily required catalyst was reduced in situ with H2
for a slurry bed type. Fluidized bed one is (99.99%, Praxair (Thailand) Co., Ltd.) by
properly for high temperature reaction; increasing the temperature from ambient
however, the catalyst has easily broken or condition to 623 K at 1 K/min. After 6 h of
got damage.7,8 FT process using a fixed bed reduction, the catalyst was cooled down to
reactor is much more convenient for 333 K at the same rate.
operation than both mentioned. However, A mixture gas contained 64%H2,
the reaction is exothermic which could be 32%CO, and 4%Ar (Linde (Thailand) PLC)
easily ruined by heated spot, temperature with H/C molar ratio of 2 was provided as
gradient, or runaway reaction.7,9 In order to feedstock for the reaction. It was introduced
avoid the drawbacks, a milli-channeled into the system after the reduction process
tubular reactor with fixed bed catalyst is by mass flow controller (Bronkhorst High-
proposed in this examination. Tech B.V.) at 20 NmL/min and pressurized
Milli-channeled tubular reactor is a to 0.5-2 MPa using back pressure regulator
reactor with the diameter within millimeter (Swagelok). Temperature was then slowly
scale. The reduction of size to millimeter increased to the desired temperature at 453-
allows for an intensification of heat transport 553 K in order to start the reaction.
in radial direction, avoids unexpected The FT products were collected and
transfer limitation, and thus isothermal separated by a cooling condenser at 278 K.
operation even of highly exothermic The gaseous products leaving the condenser
reactions become feasible. The milli- were periodically fed to GC-FID/TCD
channeled reactor has been studied in (Agilent 7890B GC) for online gas analysis
several chemical reactions, such as, and total flow rate of residual gas was also
transesterification, hydrogenation, etc., and measured. The liquid products, both aqueous
revealed promising results with the high and oily phases, were collected from the
condenser after 24 h time on stream (TOS). with the Arrhenius’ equation however, this
Offline analysis of oil phase product was study focuses on the yield of liquid HCs,
conducted in GC-FID (Agilent 6890N GC) especially gasoline and diesel (C5-C20).
with DB-5HT column.
CO conversion and selectivity of 120
453 K 493 K 553 K
CH4 and CO2 were calculated by these 100
CO conversion (%)
following equations; 80
60
x 100%
40
20
x 100%
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26
x 100% Time on stream (h)
Figure 2. CO conversion on each operating

Mass Flow Controller Thermocouple temperature as a function of time on steam
at 2 MPa and 1.10 h-1 using 5 mm ID of
reactor
MFC CO2 Selectivity CH4 Selectivity CO Conversion
Gas product
12.64
25.96 94.46
Heater
Temperature (K)
Catalyst
1.28
22.13
Back pressure
44.01
regulator
0
H 2CO/H2
+ CO
Gas 3.24
NN2
HH2
2
Cooling analyzer 8.2

Condenser
0.0 20.0 40.0 60.0 80.0 100.0

(%)
Liquid product
(a)
Figure 1. Schematic diagram of FT process 100.0
17.72
0.120
90.0
using a milli-tubular reactor 80.0 36.30
0.107 21.37
0.100 Oil quantity (goil/gcat.hr)
Weight fraction (%)
70.0 20.27 0.080

42.17
3. Results and Discussion 60.0
50.0 0.060
0.058
3.1 Influence of process parameters on 40.0
63.70 0.040
the FT performance 30.0
20.0 40.11
58.36
0.020
3.1.1 Reaction temperature 10.0
The effect of the temperature (453 K,

0.003
0.0 0.000
C20+
493 K, and 553 K) on CO conversion for the Temperature (K)

C5-C20
C1-C4
Oil quantity (gliq/h.gcat)
FT process was investigated as a function of
(b)
time on stream (TOS) shown in Figure 2.
Figure 3. Effect of reaction temperature on
The change in CO conversion from 8.2% to
CO conversion, CH4 and CO2 selectivity (a),
94.5%, CH4 selectivity from 3.2% to 26%
Oil quantity and fraction of HCs (b), at
and CO2 selectivity from 0% to 12.6%,
2MPa and 1.10 h-1 using 5 mm ID of reactor
respectively, are given in Figure 3(a).
Presumably, increased temperature caused
In Figure 3(b), the results show that
an increase in CH4 and CO2 formation by
the portion of C5-C20 is 42.2 %wt which is
methanation and water gas shift (WGS)
about 0.11goil/h.gcat when operating at
reaction.10 These are thermodynamically
493 K, 2 MPa and GHSV of 1.10 h-1. At
favors at higher temperature corresponding
553 K, the long-chained hydrocarbons could Generally, water is a by-product
be further converted to the light HCs appeared in the FT reaction which could be
because the side reactions, i.e., dealkylation kept in the pore and surface of catalyst. This
or cracking, etc., has been occurred.11 Thus, enhanced adsorption activity of reactants to
it caused less amount of liquid biofuel and the active site of catalyst increasing in
obtained more gaseous product. hydrogenation reaction and the long-chained
3.1.2 Reaction GHSV HCs was formed.12,13
The temperature was maintained at 3.1.3 Reaction pressure
493 K and the pressure at 2 MPa. The investigation of influences of
Accordingly, CO conversion decreased from operating pressure was compared between
44% to 10 % when varying GHSV from 0.5, 1 and 2 MPa at 493 K, GHSV 4.39 h-1
1.10 h-1 to 8.79 h-1 which resulted in the using reactor size 5 mm. The CO conversion
shorter reaction time.10 However, the CH4 was decreased from 20% to 15% when
and CO2 selectivity remained no different, increasing pressure. The selectivity of CH4
20-22% and 1-3% respectively, in these and CO2 were slightly changed from 21% to
conditions as presented in Figure 4(a). 23% and 1.12% to 0.99%, respectively as
shown in Figure 5(a).
2.92 CO2 Selectivity
19.71
10.09 CH4 Selectivity CO2 Selectivity CH4 Selectivity CO Conversion
CO Conversion
1.02 1.02
GHSV (h-1 )
20.29 20.98
15.1 15.1
Pressure (MPa)
0.92
21.9 0.99
32.5
22.83
20.09
1.28
22.13
44.01
1.12
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 23.15
19.41
(%)
(a) 0.0 5.0 10.0 15.0 20.0 25.0 30.0
100.0
4.15 3.04 3.16 0.02
0.250 (%)
90.0
(a)
Oil quantity (goil/gcat.hr)
Weight fraction (%)
80.0 33.20 0.191 0.200

46.21 48.57
70.0 57.59 100.0 0.250
3.80 3.92 3.16
60.0 0.150 90.0
0.135 0.199
50.0 80.0 0.191 0.200
Weight fraction (%)
40.0 0.107 0.100 44.25 49.46

70.0 57.59
30.0 62.65 60.0 0.156 0.150
50.75 51.41
20.0 39.25 0.050 50.0
10.0 40.0 0.100
0.004
0.0 0.000 30.0
51.95 46.62
C20+ 20.0 39.25 0.050
C5-C20
GHSV (h-1) C1-C4
10.0
Oil quantity (gliq/h.gcat) 0.0 0.000
(b)
C20+
C5-C20
Pressure (MPa) C1-C4
Figure 4. Effect of reaction GHSV on CO Oil quantity (gliq/h.gcat)
conversion, CH4 and CO2 selectivity (a), Oil (b)

quantity and fraction of HCs (b), at 493 K Figure 5. Effect of reaction pressure on CO
and 2 MPa using 5 mm ID of reactor conversion, CH4 and CO2 selectivity (a), Oil
quantity and fraction of HCs (b), at 493 K
In term of productivity, the amount and 4.39 h-1 using 5 mm ID of reactor
of liquid fuel was increased from
0.11goil/h.gcat to 0.19goil/h.gcat at GHSV of The liquid HCs were increased
1.1-4.39 h-1 with the maximum fraction of because the polymerization of long-chained
oil phase at 57.6%. Liquid fuel product hydrocarbons could be enhanced by the
decreased to 0.004 goil/h.gcat at higher flow pressurized reaction. In Figure 5(b), the
rate of syngas feed in as seen in Figure 4(b). liquid products were achieved at 0.156,
0.199 and 0.191 goil/h.gcat which the goil/h.gcat was achieved when using 5 mm ID
maximum portion of gasoline and diesel tubular reactor, due to this higher portion of
about 60% at pressure of 2 MPa. C5-C20 in hydrocarbon product.
3.1.4 Reactor size The heated spot occurring in the FT
Effect of reactor size on the process when using 10 mm ID tubular
performance of FT process was investigated reactor is probably the reason of
by using 5 mm and 10 mm ID tubular hydrocarbon cracking into smaller molecule
reactors. FT process in 10 mm ID tubular and led to the higher gas product
reactor resulted in the higher CO concentration. It can be seen that the smaller
conversion, CO2 and CH4 selectivity as seen tubular reactor is more effective in term of
in Figure 6(a.). The compositions of liquid biofuel production at similar
hydrocarbon products are given in Figure condition. The result could be considered as
6(b). an indication of its high performances of
CO2 Selectivity CH4 Selectivity CO Conversion heat and mass transfer properties.14
3.1.5 Stability of catalysis
16.82
The FT process was repeatedly
Size of reactor (mm)
40.08
81.79
examined within 72 hours for investigating
the stability of catalytic reaction. CO
conversion, CH4 and CO2 selectivity as
1.02
20.98
functions of time on steam (TOS) are shown
15.1
in Figure 7. During the period of 24-72
hours, the conversion slightly decreased
0.0 20.0 40.0 60.0 80.0 100.0 because of the catalyst deactivation.
(%) However, the liquid HCs production was
(a) remarkably optimized at this condition.
100.0 0.250
3.16 0.10 8.90
90.0 100.0
80.0 0.200
90.0
Weight fractrion (%)
0.191
70.0 CO Conversion (%)
80.0
60.0 0.150 CH4 Selectivity (%)
70.0
57.59
50.0 CO2 Selectivity (%)
90.99 60.0
40.0 0.100
(%)
50.0
30.0
40.0
20.0 0.050
39.25 0.035 30.0
10.0
20.0
0.0 0.000
C20+ 10.0
C5-C20
Size of reactor (mm) 0.0
C1-C4
0 10 20 30 40 50 60 70 80
Oil quantity (gliq/h.gcat)
Time on Stream (h)
(b)
Figure 6. Effect of reactor size on CO Figure 7. Stability of catalytic reaction on
conversion, CH4 and CO2 selectivity (a), Oil CO conversion, CH4 and CO2 selectivity at
quantity and fraction of HCs (b), at 493 K, 493 K, 2 MPa and 4.39 h-1 using 5 mm ID of
2MPa and 4.39 h-1 reactor
Hydrocarbon product obtained from 3.2 Comparison of FT conversion between

10 mm ID reactor mainly consisted of C1-C4 milli- and centi-tubular reactors
(91%) with small amount of C5-C20 (8.9%). Experimental results of conversions,
In contrast, FT process in 5 mm ID reactor HCs production and performance from FT
gave a hydrocarbon product which synthesis via a milli-tubular reactor in this
containing large amount of gasoline and work were compared with the previous
diesel range HCs (C5-C20, 57.6%), followed studies of other investigators those using
by C1-C4 (39.2%) and C20+ (3.2%). Thus, continuous fixed bed reactors and
higher liquid HCs yield at the value of 0.191
Table 1. Comparison of FT conversion between milli- and centi-tubular reactors
Researcher Reactor Catalyst Temperature Pressure Space velocity g /h.g
oil cat
size (mm) diameter (mm) (K) (MPa) (NL/g .h)
cat
Rafiq et al. 11 27.2 3 473 2 0.148 0.0092
Yang et al. 15 25.4 5 (Co-pellet) 490 2 1.3 0.04
25.4 5 (Co-foam) 496 2 4 0.12
Tristantini et al. 16 9.3 0.053-0.9 483 2 6.3 0.37
Liu et al. 17 6 0.15-0.25 493 1 2.4 0.24
Present work 5 1 493 2 9.6 0.19
(4.39 h-1)
milli-tubular reactors as summarized in Sust. Energ. Rev. 2011, 15, 366–378.

Table 1. The comparison results reveal that 5. Bahome, M. C.; Jewell, L. L.;
miniaturized reactor is more efficient than Hildebrandt, D.; Glasser, D. J. Appl.
conventional, centi-fixed bed, reactor. Due Catal. A: Genl. 2005, 287, 60–67.
to the increasing of heat and mass transfer in 6. Kim, Y. D.; Yang, C. W.; Kim, B. J.;
milli-tubular reactor, high liquid Moon, J. H.; Jeong, J. Y.; Jeong, S. H.;
hydrocarbon yield was achieved. This result Lee, S. H.; Kim, J. H.; Seo, M. W.; Lee,
was in accordance with works of Tristatini S. B.; Kim, J. K. Appl. Energ. 2016, 180,
and coworker and Liu and co-worker who 301–312.
also performing FT process in milli-fixed 7. Yang, L. PhD's Degree Thesis,
bed reactor. University of California. 2013.
8. Hans, S. Appl. Catal. 1999, 186, 3–12.
4. Conclusion 9. Guillermo, C.; Antonio, L.; Rafael, G.
The synthesized liquid hydrocarbon Fuel. 1995, 74, 445–451.
from syngas via FT process has been studied 10. Liu, Y.; Teng, B. T.; Guo, X. H. J. Mol.
in a milli-tubular reactor. The influences of Catal. A: Chem. 2007, 272, 182–190.
reaction temperature, pressure and GHSV on 11. Rafiq, M.; Jakoben, H.; Schmid, R.;
CO conversion, CH4 and CO2 selectivity Hustad, J. Fuel Process. Technol. 2011,
were carefully investigated. The highest 92, 893–907.
liquid hydrocarbon yield of 0.19 goil/h.gcat 12. Yu, L.; David, N.; Dominique, B.;
was optimally achieved when operated at Patrick, N.; Charlotte, P. Chem. Eng. J.
493 K, 2 MPa and 4.39 h-1 of GHSV. 2013, 222, 265–273.
13. Chunshe, C.; Jianli, H.; Shari, L.;
Acknowledgements Wayne, W.; Yong, W. Catal. Today
The authors gratefully acknowledged 2009, 140, 149–156.
financial support from National Research 14. Ying, X.; Zhang, L.; Xu, H.; Ren, Y.;
Council of Thailand (NRCT). Xuan, J. Energy Procedia 2014, 61,
1394–1397.
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Turkenburg, W. C. Energ. Convers. 89, 237–243.
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4. Damartzis, T.; Zabaniotou, A. Renew.
Bio-oils from palm oil residue via hydrothermal liquefaction process
in the environmental impact aspect
Panida Thepkhun*, Amornrat Suemanotham
Thailand Institute of Scientific and Technological Research, Khlong Luang, Pathumthani 12120, Thailand
*E-mail: panida_s@tistr.or.th
Abstract:
Palm oil is the potential feedstock for biodiesel production in Thailand. In
AEDP2015, the target of biodiesel consumption in the market has been forced to increase
from currently 7% to 20% in 2030 for achieving the energy security as well as environmental
concern. The increasing of biodiesel consumption will be the cause of palm oil production
and its residues increment. Most of residues from palm oil industry have potential to be
resources of biofuel, e.g. bio-oils, however high mount of moisture are carried. In this study,
bio-oils production from the residues via hydrothermal liquefaction process, required
moisture during operation, was investigated from an environmental impact point of view.
Raw material alternatives using two different residues, i.e. palm shell and empty fruit bunch,
were considered and compared in optimum condition. Eco-indicator 99 was used as method
to evaluate the environmental impact via SimaPro8.3 software. The environmental impacts
categories from hydrothermal liquefaction process were considered in four areas, i.e. global
warming potential, ozone layer depletion, acidification and eutrophication. The results
showed that the electric consumption during the production dominated the environmental
impacts while other chemical substances gave lower impacts. Furthermore, the sensitivity
analysis of operation factors and its production yield were considered in this study.
1. Introduction been forced to increase from currently 7% to

The annual report on energy situation 20% by 2030 for achieving the energy
showed that Thailand’s final energy security as well as environmental concern.3
consumption was 79,929 ktoe that was As palm oil has been mainly feedstock of
dominated by the commercial energy biodiesel in Thailand, the increasing of
consumption (84.2%), followed by consumption will be the cause of palm oil
renewable energy and traditional renewable production and its residues increment. Most
energy (9.0% and 6.8%, respectively). The of residues from palm oil industry have
consumption of petroleum products have potential to be resources of biofuel, e.g. bio-
been used in total commercial energy almost oils.
40 Mtoe or accounted for 49.7% of all. Its Hydrothermal liquefaction process
trend showed that the consumption (HTL) is a thermochemical conversion used
continuously increased year by year.1 The to convert biomass into liquid fuels by
petroleum products were mostly used in processing in typically conditions are 250 –
transport sector 20,996 ktoe that biofuels, 374 oC and 4 to 22 Mpa with water
i.e. ethanol and biodiesel, were used only environment for sufficient time.4,5 As the
8.32% or 1,747 ktoe.1,2 By the reason, procedure can be occurred under water
biofuels consumption has been encourage presented, it is the most important
through “Alternative Energy Development advantages of high moisture content
Plan: AEDP2015” targeted 8,712 ktoe, feedstock without the need of pre-drying
including ethanol, biodiesel, pyrolysis oil, process.5 Therefore, in this study, the HTL
compress bio-methane and others, in 2036. process was used to be a process for
Biodiesel blending ratio in the market has converting palm oil residues into bio-oils in
various conditions and their environmental by filtration under a vacuum. Bio-oil was
impact were investigated via life cycle extracted from liquid phase with
assessment tool. dichloromethane. Final product was
obtained after removed extraction solvent in
2. Materials and Methods the evaporation process.
2.1 Raw materials Comparison of reaction time from
Palm oil has been used as raw the optimum conditions of HTL process
materials in food industrials as well as in from EFB showed that the longer time of
biodiesel production for transportation fuel. reaction giving higher yield even lower
The palm oil production typically generated temperature, i.e. the yield of bio-oil from
by-products and residues which were empty EFB1 was 6.77% whereas EFB2 was
fruit bunch, palm kernel shell, palm 13.22%. The optimum conditions of PKS
mesocarp fiber, cake, and wastewater. In gave highest yield 32.26% at high
this study, empty fruit bunch (EFB) and temperature (320 oC) and followed by
palm kernel shell (PKS), which were 13.80% with 280 oC at similar reaction time
residues from palm oil industrial accounted (15 minutes). The bio-oil products as well as
for 20% and 4% of fresh fruit bunch, all input and output substances from process
respectively, were used as raw materials in were collected and recorded as shown in
HTL process. The proximate and ultimate Table 2 and Table 3.
analyses, as well as higher heating value
(HHV) of EFB and PKS were obtained from Table 2. Materials and energy use for 1 g of
the study by Suemanotham et al.6 showed bio-oil production from EFB
that volatile matters of EFB and PKS were Operation condition
83.72 and 76.93%, respectively. EFB1 EFB2
Furthermore, the initial moisture and HHV Materials
Input
of EFB were 33.0% and 19.8 MJ/kg, while EFB (g) 12.27 8.70
PKS were 25.6% and 21.0 MJ/kg, water (g) 120.70 87.04
respectively.6 dichloromethane (g) 78.49 58.19
2.2 HTL process Output
The HTL process was conducted in a dichloromethane (g) 77.48 51.32
wastewater (g) 122.44 92.87
batch type reactor equipped with an electric Others (g) 10.54 8.74
furnace. Regarding the preliminary study, Energy
the ratio of palm residue and water was set Electricity (kWh) 0.771 1.148
at 1:10. The optimized conditions of HTL
process subject to maximum yield of bio-oil Table 3. Materials and energy use for 1 g of
were studied in preliminary stage and 2 bio-oil production from PKS
conditions of each feedstock were selected Operation condition
for comparing theirs environmental impacts PKS1 PKS2
Materials
in this study (see Table 1).
Input
KPS (g) 8.55 5.58
Table 1. The optimum conditions of HTL water (g) 84.86 55.63
process dichloromethane (g) 90.49 54.45
Feedstock Condition Output
Empty fruit bunch EFB1 280 oC 15 minutes dichloromethane (g) 82.59 33.45
EFB2 260 oC 30 minutes wastewater (g) 94.10 76.49
Palm kernel shell PKS1 280 oC 15 minutes Others (g) 7.21 5.72
PKS2 320 oC 15 minutes Energy
Electricity (kWh) 0.734 0.803
The HTL reactions were occurred in
the Parr high pressure reactor. Liquid and
solid products from reaction were separated
2.3 Life cycle assessment 2.3.3 Life cycle impact assessment (LCIA)
Environmental impacts of bio-oil Life cycle impact assessment of the
production from EFB and PKS via HTL process has been evaluated by Eco-indicator
process were analyzed by life cycle 99 method via software SimaPro8.3. The
assessment (LCA) approach. LCA is wildly focusing of environmental impact categories
used as tool for analyzing the environmental investigated were global warming potential
impacts of products, technologies or services (GWP), ozone layer depletion, acidification,
with a view to their whole life cycle. The and eutrophication.
LCA methodology according to the ISO
14040-17 as outline was used as follow. 3. Results and Discussion
2.3.1 Goal and scope definition 3.1 Environmental impacts
The objective of this study was to The environmental impacts from
compare the environmental impacts of bio- functional unit of bio-oil production via
oil production via HTL process from palm HTL process from EFB and PKS were
oil residues with the scope shown in shown in Table 4. The characterized results
Figure 1. The functional unit was 1 g of bio- contributions of inputs and energy use for
oil produced. the production of 1 g bio-oil from EFM and
PKS via HTL process were illustrated by
Palm oil residue Figure 2 and 3, respectively. The results
showed that GWP emissions of the process
significantly contributed from electricity
HTL reactor
use. The highest GWP emissions occurred in
the longest reaction time batch, i.e. EFB2,
due to the electricity consumption. Apart
Separation from the GWP emissions, electricity
consumption also impacted on the
Extraction
eutrophication category thus the EFB2 and
PKS2 batches which were operated at longer
reaction time and high temperature
Evaporation generated high amount of emissions in this
category.
The impact on ozone layer depletion
category came from the dichloromethane
Bio-oil
use in the bio-oil extraction process and
recovery via the evaporation process. The
Figure 1. System boundary for bio-oil dichloromethane use also contributed in
production via HTL process of palm oil other categories, which were GWP
residue emissions and acidification. The amount of
dichloromethane consumption was
2.3.2 Life cycle inventory (LCI) depended on the lost during recovery
Emissions generated from resources process and the impact of its usage was
and materials use, and energy consumption depended on production yield.
were calculated and expressed as per Environmental burdens from
functional unit. Inventory of resources, feedstock of bio-oil production impacted on
materials, and electricity were obtained from the acidification, eutrophication, as well as
the database of the software, SimaPro8.3. GWP emissions categories that came from
Wastewater from process was not accounted the production and use of various fertilizers
in this study. during plantation as well as the
transportation of palm fresh fruit bunch from
plantation area to the mills.8 The results in
Table 4. Environmental impacts of bio-oil production from HTL process from EFB and PKS
EFB PKS
Categories
EFB1 EFB2 PKS1 PKS2
Global warming potential (kgCO2-eq) 0.48 0.76 0.50 0.55
Ozone layer depletion (kgCFC11eq) 3.01x10-6 3.03x10-6 3.01x10-6 3.03x10-6
Acidification (kgSO2-eq) 2.27x10-3 3.25x10-3 2.43x10-3 2.59x10-3
Eutrophication (kgPO4-eq) 8.68x10-4 1.50x10-3 9.74x10-4 1.08x10-3
Figure 2. Environmental impact categories of bio-oil production from EFB via HTL process
Figure 3. Environmental impact categories of bio-oil production from PKS via HTL process
this study showed that the use of PKS as burdens in all categories, particularly,
feedstock contributed more emissions than increasing in eutrophication and GWP as
EFB in all categories. 72.30% and GWP 59.74%, respectively.
3.2 Sensitivity analysis In case of PKS, the considered
The sensitivity analysis in this factor was temperature increased from 280
study was subjected to optimize production to 320 oC or accounted for 14.29% increase.
yield and environmental impacts during The temperature increment significantly
process. The EFB conditions considered affected to the production yield as increasing
reaction time twice increase that effect to the 52.54%. The environmental burdens from
increment of bio-oil yield accounted for feedstock consumption were slightly
38.55%. The environmental impacts from decreased whereas they were increased in
feedstock and water consumption decreased energy consumption part about 14% in all
in all categories about 18 and 17%, categories. The overall environmental
respectively. However the increasing of impacts were highest increase in
operation time affected to overall environmental eutrophication category (i.e. 10.78%),
followed by GWP and acidification (i.e. References
9.54 and 6.55%, respectively). 1. http://www.dede.go.th/download/state_5
3.3 Limitation of this study 9/Energybalance2016.pdf
The study of environmental impacts 2. http://www.dede.go.th/download/state_5
evaluated was conducted based on a general 9/thailand%20alternative2016.pdf
conceptual process of biomass hydrothermal 3. http://www.eppo.go.th/images/POLICY/
liquefaction to bio-oil. The parameters of ENG/AEDP2015ENG.pdf
materials consumption and the breakdown 4. Gollakota, A. R. K.; Kishore, N.; Gu, S.
of utility and electrical consumption were Renew. Sust. Energ. Rev. 2018, 81,
mainly obtained from laboratory that might 1378–1392.
not represent an industrial scale. 5. Tekin, K.; Karagöz, S.; Bektaș, S.
Renew. Sust. Energ. Rev. 2014, 40, 673–
4. Conclusion 687.
Palm oil residues, i.e. EFB and 6. Suemanotham, A.; Attanatho, L.;
PKS, were used as feedstock for producing Laosombat, T.; Sawaengsub, D.
bio-oil via HTL process. The environmental Proceeding of SEE2016 on Energy &
impacts from the process were considered. Climate Change Innovating for a
The results showed that emissions in GWP, Sustainable Future. 28-30 November
acidification and eutrophication categories 2016, Bangkok, Thailand.
were dominated by electricity consumption 7. Wenzel, H.; Hauschild, M.; Alting, L.
whereas acidification category was came Environmental assessment of products.
from dichloromethane use. The sensitivity In: Methodology, tools and cases
analysis study showed that the significant histories in product development, vol.1
factor for optimum yield and environmental Chapman & Hall: London, 1997.
burdens was temperature as occurred in the 8. Zulkifli, H.; Halimah, M.; Chan, K. W.;
highest temperature condition, i.e. PKS2 Choo, Y. M.; Mohd Basri, W. J. Oil
batch. Palm Res. 2010, 22, 887–894.
Acknowledgements
The authors wish to the financial
support from National Research Council of
Thailand.
Adsorptive removal of methyl red dye using eggshell particles
Tanadta Archvapisith, Wanida Wattanakaroon*
Sanamchandra Palace Campus, Nakhon Pathom 73000, Thailand
*E-mail: watanakaroon_w@su.ac.th
Abstract:
Presence of azo dyes in wastewater causes serious environmental impacts. Adsorption
in materials has become the effective method of choice for removal of these colored dyes.
This study aimed to investigate the feasibility of using eggshell particles prepared from
chicken eggshell waste as a low-cost adsorbent for dye removal. The eggshell was ground
into powder of different particle sizes. The morphology, charge and average size of the
eggshell particle were then characterized. The effects of particle size, dose and contact time
on the sorption efficacy of methyl red as the tested dye onto particles were evaluated. Results
stated that the adsorption capacity little increased with a decrease in the particle size. SEM
analysis revealed that the particle had an irregular shape and a highly porous structure on the
surface. The zeta potential was -13.67 mV with the size distribution of 22.23 ± 0.35 µm for
the smallest particle size obtained (less than 45 µm). The maximum percentage dye removal
was found to be 40.88 ± 1.07% at a particle size less than 45 µm for a concentration of 3 g
after 1 hour. These characteristics indicate the suitability of eggshell waste to be used as an
efficient adsorbent for the removal of dye in solutions.
1. Introduction morphology, charge and average size of the

Azo dyes are the water pollutants particle were characterized. The effects of
frequently found in industrial wastewater particle size, dose and contact time on the
that cause serious environmental impacts. sorption efficacy onto particles were then
Physical and chemical processes to remove evaluated.
color and toxicity from dye-containing
waste solutions have been extensively 2. Materials and Methods
studied.1,2 However, most of these processes 2.1 Chemicals
are expensive and availability has restricted Chicken eggshells were collected
their application. Adsorption in materials is from the refreshment stall at Silpakorn
a common and frequently used process and University. Methyl red was purchased from
has become the effective method of choice Fluka (Switzerland). All commercially
for removal of these colored dyes. In light of available reagents used were of analytical
the likely benefits of natural and nature- grade and no further purifications were
inspired biomaterials, eggshell as a natural made.
composite has been utilized in various 2.2 Preparation of eggshell particles
applications due to its unique chemical The eggshells were washed and air-
composition and substantial availability.3,4 dried for 30 minutes. After rinsing with
Studies of these materials for the removal of deionized water, the membranes were
organic dyes and inorganic ions has been separated from eggshells with forceps. The
reviewed.5-7 In this study, eggshell particles eggshells were then dried overnight at room
were prepared from chicken eggshell waste. temperature. Eggshell particles were
On the basis of their performance prepared following the method which was
evaluations, eggshell particles were used for reported by Hassan et al.8 In brief, dry
adsorption of dyes. In this respect, methyl eggshells were ground into powder using a
red was chosen as the tested dye. The commercial coffee grinder. The fine
eggshell powder was collected and soaked in adsorbed. The percentage removal was then
acetone for 2 hours with continuous stirring. calculated according to Equation (1)
Subsequently, the powder was left to settle C0  C t
Percentage removal   100 (1)
down for 1 hour. After which time a solvent C0
was withdrawn, the eggshell powder was where C0 is the initial concentration before
oven-dried at 60 C for 2 hours and left to treatment and Ct is the concentration of
for overnight. In order to prepare eggshell methyl red at time t.
particles for further experiments, the as-
prepared powder was separated using 3. Results and Discussion
standard stainless steel sieves 212, 90 and 45 3.1 Physicochemical properties of eggshell
µm in size. The sieves were arranged in particles
series on top of a sieve shaker. The Eggshells were ground into powder
separation process was run for 20 minutes at and different particle sizes were then
1.50 mm amplitude of the shaker. The collected. Figure 1 illustrates that eggshell
particles of different sizes were then particle sizes were reduced to a few
collected and stored in a desiccator until use. micrometers. The surface and internal
2.3 Characterization morphology of eggshell particles 45 µm and
The surface structure of the particles smaller was further characterized using
was observed using a scanning electron SEM. With the results presented in Figure
microscope model JSM-6610LV. The size 2a, the eggshell particles displayed size
was assessed by laser scattering particle size distribution. As seen, the particle had an
distribution analyzer Horiba Scientific LA- irregular shape and a highly porous structure
950 and zeta potential was measured using a on the surface (Figure 2b). The zeta
zeta potential analyzer Horiba Scientific potential was -13.67 mV due to the negative
(ZetaPlus). nature of the eggshell with the size
2.4 Adsorption distribution of 22.23 ± 0.35 µm for the
To investigate the efficiency of the smallest particle size obtained (less than 45
dye removal of eggshell particles, the µm). Similarly, a previous investigation by
particle size and dose and contact time were others indicated that zeta potentials of
conducted. Particles of different sizes 0.5 g nanopowdered eggshell were -15.41 mV -
were mixed with 10 ml of 25 mg/l methyl
red solution with stirring at 150 rpm for 1
hour. The mixture was then centrifuged at
2,000 rpm for 10 minutes and the
supernatant liquid containing dye was taken.
The methyl red content was measured using
a microplate reader at a wavelength of 430
nm. The relative absorbance of particles at
different sizes was estimated as the ratio of
the absorbance after adsorption to the
absorbance of the initial dye concentration
used. The effects of contact time (i.e.,
mixing period of 1, 2, 3, and 4 hours) and
particle dosage (i.e., 0.5, 1, 2, and 3 g) were
investigated using particles 45 µm and
smaller for optimization experiment, and the
adsorptive removal was performed as Figure 1. Eggshell powder particles
described above. Calibration curve of known obtained after grinding: a) 45 µm and
concentrations in the appropriate range was smaller; b) 45-90 µm; c) 90-212 µm; d) 212
used to determine the amount of methyl red µm and larger
suggesting an incipient instability of the 25
colloidal system.9
20
Percentage removal
a) b)
15
10
Figure 2. SEM micrographs of: a) eggshell 0 1 2 3 4 5

Mixing period (hours)
particles 45 µm and smaller (Scale bar = 50
µm); b) outer surface of eggshell particles Figure 3. Effect of contact time on the
(Scale bar = 1 µm) adsorption of methyl red onto eggshell
particles. The data are reported as the
Table 1. Effect of eggshell particle size on percentage dye removal versus time. Error
the relative absorbance bars indicate the standard errors of the
Particle size (µm) Relative absorbance means and the line represents the best fit to
< 45 0.86 ± 0.02 the data.
45-90 0.96 ± 0.02
90-212 1.00 ± 0.00 50
> 212 1.01 ± 0.00
40
Figures 3 and 4 represent the effects

Percentage removal
of contact time and eggshell particle dosage 30
on the adsorption of methyl red. As shown

20
in Figure 3, dye removal reached maximum
with mixing period of 1 hour and became a
10
little drop in efficiency over the tested
period. The adsorption process was rapid 0
initially due to availability of binding site on 0 0.5 1 1.5 2 2.5 3 3.5
the adsorbent surface which is controlled by Particle dose (g)
diffusion process from the bulk to the Figure 4. Effect of dosage on the adsorption
surface. Based on the observed profile, the of methyl red onto eggshell particles. The
adsorption increased with increase of data are reported as the percentage dye
adsorbent dose. The maximum percentage removal versus particle dose. Error bars
dye removal was found to be 40.88 ± 1.07% indicate the standard errors of the means and
at a particle size less than 45 µm for a the line represents the best fit to the data.
concentration of 3 g after 1 hour (Figure 4).
This trend is in agreement with the findings as an adsorbent could give economic value
of Ali Zulfikar et al. and Tsai et al.10,11 to the waste and also provide a low-cost
These results clearly suggest that the sorbent adsorbent for water treatment. Further
dose provides the adsorption surface area studies will be extended to investigate the
and the availability of more active and adsorption behavior, kinetics and
available sites. equilibrium of eggshell particles. In
addition, the development of chemical
4. Conclusion surface modification of eggshell particles
The adsorption data in this study with optimized novel properties is of interest
indicate the possibility of value-added for the applications of these materials in
utilization of eggshell for the removal of dye other suitable fields.
in solutions. The effective use of the eggshell
Acknowledgements 5. Guru, P. S.; Dash, S. Adv. Colloid
This research was financially Interface Sci. 2014, 209, 49–67.
supported by Department of Biotechnology, 6. Mittal, A.; Teotia, M.; Soni, R. K.;
Faculty of Engineering and Industrial Mittal, J. J. Mol. Liq. 2016, 223, 376–
Technology, Silpakorn University. The 387.
authors are indebted to the Faculty of 7. Abdel-Khalek, M. A.; Abdel Rahman,
Pharmacy at Silpakorn University for the M. K.; Francis, A. A. J. Environ. Chem.
facilities and equipment. Eng. 2017, 5, 319–327.
8. Hassan, T. A.; Rangari, V. K.; Rana, R.
References K.; Jeelani, S. Ultrason. Sonochem.
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2009, 105, 299–303. Int. J. Food Sci. Technol. 2014, 49,
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Qi, Y.; Zhang, G. J. Hazard. Mater. 10. Ali Zulfikar, M.; Mariske, E. D.;
2018, 346, 42–51. Djajanti, S. D. Songklanakarin J. Sci.
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Lim, K. T.; Lee, K. -H.; Seonwoo, H.; 11. Tsai, W. -T.; Hsien, K. -J.; Hsu, H. -C.;
Kim, J. Biosyst. Eng. 2016, 151, 446– Lin, C. -M.; Lin, K. -Y.; Chiu, C. -H.
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O. O.; Akpan, I. C. J. Environ. Chem.
Eng. 2016, 4, 1367–1375.
Synthesis of biodiesel by transesterification reaction of used palm oil using
modified calcium oxide doped strontium ion as a catalyst
Apisit Prokaew1, Siwaporn Meejoo Smith2, Apanee Luengnaruemitchai3,
Supakorn Boonyeun1*
1
Department of Chemistry, Faculty of Science and Technology, Thammasat University, Khlong Luang,
2
Department of Chemistry, Faculty of Science and Technology, Mahidol University, Phuttamonthon,
Nakorn Pathom 73170, Thailand
3
The Petroleum and Petrochemical College, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
*E-mail: chemistrytu@gmail.com
Abstract:
In this research studied, the modified calcium oxide catalyst doped strontium ion (3%-
50%wt.) for used in transesterification reaction of used palm oil with methanol. The modified
catalyst was synthesized by co-precipitation method between SrCl2 and Ca(NO3)2 solution
then calcined the mixture at 900 °C for 5 h to form a calcium doped strontium oxide catalyst.
The characterizations were including Thermo-gravimetric analysis (TGA), X-ray diffraction
(XRD), Scanning electron microscope (SEM), and Fourier transform infrared spectrometer
(FT-IR). The result of SEM-EDX confirmed the morphology and elements content of Sr and
Ca in the catalyst (prepared; 10.00:0.30 Ca:Sr catalyst found; Ca:Sr = 10.00:0.33). The
approximate catalyst diameter is about 12.6±5.906 µm. The results show the highest
conversion with 100% (the reaction time 3 h, catalyst loading 5% w/w, methanol to oil molar
ratio 9:1 and reaction temperature 80 °C when used 3% Sr-CaO as a catalyst). The
reusability of catalyst was studied and the results show six-time repeat reactions with a good
yield (over 80%).
1. Introduction The catalyst for biodiesel production

During the past two decades, world by transesterification can be used both
energy demand is increasing every year homogeneous and heterogeneous catalyst.6
mainly from population growth, the In the industrial scale, homogeneous
industrial, economic and social development alkaline base catalysts such as sodium
and the transportation. Most of all energy hydroxide or potassium hydroxide are
was supported by non-renewable fossil fuel usually used in the biodiesel production
such as petroleum oil, natural gas, and coal.1 process because it can produce very high
However, the result of those energies is biodiesel conversion and require mind
continuously decreased so we need to found reaction condition.7,8 However this catalyst
other alternative and sustainable energy. can cause many problems such as it difficult
Biodiesel is one of the more to separate catalyst from biodiesel product,
interesting alternative energy because it is it can produce a large amount of the toxic
renewable, biodegradable, low-level of water from the catalyst separation process
greenhouse gas emission and and catalyst can't be reused. The
2,3
environmentally friendly. Biodiesel can be heterogeneous catalyst for biodiesel
produced by using the transesterification production has been investigated because it
process between fat or oil with alcohol such is reusable, easy to separate after reaction
as methanol or ethanol.4,5 The reaction complete and more environmental friendly
produces Fatty Acid Methyl Ester (FAME) than the homogeneous catalyst.2,9 However
as the main product with trace amounts of heterogeneous catalyst still has some
glycerol. disadvantages such as it will lose surface
area after reaction complete and use long room-temperature to 1273 K at a ramping
reaction time to complete the reaction. rate of 10 K min-1 under a flow of
Therefore, to solve these problems the nitrogen.The catalysts morphology was
researchers try to improve the catalyst. The studied by JEOL, JSM-7800F equipped with
popular technique to improve catalysts is EDX ( Oxford X-MAX20 ).
doping the metal ion to the catalyst because 2.4 Transesterification reaction
it can increase the efficiency and activity of The transesterification reaction from
the catalyst, increase the surface of the used palm oil and methanol for a synthesis
catalyst, maintain high catalytic activity of biodiesel was carried out in a round-
after multiple runs, reduce leaching problem bottomed flask equipped with a cooled
and decrease the reaction time.10-14 water condenser and a mechanical stirrer
and hot plate. The biodiesel were
2. Materials and Methods synthesized by various the effects of
2.1 General methanol to oil molar ratio (1:1, 3:1, 5:1 and
Calcium Nitrate tetrahydrate 9:1), catalyst concentration (5, 10, 15 and 20
(Ca(NO3)2.4H2O was purchased from %wt), reaction time (3, 4, 5 and 7 h) on the
Scharlau Chemic S.A. Strontium Chloride conversion to biodiesel. The mixture was
(SrCl2) and Sodium carbonate (Na2CO3) heated at 80 °C and stirred until the
were purchased from Ajax completion of the reaction. After the
Finechem.Wasted Palm oil was obtained in reaction complete the catalyst was removed
the canteen in the Thammasat University. from the biodiesel by filtration and an
Methanol was analytical reagent (AR grade) excessive amount of methanol was removed
from Merck Limited. by rotary evaporator. The biodiesel
2.2 Catalyst preparation conversion was calculated by the ratio of the
The CaO doped with strontium ion integration of the 1H-NMR signals between
catalysts were prepared using co- methoxy proton (CH3O-) of methyl ester
precipitation method by integration (ACH3) at the chemical shift of 3.68 ppm and
Ca(NO3)2 and SrCl2 solution with a various ethylene proton (CH3-) of all triglyceride
molar ratio between both solutions (1:1, (ACH2) at the chemical shift of 2.30 ppm
10:1 and 10:0.3). Next, the solution of the according to the following Equation 1.15
1M Na2CO3 solution was dropping to % FAME = x 100%
mixture solution until the reaction complete
and stirring all time. After that, the catalysts Equation 1. The equation to calculate
were filtered and dried at 373K for 10 h biodiesel conversion
before calcined at 1173 K for 3 h.
2.3 Catalyst characterizations 3. Results and Discussion
The characterization of the catalysts 3.1 Catalyst characterization
were confirmed by a X-ray diffraction The TGA result of the non-calcined
(XRD) using a Bruker AXS D8 Advance; catalyst and calcined catalyst are shown in
Cu Kα1 radiation, λ=1.5406 A° measured Figure 1. The TGA result of non-calcined
from 5° to 80° (2θ) with a step size of 0.02°. catalyst showed two-step decompose. At
SEM was performed on a JEOL, JSM-6390 first step, CO2 was removed from CaCO3
Fourier transforms infrared (FTIR) spectra around 650-700 °C followed by the second
were measured on a Perkin-Elmer infrared step around 800 °C due to CO2 remove
spectroscopy spectrum GX, FT-IR applying from the SrCO3 and changed into CaO and
KBr pellet technique with 4 cm-1 resolution SrO. The TGA result of calcined catalyst
over a scanning range of 400-4000 cm-1. showed two-step decomposed. At first, the
The samples were evaluated by step was shown at 350-400 °C due to the
thermogravimetric analysis (TGA) using Ca(OH)2 decomposed and phase changed to
NERZSCH STA 449 F3 instrument from CaO followed by the second step around
650-800 °C due to some CO2 removed from 50°. However in the 10:1 CaO:Sr and 10:0.3
the catalyst. CaO:Sr, there was the only peak of CaO and
there was no peak of SrO.
Figure 1. TGA result of the calcined and

non-calcined catalyst
The FT-IR spectra of 10:0.3 CaO:Sr Figure 3. XRD pattern of all catalyst
catalyst was shown in Figure 2. The result of
the FT-IR spectra of the catalyst was shown The SEM images of calcium oxide
broadband peaks of asymmetric stretching doped with strontium ion (10:1 and 10:3
of Sr-O at 1400 cm-1 to 1500 cm-1 and CaO:Sr) are shown in Figure 4. The result
peaks of out of plane bending vibration of show at 10:1 and 10:0.3 molar CaO:Sr ratio
Sr-O at 700 cm-1 and 870 cm-1.16 The exhibited a smooth surface and porous
peaks of C=O in CO32- group also structure. The components of the catalyst
represented the peak at 2800 to 2900 cm-1 were studied by Energy Dispersive X-Ray
(C=O asymmetric stretching) and 1790 to Spectroscopy. The result found that element
1800 cm-1 (C=O stretching). A sharp peak of percentage of 10:1 CaO:Sr and 10:0.3
-OH group of the Ca(OH)2 at 3635.85 cm-1. CaO:Sr was observed closed to the
percentage of the calculated percentage in
the catalyst preparation process. The result
shown percentage of Ca = 30.08 % and Sr =
3.19 % in 10:1 CaO:Sr and percentage of Ca
= 43.8% and Sr =1.44% were shown in
10:0.3 CaO:Sr.
a b
Figure 2. FT-IR spectra of 10:0.3 CaO:Sr
catalyst
The XRD pattern of calcium oxide

doped with strontium ion was shown in
Figure 3. The catalyst was found the c d
composition of calcium oxide and strontium
carbonate and strontium oxide. The peaks of
calcium oxide were found in all catalyst and
can be detected at 2θ = 37.42° 50.91° 64.65°
67.40°. In 1:1 CaO: Sr catalyst found the Figure 4. SEM images of 10:1 CaO:Sr (a,b)
major peak of SrCO3 at 2θ = 25.28°, 25.97°, and 10:0.3 CaO:Sr catalyst (c,d)
31.67°, 34.69°, 35.50°, 43.27°, 45.96°,
46.78° and 56.61° and found small two 3.2 Transesterification
peaks of SrO at 2θ= SrO at 2θ= 34.84° and 3.2.1 Effect of reaction times
The effect of reaction time on
biodiesel conversion was showed in
Figure 5. The effect of the reaction time was
studied following the reaction condition
methanol to oil ratio 9:1, catalyst
concentration 10%wt and reaction
temperature at 80 °C by various the reaction
time at 3, 4, 5 and 7 h. The result showed
that when the reaction time increases the
percent conversion of biodiesel will
increases and constant after reaction time Figure 6. Effect of methanol to oil molar
3 h. The maximum conversion (99.27%) of ratio
wasted palm oil was achieved at 3 h.
Figure 5. Effect of reaction times

Figure 7. Effect of catalyst concentration
3.2.2 Effect of methanol to oil molar ratio
The effect of methanol to oil molar
to 10 %, 15% and 20% wt., the biodiesel
ratio was showed in Figure 6. The effect of
conversion tend to decrease comparing to
the methanol to oil molar ratio was studied
5% due to increasing of CaO, causing
following the reaction condition catalyst
integrate between biodiesel layer and
concentration 10%wt, reaction temperature
glycerol layer as well as low mass transfer.
at 80 °C, reaction time 3 h and various the
Moreover, it was found using 5%wt of
reaction time at 1:1, 3:1, 5:1, 7:1 and 9:1. It
catalyst showed the highest biodiesel
was found that increasing the molar ratio
conversion.
enhanced the biodiesel conversion.
3.2.4 Catalytic activity
Furthermore, the maximum biodiesel
The catalytic activity of all catalysts
conversion was 99.7% by using 9:1 molar
was shown in Table 1. The result was shown
ratio of methanol to wasted palm oil.
that 10:0.3 CaO:Sr gave the highest
3.2.3 Effect of catalyst concentration
biodiesel conversion when compared with
The effect of catalyst concentration
other catalysts and gave the biodiesel
was shown in Figure 7. The effect of
conversion 100% when used the optimal
catalyst concentration was studied following
condition (methanol to oil molar ratio 9:1,
the reaction condition reaction temperature
catalyst concentration 5%wt, reaction time 3
at 80 °C, reaction time 3 h and methanol to
h and reaction temperature 80 °C).
oil molar ratio 9:1 by various the catalyst
However, all of the catalysts gave biodiesel
concentration at 5%, 10%, 15% and 20% by
conversion in the range of 98 to 100%.
weight. The result showed that the highest
3.2.5 Reusability of catalyst
biodiesel conversion (99.67%) was achieved
The reusability of catalyst was
when used catalyst concentration 5%wt.
shown in Figure 8. The experiments indicate
When increasing catalyst concentration up
Table 1. Catalytic activity Science and Technology, Thammasat
Catalyst Biodiesel conversion (%) University.
CaO 99.33
1:1 CaO:Sr 99.67
10:1 CaO:Sr 99.67
References
10:0.3 CaO:Sr 100 1. Tariq, M.; Ali, S.; Khalid, N. Renew.
SrO 98 Sust. Energ. Rev. 2012, 16, 6303–6316.
2. Viriya-empikul, N.; Krasae, P.;
that CaO:Sr catalysts can be reused for 6 Puttasawat, B.; Yoosuk, B.; Chollacoop,
times and still have 80%wt with an optimal N. Bioresource Technol. 2010, 101,
condition, methanol to oil ratio 3:1, catalyst 3765–3767.
concentration 5%wt and the reaction time 5 3. Karmee, S. K.; Chadha, A. Bioresour
h. Technol. 2005, 96, 1425–1429.
4. Boey, P. L.; Maniam, G. P.; Hamidb, S.
A. Bioresource Technol. 2009, 100,
6362–6368.
5. Adashi, I. M.; Arour, M. K.; Abdul
Aziz, A. R.; Sulaiman, N. M. N. J. Ind.
Eng. Chem. 2013, 19, 14–26.
6. Boey, P. L.; Maniam, G. P.; Hamid S.
A.; Ali, D. M. H. Fuel 2011, 90, 2353–
2358.
7. Boey, P. L.; Maniam, G. P.; Hamid, S.
A. Bioresource Technol. 2009, 100,
Figure 8. Reusability of CaO:Sr catalyst for 6362–6368.
in transesterification reaction with used 8. Boro, J.; Thaku, A. J.; Deka, D. Fuel
palm oil Process Technol. 2011, 92, 2061–2067.
9. Ramadhas, A. S.; Jayaraj S.;
4. Conclusion Muraleedharan, C. Fuel 2005, 84, 335–
In this study, calcium oxide doped 340.
with strontium ion (Sr2+) was used in the 10. Li, H.; Niu, S.; Lu, C.; Li, J. Fuel 2016,
transesterification of used palm. The 176, 63–71.
synthesized catalyst can be converted from 11. Teo, S. H.; Rashid, U.; Taufiq-Yap, Y.
CaCO3 and SrCO3 to CaO and SrO after H. Fuel 2014, 136, 244–252.
calcinated at the temperature over 900 °C. 12. Faungnawakij, B.; Yoosuk, S.;
The optimal condition for biodiesel Namuangruk, P.; Krasae, N.; Viriya-
production was found to be 5% (by wt.) empikul, B. Chem. Cat. Chem. 2012, 4,
loading, 9:1 ratio of methanol to oil and a 209–216.
reaction time of 3 h at 80 °C. The catalyst 13. Chen, C. L.; Huang, C. C.; Tran, D. T.;
can be reused for 6 cycles and gave Chang, J. S. Bioresource Technol. 2012,
biodiesel conversion higher than 80% after 113, 8–13.
cycle 6. 14. Wan Omar, W. N. N.; Amin, N. A. S.
Fuel Process Technol. 2011, 92, 2397–
Acknowledgements 2405.
The financial and all experiments 15. Boro, J.; Konwar, L. J.; Thakur, A. J.;
equipment were supported by Faculty of Deka, D. Fuel 2014, 129,182-187.
Science and Technology, Thammasat 16. Lashanizadegan, M.; Mousavi, F.;
University, the energy conservation Mirzazadeh, H. J. Ceram. Process Res.
promotion funds. And National Research 2016, 17, 586–590.
Council of Thailand. Central Scientific
Instrument Center (CSIC), Faculty of
Isolation and screening of gluconic acid producing microorganisms from
flowers and fruits
Bongkot Chuenpraphai*, Phimchanok Jaturapiree
*E-mail: bchuenpraphai@gmail.com
Abstract:
Gluconic acid (GA) is produced from glucose through a simple oxidation reaction of
which a multifunctional carbonic acid regarding as a bulk chemical in food, beverage,
pharmaceutical and construction industries. This study aimed to isolate and screen GA
producing microorganisms from flowers and fruits. nine fungal genera were isolated from
flowers and fifty-four bacterial genera were isolated from fruits. Samples were tested to have
the ability for GA production. For the tests, microorganisms were grown on glucose medium
in Erlenmeyer flasks with shaking speed of 150 rpm and 37 ºC for 2-5 days. After that, GA
were examined using calcium content dissolved analysis and titration with 0.1 N KMnO4. Of
all, bacteria gave the highest yield which were 17 and 9 g/l of the culture medium,
respectively. The production and the optimization conditions of GA from sugarcane molasses
for using as a biological precursor were studied in the future.
1. Introduction the high costs and deactivation of the

Gluconic acid (GA, catalyst as well as some leaching problems
pentahydroxycaproic acid, Figure 1) is a make these chemical processes less
carbonic acid and occurs naturally in fruits, competitive than fermentation methods.6
plants, rice, meat, wine, vinegar and honey.1 Microbial conversion of glucose into
GA can also be used as the building blocks GA using submerged fermentation is of
of numerous chemical, pharmaceutical, food interest. It can utilize alternative
and beverage industries. In the construction carbohydrate sources (such as hydrol, corn
industry, GA is used as a cement additive starch, grape must, banana must, fig, cheese
because it increases the cement resistance whey, food processing residues, and
against fracture and water.2 Recent research saccharified solution of waste paper).7
has revealed the effects of GA on human Furthermore, it can also add the value of by
and animal health which boosts its use as the products from agro industries. This can lead
prebiotic in food production.3 Use of to efficient GA production processes at a
gluconic acid and its derivatives is currently cheaper price which these methods will help
restricted in many cases due to the high open up new markets for GA and its
price of glucose as the main substrate for derivatives. Production of GA is thus a new
producing GA. Up until now, GA has been challenge for the global market in bio-
produced from chemical processes via the
chemical industries. This study aimed to
reaction of catalyst and oxygen as an
oxidant under mild conditions.4,5 However, isolate and screen GA-producing
GA produced from this reaction could be microorganisms from various flowers and
toxic as a result of chemical residues from fruits.
the reaction. In addition, its low selectivity,
following components: 150 g/l glucose, 0.3
g/l NH4Cl, 0.3 g/l KH2PO4, 0.2 g/l MgSO4
ˑ7H2O, 0.05 g/l yeast extract, 0.1 mg/l
thiamine-HCl and 20 g/l CaCO3 separately
sterilized (Medium C).9 Flask cultures were
Figure 1. Chemical of gluconic acid
formula inoculated from fresh cultures of the isolated
strains and flasks were further incubated for
2. Materials and Methods 48 hours (bacteria) and for 120 hours (fungi)
2.1 Isolation and screening of organisms at 37 ºC on a shaker at 150 rpm.
For isolation of microorganisms, a 2.3 Biochemical analysis
selective acidified glucose medium Assay of total acidity: The culture
containing 100 g/l glucose, 3 g/l yeast above was centrifuged at 4000 rpm for 15
extract and two drops of Tween 40 (Medium minutes and supernatant was withdrawn for
A) was prepared. The pH of the medium was total acidity analysis. The sample was
at adjusted to 2.5-2.6 using citric acid. The prepared to insoluble calcium salt appearing
medium was autoclaved for 15 minutes at in the CaCO3-containing broth which was
121 ºC. Flower and fruit samples were put in consisted almost entirely of calcium oxalate
a medium-containing shaking and incubated as followed. It was treated with hot 20 %
at 37 ºC for 2-5 days on a shaker at 150 rpm. sulfuric acid, and the acid liberated from
calcium was estimated as oxalic acid by
Immediately after a clouding was occurred
titration with standard KMnO4 solution.
in the isolation medium, samples were
streaked on agar plates. The agar plate Soluble calcium remaining in the filtrate was
determined by precipitation of calcium as
formula contained 100 g/l glucose, 5 g/l
oxalate and subsequent titration with
yeast extract, 0.5 g/l MgSO4ˑ7H2O, 1 g/l KMnO4 solution as described above.10
KH2PO4, 5 g/l CaCO3, 20 g/l agar and 0.1 g/l Detection of gluconic acid: GA-
thiamine-HCl (Medium B). Medium was containing samples were analyzed by high
sterilized for 15 minutes at 121 ºC. Note that performance liquid chromatography
solid CaCO3 was placed in a flask, sterilized (HPLC). Gluconic acid were detected with a
separately and added to medium. The plates UV-Vis detector at a wavelength of 210 nm.
were then subjected to incubate at 37 ºC for The column used was a COSMOSIL HILIC
2-5 days. The acid-producing column 5 µm, 4.6 mm I.D. x 250 mm. The
microorganisms were isolated and stored in mobile phase was acetonitrile/10mmol/l
a YME culture medium (yeast-malt-extract- phosphate buffer (pH 7.0) at a flow rate of 1.0
agar for fungi and yeast) and incubated at 37 ml/min. The column oven was set to 30 ºC.
ºC for 2-5 days.8 Isolates were stored at 4 ºC
and sub-cultured every month. Selected 3. Results and Discussion
strains were frozen at -20 ºC or -80 ºC in a 3.1 Screening isolates and GA production
medium containing 30 % glycerine and The GA-producing microorganisms
lyophilized until use. from flowers and fruits were isolated and
2.2 Gluconic acid production screened. Ten and twenty samples from
Isolated acid-producing strains were fruits and flowers, respectively, were
tested in 250 mL shake flasks with 50 mL collected (Figure 2). Data represented in
screening medium at 150 rpm and 37 ºC. Table 1 showed that fifty-four bacterial
The screening medium contained the isolates and nine fungal isolates were tested
for their total acid production. The total acid Banana-must and grape-must as a
production from screening experiments was sole carbon source for GA production using
found to be 0.58-338.10 mg/mL medium. the three irradiated isolates (i.e., Aspergillus
Among them, Oleander, Golden Shower and niger, Penicillium puberulum and
Bougainvillea presented high superior total Penicillium frequentans) was 69.87, 63.14
acid production of 338.10, 235.20 and and 51.28 g/L of sugarcane molasses, 61.28,
210.70 g/L medium, respectively. Previous 56.37, 47.15 g/L of banana-must, and 54.25,
research reported that fungi such as 52.75 and 44.75 g/L of grape-must,
Aspergillus niger produced the high level of
respectively.8 A. niger Van tieghem was able
total acid up to 2210 ml NaOH/l medium.11
to produce the total acid of 3580 ml NaOH/l
The screening experiments were extended to
determine quantitatively the yield of medium and showed positive GA production
gluconic acid in the fifty four selected results with paper chromatography analysis.8
bacterial and nine fungal isolates using high Moreover, the pink-forming colonies,
performance liquid chromatography. The Aureobasidium Pullulans (DSM 7086)
isolate from Ervatamia provided the highest produced the highest GA of 23.5 and 67.7
production of gluconic acid (17 g/L). g/L after 8 and 27.5 hours, respectively.
Figure 2. Fruits and flowers used in this experiment: a) Pomegranate, b) Longan, c) Pineapple, d)
Dragon fruit, e) Persimmon, f) Guava, g) Orange, h) Grape, i) Banana, j) Apple, A) Bougaville pink, B)
Tridax procumbens, C) Cockcomb, D) Orange jasmine, E) Marigold, F) Butterfly pea, G) Lantana
camara, H) Adenium, I) Bird of paradise, J) Globe amaranth, K) Canna lily, L) Oleander, M) Golden
shower, N) Ervatamia, O) Bougaville Deep Pink, P) Ixora red, Q) Cactus, R) Frangipani, S) Ruellia, T)
Ixora yellow
Table 1. Microorganisms and their characteristics isolated from flowers and fruits
Sample Colony Total acidity GA Figure Cultural characteristics

(mg/ml) (g/l)
Bougaville
Fiber is full of medium and a deep
Bougaville A 7 3 dent in medium
Ervatamia
Circular, Entire, Mucoid, Conven,
Ervatamia N 20 17 Yellow
Globe amaranth
Circular, Entire, Rough, Flat,
Globe amaranth J 63 3 Opaque
Golden shower
Punctiform, Entire, Smooth,
Golden shower M 20 9 Raised, Pale Yellow
Punctiform, Entire, Smooth, Flat,

Ixora red P 93 4 Opaque
Circular, Entire, Smooth, Conven,

Ixora yellow T 9 5 Opaque
Lantana camara
Circular, Entire, Smooth, Raised,
Lantana camara G 68 3 Opaque
Punctiform, Entire, Smooth,

Orange g 78 3 Flat, Opaque
Persimmon
Persimmon e 58 2 Opaque
Pineapple
Pineapple c 166 6 Opaque
4. Conclusion 2. Pal, P.; Kumar, R.; Banerjee, S J. Chem.
Although the production of gluconic Eng. Prog. 2016, 104, 160–171.
acid is a simple oxidation process and can be 3. Canete-Rodríguez, A. M.; Santos-Duenas,
carried out by chemical methods involving
I. M.; Jiménez-Hornero, J. E.; Ehrenreich,
the catalyst is well established
commercially, development of novel, more A.; Liebl W.; García-García, I. J. Process
economical processes for the conversion of Biochem. 2016, 51, 1891–1903.
glucose alternatives to the microbiological 4. Zhang, H.; Watanabe, T.; Okumura, M.;
fermentations of GA would still be Haruta, M.; Toshima, N. J. Nat. Mater.
promising. Another way of improvement is
2012, 11, 49.
to use cheaper substrates than glucose, such
5. Biella, S.; Prati, L.; Rossi, M. J. Catal.
as by products from agro industries. This
2002, 206, 242.
work describes the isolation and screening
of GA-producing microorganisms from 6. Pal, P.; Kumar, R.; Banerjee, S.; Chem.
flowers and fruits. The production and Eng. Process. 2016, 104, 160–171.
optimization conditions of GA from 7. Singh, O. V.; Kumar, R.; Appl.
sugarcane molasses as biological precursor Microbiol. Biotechnol. 2007, 75, 713–
using our isolates could be our plan for 722.
future work. 8. Anastassiadis S.; Aivasidis A.; Wandrey
C. Appl. Microbiol. Biotechnol. 2003, 61,
Acknowledgements
110–117.
This work was supported by the
Department of Biotechnology, Faculty of 9. Takao, S. J. Appl. Microbiol. 1965, 13,
Engineering and Industrial Technology, 732–737.
Silpakorn University and by National 10. Ahmed, A. S.; Farag, S. S.; Hassan, I. A.;
Research Council of Thailand, Academic Botros, H. W. J. Radiat. Res. Sci. 2015, 8,
Year, 2018.
374–380.
References 11. Shindia, A. A.; El-Sherbeny, G. A.; El-
1. Anastassiadis, S.; Morgunov, I. G. Esawy A. E.; Sheriff Y. M. M. M. J.
Recent Pat. Biotechnol. 2007, 1 (2), 1– Mycobiology 2006, 34 (1), 22–29.
14.
Simulation and analysis of a novel dual fluidized-bed biomass gasification
integrated with CO2 capture for high-purity hydrogen production
Aranyakorn Sampavapon, Pimporn Ponpesh, Amornchai Arpornwichanop*
Abstract:
Gasification is one of thermochemical processes widely-used to convert solid biomass
to hydrogen-rich synthesis gas (syngas). However, the presence of carbon dioxide (CO2) in
syngas affects the heating value and product purity. Thus, the integration of CO 2 capture in
the gasification process can enhance the quality of the gaseous product. In this study, the
performance of a novel dual fluidized-bed gasification system with CO2 absorption using
CaO is studied via a simulation tool. The system consists of a bubbling fluidized bed where
biomass gasification and carbonation reactions occur, a circulating fluidized bed where
calcination reaction is carried out to regenerate CaO and an extra riser where the second
carbonation takes place. Simulation of the gasification system is performed by using Aspen
Plus simulator. Analysis of key parameters such as gasification temperature, riser
temperature, steam to biomass ratio (S/B) and CaO to biomass ratio (CaO/B), on the syngas
compositions and hydrogen purity is presented. The results indicated that an increase in
gasification temperature favored hydrogen production. Hydrogen purity was increased with
higher riser temperatures and then declined when the temperature was higher than 600 C.
The amount of steam had a crucial impact on hydrogen production and the addition of CaO
sorbent played an important role to reduce CO2.
1. Introduction the gasification process as well.

Nowadays, fossil fuels are the main Several processes of CO2 capture
energy sources for transportation, power and separation have been proposed for
generation and industrial processes. managing the CO2 emission. The application
However, the utilization of fossil fuels has a of a solid sorbent calcium oxide (CaO) to
great impact on the environment because of capture CO2 in the biomass gasifier has
the release of greenhouse gases. Presently, attracted much attention. The removal of
the use of alternative energy obtained from CO2 during the proceeding of the
various renewable sources such as biomass, gasification reactions can improve the H2
solar and wind, has been widely explored. yield and reduces the operating costs. It is
Gasification is a thermochemical also easy to separate CO2 in the form of
process used to convert solid biomass to CaCO3. Table 1 summarizes the reactions
hydrogen-rich synthesis gas (syngas). In this occurred in the gasifier with CaO.
process, steam is mostly used as a gasifying To further improve the purity of H2
agent1,2 because it is the effective means of in the syngas obtained from the gasifier, a
renewable H2 production3 and can increase novel calcium-looping gasification system
the concentration of H2 compared to other has been proposed by adding an extra riser
gasifying agents. Although biomass above the gasifier to remove the remaining
gasification is a promising technology for CO2 in the producer gas. In this study, the
H2 production, there is an amount of CO2 performance of a novel dual fluidized-bed
generated. Hence the mitigation of CO2 gasification system with CO2 absorption is
emission should be considered in designing analyzed when oil palm frond (OPF) is used
Table 1. Reactions in the gasifier with CaO
Reaction Name H298
(kJ/mol)
C + CO2 ↔ 2CO Boudouard 172
C + H2O ↔ CO + H2 Char 131
gasification
C + 2H2 ↔ CH4 Methanation -74.8
CO + H2O ↔ CO2 + H2 Water-gas -41.2
shift
CH4 + H2O ↔ CO + 3H2 Methane 206
reforming
CaO + CO2 ↔ CaCO3 Carbonation -178.9
CaCO3 ↔ CaO + CO2 Calcination 178.9
C + O2 ↔ CO2 Combustion -393.5 Figure 1. Schematic of a novel dual-
fluidized bed of biomass gasification with
as the biomass feedstock. The effect of key CO2 capture
operating parameters on the gasification
performance is investigated. In general, a bubbling fluidized bed gasifier where the
particle size distribution (PSD) of biomass gasification of biomass with steam as
feed affects heat and mass transfers in a gasifying agent occurred. CaO sorbent was
gasifier. However, since a thermodynamic also fed into the gasifier for in-situ CO2
approach is carried out to perform a capture via a carbonation reaction. As the
preliminary design of the gasification carbonation was an exothermic reaction,
process, the impact of the biomass PSD is heat generated could be used for the
not considered in this study. endothermic gasification reaction. CaCO3
and unreacted char were sent to the second
2. Gasification Process with CO2 Capture reactor, a regenerator, where a calcination
2.1 Process description reaction was carried out to regenerate CaO.
Figure 1 showed a schematic of a Combustion of unburned char could
novel dual-fluidized bed gasification process compensate heat required for the
with CO2 capture. The process consisted of endothermic decomposition of CaCO3 in the
three main reactors. The first reactor was a regenerator. The regenerated CaO was
moved to the next reactor, an extra riser,
Table 2. Description of Aspen Plus blocks in the process flowsheet

Aspen Plus name Block ID Description
Yield reactor – to convert the non-conventional stream OPF into
RYield DECOMP
conventional components
GASIFIC Gibb free energy reactor – to simulate the gasification and carbonation
RGibbs REGEN Gibb free energy reactor – to simulate the regeneration of CaO
RISER Gibb free energy reactor – to simulate the second carbonation
SSplit SEP-A Separator – to separate a portion unreacted carbon
SEP-D Separator – to separate the CaO/CaCO3 from the product gas
SPLIT Separator – to separate CaCO3 for regeneration
SEP-C Separator – to separate CO2 from the regenerator
FSplit CSPLIT Separator – to extract a portion of the carbon loss in ash
Heater HEATER Heater – to increase the temperature of the water
Sep SEPASH Separator – to separate ash from the gasifier
S-CAO Separator – to separate ash loss in the regenerator
Figure 2. Aspen Plus flowsheet of the novel dual fluidized-bed gasification system with CO2
absorption
Table 3. Proximate and ultimate analysis of Table 4. Model inputs used for the
oil palm frond4 simulation
Proximate (wt.%) Ultimate (wt.%) Biomass feed flow rate (kg/h) 100
analysis analysis Steam to biomass ratio 0.2-1.0
Moisture 9.82 C 45.05 CaO to biomass ratio 0.2-1.4
Ash 4.84 H 5.86 Gasification temperature (C) 500-1,000
Fixed carbon 11.88 O 48.82 Riser temperature (C) 400-700
Pressure (bar) 1
Volatile matter 83.28 N 0.23
S 0.04
description of each block in the flowsheet
was shown in Table 2.
where the second carbonation took place in Table 3 showed the ultimate and
the riser. Here, CaO absorbed the remaining proximate analysis of oil palm frond used as
CO2 in the product gas. CaO and CaCO3 the biomass feedstock in this study. The
were sent to gasifier whereas the outlet gas model inputs used to simulate the
after the condensation of water was nearly gasification process were given in Table 4.
pure H2.
2.2 Process model
Modeling of the dual-fluidized bed
gasification process was performed by using 3.1 Model validation
The developed model of the
Aspen Plus simulator. Figure 2
gasification process was validated against
demonstrated the flowsheet of biomass
published results by Chen et al.,6 where the
gasification integrated with CO2 absorption
feedstock used in their study was
in a novel dual-fluidized bed gasifier. The
bituminous coal. As seen in Table 5, the
process was assumed at steady state and
simulated results of the syngas product
isothermal conditions. Tar and other heavy
obtained from the developed model were in
products were not considered. 2% Carbon
a very good agreement with the reported
was loss in ash5 and ash was inert and did
data. Therefore, this simulation model was
not concern in chemical reactions. The
used for investigating the novel dual However, when the temperature was
fluidized-bed biomass gasification above 700 C, the H2 concentration was
integrated with CO2 capture for the high- evidently dropped down whereas CO and
purity hydrogen production process. CO2 were obviously increased. Furthermore,
at high temperatures CaO did not absorb
Table 5. Validation results CO2 in the gasifier because of the chemical
Gas component
Chen et al.6 This work equilibrium. The suitable carbonation
(kg/h) temperature could be found from the CO2
H2 471.06 471.65 equilibrium above the temperature for CaO
CO 51.21 50.96 carbonation/CaCO3 calcination reactions.7,8
CO2 59.9 59.92 It was found that H2 concentration reached
CH4 110.46 109.57 the maximum value at the gasification
temperature of 700 C.
3.2 Effect of gasification temperature 3.3 Effect of steam to biomass ratio
Figure 3 showed the effect of The influence of different steam to
gasification temperature in a range of 500- biomass ratios in the range of 0.2-1 on the
1000 C on the syngas composition. syngas composition was shown in Figure 4.
Typically, the temperature had a strong Steam to biomass ratio was an important
effect on H2 production in the gasifier. The impact on hydrogen concentration. The
more temperature rose, the higher increase in steam promoted the steam-
concentration of H2 was produced. The reforming reaction resulting in high
higher temperature supported the water gas hydrogen production and low CH4
shift and char gasification reactions. In concentration. It could be observed that at
addition the absorption of CO2 enhanced the high steam to biomass ratio, the fractions of
water gas shift reaction that supported H2 CO and CO2 went to zero due to the
production. However, the raising carbonation reaction. The removal of CO2
temperature in the gasifier from 500 to 700 by the carbonation reaction reduced CO2
C did not influence the production of H2 partial pressure in the water gas shift
because syngas used came from the exiting reaction and higher CO was reacted with
riser reactor. In the riser, the water gas shift steam and more H2 was generated. From the
and second carbonation reactions occurred results, the ratio of steam to biomass should
and H2 concentration still increased. be kept at one.
Figure 3. Effect of gasification temperature Figure 4. Effect of steam to biomass ratio

(S/B = 1, CaO/B =1 and riser temperature at
(gasification temperature 700 C, CaO/B =1
500 C)
and riser temperature = 500 C)
Figure 5. Effect of CaO to biomass ratio Figure 6. Effect of riser temperature (S/B =
(gasification temperature at 700 C, S/B =1 1, CaO/B =1 and gasification temperature at
and riser temperature at 500 C) 700 C)
3.4 Effect of CaO to biomass ratio of capture in a novel dual-fluidized bed

Figure 5 showed the influence of gasifier was performed in this work.
CaO to biomass ratio on the syngas Simulation the proposed gasification process
composition. The increase of CaO sorbent was carried out by using ASPEN Plus
increased the H2 concentration but decreased simulation. Key parameters such as
the amount of CO and CO2 due to the gasification temperature, riser temperature,
carbonation reaction. It could be explained steam to biomass ratio (S/B) and CaO to
that the added CaO reacted with CO2 biomass ratio (CaO/B), on the syngas
produced from gasification process and compositions and hydrogen purity were
lowers its partial pressure of water-gas shift investigated. It was found that the H2
reaction. When the CaO sorbent to biomass concentration reached 98 mol%. Increasing
ratio was higher than 0.8, it was observed the gasification temperature favored H2
that the product gas compositions were kept production. H2 concentration reached the
constant. The H2 concentration was reached maximum value with the gasification
its maximum at the CaO/B ratio of one. temperature at 700 C. The amount of steam
3.5 Effect of riser temperature had a crucial impact on hydrogen production
Figure 6 presented the effect of and the addition of CaO sorbent played the
operating riser temperatures (400-700 C) important role to reduce CO2. The extra riser
on syngas composition was studied. It was significantly promoted H2 production by
found that CO2 increased whereas H2 promoting water-gas shift reaction.
decreased at higher operating riser
temperatures. At high temperatures, the Acknowledgements
capability of CaO absorption was limited Support from Chulalongkorn
and the carbonation reaction went backward. Academic Advancement into Its 2nd
The appropriate riser temperature was Century Project is gratefully acknowledged.
500 C.
References
1. Pala, L. P. R.; Wang, Q.; Kolb, G.;
4. Conclusion
Hessel, V. Renew. Energ. 2017, 101,
The thermodynamic analysis of
484–492.
high-purity H2 production from oil palm
2. Wei, L.; Xu, S.; Zhang, L.; Liu, C.; Zhu,
frond gasification integrated with CO2
H.; Liu, S. Int. J. Hydrogen Energ. 2007,
32 (1), 24–31.
3. Parthasarathy, P.; Narayanan, K. S. Xiang, W. Int. J. Hydrogen Energ. 2011,
Renew. Energ. 2014, 66, 570–579. 36 (8), 4887–4899.
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Soh, A. C.; Wan, A. W. A. K. G. Chem. Fang, M.; Luo, Z. Int. J. Hydrogen
Eng. Trans. 2015, 45, 1441–1446. Energ. 2011, 36 (8), 4820–4829.
5. Doherty, W.; Reynolds, A.; Kennedy, D. 8. Wang, Z.; Zhou, J.; Wang, Q.; Fan, J.;
Biomass Bioenerg. 2009, 33 (9), 1158– Cen, K. Int. J. Hydrogen Energ. 2006,
1167. 31 (7), 945–952.
6. Chen, S.; Wang, D.; Xue, Z.; Sun, X.;
Enhancement of biodiesel properties via hydrogenation process:
a pilot plant study
Thanita Sonthisawate1*, Lalita Attanatho1, Panida Thepkhun1, Chiraphat Kumpidet1,
Nattawee Teerananont1, Piyanan Sreesiri1, Chanakan Puemchalad1, Yuji Yoshimura2,
Takehisa Mochizuki2, Yohko Abe2, Shih-Yuan Chen2, Makoto Toba2
1
Thailand Institute of Scientific and Technological Research, Khlong Luang, Pathum Thani 12120, Thailand
2
National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8565, Japan
*E-mail: thanita@tistr.or.th
Abstract:
The utilization aspect of non-food feedstock still important and necessary to the
society of endlessly-growing population with highly demand in energy. Regarding this
awareness, our recently study was focused on an alternative non-food oil crop as jatropha for
a pilot scale biodiesel production. This pilot comprises of two major sectors, i.e. a 1000
liters/day production unit and 100 liters/batch of an upgrading unit. Jatropha biodiesel
produced via this production unit, exhibited the reaction completeness with fatty acid methyl
ester (FAME) conversion more than 97wt% and yielded higher than 80%. However, the
crude oil containing high amount of unsaturated structures could not corporate and made this
jatropha biodiesel met the regulation as oxidation stability property. Hence, the upgrading
unit with the partial hydrogenation process was needed. The oxidation stability could increase
from 0.11-1.39 hours to 15-23 hours after passed through the hydrogenation process. As a
result, the high quality jatropha biodiesel obtained from this pilot plant was in accordance
with national and international specifications. Furthermore, this jatropha and others oil crop
contained high level of unsaturated compounds can be used with this pilot plant and consider
as potential feedstocks for biodiesel production.
1. Introduction significant as a source for biodiesel

Because of the increase of crude oil production.
demand, limited resource of petroleum- Jatropha oil is one of the non-edible
based diesel fuels and environmental oil that has been recognized as a potential
concerns, research and development on feedstock for biodiesel production due to its
alternative diesel fuel has become a matter high oil content and ability to be planted and
of great importance recently. Biodiesel, grown in non-fertile or waste land, with zero
which is biofuel derived from vegetable oil conflict to land use for food production.1
or animal fat, is one of the most promising Jatropha biodiesel consisted of 78wt%
biofuels to replace petrol-diesel fuel. The unsaturated fatty acid methyl ester (FAME)
choice of biodiesel feedstock may differ which including ester of oleic acid (C18:1,
from country to country and depends on 44.7wt%), linoleic acid (C18:2, 32.8wt%)
many factors such as reliability of supply, and linolenic acid (C18:3, 0.2wt%). Most of
cost and quality. The commonly used fuel properties of jatropha biodiesel could
feedstock for biodiesel production in meet the biodiesel specification, except the
commercial scale is edible oil such as palm oxidation stability.2
oil, soybean oil and rapeseed oil. However, The presence of high content of
the use of edible oil for biofuel production poly-unsaturated FAME has significant
could lead to a food-against-energy conflict effect on the oxidation stability, cetane
of interest in the foreseeable future. Thus, number and NOx emission, while high
the contribution of non-edible oil would be content of saturated FAME leads to a high
cloud point and pour point.3,4 In order to
improve the quality of jatropha biodiesel in is presented in Figure 1. Two-stage process,
terms of increasing oxidation stability at a which comprised of esterification and
low pour point and cloud point, the partial transesterification reactions, was used for
hydrogenation process to transform poly- jatropha biodiesel production. The
unsaturated FAME into mono-unsaturated esterification was carried out in stirred tank
FAME without increasing the saturated part reactor, where FFA was converted to FAME
is one of a promising way. in the presence of 0.3wt% H2SO4 catalyst at
Several researches work on jatropha 65-70 C and 12:1 to 30:1 molar ratio of
biodiesel production both in laboratory and methanol to FFA. Then the reaction mixture
pilot scales have been reported.5,6 However, was simultaneously overflowed to the
there is little information available on the transesterification reactor, where triglyceride
partial hydrogenation process for upgrading reacted with methanol in the presence of
the properties of biodiesel, especially in the 0.1wt% of KOH at 60-65 °C and 6:1 to 9:1
pilot scale.7 The data from the pilot scale molar ratio of methanol to oil.
operation could verify those laboratories
effort, is practical and would be success
once expand to be commercial scale
operation.
Thus, the objective of the present
work is to study the pilot scale process for
producing and upgrading by partial
hydrogenation process, jatropha biodiesel
which could arguably claim to be the first of
its kind to be reported.
2. Experimental
2.1 Materials
Jatropha oil was purchased from
Bioenergy Co. Ltd. (Thailand). Its main
properties were: 1.8-6.1wt% of free fatty
acid (FFA), 0.03-0.05wt% of moisture, 1.7-
6.3 mg/kg of phosphorus and 7.3-8.7 hours
Figure 1. Flow diagram of biodiesel
of oxidation stability at 110 C.
production unit
Methanol was a commercial grade
with 95% purity. Hydrogen (99.999%
The reaction mixture was transferred
purity) was obtained from Praxair Co., Ltd.
to the separation tank for separating
(Thailand). The catalysts used for biodiesel
biodiesel and glycerin layer by gravity
production process were sulfuric acid
settling. The glycerin layer was continuously
(H2SO4, 98% purity) purchased from
discharged, while crude biodiesel layer was
Labscan Co., Ltd., (Ireland) and potassium
transferred to the washing unit for removing
hydroxide (KOH, 95% purity) purchased
residual catalyst, soap, glycerol and
from Unid Co., Ltd. (Korea). A 0.5%
methanol with warm water. Wastewater was
Palladium on alumina support (Pd/Al2O3)
also concurrently separated from biodiesel
was used as catalyst for biodiesel upgrading
layer in setting tank after washing process.
process.
Then biodiesel was heated at 100-110 °C
2.2 Production of biodiesel under vacuum to remove the remaining
Jatropha biodiesel was produced in a
water. After drying process, the obtained
pilot plant that designed for continuous
biodiesel was filtered and transferred to
production process with 1000 liters/day
storage tank.
capacity. A flow diagram of production unit
2.3 Upgrading of biodiesel measured and compared with WWFC
Biodiesel upgrading process by standard. FAME compositions in the
partial hydrogenation reaction was carried biodiesel before and after the partial
out in a 100 liters batch type stirred tank hydrogenation reaction, were determined by
reactor (Figure 2). The commercial available GC according to the EN 14103 method.
Pd/Al2O3 catalyst was used and the reaction
was performed at a temperature and 3. Results and Discussion
hydrogen partial pressure of 110-115 °C and 3.1 Production of jatropha biodiesel
0.5 MPa. The reaction condition used in this
work (30:1 molar ratio of methanol to FFA
and 9:1 molar ratio of methanol to oil) gave
the completion of both esterification and
transesterification reactions. However, for
the continuous process with vary feedstock
properties, the reaction condition should be
adjustable which was in accord with the
feedstocks. FFA content in jatropha oil was
transformed to FAME after esterification
process, which resulted in the reducing of
FFA content from 1.8-6.1wt% to less than
0.04wt%. After decreasing FFA content in
Figure 2. Flow diagram of biodiesel feedstock, the following alkaline-catalyzed
upgrading unit transesterification gave reasonably good
reaction conversion with the FAME content
2.4 Characterization of biodiesel higher than 97wt%. The individual
The quality of biodiesel produced in glycerides content including monoglyceride,
pilot scale was monitored by the amount of diglyceride and triglyceride were within the
glycerol, monoglyceride, diglyceride and specification. Moreover, the glycerol content
triglyceride in biodiesel samples, which in biodiesel product was lower than the
were measured using EN 14105 method. standard limitation, which implied that both
The acid value was measured according to glycerin separation and biodiesel
EN14104 method. Properties of jatropha purification processes were efficient.
biodiesel produced in the pilot scale were
Table 1. Mass balance of jatropha biodiesel production process

Mass flow rate (kg/h) Composition (wt%)
Stream
Description In the
no. In Out Glycerides FFA Water H2SO4 MeOH FAME KOH Glycerine Soap
process
1 Jatropha oil 46.12 94.38 5.51 0.12
2 Evaporated water 0.32 100.00
3 Dehydrated jatropha oil 46.09 94.91 5.05 0.05
4 Sulfuric acid 0.11 100.00
5 Methanol 8.55 100.00
Reaction product from
6 57.74 73.69 0.04 2.88 0.19 23.20
esterification reactor
7 Potassium methoxide 7.85 93.20 6.80
8 Glycerine phase 20.74 1.33 38.66 60.01
Reaction product from
9 41.85 0.79 0.19 9.53 89.49
transesterification reactor
10 Water 12.30 100.00
11 Waste water from washing 16.23 75.70 8.71 15.59
12 Biodiesel from washing process 37.92 0.87 0.25 98.90
Water from biodiesel dehydration
13 0.08 100.00
process
14 Biodiesel product 37.00 0.87 0.03 99.10
A biodiesel yield of 80.2% based on insoluble gums and sediments. These
jatropha oil was obtained. However, the products not only affect the properties of
theoretical biodiesel yield without any loss biodiesel but also can damage to engine
should be nearly 100wt% relative to the parts.
mass of jatropha oil. Thus, the process mass Normally, biodiesel has the same
balance was determined in order to quantify fatty acid composition as the parent oil and
the yield loss and the result was presented in this fatty acid composition has an important
Table 1. The significant loss of biodiesel role in the oxidation stability. The main
yield was due to the biodiesel dissolution in FAME compositions in jatropha biodiesel
glycerin phase (18%). The glycerin produced in pilot scale are mono-
separation procedure used does not favor a unsaturated FAME at 42wt% and di-
total separation of phases and the more unsaturated FAME at 34wt%, resulting in a
efficient separation procedure would be large amounts of unsaturated fatty acid
reduce this loss. Moreover, the loss of content of 76wt%. Its high content of
biodiesel during water washing process gave unsaturated FAME resulted in a poor
the addition loss of biodiesel yield (2%). It oxidation stability of 0.11-1.39 h, which is
should be noted that high glycerol yield failed to meet the minimum oxidation
obtained was due to the presence of stability limit of 10 hours as required by
biodiesel, catalyst and excess methanol that WWFC biodiesel specification. In addition,
is not recovered in this work. For the oxidation stability reported in this work
economical viability, the excess methanol was slightly lower than those reported by
should be recovered and re-used. some other authors, who prepared jatropha
The properties of jatropha biodiesel biodiesel in laboratory scale and reported the
were compared with WWFC standard oxidation stability value in the range of
(Table 2). It can be seen that most of the 3.12-3.95 h.2,5 These results indicated that
properties were qualified under the the oxidation stability of biodiesel does not
specification, except the oxidation stability. only depend on the property of oil feedstock
Oxidation stability of biodiesel is crucial for but also on the biodiesel production process.
the reliability of biodiesel; in particular, it 3.2 Upgrading of biodiesel
provides the relevant information on fuel Jatropha biodiesel was upgraded its
storage. The oxidation of biodiesel results in properties by partial hydrogenation over the
the formation of undesired products, commercial Pd/Al2O3 catalyst at 110-115 °C
Table 2. Properties of jatropha biodiesel

Properties Unit Method WWFC Biofuel Guidelines Jatropha biodiesel
Ester % by wt EN14103 > 96.5 97.4-99.8
Density at 15 ºC kg/m3 - Reporta 874-881
Viscosity at 40 ºC Centistoke - 2.00-5.00 4.20-4.49
Flash point ºC ASTM D 93 > 100 > 140-184
Water content mg/kg EN ISO 12937 < 500 309-409
Acid value mgKOH/g EN 14104 < 0.50 0.08-0.11
Oxidation stability at 110 ºC hour EN 14112 > 10.0 0.11-1.39
Iodine value g/100g EN 14111 < 130 90.6
Linolenic methyl ester % by wt EN 14103 < 12.0 0.16-0.20
Monoglyceride % by wt EN 14105 < 0.80 0.53-0.59
Diglyceride % by wt EN 14105 < 0.20 0.12-0.20
Triglyceride % by wt EN 14105 < 0.20 0.05-0.12
Free glycerin % by wt EN 14105 < 0.02 < 0.01
Total glycerin % by wt EN 14105 < 0.25 0.18-0.19
Metal group 1 mg/kg EN 14538 < 5.0 < 4.6
Metal group 2 mg/kg EN 14538 < 5.0 < 0.2
Phosphorus mg/kg EN14107 < 4.0 < 1.0
a
There is no limitation for this density in WWFC, however, please report the result.
and 0.5 MPa in 100 liters batch reactor. unsaturated FAME in the range of 47-64%
After the reaction, polyunsaturated FAME are obtained in this present work. Partial
content decreased from 34 to 3-5.5wt%. hydrogenation of poly-unsaturated FAME
While, the mono-unsaturated FAME content strongly increased the oxidation stability of
increased from 42 to 68-70wt% and jatropha biodiesel from 0.1-1.4 to 15-23
saturated FAME content slightly increased hours (Table 3), which exceed the
from 22 to 24-26wt% as shown in Figure 3. specification limitation value of 10 hours.
Jatropha biodiesel contains mainly Cloud point and pour point of hydrogenated
mono-unsaturated FAME in the form of cis- jatropha biodiesel increased from 4 °C to
conformation. However, the formation of 16 °C and from 4 °C to 9 °C, respectively.
thermodynamically favored trans-form
C18:1 was observed during partial Table 3. Properties of hydrogenated
hydrogenation of biodiesel. It is known that jatropha biodiesel
trans-isomers are thermodynamically more Hydrogenated
Jatropha
stable than cis-isomers and the isomerization Property jatropha
biodiesel
biodiesel
of cis- to trans-isomers generally occurs Oxidation stability at
during the partial hydrogenation of 0.1-1.4 15-23
110 °C (h)
biodiesel.7 Trans-form FAME with Cloud point (°C) 4 16
relatively poor cold flow properties is not Pour point (°C) 4 9
suitable for blending with petro-diesel in
comparison to cis-form FAME. Therefore, To investigate the catalyst
the cis/trans isomerization of mono- reusability, the catalyst was repeatedly used
unsaturated FAME as well as the saturation in partial hydrogenation process for 5
of mono-unsaturated FAME to saturated cycles. Hydrogen consumption rate slightly
product should be minimized in order not to decreased with the cycle of catalyst used,
significantly increase the pour point and which possibly due to the gradual
cloud point of hydrogenated biodiesel. deterioration of catalyst activity. Thus, the
reaction time required achieving the targeted
poly-unsaturated FAME conversion slightly
Tri-unsaturated
Tri-unsaturated increased for each cycle. This result implied
that the replacement or regeneration of
Di-unsaturated
Di-unsaturated catalyst is required for the simultaneous
hydrogenation process.
Cis_monounsaturated
Cis-monounsaturated
4. Conclusion
Trans-monounsaturated
Trans-monounsaturated
The results showed that the
Saturated
Saturated
laboratory scale of jatropha biodiesel
production and upgrading processes could
0 55 10
10 15 20 25 30 35
35 40
40 45
0 15 20 25 30 45 be scaled up to pilot scale. Biodiesel
wt %
wt% production unit ensured the steady state
Jatropha
Jatropha oil
oil conditions of product in-term of FAME and
Jatropha
Jatropha biodiesel
biodiesel glycerides content with biodiesel yield of
Hydrogenated
HydrogenatedJatropha biodiesel
jatropha biodiesel 80%. Most fuel properties of jatropha
Figure 3. FAME composition of jatropha biodiesel could meet WWFC standard,
oil, jatropha biodiesel and hydrogenated except the oxidation stability property.
jatropha biodiesel However, the oxidative stability of jatropha
biodiesel could be improved to meet the
The high poly-unsaturated FAME standard specification by cooperating with
conversion in the range of 84-92% and partial hydrogenation over commercial
moderate selectivity of cis-mono- Pd/Al203 catalyst at a mild reaction
condition in pilot scale. The high quality 2. Sarin, A.; Arora, R.; Singh, N. P.; Sarin,
biodiesel produced by such a pilot plant was R., Malhotra, R. K. Energy 2010, 35,
in compliance with the WWFC. 3449–3453.
3. Papadopoulos, C. E.; Lazaridou, A.;
Acknowledgments Koutsoumba, A.; Kokkinos, N.;
The authors are very grateful to the Christoforidis, A.; Nikolaou, N.
Japan Science and Technology Agency Bioresource Technol. 2010, 101 (6),
(JST), the Japan International Cooperation 1812–1819.
Agency (JICA) and NSTDA (CPMO) which 4. Wadumesthrige, K.; Salley, S. O.; Ng,
provide research funds for this project. This K. Y. Fuel Process. Technol. 2009, 90
work is a part of the project entitled (10), 1292–1299.
“Innovation on Production and Automotive 5. Chen, Y. H.; Chen, J. H.; Luo, Y. M.;
Utilization of Biofuels from Non-food Shang, N. C.; Chang, C. H.; Chang, C.
biomass” under Science and Technology Y.; Chiang, P. C.; Shie, J. L. Energy
Research Partnership for Sustainable 2011, 36, 4415–4421.
Development (SATREPS). 6. Kywe, T. T.; Oo, M. M.: World Acad.
Sci. Eng. Technol. 2009, 26, 477–483.
References 7. Numwong, N.; Luengnaruemitchai, A.;
1. Chhetri, A. B.; Tango, M. S.; Budge, S. Chollacoop, N.; Yoshimura, Y. Chem.
M.; Watts, K. C.; Isalam, M. R. Int. J. Eng. J. 2012, 210, 173–181.
Mol. Sci. 2008, 9, 169–180.
Neural network control of the direct methanol fuel cell
for household applications
Kornkamol Eamsiri, Soorathep Kheawhom, Amornchai Arpornwichanop*
Abstract:
Nowadays, renewable energy sources have been widely utilized for electricity
generation. However, the applications of renewable power, especially in urban community,
still be limited due to its intermittent nature. Thus, a stable power source is always needed to
ensure load-supply continuity. Direct methanol fuel cells (DMFCs) are considered a
promising technology which can serve enough energy for portable and household
applications. Regarding the concept of a hybrid power system, it is necessary that each power
unit should have fast response to get the effective power management and thus, an efficient
control system is required. In this study, a neural network controller (NNC) is proposed for
controlling the DMFC, which is characterized by a highly nonlinear behavior. A dynamic
multi-layer perceptron is implemented in the NNC which is trained with the Levenberg-
Marquardt algorithm. The controller obtained can manage the control variable (cell voltage)
by adjusting the manipulated variable (methanol concentration), which is selected by
considering an input-output correlation in the electrochemical model. Performance of the
developed NNC is compared with a conventional control technique (PID controller). The
simulation results show that NNC not only achieves better control response but also requires
less computational complexity.
1. Introduction cells (DMFCs) are well-known as the most

Nowadays, power generated from promising candidates. Although, PEMFCs
renewable sources such as wind, solar, and can achieve higher performance but their
biomass has attracted growing attention as operational requirements are quite complex
alternative power sources. The growth of for the small residential applications.2 From
renewable power plants is rapidly the data regarding the household electric
increased.1 However, most of these power consumption rate, the amount of electric
sources are considered variable renewable consumption for single-person households
energy (VRE) sources, which their power has fluctuated during the day from less than
fluctuates depending on the natural 1 to greater than 3 kW.3 This means that
variability. Therefore, to enhance reliability only electricity produced from alternative
and stability of these alternative sources, a sources is likely to be sufficient for most of
system should include at least one of the the time, except for some peak-load and
stable and controllable power sources. A night periods. To guarantee the performance
fuel cell is offered as a potential back-up and stability of the hybrid power systems,
power supply because it can produce the way to control each unit to have an
electrical power indefinitely with a accurate and quick response is also
continuous source of fuels. There are several important and will be the basis of a “smart
types of fuel cells, depending on their grid”4 system. The concept of the smart grid
electrolyte and operating conditions. For is to supply electricity to consumers via the
applications required a small amount of two-way communication network that
power, both proton exchange membrane fuel means all the consumption and generation
cells (PEMFCs) and direct methanol fuel units can monitor, communicate, and also
transfer electricity to each other. From this our interested cell, which based on the
concept, if the control system is not able to model studied by several researchers.13,14
perform calculations quickly, it will affect A dynamic, isothermal, lumped parameter
the balance within the electrical system. model accounting for the kinetics of multi-
Moreover, the objectives of a control system step anodic reaction mechanism is
are to minimize the number of start-ups and formulated and summarized as a set of
shutdowns of the fuel cell, keep the state of differential algebraic equation (DAE) in
charge (SOC) in allowed levels, prevent Table 1. The simulation results of this
fuel-starvation occurring in the fuel cell, etc. dynamic model were obtained based on the
Therefore, various control strategies were parameters and assumptions given in
introduced and suggested.5,6 Also, in recent previous studies.13,14
years, many studies2,7,8 have been growing 2.2 Component models
interest in the energy management system In this study, the proposed hybrid
(EMS) for the hybrid power systems based power system consists of four power
on the artificial intelligence (AI) techniques. sources; DMFC, solar panels (PV), solar-
Among all the AI techniques, the artificial thermal collector (SC), and batteries. The
neural network (ANN) seems to have the the powers of each element are related to the
most impact on control applications.9,10 The power conservative law (no losses) as
ability of ANN to handle the nonlinear follows:
mappings with the short amount of time, and Pload (t) = PPV (t) + PSC (t) + PDMFC (t) + PBat (t) (20)
also give an accurate and efficient results, The PV and SC are used as main
makes it becomes a successful method. power sources, supported by DMFC acting
From our knowledge, most of the studies as a back-up unit. In general, the bank of the
have focused on the hybrid systems battery is also integrated into the hybrid
consisting of PEMFC, battery, and various model2,5-8,11,12, because the slow kinetics rate
VRE sources.8,11,12 Only a few studies have of fuel cell makes it is unable to provide
focused on the EMS of DMFC-hybrid sufficient power in fast dynamic required
system based-on the ANN controller.2 applications. Since batteries will provide this
Therefore, the objective of this study additional power requirement. All essential
is to control the power produced from equations of each element are given in Table
DMFC integrated with other power sources, 2.
using ANN control strategy and proposed 2.3 Neural network controller
EMS to meet the power demand in a In this work, the model reference
residential application. In the next section, a adaptive control (MRAC) structure will be
model of DMFC, a set model to estimate implemented.15,16
power produced from other sources, neural
network control strategies, and how to
implement this controller to hybrid power
system will be described. Section 3 presents
the simulation results and compares the
performance of the ANN controller to the
conventional method. Finally, conclusion is
discussed in Section 4.
2. Model and proposed control strategy

2.1 DMFC model
In order to effectively control
electricity generation from DMFCs, it
necessary to understand their mechanism to Figure 1. Schematic of the interested
get the correct model. Figure 1 illustrates DMFC model
Table 1. List of equations13,14
Electrode kinetics
  F   3 AC 1  F  
r1  k1 exp  1  a   Pt CCH OH  exp    a   Pt COH  (1)
 RT   3 K1  RT  3 
  F   1  F  
r2  k2 exp  2  a   Ru  exp    a   Ru OH  (2)
 RT   K2  RT  
Anode  2 1 2 2  
r3  k3  Pt COH  Ru OH   Pt COOH  Pt Ru  (3)
 3 K 3 
 1 AC 
r4  k4  Pt COOH  Ru OH  CCO  Pt Ru  (4)
 K4 2 
 3 
 5 F   p  2
 F   O2  
r5  k5 exp  c  1  exp   c  
 RT   p  
Cathode (5)
 RT   
 
Mass balance
AD
dCCH OH LS
3 1  Inlet AD  k A  AD AC  (6)
  CCH OH  CCH OH   AD  CCH OH  CCH OH 
Anode dt  3 3  Va  3 3 
compartment AD
C  
k LS A AD

dCCO 1
2
 Inlet
 CCO
AD
 CCO2  CCO
AC
(7)
dt  CO2 2
Va AD 2
AC
 
dCCH k LS A AD A A
3OH
 AC
CCH 3OH  CCH
AC
3OH
 AC N CH
M
3OH
 AC r1 (8)
Anode dt Va Va Va
AC
catalyst layer k LS A AD
 
dCCO A
2
AC
CCO2  CCO
AC
 AC r1 (9)
dt Va 2
Va
Charge balance
da 1
Anode   icell  6 Fr1  (10)
dt ca
dc 1
Cathode
dt

cc

icell  6 F r5  NCH
M
3OH
  (11)
Parameter calculations
Methanol M
DCH Pe exp( Pe)
flux through N M
 3OH AC
CCH (12)
exp( Pe)  1
CH3OH M 3OH
membrane d
vd M
Peclet Pe  M (13)
DCH 3OH
number
 icell k dp 
 CHM p
k  F 
 dx  k p dp
Flow velocity v  M  (14)
in membrane   DH  M k
  dx
  CH  
 RT  
Pressure dp pc  pa
gradient  (15)
dx dM
across the
membrane
 C AC K 32   C AC K 2 
 CO2   Pt3  1  CO2   Pt  1  0 (16)
Catalyst  K3 K 4  
 K 4 
 
surface AC 3
CCO K2
fraction  Pt COH  2
 Pt3 (17)
3
K3 K 4
1  F 
K2  exp   a  (18)
K2  RT 
 dM
Cell voltage U cell  U cell   a  c  icell (19)
M
Neural Net
Forward Model +
-
Neural Net
Plant
Inverse Model .
.
.
+
Reference - NARX model
Model
.
.
Figure 2. Structure of model reference .
adaptive control (MRAC)
Figure 2 shows a structure of the

MRAC, which consists of one reference Figure 3. A schematic of nonlinear
model and two neural networks work autoregressive with exogenous model
together within this structure. One network (NARX)
will be trained to predict the dynamic
behavior of plant, whereas another one will trained. The error between predicted plant
act as a controller to generate the control output and desired reference value is used to
action. In training procedures, a neural adjust weights and biases until the network
network plant model will be generated and can provide a satisfy control action.
trained off-line first. There are many types From the proposed DMFC model,
of neural network but one of the most the training sets were generated to develop a
frequently used in dynamic applications is neural network control structure. Matlab
the nonlinear autoregressive with exogenous software is implemented in learning
model (NARX), which is classified as one algorithm using the Levenberg-Marquardt
type of recurrent neural network technique.
structures.15,17 Figure 3 displays the NARX
structure, it uses the values of past predicted Table 2. The modeling for each power
cell voltage ( Vcell m
(t - n) ), and past and source6,13
current methanol feed concentration and cell Solar panel
Inlet PPV (t) = R t ηPV APV (20)
current ( CCH 3OH
,icell ) to predict the cell
   NOCT - 20  
voltage output. The objective of this plant ηPV = ηr 1- N T  Tair +   R t - Tref   (21)
model is to minimize the difference between    800 
actual plant output and predicted plant
output. After first model training, it will be Solar thermal collector
PSC (t) = R t ηSC ASC (22)
placed in series with the neural network
a1 a
 TSC - Tair  - 2  TSC - Tair 
2
controller to predict the plant response using ηSC = ηRSC - (23)
for learning algorithm of the neural network 800 800
Battery
controller. During this second model
1
Cnom 
training, the learning algorithm of a plant SOC = SOCinit - ηi Bdt
(24)
model will be turn off as it has already been
sources will be directly calculated from cell
voltage and cell current. In order to compare
the dynamic response of the system
controlled by the conventional method or
PID, and MRAC structures, step changes in
power demands are applied. From Figure 6,
the response of PID control shows the poor
voltage-tracking performance of this
nonlinear behavior. In contrast, the MRAC
method not only give faster time response,
but also has more effective control actions to
control feed concentration of methanol for
power load variation, compared to PID
Figure 4. Flowchart of the energy controller.
management system According to the proposed EMS
algorithm, Figure 7a shows that even in the
2.4 Control strategy of DMFC integrated high load variation, the hybrid power system
with the hybrid power system can supply enough power with efficient
In order to guarantee the decision-making criteria. In addition, during
performance of the hybrid power system, the all operating time battery SOC can be
EMS is needed. Besides power supplied to maintained at an allowed range (40% -
load from the main sources, the excess or 80%), as shown in Figure 7b, which
required power has to be allocated enhances the energy efficiency of the battery
appropriately. Figure 4 shows the decision- bank.
making flowchart, which is regarded under
two criteria. First, if the SOC of batteries is
less than the minimum allowed level,
batteries will be charged. Otherwise, the
batteries will be discharged. Second criteria
is the value of excess power ( Pexcess ). If the
excess power is negative, a DMFC will be
turned on. Otherwise, the batteries will be
charged.
Figure 5. Experimental and simulated
3. Results and Discussion steady state polarization curves of DMFC
3.1 Model validation for different methanol feed concentration
To validate the neural network-based
model of the DMFC, polarization curves
from experiment data13 at steady state
conditions with three different feed
concentrations will be used. As seen in
Figure 5, the simulated results agree well
with the results from experiments.
Moreover, it is clear that the methanol feed
concentration has a significant impact on
DMFC behavior.
3.2 Results
In this preliminary study, the effects Figure 6. Comparison the dynamic
of inverters and other auxiliary devices is responses of output voltage controlled by
neglected. The power produced from any PID and MRAC methods
Acknowledgements
(a) Support from Chulalongkorn
Academic Advancement into Its 2nd
Century Project is gratefully acknowledged.
References
1. http://ec.europa.eu/eurostat/statistics-
explained/index.php/Renewable_energy
_statistics
2. Chang, C. Y.; Hsu, C. H.; Wang, W. J.;
(b) Chen, C. Y. IJICIC. 2012, 8,
2101−2112.
3. https://energy-surprises.blogspot.com/
2013/03/should-i-use-electricity-from-
my-pv.html
4. Eremia, M.; Toma, L.; Sanduleac, M.
Procedia Engineering 2017, 181, 12−19.
5. Jafari, M. H.; Mohammadi, S. Presented
at the 28th International Power System
Figure 7. (a) System response during the Conference, Tehran, Iran, November 4-
day; (b) battery SOC during system 6, 2013.
operation 6. Lü, X.; Miao, X.; Liu, W.; Lü, J. Appl.
Therm. Eng. 2018, 128, 887−897.
4. Conclusions 7. El-Sharkh, M. Y.; Rahman, A.; Alam,
This study proposed a neural M. S. J. Power Sources 2004, 135,
network (NN) control with the MRAC 88−94.
structure to control the required voltage of 8. Natsheh, E. M.; Albarbar, A. SGRE.
DMFC. The performance of the proposed 2013, 4, 187−197.
NN controller was compared to the PID 9. Hussain M.A. Artif. Intell. In. Eng. 1999,
controller. From the key feature of neural 13, 55−68.
networks that can handle the nonlinear 10. Bose, B. K.; Fellow, L. IEEE Trans. Ind.
problems, it was obvious that the MRAC Electron. 2007, 54, 14−33.
structure-based NN controller was more 11. Uzunoglu, M.; Onar, O. C.; Alam, M. S.
effective over the PID controller. Besides Renewable Energy 2009, 34, 509−520.
this control method, the management of the 12. Maleki, A.; Rosen, M. A.; Pourfayaz, F.
energy distribution of a hybrid power system Sustainability 2017, 9, 1314.
was focused. Because of the high DMFC 13. Sundmacher, K.; Schultz, T.; Zhou, S.;
and battery costs, and their short life-time. Scott, K.; Ginkel, M.; Gilles, E. D.
Our proposed EMS is based on two Chem. Eng. Sci. 2001, 56, 333−341.
objectives. First is to maintain the SOC of 14. Cong, X.; Follmann, P. M.; Biegler, L.
battery, and second is to select the best T.; Jhon, M.S. Comput. Chem. Eng.
option to enhance the performance and 2005, 29, 1849−1860.
lifespan of the system. In the results, we 15. Kasparian, V.; Batur, C. ISA Trans.
showed that during the day, the hybrid 1998, 37, 21−39.
power system is able to meet the residential 16. Beale, M. H.; Hagan M. T.; Demuth, H.
load demands and maintain the SOC of B. in Neural network toolbox 5 user’s
battery at the satisfy range, with this EMS. guide; The Math Works, Inc.: 3 Apple
Hill Drive Natick, 2007.
17. Cheng, S. J.; Chang, T. J.; Tan, K. H.;
Kuo, S. L. Int. J. Comput. Inf. Eng.
2015, 9, 1218−1222.
Utilization of zeolites synthesized from water sludge for heavy metal
treatment in wastewater
Issaraporn Saenglam1, Uthai Donkwang1, Kotchakorn Yotyiamkrae1,
Warangkana Kittiwongwisan1, Bongkotchawan Pakamwong1, Nittaya Phankon1,
Orawan Simpan1, Malinee Rangkatkij1 , Suphawan Pimdee1, Thimpika Promprom1,
Malee Prajuabsuk1, Saisamon Lamlong1, Janpen Intaraprasert1, Daungdao Sattayakul1,
Pharit Kamsri2, Khemmakorn Gomonsirisuk3, Parjaree Thavorniti3, Pornpan Pungpo1*
1
2
Division of Chemistry, Faculty of Science, Nakhon Phanom University, Nakhon Phanom 48000, Thailand
3
National Metal and Materials Technology Center (MTEC), NSTDA, 111 Thailand Science Park,
Klong Luang, Pathum Thani, Thailand
*E-mail: pornpan_ubu@yahoo.com
Abstract:
Metal contamination is a major problem in industrial wastewater. Because it non-
biodegradability and toxic. The adsorbent popularly used for heavy metal adsorption of
wastewater is activated carbon because of its high surface area, resulting in high efficiency in
wastewater treatment. But activated charcoal is relatively expensive, so the synthetic zeolites
have been considered as heavy metal adsorbent. This work, water sludge was used as raw
material to synthesize zeolite using alkali fusion method. The structural characterization of
the zeolite was performed using XRD, SEM and FTIR. The adsorption efficiency to removal
Cd(II) with different adsorption parameters were investigated. The optimum parameters for
adsorbent of the removal of Cd(II) ions at 50 mg/L initial Cd(II) ions in aqueous solution
concentration were elucidated. The optimum dosage of adsorbent is 4 g/L and optimum
adsorption time is 30 minutes with 98% of Cd(II) ions in aqueous solution. The adsorption
isotherm and adsorption kinetic are corresponded well to Langmuir adsorption and pseudo
second order kinetic model, respectively. Based on the obtained results, the synthesized
zeolite is a highly efficient adsorbent for the removal of Cd(II) ions in aqueous solution.
1. Introduction applications as molecular sieves, adsorbents,

Cadmium (Cd2+), copper (Cu2+), catalysts etc.2 In the present study,
nickel (Ni2+), and lead (Pb2+) are common synthesized zeolites from water sludge for
metals found in many industrial heavy metal treatment in waste water
wastewaters. The optimization of water and include cadmium Cd(II). Batch experiments
wastewater purification processes requires were carried out to remove Cd(II) from
the development of new operations based on aqueous solutions by adsorption technique
low-cost raw materials with high pollutant using zeolite.
removal efficiency.1 Zeolites are
microporous, aluminosilicate minerals 2. Materials and Methods
commonly used as commercial adsorbents. 2.1 Synthesis zeolite
There are a group of more than 40 The synthetic zeolite was prepared
crystalline hydrated alumino silicate from water sludge using hydrothermal
minerals. Their structure is based on a three transformation. Brifly, zeolite synthesis
dimensional network of an aluminum and process was carried by water sludge mixed
silicon tetrahedral linked by shared oxygen with sodium hydroxide in ratio 1 : 2 w/w
atoms. Due to specific pore sizes and large and burn out at 600 ℃ for 6 h. Water were
surface areas, zeolites can be used in various added into mixture with ratio of 1 : 4 w/v
and shaked for 24 h. Then, the mixture was The XRD patterns of water sludge
heated at 80ºC by water bath for 3 h. The and synthetic zeolites are shown in Figure
mixture was then washed with distilled 1. In accordance with XRD of water sludge,
water and dried. The structural Fig.1a contains quartz (SiO2) and kaolinite
characterization of the zeolite was in position 2θ at 25 and 20 respectively.
performed using XRD, SEM and FTIR. Synthetic zeolite (Fig. 1b) shows quartz
2.2 Batch experiment containing unname zeolite in position 2 θ at
Synthetic zeolite was used as 2 2 and zeolite A in position 2θ at 2.
adsorbent to remove Cd(II) treatment in Confirmation of the X-ray diffraction
water by the batch adsorption technique. pattern indicated a form of the zeolite phase.
The adsorption efficiency of synthetic The obtained results show that there is still
zeolite to removal Cd(II) in aqueous solution an amorphous structure, explaining that
with different adsorption parameters was during the heat-up process, more porous
investigated. The optimum dosage of distribution effects on more specific areas.2
adsorbent and optimum adsorption time of
Cd(II) ions in aqueous solution were studied
under stirred condition. Standard technique
was used to determine Cd(II) concentration
in the solutions using a Atomic Absorption
Spectrophotometer (AAS). The adsorption
percentage of Cd(II) adsorbed on synthetic
zeolite at time (t) was calculated as:
% adsorption = [A0]-[At])/[A0]×100% (1)
Where [A0] and [At] are the initial and final

concentrations of Cd(II) solution.
To observe the effect of adsorbent

dosage on Cd(II) adsorption, different
amounts of adsorbent (varying from 0.4 to
8.0 g/L) were performed with initial heavy Figure 1. XRD patterns of water sludge (a)
metal concentration of 50 mg/L and an hour and synthetic zeolites (b)
for adsorption time. For studying the effect
of adsorption on Cd(II) adsorption, 3.2 FTIR analysis
experiments with different adsorption times The FTIR spectrum of water sludge
(varying from 15 minutes to 2 hours) were and synthetic zeolites are shown in Figure
conducted based on the optimal adsorbent 2. For water sludge, Fig. 2a shows the band
dosage obtained. To investigate the at 3,436.12 cm-1 which is attributed to the
influence of the initial heavy metal asymmetric and symmetric stretching
concentration on the Cd (II) adsorption at vibrations ν(O–H). The obtained results
room temperature, initial Cd (II) suggest the presence of an amorphous
concentrations were varied from 50-150 silicate material (glass) or possibly hydrated
mg/L. Moreover, the adsorption kinetics and aluminum silicates. The band at 1,638.92
adsorption isotherm properties were also cm-1 is attributed to the bending mode of
investigated. H2O molecules. A broad band at 1,035.36
cm-1 is due to Si–O–Si asymmetric
3. Results and Discussion stretching vibrations of silica. The bands at
3.1 X-ray diffraction analysis 797.45 and 468.79 cm-1 could be assigned to
quartz and silica, respectively.2
The FTIR spectrum of zeolites incomplete synthesized zeolites may be
synthesized (Fig. 2b) show the spectral zone caused by mixed phase resulting in unclear
of 650–745 cm-1 assigned to symmetric crystalline form (Figure 3b). More porous
νas(T–O–T) vibrations zeolite peak of the and there is a distribution of porous
sodalite framework in good agreement with dimensions.3
the peaks of 664.52, 776.26 and 796.26 cm-1
for hydroxysodalite zeolite. The bands in the
region of 420–500 cm-1 are related to
internal tetrahedron vibrations of Si–O and
Al–O of sodalite (T–O–T) bending modes of
the sodalite framework. There is an
important assignment in the range of
991.10 cm-1 with the literature for the Figure 3. SEM of water sludge (a) and
characteristic bands between 1,250 and synthetic zeolites (b)
950 cm-1 asymmetric stretching vibration for
all the zeolitic materials. The broad band at 3.4 Optimal parameters
about 3,464.73 cm-1 and a band at Batch experiments were performed
1,645.40 cm-1 are attributed to zeolitic to remove Cd(II) from aqueous solutions
water.3 using zeolite. A common adsorbent dosage
of 4 g/L, an hour adsorption time at initial
heavy metal concentration of 50 mg/L were
used for the experiments. The results show
that the synthetic zeolite was successfully
used to adsorb Cd(II) with higher adsorption
efficiency than 90%. The optimum
conditions for Cd(II) adsorption onto zeolite
were further studied. The adsorbent dosage
varying from 0.4 to 8 g/L were elucidated as
shown in Fig 5. At low adsorbent dosage
(1.2 g/L), the adsorption percentages of
Cd(II) were greater than 10 %. Based on the
obtained results, low adsorption percentage
is due to low surface areas of the adsorbent
for Cd(II) adsorption. Therefore, the optimal
adsorbent dosage for adsorption Cd(II) at 50
mg/L is 4 g/L (Figure.4).
120
100
% Adsorption
80
Figure 2. FTIR spectra of water sludge (a) 60
and synthetic zeolites (b) 40
20
3.3 Scanning electron microscopy (SEM) 0
SEM micrographs of water sludge 0 2 4 6 8 10
and synthetic zeolites are shown in Figure 3. Adsorbent dosage (g/L)
In water sludge (Figure 3a), it has smooth
Figure 4. Effect of adsorbent dosage on
surface with a round shape. SEM
adsorption efficiency of Cd (II) ions in
micrograph of the synthesized zeolite looks
aqueous solution using zeolite
like a sheet. Unstable shapes indicate that
The adsorption time for Cd(II) form of Langmuir isotherm is given by the
adsorption on zeolite was further following equation:
investigated. The adsorption percentages of
Cd(II) onto adsorbent were shown in Figure Ce/qe = 1/qmb + Ce/qm (4)
5. At 15 minutes to 90 minutes, the
adsorption efficiencies were gradually Where Ce is the equilibrium concentration
increased. At 120 minutes, the adsorption (mg/L), qe the amount adsorbed at
efficiency was slightly increased. These equilibrium (mg/g), qm the maximum
results clearly indicated that the synthetic amount adsorbed (mg/g) and b is the energy
zeolite is an efficient adsorbent to adsorb of adsorption (Langmuir constant, L/mg).
Cd(II) ions in aqueous solution and the The values of qm and b were calculated from
optimal time for dye adsorption is 30 the slope and intercept of the linear plot
minutes. Ce/qe versus Ce , respectively.
The isotherm parameters obtained
105 were summarized in Table 1. Our results
% Adsorption
100 showed that the adsorption of cationic Cd(II)

95 onto zeolite corresponds well to the
90 Langmuir adsorption isotherm for Cd(II)
85 ions in aqueous solution. Therefore, the
80 experimental data into Langmuir isotherm
0 30 60 90 120 model indicates the formation of monolayer
Adsorption time (min) coverage of Cd(II) molecules at the surface
Figure 5. Effect of contact time on of zeolite.
adsorption efficiency of Cd (II) ions in
aqueous solution using zeolite Table 1. Freundlich and Langmuir constants
for the adsorption of Cd(II) onto zeolite
3.5 Adsorption isotherm Freundlich parameters
Freundlich and Langmuir adsorption Linear equation R2 1/n KF -
isotherms were chosen to analyze the y = -0.0781x + 0.2626 225.85 1.83
2.7749
adsorption data at equilibrium process. Langmuir parameters
Adsorption equilibrium studies describe the Linear equation R2 qm b RL
interaction between zeolite and Cd(II) which y = 0.0029x +
0.8723 559.35 8.94 0.0007
assists in designing of adsorption systems. 0.0002
The Freundlich isotherm describing
equilibrium on heterogeneous system is 3.6 Adsorption kinetic
presented by the following equation:4 The next study of batch experiment
of cationic Cd(II) adsorptions is the kinetic
qe = kFC1/n (2) studies. Two type of kinetic models
including pseudo-first-order and pseudo-
The logarithmic form of the equation second-order kinetic models were selected
becomes to elucidate the adsorption mechanism of
Cd(II) onto the zeolite. The rate constant of
log (qe) = log (kF) + (1/n)log (Ce) (3) adsorption is determined from the pseudo-
first-order equation given by Lagergren and
Where kF and n are Freundlich constants Svenska:6
calculated from the slope and intercept of
the Freundlich plot. ln(qe − qt ) = ln qe − k1t (5)
The Langmuir isotherm predicts the
existence of monolayer of adsorbate at the Where qe and qt are the amount of Cd(II)
outer surface of the adsorbent.5 The linear ions in aqueous solution adsorbed (mg g−1)
at equilibrium and at time t (min), 4. Conclusion
respectively, and k1 is rate constant of In the present study, synthetic zeolite
adsorption. from water sludge was used to investigate
A pseudo second-order equation the adsorption efficiency of cationic heavy
based on equilibrium adsorption is metal (Cd2+) from aqueous solution. The
expressed as:7 zeolite has been demonstrated as excellent
adsorbent for the removal of the cationic
t/qt =1/k2qe2 + (1/qe) t (6) heavy metal (Cd2+). The best-fit adsorption
isotherm was achieved with Langmuir
Where k2 is the rate constant of second- model for zeolite. Kinetic adsorption model
order adsorption. was corresponded well to pseudo-second
order model. Based on the obtained results,
The kinetic constant parameters and the synthesized zeolite is a highly efficient
linear correlation coefficients (R2) of adsorbent for the removal of Cd(II) ions in
pseudo-first and pseudo-second-order aqueous solution.
kinetic model of Cd (II) were concluded in
Table 2. Based on the R2 of kinetic model Acknowledgements
fitting of cationic heavy metal on zeolite The Young Scientist and
surface, the adsorption data of Cd (II) were Technologist Program (YSTP) (SCA-CO-
fitted well to the pseudo-second-order 2560-4663-TH) National Metal and
kinetic model due to high correlation Materials Technology Center (MTEC) and
parameters. Moreover, the qe values Faculty of Science, Ubon Ratchathani
obtained from kinetic model is closely to qe University are gratefully acknowledged for
value from the experimental adsorption. supporting this research.
Based on the results, it can be indicated that
heavy metals interacting with surface of References
zeolite are rate determining step. 1. Hajar, M.; Mohsen, J. C. T. E. P. 2013,
15, pp 303–316.
Table 2. Kinetic constants for the adsorption 2. Denise, A. F.; Mauro, V. S. Am. J. E. P.
of heavy metal onto zeolite 2014, 2, pp 83-88.
pseudo-first-order 3. Mousa, G. J. Assoc. Arab Univ. Basic
Linear qe(exp) qe(cal) Appl. Sci. 2014, 15, pp 35–42.
R2 k1
equation (mg/g) (mg/g)
4. Freundlich, H. M. F. J. Phys. Chem.
y = -0.0067x 0.981
13.93 13.70 5.5693 1906, 57, 385–471.
+ 5.5693 0
pseudo second-order 5. Langmuir, I. J. Am. Chem. Soc. 1918,
Linear qe(exp) qe(cal) 40, 1361–1403.
R2 k2
equation (mg/g) (mg/g) 6. Langergren, S.; Svenska, B. K.
y = 7×10-5 x 0.999 Veternskapsakad. Handlingar. 1898, 24,
13.93 13.79 131.46
+ 4×10-5 5
1–39.
7. Ho, Y. S.; McKay, G. Chem. Eng. J.
1998, 70, 115–124.
The equilibrium study of methylene blue from aqueous solution using
zeolite synthesized as highly potential adsorbent from water sludge
Warangkana Kittiwongwisan1, Kotchakorn Yotyiamkrae1, Uthai Donkwang1,
Naruedon Phusi1, Chayanin Hanwarinroj,1 Malee Prajuabsuk1, Saisamorn Lumlong1,
Duangdao Sattayakul1, Jitlada Deshativong1, Pisichanun Srisuwan1, Pharit Kamsri,2
Auradee Punkvang2, Pajaree Thavorniti3, Khemmakorn Gomonsirisuk3,
Pornpan Pungpo1*
1
2
Division of Chemistry, Faculty of Science, Nakhon Phanom University,
Nakhon Phanom 48000, Thailand
3
National Metal and Materials Technology Center, NSTDA,
111 Thailand Science Park, Klong Luang, Pathum Thani 12120, Thailand
Abstract:
The synthesis of zeolite as ion exchange material, adsorbents and catalyst for
industrial applications is very importance. The aim of this work is to synthesis zeolite as
synthetic dye adsorbent from water sludge. The adsorption performance of synthesis zeolite
for methylene blue removal was examined using a batch method. Optimum parameters for
adsorption including adsorbent dosage and adsorption time were investigated. Adsorption
isotherm and thermodynamic parameters were studied. The obtained results indicated that at
initial dye concentration at 300 mg/L, optimum dosage of adsorbent is 14 g/L and optimum
adsorption time is 60 minutes with 90 % of dye adsorption. The adsorption isotherm of
methylene blue onto zeolite agrees well with the Langmuir isotherm and kinetic process
corresponded well to pseudo-second order model. Moreover, thermodynamic properties of
the adsorption, entropy (S°), enthalpy (H°) and Gibb’s free energy (G°) were determined
to be 7.44 KJ mol-1, -2.78 KJ mol-1 and -5.00 (303K) KJ mol-1. These results indicate that the
synthesis of zeolite is a promising and low-cost adsorbent for removing methylene blue dye
from wastewater due to the high adsorption capacity.
1. Introduction and waxes. Adsorption using adsorbent is

Many industries like textile, paints, potential process for dye removal with low
pulp and paper, and carpet printing generate cost. Natural zeolite and synthetic zeolite
dye-bearing effluents during their have a high adsorption capacity due to their
production processes. The effluents structure and high specific surface areas.
containing dyes are highly colored and cause The use of zeolite for wastewater treatment
water pollution. Typically dyes are broadly has been realized that is still a promising
classified as anionic, cationic, non-ionic and technique in environmental treatment
zwitter-ionic depending on the ionic charge process. In this study, synthetic zeolite from
on the dye molecules.1,2 Cationic dyes are water sludge was used as an absorbent for
more toxic than anionic dyes.3 Methylene cationic dye adsorption. Batch method was
blue, the triarylmethane dye is one of the carried out to remove cationic dyes from
commonly known cationic dyes the aqueous solutions by adsorption technique.
chemical structure is shown in Figure 1.
This dye is used for dye paper, leather, inks
2. Materials and Methods Standard technique was used to determine
2.1 Zeolite synthesis the dye concentration in the solutions using
a UV–visible spectrophotometer λmax = 665
nm. The adsorption percentage of dye
adsorbed on compared zeolite and water
sludge at time (t) was calculated as:
Figure 1. Chemical structure of methylene % Adsorption = [A0]-[At])/[A0]×100% (1)

blue
Where [A0] and [At] are the initial and final
The synthetic zeolite was concentrations of dye solution.
synthesized as shown in Figure 2. 2.3 Optimal conditions
To observe the effect of adsorbent
Water sludge dosage on dye adsorption, different amounts
Mixed with of adsorbent (varying from 8 to 16 g/L) were
NaOH power & used with initial dye concentration of 300
heated 600°C for mg/L and one hour for adsorption. For
Fused materials 6 hrs. studying the effect of adsorption on dye
adsorption, experiments with different
Added to
adsorption times (varying from 15 minutes
distilled water &
Aged materials to 6 hrs.) have been conducted based on the
aged for 24 hrs.
optimal adsorbent dosage. To investigate the
Heated at 800°C influence of the initial dye concentration on
for 3 hrs. the dye adsorption at room temperature,
Zeolite initial dye concentrations were varied from
300-500 mg/L. Thermodynamics properties
Figure 2. Diagram for zeolite synthesis were studied at room temperature, 303 K,
318 K, 333 K and 333 K. Moreover,
50 g of water sludge was mixed with adsorption isotherm and adsorption kinetics
60 g of NaOH powder. It was ground to were also investigated.
obtain a homogeneous mixture. This mixture
was heated in a crucible in air at 600 ºC for 3. Results and Discussion
6 hrs. The resultant material was cooled to 3.1 Optimal parameters
room temperature and ground again to Batch method was performed to
obtain the fused material. After that the remove methylene blue dye from aqueous
process was followed with vigorous solutions using synthetic zeolite. A common
agitation in Centrifuge tubes using shaker at adsorbent dosage of 16 g/L, one hour
room temperature to obtain the aged adsorption time at initial dye concentration
material for 24 hrs. The aged material was of 300 mg/L was used for the experiments.
heated in a water bath at 80 ºC for 3 hrs. to The obtained results show that zeolite was
obtain the product and was filtered, washed successfully used to adsorb methylene blue
with distilled water, and dried in an air oven with higher adsorption efficiency than 90%.
at 80 ºC for overnight. The optimum conditions for dye adsorption
2.2 Dye removals onto zeolite were further studied. The
The synthetic zeolite was used as adsorbent dosages varying from 8 to 16 g/L
adsorbents to remove methylene blue from were elucidated as shown in Figure 3.
an aqueous solution by the batch adsorption The obtained results show that low
technique. The effects of adsorbent dosage, adsorption percentage is due to low
initial dye concentration and adsorption time adsorbent dosage and low adsorption surface
were studied under stirred condition. areas for dye adsorption. Therefore, the
optimal adsorbent dosage for adsorption zeolite and dye which assists in the
methylene blue dye at 300 mg/L is 14 g/L. designing of adsorption systems. The
Freundlich isotherm describing equilibrium
on heterogeneous system is described by the
following equation:4
qe = kFC1/n (2)
The logarithmic form of the equation

becomes
log (qe) = log (kF) + (1/n)log (Ce) (3)

Figure 3. Effect of adsorbent dosage on
adsorption efficiency of methylene blue dye Where kF and n are Freundlich constants
using synthetic zeolite were calculated from the slope and intercept
of the Freundlich plot.
The adsorption times for dye The Langmuir isotherm predicts the
adsorption on zeolite were further existence of monolayer of adsorbate at the
investigated. The adsorption percentages of outer surface of the adsorbent. The linear
dye onto adsorbent were shown in Figure 4. form of Langmuir isotherm is given by the
At 15 and 30 minutes, the adsorption following equation:5
efficiencies were rapidly increased. At 60
minutes, the adsorption efficiency was Ce/qe = 1/qmb + Ce/qm (4)
slowly increased with more than 80% of
adsorption efficiency. At 120 minutes, the Where Ce is the equilibrium concentration
adsorption efficiency was slightly increased. (mg/L), qe the amount adsorbed at
These results clearly indicated that zeolite is equilibrium (mg/g), qm the maximum
an efficient adsorbent to adsorb methylene amount adsorbed (mg/g) and b is the energy
blue dye and the optimal time for dye of adsorption (Langmuir constant, L/mg).
adsorption is 60 minutes. The values of qm and b were calculated from
the slope and intercept of the linear plot
Ce/qe versus Ce. The isotherm parameters
obtained from methylene blue adsorption
were summarized in Table 1. Our results
showed that the adsorption of cationic dye
onto zeolite corresponded well to the
Langmuir adsorption isotherm. Therefore,
the experimental data into Langmuir
isotherm model indicate the formation of
monolayer coverage of methylene blue
molecules at the surface of synthetic zeolite.
Figure 4. Effect of contact time on 3.3 Adsorption kinetic
adsorption efficiency of methylene blue dye Two type of kinetic models
adsorption using synthetic zeolite including pseudo-first-order and pseudo-
second-order kinetic models were selected
3.2 Adsorption isotherm to elucidate the adsorption mechanism of
Freundlich and Langmuir adsorption dye onto the zeolite. The rate constant of
isotherms were chosen to analyze the adsorption is determined from the pseudo-
adsorption data. Adsorption equilibrium first-order equation given by Lagergren and
studies describe the interaction between Svenska:6
Table 1. Freundlich and Langmuir constants that dye interacted with surface of zeolite
for the adsorption of dye onto zeolite are rate determining step.
Freundlich Isotherm
Linear Table 3. Thermodynamic parameters for
R2 1/n KF
equation
y=2.9711- 5.2783x10-
adsorption of methylene blue
0.4925 2.9711 6
5.2775
Langmuir Isotherm 3.4 Thermodynamics
Linear 2 The thermodynamics parameters of
R Nm KL RL
equation
y= 0.0009+ T InKc ΔG° ΔSo ΔH°
0.9648 1,111 0.4 0.0019
0.0225 (K) (KJ/mol) (KJ/mol) (KJ/mol) (KJ/mol))
303 1.9850 -5.00
Table 2. Kinetic constants for the adsorption 318 1.9743 -5.21 7.44 -2.78
of dye onto zeolite 333 1.8895 -5.23
Pseudo-first-order 348 1.8536 -5.36
qe(cal) the adsorption of methylene blue dye onto
qe(exp)
Linear equation R2
(mg/g)
k1 zeolite is evaluated as follows. The changes
(mg/g) in Gibb’s free energy, (ΔGo) of the
y = -0.0476x + adsorption process is related to the
0.9037 21.84 19.35 0.0476
2.9628
Pseudo second-order equilibrium constant by the following
equation:8
qe(exp)
Linear equation R2 qe(cal) k2
(mg/g)
(mg/g) ΔGo = −RT ln Ka (7)
y = 0.0457x +
0.9999 21.84 21.88 0.0193
0.1081
where Ka is the equilibrium constant
calculated from the following equation.
ln(qe − qt ) = ln qe − k1t (5)
Ka= Ca/Ce (8)
Where qe and qt are the amount of dye
adsorbed (mg g−1) at equilibrium and at time where Ca and Ce are the equilibrium
t (min), respectively, and k1 is rate constant concentration of methylene blue dye (mg/L)
of adsorption. A pseudo-second-order on zeolite adsorbent and in solution,
equation based on equilibrium adsorption is respectively. The enthalpy change ( ΔHo )
expressed as:7 and entropy change ( ΔSo) were obtained
from the Van’t Hoff equation:9
t/qt =1/k2qe2 + (1/qe) t (6)
lnKc = (-ΔH°/RT)+( ΔSo/R) (9)
Where k2 is the rate constant of second-
order adsorption. where T is the absolute temperature (K); R
The kinetic constant parameters and is the gas constant (8.314 J/molK) ΔHo
linear correlation coefficients (R2) of (KJ/mol) and ΔSo (KJ/ molK)
pseudo-first and pseudo-second-order The thermodynamics parameters
kinetic model of methylene blue dye were were evaluated at three operating
concluded in Table 2. Based on the R2 of temperatures (303, 3 1 8 , 333 and 348) K at
kinetic model fitting of cationic dye on an initial dye concentration of 300 mg/L.
zeolite surface, the adsorption data of The values of InKc, ΔHo, ΔSo, and ΔGo at
methylene blue were fitted well to the the investigated temperatures are listed in
pseudo-second-order kinetic model due to Table 3. The obtained negative values of
high correlation parameters. Moreover, the ΔGo at all temperatures studied revealed the
qe values obtained from kinetic model is fact that the adsorption process was
closely to qe value from the experimental spontaneous. Negative values of ΔHo
adsorption. Based on the results indicated
indicate the exothermic of the adsorption Acknowledgements
process. The positive value of ΔSo suggests Faculty of Science, Ubon
increased randomness of the solid-solution Ratchathani University and National Metal
interface during the adsorption of dye onto and Materials Technology Center (MTEC)
adsorbent. are gratefully acknowledged for research
support.
4. Conclusion
The present study investigated the References
adsorption of cationic dye ( methylene blue) 1. Puzyn, T. in Organic Pollutants Ten
from aqueous solution using synthetic Years After the Stockholm Convention -
zeolite. Synthetic zeolite has been Environmental and Analytical Update;
demonstrated as excellent adsorbent for the Carmen, Z; Daniela, S. InTech. 2012; pp
removal of the cationic dye. The best-fit 55–86.
adsorption isotherm was achieved with 2. Tagub, M. T.; Sen, T. K.; Afroze, S.;
Langmuir model. Kinetic adsorption model Ang, H. M. Adv. Colloid Interface Sci.
was corresponded well to pseudo-second 2014, 209, 172–184.
order model. The negative ΔHo value 3. Sponza, D. T. J. Hazard. Mater. 2006,
confirmed the exothermic nature of the 138, 438–447.
adsorption interaction where as the positive 4. Freundlich, H. M. F. J. Phys. Chem.
ΔSo value showed the increased randomness 1906, 57, 385–471.
at the solid–solution interface during the 5. Langmuir, I. J. Am. Chem. Soc. 1918, 40,
adsorption process. The negative value of 1361–1403.
ΔGo indicated the feasibility and the 6. Langergren, S.; Svenska, B. K.
spontaneous nature of the adsorption of Veternskapsakad. Handlingar. 1898, 24,
methylene blue onto zeolite. The results 1–39.
obtained from this study indicated that 7. Ho, Y. S.; McKay, G.; Chem. Eng. J.
zeolite could be used as a low-cost 1998, 70, 115–124.
adsorbent for the removal of methylene blue 8. Tan. I. A. W.; Ahmad. A. L.; Hameed. B.
dye from aqueous solutions. H. J. Hazard. Mater. 2008, 154, 337–
346.
9. Chen. M.; Chen. Y.; Diao. G. W. Chem.
Eng. Data 2010, 55 (11), 5109–5116.
Effect of doped Al2O3 support in Ni-based catalyst for
ammonia decomposition
Mooklada Chaisorn, Teerapat Jerawattanakaset, Nutnicha Singhapunt,
Suparoek Henpraserttae, Pisanu Toochinda*
School of Bio-chemical Engineering and Technology, Sirindhorn International Institute of Technology,
Thammasat University, P.O. BOX 22, Pathumthani 12121, Thailand
*E-mail: pisanu@siit.tu.ac.th
Abstract:
Generation of hydrogen from carbon-free ammonia decomposition has attracted
research attention from environmental friendly aspects. The development of active Ni
catalysts at low temperatures is essential. Doping of heteroatoms into Al2O3 frameworks such
as Sr2+, Zr4+, and Ce4+ can enhance the catalyst activities. Ni over Sr-doped Al2O3,Zr-doped
Al2O3, and Ce-doped Al2O3 supports were prepared and conducted in a quartz tubular reactor
at 550-600 °C in continuous mode to evaluate ammonia conversion and hydrogen production.
The catalysts were characterized using BET, XRD and chemisorption techniques. Ni/Ce-
doped Al2O3 exhibits the highest catalytic activity (Ni/Ce-doped Al2O3> Ni/Zr-doped Al2O3 >
Ni/Sr-doped Al2O3). Different amount of dopant also affects the catalyst activities. Ni/5mol%
Ce-doped Al2O3 exhibits a higher yield of hydrogen production than Ni/1mol% Ce-doped
Al2O3. The activities of catalysts over different doped support along with the characterization
will be discussed.
1. Introduction Conventional Ni catalysts exhibit a high

Hydrogen can be used as a fuel in a NH3 conversion at around 600-700 °C.3,4
H2-fuel cell to generate electricity without The development of Ni catalyst is still
any greenhouse gas emissionsใHydrogen can required to reduce the reaction temperature
be produced from various techniques such as with a high catalytic activity. Our previous
hydrocarbon reforming, water-gas shift, and study reported that the doping of Zr4+ to
pyrolysis of hydrocarbons which cause CO partly replace Al3+ in Al2O3 support can
and CO2 emissions.1,2 Ammonia enhance the activity of Ni catalyst,
decomposition is a clean technique to compared to Ni/γ-Al2O3, for NH3
produce hydrogen without CO and CO2 decomposition from support effect.8 The
emissions.2,3 Ammonia is considered as an heteroatoms (Zr4+) partly replace the native
excellent hydrogen carrier for on-site H2 atoms (Al3+) in Al2O3 framework. This
generation. One mole of ammonia contains replacement provides a defect formation in
17.8 wt% hydrogen and can be easily Al2O3 support and alternates Ni to support
transported in the liquid phase at 298 K and interaction.9 In this study, Sr2+, Zr4+, and
pressure of 8 atm.3-5 The decomposition of Ce4+ were used as dopants to partly replace
NH3 is shown by the following reaction.3 Al3+ in Al2O3 frameworks. The objective is
to improve the activities of Ni catalysts over
2NH3↔ N2 + 3H2 ∆Hrxn = +46 kJ/mol these supports while maintaining a high
surface area and low price of γ-Al2O3. The
Ruthenium catalyst has been Ni catalysts over these supports were
reported as the most active catalyst for NH3 compared to investigate the effect of dopants
decomposition, but it suffers from high cost (different valency: Sr2+ versus Zr4+) and
and low availability.6 Nickel is an attractive dopants atomic size (same valency: Zr4+
substitution of Ru due to its low price.6,7 versus Ce4+) toward the activities of Ni
catalysts. The amount of dopant was also
studied on Ni/Ce-doped Al2O3. The results of quartz sand was mixed into 0.4 g of the
from this study can lead to a better catalyst then packed between quartz wool in
understanding of support effect to Ni the reactor to provide better temperature
catalysts and the development of active Ni distribution. The catalyst was reduced using
catalysts for NH3 decomposition. 50 vol% H2 balanced in Ar at 600 ºC for 1 h.
The reactor was flushed with 45 mL/min Ar
2. Materials and Methods at 600 ºC for 30 min to get rid of hydrogen
2.1 Catalyst and support preparations in the reactor. Pure NH3 (99.5% purity,
The Sr-doped Al2O3, Zr-doped Linde (Thailand) PLC) was directly fed at
Al2O3, and Ce-doped Al2O3 supports were 50 mL/min into the reactor for the catalyst
prepared using a sol-gel method with 1mol% activities test at 550-600 ºC and 1 at min
Sr, 1mol% Zr, 1mol% Ce and 5mol% Ce, continuous mode. The product gas was
doped in Al2O3 framework, respectively. All directly sent to an auto-sampling unit of Gas
nitrate compounds with ≥ 97% purity from Chromatography with Thermal Conductivity
Ajax Finechem Pty Ltd, Sr(NO3)2, Detector (GCMS-2010 Ultra, Shimadzu
Zr(NO3)2·6H2O, Ce(NO3)3·6H2O and Corporation, Japan) to determine the
Al(NO3)3·9H2O, were dissolved in concentration of product gas compositions.
deionized water to form Sr-Al, Zr-Al and 2.3 Analysis sections
Ce-Al salt solutions, respectively. The The surface areas of the supports
30vol% ammonia in aqueous solution were determined using Brunauer, Emmett
(Panreac AppliChem) was added into the Sr- and Teller (BET) technique (Autosorp-1C,
Al, Zr-Al and Ce-Al salt solutions drop by Quanta chrome, USA). The supports and
drop to form a sol-gel until the pH of the catalysts were characterized using an X-ray
sol-gel solutions reaches pH of 9 in 60 min. diffraction technique (XRD, X'Pert PRO
The Sr-Al, Zr-Al and Ce-Al gels were kept diffractometer, Panalytical, Almelo, the
at room temperature for 48 h. These gels Netherlands) using Cu Kα1 radiation at 40
were dried at 110°C for 24 h and then kV and 30 mA, 25°-75° 2-theta, 0.02° step
calcined at 800 °C for 4 h to obtain the Sr- size, 0.5 sec step time. The CO
doped Al2O3, Zr-doped Al2O3 and Ce-doped chemisorption by pulse injection technique
Al2O3 supports. All supports were ground using 10vol% CO in He (CO-pulse,
followed by 106 µm sieving to control the BELCAT-B, BEL Japan Inc., Japan) was
particles size of supports. 10wt% of Ni used to determine the surface area of Ni on
catalysts were prepared using an incipient the catalysts. Temperature Programmed
wetness impregnation (IWI). Desorption of carbon dioxide (CO2-TPD)
Ni(NO3)2·6H2O (≥97%, Ajax using pure CO2 was also measured using the
Finechem Pty Ltd) was dissolved in Chemisorption Catalyst analyzer (BELCAT-
deionized water to obtain the solution B, BEL Japan Inc., Japan). The basic sites of
concentration of 1.25 M. The Ni solution catalysts were calculated from the peak area
was dropped on the supportsdrop by drop to of CO2-TPD profiles to determine the
impregnate the Ni over supports. After number of basic sites on the catalysts.
impregnation, the impregnated catalysts
were dried at 110 ºC for 12 h and then, 3. Results and Discussion
calcined at 500 ºC for 4 h. The catalysts 3.1 Characterization of supports and
were ground followed by 106 µm of the catalysts
sieve to control the particle size of catalysts. Table 1 shows the surface area of
2.2 Catalytic activities test supports. The data show that Sr-doped
Hydrogen productions from Al2O3and Zr-doped Al2O3have the same
ammonia decomposition over all catalysts range of support surface area relative to γ-
were conducted using a quartz tubular Al2O3. The surface area of Ce-doped Al2O3
reactor with the inner diameter of 1 cm. 1 g slightly decreases compared to γ-Al2O3 and
further decreases with a higher amount of utilized to determine the Ni surface area and
Ce doped in Al2O3 framework. basic sites on the catalysts as shown in
Figure 2 and 3, respectively.
Table 1. Surface area of supports
Surface area.
Supports
(m2/g)
γ-Al2O3a 176
1mol% Sr-doped Al2O3 174
1mol% Zr-doped Al2O3 176
1mol% Ce-doped Al2O3 168
5mol% Ce-doped Al2O3 146
a
Data from our previous work10
Figure 2. CO-pulse profiles of catalysts
The volume of CO adsorbed over Ni

active sites on the catalysts was determined
from the peak area of CO-pulse profiles in
Figure 2. The volume of CO2 adsorbed on
the catalysts was determined from the peak
area of CO2-TPD profiles in Figure 3.
Consequently, the volume of CO and CO2
adsorbate were used to calculate the Ni
Figure 1. XRD patterns of supports (a: surface area and basic sites on the catalysts,
1mol% Sr-doped Al2O3, b: 1mol% Zr-doped respectively, as listed in Table 2.
Al2O3, c: 1mol% Ce-doped Al2O3, d: 5mol%
Ce-doped Al2O3, ■: γ-Al2O3, ●: CeO2) Table 2. Properties of catalysts
Ni
Basic sites
Figure 1 shows that all supports have Catalysts surface area
(μmol/gcatalyst)
similar XRD patterns which correspond to (m2/gNi)
the standard XRD pattern of γ-Al2O3, cubic Ni/γ-Al2O3 7.12 59
Ni/1mol%
structure; JCPDS: 10-0425 (2-theta around Sr-doped Al2O3
11.13 80
37, 46, and 67 degrees). The 5mol% Ce- Ni/1mol%
12.05 76
doped Al2O3 clearly shows sharp peaks of Zr-doped Al2O3
CeO2, cubic structure; JCPDS: 43-1002 (2- Ni/1mol%
13.36 86
theta around 28, 33, 47, and 56 degrees). Ce-doped Al2O3
Ni/5mol%
However, the other supports do not show Ce-doped Al2O3
17.25 104
sharp peaks of dopants as they contain a
small amount of dopants. Therefore, these Table 2 shows that Ni over doped
new supports can maintain the crystallinity, Al2O3 supports exhibit higher Ni surface
crystal structure and surface area similar to area and basic sites than Ni/γ-Al2O3. Figure
γ-Al2O3 at a calcination temperature of 800 3 shows that there is no significant
°C.The CO-pulse and CO2-TPD were difference in the desorption temperature of
CO2 among the catalysts. However, the Table 3. Activities of catalysts
number of basic sites is different as T
Catalysts NH3a H2b
indicated in Table 2 where Ni/Ce-doped (°C)
Al2O3 has the highest basic sites compared Ni/1mol% Sr-doped Al2O3 55.8 3769
to others. Literature reported that basic sites Ni/1mol% Zr-doped Al2O3 60.6 4157
can enhance the catalyst activities for NH3 550
Ni/1mol% Ce-doped Al2O3 74.3 5201
decomposition.3 Therefore, the doping of Ce
in Al2O3 framework can improve the
properties of Ni catalyst with low cost of Ni/1mol% Sr-doped Al2O3 86.3 5873
modification. Higher number of basic sites Ni/1mol% Zr-doped Al2O3 88.3 5928
and larger Ni surface area can facilitate the 600
H2 production from NH3 decomposition
which is discussed in section 3.2. a
NH3 conversion (%)
b
H2 formation rate (μmol/min.gcatalyst)
Ni/5mol% Ce-doped Al2O3 exhibits

higher NH3 conversion and H2 production at
all temperatures compared with Ni/1mol%
Ce-doped Al2O3. The catalytic activities go
along well with the characteristic properties
of catalysts listed in Table2. The highest
basic sites and Ni surface area of Ni/Ce-
doped Al2O3 can cause higher catalytic
activities than those of Ni/Zr-doped Al2O3
and Ni/Sr-doped Al2O3. Although, the basic
sites of Ni/Sr-doped Al2O3 are higher than
Ni/Zr-doped Al2O3, the corresponding Ni
surface area is lesser. Hence, Ni surface area
could be the main factor to boost up the H2
production from NH3 decomposition. As
Figure 3. CO2-TPD profiles of catalysts reported in our previous study, the doping
Zr4+ in Al2O3 framework can affect the
3.2 Activities of Ni catalysts over different characteristic properties of Ni catalyst
supports for NH3 decomposition resulting in improvement of the catalytic
Table 3 lists the activities of Ni activities.9 In this study, the different
catalysts in terms of NH3 conversion and H2 valency (Sr2+ and Zr4+) and the different
formation rate at 550 °C and 600 °C. atomic size with same valence (Ce4+ and
For 1mol% of dopants in Al2O3 Zr4+) as well as the amount of dopant also
supports, Ni/Ce-doped Al2O3 exhibits the affect the catalyst properties. Larger atomic
highest catalytic activities and Ni/Zr-doped size of Ce4+ doped in Al2O3 framework can
Al2O3 exhibits catalytic activities slightly increase both Ni surface area and basic sites
higher than Ni/Sr-doped Al2O3. At 550 °C, which can promote the H2 production from
Ni/Ce-doped Al2O3 exhibits significantly NH3 decomposition as well as for the higher
higher NH3 conversion than Ni/Zr-doped amount of Ce4+ doped in Al2O3 framework.
Al2O3 and Ni/Sr-doped Al2O3 around 15- Therefore, the enhancement in catalytic
20%. At 600 °C, the Ni/Ce-doped Al2O3 can activities of Ni/Ce-doped Al2O3 can reduce
achieve NH3 conversion up to 96.7% where the cost of H2 production from NH3
the corresponding H2 formation rate is 6770 decomposition in the continuous mode.
μmol/min·gcatalyst. Higher amount of dopant
also affects the catalyst activities.
The results of this study show the 1. Miltner, A.; Wukovits, W.; Pröll, T.;
feasibility of H2 production from carbon- Friedl, A. J. Clean. Prod. 2010, 18,
free NH3 decomposition. All supports show 51−62.
the same crystal structure of γ-Al2O3 and 2. Chen, J.; Zhu, Z. H.; Wang, S.; Ma, Q.;
also maintain the surface area relatively Rudolph, V.; Lu, G. Q. Chem. Eng. J.
close to that of γ-Al2O3. The doping of Ce4+ 2010, 156 (2), 404−410.
which has a larger atomic size than Sr2+ and 3. Yin, S. F.; Xu, B. Q.; Zhou, X. P.; Au,
Zr4+ to partly replace Al3+ in Al2O3, as well C. T. Appl. Catal., A: General 2004, 277
as a higher amount of Ce4+ doped in Al2O3 (1-2), 1−9.
framework can increase the Ni surface area 4. Meng, T.; Xu, Q.-Q.; Li, Y.-T.; Chang,
and basic sites of catalysts. The increase in J.-L.; Ren, T.-Z.; Yuan, Z.-Y. J. Ind.
these properties of Ni/Ce-doped Al2O3 can Eng. Chem. 2015, 32, 373−379.
promote H2 production from NH3 5. Zamfirescu, C.; Dincer, I. Fuel Process.
decomposition. The catalytic activities of Technol. 2009, 90 (5), 729−737.
Ni/Ce-doped Al2O3 with 1mol% Ce and 6. Zhang, J.; Xu, H.; Jin, X.; Ge, Q.; Li, W.
5mol% Ce exhibit significantly high NH3 Appl. Catal., A: General 2005, 290 (1-
conversions of 96.7% and 98.8%, 2), 87−96.
respectively at 600 °C. Conclusively, the 7. Zheng, W.; Zhang, J.; Ge, Q.; Xu, H.;
results from this study with the enhancement Li, W. Appl. Catal., B: Environmental
of catalytic activities of Ni/Ce-doped Al2O3 2008, 80 (1), 98−105.
can lead to the development of low cost Ni 8. Henpraserttae S.; Vacharanurak V.;
catalysts for H2 production from NH3 Leelabanyong T.; Hunhaboon P.;
decomposition in the continuous mode. Toochinda P. PACCON 2014, 968−971.
9. Henpraserttae S.; Charojrochkul S.;
Acknowledgements Klysubun W.; Lawtrakul L.; Toochinda
This work was supported by the P. Catal. Lett. (accepted).
National Research University Project of 10. Henpraserttae S.; Junpeti A.;
Thailand Office of Higher Education Kittiparikun A.; Poranapiti C.;
Commission and Thammasat University Toochinda P. PACCON 2017,
Grant. 1275−1279.
Activated carbon derived from rice husk char and its application for dye
adsorption: kinetics and isotherm studies
Nureesan Deemae, Ruethaithip Wisedsri, Nareerat Kitisripanya*
Department of General Science, Faculty of Science and Engineering,
Kasetsart University, Chalermphrakiat Sakon Nakhon Province Campus, Thailand
*E-mail: nareerat.k@ku.th
Abstract:
In this work, rice husk waste was used as a precursor for the production of a low-cost
activated carbon (AC) with phosphoric acid chemical activation. The effects of the
production variables, such as impregnation ratio, activation temperature and activation time
were investigated. The optimal activated carbon which had the highest surface area was
obtained by these conditions as follows: 30% phosphoric acid solution, 600 °C activation
temperature and 60 min activation time. The obtained activated carbon was then employed
for the adsorption of dye (Acridine Orange) and it was found that activated carbon exhibited
the highest adsorption capability. The experimental results were analyzed using Freundlich
and Langmuir isotherm models. Moreover, the equilibrium data were fitted into different
adsorption isotherm and found to fit well with Freundlich model with a maximum monolayer
adsorption capability of 476.2 mg/g, whereas the adsorption kinetics followed the pseudo-
second-order kinetics. It is inferred from the experimental result that the activated carbons
from agricultural wastes can be applied as an adsorbent substitute to commercial activated
carbon (CAC) in the removal of Acridine Orange from waste water.
1. Introduction known as a good sorbent of many metals

Dyes are widely used in various and basic dyes.3 Due to, the silica from rice
industries such as textiles, rubber, cotton, husk char can transform to high value
fabrics, paper and manufacturing of inks.1 activated carbons that are widely used for
The contamination of water by dyes are treated waste water. In view of this, the
frequently a worldwide environmental potential of using local rice husk which are
problem that can be easily recognized as its cheaper and easily available to produce
presence in water is highly visible and activated carbon with high adsorptive
undesirable. Many of the organic dyes are capacity offers interesting alternative to the
hazardous and may affect aquatic life and conventional approaches available for dye
even the food chain. Several techniques removal.4 In this present, the main study is
have been extensively developed for dye to compare the removal dye efficiency of
removal such as irradiation, photochemical commercial activated carbons (CAC) and
and membranes filtration.2 However, rice husk activated carbon (ACRH). Also,
adsorption is most suitable to solve these the influence of parameters such as initial
problems. Activated carbon is vastly used as dye concentration, contact time on the
industrial adsorbents for separation, removal efficiency was assessed. The
purification of solid and liquid due to their adsorption mechanism and kinetics were
texture being highly porous and they have a appraised using isotherm and kinetics models.
large capacity to absorb pollutants.
Commercial activated carbons are costly and 2. Materials and Methods
therefore, there is a required to search for 2.1 Adsorbent
effective adsorbents for economical waste Rice husk was collected from the
water treatment. Rice husks are usually by rice mill in the Chiang Khruea subdistrict,
product of rice milling processes which well Sakon Nakhon Province, Thailand. The 40 g
of rice husk samples were carbonized at 250 determined by shaking 10, 50, 100, 200 and
to 350 C for 2hr in a muffle furnace in 250 mg of rice husk activated carbon
order to produce charcoal. The sample was (ACRH) with 10 mL of 50 ppm AO
crushed with blender and sieved to a size solution. The suspensions were stirred at
smaller than 600 m (ASTM-E11: No.30) to 100 rpm for 2 hr to reach equilibrium. Then
obtain the charcoal of rice husk (CRH). The the mixture was filtered and the residual AO
CRH was subjected impregnation in 30% concentrations were measured by
(v/v) of H3PO4 for 6 hours. H3PO4 is a monitoring the absorbance at 496 nm max
common activating agent whose use has been using a spectrophotometer (Spectronic 200
extensively reported for preparing activated Themo Scientific). Amount of adsorbed dye
carbons from agricultural by-products.5 After samples was analyzed by the relative
that, it was cleaned with DI water until its standard curve method. The reduction in AO
pH value was approximately 7. The sample concentrations (removal %) was calculated.
was dried at 110 C for 3 hours. The dried For isotherm study, 100 mg of the adsorbent
samples were carbonized at 600, 700 and was agitated with 10.00 ml of AO solutions
800 C for 1 hour in a muffle furnace. The having initial concentrations of 50, 100, 200
charcoal was crushed and sieved to a size and 500 ppm for 3 hr at room temperature
between 150-300 m (ASTM-E11: No 50 (27 ± 1 ̊C). The adsorption capacity (qe) of
and No 100) to obtain the activated carbon ACRH was estimated using the well-known
of rice husk (ACRH). The carbon was then equation as shown in equation (1).
again dried in hot air oven at 110C, ground
and sieved to obtain desired particle size Ci  C f
qe = ×V (1)
(150 m) and stored in desiccators for future m
use. The common properties of ACRH such
as moisture, ash content and density were where, Ci and Cf are the concentration of
preliminary investigated as in described in AO at initial and final, respectively, V is the
ASTM standard. Ash content determination volume of solution and m is the mass of
was done according to the ASTM D2866-11 ACRH adsorbent. In addition, effect of
method. The moisture content was contact time and kinetic study were
determined using the “oven drying method” investigated by contacting 0.1 g of the
described in ASTM D2867-09. Apparent adsorbent, 10.00 mL volume of 50 ppm
density is essential for mass to volume solution and stirred as mentioned above. The
conversions required for designing and residue concentration was determined after
filling equipment to the ASTM 2854-09 certain time intervals (by varying time from
method. 20 min to 180 min). The adsorption capacity
2.2 Adsorbate at time t: qt (mg/g) was calculated using
Stock solutions of Acridine Orange equation (2).
(AO) was made by dissolving the dye in
Ci  Ct
distilled water. The dye, registry Number = qt = ×V (2)
494-38-2, chemical formula = C17H19N3, m
MW = 265.35 g·mol−1, max = 496 nm
(measured value) was supplied by Fluka where, Ci and Ct are the concentration of
Chemika. AO at initial and any time, respectively. The
2.3 Adsorption studies percentage removal efficiency was estimated
Batch adsorption experiments were according to equation (3).
carried out to investigate the isotherm and
kinetics studied. All the experiments were  C  Ce 
conducted at room temperature. The effect % Re moval : (%)   i  100 (3)
 Ci 
of adsorbent dosage on uptake was
where, Ci and Ce are the concentration of adsorption efficiency. Therefore, only an
AO at initial and equilibrium, respectively. activated temperature 600°C was selected to
adsorption isotherm study, which is costly.
3.1 Properties of ACRH Table 2. The efficiency of impregnation
The carbon was stimulated with ratio of H3PO4 which different activated
various concentrations of phosphoric acid at temperature on 50 ppm of AO adsorption
different temperature to yield the activated
carbon (ACRH). To verify the general Materials AO Removal (%)
properties of our ACRH product, moisture, Commercial - - -
ash content and density were analyzed that ACRH20% H3PO4 98.68a 98.24b 96.98c
shown in Table1. The standard activated ACRH30% H3PO4 99.25a 99.85b 97.63c
charcoal should have a moisture content not ACRH40% H3PO4 95.78a 95.47b 94.21c
exceeding 8%, ash content, not exceeding a
activated at temperature 600°C.
b activated at temperature 700°C.
30% and density in the range of 0.20 to 0.75
c activated at temperature 800°C.
g/mg (TISI 900-2532). All ACRH have
qualified based on the preliminary criteria
used. 3.2 Effect of adsorbent dosage
Figure 1 shows the effect of
Table 1. Some physicochemical properties adsorbent dosage range (0.01-0.25 g) on the
of the activated carbon obtained from rice removal of AO 50 ppm which increased
husk (ACRH) and commercial from 32 and 36% to 99.5 and 98.9% for
ACRH and CAC respectively, by increasing
Total the adsorbent dosage. At the beginning as
Moisture Apparent
Temp Ash
Materials
(°C)
Content
Content
Density dosage increase there is observable
(%) (g/ml) obviously increase in removal% of AO
(%)
Commercial - 0.256 1.043 0.650 which is related to baring more adsorption
600 0.187 16.56 0.678 sites. At higher adsorbent dosage the
ACRH increase in adsorption sites had a little effect
700 0.171 16.56 0.647
20% H3PO4
800 0.194 16.34 0.633 on removal percentage of AO. According to
600 0.153 16.12 0.695 this experiment the optimal dosage of 0.10 g
ACRH were selected for AO to carry out adsorption
700 0.085 15.84 0.675
30% H3PO4
800 0.123 16.53 0.651 study.
600 0.234 16.30 0.668
ACRH
700 0.227 15.87 0.643
40% H3PO4
800 0.234 16.46 0.646
 ACRH
In order to perceive that the  CAC
concentration of phosphoric acid and
activation temperature affect the absorption,
the test of 50 ppm AO was studied at
concentrations 20, 30 and 40% of H3PO4 as
shown in Table 2. From Table 2, the
maximum percentage removal of AO
obtained was 99.85% attained at 700°C and
30%H3PO4 concentration. Then, the Figure 1. Effect of adsorbent dosage on the
concentration of phosphoric acid, which adsorption of AO
30% were selected due to the adsorption
efficiency is best. Due to the variation of the 3.3 Effect of contact time and kinetic
temperature has affected not too much for studies
The effect of contact time in removal
AO by adsorption is important factor to  ACRH
determine both of equilibrium time and  CAC
kinetics of adsorption. The relation between
contact time and adsorbed amount of AO
(qt, mg/g) is shown in Figure 2. Uptake of
AO at 140 min is higher which related to the
abundance of active sites on the surface of (a)
adsorbent. AO uptake in the range 20-100
min increased from 4460 to 4986. At a
contact time 140-180 min the rate of
adsorption approximately not change which  ACRH
can be related to surface saturation with AO  CAC
molecule and equilibrium. Hence, the proper
adsorption time was judged to be 3 hr.
(b)
Figure 3. Pseudo-first order (a) and second

order(b) kinetic model for adsorption of AO
on ACRH (blue line) and CAC (red line) at
27 °C
Table 3. Parameters of Kinetic model (first

Figure 2. Effect of contact time on the and second order) for adsorption of AO on
adsorption of AO on ACRH at 27 °C ACRH and CAC at 27 °C
Adsorbent
To understand in details the exact Kinetic Model Parameter
kinetics mechanism of the adsorption ACRH CAC
process, two general kinetics models, Pseudo-first qe(mg/g) 18.73 12.48
pseudo first order and pseudo second order order k1(h-1) 2.52 1.80
were applied on the experimental data.6 The R2 0.9588 0.848
linear forms of these two models are given Pseudo-second qe(mg/g) 51.30 50.76
in equation 4 and 5, respectively. order k2(g/mgh-1) 5.74x10-3 5.90x10-3
R2 0.9999 0.9998
Pseudo first order: ln(qe  qt )  ln qe  k1t (4)
t 1 t Table 3 clearly shows that for both
Pseudo second order:  2
 (5) of adsorbent, the correlation coefficients
qt k2 qe qe
(R2) of the pseudo-second order model was
Where, qe and qt are the amounts of AO higher than those of the pseudo-first order.
adsorbed (mg/g) at equilibrium and at time, The R2 values of ACRH were 0.9588 and
respectively, k1 the pseudo first order rate 0.9999 for PFO and PSO kinetic models. In
constant for adsorption and k2 is the rate addition, it was observed that the R2 values
constant of pseudo second order adsorption. of CAC also give the same trend as ACRH,
Linear plot of these kinetic models is shown 0.848 and 0.9998 for PFO and PSO kinetic
in Figure 3. The values of k1, k2 and qe were models, respectively. These confirm that the
calculated from the slope and intercept of PSO best fits the experimental data
the linear plots. Kinetic parameters and R2 compared to the PFO. The Calculated qe
were collected in Table 3. values using PSO kinetic model closer to
that determined from Langmuir equation homogenous adsorbent surface and no
(qm) than PFO model. However, qe of both further adsorption occurs at the particular
kinetic model has the largely deviate from sites. The Langmuir equation can be
qm of Langmuir theory. Adsorption of AO expressed in equation (6)8,9
from aqueous solution by H3PO4-activated
carbons from rice husk (ACRH) follow PSO Ce 1 C
  e (6)
kinetic model because PSO model explains qe bqm qm
the intra-particle diffusion processes, surface
adsorption better than PFO kinetic model.7 Here, Ce is the equilibrium concentration
3.4 Isotherm studies
(mg/L), qe is the amount of adsorbed dye at
Effect of AO concentration was
studied for both adsorbent at 27°C, 0.01 g as equilibrium (mg/g), qm (mg/g) is the
adsorbent dosage and 2 h as equilibrium maximum adsorption capacity and b (L/mg-1)
time. Figure 4 shows an increasing AO is the Langmuir adsorption constant which is
adsorption capacity of the both adsorbent related to adsorption energy. For the
with increase in initial AO concentration. Freundlich isotherm, it based on surface
For ACRH and CAC the adsorption capacity heterogeneity, which can be written the
increase from 49.94 mg/g and 48.19 mg/g to linearized Freundlich form as shown in
465.00 mg/g and 466.00 mg/g respectively equation (7)10
when the AO concentration was increased 1
log qe  log K f  log Ce (7)
from 50 ppm to 500 ppm. This observation n
suggest that the saturation of the binding Where Kf measure the adsorption capacity of
sites on both adsorbent may not occur at the adsorbents and 1 / n is an empirical
study concentration. parameter relating the adsorption intensity,
which varies with the heterogenetic of
Table 4. The percentage removal of AO on adsorbent. Figure 5 shows Linear Langmuir
the activated carbon obtained from rice husk and Freundlich plots of AO adsorption on
(ACRH) and commercial (CAC) ACRH and CAC at 27°C. When plot of log
AO Ce qe Removal (%) qe against log Ce gives a good straight line
Conc.
(ppm)
ACRH CAC ACRH CAC ACRH CAC with R2 of 0.9319 and 0.933 of ACRH and
50 0.059 1.81 49.94 48.19 99.88 96.38 CAC, respectively, indicating the good
100 0.656 5.16 99.34 94.84 99.34 94.84 linearity of Freundlich equation. Whereas,
200 13.00 6.17 186.00 193.83 93.50 96.91
500 35.10 34.00 465.00 466.00 93.00 93.20
R2 of linear Langmuir plot give 0.7497 and
0.7268 of AO adsorption on ACRH and
CAC, respectively (Table 5). The maximum
The percentage removal of AO on
the activated carbon obtained from rice husk adsorption capacities, qm (mg/g) are 476.19
(ACRH) and commercial (CAC) was also and 909.10 for ARCH and CAC,
shown in Table 4. Adsorption isotherm respectively.
related the amounts of AO adsorbed to the
remaining concentration of AO in solution
and represents the capacity of adsorbent.
Isotherm studies were performed to study
the distribution of adsorbate (AO) between
the water and adsorbent at equilibrium
during the adsorption process.
In this work, Langmuir and
Freundlich models were applied on the
experimental data. Langmuir model assumes
monolayer coverage of dyes over a Figure 4. Effect of initial concentration
used in AO removal. The removal efficiency
of ACRH are 99.88%, 99.34%, 93.50% and
93.00% at 50, 100, 200 and 500 ppm AO
concentration. It concluded that ACRH is
an effective adsorbent due to its excellent
removal efficiency when compared with the
(a)
(b) commercial activated carbon (CAC). The
adsorption isotherm model could be better
described by Freundlich isotherm. Kinetic
adsorption models of AO well fitted pseudo-
Figure 5. Linear Langmuir (a) and second order model of both adsorbent. The
Freundlich (b) plots of AO adsorption on results showed that the ACRH is effective
ACRH and CAC at 27 °C adsorbent for AO in aqueous solutions.
Table 5. Langmuir and Freundlich Acknowledgements

parameters for AO adsorption on ACRH and The authors are thankful to the
CAC at 27 °C Faculty of Science and Engineering,
Langmuir Kasetsart University Chalermprakiat Sakon
Freundlich Isotherm Nakhon Province Campus for providing
Isotherm
b facilities support to carry out this work.
qm
Kf (1/n) R2 (L/mg) R2
(mg/g)
ACRH References
114.82 0.315 0.932 476.19 0.1680 0.750
CAC 53.95 0.774 0.965 909.10 0.0299 0.727
1. http://dyes-pigments.standardcon.com
2. Robinson, T.; McMullan, G.; Marchant,
R.; Nigam, P. Bioresour. Technol. 2011,
The increase in Kf values confirm the
77, 247–255.
increase in adsorption capacity and 1/n
3. Chuah, T. G.; Jumasiah, A.; Azni, I.;
ranged 0.315 and 0.774 for adsorbents
Katayon, S.; Choong, S. Y. T. Desalination
indicating that adsorption of AO is in
2005, 175, 305.
favorable conditions. It can be seen that
4. Ahmaruzzaman, M.; Vinod, K. G. Ind. Eng.
Freundlich adsorption isotherm could be
Chem. Res. 2011, 50 (24), 3589–13613.
used to represent the trends of adsorption on
5. Fierroa, V.; Muniz, G.; Bastac, A. H.;
Acridine orange dye in the range on
El-Saiedc, H.; Celzarda, A. J. Hazard.
concentration lower than 500 ppm. This is
Mater. 2010, 181, 27–34.
probably due to the concentration of the AO
6. Ho, Y. S.; Mckay, G.; Process Biochem.
has not advanced beyond the absorb ability
1998, 34, 451–465.
of both activated carbon type (ACRH and
7. Banerjee, S.; Chattopadhyaya, M. C. Arab.
CAC). Then, it is found that the adsorption
J. Chem. 2017, 10 (2), S1629-S1638.
of AO on both could be well described by
8. http://en.wikipedia.org/wiki/Adsorption
Freundlich isotherm.
9. http://www.chemistrylearning.com/
langmuir-adsorption-isotherm
4. Conclusion
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In this study, Rice husk has been
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made into a potentially low-cost absorbent
Effect of doped support and urea on catalytic activity and coke prevention
from Ni catalyst for ethanol steam reforming reaction
Disorn Maneewongvatana, Methichai Manatmethikun, Pornapat Tiyapornsuwan,
Sirintra Arayawate, Pisanu Toochinda, Pumiwat Vachrapong*
Thammasat University, Pathumthani 12121, Thailand
*E-Mail: pumiwat.v@gmail.com
Abstract:
Active nickel based catalyst over various supports for ethanol steam reforming (ESR)
at low temperature were developed and investigated for the catalytic activity and coke
prevention. The supports in this study are γ-Al2O3, CeO2 and Ce doped γ-Al2O3 (Ce-Al2O3).
impregnated solution. The ESR reaction was conducted in a quartz tubular reactor, at 550 C
and 750 C in continuous mode. The hydrogen production from these catalysts was
quantified by gas chromatography (GC), the Ni active surface area was analyzed by the
chemisorption analyzer, and the catalysts were characterized by the CHN analyzer to identify
the amount of coke before and after the reaction. The results show that the doping of Ce in γ-
Al2O3 can reduce the coke formation, and the addition of urea can increase the Ni dispersion
on catalyst support, which can improve the catalytic activity of ESR. Ni/Ce-Al2O3with
addition of urea, exhibits higher H2 production and lower coke formation compared to Ni/γ-
Al2O3. The role of doped support and urea in the catalyst are discussed.
1. Introduction study, the approaches of using support effect

The hydrogen energy has become an and urea in the impregnation process were
interesting choice for fossil fuel evaluated in order to increase the H2
replacement. Ethanol steam reforming production and reduce coke formation over
(ESR) is considered as an effective reaction Ni-based catalyst. From previous study, urea
to produce hydrogen since, ethanol can be was added into copper–zinc (Cu-Zn)
produced by biomass from agricultural aqueous salt solution and was able to
waste. ESR can also yield a high hydrogen increase the solubility of the solution and
content per mole of ethanol relative to other Cu-Zn metal dispersion on the support.3 In
reforming of hydrocarbons as shown in this work, the effect of urea was investigated
equation 1. towards the Ni dispersion over the support.
High conversion of ESR over Ni/Al2O3 can
CH3CH2OH+3H2O→6H2+2CO2 (1) be achieved at high temperatures (1000-
∆H298 = +174 kJ mol-1 1200 °C). Nevertheless, deactivations can
cause by coking and sintering of Ni.4 From
This reaction allows us to produce up literature, Rh/CeO2 can reduce the coke
to 6 moles of hydrogen/mole of ethanol. The formation on Rh due to the oxygen mobility
active catalyst for the ESR is Rhodium in the CeO2 framework.5-7 However, the
(Rh).1 However, the price of Rh creates a price of CeO2 is expensive and CeO2 has
problem for industrial use. Low cost nickel much lower surface area compared to γ-
(Ni) has been reported as a replacement of Al2O3. This study employed the partial
active metal Rh in many works.2 replacement of cerium (Ce) as a dopant into
Unfortunately, the low activity of Al2O3 framework to make Ce-Al2O3
Ni/Al2O3, high reaction temperature (1000- support. The objective is to reduce coke
1200 °C), and high coke formation are the formation and lower the reaction
drawbacks of the Ni-based catalysts. In this temperature of ESR down to 550 °C and 750
°C to avoid Ni sintering. The support also Table 1. The composition of supports for
maintains the cost effectiveness and high Ni-based catalysts
surface area to be suitable for commercial Catalysta
Composition of
catalyst. This study focuses on comparison support
Ni/Al2O3 γ-Al2O3
of the hydrogen production and coke
Ni/CeO2 CeO2
formation among Ni catalysts on different Ni/Ce-Al2O3 1%CeO2 in Al2O3
supports; γ-Al2O3, Ce-Al2O3, andCeO2, and a
Ni is impregnated with 10 wt% for all catalysts.
the urea effect on catalytic activity of ESR.
2. Materials and Methods The CO-pulse chemisorption
2.1 Support preparation measurement was performed to determine
The supports of the nickel based the dispersion, particle size, and surface area
catalyst were from different sources. The γ- of nickel. The CO pulse was performed at
Al2O3, and CeO2 are commercial chemicals room temperature and atmospheric pressure
from Sigma-Aldrich and Alfa Aesar, by using chemisorption analyzer (BELCAT-
respectively. The CeO2-doped Al2O3(Ce- B, Microtrac- BEL, Japan) with 10 vol% of
Al2O3) was prepared by sol-gel method with CO in He.
1 mol% of CeO2 in Al2O3. Ce-Al2O3 The metal dispersion, average
solution was formed by using 0.5 M of particle size, and metal surface area were
aluminum nitrate nonahydrate (Al2O3·9H2O, calculated by the software in the
Laboratory Reagents & Fine Chemicals) and chemisorption analyzer.
0.5M of cerium (III) nitrate hexahydrate 2.4 Catalytic activity test
(Ce(NO3)3·6H2O, Fluka) dissolved in water. The ESR was conducted in quartz
Ammonia in aqueous solution (30 vol%, tubular reactor using 0.1 g of catalyst with
Merck) was added into Ce-Al2O3 salt 0.4 g of quartz sand in order to provide
solution drop by drop until pH reaches value better temperature distribution. Before ESR
of 9 to form sol-gel solution. The Ce-Al2O3 reaction, the catalyst was reduced with 10
gel was kept at 25 °C for 48 h then dried at mL/min of H2 and 20 mL/min of Ar at 600
110 °C for 12 h. After that, it was calcined °C for 1 h. The reactor is then flushed with
at 800 °C to obtain Ce-Al2O3 support. Then Ar at 20 mL/min to get rid of the leftover
the support was grounded and sieved by 106 H2. Carrier gas (Ar) at flow rates of 30
μm sieve to control particle size of the mL/min and 10 mL/min were flowed into
support. saturator containing water at 98.4 °C, and
2.2 Catalyst preparation ethanol at 80 °C, respectively. Feed reactant
Nickel based catalysts were prepared is a mixture of 1:3 ethanol-to-water molar
by incipient wetness impregnation method. ratios. The reactions were tested at 550 °C
The amount of nickel was fixed at 10wt% of and 750 °C in a continuous mode. The
catalyst. Two types of nickel solutions were composition of produced gas was analyzed
prepared by using 1.25 M aqueous solution by gas chromatograph (GCMS-2010 Ultra,
of nickel nitrate hexahydrate Shimadzu Corporation, Japan) equipped
(Ni(NO3)2·6H2O, 99%, Lobachemie) with with thermal conductivity detector and
and without adding urea (99%, Carlo Erba) flame ionization detector.
in DI water. Nickel solutions were 2.5 Coke deposition analysis
impregnated drop by drop over the supports. The amount of coke deposited on the
The impregnated samples were dried at 110 catalyst after ESR process was determined
°C for 12 h and then calcined at 500 °C for 4 by CHN analyzer (LECO CHN-628) in
h. The composition of each catalyst is shown terms of percent weight of carbon/sample.
in Table 1.
3. Results and Discussion Table 4. Surface area of support
3.1 Characterization of Ni/γ-Al2O3 based Support
Support surface area
catalyst (m2/g support)
γ-Al2O3 176
Table 2 shows the properties of Ni/γ-
Ce-Al2O3 146
Al2O3.From Table 2, the catalyst with CeO2 52
addition of urea, yields a better dispersion
and smaller Ni particle size. This increases Table 5. Ni surface area of Ni/γ-Al2O3 and
Ni surface area, providing more active sites Ni/Ce-Al2O3
for the catalyst. Catalysta Ni surface areab
Ni/γ-Al2O3 6.39
Table 2. Properties of Ni/γ-Al2O3 Ni/Ce-Al2O3 12.46
a
Ni Ni Urea was used for all catalysts as promoter
Ni b
(m2/g-metal)
Ni/γ-Al2O3 particle surface
dispersiona
sizeb areac
Without urea 1.14 89 7.58 3.3 Catalytic activity
With urea 1.58 64 10.54 Table 6 lists the catalytic activities of
a
(%)
b
(nm)
Ni based catalysts at 550 °C and 750 °C.
c
(m2/g-metal) The results show that Ni/CeO2 exhibits the
highest H2 production rate among all
Table 3 shows the H2 production of catalysts at both temperatures. The lowest
Ni/γ-Al2O3at 550 °C. From Table 3, H2 production rate corresponds to Ni/γ-
Ni/γ-Al2O3 with the addition of urea exhibits Al2O3. Ni/Ce-Al2O3also shows a higher
a higher H2 production rate than Ni/γ-Al2O3 activity compared to Ni/γ-Al2O3; therefore,
without urea at 550 °C. Therefore, urea doped support can enhance H2 production.
could improve the activity of Ni/γ-Al2O3 Table 6 also indicates the production
catalyst at 550 °C. Due to these positive rates of CO and CO2. The amount of CO and
effects, urea was added to the studied CO2produced from ESR process can relate
catalysts. to the coke formation on the catalysts.
Ni/CeO2 exhibits the highest production of
Table 3. H2 production of Ni/Al2O3 at 550 CO and CO2 at both temperatures because,
°C both temperatures; therefore, lower coke
Ni/γ-Al2O3
H2 production rate should bedepositedon the catalyst.
(µmol/min) Coke depositions on the catalysts
Without urea 70.22 after ESR at 750 °C are shown in Table 7.
With urea 242.70
Ni/γ-Al2O3 exhibits the highest coke
formation in comparison to that of Ni/Ce-
3.2 Characterization of support
Al2O3 and Ni/CeO2. The lower coke
Table 4 shows the surface area of the
deposition on Ni/Ce-Al2O3 than that of Ni/γ-
three supports in this study. Results showed
Al2O3 reveals that doping 1% Ce into Al2O3
that γ-Al2O3 has the highest surface area
framework can reduce coke formation on the
at176 m2/g, compared to Ce-Al2O3 and
catalyst.
CeO2.The results also show that Ce-Al2O3
has a higher surface area than CeO2.
4. Conclusion
Therefore, by doping 1% Ce into Al2O3
In conclusion, adding urea to the Ni
framework, Ni/Ce-Al2O3 still retains a high
salt solution during catalyst preparation can
surface area of the support.
increase dispersion and surface area of Ni
From chemisorption results in Table
over the support, providing more active sites
5, Ni/Ce-Al2O3 exhibits a higher Ni surface
of Ni/γ-Al2O3 catalyst. In addition, doping
area compared with Ni/γ-Al2O3. Therefore,
Ce into Al2O3 framework helps to retain
doped support can increase the Ni surface
high surface area of support, reduction in
area of the catalyst.
coke formation, and increased activity of the
Table 6. Catalytic activity of ESR
Production rate (µmol/min)
Catalysta 550°C 750 °C
H2 CO CO2 H2 CO CO2
Ni/γ-Al2O3 242.7 49.75 20.2 51.67 66.49 97.98
Ni/Ce-Al 509.8 128.07 127.91 301.9 113.89 109.28
Ni/CeO2 1477.85 384.62 479.8 3395.24 1774.53 490.19
a
Urea was used for all catalysts as promoter with 2 moles of urea per mole of Ni
Table 7. Coke deposition on the surface of

Ni/γ-Al2O3, Ni/Ce-Al2O3, and Ni/CeO2 after Chem. Mon. 2010, 141 (3), 269−277.
reaction at 750 °C 4. Christensen, K. O.; Chen, D.; Lødeng,
Catalysta Coke depositionb R.; Holmen, A. Appl. Catal., A
Ni/γ-Al2O3 4.663 2006, 314 (1), 9−22.
Ni/Ce-Al2O3 3.623
5. Tuza, P. V.; Manfro, R. L.; Ribeiro,
Ni/CeO2 0.816
a
Urea was used for all catalysts as promoter N.F.; Souza, M. M. Renew. Energ.
b
(gcarbon/gcat) 2013, 50, 408−414.
6. Xu, W.; Liu, Z.; Johnston-Peck, A. C.;
catalyst. The optimum amount required to Senanayake, S. D.; Zhou, G.; Stacchiola,
dope Ce into Al2O3 framework will be D.; Stach, E. A.; Rodriguez, J. A. ACS
further studied. Catal. 2013, 3 (5), 975−984.
7. Arslan, A.; Doğu, T. Int. J. Hydrog.
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Toochinda, P. Monatshefte für Chemie-
Effect of immobilization materials and shapes on biohydrogen production
from green alga Tetraspora sp. CU2551
Cherdsak Maneeruttanarungroj1,2*
1
Department of Biology, Faculty of Science,
King Mongkut’s University of Technology Ladkrabang, Bangkok 10520, Thailand
2
Bioenergy Research Unit, Faculty of Science,
*E-mail: cherdsak.ma@kmitl.ac.th
Abstract:
Green alga Tetraspora sp. CU2551 was previously identified to contain the hydrogen-
producing capacity. Biohydrogen production conditions were optimized to increase the
production yield. However, the yield could still be enhanced using immobilization technique.
This study aims to optimize the immobilization materials and shapes that suitable for
Tetraspora sp. immobilization. The two materials chosen were alginate and agar with
modeled in 3 different shapes: sphere, block and thin-film. The results showed that alginate
could be molded into those three types whereas agar could be molded into block and thin-
film. The production results revealed that alginate matrix in sphere resulting in the highest
yield representing for 1.96 µmol H2/mg DW/hr, while block and thin-film showed the
production rate of 0.86 and 1.02 µmol H2/mg DW/hr, respectively. The agar matrix in the
block and thin-film showed the rate of 1.21 and 1.27 µmol H2/mg DW/hr, respectively. The
time-course analysis over the period of 4 to 48 hr showed the continuous increasing
production over 36 hr. Moreover, the production from alginate bead showed above 2 times
increased in the rate when compared to that in free-cell system. This study enables the
opportunities for the up-scale biohydrogen production from green microorganism.
1. Introduction pyruvate to form acetyl-CoA as shown in

H2 is served as a promising Figure 1.2
renewable fuel in the future because its
combustion reaction releases only water as
by product, which contains no greenhouse
gas such as CO2, and the reaction releases
energy in high content of 142 kJ/g or 61,000
Btu/lb.1
Biohydrogen production from green
algae has interestingly drawn attention since
hydrogen production can be coupled
together with CO2 fixation and nutrient
removal from wastewater,2,3 Hydrogenase Figure 1. H2 metabolism of green alga
enzyme catalyzes the formation of 2 protons Chlamydomonas reinhardtii
and 2 electrons to form the gas (Equation 1)
2H+ + 2e-  H2 (1) However, the practical H2 production
The first substrate, proton, comes in green algae is currently limited by the
from the equilibrium of water inside the high sensitivity and deactivated of
cells and another substrate, electron, is from hydrogenase enzyme by O2. One way to
several ways such as water splitting process overcome this problem is to modify a
through PSII system, degradation of production process. Normally, oxygenic
accumulated starch, and fermentation of photosynthesis and carbon fixation requires
light, CO2 and ordinary medium containing 2.2 Cell immobilization
all necessary nutrients to allow cell growth. In this section, an aseptic technique
These process are called ‘growth phase’. is used throughout the experiments. Algal
Then, the algal cells are transferred to H2 cells were centrifuged and re-suspended in
production called production stage. A new TAP medium to desired final OD730.
transition from growth phase to production Cells were then mixed with either sodium
phase has been performed by purging any alginate or agar-agar to make a final OD730
inert gases to remove O2 from the closed of 0.1 in both 2 immobilization matrices.
system and create anaerobic condition to The mixtures were then molded up into
establish hydrogenase enzyme activated.4 several shapes including bead, block, and
However, the cost of those inert thin-film as the following procedures:
gases causes a high production cost. Many  Bead
researches about H2 production system Sodium alginate mixture were placed
proposed an immobilization technique to into syringe in order to drop a round-shape
overcome those problems, and also enable mixture into 2% CaCl2 and place for 1 hr to
easier cells transferring from growth phase allow complete polymerization. All algal
to production phase without any specific beads were sieved to the size rage 2.8-3.0
instrument such as centrifuge. Several mm in diameter.
studies indicate that algal cell  Block
immobilization could increase a comparable Sodium alginate mixture were
production yield significantly such as poured into a plastic block with a size of 10
Chlamydomonas reinhardtii,5 Lyngbya x 10 cm until the thickness reach 3.1 mm.
perelegans,6 and Synechocystis sp. PCC Calcium chloride of 2% was later pour on a
6803.7 top of alginate surface to completely
Green alga Tetraspora sp. CU2551 covered and stand for 1 hr to allow complete
was previously reported to be a high polymerization. Whereas agar-agar matrix
potential in H2 production,8 however, free- was performed with the same strategies
cell production technique required without CaCl2 coverage. Stand the agar-agar
centrifugation in cells transferring from one gel to harden for 30 min. Both matrices were
condition to another, combined with inert cut into dices in a size of 3.1 x 3.1 x 3.1 mm
gas purging. This study proposes the cube.
immobilization of Tetraspora sp. CU2551 to  Thin-film
enhance H2 production yield. Sodium alginate or agar-agar mixture
was placed into a plastic mesh spreading the
2. Materials and Methods mixture with plastic spatula to push the cell
2.1 Algal strain and cell culture load into tiny hole of the mesh. 2% CaCl2
A unicellular green alga, Tetraspora was sprayed to completely cover the alginate
sp. CU2551 was obtained from Laboratory surface, and stand for 1 hr for
of Cyanobacterial Biotechnology (CYB), polymerization. In agar-agar matrix, without
Department of Biochemistry, Faculty of CaCl2 spraying, the film was later rested for
Science, Chulalongkorn University, 30 min to allow gel harden. These thin films
Thailand. The alga was cultured in 125 mL were cut into a size of 1.0 x 2.0 cm.
Erlenmeyer flasks containing of 50 mL of 2.3 Measurement of hydrogen production
Tris-Acetate-Phosphate (TAP) medium. The The immobilized cells equivalent to
cultures were innoculated with an initial cell dry weight of 1 mg was added into gas
OD730 of 0.1. The flasks were placed on a tight vials containing 5 mL of fresh TAP
shaking incubator with rotary speed of 140 medium. A system was blown to completely
rpm and illuminated from the top with cool- eliminate atmospheric oxygen gas with
white fluorescence lamps (light intensity of argon at 0.1 psi for 5 min. Vials were
29 µE/m2/s), kept temperature at 36 C. incubated for desired time of incubation at
the same condition as growth phase. The the matrix surface can be harvested by thin
vials were then collected from an incubator layer of the cell in alginate matrix leading to
prior to H2 measurement. The hydrogen insufficient utilize the light power to split
concentration in the head space of gas-tight water into less amount of electron. Our
vial was determined using Gas finding was similar to other study to use
Chromatograph with a column packed 1.5 m alginate matrix in immobilization of other
x 1.0 mm with molecular sieve 5A. A 400 green algae such as Chlamydomonas
µL head space volume was withdrawn and reinhardtii,5 Lyngbya perelegans,6 and
replaced with the same volume of argon gas. Synechocystis sp. PCC 6803.7
The column temperature was kept at 40 °C.
Argon was used as a carrier gas at flow rate
of 20 mL/min. The detector used thermal
conductivity (TCD) at 200 °C.

3.1 H2 production from alginate matrix
The Tetraspora sp. CU2551 was
previously reported to be high competency
to produce the gas when compare to other
algal strains. However, Tetraspora sp. can
be also enhanced a capacity in gas
production using immobilization technique.
In this section, hydrogen production of
green alga Tetraspora sp. CU2551 in Figure 2. An accumulated H2 gas
alginate was monitored over a production production from Tetraspora sp. CU2551
time of 24 hours to verify the relationship over 24 hr of time incubation by a variation
between accumulated hydrogen gas (X-axis) shapes of alginate matrix including bead,
in a unit of µmol H2/mg DW/hr and type of block, and thin-film
matrix shape (Y-axis). The results showed in
Figure 2 that green alga Tetraspora sp. 3.2 H2 production from agar matrix
could produce the highest hydrogen amount In this section, the Tetraspora sp.
in a shape of ‘bead’ when compared to other cells were immobilized by using agar-agar
shape, ‘block’ and ‘thin-film’. The as a matrix. The procedures to immobilize
maximum hydrogen production of green cells were described in section 2.2 to mold
alga Tetraspora sp. reached 1.96 the shape into bead, block and thin-film.
μmolH2/mg DW/hr when immobilized cells Unlike alginate, unfortunately, the bead
in ‘bead’ shape whereas the production from shape could not be produced for agar-agar
‘block’ and ‘thin-film’ showed the lower matrix. This was due to the fact that agar-
extended production rate of 1.02 and 0.86 agar could not be dropped into any water-
µmol H2/mg DW/hr, respectively. soluble liquid since the agar-agar bead
The results convinced the use of would immediately disperse in solvent.
‘bead’ shape in H2 production. It is because Thus, agar-agar matrix could only mold the
of light penetration power in a bead shape matrix into only 2 shapes: block and thin-
could come from in any radial direction, film.
then the light can equally penetrate into the Hydrogen production of green alga
matrix, whereas a block shape could allow Tetraspora sp. CU2551 in agar-agar was
one side to expose the maximum light also monitored over a production time of 24
intensity, the rest five sides could expose the hours to verify the relationship between
light in lower extend. This is quite different accumulated hydrogen gas (X-axis) in a unit
in thin-film shape because, light incoming to of µmol H2/mg DW/hr and type of matrix
shape (Y-axis). The results showed in Figure pattern. All successfully immobilized cells
3 that green alga Tetraspora sp. could would be used against ‘free-cell’ system to
produce the higher hydrogen amount in a confirm that immobilization could enhance
shape of ‘thin-film’ than ‘block’ shape. In a the production capacity. Other factor to
term of comparison, the maximum Y-axis reduce bias of the experiment is a size of
was kept to 2.5 µmol H2/mg DW/hr as gas-tight vial since the volume of head space
shown in Figure 2. The maximum hydrogen would be withdrawn for many time
production of green alga Tetraspora sp. intervals, thus the concentration of the
reached 1.27 μmolH2/mg DW/hr when produced gas might not be in a linear
immobilized cells in ‘bead’ shape yielding relationship. This problem could be
1.21 μmolH2/mg DW/hr which were below overcome by using a 100-mL gas-tight vial
than the maximum point as Figure 2 instead of a smaller one. About 10-fold
indicated. A production rate from agar-agar higher in head space volume would reduce
matrix was lower than the maximum an error from the experiments. Figure 4
production yield of ‘bead’ shape in alginate. showed the production results from all cell
However, agar-agar matrix resulted in types: immobilized in any forms and ‘free-
higher yield when compare to according cell’ system.
shapes of alginate matrix.
Figure 4. A time-course analysis of

accumulated H2 gas production from
Figure 3. An accumulated H2 gas Tetraspora sp. CU2551 over 48 hr of time
production from Tetraspora sp. CU2551 incubation by all testing shapes (solid lines
over 24 hr of time incubation by a variation for alginate matrix and dash lines for agar-
shapes of agar matrix including block and agar matrix) molded in this study comparing
thin-film to its ‘free-cell’ form (dot line)
3.3 Time-course analysis of H2 production Our results showed the maximum H2

Immobilization of algal cells into yield reach 67.3 μmolH2/mg DW with in
bead, block and thin-film using alginate 36 hr when immobilized algal cells with
matrix, and into block and thin-film agar- alginate as ‘bead’ shape, and this production
agar matrix resulted in high portion of rate was above 2 time higher than the rate
hydrogen yield. In this section, the hydrogen obtained from ‘free-cell’ system. These
gas production would be monitored against could be due to the fact that immobilization
time interval over 48 hr of time incubation, could reduce the blocking of light by cell
or the production capacity reach plateau stacking when cells density increasing. Our
finding corresponds to other report in cell splitting process. As a result, less electrons
stacking effect in Tetraspora sp. CU2551.9 generated in hydrogen production process.
The effect of cells stacking may lead to
decrease a penetration power of light and the Acknowledgements
light absorption of chlorophyll pigments, as This work was supported by the
a result of lower of water splitting in research grants by the Faculty of Science,
photosystem II.10 Moreover, the substrates King Mongkut’s Institute of Technology
(protons and electrons) to produce hydrogen Ladkrabang to C. Maneeruttanarungroj
by hydrogenase enzyme are reduced, (2561-01-05-52).
lowering the hydrogen yield. However, the
result remains unclear for cell References
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A unicellular green alga Tetraspora Biochem. Eng. J. 2004, 19 (1), 69−73.
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high competency strain to produce hydrogen Bioeng. 2009, 102 (1), 50−58.
gas. However, the production capacity can 6. Anjana, K.; Kaushik, A. Biomass
be enhanced by immobilization techniques. Bioenergy 2014, 63, 54−57.
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immobilization matrices and the gas Benavides, A. M. S.; Cicchi, B.;
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resulted the maximum production yield Incharoensakdi, A. Int. J. Hydrogen
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making lower down PSII activity in water
Reforming of residue gas from CBG process to synthetic gas for
bio-methanol production
Rujira Jitrwung, Kamonrat Leeheng*, Kuntima Krekkeitsakul,
Jiraporn Chalorngtham, Anantachai Wannajampa
Expert Center of Innovative Clean Energy and Environment, Thailand Institute of Scientific and Technological
*E-mail: kamonrat@tistr.or.th
Abstract:
Compressed Bio Gas (CBG) has been promoted while residue gases from the process
which it is contained high concentration of carbon dioxide (CO2) generating to environment.
The residue gases was contained 30% CH4 and 70% CO2. Therefore, the research was aimed
to utilize the residue gases to produce synthetic gas for Bio-methanol production. Hence, CO2
left from CBG process was studied via reforming reaction at 750 C and atmospheric
pressure over Ni/Al2O3 catalyst prepared by impregnation method and modified Ni/Al2O3
with Cu/ZnO/Al2O3. The effect of various Ni loading on Al2O3 (3 wt.% to 9 wt.%) was
investigated on stability of catalyst and synthesis gas composition (H2/CO ratio). Therefore,
blending (0 wt.% to 10 wt.%) Cu/ZnO/Al2O3 with 6 wt.% Ni/Al2O3 was studied the effect of
water gas shift reaction by improving H2/CO ratio and stability of catalyst. Increasing Ni
amount resulted to rising CO2 conversion (70 to 95 %), whereas maintaining CH4 conversion
(95 to 98 %) and H2/CO (≈ 1.2-1.5). However the less stability was occurred when using 9
wt.% Ni/Al2O3, it was caused that increasing coke formation on catalyst surface. Applying
Cu/ZnO/Al2O3 improved stability of catalyst (over 100 hours) and keeping synthesis gas
composition (H2/CO ratio ≈ 1.5 to 2) which is suitable for methanol synthesis.
1. Introduction CBG process, one of possible process is

Disposable agriculture and food reforming process. CBG process gives 2
waste are used for biogas. Biogas contains streams: the first stream used for CBG
mainly methane, carbon dioxide and contains 85% methane (CH4) and 15% CO2
contaminated gas such as ammonia, and the last stream is residue gas contained
hydrogen sulfide, etc. It is used as an 30% CH4 and 70% CO2. Therefore, the
alternative fuel for cooking and producing second stream or residue gas can be
electricity for counties. However, biogas transformed under steam reforming and
replaced LPG for cooking is used in a small yielded synthesis gas that is appreciate for
scale or household. In large scales for methanol synthesis.1-6 The methanol process
electricity production, it is no longer is produced from CBG can be shown in
subsidized because of unstable composition Figure 1.
gases and CO2 containing (CO2 in biogas is Basically, steam reforming of
generated to environment that one of methane is widely known for synthesis gas
greenhouse gases). As a result, biogas production (as shown in reaction (1) and (2).
producers have faced with the problem. When methane stayed with carbon dioxide,
Accordingly, Compressed Bio Gas (CBG) is the reaction goes through reaction (3) called
production from biogas, however CBG dry reforming and yielded CO and H2.7-12
process still left high concentration of CO2 CO appeared from process can be
to atmosphere. To manage this residue CO2 transformed to CO2 by water gas shift
extensively promoted as adding-value from reaction (WGS) as shown in reaction (4). To
Figure 1. Concept of reforming of residue gas from CBG process to synthetic gas for bio-
methanol production
CH4 + H2O  CO + 3H2 (1)

CH4 + 2H2O  CO2 + 4H2 (2)
CH4 + CO2  2CO + 2H2 (3)
CO + H2O  CO2 + H2 (4)
CO + 2H2  CH3OH (5)
CO + 3H2  CH3OH + H2O (6)
reform residue gas from CBG process, the

purpose is transformation of this gas to CO
and H2 because it will yield high pure (a) Overall Process (b) Diameter of reactor
methanol. As shown in reaction (5). In case
of the gas product contained CO2, the Figure 2. Flow diagram of reforming
methanol process will follow reaction (6). process of residue gas from CBG process to
synthetic gas
2.1 Reactor 110 C for overnight and sent to furnace
Fixed-bed continuous flow reactor (Thermo scientific Series II) at 800 C for
made from 304 stainless steel belonging 4 h.
dimension of 30 cm length and 1.6 cm In addition, commercial methanol
inside diameter. The reactor is installed with synthesis catalyst (CuO/ZnO/Al2O3) with
temperature control, pressure gauge and the composition of 38/38/24 wt.% from
HPLC pump which used for water feeding. Power Tech Chemical Industry Co, Ltd.
After reforming reaction, fluid product was (PTCI) was used for applying catalyst.
sent to cyclone for removal dust and Ni/Al2O3 catalyst and commercial methanol
contaminated solid. Fluid product was synthesis catalyst (CuO/ZnO/Al2O3 with the
cooled by chill water then liquid was composition of 38/38/24 wt.% from Power
trapped by water trap. After that, gas and Tech Chemical Industry Co, Ltd.) were
liquid were completely separated. Gas was reduced at 750 C for 5 h.
sent to gas chromatography (GC) for 2.3 Analytical method
analysis as shown in Figure 2. Steam reforming of residue gas for
2.2 Chemical synthetic gas production, reactant gas was
Chemical for Ni/Al2O3 catalyst was fed by the rate of 150 mL/min and 0.1
prepared by using impregnation method. mL/min of water. The reactor was operated
Al2O3 support (ultra-pure grade 99.99 % under Ni/Al2O3 catalyst controlled at 750 C
lot#ALO9070UPG8 from Inframat and atmospheric pressure over Ni/Al2O3.
Advanced Materials LLC) was calcined at Gaseous product or synthesis gases
800 C for 4 h. After that, Al2O3 support was analyzed by online Gas Chromatograph
was impregnated with Nickel (II) nitrate (GC, Agilent Technologies 7890A)
solution. The obtained mixture was dried at equipped with Thermal Conductivity
Detector (TCD) and Flame Ionization
Detector (FID). PoraPLOT (for FID), Q-HT result is related with previous research that
MolSieve 13X and Porapak Q (for TCD), Ni/Al2O3 is highly active in steam reforming
were used for gas composition separation. and improving CH4 conversion.13
After that, analytical result of
gaseous product were used for conversion
calculation as following equation.
Xi represented conversion of component i

molei,in represented inlet mole of
component i.
molei,out represented outlet mole of
component i. Figure 3. CH4 conversion of various percent
of Ni loading on Al2O3
3.1 Catalyst characterization Furthermore, increasing suitable
Pore volume, surface area and amount of Ni loading on Al2O3 can be
diameter of reforming catalysts were increased CO2 conversion as shown in
analyzed by using BET (The Brunauer- Figure 4. The result was shown that 6 wt.%
Emmett-Teller) that shown in Table 1. Ni/Al2O3 yielded higher CO2 conversion (90
to 95%) than 3 wt.% Ni/Al2O3 (70%).
Table 1. Ni/Al2O3 and Ni/Cu/ZnO/Al2O3 Conversely, 9 wt.% Ni/Al2O3 yielded the
catalyst characterization lowest CO2 conversion (50-60%) and less
Va Ab Dc stability (CH4 CO2 and H2/CO ratio were
Sample
(cc/g) (m2/g) (µm) decreased after reaction time 12 h). This
3wt.% Ni/Al2O3 0.340 68.73 12.9x10-3 phenomena can be explained that increase
6wt.% Ni/Al2O3 0.339 61.91 21.3x10-3 amount of Ni loading on Al2O3 supported to
9wt.% Ni/Al2O3 0.347 59.08 22.8x10-3 increasing active site of catalyst. However,
10wt.% 0.352 60.27 22.1x10-3
Cu/ZnO/Al2O3
increasing active site resulted to faster
On 6wt.%Ni/Al2O3 reaction rate and effected to more coke
a
formation can be seen in CH4 decomposition
V represented total pore volume of catalyst.
b
A represented surface area of catalyst. reaction (7) and CO disproportion reaction
c
D represented average pore diameter. (8).14,15 This caused to loss stability in 9
wt.% Ni/Al2O3.
The result was shown that increasing CH4  2H2 + C (7)
percentages of Ni amount effected to 2CO  CO2 + C (8)
increasing pore diameter whereas decreasing
surface areas. This caused from adding Ni The optimal amount of Ni loading on
loading effected to performance of catalyst. Al2O3 is 6 wt.% Ni/Al2O3 which yielded
3.2 Effect of various Ni loading on Al2O3 high CH4 and CO2 conversion.
The effect of Ni loading on Al2O3 (3, 3.3 Effect of various Cu/ZnO/Al2O3 with
6, and 9 wt.% Ni/Al2O3) was investigated on Ni/Al2O3
CH4 and CO2 conversion. Increasing Ni A modified catalyst was prepared by
loading on Al2O3 influenced rising in CH4 blending Cu/ZnO/Al2O3 with 6 wt.%
conversion from 95% to 98% (150 ml/min Ni/Al2O3 in ratio of 5/95 and 10/90,
of reactant rate, 0.1 ml/min of water with respectively. The modified catalyst
reaction time 25 h) and obtaining high and comparing with 6 wt.% Ni/Al2O3 was used
stable conversion as shown in Figure 3. The to study the effect of water gas shift reaction
Figure 4. CO2 conversion of various percent Figure 6. CH4 conversion of
of Ni loading on Al2O3 Ni/Cu/ZnO/Al2O3 and Ni/Al2O3
representing in CH4 conversion, CO2 However, the limitation of using

conversion, H2/CO ratio, and stability of Cu/ZnO/Al2O3 is that thermal resistance of
catalyst. Cu/ZnO/Al2O3 resulted in melting and
deformation of catalyst.
CO+ H2O  CO2 + H2 (4)
Figure 7. Synthesis gas composition and

stability of Ni/Cu/ZnO/Al2O3 catalyst
Figure 5. CO2 conversion of
Ni/Cu/ZnO/Al2O3 and Ni/Al2O3 The appropriate ratio of H2 /CO is
about 2 that is significant to obtain pure
The result in Figure 5 was shown methanol as following reaction (5).
that increasing ratio of catalyst 5/95 and
10/90 improved CO2 conversion from 65-78 CO + 2H2  CH3OH (5)
to 90%. In addition, CH4 conversion of
catalyst 5/95 (80-85%) is lower than catalyst 4. Conclusion
10/90 (95-98%) as show in Figure 6. H2 /CO CO2 utilization of residue gas from
ratio can be improved after loading 10/90 of Compressed Bio Gas (CBG) has been
catalyst from 1.5 to 2.3. studied by using steam reforming at 750 C
The stability was improved after and atmospheric pressure over Ni/Al2O3 and
applied Cu/ZnO/Al2O3 5 and 10 wt.% modified Ni/Al2O3 with Cu/ZnO/Al2O3
indicated to longer stability over 100 hours catalyst. Using Ni/Al2O3, increasing amount
comparing with Ni/Al2O3 as shown in of Ni is increasing CO2 conversion (70 to 95
Figure 7. The result confirmed that applying %), whereas maintaining CH4 conversion
Cu/ZnO/Al2O3 on Ni/Al2O3 can improve (95 to 98 %). Improving H2/CO ratio and
water gas shift reaction (4) which is one of stability of catalyst was promoted by
the major reactions for producing synthesis applying 10 wt.% of Cu/ZnO/Al2O3 into 6
gas that suited for methanol synthesis. wt.% Ni/Al2O3 (Cu/ZnO/Al2O3 : 6 wt.%
Ni/Al2O3 = 10:90). The stability of catalyst 6. Chai, M.; Machida, M.; Eguchi, K.;
was over 100 hours and H2/CO ratio rising Arai, H. Appl. Catal. A Gen. 1994, 110,
from 1.5 to 2 which appreciated for using in 239−250.
methanol production. 7. Ponelis, A. A.; van Zyl, P. G. S. Stud.
Surf. Sci. Catal. 1997, 107, 555−560.
Acknowledgements 8. Galuszka, J.; Pandey, R. N.; Ahmed, S.;
The authors would like to Catal. Today. 1998, 46, 83−89.
acknowledge the National Research Council 9. Prabhu, A. K.; Oyama, S. T. J. Membr.
of Thailand (NTCT) for the approval and Sci. 2000, 176, 233−248.
financial support in this research. 10. Prabhu, A. K.; Liu, A.; Lovell, L. G.;
Oyama, S. T. J. Membr. Sci. 2000, 177,
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The optimization of biohydrogen production from green alga KS03
Thanaporn Sirawattanamongkol1, Cherdsak Maneeruttanarungroj2,3*
1
Department of Chemistry, Faculty of Science,
2
Department of Biology, Faculty of Science,
3
Bioenergy Research Unit, Faculty of Science,
*E-mail: cherdsak.ma@kmitl.ac.th
Abstract:
Hydrogen gas is considered as a good option for a renewable energy source. Green
microorganisms contain hydrogenase enzyme catalyzing the formation of hydrogen gas in
vivo. This study aims to optimize the conditions for hydrogen production from green alga
KS03 isolated from fresh water source located in King Mongkut’s Institute of Technology
Ladkrabang (KMITL). KS03 showed the highest production yield among other hydrogen-
producing green algae. Shaking culture vial resulted in increasing the production yield which
was about 2 folds when compared to the production from non-shaking incubation. Light
intensity directly affected the hydrogen yield since high light could allow PSII generating
more substrate for hydrogenase enzyme. The highest production yield was 80.4 µmolH2/mg
dry wt. when incubated cells with OD750 of 0.1 under light intensity of 3,800 lux with
shaking. 18S rDNA data from green alga KS03 was further supported the same phylogeny as
in green algae group.
1. Introduction during its combustion in term of exothermic

Nowadays, global energy efficiency.
requirements mainly depend on fossil fuels. In the past several decades,
This will eventually lead to the depletion of researchers have discovered a hydrogen
our limited fossil energy resources. production approach through biological
Presently, the utilization of fossil fuels is the process. Microalgae are able to convert solar
main factor to global warming from the energy into chemical energy using proton
emission of pollutants especially carbon and electron catalyzed by hydrogenase
dioxide released into the atmosphere as a enzyme, through photosynthesis to produce
result of their combustion.1 So renewable hydrogen gas. Numerous strains in
energy brings energy into a new form of microalgae have been found to produce
clean and sustainable energy. Moreover, it hydrogen such as cyanobacteria,
4
does not affect to the environment such as Chlamydomonas reinhardtii, Scenedesmus
nuclear, hydro, biomass, wind capacity, and obliquus,5 and Tetraspora sp. CU2551.6
solar photovoltaic. In this study, we optimized the
Hydrogen is foreseen to play a conditions to produce hydrogen from green
pivotal role as a reliable and sustainable alga KS03 isolated from fresh water source
energy in the hydrogen driven economy of located in King Mongkut’s Institute of
the future.2 Hydrogen generates higher Technology Ladkrabang (KMITL).
heating values of 141.6 MJ kg-1 or 12.6 MJ Moreover, we identified a green algal
m-3 when compared to the fossil fuels as species by morphology observation under
traditional energy sources.3 Hydrogen has light microscope and 18S rDNA
shown to have fascinating characteristics phylogenetic tree determination. Our results
also showed that the capacity of this green
alga for hydrogen production may be Vials were incubated as following
utilized for another energy option in the conditions according to each parameters of
future. testing:
2.3.1 The effect of alga strain
2. Materials and Methods The cell cultures density was
2.1 Algal strains adjusted with final optical density of 0.1 in
Green algae, Bumilleriopsis each strain (Bumilleriopsis peterseniana,
peterseniana, KS01 and KS03 were isolated KS01, KS03, and Tetraspora sp. CU2551).
from fresh water source located in King The cells kept all vials on a rotary shaker
Mongkut’s Institute of Technology incubator at 36 °C under a light intensity
Ladkrabang, Thailand. 3,800 lux prior to measuring the hydrogen
Green alga, Tetraspora sp. CU2551 production.
was obtained from Laboratory of 2.3.2 The effect of final optical density
Cyanobacterial Biotechnology (CYB), (OD750) in shaker incubation and non-
Department of Biochemistry, Faculty of shaker incubation
Science, Chulalongkorn University, This effect was analyzed using cell
Thailand. cultures density with final optical density
2.2 Cell culture ranging from 0.1 to 0.8. Cells were
In this section, aseptic technique is incubated in a shaker incubator with shaking
used throughout the experiments using and non-shaking at a temperature of 36 °C
laminar flow cabinet. An algal colony was under a light intensity of 3,800 lux prior to
inoculated in round flat-bottom 125 mL measuring the hydrogen production
flasks containing of 50 mL of Tris-Acetate 2.3.3 The effect of light intensities
phosphate (TAP) medium.6 Then, the flasks Cells with final optical density of 0.1
were cultivated in a shaker incubator with were used to produce the hydrogen gas in a
rotary speed of 140 rpm at the temperature rotary shaker incubator at 36 °C under light
of 36 °C and illuminated from the top with intensity ranging from 0 to 3,800 lux prior to
cool-white fluorescence lamps (light measuring the hydrogen production.
intensity of 3,800 lux) for 24 hours. These 2.4 Measurement of hydrogen production
conditions were adapted from previously The incubated vials were collected
optimized condition in Tetraspora sp.CU from an incubator using in each condition.
2551 by changing the system variables.6,7 The hydrogen concentration in the head
This method is called growth phase or space of gas-tight vial was determined using
oxygenic photosynthetic stage for cell Gas Chromatograph with a column packed
growth to prepare the algal cell for hydrogen 1.5 m x 1.0 mm with molecular sieve 5A.
production stage. The column temperature was kept at 40 °C.
2.3 Conditions optimization to produce Argon was used as a carrier gas at flow rate
hydrogen of 30 mL/min. The detector used thermal
To optimize the conditions for conductivity (TCD) at 200 °C.
hydrogen production, the cell cultures were 2.5 PCR amplification
incubated in growth stage for 24 hours. Gene 18S rDNA in green alga KS03
Next, cell suspensions were centrifuge at a were amplified using polymerase chain
speed of 4,500 rpm for 4 minutes, then reaction (PCR). Forward primer 18S rDNA
discarded the supernatant. New TAP used was 5’-CAGCAGCCGCGGTAATT-3’
medium was added prior to cell resuspend, and reverse primer 18S rDNA used was 5’-
adjusted a final optical density at 750 nm in CATCTAAGGGCATCACAGACC-3’.6 The
each condition to desired value of testing. colony PCR cycles were proceeded by
The cells were transferred to a gas-tight vial, reaction mixtures of 50 µL containing DNA
capped and purged with argon gas for template (alga colony), 10 mM of each
eliminate atmospheric oxygen in head space. primer, 10 mM dNTPs, 5U of DreamTaq
DNA polymerase, 10X DreamTaq DNA sp. and KS01. Therefore, In the next
polymerase buffer and added distilled water experiments, KS03 was chosen for further
until final volume of 50 µL. The PCR study on optimization in hydrogen
program was 1 min at 95 °C for initial production.
denaturation, then followed by 35 cycles of
95 °C for 20 s (denaturation), 55 °C for 20 s
(annealing) and 72 °C for 90 s (extension).
The PCR reaction mixtures were analyzed a
size and amount of product using
electrophoresis technique in 0.7% agarose
gels and visualized by SERVA DNA stain
G. After the PCR product was purified by
FavorPrepTM GEL/PCR purification Kit, the
pure product was electrophoresed again in
order to verified DNA band before sent to
analyze DNA sequence.
2.6 Phylogenetic tree determination
DNA sequence was compared with
18S rDNA of other green alga that were
reported in NCBI database using Blastn
program. After that 18S rDNA data of a Figure 1. An accumulated H2 gas of
variety of green alga were aligned with variation strains in green algae, KS01 (),
clustal omega program and generated the KS03 (), Bumilleriopsis peterseniana (),
tree with calculated distance by Neighbor- and Tetraspora sp. CU2551 ()
joining (NJ) method. The tree was view
using Treeview program. 3.2 The effect of optical density (OD750)
during the gas production incubation
3. Results and Discussion with shaking and non-shaking
3.1 The effect of alga strain on hydrogen After KS03 was selected to improve
production the gas production yield, the variations of
In this section, hydrogen production the final optical density in culture were
of green algae including Bumilleriopsis explored. The cultures with an optical
peterseniana, KS01, KS03, and Tetraspora density (OD750) in the range of 0.1 - 0.8
sp. CU2551 were monitored over a were incubated in shaker incubator with
production time of 48 hours to verify the shaking and non-shaking incubation.
relationship between accumulated hydrogen Figure 2 shows the relationship of
gas (X-axis) and time (Y-axis). The results accumulated hydrogen (X-axis) and
showed that green alga KS03 could produce production time (Y-axis). All conditions of
the highest hydrogen amount among other final OD750 in shaking incubation (solid line)
green algal strains (Figure 1). The maximum showed increasing of the hydrogen
hydrogen production of green alga KS03 production which was about 2 folds when
reached 949.7 μmolH2/OD750 when compared to the production from non-
incubated for 24 hours. The Tetraspora sp. shaking incubation (dash line). These could
CU2551 was previously reported to be high be explained from the result of the blocking
competency to produce the gas when of light by cell stacking when increasing the
compare to other algal strains.6 However, cells density. Our finding corresponds to
Tetraspora sp. can still produce hydrogen other report in cell stacking effect in
gas at the rate of about half when compared Tetraspora sp.7 The effect of cells stacking
to the rate from KS03 even this alga can may be due to the fact that the penetration
produce the gas at higher rate from bumiller power of light and the light absorption of
chlorophyll pigments are decreased, leading µmol H2/mg dry wt. when incubated up to
to lower of water splitting in photosystem 18 hours.
II.8 Moreover, the substrate (H+ and e-) to Furthermore, a result in Figure 2 also
produce hydrogen by hydrogenase enzyme comparatively showed that the hydrogen
are reduced, decreasing the hydrogen production from final OD750 of 0.2 with
production. As a result, the cultures were shaking and from final OD750 of 0.1 with
incubated with shaking can reduce cells non-shaking showed the accumulated
stacking effect, leading to production higher hydrogen gas amount of 42 ± 2 and 40 ± 4
than the production from non-shaking µmol H2/mg dry wt., respectively, with no
incubation. significant difference. Thus, we may choose
Thus, in each type of incubation, the either one condition to applied in the future
shaking culture vial with the final OD750 of production.
0.1 can produce the highest rate 3.3 The effect of light intensities on
accumulated H2 and saturation pattern could hydrogen production
be observed after about 18 hours of Cells of KS03 were examined for
incubation because of lower cells density hydrogen production under the various light
and shaking incubation. This maximal intensities ranging from 0 to 3,800 lux in the
accumulative H2 gas amount was 84 ± 2
Figure 2. Effect of final optical density in shaking and non-shaking incubation on hydrogen
production by green alga KS03. An accumulated hydrogen gas was measured under various
final culture density, 0.1 (), 0.2 (), 0.3 (), 0.4 (), 0.5 (), 0.6 (), 0.7 (), and 0.8
() (shaking incubation was shown as solid line and non-shaking incubation was shown as
dash line).
same type of incubation. The results show in
Figure 3 to investigate the relations between
an accumulated hydrogen (Y-axis) and
production time (X-axis). Hydrogen
production of KS03 increased when
increasing the light intensity from 0 to 3,800
lux during production stage. The maximum
of hydrogen production was 80.4 μmolH2 /
mg dry wt. when incubated cells with OD750
of 0.1 in rotary shaking incubator under light
intensity of 3,800 lux at 18 hours, then the
production of hydrogen is gradually
decreasing afterward. Other light intensity
conditions could produce hydrogen, but in
lower rate. This result could be explained
that the higher intensity of light can affect Figure 3. Effect of light intensity on
the photosynthesis rate, then more substrate hydrogen production by green alga KS03.
(H+ and e-) coming to the key enzyme, An accumulated hydrogen gas was
hydrogenase. The higher electron from measured under various light intensities, 0
oxidation of water in photosystem II were lux (), 500 lux (), 1,000 lux (), 1,500
transferred through photosynthetic electron lux (), 2,000 lux (), 2,500 lux (),
transport chain into hydrogenase enzyme to 3,000 lux (), and 3,800 lux ()
increasing H2 production. In the other hand
the effect of light can be light limitation,
light saturation or light inhibition.9
However, a major problems of green
algae in hydrogen production is currently
limited by the extreme sensitivity and
deactivated of [FeFe]-hydrogenase enzyme
to O2. This is co-evolved during
photosynthesis under the light.10-12 [FeFe]-
hydrogenase enzyme sensitized oxygen in a Figure 4. Cell morphology of cells of KS03
multifaced challenge, as hydrogenase observed under a light microscope
transcription, maturation, and photocatalytic
activity are all highly vulnerable to 3.5 PCR and phylogenetic tree
inhibition by molecular oxygen.13 Relative determination
with the cyanobacterial hydrogenase enzyme The 18S rDNA was amplified and
have been showed to be much more sequenced as describe in section 2.5. A
sensitive to oxygen than green algae.13 sequence was used as a query in Blastn
Moreover, the other stain of green alga has program in order to find other related DNA
been durable to oxygen is not equal.14 sequences. All DNA sequences were align
3.4 Morphology of green algae using clustal omega prior to phylogenetic
The green alga was isolated from a tree determination using distance matrix NJ
fresh water pond near the Faculty of method. This result showed in Figure 5 that
Agricultural Technology in KMITL, 18S rDNA from green alga KS03
Thailand. Under light microscope, Figure 4 (underline) was further confirmed to the
shows the morphology of green algal KS03 same green algal group. Moreover, KS03
with the size about 5-10 µm. The cells show was nearly with Chlorella sorokiniana. We
the morphology as spherical and unicellular assume that this alga KS03 should belong to
shape. genus Chlorella sp.
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using the obtained 18S ribosomal DNA 7. Maswanna, T.; Phunpruch, S.; Lindblad,
sequence of KS03 (underline) and 9 other P.; Maneeruttanarungroj, C. Biomass
green algae strains. The number at the end Bioenergy 2018, 111, 88−95.
of each name is the accession number 8. Kim, J. P.; Kang, C. D.; Sim, S. J.; Kim,
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production. KS03 showed the maximum Show, P. L.; Phang, S. M.; Ling, T. C.;
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green algal strains. Increasing the H2 Renew. Energ. 2017, 1−15.
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industry.
Reduction of toxic gases from hydrolysis reaction of secondary
aluminium dross
Satamongkon Trekitwattanakul1,3, Achiraya Kamwongsa1,3, Mananya Pounglamjeak1,3,
Lida Simasatitkul1, Sukanya Thepwatee1, Siriporn pranee2, Samithichai Seeyangnok1,3*
1
King Mongkut’s University of Technology North Bangkok, Thailand
2
Faculty of Veterinary Technology, Kasetsart University, Thailand
3
Advanced Industrial Research and Innovation (AIRI) Group,
*E-mail: samitthichai.s@sci.kmutnb.ac.th
Abstract:
Nowadays, people use a lot of aluminium in many ways because it has several
properties such as high strength, high scratch resistance and low weight. Aluminium waste can
be recycled by annealing process which products are pure aluminium and aluminium dross.
Aluminium dross is hazardous waste which contains various compounds such as aluminium
nitrile, aluminium sulfide, aluminium phosphide and salt. When aluminium dross contacts
water or moisture in air it produces odorous gases such as ammonia, hydrogen sulfide and
hydrogen phosphide. These gases are affecting human and the environment. This research
proposes a way for trapping toxic gases from hydrolysis process by hydrochloric acid,
potassium permanganate and ethylene glycol. Hydrolysis processes use hydrochloric acid and
sodium hydroxide for accelerating the reaction. The aluminium dross as received was
characterized by X-ray diffraction (XRD). After being hydrolyzed by base and acid, gas
generated 680 and 810 mL, respectively. Alkaline solution generated ammonia 260 ppm. Acid
solution showed higher reaction rate and generated hydrogen sulfide 296 ppm and phosphide
280 ppm. The trapping toxic gases was performed by Gas detector tube. Hydrochloric acid 0.01
M 100 mL and Potassium permanganate 0.01 M 200 mL can be successful for trapping
ammonia and hydrogen sulfide, respectively.
1. Introduction Aluminium dross in industrial storage can

For centuries people have used a lot generate toxic gases for many weeks to many
of aluminium, so the demand of aluminum months because of its composition and
gets higher as well as its waste. Typically, reaction rate. Hydrolysis reaction of
aluminium can be obtained from mining aluminium dross by water takes very long
process but recycling process is more popular time because aluminium dross has thin film
than mining because it uses less energy than around particles; it needs to destroy the film
mining process and can eliminate metal before starting hydrolysis reaction.6
wastes. The entrepreneur wants to control Hydrolysis reaction of aluminium complex
toxic gases releasing from industry because needs hydroxyl group (-OH) to produce
of environmental law; wastes storage and aluminium hydroxide. Due to the reaction
species of element release. Normally, rate, water has a low Kw value (10-14) strong
aluminium dross is a non-valuable product acid and strong base solution can be as a
which disposed to landfill and classified to catalyst with high Ka and Kb value.
hazardous waste because of toxic gas that are Therefore, the reaction has more
generated by hydrolysis reaction by humid. hydroxyl group (OH-) or proton (H+) for
dissolving thin film and accelerate hydrolysis through trapping solution. The final toxic gas
reaction. That is shown in the equations 1-5.7 concentration was detected by gas detector
The aims of this research are trying to tube. The reaction experimental apparatuses
accelerate an aluminium hydrolysis reaction are shown in Figure 1.
by acidic and alkaline solution and trap toxic
gases for reducing toxicity and reducing gas 3. Result and Discussion
odor by different simple solution. 3.1 Composition of aluminium dross

2.1 Chemical reactants
Aluminum hydroxide; CHJ metal
co.;Ltd. Sodium hydroxide AR; QReC,
Hydrochloric acid AR; QReC, Potassium
permanganate AR; QReC, Ethylene glycol
AR; CDH©.
2.2 Aluminium dross preparation
An aluminium dross as received was
Figure 2. X-ray diffraction pattern of
grinded and sieved for the particle size in
aluminium dross
range of 75-250 µm by BRASS RIM; QAQC
Lab and Mechanical sieve shakers; Filtra The sieved aluminium dross was
FTL-0200, respectively. The aluminium characterized by x-ray diffraction technique.
dross sieved was characterized by X-ray Four major phases were identified;
diffraction; PANalytical; X’ Pert PRO. Aluminium nitride (AlN), Magnesium
2.3 Experiment procedure aluminate (MgAl2O4), Corundum (Al2O3)
The hydrolysis reactions were done and Brownmillerrite (Ca2FeAlO5). The
by adding alkaline solution and acid solution Aluminium dross components show that the
for accelerating the reaction. The catalytic main cause of pungent odor is aluminium
solution was added with different nitride generating ammonium gas; the
concentration and toxic gases was led reaction is shown in eq. 1.
AlN + 3H2O Al(OH)3 + NH3 (g) (1)

Al4C3 + 12H2O 4Al(OH)3 + 3CH4 (g) (2)
Al + 3H2O Al(OH)3 + 3/2H2 (g) (3)
AlP + 3H2O Al(OH)3 + PH3 (g) (4)
Al2S3 + 6H2O 2Al(OH)3 + 3H2S (g) (5)
(a) (b)
Figure 1. a) The reaction set for finding total gas generation and toxic gases concentration
b) the reaction set for finding an optimize condition to trap toxic gases
3.2 Effect of concentration on alkaline and ammonia gas to be an ammonium salt and
acid solution dissolved into aqueous solution. In this part,
Figure 3(a) shows the result of trapping solution was started with 100 mL for
different alkaline solution concentration. The each bottle and decrease the number of
sodium hydroxide solution 1, 3, 6, and 9 M bottles to finding an optimization condition.
have similar behavior with high reaction rate The trapping reaction of ammonia gas are
at the beginning for 15 minutes and shown in eq. 6, 7.8
undergoing to steady state. The gas products
are generated for 430, 500, 570, and 630 mL,
respectively. The alkaline solution generated (a)
only one of toxic gas which is an ammonia
gas at 260 ppm. Acid solution might be a
better catalytic solution. The result of
different acid solution concentration is shown
in Figure 3(b). The result shows higher
reaction rate with the volume of gas that is
dramatically increased at 5 minutes before
steady state. The concentrations of acid
solution at 1.5, 3, and 6 M of generated the (b)
gas products for 440, 810 and 950 mL,
respectively. Although 3 M and 6 M of acid
solution generated higher gas volume but the
changed is not significantly, so a higher
concentration of acid solution was not used
for the hydrolysis process. The acid solution
generated two kinds of toxic gases; there are
hydrogen sulfide and phosphine gas with 270
and 280 ppm, respectively. The summary of Figure 3. Plot of generated gas volume and
total gas generation and toxic gases reaction time a) result of alkaline solution
concentration shown in Table 1. concentration, b) result of acid solution
concentration
3.3 Toxic gases trapping
The hydrolysis of aluminum dross Table 1. Total volume gas generated and
generated three kinds of toxic gases: toxic gases concentration
Total gas Type of Toxic gas
ammonia gas, hydrogen sulfide gas and Catalytic
generated toxic gas concentration
phosphine gas with different concentrations solution
(mL) generated (ppm/L)
due to aluminum dross compositions. The NaOH 680 NH3 382
idea of trapping part is using its property:
HCl 810 H2S 365
solubility. From the set of trapping reaction
in Figure 1(b), toxic gases is allowed to pass PH3 227
through the trapping solution in a cylinder.
Ammonia gas can dissolve in water
The result show that 100 mL of 0.01
but after an hour it can release back to the
M hydrochloric acid is enough for trapping
atmosphere. Then, hydrochloric acid is an
ammonia gas, which generated from 20 g of
alternative solution for gas trapping process
aluminum dross. Moreover, the potassium
because hydrochloric acid can neutralize
permanganate solution was used to trapping
hydrogen sulfide gas. Then, hydrogen sulfide 4. Conclusion
trapping was achieved with 200 mL of 0.01 Accelerating of aluminium dross
M potassium permanganate and the reaction hydrolysis was achieved by alkaline and acid
equation is shown in eq. 8.9 solution. An alkaline and acid solution can
Another toxic gas is phosphine gas eliminate thin film on the surface of
aluminium dross particle activating the
that can dissolve in water at 17 C.10
surface of aluminum dross particles. The
Although this reaction has no cooling system,
reaction rate of aluminum dross hydrolysis
phosphine gas is soluble in such a polar process is increased with concentrations of an
solvent. However, ethylene glycol was not alkaline or an acid solution. The
achieved for trapping process but the concentration of alkaline solution at 9 M
opportunity seem higher when increasing the generated toxic gas 680 mL with ammonia
volume of trapping solution in each bottle. contained 382 ppm. For 3 M acid solution,
The volume of ethylene glycol at 560 mL is the toxic were generated at 810 mL with 365
almost successfully trapped the phosphine ppm hydrogen sulfide gas and 227 ppm
gas only 10 ppm of gas left and trapping phosphine gas. Toxic gases trapping;
result show in Table 2. ammonia gas, hydrogen sulfide gas and
phosphine gas were trapped by 0.01 M 100
mL hydrochloric acid, 0.01 M 200 mL
potassium permanganate and 560 mL
ethylene glycol, respectively.
Table 2. Results of trapping solution and final toxic gases concentration

starting
Trapping Trapping Trapping Final concentration
Toxic gas concentration
solution concentration (M) volume (mL) (ppm)
(ppm)
NH3 382 HCl 0.01 100 0
H2S 365 KMnO4 0.01 200 0
PH3 227 C2H6O2 pure 560 10
H3 + H2 O NH4+OH- (6)
NH3 + HCl NH4Cl (7)
3H2S + 2KMnO4 3S+2MnO4+2KOH+ 2H2O (8)
Acknowledgements Leonardou, S. J. Environ. Chem. Eng.

The authors would like to 2013, 1, 23-32.
acknowledge Department of Industrial 2. Biganzoli, L.; Ilyas, A.; Praagh, M.;
Chemistry, Faculty of Applied Science, Persson, K.M.; Grosso, M. Waste
Graduate Thesis Research Grant (GTRG) Management 2013, 33, 1174-1181.
from the Graduate College, King Mongkut’s 3. Ding, N.; Gao, F.; Wang, Z.; Gong, X.;
University of Teachnology North Bangkok Nie, Z. Procedia Engineering 2012, 27,
and CHJ metal co.;Ltd. for their supports in 465-474.
information and operations. 4. Koolivand, A.; Mazandaranizadeh, H.;
Binavapoor, M.; Mohammadtaheri, A.;
References Saeedi, R. Environ. Nanotechnol. Monit.
1. Tsakiridis, P.E.; Oustadakis, P.; Agatzini Manage. 2017, 7, 9-14.
5. Shin, S. K.; Kim W.-I.; Jeon, T.-W.; 7. Liu, N.-W.; Chou, M.-S. Sustain.
Kang, Y.-Y.; Jeong, S.-K.; Yeon, J.-M.; Environ. Res. 2013, 23 (1), 61-67.
Somasundaram, S. J. Hazard. Mater. 8. https://www.wikipedia.org/
2013, 260, 157-166. 9. Arunraj, P.; Bunyakan, C.;
6. Wang, E.; Chen, J.; Hu, X.; Chou, K.-C.; Chungsiriporn, J. PhD's Degree Thesis,
Hou, X. Ceram. Int. 2016, 42 (9), 11429- Prince of Songkla University, 2009.
11434. 10. https://www.wikipedia.org/
Ketalization of glycerol with acetone by using aluminum sulfate as
a solid acid catalyst
Tidarat Wahamongkol1, Jirdsak Tscheikuna2*
Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University,
*E-mail:tiwaann@gmail.com
Abstract:
Solketal is derivative of glycerol that synthesized thought ketalization with acetone
via the function of an acid catalyst. It has been used for fuel additives in gasoline, diesel and
biodiesel. Moreover, solketal used for additives in cosmetics product and solvent for cleaning
pieces of metal, electronics and polymer including to improve solubility in pharmaceutical
product. The aim of this work is to study the catalytic conversion in ketalization of glycerol
with acetone to generate the oxygenated solketal by using aluminum sulfate as a solid acid
catalyst. The influence in composition of products and operated reaction parameters such as
catalyst loading, reaction temperature and mole ratio of glycerol to acetone in the batch
reactor system were investigated. From the experiments indicate that, the glycerol conversion
increases with the increasing amount of catalyst from 1%wt to 5%wt reference to amount of
glycerol. 78% glycerol conversion and 83% solketal yield were obtained at 30 min of
reaction time and 60°C of reaction temperature by using 3%wt of aluminum sulfate. In
addition, increasing of reaction temperature and mole ratio of glycerol to acetone were
affected to drive the reaction to forwards and 82% solketal yield was obtained under obtimum
conditions.
1. Introduction example cosmetic industry, polymer

Glycerol is an oily viscous liquid, industry, textile industry, detergent industry,
colorless, odorless, water-soluble, syrup- explosives industry including to generated
sweet taste with hygroscopic properties. It is power and transport fuel. In addition,
one of the alcohols and the main component innovation of glycerol has been researching
of triglycerides that derived from transfor produced in various chemical products
esterification process in biodiesel through severally reaction process.2
production.1 Generally, the production of The ketalization of glycerol with
biodiesel generated 10%wt of glycerol as a ketone compound is an interesting reaction
by-product from the total volume that mean to produced glycerol-acetal/ketal in structure
the huge amount of glycerol occurred when of oxygenated cyclic membered ring that
biodiesel is growth up. used for pharmaceutical, solvent or fuel
Valorization of the glycerol by- additive. Solketal (Figure 1) carried out over
product from biodiesel is a critical issue for acid-catalyzed process in the ketalization
the economic viability to find the new way reaction with acetone to form a single five-
for value-add to other industrial processes. membered ring primary product (solketal),
The application of glycerol can be used six-membered ring secondary product (2,2-
directly as additives in several products on dimethyl-1,3-dioxan-5-ol) and by-product of
the markets example food and drink, water is generated during process (scheme
personal care, pharmaceutical and tobacco 1).3 It has been used for fuel additives in
humectant product. And also, indirect gasoline, diesel and biodiesel with the most
application of glycerol used as raw material interesting properties when compared to
for other chemical industries production other fuel additives that derived from
glycerol such as Glycerol-tert-butyl ether Refined glycerol (99.5%) used in this
(GTBE) and Triacetin. Moreover, solketal research was obtained from Patum
used for additives in cosmetics product and Vegetable Oil Co., Ltd. Analytical grade of
solvent for cleaning pieces of metallic, Acetone (99.5%) was used in reactant. The
electronics and polymer including to chemical for identify product, DL-1,2-
improve solubility in pharmaceutical Isopropylideneglycerol (Solketal) was
product. purchased from Sigma Aldrich and by-
Several literatures were studied in product, 2,2-dimethyl-1,3-dioxan-5-ol was
the non-catalyst, homogeneous catalyst purchased from Enamine Ltd. Aluminum
(sulphuric acid, p-toluenesulfonic acid and sulfate octadecahydrate [Al2(SO4)3.18H2O]
tin (II) chloride) and heterogeneous catalyst was used as the catalyst. All reagents were
(cation-exchange resin, zeolites, solid used without further treatment.
superacid, mixed oxides and other catalysts). 2.2 Catalyst preparation
In this study use the commercial aluminum Perior to the ketalization reaction,
sulfate as catalyst. It can be acted as a The catalyst was dried in an oven at 110°C
catalyst from Lewis acid sites on the surface, for 12 h and then calcined in a muffle
easier to apply in this reaction, easy to furnace at 500 °C for 6 h at a rate of 10
remove from product, inexpensive and °C/min for remove the hydrate form to
available in the market. These properties of Al2(SO4)3 form.
the catalyst are interesting. 2.3 Ketalization reaction
The aim of this work is to study the The catalytic reactions were carried
catalytic conversion in ketalization of out in the glass batch reactor in the total
glycerol with acetone to generate quantity of reactant preparation control at
oxygenated solketal by using aluminum 400 ml from total capacity 1L. The reactor
sulfate as a solid acid catalyst. The influence was connected to a reflux condenser and
in composition of products and operated mixing by magnetic stirrer. The electrical
reaction parameters such as molar ratio of heating was controlled by temperature
acetone to glycerol, reaction catalyst loading controller. All equipment were connected
and reaction temperature in batch reactor under operate reactor as shown in the Figure
system were investigated. 2. Acetone and catalyst were put into the
reactor in predetermined amounts. The
reactor was heated to desired reaction
temperature and liquid mixing together.
Next, Glycerol was fed into the reactor when
it reached to the reaction temperature. The
liquid-sample was taken at specified times
and then separated the catalyst by used
Figure 1. Chemical structure of solketal centrifugal separator. The sample was
analyzed for the product contents by using
gas chromatography.
2.4 Analysis
The reaction sample were analyzed
by gas chromatography (Varience 3800)
equipped with a capillary column (Agilent,
Scheme 1. Ketalization reaction of HP-Innowax, 0.32 mm diameter, 0.25 µm
glycerol with acetone over acid catalysts film thickness and 30 m length,) and flame
ionization detector (FID) were used. Helium
2. Materials and Methods was used as a carrier gas at a pressure of 10
2.1 Materials psig. The injector and detector temperature
were set at 230 °C and 260 °C, respectively.
The oven temperature was maintained at 40 remaining constant. That mean this reaction
°C for 4 min, 150 °C for 2 min, 220 °C for 2 is reversible as shown in Figure 3.
min and then increased to 250 °C for 2 min
at a ramp rate 20 °C/min of all step
increased.
Toluene was used as an internal
standard and it mixed with methanol to
prepare a stock solution. The sample was
weighted accuracy after that add an internal
standard stock solution to the sample. About
1 µL of the final sample was injected into
the column. The glycerol conversion and
selectivity to solketal were calculated by
using the internal standardization method. Figure 3. Concentration of glycerol and
All the products were confirmed by gas acetone at 60 °C, mole ratio of glycerol to
chromatography using the product standard acetone 1:1
to identify the peaks and peak area at
different retention times and calculated to The reaction product were analyzed
the correction factor of the compound. The by gas chromatography. Figure 4 presented
experimental error was checked by repeating a gas chromatogram of the product sample
the experiment 3 times. were contained solketal, 5-ol (by-product)
and no side reaction occurred. The result
showed that the amount of product occurred
corresponding to amount of glycerol and
acetone reactants were used.
Figure 4. Gas chromatogram of solketal in

a product sample
Figure 2. The glass batch reactor set up 3.2 Effect of molar ratio of acetone to
for the experiment in ketalization reaction glycerol
The molar ratio of acetone to
3. Results and Discussion glycerol is the one most important
3.1 Ketalization reaction using Al2(SO4)3 parameters affecting to ketalization reaction.
The ketalization of glycerol with All experiments were conducted at a
acetone by using Al2(SO4)3 as catalyst can reaction temperature of 60 °C with 3%wt
be described similar to a heterogeneous Al2(SO4)3 reference to amount of glycerol
Bronsted acid catalyst process.4 When the and mole ratio of glycerol to acetone in 1:1
reaction proceeds to a certain point, the to 1:4 was investigated.
concentration of glycerol and acetone were
The formation of solketal in different However, the increasing reaction
ratios of as a function of time as shown in temperature affected to increased initial rate
Figure 5. The result shown that the excess of of the ketalization reaction as expected.
acetone, glycerol conversion increased and
drastically increases the solketal formation Table 2. Result of glycerol conversion and
from 66.8% to 83.81% at mole ratio of solketal yield at equilibrium in difference
glycerol to actone1:1 to 1:4, respectively as reaction temperatures
presented in the table 1. Solketal was Temperature % Glycerol %Solketal
increased due to drive the reaction forward. (°C) conversion yield
However, the amount of acetone had no Room Temp. 87.23 88.14
40 82.87 85.81
effect on the reaction rate.5
50 79.52 82.75
60 77.87 81.66
4. Conclusion
Aluminum sulfate can be used as a
heterogeneous catalyst in the ketalization
reaction of glycerol with acetone. The
parameters that affected to the reaction such
as molar ratio of glycerol/acetone and
reaction temperature. Increasing the molar
ratio of glycerol/acetone results in the
Figure 5. Effect of the molar ratio of solketal formation was increased and drives
glycerol to acetone on solketal the reaction forward. The reaction
concentration. Molar ratio of acetone to temperature was increased affected to
glycerol 1:1 to 1:4, Temperature 60 °C, increases the initial rate of reaction.
Amount of Al2(SO4)3: 3%wt.
Table 1. Result of glycerol conversion and Acknowledgements
solketal yield at equilibrium in difference The author gratefully acknowledges
molar ratios the financial support provided by
% Glycerol %Solketal
Department of Chemical Engineering,
GLY:AC Faculty of Engineering, Chulalongkorn
conversion yield
1:1 74.80 66.80 University and also thankful to Professor.
1:2 77.87 81.66 Jirdsak Tscheikuna for invaluable all
1:4 78.84 83.81 suggestions aspects of this research.
3.3 Effect of reaction temperature References

Reaction temperature was affected 1. Quispe, C. A. G.; Coronado, C. J. R.;
stronkly to the reaction rate. The impact of Carvalho Jr, J. A. Renew. Sust. Energ.
reaction temperature on the conversion of Rev. 2013, 27, 475–493.
glycerol and solketal yield was investigated 2. Bagheri, S.; Julkapli, N. M.; Yehye, W.
from reaction at room temperature to 60°C A. Renew. Sust. Energ. Rev. 2015, 41,
at the constant mole ratio of glycerol to 113–127.
acetone at 1:2 and 3%wt of Al2(SO4)3 3. Perosa, A.; Moraschini, A.; Selva, M.;
referred to amount of glycerol. Noe, M. Molecules 2016, 21 (2); (b)
The resulted showed that the Calvino-Casilda, V.; Stawicka, K.;
increasing reaction temperature, the glycerol Trejda, M.; Ziolek, M.; Bañares, M. A.
conversion and solketal yield were J. Phys. Chem. C 2014, 118 (20),
decreased due to the higher temperature was 10780–10791.
led the reaction to a lower product yield at 4. da Silva, M. J.; Julio, A. A.; Dorigetto,
equilibrium,6 as presented in the Table 2. F. C. S. RSC Adv. 2015, 5 (55), 44499–
44506. 6. Nanda, M. R.; Yuan, Z.; Qin, W.;
5. Khayoon, M. S.; Hameed, B. H. Appl. Ghaziaskar, H. S.; Poirier, M.-A.; Xu, C.
Catal., A 2013, 464-465, 191–199. C. Fuel 2014, 117, 470–477.
Preparation of silk fibroin/chitosan film and its ability to remove
Rhodamine B dye in aqueous solution
Peeravat Tanpadid1, Nareerat Kittisripanya1, Thanonchat Imsombat2, Rapeepan Mahem1,
Tianchai Panpojan1, Sittichai Prapan1, Ruethaithip Wisedsri1*
1
Kasetsart University, Chalermprakiat Sakhon Nakhon Province Campus, Sakon Nakhon 47000, Thailand
2
Department of Chemistry, Faculty of Science and Technology,
Rajabhat Mahasarakham University, Mahasarkham 44000, Thailand
*E-mail: ruethaithip.w@ku.th
Abstract:
Water pollution is one of the major concerns recently because it creats serious
problems for water resources, thus a number of technologies have been explored to
decolorize the dye molecules in wastewater. In practical application, physical absorption is
one of the most simply, effective and economic methods for dye removal in wastewater.
In this work, silk fibroin derived from silk cocoon waste was blended with chitosan from
shrimp shell. The silk fibroin/chitosan films were prepared and then their adsorption potential
was evaluated for removal of Rhodamine B dye in aqueous solution. The surface morphology
of blend film was investigated using scanning electron microscopy (SEM) and it was found
that the cross section image of blend film showed the homogeneity of film. The interactions
between two components in the blend were determined by FTIR spectroscopy. There are the
amide band shifts suggesting that the interaction between silk fibroin and chitosan which
probably due to the interaction of hydrogen bonding. The swelling test of composite film was
carried out. Adsorption conditions such as chitosan content in the composite, contact time,
and pH were evaluated. The results show that silk fibroin/chitosan films have ability to
remove dyes in wastewater.
1. Introduction
Dyes contaminant in wastewater is its highly amino groups to chelate with
an environmental problem due to its high metal ion.2,3 Moreover, the presence of
toxic impact. Even in low concentrations, hydroxyl and amino groups in the chitosan
the dyes can affect the aquatic life and food qualifies it for adsorption of cationic and
chain. Thus, various treatment techniques anionic dyes. However, the chitosan film has
such as biodegradation, chemical oxidation, poor mechanical resistance which is
and adsorption have been used for removing important for the membrane adsorbent.
dyes from aqueous solution. Among this Many attempts have been carried out
numerous techniques, adsorption is one of to overcome this limitation. Blending
the most simply, effective and economic chitosan with others biomaterial is preferred
methods for dye removal in wastewater. because of its simplicity. Recently, blending
Adsorption based on biosorbent has received 30 wt% of silk fibroin in chitosan film
remarkable high attention because the increases the mechanical strength of the film
natural sorbent are abundant, eco-friendly due to its maximum crystallinity and
and inexpensive especially, biosorbent density.4 Thus in this work, chitosan/silk
produced from agriculture waste.1 Chitosan fibroin films were prepared and used as an
is one of the most plentiful and low cost adsorbent to remove Rhodamine B dye from
biopolymer. It is an effective adsorbent for aqueous solution. The investigation was
uptaking both of dye and heavy metals due to carried out through a series of batch
adsorption experiment. The effect of prepared and serial dilution was made to
chitosan content, pH, and contact time was obtain other lower concentrations required.
determined. The pH of the dye solution was adjusted by
adding 0.1 M HCl or 0.1 M NaOH. Samples
2. Materials and Methods of 10 mg of the chitosan/silk film were then
2.1 Materials suspended in 10 ml dye solution at
Bombyx merix silk cocoon from 20 mg L−1 and were maintained under
agricultural waste was received from Queen constant stirring speed of 150 rpm at
Sirikit Sericulture Center (Sakhonnakhon). 303 ± 2 K. The residual dye concentration
The adsorbate Rhodamine B, with molecular (Cf) was determined at 555 nm in a Cecil
weight of 479.02 g mol−1, was procured UV–vis spectrometer (Cecil-2000 model).
from Fluka. Chitosan (shrimp chitosan) The removal efficiency of dye (R) was
(COM grade, degree of deacetylation: calculated by Eq. (1).
92.13%) was received from Ta Ming
enterprises Co., Ltd. Thailand.
2.2 Silk fibroin solution preparation Eq. (1)
Silk cocoon from agricultural waste
were cut into small pieces and degummed in The amount of adsorbed dye (qt) was
aqueous solution of Na2CO3 (0.5% w/v) at calculated by Eq. (2),
80 oC for 30 min. Degummed silk fibroin
were washed with water and dried at 80 °C
for 24 h. The degummed silk was dissolved
Eq. (2)
in CaCl2/H2O/CH3CH2OH solution (molar
ratio: 1:8:2) at 80 °C. And the fibroin
where Ci and Cf are the initial and final dye
solution was dialyzed against distilled water
concentrations after adsorption equilibrium
for 3 days to yield a fibroin aqueous
(mg L−1), V is the solution volume (L) and
solution. The final fibroin concentration was
m is the mass of the solid (g).
around 5%, which was determined by
weighing the remaining solid after drying.
2.3 Chitosan/silk fibroin film
Chitosan solution was prepared by 3.1 Chemical analysis of film
IR spectroscopy is commonly used
dissolving 2 wt % of chitosan in 0.5 M
to investigate the conformation of silk
acetic acid. An equal volume of the same
fibroin and its blend because of the
concentration of silk fibroin was added to
absorption band sensitive to the molecular
prepare a mixture of silk fibroin and
conformation of silk fibroin. The structure
chitosan ratio (w/w) of 80:20, 40:60, 50:50,
of silk fibroin/chitosan film was analyzed
60:40 and 20:80, respectively. The mixture
with ATR-FTIR. As seen in Figure 1, silk
was homogenized using a homogenizer
fibroin film shows the absorption peak at
(1 h). And 15 g of mixture solution was
1617 cm-1 which assigns to Amide I bond
poured in a polystyrene petri dish and kept
and 1541 cm-1 refers to Amide II bond
at 40 oC for 2 days. The film was cut into the
which were attributed to the β-sheet
spherical shape with a diameter of 1.4 cm
conformation of silk fibroin and probably
and the sample weight was around 10 mg.
due to the interaction of hydrogen bonding
2.4 Batch adsorption experiments
between chitosan and silk fibroin.5 On the
The batch adsorptions of Rhodamine
other hand, the chitosan film showed
B dye onto chitosan/silk fibroin film
absorption bands at 1643 and 1542 cm-1
experiences were conducted by varying the
assigned to the amino group of chitosan and
operation parameters as chitosan content, pH
the amide group of chitin. And the band at
and contact time. The stock solution of
1154 and 900 cm-1 attributed to the
Rhodamine B dye (1000 mg/L) was
saccharide.6 The shift of band at 1617 cm-1
Figure 1. ATR-FTIR spectra of silk fibroin Figure 2. Thermogram of silk fibroin (SF),
(SF), Chitosan (CS) and the blend films of Chitosan (CS) and the blend films of SF/CS
CS/SF with blend ratio; a) 0/100 (pure SF) with blend ratio; a) 0/100 (pure SF)
b) 20:80, c) 40:60, d) 50:50, e) 60:40, b) 20:80, c) 40:60, d) 50:50, e) 60:40,
f) 80:20 and g) 100/0 (pure CS) f) 80:20 and g) 100/0 (pure CS)
and 1229 cm-1 leads to the conformation

change of silk fibroin.
3.3 Thermal properties
Thermal properties of the samples
were evaluated using differential scanning
calorimetric (DSC) and thermogravimetric
analysis (TGA). Figure 2 shows the
thermogram obtained from the CS/SF film at
different blending ratio. All of thermogram
exhibits endothermic peak around 100 oC
associated with the evaporation of bound
water. Silk fibroin film shows a degradation
peak around 300 oC while chitosan film
exhibits the degradation temperature around
290 oC. Increasing amount of silk fibroin
the blend film, the film has higher thermal
stability probably due to its increasing
crystallinity.7-9 Thermogravimetric curves of
CS/SF film are shows in Figure 3. The
initial weight losses at approximately 100 oC
was due to the evaporation of water.
In Figure 3a) the maximum
decomposition temperature of pure chitosan
appeared at 289 oC which is the Figure 3. Thermogram of a) silk fibroin
dehydration of the saccharide rings and (SF) and Chitosan (CS), b) The blend films
decomposition of the acetylated and of CS/SF with blend ratio; 20:80, 40:60,
deacetylated units of polymer. On the other 50:50, 60:40 and 20:80
hand, the pure silk fibroin film shows higher
decomposition temperature at 315 oC. And composition of blend film. Increasing the
in Figure 3b), the maximum temperature of amount of silk fibroin, the decomposition
decomposition shifted depending on the temperature increased.
3.2 Morphology of the blend films
The surface morphology of the silk Eq. (3)
fibroin/chitosan blend films was observed where Ws and Wd correspond to the mass of
with scanning electron microscopy. The swollen hydrogel and dry film, respectively.
Figure 4 shows the fractured surface of As seen the results in Figure 5, the chitosan
blend film at different blend ratio. The film has highest degree of swelling and it
results show that the silk fibroin/chitosan decrease when increasing the amount of silk
films did not show microscopic phase fibroin.
separation morphology. The cross section
image of the blend film showed the
homogeneity of film and the thickness of the
film is around 45 m.
Figure 5. Degree of swelling of silk

fibroin/chitosan film at different blend ratios
3.5 Effect of contact time

The effect of contact time on
adsorption is shown in Figure 6. The results
show that the blend films take 6h to reach
equilibrium. For unmodified chitosan, the
film was broken after 6h thus the adsorption
capacity is quite near zero and the low
mechanical strength is the major
disadvantages of chitosan. Blending silk
fibroin in chitosan leads to increase
mechanical strength of the film thus the
films has high ability to keep its shape.
3.6 The effect of pH
Figure 4. SEM images of a cross section of The pH of the solution influences the
the blend film at different ratios; a) pure CS, adsorption process, thus pH effect on the
b) pure SF, c) 80:20, d) 20:80, e) 60:40, f) uptake of Rhodamine B onto chitosan/silk
40:60 and g) 50:50. film was determined and the highest
percentage depends on ratio of blend film
3.4 Swelling test between chitosan and silk fibroin (Figure 7).
The blend films were weighed and
then immersed in deionized water at room 4. Conclusion
temperature. After 15 min, the films were The chitosan/silk blend films were
removed, placed on filter paper for prepared and found that silk fibroin induced
absorbing the excess of solution and the crystallinity of the film so the film has
weighed. The degree of swelling was higher mechanical strength. Increasing the
calculated according to the following amount of silk fibroin in the blend film,
equation; decomposition temperature also increased
while the percent of swelling decreased.
From the batch adsorption test, the blend
films it has ability to adsorb Rhodamine B
dye. The optimum ratio of blend film is
CS/SF 40:60. The equilibrium time it equal
to 6h. And the optimum pH is 7.
Acknowledgements
The authors are thankful to the Faculty
of Science and Engineering, Kasetsart
University Chalermprakiat Sakon Nakhon
Province Campus for providing facilities
support to carry out this work.
References
1. Jain, S. N.; Gogate, P. R. J. Environ.
Manage. 2018, 210, 226−238.
2. Gokila, S.; Gomathi, T.; Sudha, P. N.;
Anil, S. Int. J. Biol. Macromol. 2017,
104, 1459–1468.
3. Karim, Z.; Mathew, A. P.; Grahn, M.;
Mouzon, J.; Oksma, K. Carbohydr.
Polym. 2014, 112, 668–676.
Figure 6. Effect of contact time on removal 4. Park, S. J.; Lee, K. Y.; Ha, W. S.; Park,
of Rhodamine B dye (dye concentration: 20 S. Y. J. Appl. Polym. Sci. 1999, 74,
ppm, chitosan/silk blend: 1g/L, volume of 2571−2575.
sample: 10 mL, pH: 7, temperature: 30 °C) 5. Jaramillo-Quiceno, N.; Álvarez-López,
C.; Restrepo-Osorio, A. Procedia
Engineering, 2017, 200, 384–388.
6. Sionkowska, A.; Płanecka, A. J. Mol.
Liq. 2013, 178, 5–14.
7. Chen, X.; Li, W.; Yu, T. J. Polym. Sci.
B. 1997, 35, 2293–2296.
8. Kweon, H.; HA, H. C.; Um, I. C.; Park,
Y. H. J. Appl. Polym. Sci. 2001, 80,
928–934.
9. Inyinbor, A. A.; Adekola, F. A.;
Olatunji, G. A. Appl. Water Sci. 2017, 7,
2297−2307.
Figure 7. Effect of pH on removal of
Rhodamine B dye (dye concentration: 20
ppm, chitosan/silk blend: 1g/L, volume of
sample: 10ml, temperature: 30 °C, contact
time: 6 h)
The relationship between free radical scavenging activities and germination
time of peanut (Arachis hypogaea L.) extracts
Kwanyuen Leamsamrong*, Wathida Thornpho, Butsayamat Rattanadon,
Sarin Thongthummachat
Department of Chemistry, Faculty of Science and Technology, Rajabhat Maha Sarakham University,
Maha Sarakham 44000, Thailand
*E-mail: leamsamrong.kwan@gmail.com
Abstract:
Arachis hypogeae L. (peanut) has various essential properties such as type II diabetes,
cardiovascular and gastrointestinal cancer. The free radical antioxidant activities of the
peanut sprout extract in different cultivation times were determined. Seed of peanut was
germinated at room temperature for seeding, 12 h of soaking, 12, 24, 48, 72, 96, 144 and 168
h after cultivation. The peanut sprouts were extracted by using ethanol. The free radical
scavenging activities were investigated by using DPPH (2,2-diphenyl-1-picrylhydrazyl) and
ABTS+ (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). The ethanolic extract of
peanut sprouts showed the highest DPPH radical scavenging activity at 168 h after cultivation
(106.018±0.38 mg AEAC/100 g DW (dry weight) and 155.07±0.55 mg TEAC/100 g DW and
for ABTS assay showed the highest radical activity at 96 h after cultivation (234.73±0.47 mg
AEAC/100 g DW and 134.85±0.27 mg TEAC/100 g DW). A significant positive correlation,
R2=0.749 and R2=0.711 were observed between germination times and DPPH and ABTS
radical scavenging activities, respectively. The cultivation times affected on free radical
scavenging of peanut.

Peanut (Arachis hypogeae L.) is one 2.1 Chemical and reagent
important legume, which containing the Peanut seeds were obtained from
various nutrition such as protein, unsaturated local market at Muang Maha Sarakham,
fatty acid and phytochemical compound with Maha Sarakham province, Thailand. 6-
related the human health. Peanut is enrich of Hydroxy-2,5,7,8-tetramethylchromane-2-
the phenolic compound for example carboxylic acid, 2,2- azino-bis (3-
resveratrol that show the biological ethylbenzothiazo-line-6-sulphonicacid),2,
properties such as antioxidant activities, anti- 2-diphenyl-1-picrylhydrazyl, gallic acid,
inflammatory and cytoprotective effect.1,2 ascorbic acid were purchased from Sigma
The previous study was reported that the (St. Louis, MO, USA). Ethanol was
germination time of several legume sprout purchased from BDH (UK). The chemicals
can increase antioxidant activity and were used of analytical grade.
phenolic content than ungerminated 2.2 Seed germination
legumes.3-6 In this experimental, the free Peanut seed were soaked in tap water
radical scavenging activities (DPPH, ABTS at room temperature for 16 h. The soaked
and FRAP), total polyphenol (TPC) and peanut seeds were washed and rinsed. Peanut
flavonoid (TFC) contents during 12-168 h seed were germinated in dark with
germination process were determined. humidified gunnysack. The moisture of
Additionally, the relationship between gunnysack was kept by spraying with tap
germination times, antioxidant capacity, TPC water. The seed were harvested at 12, 24, 48,
and TFC were evaluated. 72, 96, 144 and 168 h during the germination
process. Sprout of peanut were dried at 60 C
in hot air oven, gentle milled, sealed in
© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) FA1
aluminum foil bag to protect moisture Trolox and ascorbic acid were used as
absorption and stored in a freezer at -20 C standard solution.
until analysis. The peanut sprout at 120 h 2.4.3 Total polyphenol contents (TPC)
was shown in Figure 1. TPC was determined according to the
method of Singleton et al.9 Briefly, 100 µL
of kernel extracts were mixed with 500 µL of
0.2 N Folic-ciocalteu reagent and 400 µL of
7.5% w/v Na2CO3 and stored at room
temperature for 30 min until the reaction
completed and the absorbance at 765 nm was
measured. The results were expressed in
terms of gallic acid equivalent antioxidant
activity (GE) in mg gallic acid per dry
weight.
2.4.4 Total flavonoid contents (TFC)
TFC was determined according to the
method of Chang et al.10 Briefly, 100 µL of
Figure 1. The peanut sprout at 120 h kernel extracts were mixed with 500 µL of
5% w/v NaNO2 and 400 µL of 10% w/v
2.3 Extraction of peanut AlCl3 and then stored at room temperature
All samples were extracted with for 15 min. The absorbance at 415 nm was
ethanol by using a shaker at 200 rpm for 3 h. measured. The results were expressed in
The solvents extract were separated and terms of quercetin equivalent (QE) in mg
filtered through a Whatman filter paper quercetin per dry weight.
No.1. The solid residue was re-extracted 2.4.5 FRAP method
twice and pooled. The solvents were Feric reducing antioxidant power
(FRAP) was the redox method. The peanut
removed by a rotary evaporator at 45 C and
sprout extracts was investigated according to
stored at -20 C until further analysis.
the method of Benzie and Strain.11 Briefly,
2.4 Antioxidant activities
100 µL of extracts were mixed with 900 µL
2.4.1 DPPH method
of FRAP reagent and incubated in water bath
The DPPH was electron transfer
4
assay. Antioxidant activity of peanut sprout at 37C for 15 min. The absorbance at 595
nm was measured. The results were
extracts was determined according to the
method of Xu et al.7 Briefly, 100 µL of expressed in terms of trolox equivalent
different concentrations of peanut sprout antioxidant capacity (TE) in mg trolox per
dry weight.
extracts were mixed with 900 µL of 0.1 mM
DPPH solution and stored in dark for 30 min.
The absorbance at 515 nm was measured. 3. Results and Discussion
Trolox and ascorbic acid were used as 3.1 DPPH and ABTS
The peanut sprout was germinated for
standard solution.
0-168 h. Free radical scavenging activity of
2.4.2 ABTS method
peanut sprout extracts of germination period
The scavenging activity of peanut
were significantly increased more than
sprout extracts was determined according to
the method of Long and Halliwell.8 Briefly, ungerminated peanut and revealed that the
highest DPPH radical scavenging activity at
100 µL of different concentrations of kernel
168 h after germination were 106.01 mg
extracts were mixed with 900 µL of ABTS+
AEAC per 100 g DW and 155.07 mg TEAC
solution (0.7 mM of ABTS mixed with 2.45
per 100 g DW. The peanut sprout extract at
mM of K2S2O8) and stored in dark for 6 min.
168 h had antioxidant activity that was 2-fold
The absorbance at 734 nm was measured.
higher than peanut seed.
For ABTS assay was significantly wheat.13 Significant result between TPC and
increased the maximum free radical activity ABTS of correlated (ABTS; r2=0.887 and
at 96 h after germination were 34.73±0.47 FRAP; peanut
mg AEAC per 100 g DW and 134.85±0.27
mg TEAC per 100 g DW. The result was
shown in Figure 2 and Figure 3, respectively,
for DPPH and ABTS method. The peanut
sprout extract at 96 h had antioxidant activity
that was 2-fold higher than peanut seed.
Figure 2. ABTS method of peanut sprouts
Table 1. TPC, TFC and FRAP method of

peanut sprout in difference germination time
(GT)
GT TPC TFC FRAP
(h) (mg GE/ g DW) (mg QE/g DW) (mg TE/g DW)
Seed 1.266 ± 0.00c 18.851 ± 0.13c 0.421 ± 0.15d
Figure 1. DPPH method of peanut sprouts Soak
0.963 ± 0.00b 19.502 ± 0.13c 0.201 ± 0.11b
12
12 0.793 ± 0.00a 14.176 ± 0.03b 0.111 ± 0.00a
3.2 TPC, TFC and FRAP d c
24 1.621 ± 0.00 16.318 ± 0.09 0.303 ± 0.12c
TPC, TFC and FRAP assay was e d
48 1.714 ± 0.00 21.342 ± 0.11 0.579 ± 0.10h
significantly increased the maximum free
72 1.823 ± 0.00f 20.900 ± 0.28d 0.512 ± 0.10e
radical activity at 96 h after germination were g f
96 2.894 ± 0.00 31.550 ± 0.54 0.624 ± 0.04i
2.894 mg GE/g DW, 31.550 mg QE/g DW and
120 2.615 ± 0.00h 9.808 ± 0.40a 0.559 ± 0.00g
0.624 mg TE/g DW, respectively. The results i c
144 2.317 ± 0.01 21.960 ± 0.07 0.512 ± 0.04e
were shown in Table 1. TPC of the peanut
168 2.242 ± 0.01j 19.781 ± 0.28c 0.521 ± 0.00f
sprout extract at 96 h was 2-fold higher than
Values are mean ± SD for n=3; identical superscripts in column
soaked seed at 12 h and 3-fold than germinated denote no significant (p < 0.05) difference between mean values
at 12 h. according independent t-test.
3.3 Correlation between germination time
(GT), antioxidant activities, TPC and TFC sprout extracts was observed (r2=0.887). TPC
Table 2 exhibits the strong correlation and antioxidant were good r2=0.887). No
among germination time and antioxidant correlation was found between germination
activities. Germination time was significantly time and TFC. Drogoudi et al14 found a strong
correlated with DPPH (r2=0.749), ABTS correlation between total phenolics and
(r2=0.803) and FRAP (r2=0.722). Similar results antioxidant activities of apricot fruit,14 peaches
have been reported in kefir,5 legumes,12 Chinese and plum15 whereas the opposite result was
reported by Scalzo et al.17 No correlation was 3. Fidrianny, I.; Puspitasari, N.; Singgih W.
found between antioxidant activity (DPPH M. Asian J. Pharm. Clin. Res. 2014,
method) and TPC. 4216.
4. Huang, X.; Cai, W.; Xu, B. Food Chem.
Table 2. Pearson’s correlation coefficient of 2014, 143, 268276.
germination time, antioxidant activities, TPC 5. Gunenc, A.; Yeung, M. H.; Lavergne, C.;
and TFC Bertinato, J.; Hosseinian, F. J. Funct.
Peanut DPPH ABTS FRAP TPC TFC Foods 2017, 32 (2), 7279.
GT 0.749** 0.711** 0.722** 0.803** 0.158
6. Khang, D. T.; Dung, T. N.; Elzaawely,
DPPH 1 0.540 0.503 0.544 0.04
ABTS - 1 0.419 0.887** 0.432
A. A.; Xuan, T. D. Foods 2016, 5,
FRAP - - 1 0.887** 0.432 2730.
TPC - - - 1 0.368 7. Xu, J. ; Yang, F. ; Chen, L. ; Hu, Y. ; Hu,
TFC - - - - 1 Q. J. Agric. Food Chem. 2003, 51 (4),
10811084.
4. Conclusion 8. Long, L. H.; Halliwell, B. Methods
The highest antioxidant activity, total Enzymol. 2001, 335, 181190.
polyphenol and flavonoid content of peanut 9. Singleton, V. L.; Orthofer, R.; Lamuela-
sprout extract were observed during 96 h of Raventós, R. M. Methods Enzymol. 1999,
germination period. From these data, the 299, 152178.
germination time was significantly increased 10. Chang, C. C.; Yang, M. H.; Wen, H. M.;
antioxidant activities and phenolic Chern, J. C. J. Food Drug Anal. 2002, 10
compound. Therefore, peanut sprout extract (3), 178182.
may be suggested as an antioxidant natural 11. Benzie, I. F. F.; Strain, J. J. Anal.
source to promote supplementary food.
Biochem. 1996, 239 (1), 7076.
12. Saleh, H. M.; Hassan, A. A.; Mansour, E.
Acknowledgements H.; Fahmy, H. A.; El-Bedawey, A. E.-F.
The financial was supported by
A. J. Saudi Soc.Agric. Sci. 2017 in press.
Research and Development Institute,
13. Chen, Z.; Wang, P.; Weng, Y.; Ma, Y.;
Rajabhat Maha Sarakham University and
Gu, Z.; Yang, R. Food Bio. 2017, 20,
Department of Chemistry, Faculty of Science
159167.
and Technology, Rajabhat Maha Sarakham
14. Drogoudi, P. D.; Vemmos, S.; Pantelidis,
University for partially supporting this
G.; Petri, E.; Tzoutzoukou, C.;
research.
Karayiannis, I. J. Agric. Food Chem.
2008, 56 (22), 1075410760.
References
1. Limmongkon, A.; Nopprang, P.; 15. Leccese, A; Viti, R.: Barolini, S. Cent.
Chaikeandee, P.; Somboon, T.; Eur. J. Biol. 2011, 6, 199204.
Wongshaya, P.; Pilaisangsuree, V. Food 16. Gil, Ml.; Tomas-Bareran, F. A.; Hess-
Chem. 2018, 239, 569578. Pierce, B.; Kadar, A. A. J. Agric. Food
2. Malhotra, A.; Bath, S.; Elbarbry, F. Oxid. Chem. 2002, 50, 49764982.
17. Scalzo, J.; Politi, A.; Pellegrini, N.;
Med. Cell.3 Longev. 2015, 1530.
Mezzetti, B.; Battino, M. Nutrition 2005,
21, 207213.
Effect of infrared drying on phytochemical content, antioxidant activity
and physicochemical properties of germinated brown rice
Hareesah Kohing, Jutarat Tasara, Weeraya Khummueng*
Department of Science, Faculty of Science and Technology,
Prince of Songkla University, Pattani Campus 94000, Thailand
*E-mail: weeraya.k@psu.ac.th
Abstract:
Infrared drying (IR) is well known as an effective drying method due to high heat and
mass transfer rates and economical cost for structure equipment. This drying method is
attractively considered because it can reduce the drying time and keep the rice quality. In this
study, the effect of infrared drying at different power (1000 and 1500 W) and temperature
(60, 75 and 95 C) on phytochemical content, antioxidant activity and physicochemical
properties of Hawm-Kradang-Ngah (HK) and Seebukabntang (SB) germinated brown rice
was investigated. HK and SB are famous local rice varieties which widely cultivar in the
south of Thailand, especially, in Narathiwat province. HK is a medium grain, red-brown
color with moderate amylose content while SB is a short grain, non-color and high amylose
content. The results showed that drying temperatures did not significantly affect the total
phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity when
compared with control rice. Additionally, the results of quality analysis showed that cooking
time and water absorption of IR drying rice were significantly different as compared with the
control rice. These studied results provided the new choice for germinated brown rice
production and applications.
1. Introduction different from convective drying because the

Germinated brown rice (GBR) is a material is dried directly by absorption of IR
functional rice product which has high energy rather than transfer of heat from the
content of phytochemicals compared to air.4 Infrared irradiation technique is easy
brown rice. During germination, significant installation and low cost for construction.5,6
changes in biochemical, nutritional and Employing the IR drying method have been
sensory were observed. known to reduce the drying time and
The major drawback in germinated energy.6,7 Therefore, the objectives of this
brown rice (GBR) is high moisture content, research was aimed to investigate the effect
which leads to infection by microorganisms, of IR drying on phytochemical content,
yellowing by non-enzymatic reaction and antioxidant activity and physicochemical
undesirable odors. To maintain quality of properties of local rice in southern Thailand,
germinated brown rice extensively, the especially, Hawm-Kradang-Ngah (HK) and
moisture content must be reduced.1,2 Seebu-kabntang (SB) rice.
Therefore, drying is an important process of
the germinated brown rice production. 2. Materials and Methods
Several, drying methods for moisture content 2.1 Materials and reagents
reduction in GBR,3 such as sun drying, hot HK and SB paddy varieties were
air drying and microwave drying etc., provided by the Rice Research Center in
However, one of the high efficient drying Pattani province, Thailand. All chemicals
methods that provide high heat and mass used in the experiment are listed below:
transfer is electromagnetic irradiation ethanol, folin–ciocalteu reagent, sodium
method, especially, infrared radiation ( IR) carbonate (Na2CO3), gallic acid, sodium
drying method. IR drying is significantly nitrite (NaNO2), aluminium chloride (AlCl3),
sodium hydroxide (NaOH), catechin, 2,2- 2.4 Determination of total phenolic
diphenyl-1-picrylhydrazyl (DPPH), 2,2'- content (TPC)
azino-bis-3-ethylbenz-thiazoline-6-sulphonic Total phenolic content were deter-
acid (ABTS), potassium peroxydisulfate mined using the Folin-Ciocalteu colori-
(K2S2O8). metric method.9 Briefly, 200 L of extract
2.2 Rice samples preparation solution was mixed with 800 L of 10%
Paddy was cleaned and soaked in Folin–Ciocalteu reagent. The mixture was
water at room temperature for 24 h and shaken for 1 min, then 2 mL of 7.5% (w/v)
incubated for 24 h. The paddy was steamed Na2CO3 solution was added, and the mixture
at temperature of 100±2 °C for 30 min. was shaken once again for 30 s. The final
Then, it was stored in a confined space for mixture was made up to 5 mL with deionized
30 min before drying with infrared (IR) at water and then allowed to stand for 2 h at
different powers ( 1,000 and 1,500 W) and ambient temperature. The absorbance of the
temperatures (60, 75 and 95 °C). The mixture was measured at 760 nm. Gallic acid
average initial moisture content of the solution was used as standard.
samples was in the range of 542% dry 2.5 Determination of total flavonoid
basis. Paddy sample was dried until it content (TFC)
reached the final moisture content at 221% Total flavonoid content were
dry basis. The samples were taken out the analyzed by the method modified by
dryer and were then ventilated by aeration Basker.10 Briefly, 0.5 mL of extract solution
until the moisture content of the paddy about was mixed with 3 mL of deionized water and
161% dry basis for prolonged shelf life.10 0.3 mL of 5% NaNO2 then incubated for 5
The paddy was husked by hulling machine min. After adding 0.6 mL of 10% AlCl3 and
into the GBR. allowing 5 min of reaction, 2 mL of 1 M
2.3 Rice samples extraction NaOH was added. The absorbance was
1.00 g of rice samples were extracted measured at 510 nm. Catechin solution was
with 10 mL of 70% aqueous ethanol at 25 °C used as standard.
in a shaking incubator at 150 rpm for 16 h. 2.6 DPPH radical scavenging analysis
The mixtures were centrifuged at 2,500 rpm The DPPH radical scavenging
for 20 min and the supernatants were activity was determined according to the
collected.The residues were re-extracted method by Iqbal.11 Briefly, 1 mL of extract
under the same conditions, and supernatants solution was mixed with 3 mL of 0. 1 mM
from both extractions were combined. 9 DPPH. After incubating for 30 min in dark,
*the hot air blower was turn off when the IR drying mode was operated
Figure 1. Infrared dryer8
the absorbance was measured at the The increased weight was calculated using
wavelength of 517 nm. DPPH radical the following equation:13
scavenging activity (%) was calculated using
the following equation: Water absorption = Weight of cooked rice/Weight of
raw rice
Scavenging activity (%) = [1-(Asample/Ablank)] 100
2.7 ABTS cation radical scavenging assay 3.1 Drying kinetics of germinated paddy
The ABTS cation radical Moisture ratio (MR) results of
scavenging activity was determined germinated paddy drying using IR at
according to the method by Shen.12 The difference temperatures (60, 75 and 95 °C)
ABTS.+ solution was produced by mixing and powers (1,000-1,500 W) were shown in
7 mM ABTS stock solution and 2.45 mM Figure 2. Using a higher drying temperature
K2S2O8 ( v/ v = 2: 1) in dark at room can decrease the MR, which results the
temperature 12-16 h. The working ABTS.+ shorter drying time. Hence, the condition of
solution was diluted by ethanol to adjust the IR drying for two rice varieties should be
absorbance to 0. 700 ± 0. 020 at the performed at 95 °C and 1,500 W to obtain
wavelength of 734 nm. The extracts or the shorter drying time. The obtained MR
standards (100 L) were mixed with 3.9 mL from the drying of germinated paddy at
working ABTS.+ solution thoroughly on a different temperatures and powers were
vortex. After reacting for about 6 min, the fitted with drying equation as shown in Table
absorbance was tested at 734 nm. ABTS 1. Accuracy of models was evaluated based
cation radical scavenging activity ( % ) was on R2 and RMSE values and the most
calculated using the same formula in section accurate model was selected with regard to
2.6. the highest of R2 and the lowest of RMSE.
2.8 Cooking properties HK and SB germinated paddy drying
2.8.1 Cooking time showed that the model proposed by
Rice sample (2 g) was boiled in 20 Approximation of diffusion, predicts the
mL of distilled water. After cooking for 10 drying process of HK and SB germinated
min, the 2-3 grains were removed from the paddy more accurately than other models.
water and placed over a Petri dish. Then, the 3.2 Phytochemical content (TPC and TFC)
grains were suddenly compressed with a The TPC contents in HK and SB of
spatula in order to visualize and count the all rice products were shown in Figure 3.
grains which do not have the opaque area TPC of HK untreated rice (without
inside core. The same procedure was germination and drying) was higher
repeated every minute until all the grain compared to the other rice products. HK is
reached to complete gelatinization.13 red-brown colored rice which frequently
2.8.2 Water absorption found flavonoid compounds especially
2 g of rice samples were added to 20 proanthocyanidin higher than non-color rice.
mL of distilled water previously heated at 95 It is water soluble and not resistant to heat
°C in a test tube covered with cotton plug and light. Therefore, during the streaming of
and placed in a water bath. The rice samples germinated paddy, heat and the steep water
were cooked according the cooking time in a partially remove the soluble phenolic
water bath as previously determined. Then, compounds including flavonoids from the
samples were cooled in water and placed at grain.14
room temperature for 1 h. The samples were
weighed.
(a) (b)
(c) (d)
Figure 2. The change of MR and drying time during (a) HK germinated paddy drying
with 1,000 W, (b) HK germinated paddy drying with 1,500 W, (c) SB germinated paddy
drying with 1,000 W and (d) SB germinated paddy drying with 1,500 W
Table 1. Constant values and drying equation of germinated paddy drying

Hawm-Kradang-Ngah Seebukabntang
Model
Constant value R2 RMSE Constant value R2 RMSE
6
Newton k=-0.0241+(3.67*10 )P 0.9767 0.0312 -6
k=-0.0251+(1.49*10 )P 0.9780 0.0296
MR=exp(-kt) +0.0004T +0.0005T
Henderson and Pabis K=0.0251+(3.73*10-6)P 0.9793 0.0293 k=-0.0265+(1.49*10-6)P 0.9841 0.0251
MR=aexp(-kt) +(4.44*10-4)T, a=1.0194 +0.0005T, a=1.0281
Logarithmic K=0.2353+(-2.88*10-4)P 0.9594 0.0411 k=-1.458+(-1.63*10-4)P 0.9855 0.0239
MR=aexp(-kt) + b +(-0.0396)T, a=(-0.0003), +(-0.0433)T, a=(-0.0010),
c=0.9820 c=0.9988
Approximation of K=0.6901+(1.01*10-4)P 0.9910 0.0193 k=0.2542+(-1.35*10-5)P 0.9879 0.0219
diffusion +0.0122T, a=(-0.0318), +(-0.0048)T, a=(-0.0812),
MR=aexp(-kt) + (1- b=0.0373 b=0.1138
a)exp(-kbt)
Midilli et al. K=0.5554+(3.72*10-6)P 0.9794 0.0293 k=0.0261+(-1.25*10-6)P 0.9856 0.0239
MR=aexp(-k(tn)) + bt +0.0004T, a=1.022, +(-0.0005)T, a=1.0197,
b=0.5797, n=0.9997 b=(-0.0007), n=1.0068
On the other hand, TPC value of SB

after germinated and IR drying was
increased when compared with untreated
rice. Due to the activation of hydrolytic
enzyme by water to degrade starch, non-
starch polysaccharide and protein to small
bio-molecules such as monosaccharide,
peptide and amino acid during germination
process.15 For TFC of HK and SB, the results
were the same trend of TPC (not showed in
Figure). Additionally, the difference of
temperature and power of IR drying did not
Figure 3. Total phenolic content in HK-GBR significantly affect TPC and TFC contents of
and SB-GBR HK-GBR and SB-GBR. However, there are
many factors that affect TPC and TFC
contents of HK-GBR and SB-GBR such as (a)
varieties, cultivation area, growing
conditions and germination conditions
including extraction methods.
3.3 Antioxidant activity

Antioxidant activity was determined
using DPPH and ABTS assay, and their
results were reported as a percentage of
scavenging using the formula as shown in
section 2.6. The antioxidant activity (DPPH (b)
assay) on both rice varieties were shown in
Figure 4a. There were no significantly
different (p>0.05) of %DPPH scavenging of
both rice varieties when IR power at 1,000
and 1,500 W with different temperatures (60,
75 and 95 °C) were used. The ABTS assay
of antioxidant activity in two rice varieties is
shown in Figure 4b. %ABTS scavenging on
HK-GBR of all rice product decrease
significantly (p<0.05) when compare to Figure 4. Antioxidant activity in HK-GBR
untreated rice. Due to the major compounds and SB-GBR (a) DPPH radical scavenging
that play an important role for antioxidant in of rice samples and (b) ABTS radical
HK-GBR such as anthocyanin, scavenging of rice samples
proanthocyanidin, etc. was removed during
the streaming and steep water. While, Due to rice, physiopropeties were
%ABTS scavenging of SB-GBR was changed during streaming and steep water
increase significantly (p<0.05) when process causing to starch granules
compared to untreated rice. The trends of modification to stronger structure which is
antioxidant activity (DPPH and ABTS assay) difficult for water to penetrate into the
were generally like that of TPC and TFC kernel.16
results, due to the high correlation between
antioxidant activity and TPC and TFC. 4. Conclusion
3.4. Cooking properties The above finding on total phenolic
Cooking is associated with changes content (TPC) total flavonoids content (TFC)
in the chemical and physical properties of the antioxidant activity and cooking properties
grain. Moreover, there are many factors that of HK-GBR and SB-GBR drying with
affect the quality of cooking such as the infrared (IR) at different power (1,000 and
chemical composition of the grain, drying 1,500 W) and temperature (60, 75 and 95 °C)
temperature and storage time, etc. The result show that IR drying have no significantly
for cooking properties in HK-GBR and SB- effect on phytochemical content, antioxidant
GBR are present in Table 2. It was found activity and physicochemical properties of
that cooking time of all rice drying products germinated paddy rice. However, in order to
was increasing when the drying temperature shorten the drying time the drying of HK-
is increase while water absorption is GBR and SB -GBR germinated paddy rice
decreasing when compared to untreated rice. should be done at 95 °C and 1,500 W
Table 2. The effect of infrared drying on cooking properties in HK-GBR and SB-GBR
Hawm-Kradang-Ngah Seebukabntang
Drying Cooking time Water Drying Cooking time Water
temperature (°C) (min) Absorption temperature (°C) (min) Absorption
Reference rice 35 2.870.01d Reference rice 30 3.580.00f
Control rice 42 2.580.01a Control rice 44 2.700.02a
Infrared (IR) drying of 1,000 W Infrared (IR) drying of 1,000 W
60 44 2.740.03c 60 48 3.230.01e
75 45 2.720.00c 75 50 3.210.01e
95 48 2.710.00c 95 53 3.210.01e
Infrared (IR) drying of 1,500 W Infrared (IR) drying of 1,500 W
60 47 2.650.04b 60 45 3.100.03d
75 50 2.580.02a 75 47 2.940.01c
95 51 2.560.01a 95 52 2.880.02b
The results are presented as meanSD (n=3). Values in each column with different letters are significantly
different (p<0.05).
of power energy. However, the other 6. Kian, J. C.; Siaw, K. C. Int. J. Food Sci.
parameters such as rice product quality and Tech. 2005, 40 (1), 23–39.
specific energy consumption (SEC) will be 7. Hebbar, U. H.; Rastogi, N. K. J. Food
further investigated. Eng. 2001, 47 (1), 1–5.
8. Tirawanichakul, S. ; Lamaepae, S. ;
Acknowledgements Tirawanichakul, Y. J. Burapha Sci. 2012,
The authors would like to thank the 17 (1), 117–129.
Faculty of Science and Technology, Faculty 9. Thammapat, P.; Meeso, N.;
of Science and Graduate School Siriamornpun, S. J. Food Chem. 2015,
Prince of Songkhla University for all 175, 218–224.
facilities and financial support. The authors 10. Baskar, R. ; Shrisakthi, S. ; Sathyapriya,
also would like to thank Rice Research B.; Sathyapriya, R.; Nithya, R.; Poongodi,
Center in Pattani province, Thailand and the P. J. Food Sci. Nutr. 2011, 2, 1128–1133.
Regular budget funds that help support 2017 11. Iqbal, S.; Bhanger, M. I.; Anwar, F. J.
budget in research. Food Chem. 2005, 93 (2), 265–272.
12. Shen, Y.; Jin, L.; Xiao, P.; Lu, Y.; Bao, J.
References S. J. Cereal Sci. 2009, 49 (1), 106–111.
1. Soponronnarit, S. ; Nathakaranakule, A. ; 13. Kaewyot, K. ; Taechapairoj, C. J. Sci
Jirajindalert, A.; Taechapairoj, C. J. Food Tech. MSU. 2013, 32 (4), 449–455.
Eng. 2005, 75 (3), 423–432. 14. Scaglioni, P. T. ; Souza, T. D. ; Schmidt,
2. Tirawanichakul, Y. ; Prachayawarakorn, C. G.; Furlong, E. B. J. Cereal Sci. 2014,
S.; Varanyanond, W.; Soponronnarit, S. J. 60 (3), 526–532.
Food Eng. 2004, 64 (4), 405–415. 15. He, D.; Han, C.; Yao, J.; Shen, S.; Yang,
3. Das, I.; Das, S. K.; Bal, S. J. Food Eng. P. Proteomics 2011, 11 (13), 2693–2713.
2003, 62 (1), 9–14. 16. Parnsakhorn, S.; Noomhorm, A. J. Agr.
4. Bal, S.; Wratten, F. T.; Chesness, J. L.; Eng Int: the CIGR E-journal. Manuscript
Faulkner, M. D. Trans. ASAE. 1970, 13 FP 08 009. August, 2008.
(5), 644-652.
5. Afzal, T. M.; Abe, T. J. Comput.
Electron. Agr. 2000, 26 (2), 137–145.
Application of irradiated chitosan for controlling chili fruit anthracnose
control and yield in chili field in Sukhothai
Prartana Kewsuwan1*, Penchan Suthanukul2, Vichai PuriPunyavanich1
1
Nuclear Research and Development Group, Thailand Institute of Nuclear Technology (Public Organization),
Ongkharak, Nakhonnayok 26120, Thailand
2
Sukhothai Horticultural Research Center, Department of Agriculture, Tha Chai, Sukhothai 64190, Thailand
*E-mail: prartanakewsuwan@gmail.com
Abstract:
Field trials were performed to evaluate the effect of foliar application of irradiated
chitosan on reduction of chili fruit anthracnose in Sukhothai province. Four field experiments
of chili plants were treated with irradiated chitosan and two fields with untreated chitosan
were used as a control. The irradiated chitosan (20 ppm) was sprayed at a regular interval of
7-10 days. The results indicated that the incidence of disease symptoms of chili fruits was
significantly lower in irradiated chitosan treatment than in a control. The plants treated with
irradiated chitosan were infected only 7.75 ± 2.70%, while 14.46 ± 1.0% of infection was
found in untreated plants. Moreover, the yield of chili fruits was increased with application of
irradiated chitosan. The results suggest that irradiated chitosan can be used as an eco-friendly
compound to induce anthracnose defense response as well as to increase the yield of chili
fruits.
1. Introduction used as plant growth promoting substances

Thai people like to consume chili in in their depolymerized form. It was reported
all forms starting from fresh green fruits that low molecular weight of chitosan had
with ripe fruits along with its dried and the potential in agriculture with regard to
powdered form. However, the production of controlling plant diseases and promoting the
chili is still not enough to meet the demand plant growth.4 Gamma-rays irradiation
due to many problems such as seed degrades the chitosan into smaller molecular
problems, planting and post-harvesting weight. Gamma radiation is an ionizing in
technology. Plant diseases have been a nature and is another alternative technique to
major reason for the crop losses. The most degrade chitosan into shorter chain chitosan
common diseases found in chili are instead of chemical and enzymatic process5.
anthracnose disease that causes from This technique was a fast process without
Colletotrichum piperatum, Colletotrichum usage of chemicals, free from chemicals
gloeosporioides and Colletotrichum leftover, processed at room temperature, and
1,2
capcisi. During several years synthetic products produced required no further
fungicides have been used to control this purification.
disease. However, in several studies it has This research aims to test the
been shown that the compounds used in efficiency of the irradiated chitosan solution
these fungicides caused strain resistance in the prevention of anthracnose disease and
representing a potential risk for the increase of the yield of chili fruits in the
environment and human health.3 Chitosan is field in Sukhothai province.
a naturally occurring polymer obtained by
the thermochemical deacetylation of chitin 2. Materials and Methods
in the presence of alkali. It is a polycationic 2.1 Preparation of irradiated chitosan
polymer that has one amino group and two solution
hydroxyl groups in the repeating glucosidic Chitosan powder was irradiated by
residue.4 Chitosan has been successfully gamma radiation from a Co-60 source at
doses up to 100 kilogray for degradation the field treated with chitosan was 1.52 ±
with a dose rate of 4.5 kilogray per hour. 0.03 ton/rai, which is about 23% higher than
The chitosan was irradiated at ambient in a control. The average yield of 1.23 ±
temperature. Then, the irradiated chitosan 0.21 ton/rai was found in a control. Figure 2
powder (2 g) was dissolved in 100 mL of and Figure 3 show the effect of irradiated
1% acetic acid solution (w/v). The solution chitosan on weight and number of infected
was left overnight. After completely and non-infected chili fruits, respectively.
dissolution, the chitosan solution was diluted The result showed that chili fruits in the
to 20 ppm using distilled water. Based on fields treated with irradiated chitosan were
the obtained data from the previous less infectious disease than in control. The
experiment (not reported), it was found that average weight of infected chili fruit treated
the most effective concentration of chitosan with chitosan was 7.62 ± 2.63%, while the
was 40 ppm. However, the result has shown 14.26 ± 1.07% of infected chili fruit weight
that there was a slightly difference between was found in a control. Also, the average
20 and 40 ppm. Therefore, irradiated number of infected chili fruits treated with
chitosan at the concentration of 20 ppm was chitosan was 7.75 ± 2.70%, while the
selected to use in this field experiment for average number of infected chili fruit in a
cost-effectiveness in commercialization. control was 14.46 ± 1.00%. The application
Then, the solutions were applied on chili of irradiated chitosan at 20 ppm significantly
field of cultivator in Sukhothai province by decreased the anthracnose on chili fruit and
using normal hand sprayer. Foliar
applications of irradiated chitosan solution
were done at every 7-10 days interval.
2.2 Application of irradiated chitosan
solution on chili field
Six chili fields at Si Satchanalai area
in Sukhothai province during November
2016 to April 2017 were randomly selected
for this experiment. Four fields of chili plant
were sprayed with the irradiated chitosan
solution and the other two fields used as a
control (no chitosan treatment). The 20 ppm Figure 1. Effect of irradiated chitosan (20
irradiated chitosan solution was sprayed ppm) on average chili fruit yield per rai
every 7-10 days interval. Other processes
were carried out to follow the usual practice.
Chili fruit disease, plant growth and chili
fruit yield were investigated after 3 months
of transplanting.

After 30 days of planting seedling,
each crop (4 fields) was sprayed with 20
ppm concentration of irradiated chitosan.
Distilled water was sprayed in the control
treatment (2 fields). The results of field
experiments are shown in figure 1-3. Figure
1 shows the effect of irradiated chitosan on Figure 2. Effect of irradiated chitosan (20
the average chili fruit yield per rai. It was ppm) on weight of non-infected and infected
found that the average chili fruit yield from chili fruits (%)
Acknowledgements
The author is grateful to Thailand
Institute of Nuclear Technology, Ministry of
Science and Technology, for financial
support with this research.
References
1. Than, P; Prihastuti, H.; Sitthisack
Phoulivong, S.; Taylor, P.; Hyde, K. D.
J. Zhejiang Univ. Sci. B. 2008, 9 (10),
764–778.
Figure 3. Effect of irradiated chitosan (20 2. Saxena, A.; Raghuwanshi, R.; Gupta, V.
ppm) on number of non-infected and K., Singh, H. B. Front. Microbiol. 2016,
infected chili fruits (%) 7, 1527–1545.
3. Sangeetha, G.; Thangavelu, R.; Rani, S.
enhanced the chili fruit yield. The chili U.; Muthukumar, A. Biological Control
plants treated with irradiated chitosan 2013, 64, 16–25.
showed healthier than those in a control. In 4. Hossaina, M. A.; Hoquea, M. M.;
addition, the incidence of anthracnose Khanb, M. A.; Islamb, J. M. M.; Naher,
symptom on chili fruit decreased compared S. Int. J. Sci. & Eng. Res. 2013, 4 (8),
with the untreated chitosan. Chitosan has 1693–1698.
potential to reduce the use of plant 5. Feng, T.; Du, Y.; Li, J.; Hu, Y.;
protection chemicals that may be harmful to Kennedy, J. F. Carbohydr. Polym. 2008,
humans and environment. 73, 126–132.
4. Conclusion
Application of irradiated chitosan at
20 ppm might significantly reduce
anthracnose of chili fruit and improve the
growth and yield of chili plant.
Effects of extraction solvents ratio and methylation time for the
determination of fatty acids from rambutan seeds kernel by
gas chromatography-flame ionization detector
Sirirat Chanvaivit*, Ticha Caechua
Department of Chemistry, Burapha University, Chonburi, Thailand
*E-mail: sirirat@buu.ac.th
Abstract:
The effect of solvents used for the extraction of fatty acids from rambutan seeds
kernel was investigated. The suitable ratio of dichloromethane-methanol-hydrochloric acid to
obtain the highest efficiency was 2:1:0.02 (v/v) followed by sonication for 10 min. The
highest peak area was received after methylation at 80 °C for 4 hours. GC-FID was
performed using DB-225 column (20 m  0.10 mm, 0.1 m film thickness). Helium was used
as carrier gas at 0.3 mL/min. Injector and detector temperatures were 240 ºC. Injection
volume was 1 µL. Split ratio was set at 100:1. The oven program started at 150 ºC for
0.5 min, rising to 200 ºC at a rate of 100 ºC/min held for 1 min and then 220 ºC at a rate of 10
ºC/min held for 12 min. The last fatty acid came out at 15.10 min. Five main fatty acids in
Rongrean rambutan seed were Palmitic acid (C16:0) 8.20±0.16%, Palmitoleic acid (C16:1n7)
0.80±0.04%, Stearic acid (C18:0) 10.68±0.12%, Oleic acid (C18:1n9) 67.88±0.30% and
Linoleic acid (C18:2n6) 12.45±0.16%. Similar composition of fatty acids was found in
Seechompoo rambutan seed as followed: Palmitic acid 8.34±0.39%, Palmitoleic acid
0.69±0.04%, Stearic acid 11.89±0.13%, Oleic acid 66.97±0.26% and Linoleic acid
12.12±0.28%.
1. Introduction for 1 hour provided extractive yield close to

Rambutan seed is a main residue soxhlet extraction for 6 hours.7 Recently,
from rambutan in syrup processing industry. sonication-assisted extraction has been
In Thailand, commercialized rambutans are applied due to high efficiency and short
namely “Rongrien” and “Seechompoo”. extraction time.8 Different extraction
Rongrien rambutan fruit has red skin with methods, conditions and solvents used for
green hair. Seechompoo rambutan fruit has extraction resulted in different compositions
yellowish to pink skin with red hair. Some and percentage of fatty acids. Moreover, the
studies reported that rambutan seeds HCl-Bligh and Dyer method extraction was
composed of protein, fat, fiber and ash.1 Fat claimed to give more total fatty acids and
and oil from waste seed become popular increase in polyunsaturated fatty acids in a
issue because it can be used as a raw short time.9 Therefore, the aim of this study
material in food, medicines and cosmetics was to evaluate the solvent ratio, sonication
industries. High essential fatty acid in and methylation time for extraction and
rambutan seeds motivated researcher to comparison of the compositions and amount
perform suitable extraction methods for of fatty acids in “Rongrien” and “Seechom-
industrial process. Previously, fat from poo” rambutan seed wastes.
rambutan seeds was extracted with n-
hexane2 through soxhlet extraction 2. Materials and Methods
technique for 6 hours3,4 or 7 hours5 and with 2.1 Plant materials
petroleum ether with the same technique for Rongrien rambutan seeds waste was
8 hours.6 Maceration technique with provided by Tropical food industry (Suan
n-hexane and shaking at room temperature luang, Bangkok, Thailand). Seechompoo
rambutan seeds waste was provided by thickness) was used. Temperature program
Prayong garden (Huangnamkhaw, Mueng, was set at 150 C for 0.5 min, then increased
Trat, Thailand). The kernels were removed to 200 C at 100 C/min held for 1 min,
from the seeds and dried in the oven at followed by rising to 220 C at 10 C/min
65 C for 5 hours. The dried rambutan seed and holding final temperature at 220 C for
were ground by 200 g WF-04A Mulry 12 min. Detector temperatures was set at
functional high speed disintegrator to a fine 240 C.
powder then kept in a sealed bottle.
2.2 Sample preparation 3. Results and Discussion
For analysis, 0.2000 grams of The fatty acids extraction from
rambutan ground seeds were extracted with rambutan seed including extraction solvents
40.00 mL of mixed solvents (dichloro- and ratio, sonication time was carried out.
methane: methanol: hydrochloric 2:1:0.002 Suitable methylation time for transformation
(v/v)), sonication for 10 min. The solution of fatty acids to methyl esters was
was transferred to separatory funnel performed before analysis by GC-FID.
containing 0.88% of 12.5 mL potassium 3.1 Effects of acid in extracting solvent
chloride and then shaken. The lower level The peak areas of fatty acids
was passed through anhydrous sodium extracted by dichloromethane: methanol:
sulfate into round bottom flask. The upper hydrochloric acid (2:1:0.01) was about
part was extracted again with 15.00 mL of 3 times higher than dichloromethane:
mixed solvents. The lower level was methanol (2:1) as shown in Figure 1.
collected in the same round bottom flask.
The solvents were removed by rotary
evaporator until dryness and purge leftover
dichloromethane with nitrogen.
The methylation step was performed
by adding methanol and sulfuric acid. The
suitable methylation times at 80 C were
investigated.10,11
All chemicals used in this studied
were of analytical grade. A PUFA No.3
(from menhaden oil) and butylated
hydroxyl-toluene (BHT) were purchased
from Sigma-Aldrich. Dichloromethane and
methanol were obtained from Fisher
Chemicals and RCI Labscan, respectively.
Hexane, hydrochloric acid and sulfuric acid
were purchased from Merck. Potassium
bicarbonate, potassium chloride and sodium Figure 1. Peak area of fatty acid in
chloride were supplied by CARLO ERBA rambutan seed extracted with dichloro
and AJAX chemicals. methane:methanol (2:1) and dichlorome
2.3 Gas chromatographic analysis thane:methanol:hydrochloric acid (2:1:0.01)
A Hewlett-Packard GC 6890 gas (n=3)
chromatography equipped with flame
ionization detector was used. Helium carrier This finding was in agreement with
gas was set at 0.3 mL/min. Injector Jensen who reported that an acidic Bligh and
temperatures was set at 240 C. Injection Dyer extraction method leaded to 10-15%
volume was 1 L of sample. A split ratio more total fatty acid and 30-50% increase in
was set at 100:1. A DB-225 column (20 m poly unsaturated fatty acids compared with
length, 0.10 mm. i.d. and 0.1 m film the official EU method.9 It might be
explained that hydrochloric acid may break sonication time was selected for further
the cell wall of biomass and release fatty study.
acids from seed.12
3.2 Effects of extraction solvents ratio
Increased adding hydrochloric acid
ratio in dichloromethane: methanol (2:1)
from 0.005, 0.01 to 0.02 resulted in
increased peak area of fatty acids as shown
in Figure 2. At low ratio of hydrochloric
acid, the cell wall may not break completely.
However, high ratio of hydrochloric acid
may erode collector part in flame ionization
detector.
Figure 3. Peak area of fatty acid in

rambutan seed extracted with dichlorome
thane:methanol:hydrochloric acid (2:1:0.02)
with various sonication times (n=3)
3.4 Effects of methylation time

Fatty acids were transformed to
methyl esters (FAMEs) and analyzed by
GC-FID. The methylation temperature was
set at 80 C. The methylation times of 2, 3,
Figure 2. Peak area of fatty acid in 4 and 6 hours were compared. As shown in
rambutan seed after extracted with Figure 4, the peak area of FAMEs increased
dichloromethane:methanol (2:1) in different when increasing methylation time from 2 to
ratio of hydrochloric acid (n=3) 4 hours. It can be seen that at 4 hours of
methylation time, fatty acids was completely
3.3 Effects of sonication time transform to FAMEs, resulting in the highest
Sonication times for 5, 10, 20 and 30 peak area. It can be seen that, at 6 hours of
minutes were studied. Figure 3 showed that methylation time, all FAMEs peak area were
sonication time for 10 minutes gave the decreased. This might be occurring due to
highest peak area. Sonication provides fatty acid methyl esters loss at high
intense shear forces and liquid jets which temperature for a long period of time.
can make solvents penetrate into plant seed 3.5 Fatty acid analysis
and release fatty acids efficiently. The major fatty acids in rambutan
Therefore, higher extraction yield at shorter seeds extract after methylation were oleic
extraction time can be received. At 5 acid (C18:1n9), linoleic acid (C18:2n6),
minutes peak area was low probably because stearic acid (C18:0) and palmitic acid
of no enough time for fatty acid liberation (C16:0) as shown in Table 1. The same type
from seed. When the sonication was more and similar amount of fatty acids in
than 10 minutes, fatty acids may be lost Rongrien and Seechompoo were presented.
during extraction. Finally, 10 minutes of
of extraction time may be useful for
industrial process. Oleic acid has a great
potential in cosmetic industries. It is claimed
to enhance percutaneous absorption of
cosmetic actives and offers excellent
delivery of lipophilic ingredients to skin.7
Moreover, oleic acid can reduce blood
pressure, increases fat burning to help with
weight loss, protects cells from free radical
damage, may prevent type 2 diabetes,
prevents ulcerative colitis and generates
brain myelin.13,14 Linoleic acid has been
used in beauty product as an anti-
inflammatory, acne reductive15 and skin –
lightening.16 It can be used for preventing
the autoxidation of lipids in vegetable oils.17
Stearic acid and palmitic acid is mainly used
Figure 4. Peak area of fatty acid in in the production of detergents, soaps,
rambutan seed extracted sonication for 10 cosmetics and candle. Therefore, fatty acids
minutes at various methylation times (n=3) in rambutan seeds are useful and it can be
used as an alternative source of fatty acids
Table 1. Percentage of fatty acids in for industry.
Rongrien and Seechompoo rambutan seeds
(n=3) 4. Conclusion
a
% Fatty acidS.D. Small amount of acid in extraction
Fatty acid Rongrien Seechompoo solvents can increase extraction efficiency.
rambutan seeds rambutan seeds The suitable ratio of dichloromethane-
C16:0 8.200.16 8.340.39 methanol-hydrochloric acid to obtain the
C16:1n7 0.800.04 0.690.04
C18:0
highest efficiency was 2:1:0.02 (v/v)
10.680.12 11.890.13
C18:1n9 67.8 0.30 66.970.26
followed by, sonication for 10 min. The
C18:2n6 12.46 0.16 12.120.28 highest peak area was received after
a
S.D. = standard deviation methylation at 80°C for 4 hours. High
percentage of oleic acid was found in both
Oleic acid was the major fatty acid in Rongrien and Seechompoo rambutan seeds.
both Rongrien and Seechompoo rambutans In rambutan plantation area, seed waste can
seeds (67.880.30 and 66.970.26% be use as raw material of fatty acids.
respectively). Extraction with
dichloromethane:methanol:hydrochloric Acknowledgements
acid (2:1:0.02) gave two times higher This work was supported by
percentage of oleic acid compared to Department of Chemistry, Faculty of
previous studies (36.790.16, 33.130.58 Science, Burapha University.
and 31.080.75%).2,4,7 However, our results
could not found arachidic acid (C:20:0). References
Compared to former studies, hexane was 1. Augustin, M. A.; Chua, B. C. Pertanika
used as an extraction solvent. As known that 1988, 11 (2), 211–215.
dichloromethane is more polar than hexane, 2. Sirisompong, W.; Jirapakkul, W.;
therefore it may not suitable for extraction Klinkesorn, U. LWT-Food Sci. Technol.
of long chain fatty acids like arachidic acid 2011, 44, 1946–1951.
from rambutan seeds. Nevertheless, the 3. Solís-Fuentes, J. A.; Camey-Ortíz, G;
finding of high oleic acid within 10 minutes Hernández-Medel, R.; Pérez-Mendoza,
F.; Durán-deBazúa, C. Biores. Technol. 11. Eder, K. J. Chromatogr. B. 1995, 671,
2010, 101, 799–803. 113–131.
4. Sonwai, S.; Ponprachanuvut, P. J. Oleo. 12. Castanha, R. F.; Salgado de Morais, L.
Sci. 2012, 61, 671–679. A.; Mariano, A. P.; Monteiro, R. T. R.
5. Winayanuwattakun, P.; Kaewpiboon, C.; Braz. Arch. Boil. Technol. 2013, 56 (4),
Piriyakananon, K.; Tantong, S.; 629–636.
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The effects of EM-Bokashi compost and vegetable wastes compost on the
growth of Hibiscus esculentus L. (lady’s finger) plant
Min Aung, Kyaw Zan Aung, Nay Thwe Kyi*
Department of Chemistry, Myeik University, Myanmar
*E-mail: dr.naythwekyi55@gmail.com
Abstract:
Application of organic composts can improve soil quality as well as sustainability of
agricultural production. This research concerns with the effects of EM-Bokashi compost and
vegetable wastes compost on the growth of lady's finger plant. The experiment comprised
two treatments viz., EM-Bokashi compost (soil:prepared EM-Bokashi = 1:1) and vegetable
wastes compost (soil:organic matter such as tea and vegetables wastes = 1:1). Increase in the
contents of moisture, organic carbon, humus and phosphorus were noticed in EM-Bokashi
compost when compared to vegetable wastes compost. The contents of nitrogen and
potassium were not much different from each other. The effects of EM-Bokashi compost and
vegetable wastes compost on the growth of lady's finger plant such as plant height,
circumferences of stem, number of fruits and leaves per plant were measured after 15 days,
30 days and 45 days. Plant height, stem circumference, number of leaves, yield and fruit size
of lady’s finger plant on EM-Bokashi compost was larger than vegetable wastes compost
because EM-Bokashi compost has high organic carbon, humus and available phosphorus
contents. In fact, the prepared EM-Bokashi compost can be used as a good source of nutrients
for the plant growth and can reduce the farmer's budget for crop fertilization.

In many countries large proportions 2.1 Sample collection
of municipal waste are not disposed properly Soil sample was collected from the
posing a potential environmental threat due campus of Myeik University, Tanintharyi
to the presence of pathogens and toxic Region, Myanmar. In this research work, the
pollutants.1 Organic municipal waste and soil from the upper position near to the
other organic material such as manure can be surface about (15 cm) was digging in zigzag
composted. Composting is an action of manner according to the sampling of soil
encouraging the breaking down of organic samples.
waste. Composting is an aerobic process 2.2 Preparation of vegetable wastes
during which the organic matter is compost
decomposed to humus-like substances. Vegetable wastes compost was
Composting can also be an anaerobic prepared by using 1:1 weight ratio of the
process, where breakdown occurs in the collected soil sample with vegetables wastes
absence of oxygen. In this case, the main as green materials and tea residues as brown
byproducts are methane, carbon dioxide and materials. A shady place under a thatched
various low organic acids and alcohols. roof to protect the compost from the sun and
Since aerobic composting is more efficient rain was chosen. The soil mixture was added
and presents fewer undesirable by products.2 to earth pot and pushed in a stick in to mark
Moreover, composting is the transformation the center of the pile. This pot was covered
of organic material (plant matter) through with plastic sheet.
decomposition into a soil-like material called After four weeks, turn the pile over to
compost. Compost is an organic fertilizer mix the different layers. The pile was
that can be made on the farm at very low watered for another week and then left for 8
cost. Compost also helps the soil stay loose weeks. Soils were incubated at 28 °C for
and easy to cultivate.3 eight weeks and rewetted to 70% of water
holding capacity. The pile was watered about and then vegetable wastes compost and EM-
three times a week. After eight weeks, Bokashi compost were added into each pot.
vegetable wastes compost was obtained. The Lady's finger was planted in the
2.3 Preparation of effective micro- prepared two composts. The seeds were
organism (EM), EM-Bokashi and EM- sowed at 2.5 cm deep on the pots. Two seeds
Bokashi compost were sowed in each planting hole. Seedlings
One bin of rice was dissolved in one emerged after 4 to 5 days of sowing. One
liter of chlorine free water and stored at healthy seedling was retained in each pot.
room temperature without direct sunlight. After 2 weeks, only one healthy seedling per
After seven days, the fermented rice water planting hold was left as shown in Figure 1.
was mixed with milk (1:10 ratio). After two The plants were watered once a day every
weeks, the yellow brown EM solution was morning. The fruit is soft and crunchy to
obtained and stored in a cool dry place. The allow a continuous yielding; the plant must
aqueous brown jaggery was added into the be planted at this stage. The effects of EM-
prepared EM solution and placed out of Bokashi compost and vegetable wastes
direct sunlight for 1-2 hours to allow the EM compost, on the growth of lady’s finger plant
to activate more fully.4 such as plant height, circumferences of stem,
Weight of ten lbs of rice bran was number of fruits and leaves per plant were
mixed with one spoon of brick powder, one measured after 15 days, 30 days and 45 days.
spoon of salt, three liters of chlorine free hot The NPK contents left in the soil were
water and 30 mL prepared EM and these determined at 30 days, and 45 days.
mixtures were added in sack. The sack was
tightly packed with rope and sit about two
weeks in dark room at room temperature.
After two weeks, gold yellow pleasant smell
bokashi was obtained.
EM-Bokashi compost was prepared
by mixing 1:1 weight ratio of the collected
soil sample and prepared EM-Bokashi. After
five days, EM-Bokashi compost was
obtained. (a) (b)
2.4 Determination of physicochemical Figure 1. Plantation of lady’s finger in (a)
properties of prepared composts vegetable wastes compost (b) EM-Bokashi
The physicochemical properties and compost
nutrients of prepared two composts were
determined by conventional methods and 3. Results and Discussion
modern techniques such as moisture content 3.1 Texture of prepared composts
by oven drying method, pH by pH meter, The texture of vegetable wastes
soil texture by particle sizes analytical compost and EM-Bokashi compost were
method, organic carbon and humus by determined and the results are shown in
titration methods, amount of soil nitrogen Table 1. All prepared composts were loam
released by Kjeldahl method, content of type. Decrease in sand and clay percentage
available phosphorus by spectrophotometer, and increase in silt percentage were noticed
content of potassium by flame photometer, in EM-Bokashi compost when compared to
contents of exchangeable magnesium, vegetable wastes compost. These increases
calcium, potassium and sodium by using might be related to the positive effect of
titration methods. microorganisms in EM-Bokashi compost.
2.5 Plantation of lady’s finger
Two experimental pots (13.5 inches
diameter  10.5 inches height) were prepared
Table 1. Texture of vegetable wastes Table 2. Moisture, pH, organic carbon and
compost and EM-Bokashi compost humus contents of prepared composts
Sample Texture Soil Sample Moist pH Organic Humus
Sand Silt Clay Total Type ure Carbon (%)
(%) (%) (%) (%) (%)
Vegetable 39.55 41.90 17.05 98.50 Loam Vegetable 1.07 5.53 3.26 5.62
Wastes Wastes
Compost Compost
EM- 38.65 46.75 13.35 98.75 Loam
Bokashi EM-Bokashi 1.78 6.11 4.60 7.93
Compost Compost
3.2 Physicochemical properties of are shown in Table 3. The macronutrients in

prepared composts vegetable wastes compost on available N
The physicochemical properties of (0.23%), P (17.10 ppm) and K (22.62
two prepared composts were determined and mg/100 g) while macronutrients in EM-
the resulted data are shown in Table 2. The Bokashi compost on N (0.21%), P (20.14
moisture percent of two composts were ppm) and K (21.99 mg/100 g) were
1.07% and 1.78%, respectively. It was found observed. Nitrogen helps plants with rapid
that the higher moisture content due to the growth, improving the quality of leaf and
organic carbon content. When the organic forage crops. Phosphorus in soil as inorganic
carbon content was increased the soil phosphate ions and sometimes as soluble
porosity was increased and the moisture organic phosphorus can be absorbed and
content was also increased. Increase in made available to meet plant. Plants need
organic carbon content (4.60%) of EM- phosphorus for strong root growth, fruit,
Bokashi compost was observed when stem, disease resistance and seed
compared to organic carbon content (3.26%) development. The increase in phosphorus
of vegetable wastes compost. The content of content of EM-Bokashi compost was
humus (7.93%) in EM-Bokashi compost was observed by the greater multiplication of
larger than vegetable wastes compost microbes. The nutrient, sometimes called
(5.62%). The higher the humus levels of the potash, is essential for vigorous growth,
soil the greater the exchange capacity. Soil disease resistance, fruit and general plant
acidification may also occur by addition of function.
hydrogen, due to decomposition of organic
matter, acid-forming fertilizers, and Table 3. Contents of macronutrients of
+ prepared composts
exchange of basic cations for H by the
roots. Basic soils have a high saturation of Sample Total N P K2O
base cations (K+, Ca2+, Mg2+ and Na+). The (%) (ppm) (mg/100 g)
Vegetable 0.23 17.10 22.62
higher the amount of exchangeable base Wastes
cations, the more acidity can be neutralized Compost
in the short time perspective. The pH showed EM- 0.21 20.41 21.99
that vegetable wastes compost were acidic Bokashi
condition and EM-Bokashi compost was Compost
nearly neutral condition. This is because of
EM-Bokashi compost has higher value of 3.4 Exchangeable cation of prepared
organic carbon and humus content. These composts
parameters controlled the pH of prepared Base saturation is the percentage of
composts. total CEC (negatively charged sites in the
3.3 Contents of macronutrients of soils) occupied by the cations: Ca2+, Mg2+,
prepared composts Na+ and K+. The ease with which plant roots
Contents of nitrogen, phosphorus, can absorb cation nutrients increases with the
and potassium as macronutrients of plants degree of base saturation. The exchangeable
cations such as Ca2+, Mg2+, Na+ and K+ of whereas EM-Bokashi compost had
prepared composts are shown in Table 4. significantly higher throughout the growing
Ca2+, Mg2+, Na+ and K+ values of vegetable period, i.e., on 45 days. The availability of
wastes compost were 5.43, 1.99, 0.42 and nutrients in two composts was not too much
0.48 meq/100 g, respectively whereas in differing from each other. However, organic
EM-Bokashi compost, they were 6.09, 2.71, carbon content and humus content in EM-
1.73 and 0.47 meq/100 g. It is still a little- Bokashi compost were much higher than the
known fact that calcium and magnesium vegetable wastes compost. This situation
amount determines how tight or loose soil is. favors the increase in the plant height and
The more calcium a soil has the looser it is; number of leaves over the vegetable waste
the more magnesium, the tighter it is, up to a compost.
point. A high calcium soil will have more 3.6 Effect of vegetable wastes compost and
oxygen drain freely and support more EM-Bokashi compost on fruit yield and
aerobic breakdown of organic matter, than a fruit size of lady’s finger
high magnesium. EM-Bokashi compost has Application of prepared composts
higher calcium content than vegetable wastes had significant effect on fruit size, fruit
compost.5 circumference and fruit yield of lady’s
finger, as shown in Table 6. Fruit yield of
Table 4. Exchangeable cations in prepared lady’s finger plant on EM-Bokashi compost
composts was higher than that of vegetable wastes
Sample Exchangeable cations (meq/100 g) compost.
Ca2+ Mg2+ Na+ K+ The EM-Bokashi compost is more
Vegetable 5.43 1.99 0.42 0.48 important than inorganic fertilizer and
Wastes
Compost vegetable wastes compost because it consists
EM-Bokashi 6.09 2.71 1.73 0.47 of relatively stable decomposed materials
Compost resulting from accelerated biological
degradation of organic matter under
3.5 Effect of vegetable waste compost and controlled aerobic conditions.
EM-Bokashi compost on plant height, From the experimental data, it was
stem circumferences and leaves observed that the length of lady’s finger in
Data on growth parameters as plant EM-Bokashi compost was longer than
height stem circumferences and number of vegetable wastes compost and
leaves at 12 days, 15 days, 30 days and 45 circumference of fruit was not too much
days are shown in Table 5. During earlier different between two composts. This
growth period, i.e., 12 days and 15 days, increase might be related to the positive
lady’s finger plant in vegetable wastes effect of compost and microorganisms in
compost had shown higher plant height, stem increasing the root surface area per unit of
circumferences and number of leaves
Table 5. Effects of vegetable wastes compost and EM-Bokashi compost on plant height,
stem circumference and number of leaves
Growth Lady’s Finger in Vegetable wastes Compost Lady’s Finger in EM-Bokashi Compost
Parameter 12 days 15 days 30 days 45 days 12 days 15 days 30 days 45 days
Plant Height 5.0 5.2 8.7 29.5 4.0 4.5 9.6 35.5
(inches)
Stem 0.6 0.7 1.2 2.3 0.4 0.4 1.1 2.5
Circumference
(inches)
Number of 4 4 10 18 3 5 11 24
Leaves
Table 6. Effects of vegetable wastes municipal and agricultural wastes going into
compost EM-Bokashi compost on fruit yield landfill. The experiments were carried out
Growth Lady’s Finger Lady’s from December 2016 to January 2017. The
Parameter in Vegetable Finger in EM- prepared vegetable wastes compost and EM-
Wastes Bokashi
Compost Compost Bokashi compost have been characterized by
Fruit Length 5.4 6.0 conventional methods and modern
(inches) techniques. The textures of two prepared
Fruit 3.3 3.0 composts were found to be loam type which
Circumference can be suitable for plants. The pH (6.11) of
(inches)
Number of 8 13 EM-Bokashi compost was nearly neutral
Fruits per condition when compared to pH (5.53) of
Plant vegetable wastes compost. The contents of
moisture (1.78%), organic carbon (4.60%)
soil volume, water-use efficiency and and humus (7.93%) of EM-Bokashi compost
photosynthetic activity, which directly were larger than contents of moisture
affects the physiological processes and (1.07%), organic carbon (3.26%) and humus
utilization of carbohydrates.6,7 (5.62%) of vegetable wastes compost. It is
indicated that EM-Bokashi compost can be
3.7 Comparison of NPK contents in used effectively as soil amended and this
prepared composts after 30 days and 45 compost also can reduce the application of
days of plantation inorganic fertilizer. Macronutrient contents
The contents of nitrogen, phosphorus such as available P content was increased but
and potassium in two prepared composts total N and K contents were not much
were found to be decreased after 30 days different in EM-Bokashi compost when
plantation. After 45 days plantation, contents compared to vegetable wastes compost. The
of phosphorous and potassium in the lady’s finger plants were planted in two
prepared composts were lower than the prepared compost soils pots. Plant in EM-
values of original and 30 days after Bokashi compost treatment had significantly
plantation. This is due to the fact that fruiting higher plant height, stem circumferences and
and seed formation requires higher number of leaves throughout the growing
phosphorous and potassium. However, total period after 30 days and 45 days plantation
nitrogen content in the prepared composts due to higher amount of organic carbon,
had the same value as after 30 days (Table humus and available phosphorous in EM-
7). Bokashi compost. Moreover, the fruit length
of lady’s finger in EM-Bokashi compost was
Table 7. NPK contents in prepared composts longer than vegetable wastes compost and
after 30 days and 45 days of plantation fruit circumference was not too much
Nutrients Vegetable Wastes EM-Bokashi Compost different between two prepared composts. A
Compost
Before 30 45 Before 30 45 slightly decrease in NPK contents EM-
days days days days Bokashi compost after plantation. The
Total N 0.23 0.20 0.20 0.21 0.18 0.18
(%)
findings indicated that EM-Bokashi compost
P (ppm) 17.10 16.18 15.80 20.41 19.20 18.11 as bio-compost can be used as fertilizer in
K2O 22.62 16.39 16.10 21.99 15.30 14.25
agriculture and horticulture.
(mg/100
g)
Acknowledgements
We wish to express our sincere
4. Conclusion gratitude to Prof. Dr. Win Naing, Rector,
The composting process is Dagon University, Prof. Dr. Si Si Hla Bu,
considered an economic and environ- Rector, Pathein University, Prof. Dr.
mentally means to reduce the wastes such as Supawan Tantayanon, President, the Council
of Science and Technology Professionals, 4. http://www.bokashicomposting2011.
Prof. Dr. Supa Hannongbua, President, the com
Chemistry Society of Thailand and Bangkok 5. Chattoo, M. A.; Ahmed, N.; Wani, M.
Bank CLVM fund for PACCON 2017 H.; Mir, S. A.; Khan, S. H.; Jabeen, N. J.
Scholarship for their encouragement. Agric. Sci. 2011, 38, 135139.
6. Epstein, E. The Science of Composting.
References Technomic Publishing Co. Inc.:
1. Darby, H. M.; Stone, A. G.; Dick, R. P. Lancaster, Pennsylvania, 1997; pp 487.
J. Sci. Soc. 2006, 70, 347358. 7. Storey, B. B.; McFalls, J. A.; Godfrey, S.
2. Bernal, M. P.; Alburquerque, J. A.; H. The Use of compost and shredded
Moral, R. Bioresour. Technol. 2009, 100, brush on right of way for erosion control,
54445453. Final Report. Texas Transportation
3. Farrell, M.; Jones, D. L. Bioresour. Institute: Texas, 1995; pp 159166.
Technol. 2009, 100, 43014310.
Chemical and physical properties of sago starch from tradition and
machine method
Rattiya Saradit*, Nutthawadee Prabtan
Department of Industrial Chemistry, Faculty of Science and Technology,
Rajamangala University of Technology Srivijaya, Thailand
*E-mail: kek315@hotmail.com
Abstract:
Sago palm (Metroxylon spp.) is one of the few tropical crops which can tolerate wet
growing conditions including peat swamps. Sago starch accumulates at the pith core of the
stem in the sago palm. Sago starch has been used for cooking of various types of dishes, such
as cookies. Sago starch has been used as textile, paper and plywood adhesives and as
stabilizer in the pharmaceutical industry. There are two ways to isolate sago starch. The
traditional method takes longer time than a machine method. In this study, the objective was
proposed to investigate chemical compositions including moisture, protein, fat and ash
content and some physicochemical properties which were gelatinization profiles of sago
starch. The results found that moisture, fat and ash content of sago starch from either two
methods were not statistically different. Sago starch prepared by the traditional method
showed protein content (0.25%) and gelatinization temperature (69.65 C) higher than
machine method which provided 0.16% protein and 67.33 C gelatinization temperature.
Machine method required lower energy for gelatinization than traditional method. The SEM
images showed that starch granules have oval shape and size ranged between 23 and 48 μm
from both methods.
1. Introduction the pith using an adze tipped with a hard

Sago palm (Metroxylon spp.) is one wood blade. The shredded pith is trampled
of the few tropical crops which can tolerate on a platform where water is added and
wet growing conditions including peat starch was allowed to pass through finely-
swamps.1 It is also one of the oldest tropical woven reed mat. The starch slurry collected
plants exploited by man specifically for its was allowed to settle and after draining the
stem starch.2 Sago palm has a main water, the solid wet flour (lamentak) is dried
advantage of the ability to thrive in the harsh by sunlight.7
swampy peat environment.3 Sago palm In the modern factories however, the
contains a large amount of starch in its trunk logs on arrival at the mills are immediately
and its productivity was calculated to be 4 processed by first being debarked, followed
times that of paddy rice. Sago starch has by maceration using a raspier. The hammer
been used in the cooking of various types of mill further disintegrated the rasped pith into
dishes such as jellies, puddings, soups, finer pieces and the starch slurry was passed
noodle, biscuit and sago pearls.4 Sago starch through a series of centrifugal sieves and
has been used as textile paper, plywood cyclone separators. The semi-dried starch
adhesives and as stabilizer in the from the rotary vacuum drum dryer is
pharmaceutical industry.5 further dried by hot air drying in the flash
Sago starch accumulates in the pith dryer.8 The different methods of starch
core of the stem of the sago palm.6 The extraction gave rise to various quality of
starch reserves are apparently at their sago starch.7
maximum just before flowering, and fruiting The Japanese researcher’s studies on
deplete these reserves.3 Traditionally, the improvement of sago starch quality found
starch is extracted manually by shredding that the low quality of sago starch is not
only due to low level of processing titrated with 0.1 N HCl solutions and the
techniques but also to other factors such as protein content was calculated (AOAC,
the freshness, maturity of raw materials 2002).
(sago logs), storage of sago logs after 2.2.4 Ash
harvesting, iron tools in starch production Ash of two sago starch samples was
and the use of high-grade water for starch determined according to the standard AOAC
processing.9 (2002) method. One gram of sago starch
In this study, the objective was sample were heated in oven for 1 hour at
proposed to investigate chemical 105 C and burnt at 550 C. Then the ash
compositions such as moisture, protein, fat content was calculated.
and ash content and some physicochemical 2.2.5 Amylose
properties which were gelatinization profiles Amylose in the sago starch samples
of sago starch of traditional method and was determined according to the
machine method. spectrophotometric measurement which was
carried out at 620 nm and amylose content
2. Materials and Methods in the starch samples was determined by
2.1 Materials standard curve. Standard solutions were
Two sago starch samples were prepared by using 0.04 g potato amylose, 1
obtained from two preparation methods. mL of 95% ethanol, 9 mL of 1 M NaOH and
Sago 1 was obtained from traditional adjusted the volume to 100 mL with distilled
method and sago 2 was obtained from water. The sago starch samples solution
machine method from Nakhon Si were prepared similarly as a standard
Thammarat. solution.
2.2 Methods 2.2.6 Scanning electron microscopy
2.2.1 Moisture (SEM)
Moisture of two sago starch samples Scanning electron microscopy
was determined according to the standard (SEM) was used for studying the shape of
AOAC (2002) method. Two grams of sago sago starch samples. The sago starch sample
starch sample were heated for 8-10 hour at was suspended in 1:1 glycerol/water
130±3 C and the moisture content was mixtures.
calculated. 2.2.7 Differential scanning calorimeter
2.2.2 Fat (DSC)
Fat of two sago starch samples was Differential scanning calorimetry
determined according to the standard AOAC (DSC) was performed using DSC7
(2002) method. Sago starch samples were differential scanning calorimeter, (Perkin
extracted with petroleum ether for 2 hour in Elmer, USA). The starch samples (3 mg)
a Soxhlet extractor and solution was dried to and 6 mL H2O was cured at 4 C for 12
obtain a content weight. hour. The cells were heated from 30 C to
2.2.3 Protein 120 C at the rate of 5 C/minute.
1.5 g of sago starch samples were 2.2.8 Viscosity
digested with K2SO4, CuSO4 and conc. Viscosity of sago starch samples was
H2SO4 in Kjeldahl’s flask at 250 C for 30 performed by rapid Visco analyzer. 3 g of
minute. When there is no bubble, sample and 25 mL H2O was cured at 60 C.
temperature is adjusted to 380 C and The cells were heated from 60 C to 95 C at
digested to obtain colorless solution. After the rate of 12 C/minute.
cooling at room temperature, sago starch
samples were separated with 200 mL H2O, 3. Results and Discussion
70 mL of 50% NaOH, 25 mL of 4% H3BO3 3.1 Moisture
and indicator in Erlenmeyer flask underlies The moisture content of sago starch
solution at condenser. The solution was samples is given in Table 1. The moisture
content of sago 1 and sago 2 is 14.20% 3.6 Scanning electron microscopy (SEM)
17.32%, respectively. There are significant SEM of sago starch showed that the
differences (p≤0.05). However, this is sago starch consists of oval granules as
consistent with that the moisture contents of shown in Figure 1 with diameters in the
sago starch ranged between 10.6% and range of 20–45 µm. There was no
20.0%.10 Under average ambient significant difference in the average
temperature and humidity conditions the diameter or shape of sago starch samples.
moisture content of most unmodified 3.7 Viscosity
starches is around 12%.10 The viscosity content is shown in
3.2 Fat Table 2. Peak viscosity, breakdown and
The crude fat content of sago starch setback of sago 1 were 4477 cP, 2300 cP
samples is given in Table 1. The quantity of and -1041 cP, respectively. Sago 2 has 5401
crude fat in the samples were low and the fat cP peak viscosity, 3331 cP breakdown and -
content of sago 2 (0.19%) higher than sago 1 1943 cP setback. The viscosity content of
(0.17%) was significant (p<0.05). While the sago 1 and sago 2 were significantly
potato starches was 0.12% and corn starch different (p≤0.05). The viscosity of sago1
0.20%.10 starts to appear at the temperature of 76.75
3.3 Protein C, while sago 2 starts to appear at higher
The crude protein content of the sago temperature (78.45 C). Pasting temperature
1 is 0.25% higher than sago 2 (0.16%) of sample starch was significantly different
significantly (p<0.05) as shown in Table 1. (p≤0.05). This result was consistent with the
The crude proteins of the sago starches study of Piyachomkwan et al. (1999) who
found in this study agreed well with the found pasting temperature of 76-77 C.14
published values between 0.13 and 0.2 %.10 Setback of sago 1 was higher than sago 2,
3.4 Ash which amylose affects the setback of
The ash content of sago 1 is 0.0461% starch.15
and sago 2 is 0.0562% as shown in Table 1. 3.8 Differential scanning calorimeter
The ash content of the sago starches is less (DSC)
than previous research, which is equal to DSC results obtained of the various
0.43%.10 The inorganic material normally starch samples are given in Table 3 and
originates in the crop from which the starch curves are shown in Figure 2. The
is isolated and also from the water used to gelatinization temperature for sago starch in
process the starch.12 The fiber contents for the column denotes the significant
all the sago starch studied were low and differences (p≤0.05). Initial temperature,
comparable to fiber contents from other peak temperature and final temperature of
starches.10,13 sago 1 was 67.33, 71.67 and 77.59 C,
3.5 Amylose respectively while sago 2 had 69.65, 73.33
The amylose content of sago 1 is and 77.61 C, respectively.
53.29% and sago 2 is 54.14% as shown in The gelatinization temperature for
Table 1. The amylose content of sago 1 and sago starches were high compared to corn,
sago 2 was not significantly different. It has pea and potato but low compared to starch
been reported by other experts that amylose from sweet potato, tania and yam.16,17 No
in most sago flour is in the range of 20% to significant differences in the gelatinization
30%. For example, the value is in the range enthalpy between sago starch samples were
of 22-31%10 or 27.2%.11 However, the observed. The gelatinization temperature
difference in the amylose content probably and enthalpy of the starches depends on the
arises as a result of cultivation of the crop, microstructure and degree of crystallinity
conditions of cultivation, seasons or within the granule and also on granule size
harvesting the sago palm at different stages and the amylose to amylospectin ratio.
of its growth.10,12,13
Table 1. Chemical properties of sago starch
Starch samples Moisture (%) Ash (%) Crude fat (%) Crude protein (%) Amylose (%)
Sago 1 14.20 0.0461 0.17 0.25 53.29
Sago 2 17.32 0.0562 0.19 0.16 54.14
Figure 1. SEM images of sago starch (a is sago 1, b is sago 2)
Table 2. Property of viscosity of sago starch

Pasting Peak Final Holding
Sample Peak time Breakdown Setback
temperature viscosity viscosity strength
starch (min) (cP) (cP)
(C) (cP) (cP) (cP)
Sago 1 4.00 76.75 4477 3458 2300 2199 -1041
Sago 2 4.20 78.45 5401 3458 3331 2070 -1943
Table 3. Gelatinization temperature of sago Normally, the smaller the granule, the higher
starch will be the gelatinization temperature.18
Sample TI (C) TP (C) TF (C) H (J/g)
starch 4. Conclusion
Sago 1 67.33 71.67 77.59 15.11
The chemical compositions of two
Sago2 69.65 73.33 77.61 14.30
TI: Initial temperature; TP: Peak temperature; TF: sago starch samples were obtained from two
Final temperature; ∆H : Enthalpy (J/g) preparation methods. The results found that
moisture, fat, ash and amylose content of
sago starch from either two methods were
not statistical differences. The amylose
content is different from other research that
is in the range of 20-30%. However, the
difference in the amylose content most
probably arises as a result of cultivation of
the crop, conditions of cultivation, seasons
or harvesting the sago palm at different
stages of its growth.10,12,13 There was no
significant difference in the average
diameter or shape of sago starch samples.
Figure 2. DSC heating curves of sago 1 and Comparison of physical and chemical
sago 2 properties of sago powder from traditional
method and mechanical method were not
different. Sago starch obtained from 8. Azudin, M. N.; Lim, E. T. K.
machine method may be better in terms of Proceedings of the Fourth International
time and energy. Sago Symposium. Sarawak, Malaysia,
1991; pp 149–52.
Acknowledgements 9. Fujii, S.; Kishihara, S.; Komoto, M.
The Department of Industrial Proceedings of the Third International
Chemistry, Science and Technology Faculty, Sago Symposium. Ibaraki, Japan, 1986,
Rajamangala University of Technology 186–92.
Srivijaya, Thungsong, Nakhon Si 10. Fasihuddin, B. A.; Williams, P. A.;
Thammarat. Doublier, J. L.; Durand, S.; Buleon, A.
Carbohydr. Polym. 1999, 38, 361–370.
References 11. Cui, R.; Oates, C. G. Carbohydr. Polym.
1. Jong, F. S. PhDs Degree Thesis, The 1997, 32, 65–72.
Natherlands Wageningen Agriculture 12. Sriroth, K. The first FAO regional round
University, Malaysia, 1995. table. Thailand, 1999, 136–172.
2. Mathur, P. N.; Riley, K. W.; Rao, V. R.; 13. Defloor, I.; Dehing, I; Delcour, J. A.
Zhou, M. Proceedings of the Sixth Starch/Stärke 1998, 50, 58–64.
International Sago Symposium. 14. Piyachomkwan, K.; Chotineeranat, S.;
Pekanbaru, Riau University, Indonesia, Chollakup, R.; Hicks, A.; Oates, G.;
1998; pp 1–6. Sriroth, K. The first FAO regional round
3. Ruddle, K. R. Proceedings of the First table, Thailand, 1999; pp 173–184.
International Sago Symposium. Kuala 15. Wang, L.; Wang, Y. J.; Porter, R. J.
Lumpur, Malaysia, 1977, 53–64. Agric. Food Chem. 2002, 50, 2695–
4. Ishizuka, K.; Hisajima, S.; Macer, D. R. 2699.
J. Proceedings of UNESCO-University. 16. Tian, S. J.; Rickard, J. E.; Blanshard, J.
Tsukuba Sci City, Japan, 1995; pp 75–6. M. W. J. Sci. Food Agr. 1991, 57, 459–
5. Singhal, R. S.; Kennedy, J. F.; 491.
Gopalakrishnan, S. M.; Kaczmarek, A.; 17. Veletudie, J. C.; Guadeloupe, L.;
Knill, C. J.; Akmar, P. F. Carbohydr. Colonna, P.; Bouchet, B.; Gallant, D. J.
Polym. 2008, 72, 1–20. Starch 1995, 47, 289–306.
6. Cecil, J. E.; Lau, G.; Heng, S. H.; Ku, C. 18. Cowburn, P. IRL Press: Oxford, 1989;
K. Tropical Products Inst.: London, 1982. pp 79–88.
7. Morris, H. S. Proceedings of the First
International Sago Symposium. Kuala
Lumpur, Malaysia, 1977, 121–42.
Effects of subcritical water treatment on functional properties of
soy protein isolate
Chutikarn Chuchaisakpichit1, Issara Slamala2*, Thongchai R. Srinophakun1*,
Kittiwut Kasemwong2
1
Department of Advance and Sustainable Environmental Engineering Program, Faculty of Engineering,
Kasetsart University, Bangkok 10900, Thailand
2
Nano Agro and Food Innovation Laboratory, National Science and Technology,
*E-mail: issara@nanotec.or.th, fengtcs@ku.ac.th
Abstract:
Subcritical water treatment (SCW) is a noticeable alternative method used to develop
the functional properties of protein due to a non-toxic, inexpensive and environmentally
friendly. This work investigated the effects of SCW on soy protein isolate (SPI) prepared
from defatted soybean. Functional properties of SPI were analyzed in terms of protein
solubility and viscoelastic properties. 5% (w/v) SPI was treated by SCW for 5, 15 and 30 min
at 120 C, compared to untreated SPI. The solubility of subcritical water treated-soy protein
isolate (SCW-SPI) for 5, 15, 30 min and untreated SPI samples were 51.10±0.40, 52.00±1.22,
56.00±2.08 and 46.57±4.29%, respectively. Because of protein aggregates dissociation, the
more treatment time, the more solubility has been found. The viscoelastic properties of all
samples exhibited the weak gel because their G′ values of all samples were higher than G′′
values. Moreover, the G′ values of SCW-SPI were decreased when increasing the time of
treatment due to collapse of complex protein networks. The results suggested that functional
properties of SPI were affected by SCW. This could be considered as an alternative process
to modify protein.
1. Introduction properties of soy protein isolate can be

Soy protein isolate is extracted from improved without acids or enzymes.
defatted soy meal which is a major by-
product obtained from cooking oil 2. Materials and Methods
producing. The protein content of soy 2.1 Materials
protein isolate contains at least 90% on dry Soy protein isolate (SPI) was
weight basis.1,2 Soy protein isolate have been obtained from Chemipan Corporation Co.,
widely used in the main part of soy-based Ltd (Thailand). The protein content of SPI
food production.3 In the past, the functional was 90% measured using Lowry’s method.6
properties of soy protein isolate were 2.2 Subcritical water treatment (SCW)
improved by physical, chemical and 5% (w/v) SPI was hydrated into
enzymatic treatment. However, these deionized water and loaded into the high
process have been realized their pressure and temperature reactor which was
disadvantages of; for example, expensive, set at 80% volume of the reactor. SCW was
non-reusable, hardly prospective and toxic.4 performed at 120 °C for 5, 15 and 30 min
Alternatively, subcritical water process and cooled down at 70 °C for safety reason
provides more attended investigation since before opening the reactor. Then, the
this process is non-toxic and solution was filtered through a filter paper
environmentally friendly. Typically, (Whatman no. 1) and spray-dried at inlet
subcritical water is liquid state under the temperature of 130 °C by Buchi B-290 mini-
high temperature and pressure (100-374 °C spray dryer (Buchi, Switzerland). The
at 0.10-22.10 MPa).5 For this reason, the mixture was fed at a feed flow rate of 10
mL/min. Finally, subcritical water treated- to untreated SPI because the high
soy protein isolate (SCW-SPI) stored at temperature water separated into hydrogen
room temperature for further analyses. and hydroxide ions. These ions performed
2.3 Protein solubility the intermolecular action during protein
1% (w/v) SPI and SCW-SPI solution aggregates. This phenomenon will cause the
were centrifuged at 10,000 x g for 10 min at smaller size of protein. It was noticed that
20 °C. The supernatant was collected and the protein aggregates dissociated and
measured by Lowry’s method6 using bovine expanded the extent of hydrogen bonding
serum albumin (BSA) as the standard. The which has a more protein-water reaction.7,8
protein solubility was calculated as grams of The solubility of SCW-SPI increased with
soluble protein/100 g of protein sample. treatment time. The highest solubility SCW-
2.4 Viscoelastic properties SPI is 30 min. Therefore, the higher
1.5 g of SPI and SCW-SPI were treatment time, the higher solubility can be
dissolved in 10 mL of deionized water and approved.
gently stirred for 1 hr at ambient
temperature. Then, the samples were stored Table 1. Solubility of untreated SPI and
overnight at 4 °C. Viscoelastic properties SCW-SPI at various times
were monitored using Modular advanced Time (min) Solubility (%)
rheometer system (HAAKE MARS 60, Untreated 46.57 ± 4.29
5 51.10 ± 0.40
ThermoFisher Scientific, Germany) and 15 52.00 ± 1.22
equipped with a parallel plate (35 mm plate 30 56.00 ± 2.08
diameter). After loading and removing the
excess sample, the gap between both of plate 3.2 Viscoelastic properties
is 1 mm. Then, the silicone oil was applied The viscoelasticity of foods is a
to seal the cap for moisture loss prevention. useful property to evaluate behavior such as
A frequency sweep test was measured for gel strength and protein coagulation.
the following step: 1) a linear heating step Oscillatory testing is a common dynamic
from 25 to 90 °C at 1.5 °C/min with a method used to study the viscoelastic
constant frequency rate at 0.1 Hz, 2) properties. A viscoelastic network is
frequency sweep step from 1 to 100 Hz at 90 described by the storage modulus (G′) and
°C, 3) a linear cooling step from 90 to 25 °C the loss modulus (G′′) which show the
at 1.5 °C/min with a constant frequency rate elastic and viscous response, respectively.16
at 0.1 Hz, 4) frequency sweep step from 1 to Figure 1 illustrates the variation of
100 Hz at 25 °C. All measurements were dynamic moduli (G′ and G″) during
operated within 1% strain amplitude and the temperature from 25 to 90 C. The G′ value
linear viscoelasticity regimen. Each sample of untreated SPI was greater than G″ value
was measured in triplicate. over a range of temperature. At low
temperature, tendency of both values
3. Results and Discussion negligibly decreased until temperature
3.1 Protein solubility
reached 80 C. Then, the values
The solubility is one of the most
continuously increased, regarding protein
important properties because protein should
swelled and formed gel. The G′ values of all
form homogeneously in food texture This
SPI-SCW were lower than G″ values at
property affects other functionalities such as
preliminary temperature because of the
emulsifying, foaming and stability.1,16
liquid nature of the solution.8 However,
Table 1 shows the effect of SCW on the
these values conversely changed after
solubility of soy protein isolate which was
temperature heated up to 60 C since the
treated at 120 °C for 5, 15, 30 min (SCW-
protein induced gel formation. Furthermore,
SPI) and untreated SPI (0 min). The
the values of G′ and G″ rapidly increased
solubility of SCW-SPI increased, compared
and these values of SCW-SPI were higher
than untreated SPI at high temperature. The
increment G′ values indicated the transition
phase from the solution state to the gel state
is defined as the temperature.9,10 Thus, the
temperature used to induce protein gelation
of SCW-SPI is lower than untreated SPI.
Furthermore, the similar behaviors of SCW-
SPI at various times were obtained.
Figure 2. G′ valves (dashed line) and G′′

valves (solid line) of 15% (w/v) SCW-SPI
solutions treated for 5 (blue) 15 (green) and
30 (yellow) compared to untreated SPI (red)
after heating at 90 C.
phobic interactions, the strength and

coagulation of gel increased.14 Moreover, the
values of SCW-SPI were slightly lower than
Figure 1. G′ valves (dashed line) and G′′ untreated SPI as the temperature cooled
valves (solid line) of 15% (w/v) SCW-SPI down, resulting in the possible collapse of
solutions treated for 5 (blue), 15 (green) and these complex networks of protein.
30 (yellow) compared to untreated SPI (red) Furthermore, protein molecules have the
during linear heating from 25 to 90 C at 1.5 lower cross-linking of aggregates.15
⁰C /min
Frequency sweep tests after heating

at 90 C are represented in Figure 2. Over
the entire frequency range of the
measurement, the G′ values of all samples
were higher than G′′ values, indicating
protein formed a weak gel (a paste-like
quality).10 The similar behavior has been
reported for starch and protein gels.12,13 This
behavior could be attributed to the cross- Figure 3. G′ valves (dashed line) and G′′
linkage formations by disulfide bonds and valves (solid line) of 15% (w/v) SCW-SPI
hydrophobic interactions in the gel solutions treated for 5 (blue) 15 (green) and
structures.12,13 The G′ values of SCW-SPI 30 (yellow) compared to untreated SPI (red)
were decreased when increasing time of during linear cooling from 90 to 25 C at
treatment and SCW-SPI for 30 min 1.5 C min-1.
produced the lowest G′ value. It
demonstrated that SCW treatment After cooling step, Figure 4 displays
interrupted the interaction of gelation when the differentiation of G′ and G′′. These
increased duration of SCW treatment. values were higher than the values of step 2
Upon cooling (Figure 3), the relation (the frequency sweep tests after heating) As
of G′ and G′′ values diversed from heating a result, protein reinforces the weak
step. G′ values of all samples were greater tridimensional network initially formed and
than G′′ values throughout the steps. These enhances theirs dynamic moduli.9 The G′
values showed the increasing trend during values of all SCW-SPI were similar and still
decreasing temperature due to the lower than untreated SPI since disulfide
enhancement of unfolded protein and hydro
bonds and hydrophobic interactions were Special thanks are also extended to Dr.
obstructed cross-linkage in the gel structure Onanong Nuchuchua and Ms.Wanwisa
by heat.12 Srinuanchai for their guidance and great
support.
References
1. Liu, K. Soybeans: chemistry, technology
and utilization Chapman & Hall: New
York, 1997.
2. Lin, S.; Huff, H. E.; Hsieh, F. J. Food
Sci. 2000, 65, 264269.
3. Krintiras, G. A.; Göbel, J.; van der Goot,
A. J.; Stefanidis, G. D. J. Food Eng.
Figure 4. G′ valves (dashed line) and G′′ 2015, 160, 3441.
valves (solid line) of 15% (w/v) SCW-SPI 4. Clemente, A. Trends Food Sci. Technol.
solutions treated for 5 (blue) 15 (green) and 2000, 11, 25462.
30 (yellow) compared to untreated SPI (red) 5. Natzle, W. C.; Moore, C. B. J. Phys.
after cooling at 25 C Chem. A. 1985, 89, 26052612.
6. Lowry, O. H.; Rosebrough, N. J.; Farr,
4. Conclusion A. L.; Randall, R. J. J. Biol. Chem. 1951,
In summary, SCW had the effect on 193, 265275.
solubility and viscoelastic properties of SPI. 7. Pelegrine, D. H. G.; Gasparetto, C. A.
The solubility of SCW-SPI increased, Food Sci. Technol. 2005, 38, 7780.
compared to untreated SPI. Moreover, the 8. Zhang, Q. T.; Tu, Z. C.; Wang, H.;
solubility of SCW-SPI was increased when Huang, X. Q.; Fan, L. L.; Bao, Z. Y.;
increasing treatment time. The viscoelastic Xiao, H. J. Food Sci. Technol. 2015, 52,
properties of SCW-SPI; represented by G′ 34123421.
and G′′, were lower than untreated SPI 9. Rocha, C.; Teixeira, J. A.; Hilliou, L.;
because the complex networks of protein Sampaio, P.; Gonçalves, M. P. Food
were disrupted. The results lead to the Hydrocoll. 2009, 23, 17341745.
conclusion that SCW could be the 10. De Maria, S.; Ferrari, G.; Maresca, P. J.
alternative method to modify functional Food Eng. 2015, 153, 3944.
properties of SPI for food processing. 11. Zhong, Q.; Jin, M. J. Dairy Sci. 2008,
91, 490499.
Acknowledgements 12. Manoi, K.; Rizvi, S. S. H. Food Res. Int.
This study was financially supported
2008, 41, 786796.
by the Thailand Graduate Institute of
13. Wang, C.; Johnson, L. A. J. Am. Oil
Science and Technology (TGIST) [grant
number TG-55-11-59-033M] under the Chem. Soc. 2001, 78, 189195.
National Science and Technology 14. Resch, J. J.; Daubert, C. R. Int. J. Food
Development Agency (NSTDA), Ministry Prop. 2002, 5, 419434.
of Science and Technology and National 15. Shim, J.; Mulvaney, S. J. J. Sci. Food
Nanotechnology Center (No. P1750064). Agric. 2001, 81, 706717.
We also acknowledged Thailand Advanced 16. Manoi, K.; Rizvi, S. S. H. Food Rer. Int.
Institute of Science and Technology and 2008, 41, 786796.
Tokyo Institute of Technology (TAIST-
Tokyo Tech) program NSTDA.
Leucaena leaves: an oyster adjective
Rattana Wongchuphan1*, Supaporn Apirattananusorn2, Patcharee Lungmann3,
Naengnoi Saengsane4
1
Program of Chemistry, Faculty of Science and Technology, Suratthani Rajabhat University,
Muang, Suratthani 84100, Thailand
2
Program of Food Science and Technology, Faculty of Science and Technology,
Suratthani Rajabhat University, Muang, Suratthani 84100, Thailand
3
Program of Biology, Faculty of Science and Technology, Suratthani Rajabhat University,
Muang, Surat Thani 84100, Thailand
4
Program of Chemistry, Faculty of Science and Technology,
Nakhon Si Thammarat Rajabhat University, Muang, Nakhon Si Thammarat 80280, Thailand
*E-mail: wongchuphanr@yahoo.com, rattana.won@sru.ac.th
Abstract:
Amylase in leucaena leaves was an expected enzyme for the function of glycogen
digestion whenever a Crassostrea belcheri oyster is chewed with leucaena leaves. Amino
acids in C. belcheri oyster and leucaena leaves were determined by chromatographic method.
The content of amylase in leucaena leaf extract was determined by dinitrosalicylic acid
(DNS) method. Strong sweetness produced in a mixture of oyster, leucaena leaves and lime
juice were experimentally proved as mimiking while chewing truly. It was found that both
leucaena leaves and C. belcheri oyster are plentiful of essential amino acids such as histidine
(141.511.80; 363.381.05 mg/100 g), isoleucine (158.450.12; 213.551.75 mg/100 g),
leucine (339.040.98; 527.751.71 mg/100 g), lysine (298.452.50; 479.062.75 mg/100 g),
methionine (53.550.77; 251.950.45 mg/100 g), threonine (258.370.93; 337.871.50
mg/100 g), tryptophan (116.082.47; 127.110.25 mg/100 g), valine (218.580.12;
259.210.25 mg/100 g). The leucaena leaves contained 12.77 U/mL of the amylase. In
various mixtures, compared to only the oyster, the content of glucose was increased
significantly in the mixture of oyster and leucaena leaves with and without lime juice. Studies
indicated the presence of the amylase-containing leucaena leaves is a key role in enhancing
sweetness of eating with the raw oyster and lime juice.
1. Introducution motto and tourists should have never missed

Surat Thani (or shortened name, to try it.
Surat) is the largest province of the South of The white-scar oyster (Crassostrea
Thailand, and located on the western shore of belcheri) belongs to the genus Crassostrea
the Gulf of Thailand.1 The city of good from Asia as being the most commercial
people is given as the meaning of Surat aquatic species in the world and one of
Thani. There are three other remarkable local notable species of five oyster species
products including the hand-woven silk cloth available in Thailand.2,3 In Surat Thani
from the coastal village Phum Riang in province, C. belcheri (or Surat oyster) is a
Chaiya district. Also, the famous source of large oyster and has been well known as a
red eggs from ducks fed with shrimps and premium and healthy food. As high in
then preserved in a soil-salt mixture is a local demand, preservation with remaining the
specialty. Fresh white-scar oysters from quality is essential for product
farms at the coastline of Kanchanadit district transportation.4,5 Oyster farming has been a
are the most famous origin of clean, large prosperous part of the culture at Tha Thong
and tasty oyster offered by nature. Thus, the estuary and Kradae canals in Kanchanadit
oyster has become a part of the provincial district for many decades. Due to suitable
physical and potential sites such as intertidal 2.2 Preparation of oyster and leucaena
brackish water for several months a year, leaf samples
these sites are considered much tastier than A number of oysters (2-4 years old)
their ocean-produced counterparts.6 Oysters were purchased from the suppliers in
range in sizes from S (1-2 years old) to L (4 Karnchandit district, Surat Thani, starting
years old). Commonly, all sizes demand to from October 2014 to February 2015 during
be consumed raw with vegetable side dish the rainy season. It will taste firm, springy
such as leucaena (Leucaena leucocephala and delicious in rainy season as sweetness is
Lam. de Wit leaves, called “Kratin”, in Thai) driven by the season.6 The clean oyster flesh
which belongs to the family Fabaceae. was homogenized and frozen at -20C prior
Leucaena is a fast-growing tree in tropical to use. Clean young leucaena leaves (5-6 cm
region and known as a nutritive plant.7 long of Yod Kratin) available in the same
Several potential uses such as nitrogen area were blended without water addition
source in leaves for crop production have and then squeezed to obtain the liquid
been established.8 Recently, leucaena can be extract.
applied for renewable energy from biomass- 2.3 Determination of enzyme activity
based gasification,9,10 wood production,11 Amylase activity in the liquid extract
feedstock from protein-abundant leaves,12 of leucaena was analyzed by dinitrosalicylic
and cosmetic manufacture from new seed acid (DNS) method with minor modification
oil.13 of Bailey’s procedure.14 The starch solution
An art of food linked with the history (1.8 mL) used as the substrate was heated in
from the past to new generations has a water bath at 50C and followed by adding
developed an economic indicator of 200 L of enzyme solution for 5 min
community and a treasury of folk wisdom. incubation. After adding 3.0 mL of DNS, the
The ease of leucaena leaves encourages the mixture was mixed well and boiled for 15
level of use as a side dish with raw oyster. min.15 The prepared sample was readily
Leucaena leaves have been well known for measured by spectrophotometer at 540 nm.
eating with C. belcheri oyster since local The reagent blank and enzyme blank were
people have found it for many decades. prepared by mixing 1.8 mL substrate, 3.0 mL
These amazing culinary experiences during DNS, and 0.2 mL of Na-citrate buffer
traveling were recommended to (enzyme solution). The unit was defined as
visitors. However, in our survey, the followed: one unit of amylase is the amount
explanation of sweetness enhancement of enzyme required to liberate 1 mol of
occurring by chewing raw oyster in the glucose from soluble starch in one minute.
presence of lime juice with leucaena leaves 2.4 Determination of amino acids
has not been provided scientifically. Hence, Qualitative and quantitative analyses
the aim of the present study was to find out for amino acids in both oyster and leucaena
what strong sweetness would be involved in leave samples were carried out by HPLC
the presence of leucaena leaves used as an method.16
oyster adjective. Amino acids and amylase 2.5 Glucose analysis of the mixtures
were also investigated. Raw oyster and leucaena leaves were
mixed with the mass ratio of 50:5.17
2. Materials and Methods Pretreatment only for the oyster was needed
2.1 General prior to make a mix.18 Non-aqueous species
All chemicals were in analytical was firstly removed by petroleum ether for
grade and supplied by Merck (Germany) and three times (30 ml each). The solvent was
Sigma-Aldrich. Amino acids in oyster and then eliminated by heat in an oven at 60C.
leucaena leaf samples were analyzed The different mixtures of C. belcheri
individually using HPLC technique. oyster (OS), leucaena leaves (LS), lime juice
(LJ) and amylase (Enz) were prepared as digesting carbohydrates like starch and
mentioned in Table 1. glycogen. As a result, amylase in leucaena
leaf liquid was higher than that in human
Table 1. Four mixtures for glucose analysis saliva by 4620, approximately 5 thousand
No. of trial Mixtures times. It was convinced that additional
OSa LSb LJc Enzd sweetness while chewing leucaena leaves
(g) (g) (mL) (mg)
and oyster in acidic medium might occur in
1 50 - - -
2 50 5 - - human mouth as carbohydrate digestion by
3 50 5 2 - amylase in that leaves. Additionally, salivary
4 50 5 2 50 amylases are also able to break down firstly
5 50 5 2 500 the polymer to small sugars which are
a
OS = oyster ultimately converted to the individual basic
b
LS = leucaena leaves
c
LJ = lime juice glucose units. Leucaena leaves were exposed
d
Enz = amylase in the other aspect of amylase in plants
suggested earlier in previous reports.20
For sugar analysis, each treatment 3.2 Amino acids
was homogenized at 6,000 rpm for 1 min. As shown in Table 2, eighteen amino
The sample was added with 15% (w/w) of acids were found in raw oyster sample i.e,
potassium hexacyanoferrate(II) and 30% alanine (Ala), arginine (Arg), aspatic acid
(w/w) zinc sulfate for 5 mL each. After well (Asp), cysteine (Cys), glutamic acid (Glu),
mixing for 15 min at room temperature, 10 glycine (Gly), histidine (His), isoleucine
mL of acetonitrile was added to the mixture (Ile), leucine (Leu), lysine (Lys), methionine
and 100 ml of final volume was carried out (Met), phenylalanine (Phe), proline (Pro),
by distilled water. The homogeneous mixture serine (Ser), threonine (Thr), tyrosine (Tyr),
was centrifuged at 5,000 rpm for 2 min. The tryptophan (Try) and valine (Val). The oyster
supernatant was filtered by 0.45 micron meat had both essential and non-essential
membrane for HPLC analysis to determine amino acids, similar to other species reported
how much glucose was existed. earlier.21
2.5 Statistical analysis
A completely randomized design was Table 2. Amino acids found in oyster and
carried out by the mean difference (95% leucaena leaf samples
Amino acids Content in Content in
confidence level) using the Duncan's New oyster sample leucaena leaf
Multiple Range Test. All data calculations (mg/100 g) sample (mg/
were performed using SPSS software version 100 g)
11.5 for Windows. Alanine 501.403.53 300.583.73
Arginine 571.873.97 484.131.40
3. Results and Discussion Aspatic acid 828.720.19 724.330.16
Cystine 133.142.34 NFa
3.1 Activity of amylase Glutamic acid 1272.500.50 538.950.13
The activity of amylase in leucaena Glycine 587.170.50 218.610.30
leaf sample was found as 12.77 U/mL. In Histidine 363.381.05 141.511.80
1977, Gillard et al. reported 2.771.12 U/L Isoleucine 213.551.75 158.450.12
(2.7710–3 U/mL) of enzyme activity in Leucine 527.751.71 339.040.98
Lysine 479.062.75 298.452.50
human saliva obtained by a number of Methionine 251.950.45 53.550.77
healthy people.19 With direct Phenylalanine 316.452.56 263.050.21
spectrophotometric method at 400 nm, Proline 406.561.41 195.310.10
p-nitrophenyl -moltoside was used as a Serine 436.650.89 348.820.52
Threonine 337.871.50 258.370.93
substrate. p-nitrophenol was obtained after
Tyrocine 332.480.71 260.810.32
hydrolyzing with alpha-amylase. In fact, Tryptophan 127.110.25 116.082.47
alpha-amylase is a common enzyme in Valine 259.210.25 218.580.12
human saliva which is responsible for a
NF = Not Found
3.3 Glucose analysis content of glucose when the amount of
The appearance of an oyster, plump amylase was risen up for 10 times,
and creamy white, indicates that an oyster is mimicking salivary amylase released more in
full of glycogen and lipids.22 The mixture 1 the presence of acidic lime juice. It was
indicated free glucose (1.87% (w/w)) in raw found that glucose was considerably
oysters in where chewing oyster long enough increased as the increase of amylase about
people can get real sweetness but not very ten times (50 to 500 mg). Amylase derived
strong.23 This is due to enzymes in oyster from both leucaena leaves and human saliva
itself will break down their tissue after the was involved in carbohydrate digestion.
death. In fact, breaking down food such as Results indicated that strong sweet taste
glycogen by our salivary enzyme fits snugly could be yielded significantly in the presence
onto our taste buds detecting sugar, resulting of amylase liberated from both leucaena
sweet favor. The amount of glucose found in leaves and salivary glands which are
mixture 1 was significantly different to that stimulated by acid solution such as lime
in the second trial of mixture 2 and 3, and in juice.
the third one of mixture 4 and 5, shown in
Table 3. 4. Conclusion
The trial of mixture 2 containing The combination of C. belcheri
oyster and leucaena leaf samples resulted in oyster, leucaena young leaves and lime juice
the significant increase of glucose, compared offers perfect sensory attribute. It was
to mixture 1. As a result, glucose analysis confirmed scientifically to account for a
indicates a key role for leucaena leaves in famous oyster cuisine in Surat Thani
strong sweetness development. Hence, it was province, Thailand. The raw oyster was
confirmed that amylase in leucaena leaves composed of smaller sugars once they are
could be associated with sweet perception. dead. Moreover, leucaena leaves and salivary
amylases released more in the presence of
Table 3. Amount of glucose in four mixtures lime juice were considered as an additional
Mixtures Amount of precursor for glycogen digestion in the
glucose human mouth. In the other word, leucaena
(% (w/w))
leaves played a major role of an oyster
1. OS 1.870.03a
2. OS + LS 2.060.10b
adjective due to it is high in amylase content.
3. OS + LS + LJ 2.110.08b Leucaena leaves can be feasible accordingly
4. OS + LS + LJ + Enz (50 mg) 2.200.06b as the other amylase-containing plant source.
5. OS + LS + LJ + Enz (500 mg) 2.580.05c
*Values are mean of duplicate measurementSD Acknowledgements
*The different superscripts (a, b, c) in the same The research project (Grant no.
column are significantly different (p < 0.05)
2556A15962004) was financially supported
by the Office of the Higher Education
Similarly, the addition of lime juice Commission, Thailand. Authors are sincerely
in trial of mixture 3 could not affect an thankful to the Sinmana Farm Stay for their
increase of glucose amount significantly beneficial experiences and interviewees for
when compared to the mixture 2. Lime juice their useful information as clear and
squeezed from fresh fruit provides citric acid convincing evidence to induce this project.
(46 g/L) that is a weak tricarboxylic acid.24 Finally, we would like to thank Suratthani
The acidic lime juice-based preparation for Rajabhat University for their facility support.
eating raw seafood just denatures the
proteins and turns the flesh white, not killing References
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rate of salivation 8 times.26 The trial of i_Province.
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Effect of subcritical water treatment on physicochemical properties
of rice bran protein
Imboon Wongthaweewatan1, Issara Sramala2*, Thongchai R. Srinophakun1*,
Kittiwut Kasemwong2
1
Advance and Sustainable Environmental Engineering, Faculty of Engineering,
Kasetsart University, Bangkok 10900, Thailand
2
  Nano Agro and Food Innovation Laboratory, National Nanotechnology Center (NANOTEC),
National Science and Technology Development Agency (NSTDA), Pathumthani 12120, Thailand
*E-mail: issara@nanotec.or.th, fengtcs@ku.ac.th
Abstract:
Subcritical water treatment is a useful technique for the extraction of active
compounds from biomass materials with short processing time, low cost and environmental-
sustainable. Defatted rice bran is a by-product from the production of rice bran oil, which still
contains protein. In this study, rice bran protein was extracted by subcritical water treatment.
The effects of temperature at 120, 140, 160 and 180 °C and time for 30, 45 and 60 min were
determined. At higher temperatures, the protein content and emulsifying activity index (EAI)
increased. Nitrogen solubility index (NSI) was in the range of 87-100%. The reaction time
affected the emulsifying stability index (ESI) values. The ESI reduced when the reaction time
was increased. The largest molecular size of protein was about 150 kDa and protein bands
were observed between 10-150 kDa. The results indicated that these temperature and reaction
times show the direct impacts on the protein extraction. Moreover, they also affected the
molecular size of rice bran protein, which would be useful in many applications.
1. Introduction compounds has been implemented in many

Rice is one of the most important researches.6,7 This technique performs using
cereal crops in the world and considered to hot water at temperature between 100 and
be the main staple food in many Asian 374 °C under high pressure to maintain its
countries.1 Thailand is in the 6th position of liquid state.8-10 It has been shown that the
rice production; after China, India, subcritical water extraction is cleaner, faster,
Indonesia, Bangladesh, and Vietnam and cheaper than the conventional extraction
respectively, with the rate of 20.26 million method.
tons. Rice bran is considered as byproduct The aim of this study is to propose the
during the rice milling process. It is the most effect of subcritical water extraction on
nutritious part of rice.2 Rice bran is rich in functional properties of rice bran protein.
protein, lipids, soluble and non-soluble fiber,
vitamins, and minerals. Rice bran offers 2. Materials and Methods
various health benefits due to its content of 2.1 Raw material
vitamin E, B vitamins, essential fatty acid, Defatted rice bran was obtained from
and antioxidant. It is a naturally lactose-free, Kasisuri Co., Ltd., Nonthaburi, Thailand.
gluten-free, and hypoallergenic.3 Rice bran 2.2 Subcritical water hydrolysis
proteins are mostly have proteins; albumin, The subcritical water extraction was
globulin, prolamin, and glutelin.4 They are conducted using a vessel made of stainless
highly nutritional and have functional steel and resistant to high pressure and
properties comparable to other cereal temperature as described in our previous
proteins.5 Recently, subcritical water as an paper. A 100 g of bran sample and 500 mL
environment friendly technique for of distilled water were placed in the vessel.
decomposition and extraction of variety The extraction was performed from 120,
140, 160 and 180 °C for 30, 45 and 60 min. was measured immediately after emulsion
After the desired conditions were achieved, formation was expressed as emulsifying
the vessel was immediately removed from activity of protein, and emulsion stability
the oven and cooled in an ice bath to room was determined as
temperature. The bran mixture was
centrifuged at 12,000 rpm for 20 min. The Emulsifying activity index (m2/g) = 2x2.203xA500 (2)
soluble products of hydrolysis were freeze 0.5xP
dried at -40 °C and stored in a desiccator
until use. A0 xDT
Emulsifying stability index (min) = (3)
2.3 Protein content DA
The protein content of the bran where A500 is the absorbance of emulsion at
extract was assessed by modifying the 500 nm, P is protein content, A0 is the
Lowry–Folin assay.11 The aqueous standard absorbance of the emulsion at 500 nm after
solutions of BSA were used for preparation homogenization, A10 is the absorbance of
of calibration curve. Alkaline solution (5 the emulsion at 500 nm after
mL) was added to the protein samples or to homogenization for 10 min, △T is 10 min
the standard BSA solution and mixed well. and △A is A0 – A10
After standing for 15 min at ambient 2.6 Gel permeation chromatography
temperature, the diluted Folin-Ciocalteu Molecular weight distributions of
reagent (1:1) was rapidly added with rice bran protein were determined by gel
immediate mixing. The mixture was stored permeation chromatography (GPC)
®
for 30 min before measuring the absorbance performed with TSKgel G4000SWXL
at 750 nm with the uv-vis spectrophotometer column (7.8 i.d.  30 cm, TOSOH, Tokyo,
and against blank containing deionized Japan) at 25 °C. The mobile phase was
water. 50 mM sodium phosphate (pH 7.2)
2.4 Nitrogen solubility index (NSI) containing 50 mM sodium sulfate, and a
Protein dispersion (1%) was made flow rate was 0.7 mL/min. Protein samples
with distilled water, centrifuged (5 mg/0.5 mL mobile phase), centrifugation
(10,000 g/10 min) and analyzed for protein at 10,000g for 20 min and filtration through
content in the supernatant following Lowry 0.45 µm nylon filter membrane. The elution
method.11 NSI was expressed as percentage was monitored at 280 nm. The protein
of total protein in concentrate. standard mixture (Sigma-Aldrich, USA),
containing thyroglobulin bovine (670 kDa),
Total protein content in supernatant
NSI (%) = x100% (1) γ-globulins (150 kDa), albumin chicken (44
Total protein content in sample
kDa) and ribonuclease (13 kDa) was used as
2.5 Emulsifying activity and emulsion molecular weight standards for calibration.
stability 2.7 Electrophoresis
Emulsifying activity (EA) and Sodium dodecyl sulfate–
emulsion stability (ES) were determined by polyacrylamide gel electrophoresis
using a method of Pearce and Kinsella.13 (SDS–PAGE) was performed in a vertical
Protein sample dispersion 1.0% (w/v) by electrophoresis unit according to the method
distilled water. Soybean oil (10 mL) and 30 of Laemmli,13 using 12% mini-PROTEAN®
mL of proteins samples was homogenized in TGX™ Precast protein gel, 10-well, 50 µL
a mechanical homogenizer at 22,000 rpm for (BIO-RAD, CA, USA). A sample buffer
2 min at room temperature to produce the was used for non-reducing SDS–PAGE. The
emulsion. The 50 μL of emulsion were protein samples (15 mg) were dissolved in
pipetted from the bottom of the container at 300 µL sample buffer, followed by heating
0 and 10 min after homogenizing and mixed in boiling water for 5 min and centrifugation
with 5 mL of 0.1% SDS. Absorbance of at 10,000 g for 10 min before
emulsions was measured at 500 nm with the electrophoresis. Protein brans were stained
uv-vis spectrophotometer. The absorbance
with Bio-Safe™ Coomassie G-250 Stain. extracted rice bran was effective to the ESI
2.8 Statistical analysis values. The ESI was reduced when the
Data were presented as mean ± reaction time increased might due to the
SEM. Comparisons among the groups using smaller of protein molecular size.
one-way ANOVA followed by Ducan’s
method. Data were considered statistically
significant when P ≤ 0.05.

3.1 Protein content
Protein content of defatted rice bran
extracted by subcritical water hydrolysis
was shown in Table 1. The results showed
that protein content increased as the
temperature increased.14 The highest protein
content was shown at 180 °C, 60 min (30.31
± 0.57%).
3.2 Nitrogen solubility index (NSI)
Rice bran protein had NSI in the
range of 86-100% (Table 1). The results
were calculated follow by the equation (1). Figure 1. (A) Emulsifying activity (Abs 500
The increased protein solubility could be nm) (B) Emulsifying stabilities bar with
due to the protein size that was smaller. different letters were significantly different
High nitrogen solubility is required for (P ≤ 0.05).
protein concentrates to be used as functional
ingredients in many types of food.15 3.4 Electrophoresis of protein
3.3 Emulsifying activity and emulsifying The electrophoresis patterns of rice
stability bran protein extraction (Figure 2) were
The emulsifying activity and determined at various times, 160 °C. The
emulsifying stabilities of rice bran protein comparison between extracted and non-
hydrolysis were shown in Figure 1. The extracted rice bran protein was found that
results were calculated, followed by the the extraction of rice bran protein at various
equations (2) and (3). The emulsifying times and temperature affected to the
activity index (EAI) was significantly decreased of molecular weight. The non-
increased (P ≤ 0.05) when the temperature extracted rice bran protein showed the
of the extraction was increased. smearing on SDS-PAGE gel might have the
The result in Figure 1 showed that contamination from other compounds.
the EAI was stable at 160 °C and 180 °C, The bands of extracted protein at
but significantly higher compared to 120 °C various times and temperatures were
and 140 °C (P ≤ 0.05). According to the observed between 10-150 kDa. There is a
high temperature affected to the molecular study determined the molecular weight
size of protein, which had smaller molecular range of RBP hydrolysates between 1 and
size than the low temperature. The 150 kDa,16 which similar to the degree of
emulsifying stability index (ESI) was hydrolysis of rice bran protein in this study.
showed in Figure 1B. The reaction time of
Table 1. The protein contents and nitrogen solubility index of rice bran protein
Temperature (˚C) Reaction time (min) Protein content (%) NSI (%)
120 30 10.90 ± 0.08a 86.54 ± 1.74a
45 10.86 ± 0.13a 90.10 ± 2.11ab
a
60 10.70 ± 0.68 100.70 ± 3.25c
b
140 30 12.47 ± 0.35 100.82 ± 4.69c
b
45 12.47 ± 0.36 95.07 ± 0.50bc
60 13.59 ± 0.06b 95.22 ± 0.63bc
160 30 15.24 ± 0.20c 95.26 ± 3.68bc
d
45 22.01 ± 0.91 87.16 ± 0.68a
e
60 23.47 ± 0.86 96.08 ± 0.23bc
de
180 30 22.72 ± 0.02 99.61 ± 0.88c
f
45 26.22 ± 0.13 94.80 ± 1.60bc
g
60 30.31 ± 0.57 96.43 ± 1.23bc
a-g
Different letters in the column indicate significant (P ≤ 0.05) difference among groups
The chromatogram of size A

exclusion was measured by gel permeation
chromatography (GPC) (Figure 3). The
result could not observe since the B
molecular size of rice bran proteins was
lower than the standard peaks. Trends of C
molecular size were smaller when the
extraction time was increased.
D
Figure 3. (A) Protein standard mixture 15-

600 kDa, (B) RBP 120 °C, (C) RBP 140
°C, (D) RBP 160 °C, (E) 180 °C for 30
min
decreased with time reduced. Rice bran

protein had NSI in the range of 86-100%.
Reaction time and temperature affected to
the molecular size of rice bran protein.
Figure 2. SDS-PAGE of RBP and defatted
rice bran, Lane 1 is the marker, 160 °C for Acknowledgements
30 min (Lane 2), 160 °C for 45 min (Lane The authors gratefully thank the
3), 160 °C for 60 min (Lane 4) and support of this work provided by Kasetsart
defatted rice bran that non-extracted (Lane University, TAIST-Tokyo Tech and
5) National Science and Technology
Development Agency program. We would
4. Conclusion like to extend special thanks to Kasisuri
The rice bran protein was prepared Co., Ltd., Nonthaburi, Thailand for
by subcritical water hydrolysis. The supplying raw material (defatted rice
highest protein content was shown at 180 bran). The authors also thank Assoc. Prof.
°C, 60 min (30.31±0.57%). At high Shimoyama Yuzuke of Tokyo Institute of
temperature, the protein content and EAI Technology and staff of Nano Agro and
were increased, while the ESI was
Food Innovation laboratory for their help 9. Asl, A. H.; Khajenoori, M. in Mass
and valuable suggestions. Transfer-Advances in Sustainable
Energy and Environment
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Optimization of extraction condition of
microcrystalline cellulose from rice bran residue
Supatsorn Chupong1, Pakorn Opaprakasit1, Kittiwut Kaseamwong2*
1
School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of
Technology (SIIT), Thammasat University, Phathum Thani 12121, Thailand
2
National Nanotechnology Center, National Science and Technology Development Agency, Thailand Science
Park, Klong Luang, Pathumthani 12120, Thailand
*E-mail: kaseamwong@icound.com
Abstract:
Microcrystalline cellulose (MCC) that was extracted from Rice bran residue (RBR)
contains a high crystalline structure. It can use as a valuable additive in several fields. This
work aimed to determine the optimum conditions for MCC extraction from the RBR. RBR
was extracted by alkaline treatment and bleaching process to produce rice bran cellulose
(RBC). Latterly, the crystalline structure was extracted to form of MCC by acid hydrolysis.
The results suggest that two main factors effecting the qualities of RBC are sedimentation
time in the alkaline treatment and NaClO2 concentration. The characteristic of RBR, RBC and
MCC were investigated by Scanning Electron Microscope (SEM), Fourier Transform
Infrared Spectroscopy (FTIR) and X-ray diffraction (XRD). The results revealed that the
proper conditions are 24 h sedimentation time and 2% (w/v) of NaClO2 since it can change
RBC into white color. FTIR spectra showed a characteristic peak at 1604, 1515 and 1320
cm-1 that corresponded to the benzene ring, aromatic ring of lignin and C-O stretching
vibration of hemicelluloses, respectively which disappeared in RBC and MCC. In XRD result
showed the increase in crystallinity of RBC comparing to RBR and that of crystallinity is
higher after converting RBC into MCC.
1. Introduction thus it can make more benefits by extracting

Rice production in Thailand is a microcrystalline cellulose.
significant portion of Thai economic, which Microcrystalline cellulose (MCC) is
is a priority crop in Thailand. Thailand has one of the cellulose derivatives and prepared
been ranked 6 th in rice exporter of the world. by breaking β-1,4 glycosidic bond by acid
Many rice brans are generated during the hydrolysis method. This procedure is widely
milling process in rice milling industry used in general, due to shorter reaction time
which it consists of high amount nutrition than others process and low cost. These
and energy content such as fat, protein, methods are not much different in each
carbohydrate, soluble dietary fiber, the total research work, but the concentration of acid
dietary fiber and etc. 9 0 % of rice bran was and hydrolysis times are quite important for
sold cheaply as an animal feed. Moreover, the properties of MCC. The conditions of
the people try to increase the efficiency and hydrolysis can affect the properties of
the value of them by many ways such as MCC.1 In the extraction process, when the
extraction rice bran oil to produce structure contacts with acidic solutions the
supplementary food. After this process, it amorphous regions and the crystalline
also generated a high amount of rice bran regions are separated and the amorphous
waste and it was dumped or burned in the regions were digested by acid solution. The
disposal process which is a cause of diameter of MCC fiber is approximately 20
environmental problems. Even though it has μm to 30 nm, depending on the source and
no protein or vitamins but its structure the extraction process.
remains carbohydrate and total dietary fiber,
MCC structure contains high mixed with 1,500 mL of 4 % NaOH solution
crystalline structure. In general, the degree in an Erlenmeyer flask under constantly
of polymerization is less than 400 and 10% stirred at room temperature for 30 min, 24
of MCC is less than 5 μm. It has a white hr. and 48 hr.
color, odorless, inert substance, renewable The mixture was autoclaved with
and biodegradable material. Therefore, high-pressure steam sterilizer (TOMY SX-
MCC can be used in a wild range of 700, Japan) as a sterilizing liquid at 121°C
applications such as a texturing agent in for 45 min afterwards wait for sedimentation
food, industry binder and water retainer in about four different time that is no-retention
the pharmaceutical industry as well as a time, 30 min, 24 hr and 48 hr Finally, the
reinforcement agent in the development of remaining solid was filtered with a vacuum
polymer composites.2 Trache et al.3 studied pump (JEIO TECH, Korea) and neutralized
MCC isolation and bio-composites by washing several times with excess
application. They mainly focused on the distilled water.4
MCC isolation procedures and reported that 2.2.2 Bleaching treatment
MCC has many benefits and can use in The bleaching process was modified
several areas both of powder or colloidal from LaCourse et a1. (1994) RBR which
form. Types and age of the plant mainly received by alkaline treatment process was
affected the amount of cellulose, different mixed in varies the concentration of aqueous
source and extraction in varies conditions chlorite solution also in different
like a temperature, reaction time and concentrations about 1%, 2%, and 3%, then
concentration of reagent which can cause adjusted pH with 4% of acetic acid the value
different properties such as porous, moisture is approximate 5±0.2 (SUNTEX SP-2200,
content, bulk density, particle size and Microprocessor pH meter, Taiwan).
amount of crystallinity. Afterwards heated to 80 °C with constant
The aim of this work is to increase agitation by a magnetic stirrer (IKA RCT
the values of rice bran residue as an basic, Malaysia) then filtered by a suction
agricultural waste by using acid hydrolysis filtration. The solid product was mixed with
method to produce microcrystalline ethanol at a ratio of 1:3 (sample over liquor)
cellulose (MCC) including investigation of for a few hours to remove the remaining
the optimum conditions for extraction MCC water, before filtered and washed again with
and study of characteristic as crystallinity, acetone. The product was finally oven dried
chemical structures, bulk and tapping in a hot-air oven (BINDER, Germany) at
densities and amount of cellulose. 50 °C overnight. Latterly, dazzling white of
a powder product which is α-cellulose was
2. Materials and Methods received.
2.1 General 2.3 Synthesis of MCC
Rice bran residue (RBR) was Acid hydrolysis method is
supplied by Kasisuri Co., Ltd, a rice bran oil commonly used in the extraction process of
production company in Bangkok, Thailand. micro or nano-structure extraction and also
Sodium hydroxide pellets (AR grade, used in the microcrystalline cellulose
Quality Reagent Chemical), sodium chlorite extraction as well. This method slightly
80%, hydrochloric acid 37% (technical modified from Ohwoavworhua et al. 30 g of
grade, Sigma-Aldrich), acetic acid glacial α-cellulose from bleaching process was
with more than 80% acid by mass placed into 1000 mL of a Pyrex glass beaker
(Mallinckrodt Chemicals) and then hydrolyzed with 500 mL of 2.5 N
2.2 Synthesis of α-cellulose hydrochloric acids at 105 °C for 15 min with
2.2.1 Alkaline treatment continuous stirring, resulting in white cloudy
This method was developed by suspendable slurry. Then the hot acid mixed
Deepa et al. (1997) 60 g of rice bran residue with 1.5 L of cold tap water that was
followed by vigorous stirring and allowed to 2.7 X-ray diffraction measurement
stand overnight. Then it was washed with The XRD patterns of rice bran, α-
excess water for adjusting pH until neutral, cellulose, and MCC were measured at 40 kV
filtered with a vacuum pump and dried at and 30 mA. The scanning region of the two-
50°C overnight before milling and sieving. theta angle (2𝜃) was conducted from 5° to
The fraction passing through 0.710 mm 40° with a scan rate of 1°/min.
sieve was obtained and stored at room 2.8 Bulk and tapping densities
temperature in a desiccator. The samples were poured into a
2.4 Cellulose and lignin measurement graduate cylinder. The cylinder was lightly
Dry MCC powder was sieved at 0.1 tapped two or three times to collect the
mm and weight. 1 wt% of MCC was taken powder on the cylinder wall. The volume
to a crucible glass before adding 100 mL of was read from the cylinder and bulk density
acid detergent solution at room temperature calculated by the equation below this.
and n-octanol was dropped one or twice into
the sample. Then solution was boiled and
refluxed for 60 min. Afterward, it was For tapping density, the cylinder was tapped
washed with hot water for 3 times and the until the volume no change before reading
two last time washed with acetone. Lately, the value and calculate a bulk density.
samples was dried in an oven about 105 °C
for 8 hr and cooled down in a desiccator 3. Results and Discussion
before weight. 3.1 Comparing the color of α-cellulose
The time after autoclave is the main
factors that affect the color of α-cellulose.
When compared batch 4, 5 and 6 all of this
2.5 Scanning electron microscopy sample used the same concentrations of
measurement NaClO2 and same time before autoclave but
The morphology of rice bran difference time after autoclave the results
cellulose, α-cellulose and MCC was show that α-cellulose have a different color.
determined on a HITACHIS-3400N In batch 4 used the least time after autoclave
scanning electron microscope, Illinois, USA the sample has a quite yellow than others.
with a voltage of 10 kV. The sample was Moreover when compared these sample with
mounted on a stub with carbon tape and gold batch 8 at the same time both of before and
coated by Hitachi E-1010 an ion sputter after autoclave but batch 8 used a higher
prior to a current of 15 mA for 1.30 min. NaClO2 concentration which is 3%. The
2.6 Fourier transform infrared results still showed the sample in batch 4 is
spectroscopy measurement higher white than batch 8. Thus, this results
Chemical structures of rice bran can be concluded that the NaClO2
residue, α-cellulose, and MCC were concentration cannot make the sample in
investigated by Fourier transform infrared term of a white color unless leave the
spectroscopy (NICOLET 6700, Thermo sample after autoclaving for increase the
Scientific, USA). Before examining, the reaction time. NaClO2 concentration has a
sample was taken to an incubator for 4 hr. to slight effect on the color of the sample that
removal humidity of the sample. The sample observed from batch 3 and batch 7 as shown
was grinded by mortar and pestle and then in Table 1.
mixed with KBr and the average about 148 3.2 Cellulose and lignin content
mg. The mixture was compressed by using a In rice bran residue are the lowest
hydraulic machine at a pressure of 9.5 tons ADF value because of a structure contains
for 90 sec. All spectra were recorded from high impurities or high amorphous than
4000-400 cm-1. others.
Table 1. The color of MCC in different time after autoclave can be removed the
extraction conditions amorphous region in the samples.
Before- After- NaClO2
Batch
autoclave autoclave (%) Table 2. Acid detergent fiber (%ADF)
Sample %ADF
1 24 hr. - 1 Raw materials 20.575
α-cellulose batch 3 77.6595
2 48 hr. 24 hr. 1 MCC batch 4 93.1436
3 24 hr. 48 hr. 1 MCC batch 5 95.4672
3.3 Scanning electron microscopy SEM

4 30 min - 2
The morphological structures of
different samples are investigated, as shown
5 30 min 24 hr. 2 in Figure 1. Rice bran residue has irregular
fibrils shape like a honeycomb. This
6 30 min 48 hr. 2 structure contained with the impurities
components like fat, protein, pectin, wax
7 24 hr. 48 hr. 2
and others. After alkaline treatment and
bleaching process, the morphology changed
into rod-like fibers occurred in sticky
8 30 min - 3 packed, as shown in Figure 1b since the
impurities component were removal after
9 48 hr. - 3 the chemical treatments.
It has reported that lignin can acts as
a binder in the fiber components because it
For α-cellulose batch 3, 4 and 5 it can form a bridge bond with the cellulose
showed the highest value in batch 4 ester and can be removed by boiling the
followed by batch 5 and the lowest in batch product in 0.4% NaOH solution but for this
3 which is the results from NaClO2 experiment used 4% of NaOH which is the
concentration since batch 3 used l% expectation that a majority of the lignin and
NaClO2. Therefore, it has the low efficiency hemicellulose content would be removed. In
to eliminate lignin content in the sample. the other words during the alkaline
Batch 4 and batch 5 used the same treatment, mainly hemicellulose was
concentrations but different reaction time removed and bleaching process can remove
after autoclave. The results showed batch 4 the rice bran fiber-bundles and separated
has higher %ADF, even though it used into individual fibers.5
lower reaction time because of %ADF not For the MCC structure as shown in
only reported the amount of cellulose but Figure 1c, the morphology has a shortening
also reported lignin content in the sample. of fibers when compared with α-cellulose. It
Therefore, comparison in MCC extractions has reported that no significant differences
was required to obtain more reliable results. between MCC from a different source of
Despite the value of %ADF in batch 4 was raw materials except for the plants that have
higher than batch 5 but when compared with a large amount of pith as a cotton stalks
the results in MCC structure, the results are MCC and the type of acid hydrolysis have
opposite with α-cellulose. It is found that no significant difference to the morphology
batch 4 has lower %ADF than batch 5 as of MCC.
shown in Table 2, meaning than the reaction
3400 1320
(a) a
2900
0. 60 B R 1
1604-1515
0. 50
Abs
0. 40
0. 30
0. 20
c e llulo s e2
0. 60
0. 50
b
Abs
0. 40
0. 30
0. 20
mc c2
0. 50
c
0. 40
(b)
Abs
0. 30
0. 20
40 00 35 00 30 00 25 00 20 00 15 00 10 00 50 0
W aven um bers (c m -1)
Wavenumber (cm-1)
Figure 2. FT-IR spectra of (a) rice bran, (b)

α-cellulose and (c) MCC
3.5 X-ray diffraction (XRD)

(c) The X-ray diffraction pattern of
MCC from batch 3, 4, together with batch 5
is shown in Figure 3. All of the samples
have two main peaks around 15 and 22°
which corresponds to the structure of
cellulose type I.6 However difference in the
intensity and the value of crystallinity index
with highest in MCC batch 5, followed by
Figure 1. SEM images of (a) rice bran batch 3 and batch 4, respectively was
residue (b) α-cellulose and (c) MCC observed. Even though, batch 4 used higher
NaClO2 concentration than batch 3, less
3.4 Fourier transform infrared reaction time after autoclave was used.
spectroscopy (FTIR) These results showed that the reaction time
A spectrum of rice bran residue, α- after autoclave was an important factor that
cellulose and MCC was shown in Figure 2. made MCC has a high Crl than NaClO2
The wavelength of 3400 cm-1 indicated the concentration. Moreover, the result showed
large mount number of various type of the increase in crystallinity of α-cellulose
hydrogen bond which formed by –OH comparing to rice bran residue and it is
groups. Beside, the field of 2,900 cm-1 higher after converting α-cellulose into
showed the band of –CH2 group vibrations MCC as shown in Figure 4.
including symmetric and asymmetric
structures which it tends to decrease in α-
cellulose, following by MCC structures. The
significant portions at 1604-1515 and
1320 cm-1 are due to its benzene ring, the
aromatic ring of lignin and the C-O
stretching vibration of hemicellulose,
respectively. Therefore, this figure can be
confirmed that when rice bran was treated
by the chemical reaction, all of these peaks
will disappear in α-cellulose and
microcrystalline, meaning that the chemical
reaction can remove the all of this Figure 3. XRD patterns of MCC in different
component. conditions
chemical reaction is the main factor that
affects to a crystallinity index of MCC.
The optimum condition is 2%
NaClO2 concentration and 24 hr after
autoclaving for alkaline treatment. At this
condition, it results white color α-cellulose
and highest crystallinity index after
converted to MCC.
Acknowledgements
This project was supported by the
Figure 4. XRD patterns of rice bran residue, Advanced and Sustainable Environmental
α-cellulose, and MCC Engineering of TAIST-Tokyo Tech
program, National Nanotechnology Center
3.6 Bulk and tapping densities and Sirindhorn International Institute of
The porosity of MCC is the Technology, Thammasat University.
important factor in MCC application. Table
3 shows the bulk and tapped densities References
between MCC comparing α-cellulose along 1. Hindi, S. S. Z. Nanosci. Nanotechnol.
with rice bran residue. It is higher bulk and Res. 2017, 4 (1), 17–24.
tapping density in rice bran residue than 2. Chuayjuljit, S.; Su-uthai, S.;
α-cellulose and MCC because rice bran Charuchinda, S. Waste Manag. Res.
residue contained a large amount of 2010, 28 (2), 109–117.
amorphous regions. 3. Mathew, A. P.; Oksman, K.; Sain, M. J.
Appl. Polym. Sci. 2005, 97 (5), 2014–
Table 3. Bulk and tapping densities of Rice 2025.
bran residue, α-cellulose, and MCC 4. Deepa, B.; Abraham, E.; Cherian, B.
Sample Bulk density Tapping density
M.; Bismarck, A.; Blaker, J. J.; Pothan,
(g/cm3) (g/cm3) L. A.; Leao, A. L.; de Souza, S. F.;
Raw materials 0.28 0.30 Kottaisamy, M. Bioresour. Technol.
α-cellulose b.5 0.24 0.26 2011, 102 (2), 1988–1997.
MCC b.5 0.21 0.25 5. Johar, N.; Ahmad, I.; Dufresne, Ind.
Crops Prod. 2012, 37 (1), 93–99.
4. Conclusion 6. Li, H.; Li, Q.; Hao, J.; Xu, Z.; Sun, D.
Rice bran residue could be used for Colloids. Surf. A Physicochem. Eng.
the preparation of MCC by acid hydrolysis Asp. 2016, 502, 107–113.
method. However, the reaction time in a
The effects of lime lemongrass and oregano essential oils treatment on
quality preservation of salted fish (Rastrelliger brachysoma)
Lanchakon Chanudom1*, Ausnee Sakarasa1, Nursofwanee Uma1, Nutwara Ongsara2
1
Faculty of Science and Technology, Nakhonsithammarat Rajabhat University,
Nakhonsithammarat 80280, Thailand
2
Science Center, Nakhonsithammarat Rajabhat University, Nakhonsithammarat 80280, Thailand
*E-mail: lanchakon@gmail.com
Abstract:
Thai salted fish or Pla-kem is a traditional food and popularly taken as side dishes.
This work lime, lemongrass and oregano essential oils were evaluated for their effectiveness
on post mortem quality of salted fish. Salted fishes were prepared with 60% w/w of NaCl, air
dried and covered with coated paper which sprayed by 4 µL/L of each essential oil, physical,
chemical, microbiological changes and sensory evaluation were determined for 15 days. The
results showed that salted fish coated with all essential oils coated paper have no change on
physical appearance but effect on chemical and microbiological changes. The fat content in
salted fishes covered with essential oils (lime, lemongrass and oregano) coated paper were
significantly decreased to 0.25±0.01, 0.24±0.01 and 0.23±0.01% lipid (p≤0.05) when
compared to control on 15th day and were retained the changes of protein. Moreover, salted
fish covered with oregano coated paper were inhibited growth of total bacteria through the
experiment period, while total fungal, lactic acid bacteria, total coliform and E. coli were
ranged on standard. Salted fish covered with lime coated paper had the highest scores on
customer acceptance test. Indicated that essential oil might be a potential for maintaining
quality of salted fish.
1. Introduction Essential oils - obtained from plant

One of the oldest and commonly materials- such as leaves, seeds, flowers,
used processing techniques for fish buds, roots and other plant parts are
preservation all over the world is salting aromatic oily extracts. As an alternative to
because of simplicity of the process and low chemical and synthetic preservatives,
production cost.1 Thai salted fish or Pla-kem essential oils can be used in food, perfumes
is a traditional food and popularly taken as and pharmaceuticals and meet the demands
side dishes. In the production of salted fish, of consumers for natural products.4 The
the raw fish is filleted and then heavily antimicrobial properties of essential oils
salted. Salt is effective as a preservative derived from various plant organs have been
because it reduces the water activity of fish empirically recognized for centuries,
muscle, consequently bacterial growth and although scientific confirmation has been
enzymatic spoilage are inhibited.2 However, reviewed recently5 such as oregano essential
those spoilage in salted fish were continued oils was inhibited Listeria monocytogenes,
during preservative process therefore the Salmonella typhimurium, Bacillus cereus,
way to extending the shelf life is in need. Yersinia enterocolitica, Pseudomonas
Targeting to the reduction of the use of aeruginosa, Staphylococcus aureus and
chemical additives in food industry, growing Escherichia coli.6 Lime essential oil has an
interest has risen recently on the use of antimicrobial activity on E. coli, S.
natural food additives with antimicrobial typhimurium, S. aureus, B. cereus and L.
and antioxidant properties that do not have monocytogenes7 and lemongrass essential oil
any negative effects on the human health.3 was inhibited the growth of E. coli, S.
typhimurium, P. aeruginosa, L.
monocytogenes and S. aureus.8 However, weight became constant. The fat content was
the treatment of essential oil more than 4 measured by extracting samples with
µL/L caused some sensory damages, petroleum ether using a Soxhlet apparatus.
including off-flavor in some fish product.4 The ash content was determined by
In this work, oregano lime and incineration in a muffle furnace maintained
lemongrass essential oils were evaluated for at 600 oC until a constant final weight was
their effectiveness in maintaining post achieved. The crude protein was calculated
mortem quality of salted fish by sprayed it by converting the nitrogen content
on food grade paper and applied it through determined by Kjeldahl's method. Salinity
the products. Physical changes, pH, was evaluated using salinometer. Analyses
moisture, ash, lipid, protein, s a l i n i t y, were conducted in triplicate, and all reagents
microbiological and sensory evaluation were were of analytical grade.
determine during storage at room 2.2.3 Microbiological properties
temperature for 15 days. Total bacterial and total fungal were
determined following AOAC method.9 Total
2. Materials and Methods lactic acid bacteria were modified following
2.1 Preparation of salted fish and by O' Connor-Shaw et al. (1994)10 and total
essential oil coliform and E. coli were determined by
Rastrelliger brachysoma fish was MPN (Most propable number).
brought from Nakhonsithammarat local 2.3 Sensory evaluation
market with the size of 200±20 g per each. Sensory analysis was evaluated at
The fishes were filleted by hand and cleaned the last day of storage time (day 15).
with tap water and then prepared with 60% Panelists scored for color, odor, texture and
w/w of NaCl and air dried for 1 week. Each general acceptability, using a nine-point
essential oil (oregano, lime and lemongrass) hedonic scale (1, dislike extremely to 9, like
was prepared at a concentration of 4 µL/L extremely).
and 3 mL was sprayed on 112 inch of food 2.4 Statistical analysis
grade paper and air died. Essential oils were The experiment followed a
commercially available from completely randomized design (n=3). Data
Nakhonsithammarat local market. were subjected to one-way analysis of
Salted fishes were covered with each variance (ANOVA). Mean separations were
essential oil coated paper and storaged at assessed by Duncan's multiple range test.
room temperature for 15 days. Differences at p < 0.05 were considered
2.2 Determination of physical, chemical significant.
and microbiological changes on salted fish
during storage 3. Results and Discussion
Physical, chemical and 3.1 Effect of essential oil treatment on
microbiological changes of salted fishes physical and chemical properties
were determined on 0, 5, 10 and 15 days of Physical changes of salted fishes
storage times. after covered with essential oil coated paper
2.2.1 Physical properties were considered on color and odor. After 10
Color and odor changes of salted days of storage time, the salted fishes were
fishes were determined following the not different from the 1st day of storage. At
observation process during the storage time. 15 day of storage time, the color and odor of
2.2.2 Chemical properties salted fish were differently changed on
Chemical properties of salted fishes oregano coated paper treatment (data not
were carried out by the following AOAC show). The results of chemical analysis of
methods.9 The pH was determined by using salted fish are summarized in Figure1.
pH meter. The moisture content was Changes in the pH values of salted fish over
determined in an oven at 103 oC until the storage time are shown in Figure 1A. The
pH values of all treatments gradually 3.2 Effect of essential oil treatment on
increased during 15 days of storage time. microbiological properties
Salted fishes cover with all essential oil Table 1 was demonstrated on the
coated papers were not significantly effects of the different treatments on the
different (p≤0.05) to the control sample. The count of total bacteria, total fungi, lactic acid
rising pH value during storage was due to bacteria (LAB), total coliform and E. coli
the accumulation of the alkaline compounds, compared to control. Salted fish covered
such as ammonia and trimethylamine mainly with oregano coated paper were inhibited
derived from the microbial action during the growth of total bacteria throughout the
fish muscle spoilage.11 The result was experiment period, while total fungal, total
similar with Bensid et al.,12 who showed the lactic acid bacteria, total coliform and E.
same change trend in pH value in anchovy coli in all groups were ranged on standard.
under the icing with plant extracts treatment The absence of coliforms and other
during chilled storage. pathogenic bacteria could be attributed to
Protein, ash, moisture and fat content the salt added during fermentation, proper
in salted fish were demonstrated in Figure hygiene and good manufacturing practices
1B-E. Percentage of salted fish protein in during processing of fish.15 Moreover, the
control and treatment (lime, lemongrass and effect of essential oil can be enhanced by
oregano coated paper) groups was average at sodium chloride content in high salted food.
30.54±0.12%, 28.69±0.24%, 29.91±0.09% That is because essential oil can increase in
and 29.86±0.54%, respectively on 1st day the permeability of bacterial cells, and then
and decreased to 34.91±0.38%, 26.65 sodium chloride decreased the activity of
±0.19%, 28.60±0.31% and 28.62±0.69%, decarboxylase.16
respectively on 15th day of storage time. The 3.3 Effect of essential oil treatment on
protein levels in raw and cooked short- sensory evaluation
bodied mackerel (Rastrelliger brachysoma) The sensory assessment of salted fish
in Thailand were claimed at 21.1 and 23.4 was evaluated at day 15 of storage time. The
g/100 g, respectively.13 At the last day of highest hedonic value on color and odor was
storage times, ash contents in control and found in control group and salted fish
treatment groups was not different (p≤0.05) covered with lemongrass coated paper at
while the moisture of salted fish covered 6.97±1.22 and 6.70±1.15 respectively. The
with oregano essential coated paper was highest score on texture and general
significantly decreased when compared to acceptability were recorded for salted fish
control group. The lipid content in salted covered with lime coated paper at 6.47±1.46
fishes covered with essential oils (lime, and 6.70±1.53, respectively. However, in
lemongrass and oregano) coated paper were every sensory assessment, there was no
significantly decreased to 0.25±0.01, significantly differences between groups
0.24±0.01 and 0.23±0.01% lipid (p≤0.05) (data not show).
when compared to control group on 15th
day. Salinity in salted fish in all groups was 4. Conclusion
slightly increased due to the storage time. The results indicated that plant
The differences depend on the fish size, the essential oil treatment can be retained
state of nutrition, the reproductive cycle of physical, microbiological, chemical changes
the animal, as well as other environmental in salted fish and it was accepted by sensory
conditions, among which the feeding pattern score value. Thus, essential oil treatment
has a key role in the compositional could be a potential application for
variations.14 maintaining the quality of salted fish.
A B
C D
E F
Figure 1. The effects of essential oils treatment on pH (A), protein (B), ash (C), moisture
(D), fat content (E) and salinity (F) during storage times; each data point is the mean of three
replicate samples. Standard deviations are indicated by bars and * represent the significantly
differences at p≤0.05 when compared to control group at the same period of storage time.
Table 1. The effects of essential oils treatment on microbiological properties during storage
times
Microbiological Day Treatment
properties Control Lime coated Lemongrass Oregano
coated coated
Total bacteria 0 >300 >300 >300 >300
(cfu/g) 5 2.63x108 2.83x108 2.92x108 2.26x108
10 2.26x108 2.87x108 2.90x108 2.53x108
15 2.24x108 2.80x108 2.80x108 2.15x108
Total fungi 0 <30 <30 <30 <30
(cfu/g) 5 <30 <30 <30 <30
10 <30 <30 <30 <30
15 <30 <30 <30 <30
Lactic acid 0 - - - -
bacteria (cfu/g) 5 - - - -
10 - - - -
15 - - - -
Total coliform 0 <3 <3 <3 <3
(MPN/g) 5 <3 <3 <3 <3
10 <3 <3 <3 <3
15 <3 <3 <3 <3
E. coli (MPN/g) 0 - - - -
5 - - - -
10 - - - -
15 - - - -
Acknowledgements 7. Sanchez, D; Contreras, J. C; Nevarez,

The research leading to these results G. V; Aguilar, C. N. CyTA J. Food.
has been facilitated by Biological program, 2015, 13 (1), 17 – 25.
Faculty of Science and Technology, 8. Ahmad, M; Benjakul, S; Sumpavapol,
Nakhonsithammarat Rajabhat University. P; Nirmal, N. P. Int. J. Food Microbiol.
2012, 155 (3), 171 – 178.
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Technol. 2013, 53, 387–394. Analytical Chemists. Washington, DC,
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Comparison between mimic olfactory tool and sensory evaluation
for grading of jasmine rice
Wimonsiri Tanomsridachchai1, Siriwit Buajarern1, Adisak Romputtal2,
Yuthana Tantirungrotechai1*
1
2
Department of Physics, Faculty of Science and Technology, Thammasat University,
*E-mail: yt203y@gmail.com
Abstract:
We developed the electronic nose (e-nose) with five gas sensors including temperature
sensor to classify the Jasmine rice that is adulterated with low quality rice in order to reduce
the selling price. To produce the pattern recognition of fragrance of Jasmine rice, the e-nose
was trained. The different Jasmine rice concentrations (0%, 5%, 10%, 15%, 20%, 40%, 60%,
80%, 85%, 90%, 95%, and 100%) were systematically studied. The features of collected data
from e-nose were extracted. We used several statistical methods: principal component analysis
(PCA), cluster analysis (CA), and linear discriminant analysis (LDA) to distinguish the
concentrations of Jasmine rice. The data analysis indicates that the LDA method provides the
best discrimination of Jasmine rice compared to the others. Testing the sense of smell in
sensory evaluation by serving 8 concentrations of Jasmine rice to 8 trained sensory panels was
also investigated. The result showed that four groups of concentration were classified.
Compared with E-nose, the performance of e-nose was better than sensory evaluation but they
were still in good agreement.
1. Introduction classify pure rice and mixed rice.5 As the

Thai Hom Mali or Jasmine rice is aroma of Jasmine rice consists of several
more expensive than other types because of volatile compounds in different
its special properties, including soft texture, concentration, Gas Chromatography – Mass
unique flavor, and fragrance. Adulteration of Spectrometry (GC-MS) has also been used.6
Jasmine rice by lower grade, such as white However, there is no single compound which
rice is done in order to reduce the cost.1,2 A can be used for determining the rice
method for estimating of rice quality is specification.7 Smell is a group of several
therefore required. Thai agricultural standard chemicals in different concentration. It can be
specify observation of color change after analyzed using sensory evaluation or sensory
degradation in base solution, dying, and analysis which is a scientific method used for
analysis of amylose content for rice analysis.3 food qualification. However, there are many
However, it is difficult to identify the factors affecting to the performance of
adulterated rice by naked-eye inspection, and human’s sensory.8 To replace the human
other techniques require expensive olfactory sense, electronic nose or E-nose is
instruments. Many alternative methods have widely used9 for many applications including
been proposed. Whitworth et al. used a food analysis.
combination of three techniques: Near In this work, we compared the use of
Infrared spectroscopy, visco analysis, and the electronic nose system with sensory
image analysis to discriminate between evaluation to identify the percentage of
Basmati rice and other long grain rice.4 Jasmine rice.
Fluorescent simple sequence length
polymorphism (SSLP) has been applied to
2.1 Sample preparation 3.1 Classifiation by electronic nose
Jasmine rice was mixed with white The signal responses from five gas
rice at percentages by weight of 0%, 5%, sensors were collected and analyzed using
10%, 15%, 20%, 40%, 60%, 80%, 85%, 90%, principal component analysis (PCA), cluster
95%, and 100%. The volatile compounds in analysis (CA), and linear discriminant
the samples were extracted before analysis (LDA). For selecting the suitable
measurement. features, unsupervised methods such PCA
2.2 Methods and CA are used as preliminary analysis.9
2.2.1 Electronic nose system The PCA output is shown in Figure 2. For
The electronic nose used in this study ease of visualization, these were reduced to
comprised five gas sensors with individual first two dimensions or components: PC1 and
levels of sensitivity. A temperature sensor PC2. Nine from 12 groups can be explained
was also included to ensure that all tests were by the total variance of 91.88% (84.962% and
conducted within the same temperature 6.918% of first two dimensions). However,
range. The electronic nose system is shown some percentages were still overlap with the
in Figure 1. The assembly process of E-nose others.
is reported in detail elsewhere. The groups of rice sample can be
divided into five clearly clusters by CA based
on distance between objects (Euclidean
distance) as indicated in dendrogram in
Figure 3.
The LDA supervised classifier was
used for distinguishing the different
Rice Sensor Data percentages of Jasmine rice by creating the
sample array analysis linear functions from original variables to
new dimensions. Then, the relation of
Figure 1. Schematic of developed electronic distance between within and between groups
nose system was analyzed. Table 1 shows the
summarization of classification using LDA.
The rice odor was measured by E- It indicates that the predictions of Jasmine
nose for 3 min after storage for 5 min. Each rice concentrations from LDA analysis were
sample was measured for 5 times at 25±2 ºC. related with the real samples. It means LDA
The ambient air was used as reference gas provided the 100% classification accuracy
and to clean the system between sample for all concentrations.
measurements. 3.2 Identification of jasmine rice ratio by
2.2.2 Sensory analysis sensory evaluation panel
A panel of eight participants was The several percentages of Jasmine
selected for sensory analysis. The panelists rice were ranked by eight trained participants
were trained to recognize the Jasmine aroma based on rice aroma. Fisher’s Least-
using standard chemical at different Significant Different (LSD) was used for
concentrations. The panel was then asked to grouping the ratios of Jasmine rice. We found
distinguish between samples in which that rice sample was classified into five
Jasmine rice was mixed with white rice at groups from eight groups as shown in Table
percentages of 0%, 10%, 20%, 30%, 70%, 2. It means the panel cannot identify some
80%, 90%, and 100%. concentrations.
Table 2. Classification result from sensory 3.3 The comparison between E-nose and
test using Fisher’s LSD for rank sum at 80% sensory evaluation for classifying the
confidence level percentages of Jasmine rice
Groups Percentage of Jasmine rice Although the use of E-nose with PCA
1 0% and CA cannot identify all of concentrations,
2 10%, 20%, 30%
the combination of E-nose and LDA can be
3 70%
4 80%, 90% provided the best classification (100%
5 100% accuracy). While sensory analysis can
identify some concentrations.
Concentration of
Jasmine rice
Cluster diagram o
10% 5%
20%
0%
Figure 2. PCA result for rice sample in different percentages of Jasmine rice
Table 1. Summarization of classification of Jasmine rice ratios using LDA
Predicted group membership
Class total
0% 5% 10% 15% 20% 40% 60% 80% 85% 90% 95% 100%
0% 5 0 0 0 0 0 0 0 0 0 0 0 5
5% 0 5 0 0 0 0 0 0 0 0 0 0 5
10% 0 0 5 0 0 0 0 0 0 0 0 0 5
15% 0 0 0 5 0 0 0 0 0 0 0 0 5
20% 0 0 0 0 5 0 0 0 0 0 0 0 5
40% 0 0 0 0 0 5 0 0 0 0 0 0 5
60% 0 0 0 0 0 0 5 0 0 0 0 0 5
80% 0 0 0 0 0 0 0 5 0 0 0 0 5
85% 0 0 0 0 0 0 0 0 5 0 0 0 5
90% 0 0 0 0 0 0 0 0 0 5 0 0 5
95% 0 0 0 0 0 0 0 0 0 0 5 0 5
100% 0 0 0 0 0 0 0 0 0 0 0 5 5
Figure 3. Dendrogram of rice classification using Euclidean distance in cluster analysis
4. Conclusion Thammasat University, for research

In this study, we developed an funding.
electronic nose and used it to distinguish
between different percentages by weight of References
Jasmine rice. Multivariate analysis was 1. http://www.dailynews.co.th/economic/
used to analyze the data: PCA, LDA, and 363218 (accessed Jul 20, 2016).
CA. The LDA was demonstrated to give the 2. National Bureau of Agricultural
best resolution when distinguishing Commodity and Food Standards:
between the samples on the basis of aroma. ACFS http://www.acfs.go.th/
The proposed E-nose also provides the (accessed July 20, 2016).
Jasmine rice classification more accurate 3. Thai Hom MaLi Rice. Thai
than sensory analysis. Agricultural Standard. National
Bureau of Agricultural Commodity
Acknowledgements and Food Standards (ACFS)
We would like to thank Dr. Suteera http://www.acfs.go.th/ (accessed Jul
Vatthanakul, Thammasat University, 20, 2016).
Thailand, for suggestion in sensory 4. Whitworth, M.; Greenwell, P.; Fearn,
evaluation. W.T. gratefully appreciated T.; Osborne, B. G. Physical technique
Faculty of Science and Technology,
for establishing the authenticity rice. 1999, 14 (2), 209–233.
CCFRA R&D Report, 1995, 16. 8. Śliwińska, M.; Wiśniewska, P.;
5. Bligh, H. F. J. Int. J. Food Sci. Technol. Dymerski, T.; Namieśnik, J.;
2000, 35 (3), 257–265. Wardencki, W. J. Agric. Food
6. Petrov, M.; Danzart, M.; Giampaoli, Chem. 2014, 62 (7), 1423–1448.
P.; Faure, J.; Richard, H. Sciences des 9. Sipos, L.; Kovács, Z.; Szöllősi, D.;
Aliments 1996, 16 (4), 347–360. Kókai, Z.; Dalmadi, I.; Fekete, A. J.
7. Yau, N. J. N.; Liu, T. T. J. Sens. Stud. Chemometrics 2011, 25 (5), 275–286.
Antioxidation, antibacterial and cytotoxicity activities of Hom tong
banana flower extract
Atchara Patamakhachorn1, Piyaporn Nongharn1, Supakorn Boonyuen1*
Pariya Na Nakorn2*, Audjima Poomkleang2, Papassorn Kindklao2
1
Department of Chemistry, Faculty of Science and Technology, Thammasat University, Paholyotin,
Klong-Nung, Klong-Luang, Pathumthani 12120, Thailand
2
Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Paholyotin,
Klong-Nung, Klong-Luang, Pathumthani 12120, Thailand
Abstract:
The antioxidant capacities of the extracts from different parts of Hom tong banana
(Musa sapientum) flower, including stick, florets, bract, peel, banana flower and banana bud
were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Total phenolic content
and total flavonoid content (TPC) were measured using Folin-Ciocalteu and Downs methods,
respectively. Peel extract presented the highest total phenolic compounds (6.67±0.00 g GAE
100 g-1 dry wt.). The lowest IC50 values was found from peel and bract extracts with 25±0.04
mg/L and 30.70±0.03 mg/L, respectively. Banana bud extract showed the highest total
flavonoids content (27.30±0.07 g QE 100 g-1 dry wt.). The cytotoxicity of all extracts were
studied toward a human cell derived cell line (using MTT assay). There was no toxicity for
all extract at the concentration less than 100 μg/mL. Flower and peels extracts of Hom tong
banana were potentially source of natural antioxidant and antibacterial (E. coli and S. aureus)
agent which can be further investigated for medicinal usage.
1. Introduction diseases and cancers.4 Banana is a valuable

Antioxidant is a molecule that inhibits food source of nutrients with high-quality
the oxidation of other molecules. Oxidation is proteins, vitamins (A, B, C and E), minerals
a chemical reaction that can produce free and bioactive compounds.5 The phenolic
radicals, leading to chain reactions that may compounds are secondary metabolites in
damage cells. Banana tree is a useful plant. plants that have been major antioxidant.
Which is widely cultivated in Thailand. Flavonoids are a subset of phenolic
Banana is classed as one of the most popular compounds.6
fruits in the world market. Its tropical fruit Despite the development of several
has a strong ability to protect itself from the modes of anticancer therapies, skin cancer
oxidative stress caused by intense sunshine cure is still facing many obstacles. It is for
and high temperature by increasing its this reason that considerable research has
antioxidant levels.1 been carried on in the hope of discovering
According to the FAO (Food and anticancer agents that are highly specific
Agriculture Organization of the United against both proliferative and non-
Nations), banana (Musa spp.) is amongst the proliferative tumorigenic cells, with no
world’s leading crops, after rice, wheat, and major effect on healthy tissues.7
maize. The worldwide production of banana The aim of this work was therefor to
tends to increase and was 102 million tons in evaluate the antioxidant capacity, total
2010.2,3 Banana flower and banana peels phenolic content, total flavonoid content,
provide a major bioactive molecules. cytotoxicity and antibacterial activities of
Therefore the consumption of flower and Hom Tong banana flower and peel.
peel provide numerous benefit for human
such as the prevention of cardiovascular
2. Materials and Methods µL of 7.5% sodium carbonate. The mixture
2.1 Chemical was then kept in the dark for 30 min. The
2,2-Diphenyl-1-picrylhydrazyl absorbance of the mixture was immediately
(DPPH), Folin-Ciocalteu and all solvents measured at 765 nm using
used for plant extraction were of analytic spectrophotometer.11 Gallic acid was used a
grade. standard12 to obtain the standard curve. The
2.2 Preparation of the plant material results were expressed as a gram of Gallic
The banana was picked on 6 March acid per 100 gram dry weight of the sample
2017 and cut parts of banana bud (bract, (g GAE 100 g-1 dry wt.).
banana flower, florets, stick, banana bud and 2.5 Determination of total flavonoid
peels) into small pieces. After that banana content (TFC)
flower was dried and mashed into powder. The total flavonoid content of the
Water extraction of the sample was carried banana flower was carried out by mixing
out by boiling 40 g of banana in a 100 mL of each sample (2000 µL) with distilled water
distilled water for 2 h. The extracts were (400 µL). After that, 400 µL of sodium
then filtrated and then freeze-dried. nitrite solution was added and the mixture
was kept in the dark for 6 min. 600 µL of
bract
10% aluminum chloride, 4000 µL of 5%
stick peel sodium hydroxide solution was then added
and the solution was kept in the dark for 15
min. The absorbance of the mixture was
immediately measured at 510 nm using
spectrophotometer.6 The calibration curve of
florets
Banana flower the total flavonoid content was prepared by
using standard solution of quercetin (0, 10,
Figure 1. Part of banana bud 20, 50, 100 and 150 mg/L. The total content
of flavonoids was expressed as gram of
2.3 DPPH radical scavenging capacity quercetin equivalent per 100 gram dry
The antioxidant activity of the weight of sample (g QE 100 g-1 dry wt.).
banana flower extract was determined using 2.6 Antibacterial activity assay
DPPH radical scavenge assay.8 In this assay, For the determination of the
equal volume of DPPH solution (0.08 mM) antibacterial activity, the disk diffusion
2 mL was mixed with six different method was used Briefly, a suspension
concentrations of samples (0, 25, 50, 75, (0.1x108 cells/mL) of the tested organisms
100, 125 mg/L), Blank was prepared using [Escherichia coli (TISTR780) and
methanol instead of sample. The mixtures Staphylococcus aureus (MSSA, TISTR885)
were then incubated in the dark at room was spread on the nutrient agar solid media
temperature for 30 min. The absorbance at plate. Filter paper disk (6 mm in diameter)
515 nm was measured by uv-vis was soaked with 10 µL of extract (1000
spectrometer The %FRSA were calculated mg/L, dissolved in methanol) and placed on
by following this formula. the inoculated plates. After being kept at 4
Abs(blank)− Abs(sample) o
C for 2 h, they were incubated at 37 oC for
%FRSA = × 100
Abs(blank) 24 h. The zone diameter of inhibition was
The inhibition concentration at 50% measured.
(IC50) were calculated for all the sample.9,10 2.7 Cytotoxicity against HaCat cell line
2.4 Total phenolic content (TPC) using MTT assay
Total phenolic contents in all MTT (Sigma-Aldrich, Oakville, ON,
samples were determined by Folin-Ciocalteu Canada) was dissolved at a final
assay. Sample (200 µL) was mixed with 500 concentration of 5 mg.mL-1 in HEPES
µL of 10% Folin-Ciocalteu reagent and 800 buffer saline supplemented with D-glucose
(HBS/G: 20 mM HEPES, pH 7.4, 140 mM one mL of MTT solution. After 3 h
NaCl, 4 mM KCl, 1.8 mM CaCl2, 0.8 mM incubation (37 °C, 5%CO2), the formed
MgCl2 and 20 mM Dglucose). Each culture formazan crystals were dissolved in 1 mL of
well was delicately washed once with dimethyl sulfoxide (DMSO) and
phosphate buffered saline solution [PBS: absorbances were read at test 550 nm and
NaCl 0.8%, KCl 0.02%, KH2PO4 0.02%, reference 664 nm wavelengths.14
Na2HPO4 0.12%, pH 7.3] before adding.
Table 1. Antioxidant activity (IC50) using DPPH• radical, total phenolic content and total
flavonoid content of Hom Tong banana flower and peel extracts (n=3)
TPC TFC
Extracts IC50 (mg/L)
(g GAE 100 g-1 dry wt.) (g QE 100 g-1 dry wt.)
Bract 30.70 ± 0.03 6.02 ± 2.30 27.30 ± 0.07
Flower 84.49 ± 0.29 0.36 ± 1.22 12.33 ± 2.14

Florets 91.33 ± 0.35 3.15 ± 0.70 15.09 ± 2.16
Stick 75.46 ± 0.56 6.67 ± 0.00 22.57 ± 0
Banana bud 43.17 ± 0.51 5.99 ± 0.81 22.36 ± 4.28
Peels 25.00 ± 0.04 3.41 ± 0.20 14.59 ± 0
Ascorbic (vitamin C) 0.64±0.01 - -
3. Results and Discussion Table 2. Cytotoxicity against HaCat cell

Table 1 shows the antioxidant line of Hom Tong banana flower and peel
activities (IC50, mg/L), total phenolic extracts
content and total flavonoid content of %Toxicity (µg/mL)
different parts of banana flower (bract, Extracts
banana flower, florets, stick, whole banana HaCat cell line
flower and peel). The DPPH radical Bract <100
scavenging capacity was used for study the
antioxidant activity. This method is based on Flower <100
the ability of DPPH radicals to react with Florets <100
hydrogen donor species such as phenols and
flavonoids present in the analyzed sample.10 Stick <100
It was found that peel extract showed the
Banana bud <100
highest antioxidant activity with IC50 of
25.00 ± 0.04 mg/L. Stick extract showed the Peels <100
highest total phenolic 6.67 ± 0 . 7 0 g GAE
100 g-1 dry wt.), followed by a bract. The
highest total flavonoid content was found in The inhibition zone of Hom Tong
the bract extract (27.30 ± 0.70 g QE 100 g-1 banana flower and peel extracts agent
dry wt.) Escherichia coli and Staphylococcus aureus
The cytotoxicity of banana bud and is presented in Table 3. The standard disc of
peel extracts were studied toward a HaCat drug penicillin was served as positive
cell line (Table 2) There was no toxicity for control and DMSO solvent was used for
the extract at the concentration less than 100 control experiments. The bract showed the
μg/mL. greatest value of inhibition zone of 8.5±1.2
and 9.0±1.1 mm. agent Escherichia coli and Central Scientific Instrument Center (CSIC)
Staphylococcus aureus respectively, of Faculty of Science and Technology,
whereas, banana bud showed the lowest Thammasat University.
inhibition zone of both bacteria.
References
Table 3. The inhibition zone of banana 1. Shaida, F. S.; Nor, A. M.; Yusoff, I. M.;
crude extracts of Hom Tong banana flower Eng, M. S.; Azliana, A. B.; Kheng, L O.
and peel extracts (n = 3) J. Food Compos. Anal. 2011, 24, 1-10.
Inhibition zone (mm) 2. Chonthira, S.; Regin, S.; Ken, S.;
banana crude Emmerich, B.; Perry, K. W. Food
extracts Escherichia Staphylococcus
coli aureus Chem. 2017, 143, 33-39.
3. Shinichi, S.; Yumiko, Y.; Kazuyoshi, O.
bract 8.5±1.2 9.0±1.1
Food Chem. 2002, 79, 351-354.
banana flower 7.5±1.4 8.5±1.0 4. Damila, R.; Eliza, M. R.; Sheisa, C.;
Sargi, E. M.; Schmidt, G. B.; Marcos,
florets 7.5±0.8 8.5±1.2
N.; Eberlin, A.; Sawaya, J. V. Food Res.
Stick 7.5±1.0 7.5±1.0 Int. 2015, 77, 392-399.
banana bud 7.0±1.1 6.5±0.9 5. Hongyan, L.; Zeyuan, D.; Ronghua, L.;
Honghui, Z.; Jamie, D.; Massimo, M.;
peels 8.0±0.8 8.5±0.9 Yong, S.; Rong, T. J. Food Compos.
DMSO 6.0±1.0 6.0±1.0 Anal. 2014, 37, 75- 81.
6. Lilia, M.; Juan, R.; Marlene, C.; Sadoth
penicillin 23±0.9 -
S.; Gerardo, B. Food Chem. 2017, 230,
174-181.
4. Conclusion 7. Saly, S.; Tania, Y.; Souad, H.; Walid,
The antioxidant activity of Banana H.; Marc, K. Exp. Toxicol. Pathol.
bud and peel of Hom tong banana (Musa 2017, 69,173-178.
sapientum) extracts from various part of 8. Xinfeng, Z.; Meng, X.; Jingwei, Z.;
banana bud. The banana peel possessed the Lingshang, W.; Jingjing, L.; Jinping S.
highest antioxidant activity with the IC50 Ind Crop. Prod. 2017, 102, 164-172.
value of 25.00 ± 0.04 mg/L for DPPH assay. 9. Emiliano, M.; Pablo, M.; Stefani,
Stick extract showed the highest total Roxana A. Food Sci. Tech. Brasil 2016,
phenolic content, followed by bract extract. 74, 353-362.
The highest total flavonoid content was 10. Nabil, A.; Naima, O.; Fatiha, B.; Ind.
found in the bract extract. Crop Prod. 2015, 88, 112-119.
Flower and peel extracts had no 11. Hui, Y.; Gangqiang, Y.; Minoru, S.;
toxicity for the extract at the concentration Toshiyasu, Y.; Toshiki, N.; Yinci, X.;
less than 100 μg/mL. Flower and peel of Food Chem. 2017, 232, 379-386.
Hom tong banana were potentially source 12. Nemanja, S.; Tatjana, M.; Bojan Z.;
of natural antioxidant which can be further Vesna, S.; Violeta, M.; Jovana, J.;
investigated for medicinal usage. Ljiljana, C.; Branislava, K; Nirit, B.;
The bract extract showed the greatest Life Scis. 2016, 78, 21-28.
value of inhibition zone for E. coli and S. 13. Shaida, F. S.; Ibrahim M.; Eng, M. S.;
aureus. Azliana, A. B. S.; Kheng L. O. J. Food
Compos. Anal. 2011, 24, 1-10.
Acknowledgment 14. Mariela, G. P.; Lyvia, F.; Mircea, A.
The financial and all equipments for M.; Joanne, P. Anal. Biochem. 2017,
experiments were supported by Department 535, 43-46.
of Chemistry, Faculty of Science and
Technology, Thammasat University and
The preliminary study of volatile oil in Melaleuca cajuputi by GC-MS
Mayoon Lamsub1*, Sirirat Phaisansuthichol2, Orasa Choola-Aied3
1
Department of Chemistry, Faculty of Science and Technology, Nakhon Si Thammarat Rajaphat University,
Nakhon Si Thammarat 80280, Thailand
2
Program in Chemistry, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand
3
King Mongkut’s Institute of Technology Ladkrabang, Prince of Chumphon Campus,
Chumphon 86160, Thailand
*E-mail: mayoon105@gmail.com
Abstract:
Melaleuca cajuputi, commonly known as cajuput or white Samet, is a plant in the
myrtle family, “Myrtaceae”. It is found in many areas of the south and east of Thailand. The
leaves of Melaleuca cajuputi possess antibacterial, anti-inflammatory and anodyne
properties. Essential oil is produced from the leaves by solvent extraction and steam
distillation. The preliminary examination for the volatile oil components from Samet, was
performed using Gas Chromatography-Mass Spectrometry (GC-MS) with electron impact
(EI). The result of GC-MS analysis showed that Samet essential oil consists of 34
components in wetted leaves and 36 components in dried leaves.
1. Introduction used as safe alternative botanical insecticide

Melaleuca cajuputi, a tree species for controlling stored product insects.3,4
within the family “Myrtaceae”, (Figure 1)
naturally grows in Myanmar and Thailand
through Southeast Asia to northern
Australia. Melaleuca cajuputi is able to
grow in a wide range of environment
disadvantages including high acid soil,
saline soil, acid soil and water-locked soil
such as canal, river, lagoon and swamp.1,4
Melaleuca cajuputi is an important
economic tree in southern Thailand. It is a
multipurpose tree that every part is usable
and the local people have been recognized
its usefulness for a long time. Stems of
Melaleuca cajuputi are used for structural
post, fuelwood, charcoal production, fence,
platform, fishing rod, agricultural pole and Figure 1. Melaleuca cajuputi
stake etc.2-4
The leaves of Melaleuca cajuputi Moreover, this essential oil of
possess antibacterial, anti-inflammatory and Melaleuca cajuputi has been used as a
anodyne properties and are reputed to have perfume and a popular remedy for the
insect-repellent properties. It is also used as treatment of colic, cholera, headaches,
flavor in cooking and as a fragrance and toothaches, various skin diseases,
freshening agent in the soap, cosmetic, germicides and in the treatments of several
detergent and perfume. So, leaf essential oil ailments.3
of Melaleuca cajuputi is considered to be It is believed that there are some
benefits of essential oil of Melaleuca
cajuputi. Researchers are interested in with HP-5MSI capillary column (30 m x
studying the components of such essential 0.25 mm i.d., 0.25 µL film thickness) as
oil using specialized chemistry techniques
separation unit. The oven temperature was
such as Column Chromatography, High-
performance Liquid Chromatography or programmed as follows: initiated 60 °C
HPLC, Thin layer Chromatography and Gas increased at 3 °C min-1 maintained at 200 °C,
Chromatography–mass Spectrometry or increased at 15 °C min-1 and maintained at
GC-MS etc.5,6 280 °C then held for 5 min. The injector
However, it was reported that each temperature was set to 250 °C. The MS
Melaleuca cajuputi essential oil will provide transfer line temperature was held at 280 °C.
different chemical compositions and Helium was used as a carrier gas with a
different methods of analysis will also constant flow rate of 1.0 mL min-1. The
provide different components of essential ionization mode was selected with electron
oil.
impact at 70 eV.
In Thailand, Melaleuca cajuputi can
be found in the swamp forest, especially at 2.2 Sample preparation
Kreang(Phu Kuan Kreang) Sub-District, Melaleuca cajuputi samples ( leaves)
Cha-Uat District, in Nakhon Si Thammarat, were collected from Kreang ( Phu Kuan
south of Thailand. There are a lot of Kreang) Sub-District, Cha-Uat District, in
Melaleuca cajuputi can be found there. Nakhon Si Thammarat, south of Thailand.
Therefore, the preliminary study of The leaves samples were dried at 60 °C for 4
volatile oil in Melaleuca cajuputi by GC-MS hours (Figure 2) and then were ground to a
uses the collected samples from Kreang particle size of 1.00 mm ( for dried leaves
(Phu Kuan Kreang) sub-district, Cha-Uat samples).
district, Nakhon Si Thammarat province in The essential oils were extracted
the south of Thailand. from dried leaves samples and wetted leaves
GC-MS or Gas chromatography- samples. A 1000 mg of the plant sample was
mass spectroscopy is one of the so-called freshly cut and extracted using 1000 mL
hyphenated analytical techniques. As the water for 5 hours. The cooled volatile oils
name implies, it is actually two techniques were filtrated through filters, dried with
that are combined to form a single method anhydrous magnesium sulfate and stored at
of analyzing mixtures of chemicals. Gas 4 °C until removed from storage to
chromatography separates the components analytical procedure.
of a mixture and mass spectroscopy
characterizes each of the components 3. Results and Discussion
individually. By combining the two 3.1 Chemical components of the essential
techniques, an analytical chemist can both oil
qualitatively and quantitatively evaluate a Essential oils isolated from leaves of
solution containing a number of chemicals.5 Melaleuca cajuputi, commonly known
as cajuput or white samet using steam
2. Materials and Methods distillation were analyzed by GC-MS. The
2.1 Materials and equipment compounds identified by mass spectrum
Anhydrous magnesium sulfate and were matched with a mass spectral library
dichloromethane used in the experiment,
were analytical grade, purchased from collection. The chromatograms from fresh
Merck (Germany). Deionized water was and dried leaves of Melaleuca cajuputi are
purified by Milli-Q purification system shown as major components in Figure 3 and
(Millipore, USA). GC-MS (Agilent Figure 4, respectively. The percentage of
Technologies. Wilmington, DE, USA) was chemical components of Melaleuca cajuputi
used as an analytical instrument equipped are shown in Table 1.
Figure 2. The leaves samples after drying at 60 °C for 4 hours
Figure 3. Chromatogram of the essential oil from fresh leaves of Melaleuca cajuputi
Figure 4. Chromatogram of the essential oil from dried leaves of Melaleuca cajuputi
Table 1. The chemical composition of Melaleuca cajuputi
Fresh leaves Dried leaves

Compounds
RT %Area RT %Area
Alpha-pinene 5.14 4.38 5.15 4.92

Cymene 8.07 4.16
gamma-Terpinene/
9.36 9.15 9.36 6.95
1,4-Cyclohexadiene
Alpha-terpinolene 10.52 10.05 10.52 8.04
beta-Elemene 23.41 3.74 23.43 5.87
Caryophyllene 24.50 18.98 24.54 9.29
alpha.-Caryophyllene 25.85 7.55 25.87 9.29
3,3,6,9,9-Pentamethyl-2,10-
35.78 11.54 35.76 7.57
diazabicyclo-1-decene-2-oxide
4. Conclusion of Nakhon Si Thammarat Rajaphat University,

The preliminary examination for the Nakhon Si Thammarat, Thailand.
volatile oil components from Samet, was
performed using Gas Chromatography-Mass References
Spectrometry (GC-MS) with electron impact 1. Nuyim, T.; The First Thai-Biomass
(EI). The chemical components of the different Utilization Symposium. 2002, 43-66.
oils were identified by retention time and mass 2. Schmidt, L. H.; Thuy, L. T. T.; Forest &
spectrum in comparison with a mass spectral Landscape Denmark. 2004, 92, 2-3.
library collection. The result of Gas 3. Rini P.; Yoshito O.; Titis B. W.;
Chromatography-Mass Spectrometry (GC- Kasmudjo. Wood Research Journal
MS) analysis showed that Samet essential 2012, 22, 94-99.
oil consists of 34 components in fresh leaves 4. Ko, K.; Juntarajumnong. W.;
(Figure 3) and 36 components in dried Chandrapatya. A. Thai Journal of
leaves (Figure 4). Agricultural Science 2009, 42, 1, 27-33.
5. http://cires1.colorado.edu/jimenez/CHEM-
Acknowledgements 5181/Labs/Gas_Chromatography.pdf
This work was financially supported 6. https://www.intechopen.com/books/adv
by the Research and Development Institute ances-in-gas-chromatography/gas-
chromatography-mass-spectrometry
Free radical scavenging activity, total phenolic and flavonoid contents of
herbal extracts
Jirapa Rattpan, Narumon Wuttiprom, Panadda Kulla, Supaporn Sangsrichan*
Department of Chemistry, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand
*E-mail: supaporn-s@mju.ac.th
Abstract:
Some herbal plants contain phytochemicals that may have effects on the human body.
This study aimed to determine free radical scavenging activity, total phenolic contents and
total flavonoid contents of five herbal plants; Annona squamosa L., Azadirachta indica A.,
Chromolaena odorata L. and Tinospora crispa L. leaves and Alpinia galanga L. rhizomes.
The plants were extracted with 50% ethanol and evaporated to dryness. The crude extracts
were furthered analyzed. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging
activity was found IC50 ranging from 5.5-47.6 ppm and the highest free radical scavenging
activity was A. squamosa L. leaf extract. Total phenolic content and flavonoid contents were
found ranging from 5.0-49.0 mg gallic acid equivalent (GAE)/g crude extract and 3.7-73.9
mg catechin equivalents (CE)/g crude extract, respectively. A. squamosa L. gave the highest
total phenolic and flavonoids contents.
1. Introduction such as nimbidin, nimbolide, mahmoodin,

Medicinal plants are the most margolone, margolonone, and
important source of folk medicine. The use isomargolonon, all of which show good
of plants in traditional medicine for treating antibiotic properties.5
various ailments remains an integral part of C. odorata is for medicinal and
the culture and traditions of a majority of the ornamental purposes. The plant is also
world's population.1 In addition, factors such popularly used for wound healing due to its
as the availability, affordability and antimicrobial properties. Concentrations of
accessibility of medicinal plants have led to 0.25 mg/mL and 0.125 mg/mL of ethanolic
their high demand and usage. Secondary extract of C. odorata exhibited antimicrobial
metabolites such as essential oils, alkaloids, effects against some human pathogens.
flavonoids, tannins, saponins generally Several researchers have reported the wider
produced by plants for their defense use of C. odorata as an effective therapy
mechanisms have been implicated in the against diarrhea, malarial fever, tooth ache,
therapeutic properties of most medicinal diabetes, skin diseases, dysentery, and
plants.2 colitis. The medicinal values of C. odorata
Annona is in the family Annonaceae. are attributed to some specific phenolic
A. squamosa Linn. One of the important compounds that have been isolated from it.
medicinal plants, commonly called “custard However, C. odorata also contains a
apple,” or sugar. It has been reported to carcinogenic substance called “pyrrolizidine
contain phytochemical and pharmacological alkaloids.”
activities. One of traditional Asian herbal
Azadirachta indica commonly remedies is T. crispa. It is a climbing plant
known as Neem, belongs to Meliaceae used as a traditional herbal medicine in
family commonly seen in South Asia, is South Asia. The use of T. crispa as herbal
established as a traditional antibiotic for a medicine has increased over recent years,
broad spectrum bacterial pathogens. Neem and several studies have assessed its
leaf extracts contain organic compounds
hypoglycemic, antioxidant and analgesic was added to 3 mL of DPPH reagent and
properties. allowed to incubate at room temperature for
Alpinia galanga (L.) Swartz is 30 min. The absorbance was measured at
traditionally used in the treatment of various 517 nm. The percentage of DPPH radical
ailments across India, China, and Southeast scavenging activity was calculated by the
Asian countries. It is a reputed drug in following formula:
indigenous system of medicine and largely
used as antibacterial and antiseptic. The %Inhibition
rhizome has been used as a domestic remedy = [(Acontrol-Asample)]/Acontrol × 100
against bacterial infections. Phenolic acids
and flavonoid also function as reducing Where Acontrol and Asample is the absorbance
agents, free radical scavengers. This study, of the control and sample, respectively.
hence aimed to determine free radical The sample concentration providing 50%
scavenging activity, total phenolic and inhibition (IC50) was calculated by plotting
flavonoid contents of five herbal plants inhibition percentages against concen-
Annona squamosa L., Azadirachta indica trations of the sample.
A., Chromolaena odorata L. and Tinospora 2.4 Total phenolic content determination
crispa L. leaves and Alpinia galanga L. The amount of total phenolic in the
rhizomes. herbal extracts was determined by using
Folin-Ciocalteu's reagent according to the
2. Materials and Methods method of Slinkard and Singleton3, with
2.1 Herbal plants some modifications. Gallic acid was used as
Herbal plants of 5 species; Annona a positive control. Samples (500 µL, three
squamosa L., Azadirachta indica A., replicates) were introduced into 5 mL
Tinospora crispa L. and Alpinia galanga L. volumetric flask, and then 1.0 mL of 10%
were collected from Chiangkham District, Folin-Ciocalteu's reagent and 1.0 mL of 5%
Payao Province, except Chromolaena sodium carbonate solution was added,
odorata L. from Sansai District, Chiang Mai leaved at room temperature for 2 hr. then
Province, Thailand during January-March adjusted the volume to 5 mL. The
2019. absorbance was measured at 765 nm against
2.2 Plant extraction a blank prepared as described above with
The preparation of extracts from distilled water, Folin-Ciocalteu’s reagent,
leaves of Annona squamosa L., Azadirachta and sodium carbonate solution. The content
indica A., and Chromolaena odorata L. of total phenolic was expressed as
rhizomes Alpinia galanga L. and trunk of milligrams of gallic acid equivalents per
Tinospora crispa L.. Plant samples of were gram of crude extract (mg GAE/g crude
cleaned by water. The fresh plants were cut extract).
into small pieces and macerated with 50% 2.5 Total flavonoid content determination
ethanol (1:3, kg/L) for 12 weeks at room Aluminum chloride colorimetric
temperature with occasional shaking and method4 was used for flavonoid
evaporated to dryness. The crude extracts determination, with some modifications. In
were furthered analyzed. brief, 1 mL (three replicates) of sample
2.3 Free radical scavenging activity solution was mixed with 4 mL of distilled
A free radical scavenging ability was water. 300 µL of sodium nitrite was added.
determined by 2, 2-diphenyl-1- After 5 min, 300 µL aluminum chlorides
picrylhydrazyl (DPPH) using the method of was added and allowed to stand for 6 min.
Diaz,6 Tanzadehpanah7 and Nordin,8 with Then, 2 mL of sodium hydroxide was added
some minor modifications. Crude extracts and the mixture was shaken to mix well. The
were dissolved in methanol at 20, 40, 60, 80, absorbance was measured at 510 nm using
100 mg/mL and 200 µL of sample solution UV spectrophotometer. The results were
expressed in millrams of catechin flavonoid contents were found ranging from
equivalents per gram of crude extract (mg 3.7-73.9 mg catechin/g crude extract. A.
catechin/g crude extract). squamosa L. gave the highest total
2.6 Statistical analysis flavonoid.
All results are presented as mean ±
standard deviation. Significant differences 4. Conclusion
between antioxidative activities. The Plants are an important source for the
contents of phenolic and flavonoid of development of new chemotherapeutic and
selected herbal plants were subjected to one- antioxidant agents. This study indicates
way analysis of variance (ANOVA) test of quantitative free radical scavenging activity,
Duncan with p ≤ 0.05. total phenolic contents and total flavonoid
contents of five herbal plants. The crude
3. Results and Discussion extracts from A. squamosa leaves showed
3.1 Crude yield strong antioxidant activity and also gave the
Crude extract yields by ethanolic highest of total phenolic contents and total
maceration of 5 herbal plants are shown in flavonoid contents. Therefore, A. squamosal
Table 1. Crude yields were ranging from L. leaves extract could be used as a
4.15 to 8.76 mg/kg. A. galanga L. gave the medicine for the treatment diseases. Future
highest crude extracts. research should be addressed on the
3.2 DPPH free radical scavenging activity application of using A. squamosa L. leaves
The DPPH radical was used for the as natural food preservative and to protect
evaluation of free radical-scavenging against peroxidative damage in living
properties of 5 herbal plants. The DPPH free systems related to aging and carcinogenesis.
radical scavenging activity were found IC50
ranging from 5.5-47.6 ppm and the highest
free radical scavenging activity was A.
squamosa L. leaf extract. The results of the
investigation are shown in Figure 1.
3.3 Total phenolic content determination
The quantitative determination of
total phenol was carried out using Folin-
Ciocalteu’s reagent in terms of gallic acid
equivalent. Total phenolic content is
expressed as mg gallic acid equivalent per
gram crude extract (Table 2). Total phenolic
content were found ranging from 5.0-49.0
mg GAE/g crude extract. A. squamosa L.
gave the highest total phenolic.
3.4 Total flavonoid content determination
Quantitative of total flavonoid
content is performed by methods aluminum
chloride. The flavonoid in the presence of
aluminum chloride have an intense yellow Figure 1. The DPPH free radical
fluorescence when observed UV scavenging activity of 5 herbal plants
spectrophotometer. Total flavonoid content
was expressed as mg catechin equivalent
(CE) per gram crude extract (Table 2). Total
Table 1. Crude yields extraction
Plant Scientific name Local name Family Parts used Crude yield (%)
1 Annona squamosa Sugar-apple Annonaceae leaves 8.24
2 Azadirachta indica Neem Meliaceae leaves 6.94
3 Chromolaena odorata Siam weed Asteraceae leaves 8.75
4 Tinospora crispa Guduchi Menispermaceae trunk 4.15
5 Alpinia galanga Thai galangal Zingiberaceae rhizomes 8.76
Table 2. Total phenolic and flavonoid 2. Ghribia, L.; Ghouilaa, H.; Omrib, A.;
contents of selected herbal plants Besbesb, M.; Janneta, H. B. Asian Pac.
Plants sample Total phenolic Total flavonoid J. Trop. Biomed. 2014, 4, S417–S423.
content (mg content (mg CE 3. Zheng, W.; Wang, S. Y. J. Agric. Food.
GAE/g extract /g extract wt.)
wt.)
Chem. 2001, 11, 5165–5170.
Annona 49.0±2.176d 73.9±0.315e 4. Zhishen, M. T.; Jianming, W. J. Food.
Azadirachta 8.01±1.709 b
5.14±0.229b Chem. 2014, 64, 49–54.
c
Chromolaena 40.6±0.992 40.8±0.421d 5. Subramani, K.; Murugan, V.;
a
Tinospora 5.05±0.464 3.72±0.235a Kolathupalayam, B.; Rangaraj, S.;
Alpinia 6.63±1.014a,b 7.30±0.121c
Palanisamy, M.; Venkatachalam, R.;
Notes: data expressed as mean value ± standard
deviation. GAE: gallic acid equivalent; CE: catechin Suresh, V. J. Alloys Compd. 2017, 723,
equivalent; wt.: weight. Different letters in the same 698–707.
line means significant differences at p ≤ 0.05. 6. Coloma, A. G.; Bailen, M.; Diaz, C. E.;
Fraga, B. M.; Diaz, R. M.; Zuniga, G.
Acknowledgements E.; Contreras, R. A.; Cabrera, R.;
This work is financially supported by Burillo, J. Ind. Crop. Prod. 2012, 37,
the National Research Council of Thailand 401–407.
2560 (NRCT) 196 Paholyotin rd., 7. Tanzadehpanah, H.; Asoodeh, A.;
Chatuchak, Bangkok 10900. Chamani, J. Food. Res. Int. 2012, 49,
105–111.
References 8. Leong, S. W.; Abas, F.; Kok, W. L.;
1. Samejo, M. Q.; Sumbul, A.; Shah, S.; Shaari, K.; Nordin, H. L. Bioorgan.
Memon, S. B.; Chundrigar, S. J. Pharm. Med. Chem. 2016, 24, 3742–3751.
Res. 2013, 7, 181–183.
Antioxidant activity and stability of total carotenoid and lycopene in
coconut oil with extracts from gac fruit aril and tomato
Pensri Penprapai*, Supamas Intharit
Faculty of Science and Technology,Rajamangala University of Technology Srivijaya,
Nakhon Sri Thammarat 80110, Thailand
*E-mail: pensir_srivub@hotmail.com
Abstract:
Coconut oil with extracts from gac fruit (COG) and coconut oil with extracts from
tomato (COT) contains high total carotenoid such as lycopene and beta-carotene and can be
used in cosmetic products or dietary supplement. The objectives of this work were to study
the antioxidant activity and stability of carotenoids in COG and COT during storage. The
antioxidant activity was assayed by DPPH (1,1-Diphenyl-2-picrylhydrazyl) and ABTS(2,2’-
Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) calculated as IC50 (the concentration of an
antioxidant at with 50% inhibition of free radical activity), The IC50 by DPPH assay of COG
and COT were found to be 0.05 and 0.13 mg/mL, respectively. The IC50 by ABTS method of
COG and COT were 0.007 and 0.036 g/mL, respectively. The results showed that COG had
the higher antioxidant activity than COT. The stability of lycopene and carotenoid in COG
and COT was studied during storage in the dark and light at room temperature for12 weeks.
We found that lycopene and carotenoid in COG and COT during storage in the dark was
reduced less than storage under the light. Moreover, COG had the higher lycopene and
carotenoids than COT. This concluded that COG had the higher antioxidant activity, stability
of lycopene and carotenoids than COT.
1. Introduction the body. Lauric acid (C12:0) (45%), was

Gac fruit (red aril) and tomato had found in coconut oil in major amounts. It
high levels of carotenoids, especially beta- shows various biological activities such as
carotene and lycopene. Carotenoids play a antiviral, antibacterial and antiprotozoal
crucial role in human health. It exerts anti- properties. Moreover, coconut oil leads to a
inflammatory and anti-oxidant effect.1 normalization of body lipids, protects
Consumption of carotenoid can increase against alcohol damage to the liver and
plasma beta-carotene and retinol levels.2 improves the immune system by anti
Carotenoids, especially beta-carotene and inflammarory. The objectives of this work
lycopene are non polar compounds. In the were to study the antioxidant activity and
conventional method, carotenoids were determine stability of the carotenoids in
extracted with non-polar organic solvent coconut oil with extracts from gac fruit and
such as hexane which was dangerous to tomato during storage.
environment and experimenter. To solve this
problem, coconut milk was used as a solvent 2. Materials and Methods
to extract beta-carotene and lycopene from 2.1 Materials
gac fruit aril and tomato to obtain coconut Coconut milk was extracted from
oil enriched with lycopene and beta mature coconuts (10-12 months old). It was
carotene. purchased from local markets in Thongsong,
Coconut oil is one of vegetable oil Nakhon Si Thammarat, Thailand. All
which is rich in saturated fatty acids (93%).3 chemicals as analytical grade were procured
It contains medium chain fatty acids (60%) from J.T. Baker chemicals, USA.
which burnt for energy rather than stored in
2.2 Preparation of coconut oil with Reaction flask was shaken for 10 s in vortex
extracts from gac fruit aril and tomato mixer and it was allowed to stand in the dark
500 Grams of gac fruit aril or 1,000 for 30 min. The absorbance of this mixture
g of tomato and 500 mL of water were was measured by spectrophotometer
mixed. This mixture slurry was filtered (Biochrom S22) in a 1 cm quartz cell at 30
through a cheesecloth to obtain solution min against a blank of ethyl acetate without
from gac fruit aril. The solution was added DPPH radical. %Inhibition was determined
into a flask (10 L) containing coconut milk from differences in absorbance of DPPH
(1 kg of coconut endosperm: 3L of water). solution with or without sample (control).
This mixture was stored at 4 oC for 12 h to
coagulate. After that this mixture was melt % Inhibition = (Acotrol - Asample) x 100
to obtain coconut oil with extracts from gac Acotrol
fruit aril (COG) and that with extract from
tomato (COT). These oil samples were kept The graph between %inhibition vs.
in amber flask. concentration of sample or standard
2.3 Determination of total phenolic antioxidant was plotted to obtain linear
content equation. The concentration of sample or
2 g of oil sample was added into 25 standard antioxidant at with 50% inhibition
mL of hexane. After that the mixture was of free radical activity was determined.
shaken. The absorbance of this mixture was 2.6 ABTS method
measured at 725 nm by spectrophotometer The antioxidant activity of sample
(Biochrom S22). Gallic acid was used as a oils was determined by the ABTS radical
standard. cation (ABTS.+) delocourisation assay. The
2.4 Determination of lycopene ABTS.+ working solution was prepared from
10 g of oil sample was dissolved in ABTS (7 mM) (8 mL) and potassium
hexane:acetone:ethanol (25:12.5:12.5). After persulfate (2.45 mM) (12 mL). The mixture
that, this solution was transferred to was allowed to stand in the dark at 4 oC for
separatory funnel and added with 10 mL of 16-18 h before use. The solution was diluted
methanol:water (80:10 v/v). This mixture in ethanol to give an absorbance at 750 nm
was shaken for 3 min to obtain the upper of 0.800±0.200 before use. The stock
layer and followed by repeating 2 times. solution of oil sample and standard
The absorbance of solution from upper layer antioxidant were prepared. ABTS.+ working
was measured at 663, 645, 505 and 453 nm solution and sample were mixed. The
by spectrophotometer (Biochrom S22). mixture was shaken vigorously and left for
Lycopene was calculate from equation (1) 30 min and the absorbance was measured at
750 nm. The solution for determining
Lycopene (mg/100 g oil) = -0.0458A663 + antioxidant activity was prepared as
0.204A645 + 0.372A505 - 0.0806A453 ….(1) following: 1) sample solution was the
mixture between the oil sample and ABTS
2.5 Radical scavenging activity (RSA) working solution, 2) blank solution was the
toward DPPH radicals mixture between the oil sample and ethanol,
Radical scavenging activity of COG 3) positive solution was the mixture between
and COT were examined by reduction of ethanol and ABTS working solution, 4)
DPPH radical in ethyl acetate. One gram of negative solution was the ethanol.
oil was dissolved in ethyl acetate in 10 mL Absorbance of these solution were measured
volumetric flask, and then 1 mL of this at 750 nm at 30 min. The percentage of
solution was transferred into the second 10 inhibition was calculated by using the
mL volumetric flask containing DPPH following equation:
radical solution, which was freshly prepared %Inhibition = [(Abs controlc - Abs
in ethyl acetate at a concentration of 10-4 M. samplee)/Abs controlc] x 100,
where Abs controlc is Abs positive – Meanwhile, the ABTS radical cation
Abs negative, and Abs samplec is Abs undergoes the reduction process by
sample – Abs bank. The graph between hydrogen donating antioxidant and can be
%inhibition vs. concentration of sample or spectrophotometrically measured. It is well
standard antioxidant was plotted to obtain accepted that the carotenoid content and
linear equation. The concentration of a lycopene in COG and COT influence the
sample or standard antioxidant with 50% antioxidant activities. In agreement, our
inhibition of free radical activity was findings showed that radical scavenging of
determined. DPPH and ABTS were positively correlated
2.7 Statistical analysis to the carotenoid content and lycopene.
All experiments were carried out in carotenoid content and lycopene in COG
triplicate and expressed as mean ±standard and COT play important roles in inhibitory
deviation (SD). effect of ROS and antioxidant agent.
In this work, the antioxidant activity
3. Results and Discussion of resulting coconut oil was assayed with
3.1 Yield of COG and COT DPPH (2,2-diphenyl-1-prohydrozyl), and
Coconut milk is an oil-in-water ABTS and was shown in Table 2.
emulsion of coconut oil droplets stabilized Antioxidant activity with ABTS and DPPH
by coconut proteins such as cocosin, calculated in IC50. The results showed that
globulins and albumins act as emulsifier and COG show the higher antioxidant activity
phospholipids. In this work, COG and COT calculated in IC50, 0.007 g/mL and 0.052
is produced by freezing as shown in Figure g/mL for ABTS and DPPH radical
1. scavenging assay, respectively than COT
(0.036 and 0.135 g/mL).
Table 2. IC50 of oil sample at 30 min by

ABTS and DPPH assay
Oil sample IC50(g/mL) IC50 (g/mL)
COT (ABTS) (DPPH)
COG
COG 0.007±0.000 0.052±0.004
COT 0.036±0.364 0.135±0.002
-
Vitamin C 0.0024±0.000
(a) (b)
Figure 1. COG (a) and COT (b) 3.3 Stability of lycopene and total
carotenoids
Table 1. Yield of virgin coconut oil (VCO), The stability of lycopene and total
COG, COT carotenoids in COG and COT was studied
Oil sample %yield during storage in the dark and light at room
VCO 18.67±1.76
COG 16.80±1.95 temperature for 12 weeks as shown in Table
COT 17.61±0.64 3 and 4. We found that lycopene and
carotenoid in COG and COT during storage
3.2 Antioxidant activity in the dark was reduced less than storage in
Approaches can be used to study the light. Many studies have reported that
radical scavenging activity (RSA) of carotenoid and lycopene can degrade due to
compounds as DPPH and ABTS. light or heat during storage.4,5 Moreover,
Scavenging of DPPH radicals is mechanized coconut oil with extract from gac fruit had
by the donation of hydrogen atom to the the higher lycopene and total carotenoids
unpaired electron of nitrogen bridge causing than that with extract from tomato.
the purple color turn to yellowish.
Table 3. Stability of lycopene and total carotenoid in COG
Time (week) Lycopene (mg/100g oil) Total carotenoid (mg/1g oil)
light dark light dark
0 2.37±0.01 2.30±0.01 0.85±0.01 0.87±0.00
3 2.08±0.01 2.03±0.01 0.80±0.00 0.81±0.00
6 1.67±0.01 1.84±0.01 0.76±0.00 0.82±0.00
9 1.19±0.01 1.92±0.01 0.65±0.02 0.80±0.02
12 0.80±0.01 1.85±0.01 0.38±0.29 0.80±0.01
Table 4. Stability of lycopene and total carotenoid in COT

Time (week) Lycopene (mg/100g oil) Total carotenoid (mg/g oil)
light dark light dark
0 0.50±0.01c 0.49±0.01c 0.10±0.00a,b 0.10±0.00a,b
3 0.39±0.01b,c 0.35±0.01b 0.07±0.00a 0.07±0.00a
6 0.39±0.01b,c 0.43±0.01b,c 0.08±0.00a,b 0.09±0.01a,b
9 0.23±0.01a 0.35±0.01b 0.06±0.00a 0.12±0.00a,b
12 0.17±0.01a 0.45±0.01b,c 0.06±0.00a 0.09±0.00a,b
This concluded that coconut oil with 1. Müller, J.; Sprenger, J.; Hempel, J.;
extracts from gac fruit had the higher Kreiser, F.; Carle, R.; Ralf, M. S. Food
antioxidant activity and stability of lycopene Res. Inter. 2016.
and total carotenoids than that with extract 2. Vuong, L. T. Food Nutr. Bull. 2000, 21
from tomato. Lycopene and carotenoid in (2), 173–181.
COG and COT stored in the dark had higher 3. Marina, A. M.; Che, Y. B.; Nazimah, S.
stability. J. Am. Oil Chem. Soc. 2009, 86: 991-
999.
Acknowledgements 4. Aoki, H.; Kieu, N. T. M.; Kuze, N.;
This work was supported with grants Tomisaka, K.; Chuyen, N. V. J.
from the office of the National Research Biotech. 2002, 66 (11), 2479–2484.
Council of Thailand (2560) and the Faculty 5. Cao, L.; Phan, H.; Osorio, F. J.; Waché,
of Science and Technology, Rajamangala Y. Food Res. Inter. 2011, 44, 2252–
University of Technology Srivijaya. 2257.
Survey of benzophenones residues contaminated in food packaging paper
in Bangkok, Thailand; using GC-MS and IDMS techniques
Neungrutai Saesaengseerung
Department of Science Service, Division of Chemicals and Consumer Products,
Household Products Section, Ratchathewi, Bangkok 10400, Thailand
E-mail: sneungrutai@dss.go.th
Abstract:
Benzophenone (1) and its derivatives, including 4-methylbenzophenone (2), 4-
hydroxybenzophenone (3), 2-hydroxybenzophenone (4), 4,4’-bis(dimethylamino)benzo-
phenone (5) and 4,4’-bis(diethylamino)benzophenone (6) are known as carcinogenic
compounds. They are photo-initiators used in UV cure ink for printing on food packaging
paper. Contamination of these residues in the paper may cause human health problems.
During year 2015 – 2016, benzophenones and its five derivatives in eighty food packaging
papers with various types, such as baking, cup, dish and food box paper, collected from
grocery stores and supermarkets in Bangkok, were analysed. 95% ethanol was used as an
extraction solvent and sonication was performed for 30 min. The supernatant was filtered and
extraction procedure was repeated further 2 times. The combined extracts were dried by
rotary evaporation and dissolved in dichloromethane:cyclohexane (1:1) prior to gas
chromatography mass spectrometry (GC-MS) with isotope dilution mass spectrometry
(IDMS) analysis. The results indicated that only three compounds were found to exceed the
limit declared by the European paper and board food packaging chain, in year 2012. A
benzophenone (1) was detected in 7.5% of all samples in the range of 0.15 - 3.5 mg/dm2,
while compounds (5) and (6) were detected in 2.5% and 1.25% of all samples, respectively,
in the range of 0.08 – 0.11 mg/dm2.
1. Introduction
Benzophenone (1) and derivatives
(2)-(6) (Figure 1) known as carcinogenic
compounds have been widely used as photo-
initiators for UV-cured ink of paper
packaging printing. They cannot be
removed completely after the printing Figure 1. Chemical structure of compounds
process. Also, they are not completely tested in this study, consisted of
diminished from recycled printed paper and benzophenone (1), 4-methyl benzophenone
board. These compounds may persist in any (2), 4-hydroxybenzophenone (3), 2-
packaging made from these recycled hydroxybenzophenone (4), 4,4’-
materials even if that packaging itself has bis(dimethylamino)benzophenone (5) and
not been printed with UV-cured inks 4,4’-bis(diethylamino)benzophenone (6)
containing the toxic compounds.1-3
At the present time, printing inks Several survey studies revealed the
and/or paper as well as cardboard are not contamination of those compounds in food
covered by specific European legislation on packaging paper. For example, in year
food contact materials. However, there was 2010, Koivikko R. et al.,3 analyzed 46 food
a guideline published by the European paper packaging papers by HPLC-DAD and GC-
and board food packaging chain, in year MS and found the contamination of
2012. This guideline declared the limit of substance (1) in the range of 0.57-3.99
substances (1) - (6) in food contact paper.4 mg/dm2 from more than half of the samples
and substance (2) in 30% of tested samples. of cyclohexane:dichloromethane (1:1) in a
Another survey 2 was published in year vial. The stock solution (5 g) was
1997. The study was conducted on analysis transferred into a volumetric flask (100 mL)
of more than 1900 food package paper and adjusted the volume using mixture of
samples in the United kingdoms and cyclohexane:dichloromethane (1:1) to obtain
substances (5) and (6) were detected at very intermediate standard mixture (1) - (6)
low concentration, causing no risk to human solution (50 mg/L).
health. The information obtained from these Stock solution of internal standard
surveys was advantage to consumer health. (1000 mg/L) was prepared by dissolving 50
In Thailand, contamination of benzophenone mg of benzophenone-D10 (7) in 50 g
and its derivatives has not been studied yet. cyclohexane:dichloromethane (1:1) and
As a result, in this study, analysis of diluted to intermediate internal standard
benzophenones residues contaminated in solution (50 mg/L) with the same solvent.
food packaging paper was performed using To prepare the standard solution for
extraction and GC-MS and IDMS calibration curve in the ranges of 0.1 - 10.0
techniques guided by the previous work.5,6 mg/L, intermediate standard mixture (1) -
(6) solution (50 mg/L) at volumes of 0.05,
2. Materials and Methods 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.5 and 5.0mL
2.1 Food packaging paper samples was pipetted into separately volumetric flask
The paper samples (without food) for (25 mL), added with 0.2 mL of intermediate
the study were collected from grocery stores internal standard solution (50 mg/L) and
and supermarkets, in Bangkok, Thailand. adjusted volume using cyclohexane:
All eighty paper samples consisted of baking dichloromethane (1:1) to obtain concertation
package paper (n = 21), trays, dishes and at 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0 and
bowls (n = 18), food bags (n = 2), baking 10.0 mg/mL, respectively. The solution was
papers cups (n = 20), molded pulp fruit trays transferred to 2 mL GC vial prior to GC-MS
(n = 3), drinking cups (n = 13) and tea filter analysis. Calibration curves were
papers (n = 3). constructed by plot between Y-axis
2.2 Chemicals and standards (quantification ion peak area for each
Standards including benzophenone compound (1) - (6) (see Table 1, Asub) to
99.9% (CAS no. 119-61-9), 4- area peak of internal standard (m/z = 110,
methylbenzophenone 99.9% (CAS no. 134- (7)) and X-axis (concentration of standard
84-9), 4-hydroxybenzophenone 99.9% (CAS (1) - (6)).
no. 1137-42-4), 2-hydroxybenzophenone
(CAS no. 117-99-7), 4,4’-bis(dimethyl- Table 1. Quantified ion and qualification
amino)benzophenone 99.5% (CAS no. 90-94- ion of substances (1) - (7), obtained from
8) and 4,4’-bis(diethylamino)benzophenone injection of each standard compound (10
99.7% (CAS no. 90-93-7) were purchased mg/L)
from Sigma Aldrich. Internal standard Substances Quantification Qualification ion
benzophenone-D10 99.5% (CAS no. 22583- ion (amu) (amu)
(1) 105 182, 77
75-1) was purchased from AccuStandard®,
(2) 118 196, 181
Inc. Cyclohexane 99.9% (CAS no. 110-82- (3) 121 198, 197
7) and dichloromethane 99.9% (CAS no. 75- (4) 121 198, 197
09-2) were supplied from Fisher. Ethanol (5) 148 268, 224
absolute 99.9% (CAS no. 64-17-5) was (6) 309 324, 132
(7) 110 192, 82
supplied from Merck.
2.3 Standard solution
Stock solution (1000 mg/kg) of 2.4 Sample preparation5
standard mixture (1) - (6) was prepared by Each paper sample was cut into
dissolving each standard at 50 mg with 50 g small pieces (0.25 - 1 cm2), weighed (0.1 - 2
g) and placed into an amber vial with cap. 2.7 Substance quantification
The sample was added with internal Detection (LOD = 0.25, 0.38, 3.8,
standard solution (50 mg/L, 0.2 mL) and 0.88, 0.50 and 0.25 mg/kg for substances (1)
sonicated for 30 min (Sonorex, super RX - (6); respectively) and quantification limits
255H, Germany) in 95% ethanol (20 mL). (LOQ = 1.3, 6.5, 9.0, 11.1, 9.2 and 9.2
The supernatant was filtered through a plug mg/kg substances (1) - (6); respectively)
of defatted cotton wool in a Pasteur pipette. were obtained from the result of an in-house
The procedure was repeated for another two method validation by previous study.6
times and the combined extracts were 2.8 Internal quality control
reduced to dryness by a rotary evaporator Calibration curve was prepared for
(Buchi, Switzerland). Extract was dissolved every analytical batch with the limit of
in cyclohexane: dichloromethane (1:1) and acceptance (correlation coefficient, r2 
transferred to volumetric flask (25 mL) prior 0.990). The food packaging paper samples
to GC-MS analysis. were analyzed in analytical batches of about
2.5 GC-MS analysis5,6 10 samples. Each batch contained two
Instrument for analysis was carried solvent blanks, duplicate of sample extract
out using a GC-MS (Thermo ScientificTM, and one sample spiked with 50 mg/L 0.5 mL
Italy) comprising a TraceTM 1300 gas of benzophenone (1). If the recovery of the
chromatograph, an AI 1310 autosampler, spike did not fall in the range 80-120%, the
ISQ LT mass spectrometer. The batch would be rejected. The rejected batch
XcaliburTM2.2 software was used for was repeated again.
processing data. Analysis was in both full
scan mode (50 - 350 amu) and the selected 3. Results and Discussion
ion monitoring mode following all ions in 3.1 GC-MS with IDMS chromatogram
Table 1. The carrier gas was helium with Chromatogram obtained from full
the flow of 1.0 mL/min. The injector scan mode GC-MS analysis of standard
temperature was 300 C and injection was solution mixed between benzophenone (1),
performed in the splitless mode onto a TG- derivatives (2) - (6) and isotopic internal
17SilMS capillary column (Thermo standard (7) is shown in Figure 2. Using the
Scientific; 30m × 0.25 mm id., 0.25 µm film NIST/EPA/NIH mass spectral library
thickness). The column was held at 150 C version 2.0, build in May 19, 2011, mass
for 2 min after injection and then spectrum of each peak in the chromatogram
programmed to rise at 20 C/min to 290 C was identified. It was found that derivatives
with a final hold time of 15 min. (2), (3), (4), (5) and (6) could be well
2.6 Substance identification separated at 7.02, 8.84, 6.84, 17.76 and
To considerate the presence of 21.02, respectively, while co-elution
substances (1) - (6) in a food paper sample, between benzophenone (1) and
all the following criterial had to be met for benzophenone-D10 (7) was obtained at 6.30
the analytical batch. min. It is possible that starting temperature
 All quantification and qualification ion of of GC condition, 150 C for 2 min, was too
each substance following Table 1 were high to separate two isotopic compounds (1)
detected. and (7) when using only full scan mode for
  0.02 min difference between the GC-MS. Results of GC-MS with IDMS
retention time for quantification ion and technique analyzed with SIM mode showed
qualification ion of each substance. a separation between a benzophenone (1)
  0.1 min difference in the retention time and a benzophenone-D10 (7) at 6.29 and
for quantification and qualification ion of 6.25 min, respectively.
each substance following Table 1
between standards and samples.
Table 2. Data of calibration curve of
benzophenone and its derivatives obtained
from GC-MS with IDMS technique analysis
Substances r2a Linear range
(mg/L)
(1) 0.9995 0.02-10.49
(2) 0.9989 0.02-10.45
(3) 0.9969 0.31-10.31
(4) 0.9986 0.37-12.66
(5) 0.9960 0.53-10.53
(6) 0.9955 0.57-10.54
a 2
r was correlation coefficient of the calibration
Figure 2. GC-MS chromatogram obtained equation.
from full scan mode (50.0-350.0 amu)
analysis of 5 ppm benzophenones (1) - (6) 3.2 Analysis of samples
standard mixture solution and isotopic Results of 80 food paper samples
internal standard (7) extracted by 95% ethanol and analyzed by
GC-MS with IDMS techniques are shown in
An evidence of the separation was a Table 3. Most abundant photo-initiator
illustrated by obtaining of benzophenone (1) found in this survey was benzophenone (1),
mass ion, involving m/z = 182, 105 and 77, which was detected in 35 samples (43.75%)
at 6.29 min and obtaining of benzophenone- of the total 80 samples, while 4,4’-
D10 (7) mass ion, involving m/z = 192, 115 bis(dimethylamino)benzophenone (5) and
and 82, at 6.25 min (Figure 3). 4,4’-bis(diethylamino)benzophenone (6)
were detected in 2 samples (2.5%) and 1
sample (1.25%) of all sample, respectively.
Consideration of benzophenone (1)
contamination, it was found that the baking
package paper (n = 21) was the largest type
contaminated with benzophenone (1) in 20
samples, whereas the trays/dishes/ bowls (n
=18), food bags (n = 2), baking paper cups
(n = 20) and molded pulp fruit trays (n = 3)
were found contaminated with
benzophenone (1) in 6, 2, 4 and 3 samples,
Figure 3. GC-MS chromatogram obtained respectively. Notably, there was no
from SIM mode analysis, following 2.5, of 5 benzophenone (1) contamination in drinking
ppm benzophenones (1) - (6) standard cups (n = 13) and tea filter papers (n = 3)
mixture solution and isotopic internal made from virgin pulp fiber, while
standard (7) contamination obtained from other paper
types, especially samples which the printed
Although the difference between a surface was glossy cover, such as sample no.
retention time of benzophenone (1) and 11 and 19 and 24 (Figure 4), could be
isotopic internal standard (7) was about detected at high level because the samples
0.04 min, the calibration curves of were possibly printed with UV-cured ink or
substances (1) - (6) were created with the made from recycle paper.1
limit of acceptance (r2  0.990). The linear
range of the calibration curves of substances
(1) - (6) is shown in Table 2.
Table 3. Level of substances (1) - (6) were found in 80 food paper samples
Sample type Sample No. Ga Detected Conc. mg/kg
g/m2 substances (mg/dm2) b
Baking package 1 51.8 n.d.c n.d.c
paper 2 50.0 (1) 2.4 (0.001)
(n = 21) 11 384.4 (1) 897.6 (3.5)
12 479.1 (1) 5.1 (0.02)
19 350.1 (1) 92.1 (0.32)
(6) 32.4 (0.11)
83 397.1 (1) 2.6 (0.01)
84 353.5 (1) 1.7 (0.006)
79, 80, 81, 88, 82,85, 86, 87, 89, 90, 91, 92, 93, 94 -d (1) < 1.3 (-)e
Trays/Dishes 3 237.1 (1) 1.5 (0.004)
/ bowls (n = 18) 4, 43 -d (1) < 1.3 (-)e
24 255.4 (1) 107.5 (0.27)
44 307.1 (1) 49.0 (0.15)
45 300.3 (1) 7.8 (0.02)
38, 40, 41, 42, 46, 47, 63, 64, 65, 66, 67, 68 -d n.d.c n.d.c
Food bag 5, 6 -d (1) < 1.3 (-)e
(n = 2)
Baking paper 7, 8 -d n.d.c n.d.c
cups (n = 20) 9, 59 -d (1) < 1.3 (-)e
10 267.2 (1) 58.2 (0.16)
48, 49, 50,51, 52, 53,54, 55, 56, 57, 58, 6061, 62 -d n.d.c n.d.c
95 52.9 (1) 10.7 (0.006)
molded pulp 18 364.3 (1) 78.9 (0.29)
fruit trays (5) 21.5 (0.08)
(n = 3) 96 229.8 (1) 25.7 (0.06)
(5) 42.7 (0.10)
97 111.7 (1) 25.1 (0.03)
Drinking cups 36, 37, 39, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 -d n.d.c n.d.c
(n = 13)
tea filter papers 98, 99, 100 -d n.d.c n.d.c

(n = 3)
a
Grammage of paper (as g/m2) measured following ISO 536: 2012.7
b
Concentration of substance was detected in mg/kg unit and the unit was converted to mg/dm 2 using formula
given in the paper of the European paper and board food packaging chain.4
c
Benzophenone (1) and its derivatives (2) - (6) was not detected because the test result were less than LOD.
d
Gramamage was not determined.
e
Convertion unit from mg/kg to mg/dm2 was not determined.
Consideration of benzophenone (1) packages, such as papers of plastic films

contamination concentration, only six have not enough good barrier property.8,9
samples (calculated as 7.5% of all samples), For the other contamination, 4,4’-
including sample no. 10, 11, 18, 19, 24 and bis(dimethylamino)benzophenone (5) was
44 (Figure 4) were contaminated in the found exceed the limit (0.0016 mg/dm2)4 in
range of 49.0 - 897.6 mg/kg (0.15 - 3.5 2 molded pulp fruit trays sample no. 18 and
mg/dm2, Table 3), which exceed the limit 19 at 0.08 and 0.10 mg/dm2, respectively.
(0.1 mg/dm2) declared by the European Finally, 4,4’-bis(diethylamino)benzophenone
paper and board food packaging chain, in (6) was surprisingly detected in sample no.
year 2012.4 Although sample no. 11 and 19 19 at 0.11 mg/dm2 which exceed the limit
were used as secondary packaging, it is (0.0016 mg/dm2).4
possible that benzophenone (1) could
migrate to food with gas-phase diffusion
mechanism if the cooperated primary
Acknowledgements
This work was a part of a research
project named “Survey of restricted organic
substance in food contact materials: paper
and board for the safety of consumers”
funded and supported by Department of
Science Service, Ministry of Science and
Technology, Thailand.
References
1. Anderson, W. A. C.; Castle L. Food
Add. Contam. 2003, 20, 607–618.
2. Castle, L.; Damant, A. P.; Honeybone,
C. A.; Johs, S. M.; Jickells, S. M.;
Sharman, M.; Gilbert, J. Food Add.
Figure 4. Appearance of paper samples Contam. 1997, 14, 45-52.
were tested by GC-MS and IDMS 3. Koivikko, R.; Pastorelli, S.; Rodríguez,
techniques and found an exceed limit of A.; Paseiro, R.; Paseiro, P.; Simoneau,
benzophenones, involving compound (1), C. Food Add. Contam. 2010, 27, 1478-
(5) and (6). The number shown under each 1486.
sample pictures refers to the sample no. in 4. http://www.cepi.org/system/files/public/
the Table 3. In the sample no. 19 and 24, documents/publications/foodcontact/20
the mark of packaging brand was hidden 12/Industry%20guidelineupdated2012fi
with a blue area labeled with the alphabet a. nal.pdf. (Accessed June 26, 2013).
5. Saesaengseerung, N. Bull. Appl. Sci.
4. Conclusion 2016, 5, 97, 97–102.
The survey was carried out with 80 6. Saesaengseerung, N.; Nuamthanom, A.;
samples of food paper packaging. Results Sriyotai, W.; Singra, P.; Phothisuwan,
showed that primary packaging such as A., Project report, Department of
drinking cups and tea filter papers were not Science Service, Bangkok, Thailand,
contaminated with studied benzophenones. 2017.
However, some of primary packaging, such 7. International Standard, ISO 536-2012.
as sample no. 10, 24 and 44 was Paper and board – Determination of
contaminated with benzophenone (1) exceed grammage.
the limit.4 Also, exceed the limit of 8. Johns, S. M.; Gramshaw, J. W.; Castle,
benzophenone (1) contamination were L.; Jickells, S. M. Deutsche
detected in secondary packaging, such as Lebensmittel-Rundschau. 1995, 91, 69–
sample no. 11 and 19 (3.5 and 0.32 mg/dm2) 73.
so high barrier primary packaging were 9. Biedermann, M.; Ingenhoff, J. E.;
required to protect the gas phase migration Zurfluh, M.; Richter, L.; Simat, T.;
of benzophenone (1) to food. For substances Harling, A.; Altkofe, W.; Helling R.;
(5) and (6), although they were found in Grob, K. Food Add. Contam. 2013, 30,
2.5% and 1.25% of all sample, concentration 885-898
was exceed the limit cause of human health.4
The detection of organophosphate insecticide using immobilized whole cells
from the clone carrying mpdB gene expressing methyl parathion hydrolase
Chadaporn Pootawee, Witsanu Senbua, Chaiyong Taechapairoj, Jesdawan Wichitwechkarn*
*E-mail: wjesdawan@gmail.com
Abstract:
Because of high contamination in agricultural products and the environment of
organophosphate (OP) insecticide, we are interested in studying a simple and convenient
method for OP detection, using methyl parathion (MP) as a representative insecticide. Methyl
parathion degrading, mpdB, gene from MP-degrading Burkholderia cepacia was previously
cloned and expressed in Escherichia coli as a fusion protein of methyl parathion hydrolase
(MPH) and glutathione-S-transferase (GST) [MPH-GST]. The MPH-GST was capable of
degrading MP and other phenyl-substituted OP to a yellow product p-nitrophenol (PNP).
Whole cells from this clone were used to detect MP by spectrophotometrically measuring
PNP at OD410. The cells were optimally immobilized onto the polystyrene-microtiter plate,
using physical adsorption followed by cross-linking with glutaraldehyde. Scanning electron
microscope analysis revealed numerous cells of rod shape binding to the plate surface. The
temperature at which the enzyme activity was most stable was 25°C. This bio-component
system could be used to detect MP repetitively up to 46 rounds. The MP detection limit of
this system was 2 µg/mL, and the linear range was 2-200 µg/mL. This whole cell
immobilization system has a great potential to be utilized for the detection of OP insecticides
with ease, convenience and low cost.
1. Introduction and inexpensive method is still in need for

Extensive use and contamination of screening detection in agricultural products
organophosphate (OP) insecticide, a group and the environment.
of neuro-toxic compounds, in agricultural Here we used our clone of mpdB
field has been a threat to human health and (MP-degrading) gene from MP-degrading
the environment. OP exerts its detrimental Burkholderia cepacia, capable of degrading
effects by the inhibition of the enzyme MP and other OPs, for the detection of OP
acetylcholinesterase (AChE) that normally insecticides. This clone, namely BpGP, was
hydrolyses acetylcholine, causing the expressed in E. coli as MPH-GST fusion
accumulation of this neurotransmitter.1 In protein which could degrade MP and other
order to effectively monitor and control the phenyl-substituted OPs to a yellow product,
OP contamination, a sensitive, simple, and PNP. Our objective is to use the
rapid method for the detection of the immobilized whole cells from this clone to
insecticide is required. Conventional detect OPs, using MP as a representative
methods such as gas chromatography and insecticide.
high performance liquid chromatography are
expensive, time-consuming, laborious, 2. Materials and Methods
require elaborated equipment and well- 2.1 Bacteria and construction of the mpdB
trained technician, and hence, are not clone
suitable for field use. Attention has been MP-degrading Burkholderia cepacia
drawn to the development of biosensors for was previously isolated from soil in
OP detection such as AChE2-4 and OPH5- Thailand.11 The mpdB gene was isolated and
7
/MPH8-10 biosensors. Nevertheless, simple purified from the bacteria,12 and cloned into
an expression plasmid pGEX 6P-1 which For reusability on MP detection of
was then transformed into E. coli BL21,13 the immobilized cells, 50 µg/mL of MP was
yielding BpGP clone. The clone was grown added to the wells at each round of
in LB medium with 100 µg/mL of ampicillin detection. The reactions were allowed to
at 37 oC and was induced by 1 mM IPTG in occur for 5 min at room temperature before
the presence of 0.5 mM CoCl2 as previously measuring OD410 by using the microplate
described.13 MPH-GST expressed from the reader (Tecan, Switzerland). After each
clone was tested for its MP degradation round of reaction, the cell suspensions were
activity by MPH microtiter plate assay gently removed and the wells were washed
and/or spectroscopic measurement at 410 with 50 mM phosphate buffer, pH 8.0,
nm as previously described.13 before starting the new round. These were
2.2 Cell immobilization performed for 100 rounds. For temperature
Cell immobilization was performed effect on the stability of the system for MP
onto the well surface of 96-well polystyrene detection, the reactions were allowed to
microtiter plate, using adsorption method, occur at different temperatures (4, 25, 37
followed by cross-linking with and 50 oC) and the OD410 was measured
glutaraldehyde.14,15 Cells from the culture of every day for 30 days. All reactions were
clone BpGP were resuspended in 50 mM done in triplicate.
PBS, pH 8.0 and were added to the center of 2.4 Effect of MP Concentration on
each well. The plates were kept at 4oC until Spectrophotometric Measurement
the cells were dried or the volume reduced To determine the effect of MP
as much as possible. The cell suspension concentration on the OD410 value, various
volume was varied as 10, 20, 30, 40, and 50 concentrations of MP (2-250 µg/mL) were
µl. Glutaraldehyde of different percentages added to individual wells. After 5 min
(0.2, 0.5, 1, 2 and 5% v/v) was separately incubation at room temperature, OD410 of
added to each well at varying volumes (8, 10 the reaction mixture in each well was
and 12 µl), and the plates were left at room measured. The reactions were performed in
temperature (28 C) for 3.5 hours. The wells triplicate. The detection limit and linear
were then gently washed with 50 mM range for MP detection were determined as
phosphate buffer, pH 8.0 and kept at 4 oC previously described.15
until use.
Electron microscopic detection of the 3. Results and Discussion
immobilized cells on the plate surface was 3.1 Cell immobilization
done using scanning electron microscope Clone BpGP used here contains
(SEM) (Hitachi, USA). The wells were cut mpdB gene which was previously cloned
to the height of 2 mm and, after cell into plasmid pGEX 6P-1.13 Its gene product
immobilization, were coated with gold was expressed as MPH-GST fusion protein
particles before viewed under SEM. The which possessed MP-degrading activity. The
magnifications used were 1,000x and MP degradation reaction by MPH is shown
10,000x. Immobilized cells were counted in Figure 1.
using total plate count technique, by Whole cells of clone BpGP were
washing the wells with 50 mM phosphate immobilized onto the polystyrene surface of
buffer, pH 8.0, and the washed-out cell 96-well microtiter plate by adsorption,
suspension were diluted and spreaded onto followed by cross-linking method using
LB plates containing 100 µg/mL of glutaraldehyde. From optimization tests, it
ampicillin. The culture plates were appeared that 40 µl of the cell suspension
incubated at 37 oC overnight and the number and 2% glutaraldehyde yielded highest
of cells were determined as CFU/mL. OD410, and hence were optimal conditions
2.3 Reusability and stability of the for the MP-degradation reaction (Figure 2).
immobilized cells for the detection of MP
Glutaraldehyde at the volume of 10 µL was
also shown to give highest OD410 (Figure 3).
Figure 2. Optimization of cell loading and

glutaraldehyde concentration for
immobilization
Figure 1. MP degradation reaction by MPH

With all these optimal conditions, it
was found that only negligible number of
cells was washed out during washing after
immobilization, being only 930 CFU/mL as
compared to 1.1x109 CFU/mL of the start
bacterial cell number used for the Figure 3. Optimization of glutaraldehyde
immobilization. SEM analysis of well volume and cell suspension volume for
surface with immobilized cells showed immobilization.
highly dense bacteria attached to the matrix
(Figure 4A) while only debris from well 3.2 Reusability and stability
cutting was found on empty well (Figure This immobilization system was
4C). Figure 4B clearly revealed rod-shaped tested for the reusability of MP detection for
bacteria, characteristic of E. coli, on the 100 rounds. It appeared to be reused for 46
immobilized well when viewed at 10 times rounds. As shown in Figure 5, the relative
higher magnification (10,000x). activity remained at approximately 70-100%
for the first 22 rounds, and reduced to about
40-60% after that until round 46. From
round 47 on, the relative activity dropped to
about 42-45% and remained constant
throughout the experiment.
Figure 4. Scanning electron micrographs of well surfaces with (A and B) and without (C)
immobilized cells. A and C were performed at 1,000x; B was performed at 10,000x
For stability test to determine the
effect of temperature, it appeared that 25 oC
was the temperature at which the
immobilized cells were most stable and
attained highest activity. From Figure 6, it
can be seen that at 25 oC the relative activity
gradually increased from day 4-11 to about
twice as much, after which it finally dropped
to about 60% until day 18, and remained
constant thereafter. This activity peak was
seen only at 25 oC. This is advantageous for Figure 6. Effect of temperature on stability
the practical use of this cell immobilization of the immobilized cells for the detection of
at room temperature. The temperature of 4 MP
o
C was the second most suitable temperature
for the stability, while at 37 oC and 50 oC, 3.3 Effect of MP concentration
the activities were too low. The effect of MP concentration was
investigated (Figure 7). When the OD410 was
measured with varying concentrations of
MP (2-250 µg/mL), it was shown that the
linear range of the system was 2-200 µg/mL
and the detection limit was 2 µg/mL.
Figure 5. Reusability of the immobilized

cells for the detection of MP
Figure 7. Effect of MP concentration
4. Conclusion 2. Palchetti, I.; Cagnini, A.; Carlo, M. D.;
Here, whole cell immobilization on Coppi, C.; Mascini, M.; Turner, A. P. F.
polystyrene microtiter plate, which can be Anal. Chim. Acta 1997, 337 (3), 315-
easily utilized for OP insecticide detection 321.
was described. The immobilization of the 3. Andreou, V. G.; Clonis, Y. D. Bios.
whole cells was successful with physical Bioelectron. 2002, 17 (1-2), 61-69.
adsorption, followed by cross-linking 4. Andres, R. T.; Narayanaswamy, R.
method using glutaraldehyde. Because of its Talanta 1997, 44 (8), 1335-1352.
ability to be repetitively used, its stability at 5. Ji, X.; Zheng, J.; Xu, J.; Rastogi, V. K.;
room temperature, its wide linear range, and Cheng, T. C.; DeFrank, J. J.; Leblanc,
low detection limit, this bio-component R. M. J. Phys. Chem. B 2005, 109 (9),
system therefore has a great potential as a 3793-3799.
selective means for practical detection of OP 6. Simonian, A. L.; Good, T. A.; Wang, S.
insecticides with ease, simplicity, and low S.; Wild, J. R. Anal. Chim. Acta 2005,
cost, which is needed for screening OP 534, 69-77.
contamination in agricultural products and 7. Lee, J. H.; Park, J. Y.; Min, K.; Cha, H.
the environment. OP detection by the system J.; Choi, S. S.; Yoo, Y. J. Biosens.
in real samples that tend to be contaminated Bioelectron. 2010, 25, 1566-1570.
with OP such as apple, grape, dragon fruit, 8. Lan, W.; Chen, G.; Cui, F.; Tan, F.; Liu,
cowpea, Chinese kale, red pepper, basil leaf, R.; Yushupujiang, M. Sensors 2012, 12,
soil and water in agricultural areas should be 8477-8490.
further investigated. 9. Zhao, Y.; Zhang, W.; Lin, Y.; Du, D.
Nanoscale 2013, 5 (3), 1121-1126.
Acknowledgements 10. Liu, G.; Guo, W.; Yin, Z. Biosens.
This research was supported by a Bioelectron. 2014, 53, 440-446.
grant from Graduate School and the 11. Keprasertsup, C.; Upatham, S.;
government budget through the Department Sukhapanth, N.; Prempree, P. Sci.
of Biotechnology, Faculty of Engineering Asia 2001, 27, 261-270.
and Industrial Technology, Silpakorn 12. Wongthianlai, S. Master’s Degree
University. We are grateful to Associate Thesis, Silpakorn University, 2004.
Professor Dr. Mana Sriyudthsak, at the 13. Ekkhuntham, A.; Jongsareejit, B.;
Department of Electrical Engineering, Yamkunthong, W.; Wichitwechkarn, J.
Chulalongkorn University, for his helpful World J. Microbiol. Biotechnol. 2012,
academic advice. We thank Mr. Nutthawut 28, 1739-1746.
Pheungphasutadol for his help on figure 14. Kumar, J.; D’Souza, S. F. Biosens.
preparation for the manuscript. Bioelectron. 2011, 26, 4399-4404.
15. Kumar, J.; D’Souza, S. F. Biosens.
References Bioelectron. 2010, 26, 1292-1296.
1. Abou, M. B. Arch. Environ. Health
2003, 58, 484-497.
Comparative study of total phenolic content and antioxidant activity of
coconut oil with Curcuma zedoaria Roscoe and Curcuma longa L. extracts
Norasing Penprapai1*, Amporn Ratanamusik1, Pensri Penprapai2, Manoch Chumchareon3
1
Faculty of Agriculture, Rajamangala University of Technology Srivijaya,
2
Faculty of Science and Technology, Rajamangala University of Technology Srivijaya,
3
Faculty of Science and Fisheries Technology, Rajamangala University of Technology Srivijaya,
Trang 92150, Thailand
*E-mail: norasing_p@hotmail.com
Abstract:
Coconut oil with extracts of Curcuma longa L. (COL) or Curcuma zedoaria Roscoe
(COZ) can be used in food for aquatic animals because its high total phenolic compound as
antioxidant and medium chain triglyceride which is easy to absorb. The objectives of this
work were to determine the antioxidant activity and the total phenolic content of the obtained
coconut oil, and applied COL. and COZ in feed to raising Pacific White Shrimp. The
antioxidant activities of resulting coconut oil were assayed with 1,1-Diphenyl-2-
picrylhydrazyl (DPPH) and lipid peroxidation method. DPPH radical scavenging activity
calculated as IC50 were found as 0.41 and 0.14 µg/mL for COL and COZ, respectively while
%antioxidant activities (AA) from lipid peroxidation method of COL and COZ were
observed as 82.52 and 92.20%, respectively. The results showed that COZ had the higher
antioxidant activities than COL. Total phenolc contents as determined by the Folin-Ciocalteu
method, were found as 1.29 and 1.52 mg GAE/g oil for COL and COZ, respectively. In this
work, COL and COZ were applied in feed to study growth performance of Pacific White
Shrimp. It was found that feed with COL affected growth performance of Pacific White
Shrimp higher than feed with COZ.
1. Introduction antiviral, antibacterial and antiprotozoal

Extracts of Curcuma longa L (COL). properties. Moreover, coconut oil lead to a
and Curcuma zedoaria Roscoe (COZ) have normalization of body lipids, protect against
shown various bioactivities, including alcohol damage to the liver, improves the
antimicrobial against bacteria and fungal, immune system and anti-inflammatory
antioxidant, anticancer, and anti-inflamatory response. The objectives of this work were
activities.1 C.longa L. and C. zedoaria to produce coconut oil with extract of C.
Roscoe contains curcuminoids including zedoaria Roscoe (COZ) and that with
curcumin, bis-demethoxycurcumin and extract of C. longa L. (COL) and to
demethoxy-curcumin with high antioxidant determine the antioxidant activity and the
activity. These compounds are natural total phenolic content of coconut oil
antioxidants and can be used for food, obtained. Moreover, COL and COZ were
cosmetic and other applications.2 applied in feed to study growth performance
Coconut oil is one of vegetable oil and survival rate of Pacific white shrimp.
which is rich in saturated fatty acids (93%).3
It contains medium chain fatty acids (60%), 2. Materials and Methods
especially lauric acid (C12:0) (45%), which 2.1 Materials
are burnt for energy rather than stored in the Coconut milk was extracted from
body. Lauric acid, found in coconut oil in mature coconuts (10-12 months old). It was
major amounts, is known for its unique purchased from local markets in Thongsong,
Nakhon Si Thammarat, Thailand. All longa L. extracts were examined by
chemicals as analytical grade were procured reduction of DPPH radical in ethyl acetate.
from J.T. Baker chemicals, USA. One gram of oil was dissolved in ethyl
2.2 Production of COZ and COL acetate in 10 mL volumetric flask, and then
One kilogram of fresh rhizome of C. 1 mL of this solution was transferred into
zedoaria Roscoe and C. longa L., and 500 the second 10 mL volumetric flask
mL of water were mixed and blended using containing DPPH radical solution, which
a blender. This mixture slurry was filtered was freshly prepared in ethyl acetate at a
through a cheesecloth to obtain solution. concentration of 10-4 M. Reaction flask was
Then this solution was added into a flask (10 shaken for 10 s using vortex mixer and it
L) containing coconut milk (5 kg of coconut was allowed to stand in the dark for 30 min.
endosperm: 5L of water). This mixture was The absorbance of this mixture was
stored at room temperature for 24 h to allow measured by spectrophotometer (Biochrom
some bioactive compounds of fresh rhizome S22; Libra S22, England) in a 1 cm quartz
to be extracted into the oil layer. The oil cell against a blank of ethyl acetate without
layer was separated from the cream and DPPH radicals. After that, %Inhibition was
water. Coconut oil with extracts of C. determined from differences in absorbance
zedoaria Roscoe and C. longa L. were of DPPH solution with or without sample
referred to as ‘COZ’ or ‘COL’, respectively. (control).
These oil samples were kept in amber flask.
2.3 Extraction of phenolic compounds % Inhibition = (Acotrol - Asample) x 100
5 g of oil sample was dissolved in 25 Acotrol
mL of hexane and transferred to a separatory
funnel. 25 mL of the methanol-water The graph between % inhibition vs.
mixture (80:10 v/v) was added. After 2 min concentration of sample or standard
of shaking, the lower methanol-water layer antioxidant was plotted to obtain linear
was removed. The extraction was repeated equation. The concentration of samples or
twice and the methanol-water phase was standard antioxidant at with 50% inhibition
combined. The methanol-water extract was of free radical activity was calculated.
evaporated in a rotary evaporator under 2.6 The rearing Pacific white Shrimp
vacuum at 40 C. The dry residue was then Pacific white shrimp was reared in
diluted in 1 mL of methanol. circle concrete ponds diameter of 1.5 m.
2.4 Determination of total phenolic Mean weight of Pacific white shrimp start at
content 1.0-2.0 g/body. Pacific white shrimp was
The content of total phenolic obtained from farm in Sikao district, Trang.
compounds in oil sample was determined by Sea water was added in circle concrete
Folin-Ciocalteau reagent. The reaction ponds approximately 400 L water/pond. Sea
mixture contained 1 mL of methanol-water water had salinity of 5-8 ppt. Pacific white
extract, 1 mL of freshly prepared diluted shrimp was reared for 28 days (4 weeks).
Folin-Ciocalteau reagent and 8 mL of Feeding was performed at 6 am, 11 am, 5
sodium carbonate solution. The mixture was pm and 10 pm. Density of Pacific white
kept in the dark at ambient condition for 30 shrimp/ponds was 62 body/m2. The
min. The absorbance at 725 nm was experiment was done using completely
measured by spectrophotometer (Biochrom Randomized Design (CRD), 3 treatment,
S22; Libra S22, England). Gallic acid was replication, T1 used Virgin coconut oil, T2
used as a standard. Results were expressed used COZ and T3 used COL.
as mg of gallic acid per g of oil. Feed ingredients: 48.90% fish meal,
2.5 DPPH radical scavenging activity 24.21% soy bean meal, 3.68% broken corn,
Radical scavenging activity in 3.68% rice bran, 3.60% broken rice, 3.40%
coconut oil with C. zedoaria Roscoe and C. squid oil, 2.0% compound vitamin, 3.0%
premix, 4.0% alfa starch, (T1, T2, T3, used hydrogen atom to the unpaired electron of
3% COZ or COL or VCO ). nitrogen bridge causing the purple color turn
to yellowish. It is well accepted that the
Table 1. Proximate chemical composition phenolic content significantly influence the
analysis (%dry matter basis of test feed 3 antioxidant activities.4-6 In agreement, our
formulae). findings showed that radical scavenging of
Treat Protein Crude Moisture Ash Energy DPPH and lipid peroxidation method were
ment (%) Fat (%) (%) (%) (Kal/100g)
T1 54.46 3.44 12.05 33. 248.50 positively correlated to the total phenolic
05 content. Phenolic compound such as
T2 54.20 3.37 12.37 33. 247.13 curcumin, and its derivatives play important
16 roles on antioxidant effect.
T3 55.11 3.92 9.66 33. 255.72 IC50 values of DPPH radical
36
scavenging in COZ and COL were found as
0.41 and 0.15 mg/mL, respectively. The
Weight gain (WG) and specific growth rate
results showed COZ and COL showed
was calculated in equation (1) and (2)
higher antioxidant activity as determined by
DPPH assay and lipid peroxidation method
Weight gain (WG), (g/body) = W1-W0 …(1)
than VCO.
Specific growth rate (SGR), (%/day)
= (lnW1 - lnW0) x 100 …… (2)
Table 3. IC50 of oil sample at 30 min by
T
DPPH assay and %AA by lipid peroxidation
W1 = Final wet weight
method
W0 = Initial wet weight Sample oil %AA at 0.001 IC50 (mg/mL)
T = Time interval in days. mg/mL DPPH
3. Results and Discussion VCO 21.55±.53a 4.29±0.41a

3.1 Total phenolic content COZ 92.20±1.32c 0.15±0.01b
The total phenolic contents in COZ COL 82.53±.93d 0.41±0.01b
and COL were found as 1.29 and 1.52 mg/g
Values are mean±SD of three independent
oil, respectively. The total phenolic contents experiments.
of COZ and COL were not significantly Data points with different superscript within the same
different. However, these values were rows differ significantly at P
different in comparison with virgin coconut ≤0.05
oil (VCO). The result indicated that C. longa
L. might contain the similar content of 3.3 Growth performance of Pacific white
phenolic compound as C. zedoaria Roscoe shrimp
C. longa have curcumin and phenol
Table 2. Total phenolic contents of VCO, and hydroxyl ion in molecular.7 It can
COZ and COL inhibit synthetic pro stagrandin (PG
Oil sample Total phenolic content (mg/ g synthetase) at 200 mg /L. It was used in
oil) form of powder of rhizome. In this research,
VCO 0.22 ±0.03a C. zedoaria Roscoe or C. longa L. was
COZ 1.29±0.14b extracted in coconut oil in liquid form which
COL 1.52±.103b
Values are mean±SD of three independent
easy to absorb. Coconut oil is one of
experiments. Data points with different superscript vegetable oil which is rich in saturated fatty
within the same rows differ significantly at P ≤ 0.05 acids (93%). It contains medium chain fatty
acids (60%) which burnt for energy rather
3.2 Antioxidant activity than stored in the body. Lauric acid (C12:0)
Antioxidant activity of samples is (45%) in coconut oil is major amounts. It
shown in Table 3. Scavenging of DPPH showed antiviral, antibacterial and
radicals is mechanized by the donation of antiprotozoal properties. We found that
Pacific white shrimp has the highest specific Council of Thailand (2560) and the Faculty
growth rate and weight gain with T3. of Agriculture, Rajamangala University of
Technology Srivijaya.
Table 4. Initial weight and final weight of
Pacific white shrimp References
Treatment Initial weight Final weight 1. Jarikasem, S.; Thubthimthed, S.;
T1 2.07 ±0.80 2.17±0.22 Chawanannoraseeth, K.; Suntrontarasat,
T2 1.78±1.08 1.90±0.23
T. Proceedings of WOMAP III 2005, 3,
T3 1.84±0.96 2.20±0.10
37-41.
2. Martinez, H. A.; Paula, J. T.; Kayano,
Table 5. Weight gain and specific growth
A. C. A. V.; Queiroga, C. L.;
rate of Pacific white shrimp
Magalhaes, P. M.; Costa, F. T. M.;
Treatment Weight gain Specific Cabral, F. A. J. Supercrit. Fluids 2017,
growth rate 119, 122-129.
T1 0.645±0.431 1.254±0.986 3. Marina, A. M.; Che, Y. B.; Nazimah, S.
T2 0.626±0.262 1.546±0.639 J. Am. Oil Chem. Soc. 2009, 86, 991-
T3 0.899±0.535 2.027±1.582 999.
4. Othman, A.; Mukhtar, N. J.; Ismail, N.
4. Conclusion S.; Chang, S. K. Int. Food Res. 2014, 21
Total phenolic content of COZ and (2),18.
COL were higher than VCO. Total phenolic 5. Qader, S. W.; Abdulla, M. A.; Chua, L.
content correlated well with antioxidant S.; Najim, N.; Zain, M. M.; Hamdan, S.
activity. COZ had the highest antioxidant Molecules 2011, 16 (4), 3433-3443.
activity calculated in IC50 by DPPH assay. 6. Wong, S. P.; Leong, L. P.; William, J.H.
COZ and COL had higher antioxidant Food Chem. 2006, 99 (4):775-783.
activity than VCO. Growth performance of 7. Supamattaya, K.; Suntornchareonnon,
Pacific white shrimp with treatment 3 was N.; Boonyaratpalin, M.; Ruangsri, J.;
the highest. Tatanon, T.; Klowkliang, T.
Songklanakarin J. Sci. Tech. 2005, 27
Acknowledgements (1), 55-70.
This work was supported by grants
from the office of the National Research
Nanoemulsion of Syzygium aromaticum (L.) Merr. & L.M.Perry. oil extract
in water prepared by emulsification process
Rungnapa Pimsen1*, Thiprada Thongrak1, Paweena Porawatkul1, Arnannit Kuyyogsuy1,
Nongyao Tepaya1, Monthakharn Thongsom2, Sudkamon Lasopa3
1
Department of Chemistry and Nanomaterials Chemistry Research Unit, Faculty of Science and Technology,
Nakhon Si Thammarat Rajabhat University, Nakhon Si Thammarat 80280, Thailand
2
Department of Biology, Faculty of Science and Technology,
Nakhon Si Thammarat Rajabhat University, Nakhon Si Thammarat 80280, Thailand
3
Department of Chemistry, Faculty of Science and Technology, SaKhonNaKhon Rajabhat University,
Sa Khon Na Khon 47000, Thailand
*E-mail: rung_cha@yahoo.com
Abstract:
Syzygium aromaticum (L.) Merr. & L.M.Perry. oil (clove essential oil) loaded
nanocapsules were prepared from oil-in-water emulsions using chitosan (ChS) as shell
material. A sodium dodecyl sulfate (SDS) was used to stabilize the charge of nanocapsules by
ionic interaction. The nanocapsules were prepared by ultrasonic emulsification with
sonication time of 5 min and 37 kHz frequency. The effects of ChS (50 to 150 ppm), SDS (5
x 10-3 to 20 x 10-3 M) and clove essential oil (0.5 to 1.5 g) concentration on particle size, and
colloidal stability of resulting emulsions were investigated using a dynamic light scattering
(DLS) and zeta potential measurements, and scanning electron microscopy (SEM). The zeta
potential of nanocapsules was in the range of -23.51-26.39 mV with particle size of 200-350
nm. The SEM image of the nanocapsules showed the spherical shape and uniform size
distribution. Based on antimicrobiological activity test, the developed nanocapsules could
inhibit both Escherichia coli and Staphylococcus aureus with inhibition zone of 9.0±0.0 and
9.5±0.5 mm, respectively. Moreover, this nanocapsule is proved to be cost-effective and
environmentally friendly.
distribute lipophilic components to water
1. Introduction phase of foods.
Essential oils are obtained from plant Chitosan is a biopolymer which has
extract and commonly exploited as additive confirmed to be an efficiency carrier with
in foodstuffs for antimicrobial activity essential characteristics such as its
purpose. The researcher are considering the biodegradability, biocompatibility,
utilization of the essential oil extracted as availability, safety, and innate antimicrobial
potential green chemical preservatives in potential.4
food industry such as thyme(Thymus This study aimed to evaluate the
vulgaris), lemongrass (Cymbopogon physical, mechanical and antimicrobial
citratus) and sage (Salvia officinalis).1–4 activity of Syzygium aromaticum (L.) Merr.
However, the compatibility of the extracted & L.M.Perry. oil filled nanocapsules using
and food media is restricted because of their sodium dodecyl sulfate and chitosan as shell
hydrophobic property with difficult material.
dispersion in water. Nanoemulsions have
been introduced to improve water 2. Materials and Methods
compatibility of essential oil extracted. The 2.1 Materials
oil extracted was encapsulate using Chitosan was purchased from
surfactant for stabilize the charge of Bonafides marketing, Thailand. The degree
nanocapsules by ionic interaction to of deacetylation was ca. 85%. SDS was
purchased from GFS, USA (≥98.5%).
Syzygium aromaticum (L.) Merr. & 3.1 Stability of clove essential oil
L.M.Perry. oil or clove essential oil was nanocapsules (%Creaming index)
purchased from Thai China Flavours & The stability of nanoemulsion was
Fragrances industry Co., Ltd. Acetic acid investigated using the creaming index under
(Sigma Aldrich, Germany, 99.9%) of 1% phase separation of stabilized emulsions
v/v was utilized as a medium. Distilled within 15 days. The creaming index of
water was used throughout this study. nanocapsules is reported (see Eq.1)
2.2 ChS/SDS-stabilized emulsions of clove
essential oil preparation
The mixtures of preset amount of
ChS, SDS, and clove essential oil, in 1%
acetic acid solution with the total volume of
50 ml were transferred into the glass bottle.
Then, the mixtures were ultrasonicated
(ultrasonicator, Elma S 60 H Elmasonic,
Germany) for 5 min with 37 kHz frequency.
2.3 Characterization of clove essential oil
nanoemulsions
The particle size of the formulated
clove essential oil emulsions was evaluated
using a dynamic light scattering (DLS)
method with a laser particle size analyzer at Figure 1. Creaming index percentage of
25 ◦C. The electrostatic or charge repulsion clove essential oil nanocapsules using ChS
of the prepared clove essential oil emulsions at 100 ppm and clove essential oil at 1 g
is known to affect stability. The surface
charge of prepared emulsions was It was found that the increasing ChS
determined by zeta potential analyzer, can cause the percentage of creaming index
Model ZetaP ALS, Brookhaven, USA. The reduction (Figure 1). As a result, it was
morphology of emulsion was characterized possible that the complex of chitosan and
by a scanning electron microscope (SEM) sodium dodecyl sulfate stabilized clove oil
SEM-Hitachi SU8020, Japan. in water emulsion, thus providing a superior
2.4 Clove essential oil emulsions stability resistance to droplet combination.
The clove essential oil emulsion was Accordingly the droplet size and creaming
transferred into a glass bottle. The height of progression was diminished.6
clove essential oil emulsions was fixed at 10 3.2 Zeta potential analysis of clove
cm. Then, the bottle of clove oil emulsion essential oil nanocapsulation
was left at room temperature for 15 days. The zeta potential with various SDS
The emulsion was separated into a milky concentrations was determined for the three
layer (cream) at the top and a translucent ChS concentrations at the three amounts of
layer (serum) at the bottom. The total height clove oil. At 5 × 10−3M SDS, positively
of the emulsion of each bottle (E) and the charged nanoemulsion droplets were found.
height of the serum layer (S) were At low SDS concentration, SDS fully
determined. The creaming index was interacted with ChS molecules at interfaces.
calculated by following equation: When SDS concentration increased up to 20
x 10-3 M, negative charged nanoemulsions
Creaming index = S/E×100 … Eq.1 were obtained (Table 1). As a result, the
ChS/SDS shell was re-dissolved in the
presence of excess surfactant.1
Table 1. Zeta potential of the nanocapsules The particle size distribution of the
in the various SDS concentration 5 x 10-3, nanoemulsion was investigated using
10 x 10-3 and 20 x 10-3 M dynamic light scattering (DLS) to determine
Zeta potential (mv) the average particle size and particle size
CS SDS distribution. At SDS 5 x 10-3 M provided a
concentration concentration Clove essential oil
amount (g)
particle size of nanoemulsion about 200 nm.
(ppm) (M)
0.5 1.0 1.5 However, with the addition of sodium
dodecyl sulfate up to 10 x 10-3 M, the greater
particle size was obtained, which were
50 5 x 10-3 M 11.47 8.27 -
6.48
around 350 nm. Although the description of
100 12.92 7.25 -8.63 nanoparticles as having sizes less than 0.1
μm or 100 nm, especially in the area of
150 12.86 10.99 -7.00 nanoencapsulation for drug delivery
relatively large (size > 100 nm)
50 10 x 10-3 M 22.70 13.61 -5.03
nanoparticles may be required for charging a
100 22.32 12.60 -3.74 sufficient amount of drug onto the particles.7
The appropriated ChS/SDS complexes
150 26.39 14.56 -1.07 determined nanoemulsion stability and
particle size. Moreover, the nanoemulsion
50 20 x 10-3 M - - - may possibly be disrupted by either
23.51 21.19 23.43 coalescence or combination, leading to the
100 - - -
decrease in surface area of the
22.51 22.71 20.12
150 - - - nanoemulsion.
23.26 21.82 18.51
3.4 Antibacterial activity of clove
3.3 Morphology of clove essential oil essential oil nanocapsules
nanocapsules The assessed antibacterial activity of
Morphology of clove essential oil the pure and emulsified clove essential oils
nanocapsules with 100 ppm chitosan and 1 g exhibited inhibitory effects against
of clove oil was obtained from a scanning Escherichiac coli (Gram-negative) and
electron microscope (SEM) images (Figure Staphylococcus aureus (Gram-positive) with
3). The nanocapsules with SDS 5X10-3M inhibition zone of 9.0±0.0 and 9.5±0.5 mm,
had a spherical shape with different particle respectively (Figure 3). The result confirmed
size as shown in Figure 2.1(a). The that high levels of eugenol in clove essential
increasing SDS provided regular spherical oil are responsible for its antimicrobial
particles of nanoemulsion as shown in activities. Generally, it can react with cell
Figure 2.1(b) because of the electrostatic membrane phospholipids of bacteria,
interaction between ChS and SDS adequate leading to the inhibition of several of Gram-
to stabilize the nanoemulsion.1,6 negative and Gram-positive.
Figure 2. SEM image of clove essential oil

a) ChS 100 ppm with SDS 5X10-3 M, b)
ChS 100 ppm with SDS 10X10-3 M
inhibition zone of 9.0±0.0 and 9.5±0.5 mm,
respectively. Having these physical
properties, this nanoemulsion has a potential
to apply for food industrial purposes.
Acknowledgements
Nanomaterials Chemistry Research
Unit, Department of Chemistry, Faculty of
Figure 3. Antibaterial activity of clove Science and Technology, Nakhon Si
essential oil nanocapsules a) Escherichia Thammarat Rajabhat University, Nakhon Si
coli, b) Staphylococcus aureus at 50% Thammarat, Thailand.
dilution of DMSO, emulsified clove
essential oils (1-9), pure clove essential oils References
(10), amoxicillin (11) 1. Sunintaboon, P.; Pumduang, K.;
Vongsetskul, T.; Pittayanurak, P.;
4. Conclusion Anantachoke, N.; Tuchinda, P.; Durand,
The ChS/SDS-stabilized clove oil A. Colloids Surf. A Physicochem Eng
nanoemulsions were effectively prepared Asp. 2012, 414, 151–159.
using ultrasonic emulsification process with 2. Randazzo, W.; Jiménez, A.; Settanni,
sonication time of 5 min and 37 kHz L.; Perdones, A.; Moschetti, M.;
frequency. The nanoemulsion was formed Palazzolo, E.; Guarrasi, V.; Vargas, M.;
under ionic interaction between positively Germanà, M. A.; Moschetti, G. Food
charged ChS and negatively charged SDS as Control 2016, 59, 750–758.
stabilized nanoemulsion. The creaming 3. Acevedo, A.; Salvia, L.; Rojas, M. A.;
index was used to describe the Martín, O. Food Hydrocoll. 2015, 47,
nanoemulsion stability. The addition of 168–177.
ChS/SDS could lower the percentage of 4. Hu, J.; Wang, X.; Xiao, Z.; Bi, W. LWT
creaming index due to their resistance to - Food Sci. Technol. 2015, 63 (1), 519–
droplet combination. DLS was used to 526.
determine the average particle size of the 5. Cui, H.; Bai, M.; Rashed, M. M. A.;
nanoemulsion which were performed around Lin, L. Int. J. Food Microbiol. 2018,
200-350 nm with zeta potential of -23.51- 266, 69–78.
26.39 mV. The morphorlogy of the 6. Babak, V. G.; Desbrieres, J. Colloid
nanocapsules displayed the consistent Polym. Sci. 2006, 284 (7), 745–754.
spherical form. Moreover, the prepared 7. Pan-in, P.; Wanichwecharungruang, S.
nanocapsules could inhibit both Escherichia Burapha Sci. J. 2013, 18 (2), 107-112.
coli and Staphylococcus aureus with
ไม่มีแหล่งข้อมูลในเอกสารปัจจุบนั
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A reactor design for the synthesis of aluminium phosphide for

insects elimination in rice mill
Tanawat Sakoltanaviroj1, Samitthichai Seeyangnok2, Kraipat Cheenkachorn1*
1
Department of Chemical Engineering, Faculty of Engineering, King Mongkut’s University of Technology of
North Bangkok, Bangkok 10800, Thailand
2
Department of Industrial Chemistry, Faculty of Applied Science, King Mongkut’s University of Technology of
North Bangkok, Bangkok 10800, Thailand
*E-mail: kraipat.c@eng.kmutnb.ac.th
Abstract:
Rice is a prominent national food and one of the top economic crops of Thailand. The
important step in the rice industry is a preservation for the best quality of the rice for a longer
period of time. Fumigant chemicals are used to prevent insects during the storage. One of the
most widely used fumigant chemicals is aluminium phosphide (AlP) that is synthesized from
aluminium and phosphorus via phosphidation reaction. The product of AlP has been used as a
reactant to react with moisture in the air to release phosphine gas (PH3) for insect elimination.
There are currently no research studies on the state of AlP synthesis, including the design of
the AlP synthesis reactor. This research focuses on the synthesis of AlP. A reactor was
designed to synthesize AlP which is made by Austenitic stainless steel 314 type and it has a
closed reactor and high temperature resistance. The reaction time and reactant ratios to
achieve the optimal crystalline products of AlP. It was shown that the reaction temperature of
500 °C and ratio of Al:P = 1:0.8 by mole gave the highest percentage of AlP of 63.44% (wt).
1. Introduction result in a respiratory tract. It is widely used

An important step in the rice industry to control or eliminate insects.
is preservation for the best quality of the rice
for a long period of time. Therefore, AlP + 3H2O → Al(OH)3 + PH3 (2)
fumigants are used to prevent moths and
insects during the storage. Due to gaseous Generally, AlP is commercially
phase, it can spread into the desired area in available in the form of tablets which
which the common pesticides are not contain approximately 1 gram of AlP per
accessible. Additionally, no residue remains tablet. The recommended dosage is about 2-
in the stored rice that contaminates the rice 3 tablets per a ton of a well-closed storage
quality.1 The most widely used fumigants is for not less than 7 days.4 After the
aluminium phosphide (AlP) which is disclosure, the gas will decay within 12
synthesized by the reaction between the hours without a trace of contamination left.
aluminium and phosphorus, called Previously, methyl bromide (CH3Br) was
“Phosphidation” as shown in equation (1).2 popularly used as a fumigant.5 However, it
was listed as an ozone depleting substance
4Al + P4 → 4AlP (1) under the Montreal Protocol in 1992, and all
developed countries agreed to reduce the
The product of AlP has been used as amount of methyl bromide in 2005.6 So, the
a reactant to react with moisture in the air to replacement of AlP over CH3Br becomes
release phosphine gas (PH3) as shown in widespread in use as a main fumigant.
equation (2).3 Phosphine gas is toxic which According to the Codex Alimentarius
© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) IN1
Commission (Codex) standards, the maximum temperature and pressure resis-
maximum residue levels to a safe level is tance is 600 °C and 20 bars, respectively.
less than 0.1 mg/kg.7 Although AlP has long The dimension of reactor is 10-cm diameter
been used for Thai rice export, the over- and 39.5-cm height. The reactor is installed
dosage of contamination has never been with auxiliary equipment: (1) a temperature
reported from their clients. controller to connect with a thermocouple to
Though Thailand is one of the measure a temperature inside the reactor and
world's top rice exporters, the fumigant is a heating coil; (2) a pressure gauge to
not domestically produced. It has been measure a pressure inside the reactor; (3) a
reported that the imported amount of tubing system to feed and purge nitrogen gas
fumigants was 0.94 million kilograms in for an oxygen removal from the reactor; and
2012, which has a value of 154.38 million (4) a cooling water system to cool down a
baht. From 2007-2012, among the imported reactor with a cooling water flowing through
fumigants, AlP showed the highest amount a ¼-stainless steel five-round spiral bended
of consumption of 562.81 tons per year.8 It tube which is installed inside the reactor.
is interesting to synthesize AlP domestically
to reduce the reliance on the imported Table 1. The condition of AlP synthesis
chemicals. Ratio of Reaction Reaction
Since the synthesis of AlP has not (Al:P) Temperature Time
by mole (°C) (h)
been extensively studied, the details of the 1 : 1.05 350 3
reaction and its process are not widely
1 : 0.97 350 3
available. Though some patents have been
1 : 0.8 500 1
reported on the synthesis a metal phosphide,
1 : 0.8 500 3
there was a lack of information which was
1 : 0.8 500 5
not reported including the reactor design, the
variation of influencing factor, the product
characterization and phosphine release rate. 2.3 AlP synthesis method
Therefore, this research focuses on the Aluminium powder and red
synthesis of AlP. The reactor design for the phosphorus powder were initially weighed
AlP synthesis was investigated including by a two-digit balance scale according to the
influencing factor. The product characteri- specified ratio. To obtain a uniform
zation was done to confirm that is AlP composition of a powder mixture, the
component. Phosphine release was con- reactants were manually stirred prior to
ducted for testing a performance of AlP. being fed into the reactor. The reactor was
then placed and tightly locked on the
2. Materials and Methods platform. The thermocouple and pressure
2.1 Materials gauge were installed to monitor the
AlP was synthesized by the reaction condition within the reactor. A gas-leakage
of the aluminium powder (CNPC powder, test was conducted for every joint and
China) to the red phosphorus powder (CN connection by soap solution method.
PC powder, China) by the ratio, temperature Subsequently, nitrogen gas was purged to
and reaction time as shown in Table 1. the reactor at 3-5 bars for 5 min. The reactor
2.2 A reactor design was turned on with a heating rate of 10
A schematic diagram of AlP synthe- °C/min and stirred at 20 rpm until the
sis reactor is shown in Figure 1. A reactor is temperature of reactor reached a target one.
designed as a pressure vessel with 4-blade After the reaction time was finished, the
turbine. To resist high temperature and reactor was cooldown at the cooling rate of
prevent corrosion of this reaction, the 10 °C/min. When the temperature of the
reactor is made of Austenitic stainless steel- reactor was decreased to approximately 70
314 with a thickness of 10 mm. The °C, nitrogen gas was fed to remove residual
phosphorus on a gaseous phase in the 3. Results and Discussion
reactor. A product was collected and stored 3.1 AlP characterization
for an analysis. The results from the X-Ray
2.4 Characterization diffraction (XRD) reveals the phase
The materials were characterized composition of the product. The areas under
using an X-ray diffraction (XRD, BRUKER the graph indicate the crystalline products
AXS model D8 Advance, Germany) which percentage of the compound of each
operated at 40 kV and 30 mA. The component. Figure 2 (left) shows the XRD
measurement operated with the angle from pattern of AlP from the reactions at a
10 to 80° of 2-theta angle, a step size of temperature of 350 °C, reaction time of 3 h,
0.02°, and a scan speed of 1 °/min. and two different ratios of Al : P = 1:0.97
2.5 Gas release rate and concentration and 1:1.05. Normally, the reference peak
AlP sample of 3 g was placed in a positions of AlP at 2-theta are 28.496
suction flask with a magnetic stirrer bar. A (strongest line), 47.395, 56.235, 69.275, and
silicone rubber tube was used to connect 76.542 which are marked with *. As shown
between the suction flask and an up-side- in the figure, the higher Al:P ratio results in
down cylinder for measuring a volume of higher intensity of AlP formation. It is
gas. Water of 50 mL was poured into the clearly seen that the Al:P of 1:1.05 showed
suction flask and stirring occurred at 200 higher intensity of the 2-theta peaks at
rpm for a uniform hydrolysis reaction. Gas 28.496, 47.395, 56.235, 69.275 than those of
release rate was measured using a Al:P = 1:0.97. It can be seen that if the
water-replacement technique. phosphorus was put in a higher ratio than
A gas sample was diluted with a aluminium, it would increase the chance of
ratio of 1:400 (gas to air by volume) prior to reaction to get AlP, but if the reaction
being analyzed for a phosphine temperature of 350 °C, aluminium phos-
concentration using by a gas detector with a phate (Al(PO4)) would be more produced.
phosphine tube (Scientific promotion,
Thailand).
stirrer
Pressure
Thermocouple Gauge
T P Cooling water
(inlet)
N2 & O2 N2 & O2
(inlet) (outlet) Cooling water
(outlet)
Feed Product
AlP synthesize reactor
Figure 1. A schematic diagram of AlP synthesis process
Table 2. The crystalline products percentage of each the components of AlP synthesized at
various condition
Samples AlP (%) γ-Al2O3 (%) Corundum (%) Al (%) Al(PO4) (%)
(α-Al2O3)
Al : P = 1 : 1.05 35.00 - 29.70 13.70 21.50
(350 °C, 3 h)
Al : P = 1 : 0.97 18.70 13.70 8.20 55.10 4.40
(350 °C, 3 h)
Al : P = 1 : 0.8 47.23 - 22.50 30.27 -
(500 °C, 1 h)
Al : P = 1 : 0.8 62.14 - 19.43 18.43 -
(500 °C, 3 h)
Al : P = 1 : 0.8 63.44 - 25.33 11.23 -
(500 °C, 5 h)
Figure 2. The XRD pattern of AlP at temperature of 350 °C (left) and 500 °C (right)
Figure 2 (right) show the effect of Therefore, the oxygen gas is not completely
reaction time on AlP yield at a temperature eliminated, the oxygen remains in the
of 500 °C, in ratios of Al:P = 1:0.8 and reactor that react with aluminium to get γ-
reaction time of 1 h, 3 h, and 5 h. The longer Al2O3 or in form α-Al2O3 “Conrundum”;
of reaction time result in higher intensity of and (4) Aluminium phosphate (Al(PO4)) is
AlP formation. It is clearly seen that the formed only at 350 °C. Synthesis of
reaction time of 5 h has the highest intensity compounds have aluminium and phosphorus
that followed by 3 h and 1 h, respectively. A as a reactant at temperature range of 200-
longer the reaction time affect the more 350 °C produces Al(PO4) but temperature
contract time of reactant which resulting in range of 300-600 °C produces AlP.
the high crystalline products percentage of 3.2 The phosphine release rate and concen-
AlP. tration
In addition, there are other The phosphine release rate is shown
components that occur as shown in Table 2 in Figure 3. The graph shows the volume of
including: (1) Al powder occurs because the the phosphine gas at different times. In
aluminium had a melting point of 650 °C range of 0-4 mins, it was found that the
but this experiment was operated at 350 °C, phosphine release rate of AlP at 500 °C in
so some aluminium powder was not the ratio of Al:P = 1:0.8 for 3 h was highest,
reacting; (2) γ-Aluminium oxide (γ-Al2O3) followed by 1 h and 5 h, respectively. Since
and (3) Corundum (α-Al2O3) occurs because the synthesized sample has aluminium oxide
the nitrogen gas is designed on the top of the (Al2O3) component which covers AlP in
reactor and the specific gravity of the form of film. So, if the synthesized sample
nitrogen gas is lower than the oxygen gas has a lot of Al2O3, it will reduce the gas
which the nitrogen gas floats above it. release rate. In range of 5 mins or more, it
was found that the phosphine release rate of found that the crystalline products
AlP at 500 °C in the ratio of Al:P = 1:0.8 for percentage of AlP is the highest for 5 h of
5 h was highest, followed by 3 h and 1 h, reaction time.
respectively. That is direct varies with the The phosphine release is the highest
amount of AlP obtained in the synthesis. for 3 h of reaction time in 0-4 min and for 5
Furthermore, it is also found that Al:P = h of reaction time in 5 min or more. The
1:0.8 for 5 h takes the longest time to release phosphine concentration is the highest at
phosphine gas until it is exhausted, followed 560,000 ppm for 5 h of reaction time.
by 1 h and 3 h, respectively. Since the Al2O3
film is delayed releasing of phosphine gas in Acknowledgements
0-4 min period, it takes a longer to release. The authors would like to express
In the part of phosphine their gratitude to Mr. Pattra Kongsupapkul
concentration, it was found that the and Mr. Supawat Watcharasuwanseree for
phosphine concentration of Al:P = 1:0.8 at his assistance.
500 °C for 5 h was highest at 560,000 ppm,
followed by 3 h at 360,000 ppm and 1 h at References
160,000 ppm, respectively. That is direct 1. Bumbrah, G. S.; Krishan, K.; Kanchan,
variation with the amount of AlP obtained in T.; Sharma, M.; Sodhi, G. S. Forensic
the synthesis. Sci. Int. 2012, 214 (1–3), 1–6.
2. White, W. E.; Bushey, A. H. J. Am.
Chem. Soc. 1994, 66 (10), 1666–1672.
3. Wiberg, N.; Holleman, A. F.; Wiberg, E.
Inorganic Chemistry; Elsevier Science
Publishing Co Inc: San Diego, United
States, 2001.
4. Neo Agro Fume Co., Ltd. Phosphine
http://www.neoagro.co.th/pdf/Phosphine
.pdf.
5. Fields, P. G.; White, N. D. G. Annu. Rev.
Entomol. 2002, 47, 331-359.
6. United Nations Environment Program-
Figure 3. The released gas volume in Technology and Economic Assessment
various time Panel (TEAP). Montreal Protocol on
substances that deplete the ozone layer;
4. Conclusion 2000.
AlP is synthesized with a pilot scale 7. Suwannakoot, A. Fumigation with rice
reactor in form of the pressure vessel. The trading business
reactor is made of Austenitic stainless steel- http://www.doa.go.th/pibai/pibai/n16/v_
314 and the maximum temperature and 6-july/ceaksong.html.
pressure resistance is 600 °C and 20 bars, 8. Thai rice exporter association.
respectively. Thailand’s rice export situation in 2016
The effect of reaction temperature and the trend and direction of Thai rice
was found that the crystalline products export in 2017; 2016.
percentage of AlP at 500 °C is higher than
350 °C. And the effect of reaction time was
Synthesis, characterization and anticancer studies of acesulfame mixed
with triphenylphosphine silver(I) complexes
Bussaba Boonseng1*, Jaruwan Chatwichien1, Ratanon Chotima1, Teerawat Khudkam1,
Taweesak Pila2
1
Department of Chemistry, Faculty of Science, Naresuan University, Phitsanulok, Thailand
2
School of Molecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology,
Rayong, Thailand
* E-mail: bussabab@nu.ac.th
Abstract:
Silver(I) complexes incorporated with the mixed ligands of acesulfame (ace) and
triphenylphosphine (PPh3) in different ratio, were prepared. The synthesized complexes of
[Ag(ace)]n (1), [Ag(ace)(PPh3)] (2), [Ag(ace)(PPh3)3] (3) and [Ag(PPh3)4](ace) (4) were
characterized by IR, MS, and NMR spectroscopic methods. The crystal structures of
complexes 2 and 4 were also determined by X-ray crystallography. The complexes were
investigated in their biological reactivity with A549 cancer cells and activities were evaluated
by IC50 method. [Ag(ace)]n (1), [Ag(ace)(PPh3)] (2) and [Ag(ace)(PPh3)3] (3) exhibited the
anticancer activity with the IC50 value of 42.037 M, 1.655 M and 1.542 M which were
higher than the standard Epotoside (IC50 5.623 M). The results indicated that the mixed
ligands could perform more excellent inhibition of biomolecular growing in comparison to
the using of only acesulfame or triphenylphosphine ligand.
1. Introduction revealed that introduction of such compound

The chemistry involved biological to the complexes could reduce the aftereffect
studies have been recently interested in caused to human body.7
which anticancer activity evolves into one of
the most attractive application. Silver
sulphadiazine was found to be the most
effective complex in antibacterial and
antifungal studies about 50 years ago.1
Thereafter, a number of silver(I) complexes Figure 1. Structure of acesulfame anion
bearing diverse ligands have been reported
in their biological activity.2-4 The work by Thereafter, the literature of
Nomiya and co-workers was proposed that platinum(II) and silver(I) complexes with
silver(I) complexes performed their activity acesulfame-K was recently described. The
via the replacement on nitrogen or oxygen silver(I)–ace complex exhibited good
atoms of silver complexes by sulfur atom in antimycobacterial activity against M.
the active site of proteins.5,6 Consequently, tuberculosis and also antibacterial against
the ligands containing such donor atoms like Gram negative (E. coli and P. aeruginosa)
nitrogen and oxygen have become much and Gram positive (E. faecalis)
paid attention in this research field. microorganisms.8 In 2011, Nawaz and co-
Acesulfame-K, considered as an workers reported the synthesis of
anionic ligand, is one of the most utilized heterocyclic thiones mixed with
artificial sweetener presently. Anionic triphenylphosphine (PPh3) complex and
structure of acesulfame (ace) is presented in examined on antimicrobial activity. The
Figure 1. At low concentration, the results indicated that the synthesized
compound is found to have low toxicity. complex exhibited more excellent activity
Furthermore, the previous research also than using of only thione compounds.9 In
addition, silver(I) ion complexes have also were operated at the frequency of 400 MHz
been studied against cancer cells recently. In (Bruker). Mass spectrometric analysis was
2013, Ali and co-workers reported the performed using the ESI technique (Agilent
studies of anticancer activity against four 6540 LC/MS system). The diffraction of the
human cancer cell lines; colon carcinoma single crystal was collected with a Bruker
(HT29), breast adenocarcinoma (MCF7), D8 VENTURE diffractometer using Mo K
hepatocellular carcinoma (HePG2) and lung radiation (k = 0.71073 Å). The structures
adenocarcinoma (A549) in which the were solved by ShelXS. The Olex2 and
complexes showed better activity than the Mercury3.8 were used for materials and
ligands.10 Few years later, Kaplan et al. molecular graphics.
reported the study of time-course cytotoxic 2.2 Synthesis of silver(I) complexes
effects against lung cancer A549 cells by [Ag(ace)]n (1) was provided
silver complex via MTT assay. The results 8
according to the literature. [Ag(ace)(PPh3)]
indicated that silver substrate performed (2) was prepared by adding aqueous solution
inhibitory effects on A549 cells in a dose of AgNO3 into PPh3 solution in EtOH. After
and time dependent.11 Lung cancer is the stirring at 80 oC for 2 h, ace in water was
most common cause of cancer-related death added into the mixture with continuously
amongst both men and women12 and the stirring at room temperature for 5 h. The
statistics reported by World Health obtained product was filtered and
Organization (WHO) revealed that about recrystallized with EtOH. [Ag(ace)(PPh3)3]
more than 50 per cent of lung cancer cases (3) and [Ag(PPh3)4](ace) (4) were also
occurred in less developed countries.13 synthesized with the same procedure using
Consequently, lung cancer has become our three and four equivalents of PPh3 instead.
desired target. In this study, the complexes The single crystals were achieved by slow
of silver(I) incorporated with the mixed evaporation of ethanolic solution of the
ligands of PPh3 and ace in different ratio desired products at room temperature in the
were prepared and determined the structures absence of light about a week.
by spectroscopic techniques and single 2.3 Anticancer studies
crystal X-ray crystallography. The products The synthesized complexes were
were examined in the inhibition of lung studied in anticancer activity against A549
cancer cells (A549) and further evaluated by cancer cells and evaluated by IC50 method.
IC50 method. Introduction of ace which have The cells were propagated in modified
been already known as a generally used Eagle's medium (MEM) supplemented with
sweetener and PPh3 which could increase 10% fetal bovine serum and 1% Penicillin
the biological activity is expected to reduce Streptomycin. All cells were maintained at
toxicity to the living cells caused by the 37 oC in a humidified atmosphere with 5%
studied complexes and to enhance the CO2 and were sub-cultured two times a
inhibition of such cancer cells. week. The microtiter plates were incubated
at 37 oC in a moistened incubator with 5%
2. Materials and Methods CO2 for 48 h. After incubation, the samples
2.1 General dissolved in DMSO or water with
All the chemicals used in this study concentration of 50 mM were added, and
were at least analytical grade. Acesulfame-K incubation was continued for 48 h. Then,
and triphenylphosphine were supplied by media were aspirated and MTT solution (1%
Fluka. Silver nitrate (AgNO3) and ethanol in growth media) was added to each well
were obtained from Labscan and Merck. and incubated at 37 oC for 4 h. The
The IR spectra were recorded on a Perkin- absorbance was measured at the wavelength
Elmer FT-IR spectrophotometer in wave of 570 nm and the results were compared
number region 4000-400 cm-1. The 1H and with standard Epotoside.
31
P NMR spectra of the complexes in CDCl3
3. Results and Discussion 3.2 X-ray crystallographic studies
3.1 Spectroscopic studies Colorless crystals of both 2 and 4
IR spectroscopic bands for were grown by a slow evaporation of
acesulfame-K ligand appeared at the ethanol at room temperature and
frequency of 1651 cm-1, 1588 cm-1, 1287 crystallographic data of complexes 2 and 4
cm-1 are related to the vibration of C=O, are shown in Table 2. Complex 2 was
S(=O)2 and C-N groups, respectively. The crystalized in the triclinic crystal system
notable change in IR spectrum of complex 4 with P-1 space group. The bond angles
was not observed which might be P1−Ag1−O1, P1−Ag1−N2 and N2−Ag1−O1
contributed to no formation bonding with the values of 95.35(3)o, 136.32(4) o and
between ace and the metal center. A low 101.13(5)o displayed a geometry of distorted
frequency shift in (C=S) and a high trigonal planar around the silver atom as the
frequency shift in the (C–N) bands in structure shown in Figure 2.
complexes 1-3 indicate the coordination of
the ligands to silver(I) ion. Complexes 2-4 Table 2. Crystallographic data of complex 2
were observed in 31P NMR spectra to and 4
examine the change of phosphorus signal Complex 2 4
upon the coordination to silver ion. The Formula C22H19AgNO4 C76H65AgNO4
PS P4S
observed phosphorus atom was found to Formula weight 532.31 1406.33
resonate the chemical shift of -5.25 ppm in Temperature/K 120.06 100.0
the free PPh3. Crystal system triclinic Monoclinic
Space group P-1 C2/c
Table 1. 31P NMR and IR data of free a/Å 10.0298(8) 32.0063(18)
b/Å 10.2434(8) 22.2266(14)
ligands and complexes 1-4 c/Å 10.7526(8) 23.5928(14)
Compounds 31P IR (cm-1) α/° 102.919(3) 90
No. (ppm) C=O S(=O)2 C-N β/° 94.618(3) 124.719(2)
PPh3 -5.25 - - - γ/° 94.871(3) 90
acesulfame - 1651 1588 1287 Volume/Å3 1067.22(14) 13795.5(15)
(1) - 1644 1563 1312 Z 2 8
(2) 15.81 1643 1566 1342 ρcalc g/cm3 1.6564 1.3541
(3) 5.56 1638 1557 1323 μ/mm-1 1.146 0.469
(4) 3.27 1652 1589 1290 2Θ range for data 4.96 to 60.74 4.48 to 36.32
(1) : [Ag(ace)]n collection/°
(2) : [Ag(ace)(PPh3)] Reflections 52850 56712
(3) : [Ag(ace)(PPh3)3] collected
(4) : [Ag(PPh3)4](ace) Independent 6436 4883
reflections
The signal has shifted to 15.81 ppm Rint 0.0531, 0.0965,
Goodness-of-fit 1.046 1.052
in 2, to 5.56 ppm in 3 and 3.27 ppm in on F2
complex 4 which all are confirmed the Final R R1 = 0.0292, R1 = 0.0356,
complex formation with Ag-PPh3 bond in [I>=2σ (I)] wR2 = 0.0537 wR2 = 0.0788
different manners. However, the 1H NMR
spectra could not verify this complex
formation as the signals of protons in ace are The bond O1−C1; 1.237(2) Å is
all obscured by abundant protons of phenyl lengthened whilst the distance of C1−N1;
rings in PPh3. The results of 31P NMR and 1.366(2) Å is slightly decreased related to
IR are summarized in Table 1. The complex the free ligand.14 The complex displayed the
formation of 3 was also approved by mass deviation from the planar with the torsion
spectrometric technique. The m/z+ was angles of P1−Ag1−O1−C1: 103.0(2)o, P1−
detected at 1180.9195 as a minor peak. Ag1−N2−C4; 146.1(1)o and P1−Ag1−N2
−S2; -36.3(1)o, respectively. Some selected
bond length and angles of complex 2 is
presented in Table 3.
Figure 3. Ellipsoid plot of complex 4 with

ellipsoids at the 50% probability level;
hydrogen atoms are omitted for clarity
3.3 Anticancer studies

The prepared complexes were
Figure 2. Ellipsoid plot of complex 2 with examined in the anticancer activity against
ellipsoids at the 50% probability level; A549 cancer cells to compare with starting
hydrogen atoms are omitted for clarity materials and the standard Epotoside. The
IC50 measured against on A549 cancer cells
Table 3. Selected bond length and angles of by complexes 1-3 are 42.037, 1.655 and
complex 2 1.542 μM. The results indicated that silver(I)
Complex 2 complexes with mixed ligands perform more
Ag1−P1 2.3918(5) O1−C1 1.237(2) excellent activity than the complex
Ag1−O1 2.484(1) C1−N1 1.366(2) containing only ace ligand. Both complexes
Ag1−N2 2.220(1) N1−S1 1.593(1) 2 and 3 also provided greater IC50 compared
P1−Ag1−O1 95.35(3) O1−C1−N1 120.1(2)
P1−Ag1−N2 136.32(4) C1−N1−Ag2 134.0(1)
to the standard Epotoside. Unfortunately,
N2−Ag1−O1 101.13(5) Ag1−O1−C1 121.7(1) IC50 of complex 4 could not be evaluated
P1−Ag1−O1−C1 -103.0(2) due to the poor solubility of the sample in
P1−Ag1−N2−C4 146.1(1) DMSO or water. The results of all biological
P1−Ag1−N2−S2 -36.3(1) studies are shown in Table 4. However, the
N2−Ag1−O1−C1 117.7(2) effect of mixed ligands ratio and structural
O1−Ag1−N2−C4 -36.0(1)
O1−Ag1−N4−S2 146.36(8)
geometry on biological activity should be
further investigated.
Complex 4 exhibits the coordination Table 4. IC50 values of studied compounds
of four PPh3 molecules toward the metal Compounds IC50 (M)
center whereas ace anion performs as a AgNO3 N/A*
counterion to neutralize the cationic acesulfame-K N/A
complex (Figure 3). The obtained crystal PPh3 N/A
[Ag(ace)]n (1) 42.037±3.9235
acquires the crystal system of monoclinic
[Ag(ace)(PPh3)] (2) 1.655±0.0348
with C2/c space group. The angles P1 [Ag(ace)(PPh3)3](3) 1.542±0.0214
−Ag1−P2, P1−Ag1−P3 and P1−Ag1−P4 [Ag(PPh3)4](ace)(4) -
with 108.95(6)o, 110.34(6)o and 109.48(6)o Epotoside 5.623±0.7816
exhibit an almost symmetrical tetrahedral * The IC50 value cannot be determined.
structure.
4. Conclusion M. A.; Mubarak, M. S.; Peters, D. G.
In summary, silver(I) complexes Polyhedron 2015, 85, 450-456.
with the mixed ligands of triphenyl 5. Nomiya, K.; Yokoyama, H. J. Chem.
phosphine and acesulfame in different ratio Soc. Dalton 2002, 12, 2483-2490.
are prepared and characterized. The 6. Nomiya, K; Noguchi, R; Oda, M.
biological study with cancer cells revealed Inorg. Chim. Acta 2000, 298, 24-32.
great activity of the synthesized complexes. 7. Cong, W. N.; Wang, R.; Cai, H.;
The capability is increased when Daimon, C. M.; Scheibye-Knudsen, M.;
triphenylphosphine was introduced to Ag- Bohr, V. A.; Turkin, R.; Wood, W. H.;
ace complex. Anyhow, the effect of Becker, K. G.; Moaddel, R.; Maudsley,
ace:PPh3 proportions on biological activity S.; Martin, B. Plos. One. 2013, 8 (8).
is still ambiguous since the obtained results 8. Cavicchioli, M.; Massabni, A. C.;
were not significantly different. Heinrich, T. A.; Costa-Neto, C. M.;
Abrao, E. P.; Fonseca, B. A. L.;
Acknowledgements Castellano, E. E.; Corbi, P. P.; Lustri,
The authors would like to thank W. R.; Leite, C. Q. F. J. Inorg.
Naresuan University for the experimental Biochem. 2010, 104 (5), 533-540.
equipment and financial support. 9. Nawaz, S.; Isab, A. A.; Merz, K.;
Vidyasirimedhi Institute of Science and Vasylyeva, V.; Metzler-Nolte, N.;
Technology (VISTEC) is acknowledged for Saleem, M.; Ahmad, S. Polyhedron
the X-Ray single crystal crystallographic 2011, 30 (9), 1502-1506.
study. 10. Ahmad, S.; Isab, A. A.; Ali, S.; Al-
Arfaj, A. R. Polyhedron 2006, 25 (7),
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2. Chandra, S.; Ruchi. Spectrochim. Acta. Polyhedron. 2004, 23 (16), 2549-2559.
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3. Kumar, G.; Kumar, D.; Devi, S.; Johari, D. M.; Zhang, Y. Life Sci. 2004, 75
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2010, 45 (7), 3056-3062. 13. http://globocan.iarc.fr
4. Charef, N.; Sebti, F.; Arrar, L.; Ourari, 14. Velaga, S. P.; Vangala, V. R.; Basavoju,
A.; Djarmouni, M.; Boussoualim, N.; S.; Bostrom, D. Chem. Commun. 2010,
Baghiani, A.; Khennouf, S.; AlDamen, 46 (20), 3562-3564.
Synthesis, ESR study, and antibacterial activity of Cu(II) and Ag(II)
tetrakis(4-alkyloxyphenyl)porphyrin complexes
Tossapon Prohmsatit1, Autthavit Nuchthanom1, Ausjima Poomkleang2,
Pariya Na Nakorn2, Soraya Pornsuwan3, Supakorn Boonyuen1*
1
2
Department of Biotechnology, Faculty of Science and Technology, Thammasat University,
3
Department of Chemistry, Faculty of Science, Mahidol University, 272 Rama 6 Road, Ratchthewee
Abstract:
The novel tetrakis(4-methyloxyphenyl)porphyrin (TOMPP), tetrakis(4-
butyloxyphenyl)porphyrin (TOBPP), and their copper and silver complexes have been
synthesized. All synthesized compounds have been fully characterized by mass spectrometry,
CHN elemental analysis, 1H, 13C NMR, FT-IR spectroscopy, ESR spectroscopy, UV-visible
and fluorescence spectroscopy. The absorption spectra of free base porphyrin exhibited one
S-band (420 nm) and four Q-bands (between 500 – 700 nm) in dichloromethane. The
absorption band of copper porphyrin and silver porphyrin complexes displayed only one S
band (419 and 427 nm) and one Q bands (between 550 – 600 nm). Free base porphyrin (ex =
534 nm) and metalloporphyrins (ex = 556 nm) showed their emission bands at 654 nm and
around 604-607 nm, respectively. The appearance of the ESR signal from silver porphyrin
confirmed the unpaired electron on the silver(II) ion. The g value of each complexes was
calculated. For example, AgTPP has g value of 2.113. Moreover, the antibacterial activity
from all synthesized compounds has been investigated. The inhibition zone of all synthesized
compounds for E. coli and S. aureus are over than 6.5 mm and 6.8 mm, respectively. All
porphyrin complexes were found to be effective in inhibiting bacterial growth and can be
used in medical application in the future.
1. Introduction interaction of metal pπ or dπ orbitals.3

Porphyrins are one of the chemical Nowadays, there are several routes to the
units essential for life processes. Porphyrins synthesis of meso-substituted porphyrin.4-6
play a vital role in biological systems, and Porphyrin contains 22 conjugated π-
their synthesis is of interest in biological, electrons, whereas 18 π-electrons are
material, and inorganic chemistry.1 Dilation required for a conjugated aromatic network.
may result in the distortion of the planar Porphyrin can undergo additional reaction
macrocyclic maximizing the binding without loss of the aromatic properties. The
strength towards the metal fragment. The functionalized porphyrin finds multiple
metalloporphyrins have been synthesized. applications in biology, medicine, and
The normal coordination geometry around materials. The meso-positions are the most
the metal ion in the former species would be electronically reactive, and preferential sites
square planar.2 The natural bonding between for substitution and addition.7 The meso-
a central metal and the porphyrin ligand substituted porphyrins are of particular
originates from two types of primary interest because the meso-substituted
interaction: σ-coordination of nitrogen lone porphyrins have applications including
pairs directed towards the metal atom and π- catalysis,8 electron transfer, 9 photodynamic
therapy,10 and molecular recognition. 7.42. Mass m/z (ESI) calcd for C48H38N4O4
Although meso-substituted porphyrins are :734.84 Found 735.4 [M+H]+. UV-
not a naturally occurring compound, it has vis(CH2Cl2): (𝜆abs(nm), 𝜀(103M-1cm-1)):
provided chemists and other scientist with a S-band; (421, 296.9), Q-band; (518, 11.9),
multitude of applications and fundamental (555, 8.2), (595, 3.9), (650, 5.2).
studies. Their syntheses are based on The TOBPP was obtained in 9%
condensation of pyrrole and aldehyde under yield. 1H NMR (400 MHz, CDCl3): δ 8.9
various reaction conditions. (8H, pyrrole, 𝛽-H), 8.1 (8H, phenyl, o-H), 7.4
In the present work, the Adler-Longo (8H, phenyl, m-H), 4.2 (-OCH2), 1.9 (-
synthesis method was applied to the free OCH2CH2), 1.3 (-CH2CH3), 0.9 (-CH3). 13C
base porphyrin synthesis. The free base NMR (100 MHz, CDCl3): 159.09, 135.32,
porphyrins and metalloporphyrin complexes 134.74, 130.70, 128.76, 119.7, 112.84, 68.14,
were prepared and purified. Then, their 31.49, 23.98, 13.84 ppm. IR (KBr): 3320,
properties were characterized by IR 2931, 2868, 1607, 1509, 1245, 1174, 966,
1 13
spectroscopy, H and C NMR 802 cm-1. Elemental analysis; calcd (%) for
spectroscopy, mass spectrometry (MS), C60H62N4O4: C 79.79, H 6.92, N 6.20; found:
CHN analysis, UV-Vis and fluorescence C 79.44, H 6.55, N 5.83. Mass m/z (ESI)
spectroscopy and electron spin resonance calcd for C60H62N4O4: 903.16. Found 903.6
spectroscopy (ESR). [M+H]+. UV-vis (CH2Cl2): (𝜆abs(nm),
between main sections) 𝜀(103M-1cm-1)): S-band; (422, 530.6),
2. Materials and Methods Q-band; (519, 22.9), (556, 17.3), (595, 8.6),
2.1 Synthesis of free base porphyrins (651, 11.6).
The tetrakis(4-methoxyphenyl)por 2.2 Synthesis of metalloporphyrin complexes
phyrin and the tetrakis(4-butyloxyphenyl) The metalloporphyrins were
porphyrin were synthesized by following a synthesized by refluxing each free base
published procedure with slight porphyrin with an excess copper acetate and
11
modification. Pyrrole and aldehyde (𝜌- silver nitrate in mixed solvent between DMF
anisaldehyde and butyloxybenzaldehyde) and dichloromethane for 5 hours. After
refluxed in propionic acid at 383 K for 2 h. refluxing, the reaction mixture was cooled to
After refluxing, the reaction mixture was room temperature. The mixture was kept in
cooled to room temperature and added 40 mL refrigerator overnight after adding distilled
of ethanol. Then kept it in the refrigerator water. Finally, the purple crystals were
overnight. The purple crystals were filtered, filtered, washed, and dried by vacuum
washed, and dried by vacuum filtration with filtration with cold methanol. The reaction
cold ethanol to removed traces of propionic products were purified by silica gel column
acid. The crude products were purified by chromatography and eluted with the
silica gel column chromatography and eluted increasing polarity by the mixture of
with the increasing polarity by the mixture of dichloromethane and hexane to afford the
dichloromethane and hexane to afford the metalloporphyrins
free base porphyrins as a purple crystal. The tetrakis(4-methoxyphenyl)phe-
The TOMPP was in 26% yield. 1H nylporphyrinatocopper(II) [CuTOMPP] was
NMR (400 MHz, CDCl3): δ 8.8 (8H, pyrrole, obtained in 92% yield. IR (KBr): 2929, 2835,
𝛽-H), 8.1 (8H, phenyl, o-H), 7.2 (8H, phenyl, 1607, 1499, 1248, 1173, 804 cm-1. Elemental
m-H), 4.1 (12H, -OCH3). 13C NMR (100 analysis; calcd (%) for CuC48H36N4O4: C
MHz, CDCl3): 158.75, 134.68, 133.99, 60.14, H 4.07, N 5.55; found: C 60.42, H
129.97, 127.89, 118.83, 111.44, 54.65 ppm. 4.17, N 6.22. Mass m/z (ESI) calcd for
IR (KBr): 3316, 2931, 2834, 1607, 1509, CuC48H36N4O4: 796.38. Found 797.4 [M+H]+.
1248, 1174, 966, 804 cm-1. Elemental UV-vis (CH2Cl2): (𝜆abs(nm), 𝜀(103 M-1cm-1)):
analysis; calcd (%) for C48H38N4O4: C 78.45, S-band; (419, 402.5), Q-band; (541, 18.9),
H 5.21, N 7.62; found: C 76.42, H 5.75, N (653, 3.6).
The tetrakis(4-butyloxyphenyl)phe- 3.1 UV-visible spectroscopy
nylporphyrinatocopper(II) [CuTOBPP] was The absorption spectra of all
obtained in 71% yield. IR (KBr): 2930, 2869, porphyrins and metalloporphyrins were
1607, 1502, 1246, 1174, 799 cm-1. Elemental recorded in dichloromethane at room
analysis; calcd (%) for CuC60H60N4O4: C temperature. The absorption data and molar
74.77, H 6.23, N 5.82; found: C 74.51, H extinction coefficient (ε) of all synthesized
6.25, N 5.76. Mass m/z (ESI) calcd for compounds are summarized in Table. 1. The
CuC60H60N4O4: 964.70. Found 965.6 [M+H]+. absorption spectra of the free base
UV-vis (CH2Cl2): (𝜆abs(nm), 𝜀(103 M-1cm-1)): porphyrins exhibited strong absorption
S-band; (419, 415.4), Q-band; (541, 24.5), (Soret band) between 417-422 nm and four
(578, 9.6), (618, 7.2). weak adsorptions (Q band) around 500-700
The tetrakis(4-methoxyphenyl)phe- nm, both of which can be assigned to π⟶π*
nylporphyrinatosilver(II) [AgTOMPP] was electronic transitions. The very intense Soret
obtained in 57% yield. IR (KBr): 2934, band assigned to the S0⟶S2 transition is
2833, 1606, 1548, 1250, 1175, 809 cm-1. found at shorter wavelength. In contrast, the
Elemental analysis; calcd (%) for Q bands assigned to the S0⟶S1 transitions
AgC48H36N4O4 (840.69): C 62.47, H 4.53, N appear at longer-wavelength.12 From the
6.13; found C 62.63, H 4.10, N 5.92. Mass UV-Vis spectroscopy data, the Soret band
m/z (ESI): found 841.6 [M+H]+. UV-Vis and Q band of the para-substituted
(CH2Cl2): (λabs(nm), Ɛ (103M-1cm-1)): S- porphyrin move to a small red shift when
band; (427, 442.21), Q-band; (542, 22.95). compared with TPP. The free base
The tetrakis(4-butyloxyphenyl)phenyl porphyrins displayed one Soret band with
porphyrinatosilver(II) [AgTOBPP] was four Q bands. When coordinated with a
obtained in yield 18% yield). IR (KBr): 2932, copper(II) and silver(II) ion the number of Q
2834, 1609, 1506, 1248, 1176, 805 cm-1. bands decreased to only one peak due to the
Elemental analysis; calcd (%) for symmetry change of metalloporphyrins. The
AgC60H60N4O4 (1009.01): C 71.42, H 5.99, N copper(II) and silver(II) complexes exhibit
5.55; found C 74.51, H 6.25, N 5.76. Mass m/z the Soret band at 419 nm and 427 nm,
(ESI): found 1010.1 [M+H]+. UV-Vis respectively. Thus the energy gap (HOMO
(CH2Cl2): (λabs(nm), Ɛ (103M-1cm-1)): S-band; and LUMO) of the metalloporphyrins are
(427, 356.56) and Q-band; (543, 25.00). higher than free base porphyrin for
between main sections)
Figure 1. UV-Visible absorption spectra of TOMPP, CuTOMPP, and AgTOMPP in CH2Cl2
Table 1. Absorption spectra data of all synthesized complexes
Dichloromethanea
Porphyrin Q band (nm), 𝜀(M-1cm-1)
S band (nm), 𝜀(M-1cm-1)
Q1 Q2 Q3 Q4
TOMPP 421, 296998 518, 11941 555, 8266 595, 3980 650, 5205
TOBPP 422, 530607 519, 22955 556, 17310 595, 8655 651, 11665
CuTOMPP 419, 402504 - 541, 18914 - -
CuTOBPP 419, 451480 - 541, 24599 - -
AgTOMPP 427, 485076 - 542, 22951 - -
AgTOBPP 427, 465081 - 543, 25004 - -
copper(II) and silver(II) porphyrins exhibited lower energy gap at 1.90 eV.
complexes, respectively. Related to the donating electron of the long
3.2 Fluorescence spectroscopy chain substituent group at the para- position.
The emission spectra of the free base
porphyrins, and metalloporphyrins were Table 2. The emission spectra data of all
recorded in dichloromethane using synthesized compounds
fluorescence spectroscopy. The spectra of Porphyrin
Dichloromethane
both free base porphyrins are shown in 𝜆ex (nm) 𝜆em(nm) Egap(ev)
Figure 2. All free base porphyrins exhibited TOMPP 534 654 1.90
TOBPP 534 654 1.90
the emission peak at 654 nm, when excited CuTOMPP 556 604 2.16a
at 534 nm. The same values of 𝜆em were CuTOBPP 556 604 2.17a
obtained by exciting the sample at the AgTOBPP 557 607 2.14a
wavelength of maximum absorption of the Q AgTOMPP 557 604 2.14a
band (first Q band).13 The fluorescence a
Calculated by averaging wavelength between Q-
emission spectra of the metalloporphyrin band and emission band
complexes were obtained around 604 - 607
nm when excited at 556 nm. All synthesized 3.3 Electron spin resonance spectroscopy
porphyrin compounds exhibited an The electron spin resonance
interesting trend in the blue shift of their spectroscopy (ESR) is a good tool to
corresponding emission band with free base investigate silver porphyrin complexes,
porphyrins due to the interaction between providing valuable information about
the metal ion in the porphyrinic substituents oxidation state, ligand type, and symmetry.
and the solvent molecules.14 However, the The ESR spectrum of the both silver(II)
emission spectra show a slight difference porphyrin complexes (AgTOMPP and
due to electron donating ability of alkoxy in AgTOBPP) are shown in Figure 2. The ESR
the chain.15 The emission data of all signal shows the only structure having one
compounds were summarized in Table 2. strong absorption peak centered at 3432.991
The energy difference between HOMO and G and 3441.789 G, respectively. The g
LUMO was calculated in terms of the values are calculated at 2.08 and 2.07,
energy gap (Egap) The estimated energy gap respectively. These values are in agreement
determined from intersection of UV-Vis with those reported earlier.17 The ESR signal
absorption (Q4 band) and fluorescence of silver porphyrin complexes confirmed that
emission spectra follows the equation Egap = these compounds must contained silver(II) ion
hc/𝜆.16 However, the estimated energy gap in the structure.
of copper(II) porphyrin complexes could not
be calculated because the absorption spectra
of neither compound showed those Q4 band.
The estimated energy gap of all free base
porphyrins are summarized in Table 2. The
long chain substituted free base porphyrins
Table 3. Antibacterial screening data for the
synthesized porphyrins
Inhibition zone (mm)
Complex
S. aureus E. coli
TOMPP 8.0 6.5
TOBPP 6.8 6.5
CuTOMPP 14.0 8.0
CuTOBPP 8.0 6.5

Figure 2. ESR spectrum of AgTOMPP and
AgTOMPP 7.5 7.5
AgTOBPP
AgTOBPP 11.5 10.0
3.4 Biological activity 6.8 6.5
DMSO
The current study, the free base
porphyrin, and metalloporphyrin were Penicilin 12.5 8.0
evaluated for their antibacterial activity
against Staphylococcus aureus (ATCC 4. Conclusion
25923)and Escherichia coli (ATCC 25922) The free base porphyrins (TOMPP,
by disc diffusion method at concentration 1 and TOBPP) and metalloporphyrins have
mg/mL. The antibacterial been successfully synthesized and their
screening data (Table 3) show that all identities were confirmed by mass
synthesized porphyrin complexes were spectrometry (MS), CHN elemental
found to be sensitive against both analysis, 1H, 13C NMR, FT-IR spectra, UV-
Staphylococcus aureus (ATCC 25923) and Visible and fluorescence spectroscopy, and
Escherichia coli (ATCC 25922) the electron spin resonance spectroscopy. The
inhibition zone was greater than control free base porphyrins was obtained at optimal
solvent (DMSO). In addition, the condition (refluxing in propionic acid) lower
commercially available standard drug than 20% yield. The reactions of free base
penicillin was used as a positive control the porphyrins with metal ions (Cu2+ and Ag2+)
zone of inhibition against. The antibacterial gave copper(II) porphyrin, and silver(II)
screening data indicate that the porphyrin complexes more than 50% yield.
metalloporphyrin complexes are more potent The pure ligand gave the lower product
antibacterial against than free base yield than metal complexes. The UV-Vis
porphyrin. In free base porphyrin, the absorption spectra for free base porphyrins
TOMPP showed the greatest zone of exhibited a single S-band with four Q-band,
inhibition of 8.0 mm against S. aureus, while the metalloporphyrin complexes show
while the inhibition zone of E. coli was a single S-band couple with only one Q-
observed at 6.5 mm. In metalloporphyrin band. Excited the synthesized complexes,
complexes the CuTOMPP showed the the products show the fluorescence spectra,
maximum zone of inhibition of S. aureus emitted at 654 nm for free base porphyrins
and E. coli at 14.0 and 8.0 mm, respectively. and emitted range of 605-607 nm for
However, the metalloporphyrin complexes metalloporphyrin complexes. The ESR
showed more potent antibacterial against signal of silver porphyrin complexes
than free base porphyrins due to faster (AgTOMPP and AgTOBPP) confirmed that
diffusion of metal complexes as a whole these compounds must contained silver(II) ion
through the cell membrane which combined in the structure with the g value 2.08 and
effect of metal atom and the ligand.18 2.07, respectively.
The antibacterial activity indicates 7. Temelli, B.; Unaleroglu, C. Tetrahedron
that the synthesized compounds are found to 2009, 65, 2043-2050.
be sensitive against both of Staphylococcus 8. Zhang, Z.; Duan, Y.; Zhang,L. ; Yu, M.;
aureus (ATCC 25923) and Escherichia coli Li, J. Inorg. Chem. Commun. 2015, 58,
(ATCC 25922). The TOMPP and 53-56.
CuTOMPP show higher zone of inhibition 9. Xu, Z.; Mei, Q.; Hua, Q.; Tian, R.;
than other compound in each series. Weng, J.; Shi, Y.; Huang, W. J. Mol.
Struct. 2015, 1094, 1-8.
Acknowledgements 10. Thomas, A. P.; SaneeshBabu, P. S.;
We are grateful to the Department of Asha Nair, S.; Ramakrishnan, S.;
Chemistry, Faculty of Science and Ramaiah, D.; Chandrashekar, T. K.;
Technology, Thammasat University and Srinivasan, A.; Radhakrishna Pillai, M.
Science Achievement Scholarship of J. Med. Chem. 2012, 55, 5110-5120.
Thailand (SAST), for financial and all 11. Alan, A.; Longo, F. R.; Shergalis, W. J.
experiments support. Faculty of Science, Am. Chem. Soc. 1964, 86, 3145 - 3149.
Mahidol University for ESR measurement. 12. Fodor, M. A.; Horváth, O.; Fodor, L.;
Grampp, G. Wankmüller, A. Inorg.
References Chem. Comm. 2014, 50, 110-112.
1. Domingues, R.; Marques, M.; 13. Maestrin, A.; Tedesco, A.; Neri, C.;
Domingues,P.; Neves, M.; Cavaleiro, J.; Gandini, M.; Serra, O.; Iamamoto, Y. J.
Correia, A. J. Am. Soc. Mass Spectrom. Braz. Chem. Soc. 2004, 15, 708-713.
2000, 12, 381 - 384. 14. Boscencu, R.; Molecules. 2012, 17 (5),
2. He, H.; Dubey, M.; Zhong, Y.; Shrestha, 5592-5603.
M.; Sykes, G. Eur. J. Inorg. Chem. 15. Lu, F.; Zhang, J.; Zhou , Y.; Zhao, Y.;
2011, 3731–3738. Zhang, B.; Feng, Y. Dyes Pigm. 2016,
3. Li, J.; Tang, T.; Li, F.; Li, M. Dyes 125, 116–123.
Pigm. 2008, 77, 395-401. 16. Gamboa, M.; Campos, M.; Torres, L.
4. Jiblaoui, A.; Leroy-Lhez, S.; Ouk, T. S.; A. J. Chem. Thermodyn. 2010, 42, 666-
Grenier, K.; Sol, V. Bioorg. Med. 674.
Chem.Lett. 2015, 25, 355-362. 17. Fritz, K.; Koski, W.; Caughey, W. J.
5. Lee, C.-H.; Park, J.-Y.; Kim, H.-J. Bull. Am. Chem. Soc. 1961, 83, 1607 – 1609.
Korean. Chem. Soc. 2000, 21, 97 - 100. 18. Bajju, G. D.; Kundan, S.; Bhagat, M.;
6. Fedulova, I. N.; Bragina, N. A.; Gupta, D.; Kapahi, A.; Devi1, G.
Novikov, N. V.; Ugol’nikova, O. A.; Bioinorg. Chem. Appl. 2014, 1-13.
Mironov, A. F. Russ. J. Bioorg. Chem.
2007, 33, 589-593.
Fe2O3 Particles deposition onto stainless steel
Taweechai Watcharapiboon, Varong Pavarajarn*
Center of Excellent in Particle Technology, Department of Chemical Engineering,
Faculty of Engineering, Chulalongkorn University, Thailand
*E-mail: varong.p@chula.ac.th
Abstract:
This research investigates the deposition behavior of suspended iron(III) oxide, Fe2O3,
particles in liquid onto a stainless-steel plate. Fe2O3 is a common compound suspending in
water and adhering to surface of liquid pipes in unit operations. Previous studies showed that
in stationary systems, large particles (diameter range of 2-2.5 µm) were unable to adhere to
surface of stainless steel. However, small particles (diameter range of 0.2-0.8 µm) could
adhere and deposit on the surface of stainless steel. This work aims to examine the deposition
behavior of Fe2O3 particles in the flow system. Fe2O3 particles concentration and velocity of
the suspended liquid were investigated in this study. The scanning electron microscope
(SEM) and ZeGage™ optical profilers were used to investigate the structure of Fe2O3
particles coating on the surface of stainless steel.
1. Introduction and operating temperature were major cause

Fe2O3 or Hematite can be found in of fouling on the surface of heat exchanger.4
nature. It is a key feedstock for several Therefore, this work has examined
industrial sections. It consists of Fe around the deposition behavior of Fe2O3 particles on
70% which can be extracted easily. It has the stainless-steel surface. Particles
been considered as low cost and suitable concentration, and velocity of the suspended
structure for extracted Fe+. In addition, liquid have been investigated to enhance
Fe2O3 can also apply to various applications understanding of operating-parameters on
including red pigment, polished powder, and the deposition behavior.
supercapacitors.1
Supercapacitors are energy storage 2. Materials and Methods
equipment composed of Fe2O3 thin film 2.1 Sample preparation
coated on stainless steel using liquid phase 1x1 cm stainless steel plates were
deposition (LPD) method. This method is polished by sandpaper to enhance uniformly
environmental friendly and relatively low smooth surface. After being washed with de-
cost.2 ionize water, double-sided tape was applied
Stainless steel has been widely used on the half of each stainless steel plate as
owing to its properties such as hardness, shown in Figure 1a.
corrosive, impact resistant, electrical
Double Particles
conductivity and heat transfer. It can be sided tape deposit
applied to several applications including
household products and industrial
equipment. Dispersions of fine particles in Remove
double
turbulent flow regime have an effect on Surface of sided
variety of instruments such as air stainless-steel tape
conditioners, heating boilers and heat a b
exchangers.3 Figure 1. Stainless steel plate with double-
Previous studies pointed out that sided tape a) before deposition and b) after
velocity of fluid, concentration of solution deposition of particles
2.2 Solution preparation It was found that the particles could
Fe2O3 purchased from HIMEDIA. deposit onto the surface of stainless steel
Particle size of Fe2O3 is 0.1-10 µm analyzed plate placed at pipe wall. The wall area was
by master sizer. Fe2O3 was used to prepared considered as the area that has the highest
283 and 115 ppm solution. Then, the shear stress in the system so the locomotion
solution was mixed with ethylene glycol at of particle in the system was restricted.
15% by weight to decelerate the rate of When velocity of 115 ppm solution is 0.1
precipitation. m/s, the thickness of Fe2O3 thin film are 0.2
2.3 Spectroscopic measurement µm as shown in Figure 4. Monolayer of
Stainless steel plates with double- Fe2O3 particles are deposited onto stainless
sides tape were immersed in Fe2O3 solution steel plate by adhesive force. It was
that was flowed in the system as shown in observed that attraction between particles to
Figure 2. Velocity of the solution was varied the stainless steel plate was more stable than
in the range of 0.02, 0.05, and 0.2 m/s. attraction between particle to particle
Every 3 days, one of stainless steel plate considered as double-layer. In addition, as
(Figure 1b) was collected to measure its shown in Figure 5, the rate of deposition
thickness by ZeGage™ optical profilers. was increased with increasing of time.
3.1 Effect of velocity
sample From Figure 4, the thickness of thin
layer decreases with increasing of solution
velocity because the increasing of velocity
results in increasing of shear force on the
surface of stainless steel. This is the
evidence to support that adhesive force
solution between stainless-steel plate to particle is
stronger than that of between particle to
particle.4
Figure 2. Diagram of experiment setup

The SEM results showed that
concentration of Fe2O3 solution influences
particle dispersion as shown in Figure 3. The
particles were crystalline and clustered
together. The lower concentration, 115 ppm
solution posed better dispersion than the
higher 283 ppm one.
a b
Figure 4. Thickness of thin layer of particle
versus velocity of solution at 15 days
3.2 Effect of concentration

As shown in Figure 5, thickness of
the thin layer increases with the increasing
Figure 3. SEM images of Fe2O3 particles a)
concentration of solution because the force
115 ppm solution and b) 238 ppm solution
between stainless steel plate and particle is
stronger than that of between particle and
particle.4 In addition, increasing of solution
concentration results in particle 4. Conclusion
accumulation. The accumulation leads to the Fe2O3 particles deposited onto
larger particle size and higher gravitational stainless steel plate by physical force.
force onto the particle resulting in growth of Thickness of the thin layer decreases with
the thin layer thickness. On the other hand, increasing solution velocity and increases
the thin layer thickness decreases with with increasing solution concentration.
decreasing of solution concentration.
Acknowledgements
The authors are grateful to PTT
global chemical for supporting ZeGage™
optical profilers.
References
1. Department of Mineral Resources
Home Page. http://www.dmr.go.th/main
.php?filename=hematite (accessed
March 24, 2018).
2. Khatavkar, S. N.; Sartale, S. D. J. Solid
State Electrochem. 2017, 21 (9), 2555-
2566.
3. Cleaver, J. W.; Yates, B. Chem. Eng.
Figure 5. Particle thin layer thickness versus Sci. 1975, 30 (8), 983-992.
days (1) at concentration 115 ppm and (2) at 4. Kho, T.; Zettler, H. U.; Müller-
concentration 283 ppm Steinhagen, H.; Hughes, D. Chem. Eng.
Res. Des. 1997, 75 (7), 635-640.
Studies on the preparation and bioactivity of highly porous bioactive glass
Monthita Sasana, Radchada Buntem*
Department of Chemistry, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand
*E-mail: radchadab@yahoo.com
Abstract:
Hydroxyapatite, the main component in human bone, can be generated from the
surface transformation of bioactive glass. In this research highly porous bioactive glass was
prepared by mixing deionized water, nitric acid, ethanol, tetraethyl orthosilicate,
triethylphosphate and calcium nitrate to obtain BG-sol. The sol was added to the fish scale
gelatin solution and the mixture was then stirred for 2 days. The wet gel was dried and
calcined at 1000 °C for 3 h to obtain highly porous bioactive glass (HPBG). A HPBG disc,
prepared from 0.4 g HPBG powder, was kept in 80 cm3 simulated body fluid (SBF) at 37.5±
0.5 °C for various periods: 8, 16, and 24 days. The SBF solution was replaced every two
days. The Ca2+ concentrations in collected SBF solutions were analyzed by atomic absorption
spectroscopy. The IR spectrum of the HPBG showed the absence of NO3- and the presence of
strong Si-O-Si peak. While the appearance of hydroxyapatite on the surface of the HPBG
after bioactivity test was observed as evidenced by FTIR and SEM-EDX analyses. The
fluctuation of Ca2+ concentration in SBF solutions collected at different time interval
corresponds to the proposed mechanism of Ca2+ exchange process.
1. Introduction poly(methylmethacrylate),6 poly (4-

Bioactive glass, osteoconductive, vinylpyridine), 7
chitosan, 8
chitosan-
and biodegradable biomaterial, was 9
polyacrylic acid (CS-PAA) were used to
1
developed by Hench as a bone repair bind with silica colloidal particle to form the
material. The bone bonding ability of the silica/polymer hybrids. The hydrogen
glass has been attributed to its ability to bonding interactions are responsible for the
generate hydroxycarbonate apatite (HCA) attachment between silica surface and
on the surface.2 From literatures, they polymeric chain preventing particle
showed that bioactive glass was superior to aggregation. Gelatin, a hydrolyzed form of
hydroxyapatite coating on implant surface.3 collagen, can be extracted from various parts
It was evidenced by in vivo studies about its of fish especially the hard scale.10,11 This
non-toxicity, non-inflammation, and non- denatured protein provides various
foreign-body response.4 The benefits for the functional groups for interacting with silica
synthetic method via sol gel are the low surface.12 In this research, the bioactive
temperature process, the obtained glass containing fish scale gelatin was
mesoporous texture of the product and the prepared via sol gel process followed by
ability to mix with another dissolved calcination to obtain highly porous bioactive
chemicals for various modification glass (HPBG). The in vitro bioactivity was
purposes. The porous inorganic oxide like performed in a simulated body fluid (SBF).
silica can be prepared by the pyrolysis of the The surface transformation was
silica/organic polymer hybrid materials. characterized by Fourier Transform Infrared
Various polymers such as polystyrene,5 Spectroscopy and Scanning Electron
Microscopy (SEM). The released amount of K2HPO4·3H2O (0.2310 g), MgCl2·6H2O
Ca2+ was analyzed by Atomic Absorption (0.3110 g), 1.0 M HCl (39.0 mL), CaCl2
Spectroscopy (AAS). (0.2920 g), Na2SO4 (0.0720 g) and
Tris(HOCH2)3CNH2 (6.1180 g) to 1 L of
2. Materials and Methods distilled water. The pH of the solution was
Tetraethylorthosilicate (TEOS), adjusted to 7.4 by the addition of 1 M HCl.
triethylphosphate (TEP) and The HPBG disc was immersed in 80 mL of
Ca(NO3)2·4H2O were obtained from Sigma- SBF solution and kept for incubation at 37
º
Aldrich and used as received. Gelatin was C for 8, 16, and 24 days. The SBF solution
extracted from fish scale according to the was replaced every 2 days to avoid depletion
published method.13 All solvents used were of the ionic species in the SBF due to the
of analytical grade and dried over molecular nucleation of biominerals on the samples.
sieves. The functional groups of the samples The Ca2+ concentration in the collected SBF
were analyzed using Spectrum 100 FT-IR solution was analyzed by Atomic
spectrophotometer (Perkin Elmer). While Absorption Spectrophotometry (AAS). After
the surface morphology of the samples were the precise time duration, the samples were
measured using Scanning Electron removed, washed with deionized water to
Microscope (SEM: JSM-6480LV (JEOL) remove the adsorbed minerals, dried at 60
with Energy Dispersive X-ray (EDX: C for 6 h to obtain HPBG_8, HPBG_16,
Oxford instruments). The calcium ion and HPBG_24 and analyzed using FT-IR
concentrations in simulated body fluid were and SEM/EDX for mineralization.
analyzed using atomic absorption
spectrometer (AAS: Perkin Elmer, AAnalyst 3. Results and Discussion
800). 3.1 Characterization of highly porous bio-
2.1 Preparation of highly porous bioactive active glass (HPBG)
glass (HPBG) Mixing BG-sol with gelatin solution
Deionized water (0.1918 mol), produces BG-FSG. After being calcined, the
HNO3 (3.20  10-3 mol), ethanol (0.1920 HPBG was obtained. The synthetic process
mol), and TEOS (0.0480 mol) were mixed of HPBG is shown in Figure 1. The silica
and the mixture was stirred for 30 min. Then particles were formed after mixing TEOS
TEP (4.7982  10-3 mol) was added while with water, acid and ethanol. Calcium and
stirring. After 30 minute, Ca(NO3)2·4H2O phosphate ions were then adsorbed on the
was added (0.0271 mol). The mixure was silica surface. The added gelatin acted as
stirred for another 3 h to obtain BG sol. It polymer matrix for particle attachment
was then slowly mixed with 3% w/v gelatin either through hydrogen bonds or ester
solution and the mixture was stirred for 2 bonds and helped reduce the aggregation of
days. The mixture was left dry at room silica particles. After calcination, the gelatin
temperature, subsequently dried in the oven was decomposed and some of the calcium
at 60 C to obtain BG-FSG. The dry BG- and phosphate ions were then diffused into
FSG was calcined at 1000 C for 3 h to silica matrix.
obtain HPBG. The HPBG was analyzed by The FTIR spectra of BG-FSG and
FT-IR and SEM/EDX. HPBG are shown in Figure 2. For BG-FSG,
2.2 In vitro bioactivity study the characteristic bands of gelatin are related
0.4 g of HPBG powder was pressed to C=O stretching (amide I) at 1651 cm-1,
to circular disc using pressure of 5 tons for N-H bending (amide II) at 1552 cm-1, the
10 min. It was then immersed in a simulated broad band above 3200 cm-1 corresponding
body fluid (SBF) prepared according to the to the free and bounded hydroxyl and amino
previous method14 by adding NaCl (8.0350 groups, and the absorption band at 1333
g), NaHCO3 (0.3550 g), KCl (0.2250 g), cm-1 attributable to CH2 wagging of
proline.15
Figure 2. FTIR spectra of BG-FSG and
HPBG
For HPBG, the gelatin and nitrate

peaks disappear because of their thermal
degradation and the bands of SiO2 are
shifted to lower wavenumber and cover a
broader wavenumber range. This can be due
to the incorporation of Ca2+ into the silica
network.24 The spectrum also suggests the
formation of some crystalline phases. All
typical peaks of pseudowollastonite
Figure 1. Synthetic process of HPBG (Ca3(Si3O9), 714 cm-1) are observed together
with two bands at 605 and 564 cm-1 which
The C-N stretching (amide III) of can be ascribed to the presence of tricalcium
gelatin is obscured due to the strong signals phosphate (TCP) and calcium phosphate
related to the asymmetric and symmetric silicate [Ca15(PO4)2(SiO4)6].25-27
stretching of nitrate ions at 1423, 1384 and 3.2 In vitro bioactivity study
1357 cm-1.16,17 The sharp peak at 823 cm-1 The FTIR spectra of HPBG before
and the weak peak at 738 cm-1 can be and after being immersed in SBF are shown
ascribed to the bending modes of the nitrate in Figure 3. In all immersed samples, the
ions.16-18 The thermal degradation of nitrates bands at 3430 and 1635 cm-1 are
occur only after heat treatment at a consecutively assigned to the O-H stretching
temperature beyond 350 C, as nitrate by- and O-H bending modes of water and silanol
products (gaseous NO2 and O2) are driven (Si-OH) group. The presence of C – O
off and calcium ions enter into the network stretching and bending bands of CO32- at
by diffusion.19 However, the presence of 1420, 872 cm-1 indicates the formation of
SiO2 is proven by the bands at 1087 cm-1 carbonatehydroxy apatite on the surface of
and at 804 cm-1, assigned to the asymmetric HPBG. These bands are more intense when
and symmetric stretching modes of SiO4 the immersing time is increased. However in
tetrahedra, respectively, and by the band at the case of 8 day immersion, the band at 872
963 cm-1 due to Si–OH groups.20-22 cm-1 cannot be observed. In addition the
Moreover, the broad band centered at 1160 appearance of the double band at 604 and
cm-1 are due to the asymmetric stretching 565 cm-1 assigned to P-O bending of apatite
vibrations of the P–O groups in Q2 and Q1 confirms the surface transformation. The Si-
structural units respectively23 where Q is the O-Si stretching at 1092 cm-1 is overlapped
PO43- ion and the superscript designates the with the P – O asymmetric stretching at 1087
number of bridging oxygen bonds formed cm-1. While Si-OH stretching is observed at
with other PO43- ions. 959 cm-1 in the case of 8 day immersion.
The increased immersing time obscures the
signal and only a small shoulder is observed. details in Figure 5. The exposure time
Band at 801 cm-1 is assigned to SiO4 affects the Ca/P molar ratio. The longer
asymmetric stretching while Si – O – Si exposure time increases the Ca/P of the
bending at 475 cm-1 is found in all immersed crystals on HPBG surface; 1.29 for
samples. HPBG_8, 1.38 for HPBG_16 and 1.59 for
HPBG_24. However the Ca/P molar ratios
of apatite on all immersed HPBG surfaces
are lower than the theoretical Ca/P molar
ratio (= 1.67) of pure apatite
(Ca5(PO4)3(OH)). It is quite common for the
nucleation of calcium deficient
hydroxyapatite on the bioactive glass
surface.28 In the literature, it is claimed that
carbonate hydroxyapatite was initially
formed due to the presence of carbonate ion
in SBF solution29,30 and the dissolution of
atmospheric CO2 in water generating
carbonic acid with subsequent dissociating
into carbonate ion in basic solution.31 The
immersed HPBG samples release Ca2+ ions
(as evidenced by AAS analysis) which can
be exchanged with H+ in the solution or can
react with OH- ions to form Ca(OH)2. This
results in the pH increase inducing a higher
amount of carbonate ions.
Figure 3. FTIR of HPBG in 4000 – 450

cm-1 after being immersed in SBF for 8, 16
and 24 days
The results of SEM analysis after

immersing in SBF are in agreement with the
FTIR data. The formation of globular
crystals is observed on the surface of all
samples (Figure 4) and it increases with the
exposure time to SBF. On the surface, the Figure 4. SEM images of (a) HPBG before
flake-like apatite is formed with the long soaking in SBF (b)-(d) HPBG after soaking
oval and circular-shape holes on the surface. in SBF for 8, 16 and 24 days; The insets are
For HPBG_8, the lengths of the oval holes higher magnification. (Scale bar indicates
range from 0.8571 m to 2.8571 m and the 10 m.)
diameters of the circular holes are in the
range of 0.4286-0.5714 m. When the SBF
exposure time is increased the trend in the
size of holes is decreased due to the growth
of apatite crystals.
The EDX analyses were performed
on the globular surfaces of all samples as the
Figure 6. Variation in concentration of Ca2+
with soaking in SBF
4. Conclusion
Highly porous bioactive glass was
successfully prepared by the sol-gel method
and calcination. Adding gelatin helps reduce
the aggregation of silica particles and after
being calcined the highly porous bioactive
glass was obtained. Study on in vitro
bioactivity in SBF solution shows the
formation of hydroxyapatite on the HPBG
Figure 5. EDX of immersed HPBG for 8, surface as evidence from various analyses.
16, and 24 days The transformation consists of Ca2+
exchange between HPBG surface and SBF
3.3 The Release of Ca2+ from HPBG yielding flake-like apatite and circular-shape
Atomic absorption spectroscopy was holes. The second day of SBF immersion,
used to measure the released concentrations the highest Ca2+ release was observed. The
release rate was decreased and the
of Ca2+, in SBF solution to study the
equilibrium was reached within 8 days.
dissolution behavior of samples during From SEM analysis, Ca/P molar ratio of the
bioactivity study. It is found that immersed HPBG was found to be lower than
concentration of Ca2+ increases in first 2 that of pure hydroxyapatite confirming the
days (Figure 6). This is attributed to the formation of calcium deficient
exchange of Ca2+ from HPBG with H+ or hydroxyapatite.
H3O+ ions from the SBF solution forming
Acknowledgements
silanol groups (Si–OH) on the glass surface. Many thanks to Science and
These silanol groups act as nucleation agent Mathematics talented teacher production by
for hydroxyapatite formation. After 2 days, the institute for the Promotion of Teaching
concentration of Ca2+ decrease continuously. Science and Technology for the Master
This decrement of Ca2+ indicates the apatite degree scholarship and Department of
formation on HPBG. Chemistry, Faculty of Science, Silpakorn
University for the research facilities.
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A dinuclear cadmium(II) complex based on 2-hydroxybenzoate and 2-
aminopyrimidine ligand: synthesis, characterization, and crystal structure
Sudarat Thummatudtho1, Natthakorn Phadungsak1,2, Sukanya Mingphimai1,
Supakorn Boonyuen1*, Kittipong Chainok2
1
2
Materials Innovation and Technology, Faculty of Science and Technology, Thammasat University,
*Email: chemistrytu@gmail.com
Abstract:
One novel dinuclear Cd(II) complex, [Cd2(2-OHbza)4(apm)4] (where 2-OHbza = 2-
hydroxybenzoate, apm = 2-aminopyrimidine) has been prepared from a reaction of
cadmium(II) nitrate with 2-OHbza in ethanol followed by a reaction with apm in ethanol and
water. The [Cd2(2-OHbza)4(apm)4] complex was characterized by CHN elemental analysis,
FT-IR spectroscopy and single crystal X-ray diffraction. The complex revealed monoclinic
space group P21/c. The environment of Cd(II) ions is distorted pentagonal bipyrimidal by five
oxygen atoms from three 2-OHbza ligands and two nitrogen atoms from two apm ligands.
The dinuclear complex connected by two tridentate bridging 2-OHbza anions that is
intramolecular hydrogen bonds between hydroxy and carboxylate groups from 2-OHbza
ligands and - stacking interactions between parallel apm ligands are observed.
1. Introduction bonding modes.6-8 2-Aminopyrimidine has

In recent years, there has been endo- and exocyclic nitrogen donors for
increasing research interest in the design and coordination. The amino nitrogen atom is
synthesis of complexes.1,2 The cadmium and known to be more basic in comparison to the
2-hydroxybenzoate with 2-aminopyrimidine pyrimidine ring nitrogens.9,10 2-Hydroxy
has been of great interest for their co- benzoic acid is the most popular model
ordination behavior. Among d10 metals, the systems for studying intramolecular hydro-
heavy metal cadmium forms a plethora of gen bonds.11-13 The aim of this study is to
complexes with wide variety of coordination synthesize and structural characterize of
numbers ranging from four to eight.3,4 cadmium(II) heteroligand complexes, con-
Moreover the d10 metal ions also find taining organic ligands 2-aminopyrimidine
potential applications as photoluminescence (apm) and 2-hydroxybenzoic acid (2-
materials cadmium carboxylates are of OHbza). The 2-aminopyrimidine ligand has
special interest for their interesting material widely been used for constructing polymeric
properties as well as for their versatile co- chains as a dinuclear complex of [Cd2(2-
ordination behavior. Cadmium have OHbza)4(apm)4].
interesting polymeric structure.5 It is
important to understand how the chemistry
of bonding in the complex form.6
2-Aminopyrimidine is of particular
interest owing to its demonstrated ability to
form very stable hydrogen bonded chain
arrays via its stereochemically associative
Figure1. Chemical structures of 2-hydroxy
amino and hetero ring nitrogens and to
benzoic acid and 2-aminopyrimidine ligand
coordinate to metal centers through various
2.1 General 3.1 Description of structures
FT-IR spectra were recorded on The novel synthesized complex
Perkin-Elmer infrared spectrophotometer [Cd2(2-OHbza)4(apm)4] was successfully
(spectrum GX) using KBr pellets (range synthesized. However, the complex did not
4,000-400 cm-1). Thermogravimetric dissolved in any solvents. Further study by
analyses by using a 2050 thermogravimetric single-crystal X-ray diffraction shows the
analyzer (TGA Instruments) were heated at crystallographic data in Table 1. The
10 oC per min, under nitrogen atmosphere. complex revealed monoclinic space group
CHN elemental analyses was carried out on P21/c. The environment of Cd(II) ions is
Perkin Elmer (2400) elemental analyzer. distorted pentagonal bipyrimidal by five
2.2 Synthesis of [Cd2(2-OHbza)4(apm)4] oxygen atoms from three 2-OHbza ligands
Cd(NO3)2· 4H2O 1 mmol was and two nitrogen atoms from two apm
dissolved in water (2 mL) and kept stirring ligands. The dinuclear complex connected
for 2 min to obtain a clear solution. 2- by two tridentate bridging 2-OHbza anions
Hydroxybenzoic acid (2 mmol) in ethanol (2 that is intramolecular hydrogen bonds
mL) was added into the reaction solution between hydroxyl and carboxylate groups
and continued to stir for 5 min. After from 2-OHbza ligands and - stacking
adjusting the pH to pH7, 2-aminopyrimidine interactions between parallel apm ligands
(2 mmol) was then added into the reaction are observed.
solution and kept stirring for 5 min. After
slow evaporation of reaction solution for 4 Table 1. Crystallographic data for [Cd2(2-
days, yellow crystals of complex were OHbza)4(apm)4]
formed and collected by filtration, then Compound [Cd2(2-OHbza)4(apm)4]
washed with water and dried. (48% yield)
Anal. Calc. for C36H30N4NiO4: C, 68.0; H, Empirical formula C44H40Cd2N12O12
4.5; N, 7.0 Found: C, 67.5; H, 4.6; N, 6.9%.
Formula 1153.68
IR (KBr, cm-1): 3163 v(N-H), 2925 v(-CH), weight(g/mol)
1568 v(C=O), 1650 v(C=N), 1223 v(C-O), Temperature (K) 296.15
529 v(M-O). Crystal system monoclinic
2.3 X-ray crystallographic analysis Space group P21/c
X-ray diffraction data were collected a (Å) 11.1654(7)
b (Å) 10.5903(6)
on a Bruker D8 QUEST CMOS c (Å) 19.3149(12)
diffractometer equipped with graphite- α( ) 90
monochromatic Mo-Kα radiation (λ = β( ) 93.912(2)
0.71073 Å) at Faculty of Science and γ( ) 90
Technology, Thammasat University, V (Å3) 2278.6(2)
operating at 296 ± 2 K. Empirical absorption Z 2
corrections were applied to all data using µ (Mo K ) (mm-1) 1.011
SADABS.9 The structures were solved by Reflections collected 5021
SHELXT-201510 and refined by full-matrix Independent 6395
reflections (Rint) 0.0742
least-squares based on F2 using the
R[I 2 (I)] 0.0359
SHELXL-9711 with Olex212 as a graphics wR2 (F2) 0.0885
interface. Anisotropic displacement
parameters were refined for all non-
hydrogen atoms. Hydrogen atoms were
added theoretically and were riding on their
parent atoms.
Table 2. Selected bond length and angles of [Cd2(2-OHbza)4(apm)4]
Cd1-O1 2.3509(18) Cd1-O5 2.3540(15)
Cd1-O2 2.3743(17) Cd1-N1 2.3796(18)
Cd1-O4 2.5672(18) Cd1-N4 2.3709(18)
Cd1-O41 2.4014(16) Cd1-C1 2.719(2)
O1-Cd1-O2 55.42(6) N1-Cd1-C1 90.33(7)
O1-Cd1-O4 165.17(5) N4-Cd1-O2 94.27(6)
O1-Cd1-O41 90.02(6) N4-Cd1-O4 85.52(6)
O1-Cd1-O5 141.27(6) N4-Cd1-O41 92.82(6)
O1-Cd1-N1 92.17(6) N4-Cd1-N1 177.33(6)
O1-Cd1-N4 90.01(6) N4-Cd1-C1 92.32(6)
O1-Cd1-C1 28.23(6) C1-O1-Cd1 91.97(14)
O2-Cd1-O4 138.98(5) C1-O2-Cd1 92.03(14)
O2-Cd1-O41 144.63(6) Cd11-O4-Cd1 103.89(6)
O2-Cd1-N1 88.25(6) C8-O4-Cd11 165.95(16)
O2-Cd1-C1 27.19(6) C8-O4-Cd1 89.22(14)
O41-Cd1-O4 76.11(6) C8-O5-Cd1 98.68(13)
O4-Cd1-C1 165.87(6) C15-N1-Cd1 125.96(15)
O41-Cd1-C1 117.97(7) C18-N1-Cd1 117.59(14)
O5-Cd1-O2 86.36(6) C18-N1-C15 115.8(2)
O5-Cd1-O4 52.64(5) C16-N2-C15 117.1(2)
O5-Cd1-O41 128.66(6) C19-N4-Cd1 126.98(15)
O5-Cd1-N1 92.63(6) C22-N4-Cd1 117.43(14)
O5-Cd1-N4 86.63(6) C22-N4-C19 115.53(19)
O5-Cd1-C1 113.34(6) C20-N5-C19 116.8(2)
N1-Cd1-O41 85.63(6) O1-C1-Cd1 59.79(12)
N1-Cd1-O4 91.99(6) O2-C1-Cd1 60.78(13)
Figure 2. Perspective view of the coordination Figure 4. Selected intramolecular [Cd2(2-

environment of cadmium atoms in [Cd2(2- OHbza)4(apm)4] – stacking interactions in
OHbza)4(apm)4] 1D chain of complex
Figure 3. Selected intramolecular [Cd2(2- Figure 5. Selected intramolecular [Cd2(2-

OHbza)4(apm)4] hydrogen bonding and OHbza)4(apm)4] hydrogen bonding and –
interactions in 1D chain of complex stacking interactions in 2D of complex
3.2 Thermogravimetric analysis Acknowledgements
Thermal gravimetric analyses (TGA) The financial and all experiments
of [Cd2(2-OHbza)4(apm)4] were recorded for equipment were supported by Department of
the polycrystalline samples in the Chemistry, and Physics, Faculty of Science
temperature range 25-800 C. The TGA and Technology, Thammasat University.
curves showed two declined steps. The first Central Scientific Instrument Center (CSIC)
step demonstrated the release of 2- of Faculty of Science and Technology,
hydroxybenzoic acid weight loss in the Thammasat University.
temperature range 222-261 oC (found,
58.66%). The second step indicated of the References
framework weight loss in the temperature 1. Jahromia, S. M.; Montazerozohoria, M.;
between 455 and 529 oC (found, 24.42%). Masoudiaslb, A.; Houshyarc, E.;
Then the structure rapidly decomposes to Jooharid, S.; Whitee, J. M. Ultrason.
metal oxide. Sonochem. 2018, 41, 590-599.
2. Salinas-Castillo, A.; Calahorro , A. J.;
Choquesillo-Lazarte, D.; Seco, J. M.;
Rodríguez-Diéguez, A. Polyhedron
2011, 30, 1295–1298.
3. Kuriakose, D.; Aravindakshan, A. A.;
Prathapachandra Kurup, M. R.
Polyhedron 2017, 127, 84–96.
4. Peng Li, C.; Du, M. Inorg. Chem.
Commun. 2011, 14, 502–513.
Figure 6. The TGA curves of [Cd2(2-
5. Evans, O. R.; Lin, W. B. Acc. Chem.
OHbza)4(apm)4]
Res. 2002, 35, 511.
4. Conclusion 6. Shahbazi, M.; Mehrzad, F.; Mirzaei,
A complex of [Cd2(2-OHbza)4 M.; Eshtiagh-Hosseini, H.; Mague, J.
(apm)4] was successfully synthesized in T.; Ardalani, M.; Shamsipur, M. Inorg.
48% yield. The complex was characterized Chim. Acta. 2017, 458, 84–96.
by CHN elemental analysis and, FT-IR 7. Luo, G. G.; Huang, R. B.; Chen, J. H.;
spectroscopy. Structural analysis of desired
Lin, L. R.; Zheng, L. S. Polyhedron.
crystalline product by Single Crystal X-ray
diffraction, confirmed the dinuclear title 2008, 27, 2791.
complex crystallized in the monoclinic 8. Van, A.; Gerard, A.; Mutikainen, I.;
space group P21/c with two formula units Turpeinen, U.; Reedijk, J. Polyhedron
per unit cell. The bridging 2-OHbza anions 2004, 23, 993–998.
between two Cd(II) ions were found, 9. Akyuz, S. J. Supramol. Chem. 2002, 2,
whereas each Cd(II) ion from a seven co- 401–404.
ordination unit. The distorted pentagonal
10. Stringfield, T. W.; Shepherd, R. E.
bipyrimidal by five O atoms from three 2-
OHbza ligands and two N atoms from two Inorg. Chem. Commun. 2001, 4, 760–
apm ligands were observed. The – 765.
stacking interactions between parallel apm 11. Steinwender, E.; Mikenda, W. Monatsh.
ligands are observed. Chem. 1994,125, 695–705.
12. Mikenda, W.; Steinwender, E.;
Mereiter, K. Monatsh. Chem. 1995,
126, 495–504.
13. Lampert, H.; Mikenda, W.; Karpfen, A.; 15. Sheldrick, G. M. Acta Cryst. 2015, C71;
J. Phys. Chem. 1996, 100, 7418-7425. 3–8.
14. Bruker (2014). APEX2, SADABS and 16. Dolomanov, O. V.; Bourhis, L. J.;
SAINT. Bruker AXS Inc., Madison, Gildea, R. J.; Howard, J. A. K.;
Wisconsin, USA. 10. Sheldrick, G. M. Puschmann, H. J. Appl. Cryst. 2009, 42,
Acta Cryst. A 2015; 71, 3–8. 339–341.
Effect of additional phosphine in n-butanol production catalyzed by
Fe(II)-N-heterocyclic carbene complexes
Nattapong Siengdang1, Watsapon Yimkaew2, Bussaba Boonseng3, Ratanon Chotima3*
1
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
2
Department of Material Science, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
3
Department of Chemistry, Faculty of Science, Naresuan University, Muang, Phitsanulok 65000, Thailand
*E-mail: ratanonc@nu.ac.th
Abstract:
n-Butanol is considered as one of the alternative biofuels which can be synthesized
via Guerbet’s reaction using a metal complex as a homogeneous catalyst. A series of
complexes between Fe(II) and N-heterocyclic carbenes, 1,3-bis(2,4,6-trimethylphenyl)-4,5-
dihydroimidazol-2-ylidene (SIMes), 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (IMes)
and 1,3-Bis(2,6-diisopropylphenyl)imidazol-2-ylidene (SIPr), was synthesized in order to use
as catalysts in the production of n-butanol from ethanol. Triphenylphosphine was also
introduced to the complexes which expected to enhance the catalytic efficiency. The
complexes were characterized by nuclear magnetic resonance spectroscopy (1H-NMR and
31
P{1H}-NMR). The spectrum revealed that there were both N-heterocyclic carbenes and
triphenylphosphine in the synthesized complexes, but the intrinsic structure of the complexes
could not be confirmed. The amount of products was increased when lengthened the reaction
time. The catalytic production of n-butanol from ethanol was performed at 80 ˚C and 150 ˚C,
and monitored by gas chromatography (GC). The chromatogram of the catalytic reaction
showed only trace amount of n-butanol production.
1. Introduction Usually, in industrial process, it was

Fossil fuels such as coal, oil and synthesized from propylene which extracted
natural gases are the main sources of energy from petroleum resource. The synthesis of
used in transportation and industrial n-butanol using sustainable bio-resources
processes. However, these natural resources such as ethanol remains a great challenge.
could not be used last forever. Alternative Most recently, ruthenium complexes6 have
energies have been researched and been used as a homogeneous catalyst to
developed over a past few decades to ensure selectively make n-butanol or isobutanol via
the sustainable energy status in the future.1,2 the so-called Guerbet reaction.7
Ethanol is one of the biofuels used in many Therefore, this work is focusing on
vehicles, by mixing ethanol with either development of these homogeneous
gasoline or diesel.3 Nonetheless, there are catalysts to use in this reaction. Our previous
significant drawbacks when compared with report8 suggested that iron(II) complexes
conventional gasoline such as low energy could potentially be used as catalysts. The
content, corrosive and well soluble in water. advantages of using iron(II) as metal are less
This could causes some serious problems expensive, less toxicity and more abundant
with the vehicle engines, pipelines or in nature. Herein, we studied the effect of
storage tanks.4 Recently, n-butanol has been addition of triphenylphosphine to the
studied and attempted to use as an reaction. The improvement of optimal
alternative fuel. It was found that several conditions of synthetic and catalytic
energy-related properties are as similar as strategies have also been studied.
gasoline. Over the past decade, a lot of effort
has been put on synthesizing n-butanol.5
All chemicals were used as
purchased unless otherwise stated. All
reactions are processed under nitrogen
atmosphere unless otherwise stated. One of
the NHC ligands, IMes.HCl, was freshly
synthesized as reference procedure.9
2.1 Synthesis of IMes.HCl (1,3-bis-(2,4,6- Figure 2. Synthesis of iron(II) complexes
trimethylphenyl)imidazolium chloride)
IMes.HCl can be synthesized in two Complex 1 (iron(II) with SIMes ligand)
main steps according to the procedure.9 The A mixed solution of SIMes∙HCl
yellow solid was obtained (77.8 mg, (68.5 mg, 0.2 mmol) and C4H9OK (22.4 mg,
64.62%) and identified by 1H-NMR. 0.2 mmol). 1 equiv. of FeCl2·4H2O (39.0
mg, 0.2 mmol) and 1 equiv. of PPh3 (52.4
mg, 0.2 mmol) to obtained the yellowish
solid (72.52% yield). 1H NMR (400 MHz,
CDCl3): δ 7.31-7.69 (m, Ar-H), 6.98 (s, 4H,
Ar-H), 4.64 (s, 4H, NCH2CH2N), 2.41 (s,
6H, CH3), 2.30 (s, 12H, CH3). 31P{1H}-
NMR (400 MHz, CDCl3): δ 29.28 (s, 1P,
Fe-P), 5.25 (s, 1P, free PPh3).
Figure 1. The synthesis of IMes.HCl Complex 2 (iron(II) with IMes ligand)
A mixed solution of IMes∙HCl
2.2 General procedure for the synthesis of (61.08 mg, 0.2 mmol) and C2H5ONa (13.61
iron(II) complexes mg, 0.2 mmol). 1 equiv. of FeCl2·4H2O
A two-necked round bottom flask (39.5 mg, 0.2 mmol) and 1 equiv. of PPh3
was charged with imidazolium or (52.0 mg, 0.2 mmol) to obtained the
imidazolinium salt (IMes∙HCl, SIMes∙HCl yellowish solid (82.68% yield). 1H NMR
or SIPr∙HCl), alkoxide base (C2H5ONa or (400 MHz, CDCl3): δ 7.60-7.49 (m, Ar-H),
C4H9OK) in acetonitrile. The mixture was 7.12 (s, 2H, NCHCHN), 7.00 (s, 4H, Ar-H),
stirred for 20 min. Subsequently, an 2.34 (s, 12H, CH3) 1.98 (s, 6H, CH3).
equivalent of FeCl2·4H2O was added to the 31
P{1H}-NMR (400 MHz, CDCl3): δ 29.39
flask then stirred at 20 – 25 °C for an hour (s, 1P, Fe-P), 5.27 (s, 1P, free PPh3).
under nitrogen atmosphere. The solution of Complex 3 (iron(II) with SIPr ligand)
triphenylphosphine in acetonitrile was then A mixed solution of SIPr∙HCl (85.01
added to the mixture and stirred for 30 to 60 mg, 0.2 mmol) and C2H5ONa (13.61 mg, 0.2
minutes. Solvent was removed by rotary mmol). 1 equiv. of FeCl2·4H2O (39.8 mg,
evaporator. Crude product was extracted by 0.2 mmol) and 1 equiv. of PPh3 (mg, mmol)
liquid-liquid extraction using 15 mL of to obtained the yellowish solid (79.85%
dichloromethane (5 mL×3 times), dried over yield). 1H NMR (400 MHz, CDCl3): δ 7.75-
sodium sulfate, filtered with Celite and the 7.50 (m, Ar-H), 7.45 (t, J = 8.0 Hz, 2H, Ar-
solvent was removed. Lastly, the purified H), 7.31 (d, J = 8.0 Hz, 4H, Ar-H), 4.21 (s,
product was determined by using 1H-NMR 4H, NCH2CH2N), 2.95-2.80 (m, 4H,
and 31P{1H}-NMR spectroscopy and further CH(CH3)2), 1.25, 1.20 (both d, J = 7.2 Hz,
confirmed by mass spectrometry. The each 12H, CH3). 31P{1H}-NMR (400 MHz,
synthetic route of the complexes is shown in CDCl3): δ 29.12 (s, 1P, Fe-P), 5.28 (s, 1P,
Figure 2. free PPh3).
2.3 Catalytic conversion of ethanol to n- Consequently, the addition of
butanol triphenylphosphine could possibly be
Catalyst (0.2 mol%) and 5 mol% of separated the dimer to a monomeric
sodium ethoxide (NaOC2H5) were added in complex as shown in Figure 3.12
1 mL of ethanol which purified through
distillation before use. The process was
carried out using a test tube with a screw cap
under an inert atmosphere (N2) at 80 and
150 oC for four hours. The product was Figure 3. Possible reaction of generating the
monitored by gas chromatography (GC). complexes
3. Results and Discussion Variation of NHCs, bases and time

3.1 Synthesis and characterization of the Three different types of NHC were
complexes selected, SIMes.HCl, IMes.HCl, and
As our previous report,8 some SIPr.HCl as shown in Figure 4.
selected N-heterocyclic carbene ligands
were used to make the complexes. However,
catalytic conversions from ethanol to n-
butanol were unsuccessful. In this work,
triphenyl phosphine was chosen to assist the
reaction as not because of its strong electron Figure 4. N-heterocyclic carbenes used in
donor ability but also its steric structure this work
which could possibly help the reaction to
occur. The synthetic route of the complexes IMes.HCl was the only ligand which
are similar to our previous work with the freshly synthesized in the laboratory. The
addition of triphenylphosphine in the final first step is the condensation between
step. The 1H-NMR spectrum suggest the glyoxal and two equivalent of 2,4,6-
difficulty in interpretation due to many trimethylaniline to produce the bright yellow
aromatic proton on both ligands. Therefore, solid of the imine compounds. In the next
31
P{1H}-NMR was used to monitored the step, chloroethyl methyl ether was the
products. According to Seewald and co- substrate to close the five-membered ring.
workers’ report,11 the bond between Controlling temperature to around 20 to 25
triphenylphosphine and iron(II) gives the °C is the very important key condition to the
phosphorus chemical shift at 31.9 ppm precipitation of the product. The 1H-NMR
(C6D6). Moreover, the chemical shift of a spectrum was proved to be in consistent
free triphenylphosphine appears around -4 to with the commercial compound.
-5 ppm. In this work, there are two singlet For bases variation, two alkoxide
peaks appear in phosphorus spectrum, the bases are used, sodium ethoxide and
first one locates around -5 ppm which can be potassium tert-butoxide. It was noticed that
identified as free triphenylphosphine. In both bases have the ability to generate the
addition, the phosphorus spectrum suggested carbene but it was also depended on types
that there was a significant amount of and time. The longer the reaction was
triphenylphosphine left in final product. processed, the higher the percentage yield of
Another peak appears around 29 ppm in all the product occurred when controlling other
complexes which could be identified as the factors. However, the solubility of all
Fe-P bond. This can be suggested the starting materials also play the significant
possible formation of the desired complexes. role to the amount of the product. In early
However, considered the mass spectrum in attempts, with different solvents such as
our previous report,8 it suggested the THF, little amount of complexes has been
formation of dimeric iron(II) complexes. produced. Therefore, the best condition so
far for generating this complexe is alkoxide
bases, 60 minutes reaction time in
acetonitrile.
3.2 Study of catalytic testing
These iron(II) complexes were used
as catalysts in the reaction attempting to
convert ethanol to n-butanol. Some previous
works using ruthenium catalysts showed
very high selectivity but relatively low
yield.10 Our previous report8 showed that
iron(II) complexes had less catalytic activity
than ruthenium(II) catalysts.
To use homogeneous catalysts with Figure 6. GC Chromatogram of catalytic
ethanol as a starting material, n-butanol can reactions compared to standard solutions
be synthesized via “Guerbet Reaction”. 0.2
mol% of the catalyst was used in the Two obvious peaks are acetone and
reaction together with 5 mol% of sodium ethanol (starting materials), respectively.
ethoxide at 80 and 150 °C. All reactions was Other spots are too little to be analyzed.
run in a tube with a screw cap. The reaction There were some reports suggest that a few
is shown in Figure 5. types of aldehyde compounds could be
generated such as crotonaldehyde or
butylradehyde. Therefore, in this case,
addition of triphenyl phosphine could not
improve catalytic efficiency. However,
many reports suggested that in this type of
Figure 5. Catalytic conversion of ethanol to reaction, higher catalyst loading is needed.
n-butanol using iron(II) complexes as the Especially when using iron which normally
catalyst; In this case, sodium ethoxide was has less catalytic capability than ruthenium.
used as a base.
4. Conclusion
Table 1. The selected catalytic reactionsa A series of complexes between
Entriesb,c NHCs Temperature Yield iron(II), N-heterocyclic carbenes and
(°C)
triphenyl phosphine was synthesized. The
RC1 SIMes 80 N/A
RC2 SIMes 150 N/A certain structures are yet to be confirmed by
RC3 IMes 80 N/A spectroscopic methods. More precise
RC4 IMes 150 N/A structures are in the processes of
RC5 SIPr 80 N/A characterization. Catalytic conversion of
RC6 SIPr 150 N/A
a
ethanol to n-butanol was attempted using
All complex catalysts are [FeCl2(NHC)PPh3].
b
All reactions used sodium ethoxide as base. several catalytic conditions. However,
c
Reaction time is 4 hours. successful products had not been
determined.
Conditions were screening in factors
of times, bases, and temperature. However, Acknowledgements
very little amount of the product was We would like to sincerely thank the
observed when monitored the reaction by Department of Chemistry, Faculty of
GC as seen in Figure 6. All results are Science, Naresuan University and NRCT for
summarized in Table 1. financial funding and facilities support. NS
would like to thank the Development and
Promotion of Science and Technology
Talents (DPST).
References 7. Guerbet, M.; C. R. Acad. Sci. Paris
1. International Energy Agency Home 1899, 128, 511. Viebel, S.; Nielsen, J. I.
Page. http://www.iea.org (accessed Tetrahedron 1967, 23 (4), 1723–1733.
March 24, 2018). 8. Yimkaew, W.; Sombut, P.; Boonseng B.;
2. Ragauskas, A. J.; Williams, C. K.; Chotima R. Proceedings of PACCON
Davison, B. H.; Britovsek, G.; Cairney, 2017, Bangkok, Thailand, Feb 2-3, 2017,
J.; Eckert, C. A.; Frederick Jr., W. J.; 509–514.
Hallett, J. P.; Leak, D. J.; Liotta, C. L.; 9. Gautier, A.; Cisnetti, F.; Diaz-González,
Mielenz, J.; Murphy, R. R.; Templer, R.; S.; Gibard, C. Protocol Exchange 2012.
Tschaplinski, T. Science 2006, 311, 10. Wingad, R. L.; Gates, P. J.; Street, S. T.
484–489. G.; Wass, D. F. ACS Catal. 2015, 5 (10),
3. Niven, R. K. Renewable Sustainable 5822–5826
Energy Rev. 2005, 9 (6), 535–555. 11. Seewald, O.; Flӧrke, U.; Henkel, G. Acta
4. Dowson, G. R. M.; Haddow, M. F.; Lee, Crystallogr. Sect. E. 2005, 61, 1829–
J.; Wingad, R. L.; Wass, D. F.; Angew. 1830.
Chem. Int. Ed. 2013, 52, 1–5. 12. Wass, D. F.; Hey, T. W.; Rodriguez-
5. (a) Koda, K.; Matsu, T.; Obora, Y.; Ishii, Castro, J.; Russell, C. A.; Shishkov, I.
Y. Chem. Lett. 2009, 38, 838–839. (b) V.; Wingad, R. L.; Green, M.
Wingad, R. L.; Bergström, E. J. E.; Organometallics 2007, 26, 4702–4703.
Everett, M.; Pellow, K. J.; Wass, D. F. Chotima, R.; Dale, T.; Green, M.; Hey,
Chem. Comm. 2016, 52, 5202–5204. T. W.; McMullin, C. L.; Nunns, A.;
6. Wingad, R. L.; Gates, P. J.; Street, S. T. Orpen, G. A.; Shishkov, I. V.; Wass, D.
G.; Wass, D. F. ACS Catal. 2015, 5 (10), F.; Wingad, R. L. Dalton Trans. 2011,
5822–5826. 40, 5316–5323.
A three-dimensional cobalt(II) coordination polymer with formate ligands:
synthesis and crystal structure
Sukanya Mingphimai1, Natthakorn Phadungsak1,2, Supakorn Boonyeun1*,
Kittipong Chainok2
1
2
Materials Innovation and Technology, Department of Physics, Faculty of Science and Technology,
Thammasat University, Pathum Thani 12120, Thailand
Abstract:
One three-dimensional (3D) cobalt(II) coordination polymer was constructed by
reaction of formate ligands, the [Co2(HCO2)4(H2O)4]n has been synthesized under refluxed
conditions. The coordination polymer was characterized by IR spectroscopy, elemental
analyses, thermogravimetric analysis, single crystal and powder X-ray diffraction. The
[Co2(HCO2)4(H2O)4]n coordination polymer was crystallized in the monoclinic space group
P21/c. The structure of cobalt(II) coordination polymer is a distorted octahedral, in which
metal ions are connected by formate ligands and water molecules. The 2D structure of
coordination polymer is further connected to formate ligands, leading to a 3D structure.
1. Introduction adsorption, fluorescent sensing, magnetism

Recently, dicarboxylic acid has been and catalysis applications.14
widely used in the industry. Dicarboxylic This paper aims to synthesize
acids are used in the preparation of [Co2(HCO2)4(H2O)4]n ,and to characterize
copolymers such as polyamides and the structures of the mentioned coordination
polyesters.1-4 Many previous studies focus polymer.
on metal dicarboxylic acid, which adducts
mainly aromatic dicarboxylic acids; for 2. Materials and Methods
example, phthalic acid, isophthalic acid, 2.1 General
terephthalic acid, diphenic acid, biphenyl- All chemicals and solvents in this
2,2-dicarboxylic acid and naphthalenedi- work are commercial grade material and
carboxylic acid.5-8 These ligands are were used without further purification. FT-
oxidized meta-xylene by chromic acid, or by IR spectra were recorded on Perkin-Elmer
fusing potassium meta-sulphobenzoate, or infrared spectrophotometer (spectrum GX)
meta-brombenzoate with potassium using KBr pellets (range 4,000-400 cm-1).
formate.9,10 However, formate anion (HCO2-) Elemental analyses were studied by
is a simple carboxylate, produced from weighing 5-10 mg sample in a tin capsule.
hydrolysis of N,N-dimethylformamide.11 After folding the capsule (looking rather like
As for the spacer, the crystal-to- wrapped tin foil) the sample is placed in the
amorphous transformation of metal organic auto sampler. The powder X-ray diffraction
framework (MOFs), supramolecular (PXRD) data were collected on a Bruker D8
framework and coordination polymer are Quest XRD diffractometer using
interested with a potential to apply as monochromatic Cu Kα radiation in the 2θ
adsorbent material for moisture and organic range of 5-60o, with a 0.02o step scan 0.1 s
vapor.12 A novel coordination polymers can per step. Thermogravimetric analyses were
be designed and synthesized as by working performed using a 2050 thermogravimetric
on the chemical identity.13 Each analyzer (TGA Instruments) with a heating
coordination polymers can be used as gas
rate of 10 oC/min, and purge gas was adding Co(NO3)2.4H2O, isophthalic acid and
nitrogen 2-aminopyrimidine in the mixture of
2.2 Synthesis of compound methanol and n,n-dimethylformamide
Co(NO3)2.4H2O (1 mmol) was solution. The resulting mixture gave the new
dissolved in mixed solvent H2O:DMF (3:3 crystal with four formate and four water
mL). Then the solution of isophthalic acid (1 molecules in the coordination polymers. The
mmol) was added into the first solution. formates came from hydrolysis of n,n-
After that the solution of 2-aminopyrimidine dimethylformamide.11 The structure of
(1 mmol) was added into the mixture. The coordination polymer is further connected to
mixture was refluxed at 100 oC for 24 h. formate ligands, leading to a 3D structure.
After cooling down to room temperature for Infrared spectroscopy is a technique
two days, pink crystals of complex were used to study the functional groups of
collected by filtration followed by washing coordination polymer. The IR spectra were
with cold methanol and dried. (51.16% observed include the stretching of formate
yield) Anal. Calc. for C2H6CoO6: C, 12.98; anion at 1579.08 cm-1,11 O-H stretching of
H, 3.27 Found: C, 12.99; H, 3.29%. IR H2O at 3210.08 cm-1, 3300.04 cm-1, C-O
(KBr, cm-1): 2896.35 v(-CH), 3300.04- stretching of formate around 1356.67,
3210.08 v(H-O), 1377.44 v(C-O), 1579.08 1377.44 and 1397.25 cm-1. The M-O
cm-1 v(C=O), 572.03 v(M-O). stretching around 572.03 cm-1. These results
2.3 X-ray crystallographic analysis corresponded to IR spectrum of dehydration,
X-ray diffraction data were collected
which are identical with spectrum of
on a Bruker D8 QUEST CMOS
diffractometer equipped with graphite- rehydration as shown in Figure 5.
monochromatic Mo-Kα radiation (λ = 3.1 Description of structures
0.71073 Å) at Faculty of Science and Single-crystal X-ray diffraction
Technology, Thammasat University, shows the crystallographic data in Table 1.
operating at 296 ± 2 K. Empirical absorption The coordination polymer crystallized in the
corrections were applied to all data using monoclinic system, space group P21/c. The
SADABS.9. The structures were solved by
coordination geometry of cobalt(II)
SHELXT-201510 and refined by full-matrix
least-squares based on F2 using the coordination polymer is a distorted
SHELXL-9711 with Olex2 1.2 as a graphics octahedral, in which Co(II) ions are
interface. Anisotropic displacement coordinated to formate ligands and water
parameters were refined for all non- molecules as shown Figure 1. The molecules
hydrogen atoms. Hydrogen atoms were are further linked with other formate
added theoretically and were riding on their ligands, leading to a 3D structure (Figure 2).
parent atoms.15-17
The selected bond length and angles are
3. Results and Discussion given in Table 1.
The Co(II) coordination polymer was The Selected hydrogen bonding
synthesized by direct method. The new intermolecular interactions in coordination
coordination polymer was started with polymer are shown in Table 3.
Table 1. Selected bond length and angles of coordination polymer
Bond Length/ Å Angle/o
Co1-O1 2.0687 (18) O1-Co1-O1i 180.00 O4-Co2-O4iv 180.00
Co1-O1i 2.0687 (18) O1i-Co1-O2ii 89.48 (7) O5iv-Co2-O4 90.48 (10)
Co1-O2ii 2.0934 (17) O1-Co1-O2ii 90.52 (7) O5-Co2-O4 89.52 (9)
Co1-O2 iii
2.0934 (17) i
O1 -Co1-O2 iii
90.52 (7) iv
O5 -Co2-O4 iv
89.52 (9)
Co1-O3 2.1374 (18) O1-Co1-O2 iii
89.48 (7) O5-Co2-O4 iv
90.48 (10)
Co1-O3i 2.1374 (18) O1i-Co1-O3 87.25 97) O5-Co2-O5iv 180.00
Co2-O4iv 2.1432 (19) O1-Co1-O3 92.75 (7) O5-Co2-O6iv 90.91 (10)
Co2-O4 2.1432 (19) O1-Co1-O3 i
87.25 (7) iv
O5 -Co2-O6 iv
89.09 (10)
Co2-O5 2.043 (2) i
O1 -Co1-O3 i
92.75 (7) iv
O5 -Co2-O6 90.91 (10)
Co2-O5iv 2.043 (2) O2iii-Co1-O2ii 180.00 O5-Co2-O6 89.09 (10)
Co2-O6 2.118 (2) O2iii-Co1-O3ii 93.23 (7) O6iv-Co2-O4 88.61 (8)
Co2-O6 iv
2.118 (2) ii
O2 -Co1-O3 93.23 (7) iv
O6 -Co2-O4 iv
91.39 (8)
iii
O2 -Co1-O3 86.77 (7) O6-Co2-O4 91.39 (8)
O2ii-Co1-O3i 86.77 (7) O6-Co2-O4iv 88.61 (8)
O3-Co1-O3i 180.00 O6iv-Co2-O6 180.00
Symmetry codes: (i) -x, -y+1, -z+1; (ii) -x, y-1/2, -z+1/2; (iii) x, -y+3/2, z+1/2; (iv) -x+1, -y, -z+1;
(v) -x, y+1/2, -z+1/2
Table 2. Crystallographic data for

coordination polymer
Compound [Co2(HCO2)4(H2O)4]n
Empirical formula C2H6CoO6
Formula 185.00
weight(g/mol)
Temperature (K) 296.15
Crystal system monoclinic
Space group P21/c
a (Å) 8.6820(9)
b (Å) 7.1623(8)
Figure 1. Structure of the coordination
c (Å) 9.2779(11)
polymer, showing atom-labeling; hydrogen
α( ) 90
bonds are shown as red dotted lines
β( ) 90.407(4)
γ( ) 90
V (Å3) 572.23(11)
Z 4
Dcalc (gcm-3) 2.1472
µ (Mo K ) (mm-1) 2.958
Reflections collected 14312
Independent 1744
reflections (Rint) 0.0676
R[I 2 (I)] 0.0387
wR2 (F2) 0.1011 Figure 2. The three-dimensional crystal
packing model
Table 3. Selected hydrogen bonding temperature range of 143-181 oC (found,
intermolecular interactions in coordination 24.51%). The second step indicated of the
polymer (Å) framework weight loss in the temperature
D ̶ H‧‧‧A
D̶
H‧‧‧A D‧‧‧A D ̶ H‧‧‧A between 281 and 305 oC (found, 45.72%).
H
Then, the structure rapidly decomposes to
0.70
O6 ̶ H6A‧‧‧O3iv 2.104 2.755 156.403 metal oxide (Figure 4).
(4)
0.70
O6iv ̶ H6Biv‧‧‧O1 2.088 2.790 178.718
(4)
0.79
O5 ̶ H5A‧‧‧O2ii 1.990 2.778 172.735
(5)
0.79
O5iv ̶ H5Biv‧‧‧O4 1.960 2.744 174.352
(5)
Symmetry codes: (ii) -x, y-1/2, -z+1/2; (iv) -x+1, -y,
-z+1
3.2 Powder X-ray diffraction

The powder X-ray diffraction is an Figure 4. The TGA curves of
analytical technique for phase identification [Co2(HCO2)4(H2O)4]n
of a crystalline material. The PXRD patterns
of [Co2(HCO2)4(H2O)4]n coordination 3.4 Structural phase transformation by
polymer shows the simulated PXRD pattern
thermal de/rehydration process
for the single crystals of material which
obtained from single crystal X-ray The coordination polymer features
diffraction, and also match with patterns coordinated water molecules in the structure.
obtained for polycrystalline. The results The stable dehydration was confirmed by
show that the simulated PXRD pattern has TGA profiles.18 Dehydration and
the same pattern with the PXRD pattern of rehydration process could occur from losing
polycrystalline. That has the phase of water molecules of samples and adsorption
crystalline (Figure 3). water vapor at room temperature. The
crystalline samples were heated at 140 oC in
the oven for 1 h, then samples lose their
crystallinity and change the color from pink
to purple. After that, the noncrystalline
solids were placed in air at room
temperature for 1 hours, and the color of
solids returned to the original samples as
shown in Figure 6. The coordination
polymer reveals water-induced reversible
crystal-to-amorphous transformations with
Figure 3. The PXRD patterns of chromophism confirmed by spectroscopic
[Co2(HCO2)4(H2O)4]n, simulated (black techniques.
line), and measured (blue line) pattern
3.3 Thermogravimetric analysis

Thermal gravimetric analyses (TGA)
of [Co2(HCO2)4(H2O)4]n was recorded for
the polycrystalline samples in the
temperature range of 25-800 C. The TGA Figure 5. The infrared spectrum of
curves showed two declined steps. The first [Co2(HCO2)4(H2O)4]n, the as-synthesized
step demonstrated the release of coordinated (blue line), and the rehydrated form (red
water molecules weight loss in the line)
Figure 6. Changes in color during de- and rehydration processes in bulk samples
4. Conclusion 4. Organic Chemistry, Schmidt, J. Oliver

In summary, new coordination and Boyd: London, 1955, 159 (2), 283–
polymer constructed by formate ligands and 284.
water molecules, have been successfully 5. Farmer, E. E. Plant Mol. Biol. 1994, 26
synthesized and characterized by IR (5), 1423–1437.
spectroscopy, elemental analyses, 6. Wiwanitkit, V. S. S.; Suwansaksri, J.
thermogravimetric analysis, single crystal Toxicol. Pathol. 2007, 35 (2), 268–269.
and powder X-ray diffraction. The structure 7. Weaver, V. M.; Davoli, C. T.; Heller P.
of coordination polymer shows a distorted J. Environ. Health Perspect. 1996, 104
octahedral, in which metal ions are (3), 318–323.
connected by formate ligands and water 8. Sati, S. C.; Sati, N.; Sati, O. P.
molecules. The molecules are connected by Pharmacogn. Rev. 2011, 5 (10), 174–
formate ligands, leading to a 3D structure. 183.
The crystal-to-amorphous transformation is 9. Jang, S. P.; Poong, J. I.; Kim, S. H.;
revealed the color of solids returned to the Lee, T. G.; Noh, J. Y.; Kim, C.; Kim,
original samples between dehydration and Y.; Kim, S. J. Polyhedron 2012, 33,
rehydration processes. 194-202.
10. Hwang, I. H.; Jo, Y. D.; Kim, H.; Kim,
Acknowledgements K. B.; Jung, K. D.; Kim, C.; Kim, Y.;
The financial and all experiments Kim, S. J. Inorg. Chim. Acta 2013, 402,
equipment were supported by Department of 39-45.
Chemistry, and Physics, Faculty of Science 11. Wang, Y.; Cao, R.; Bi, W.; Li, X.;
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Central Scientific Instrument Center (CSIC) Mesoporous Mater. 2006, 91, 215-220.
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Thammasat University. J.; Chainok, K.; Pakawatchai, C.;
Youngmea, S. Inorg. Chem. Commun.
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Synthesis, crystal structure and alcohol sensing properties of a cobalt(II)
containing by benzoylacetonate and imidazole ligands
Pornsan Lueangsriprech1, Supakorn Boonyuen1*, Kittipong Chainok2,
Natthakorn Phadungsak1,2, Sumana Kladsomboon3
1
2
Materials Innovation and Technology, Department of Physics, Faculty of Science and Technology,
3
Center for Research and Innovation, Faculty of Medical Technology, Mahidol University,
Abstract:
The cobalt(II) complex of benzoylacetonate (bzac) and imidazole (Im),
[Co(bzac)2(Im)2]·2MeOH was successfully synthesized and characterized by various
spectroscopic techniques. The title complex crystallizes in the monoclinic system with space
group P21/c. The Co(II) center adapts a distorted octahedral geometry with four oxygen
atoms from two different bzac ligands and two nitrogen atoms from two different Im ligands.
In the crystal, molecules are linked into a one-dimensional chain via bzac-methanol O-H···O
and Im-methanol N-H···O hydrogen bonds. The alcohol sensing property was detected by
electronic nose (E-nose) from principle of optical changes. The result from principal
component analysis (PCA) showed a good selectivity and reproducibility.
1. Introduction were analytical grade from RCI Labscan

β-Diketones complexes are Thailand. Elemental analysis was carried out
considered as evolution materials to a novel using a Perkin-Elmer analyzer (2400). FT-
materials in catalysis, gas storage, functional IR spectra were recorded on Perkin-Elmer
materials, magnetism and sensors.1-4 The infrared spectrophotometer (spectrum GX)
field of mixed-ligands chemistry with metal using KBr pellets. UV-Vis absorption
is developing rapidly because of its physical measurements were carried out on a
and chemical properties depending on metal Shimadzu UV-spectrometer (UV-1700).
center, ligands, structure, bonds and
2.2 Synthesis of [Co(bzac)2(Im)2].2MeOH
interactions.5 The [Co(bzac)2(H2O)2] (2 mmol),
Current studies show the design and dissolved in 10 mL of hot methanol, was
synthesis of [Co(bzac)2(Im)2] (bzac = added dropwise to a methanolic solution (20
benzoylacetonate, Im = Imidazole). The mL) of the ligand (Imidazole 4 mmol). The
structure determination was studied by solution was refluxed for 1 h with constant
single crystal X-ray spectroscopy. The stirring. After cooling to room temperature
characterizations were analyzed from for four days, orange block crystals of
elemental analysis, FT-IR and UV-Vis
[Co(bzac)2(Im)2].2MeOH were collected by
spectrophotometry. Furthermore, the filtration, washed with methanol and then
complex was measured the gas sensing dried. (72.52% yield) Anal. Calc. for
application by electronic nose (E-nose). C28H34N4O6Co: C, 57.83; H, 5.89; N, 9.63
Found: C, 57.80; H, 5.88; N, 9.64%. IR
2. Materials and Methods (KBr, cm-1): 3133.01 (-NH str.), 2856.28 (-
2.1 General CH str.), 1593.22 (C=O str.), 1069.79 (C-O
All starting materials were obtained str.), 534.80 (M-O str.), 442.55 (M-N str.).
commercially from Aldrich. All solvents
2.3 X-ray crystallographic analysis transition and π-π* transition, respectively.
X-ray diffraction data were In both complexes, the d-d transitions were
collected on a Bruker D8 QUEST CMOS not observed presumably due to low
diffractometer equipped with graphite- intensity.12
monochromatic Mo-Kα radiation (λ =
0.71073 Å) at Faculty of Science and Table 1 Physical, IR and UV-Vis
Technology, Thammasat University, spectroscopic data for Co(II) complex
operating at 296 ± 2 K. Empirical absorption λmax
IR(cm-1)
corrections were applied to all data using (nm),
Complex Color
(ԑ) in
SADABS.9 The structures were solved by N-H C=O C-O MeOH
SHELXT 2015 and refined by full matrix 316
least squares based on F2 using the 1 Orange 3133 1593 1069
(16730)
SHELXL 97 with Olex2 as a graphics
interface.10,11 3.2 Structural description of Co(II) complex
2.4 Gas sensing application The [Co(bzac)2(Im)2]∙2MeOH com-
The [Co(bzac)2(Im)2]∙2MeOH was plex crystallize in the monoclinic crystal
dissolved in dichloromethane solution at the system, space group P21/c with
concentration 5 mg/mL and then coated onto crystallographic data in Table 3. The
a glass slide. The complex was tested by coordination environment of the cobalt ion
dynamic measurement the electronic nose is shown in Figure 1. The cobalt ion is
with the flow of different 100% alcohol coordinated to four oxygen atoms from two
vapors such as methanol, ethanol and bzac ligands, two nitrogen atoms from two
isopropanol at 25 °C. Alcohol vapor was Im ligands. The cobalt atoms exhibit a
purged in a chamber by switching between distorted octahedral, as seen in Table 4. The
the alcohol samples and the reference gas molecular structure of this complex is
(nitrogen).The principal component analysis stabilized by intermolecular H-bonding from
(PCA) method analyzed the collected data H atom of imidazole to O atom of methanol
from an electronic nose. and H atom of imidazole to O atom of
benzoylacetonate, leading to one-
3. Results and Discussion dimensional chains as shown in Figure 2 and
3.1 Synthesis, FT-IR and UV-Vis spectro- Table 2. The resulting in the formation of
scopic studies of Co(II) complex 1D supramolecular structure shows the
The addition of imidazole solution in hydrogen bonds O(4)-H(4)⋯O(2) and N(4)-
methanol to a [Co (bzac)2(H2O)2] H(4)⋯O(4) are present as the intra-molecule
methanolic solution yielded the expected hydrogen bond.
complex in moderate to excellent yield
(72.52%) as orange solids. Table 2. Intra/intermolecular interactions
The infrared spectral band (Table 1) hydrogen bonds in [Co(bzac)2(Im)2] 2MeOH
revealed (C-O) stretching between 1050- Donor HAcceptor O(4)-H(4) N(4)-H(4)
1150 cm-1, confirmed the coordination of β– O(2) O(4)
diketonate in complexes. In addition, D – H (Å) 0.81(9) 0.85(2)
stretching for the (N-H) of the imidazole HA (Å) 2.02(7) 1.96(2)
ligands were observed at 3133.01 cm-1.
DA (Å) 2.82(18) 2.81(2)
Strong bands at 534.80 cm-1 can be assigned
to (Co-O) vibrations. All results are in an D – HA() 169.0(2) 169.2(2)
agreement with previous result.12 The UV-
visible spectra of [Co(bzac)2(Im)2] complex
were recorded in MeOH. The absorption
bands at 316-323 nm and 244 nm
correspond to n-π* charge transfer (CT)
Table 3. Crystallographic data for
[Co(bzac)2(Im)2]∙2MeOH
Complex [Co(bzac)2(Im)2]∙2MeOH
Empirical formula CoC28H34O6N4
Formula weight(g/mol) 581.52
Temperature (K) 296.15
Crystal system Monoclinic
Space group P21/c
a (Å) 9.6420(3)
b (Å) 8.6450(2)
c (Å) 17.6980(4)
α( ) 90
β( ) 98.87(2)
Figure 1. A molecular structure of γ( ) 90
[Co(bzac)2(Im)2] 2MeOH V (Å3) 1458.6(5)
Z 2
Dcalc (gcm-3) 1.3233
R1 , wR2 (all data) 0.0450,0.0829
Figure 2. View of a one dimensional chain formed by classical O H···O and N H···O
hydrogen bonds in [Co(bzac)2(Im)2] 2MeOH
Figure 3. The PCA two dimensional plot of the optical response of the [Co(bzac)2(Im)2] 2MeOH
film to alcohol; a) methanol; isopropanol and b) ethanol; isopropanol
Table 4. Selected bond length and angles of [Co(bzac)2(Im)2]∙2MeOH
Co-O(1) 2.0820(4) Co-O(4) 2.0600(4)

Co-O(2) 2.0820(4) Co-N(1) 2.1440(7)
Co-O(3) 2.0600(4) Co-N(4) 2.1440(7)
O2-Co1-O1 88.357(4) N4-Co1-O3 90.410(5)

O3-Co1-O1 180.000(0) N4-Co1-O4 89.848(5)
O3-Co1-O2 91.643(4) N4-Co1-N1 180.000(0)
O4-Co1-O1 91.643(4) C2-O1-Co1 126.029
O4-Co1-O2 180.000(1) C4-O2-Co1 126.902
O4-Co1-O3 88.357(4) C12-O3-Co1 126.209
N1-Co1-O1 90.410(5) C14-O4-Co1 126.902
N1-Co1-O2 89.848(5) C21-N1-Co1 129.525
N1-Co1-O3 89.500(5) C24-N1-Co1 128.323
N1-Co1-O4 90.152(5) C25-N4-Co1 129.525
N4-Co1-O1 89.590(5) C28-N4-Co1 128.323
N4-Co1-O2 90.152(5)
3.3 Gas sensing application mononuclear cobalt(II) species and

PCA method was used to compare represented a very effective alcohol sensor.
the efficiency of three gas sensor or alcohol
classification as shown in Figure 3. The Acknowledgements
[Co(bzac)2(Im)2] 2MeOH complex showed a The financial and all experiments
good data set of separation of methanol and equipment were supported by Department of
ethanol from isopropanol, when the Chemistry, Physics and Biotechnology,
combination of PC1 and PC2 are over 80%. Faculty of Science and Technology,
The separation between methanol and Thammasat University. Central Scientific
isopropanol offered the excellent PC 95.1% Instrument Center (CSIC) of Faculty of
and explicated to alcohol sensor. The Science and Technology, Thammasat
bonding between Co(II) complex and University.
alcohol molecule formed by the interaction
of metal ion with an electron donor of References
alcohol molecule. Different alcohols made 1. Almeida, J. d. C.; Paixão, D. A.;
different changes of the absorption spectra Marzano, I. M.; Ellena, J.; Pivatto, M.;
of the complex. Lopes, N. P.; Ferreira, A. M. D. C.;
Pereira-Maia, E. C.; Guilardi, S.; Guerra,
4. Conclusion W. Polyhedron 2015, 89, 1–8.
The new coordination complex of 2. Adhikary, C.; Koner, S. Coord. Chem.
[Co(bzac)2(Im)2] 2MeOH was synthesized Rev. 2010, 254 (23), 2933–2958.
using cobalt(II) acetate tetrahydrate complex 3. Yoshida, T.; Suzuki, T.; Kanamori, K.;
precursor, benzoylacetonate and imidazole Kaizaki, S. Inorg. Chem. 1999, 38 (6),
as co-ligand. The crystal structure of 1059-1068.
complex showed a distorted octahedral 4. Ghosh, A. K.; Mitra, M.; Fathima, A.;
geometry, which determined by X-ray Yadav, H.; Roy Choudhury, A.; Nair, B.
diffraction. The complex was new
U.; Ghosh, R. Polyhedron 2016, 107, 1– 10. Sheldrick, G. M. Acta Cryst. A 2015, 71,
8. 3–8.
5. Harding, P.; Harding, D. J.; Phonsri, W.; 11. Dolomanov, O. V.; Bourhis, L. J.;
Saithong, S.; Phetmung, H., Inorg. Gildea, R. J.; Howard, J. A. K.;
Chim. Acta 2009, 362, 78-82. Puschmann, H. J. Appl. Cryst. 2009, 42,
6. El-Sonbati, A.; Diab, M. A.; Belal, A. 339–341.
A.; Morgan, S. M. Spectrochim. Acta 12. Harding P; Harding D. J.; Sirirak, J;
Part A 2012, 99, 353-360. Adams, H. Transition Met. Chem. 2012,
7. Sheikha, J.; Junejab, H.; Ingleb, V.; 37, 639-644.
Alib, P.; Haddac, T. B. J. Saudi. Chem. 13. Almeida, J. D. C.; Paixão, D. A;
Soc. 2013, 17, 269–276. Marzano, I. M; Ellena, J; Pivatto, M;
8. Ghosha, A. K.; Mitraa, M.; Fathimab, Lopes, N. P; Ferreira, A. M. D. C;
A.; Yadavc, H.; Choudhuryc, A. R.; Pereira-Maia, E. C; Guilardi, S; Guerra,
Nairb, B. U.; Ghosha, R. Polyhedron W. Polyhedron 2015, 89, 1-8.
2016, 107, 1–8. 14. Özbek, M.; Yüksek, M.; Optik, H. N.
9. Bruker (2014). APEX2, SADABS and Inorg. Chimica Acta 2016, 127, 7710–
SAINT. Bruker AXS Inc., Madison, 7716.
Wisconsin, USA.
Ag2O-ZnO photocatalyst supported on hydroxyapatite from bovine bones
for photo-reforming of glycerol
Damrong Adam1*, Netnapid Ongsuwan2, Saowapa Chotisuwan1
1
Department of Science, Faculty of Science and Technology, Prince of Songkla University,
Rusamilae, Pattani 94000, Thailand
2
Department of Food Science and Nutrition, Faculty of Science and Technology,
Prince of Songkla University, Rusamilae, Pattani 94000, Thailand
*E-mail: damrong3787@gmail.com
Abstract:
Good organic adsorption materials such as hydroxyapatite (HAp), has been
synthesized from bovine bones and used as supporting material for photocatalyst. In this
work, ZnO photocatalyst and Ag2O semiconducting material were chosen to improve
photocatalytic reaction. Pure HAp from bovine bones was prepared by calcination at high
temperature of 900 oC. Ag2O-ZnO/HAp photocatalyst were prepared by hydrothermal
method by using 3.0, 5.0, or 7.0wt% ZnO and fixed amount of Ag2O at 1.0wt%. The results
showed that HAp resembles white-yellow powder, porous and high roughness surface. X-ray
diffraction pattern showed 2θ positions at 31.8º, 32.2º and 32.9º, corresponding to primary
peaks of the HAp crystals. FT-IR spectra showed absorption peak at 3571 cm-1 represented
hydroxyl groups and absorption peaks at 1045, 961, and 563 cm-1 corresponding to phosphate
groups. The chemical analysis of HAp showed that the molar ratio of Ca:P was 1.56. The
catalytic activity testing for glycerol photo-reforming was performed in 5.0%vol glycerol
under N2 atmosphere with 50 mg of photocatalyst. The highest percentage of glycerol
conversion, 36%, was obtained by using Ag2O-ZnO/HAp photocatalyst containing 5.0wt%
ZnO and 1.0wt% Ag2O. The confirmation of hydrogen gas from glycerol photo-reforming
reaction was determined by using GC-TCD chromatography.
1. Introduction Among all photocatalysts, ZnO has

Nowadays, the most effective been comprehensively studied due to its
advanced oxidation processes using non-toxicity and good photocatalytic
semiconductor metal oxide photocatalysts, properties.5 However, ZnO has wide band
have been studied extensively due to gap energy (3.37 eV), which is able to
environmental friendly and green process absorb photons only in the UV spectrum and
for several applications.1 One of interesting ZnO has rapid recombination rate of
application is the production of hydrogen photogenerated electrons (e-) and holes
from chemicals and photo-chemical trans- (h+).5,6 It is possible to use visible light to
formations.2 The photocatalytic hydrogen activate photo-activity of ZnO and can be
production is a less energy-consuming inhibited the re-combination of
reaction because it can be performed under photogenerated electron/hole pairs, by
mild conditions and different processes modifying the surface of ZnO with transition
using fossil fuels and biomass-derived and noble metal oxide nano-particles such as
materials including alcohols, sugars, silver oxide (Ag2O) and nickel oxide (NiO).7
glycerol, glucose and so on as hydrogen Ag is attracted much attention due to its low
sources.3 Using glycerol is interesting for cost, non-toxicity, high electrical and
effective materials recycling, which it is a thermal conductivity.7,8 However, there is a
byproduct from transesterification reaction recovery problem of Ag containing ZnO
process to produce biodiesel.4 photocatalyst particles and also being photo-
corrosive in aqueous media.9 These
© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) MN1
disadvantage can be eliminated by using and followed by dropwise of 10 M NaOH
support materials which is rigid substrates solution to adjust pH to 12–13 and then
e.g. plastic, glass and ceramic biomaterials white precipitate was observed. The mixture
such as hydroxyapatite or HAp was transferred to Teflon-lined stainless
(Ca10(PO4)6(OH)2).10 HAp has high capacity steel autoclave, which was sealed and
for metal ions retention and good organic maintained at 160 °C for 1 h. The white
adsorption properties.11 solid was separated from the solution by
The aim of this research was to centrifugation at 7500 rpm for 10 min and
prepare coupled Ag2O-ZnO photocatalysts washed the solid product with deionized
sup-ported on HAp by hydrothermal method water and ethanol. The final products were
and test their catalytic performance in photo- dried at 80 °C and calcined in muffle
catalytic hydrogen production from a furnace at 400 °C for 3 h.
mixture of glycerol solution under LED 2.4 Characterization of photocatalysts
daylight irradiation conditions. The HAp and photocatalysts were
characterized by various techniques. The
2. Materials and Methods powder X-ray diffraction (XRD) (Model
2.1 Materials X’Pert MPD, Phillips, the Netherland) was
Silver nitrate (AgNO3, 99%), zinc equipped with Cu Kα as X-ray source. The
acetate dihydrate (Zn(CH3COO)2•2H2O, diffraction patterns were collected in the
99.5%), sodium hydroxide (NaOH), acetic scanning range of 2θ = 5°-90°. Fourier
acid (CH3COOH), ethanol and acetone were transform infrared (FTIR) spectra of
AR grades and were used as received. prepared photocatalysts were recorded by a
2.2 Hydroxyapatite preparation FTIR Spectrometer (Bruker Tensor 27) in
Bovine femur bones were obtained the range 400-4000 cm-1 at room
from local butcher soups which were already temperature. The specific surface areas were
boiled for 6-7 h. The bones were washed measured by nitrogen adsorption-desorption
and cleaned well with deionized water, then isotherms at 77 K according to the
immersed in acetone for 2 h, washed with Brunauer-Emmett-Teller analysis (BET,
water several time afterwards and finally ASAP 2460). The morphology was studied
dried in hot air oven. After that, the dried by Scanning Electron Micro-scope (SEM
bovine bones were crushed into small Quanta400), equipped with Energy
pieces, and calcined in muffle furnace at dispersive X-ray (EDX) analysis to provide
temperature 900°C for 3 h, with heating rate information relating to elemental com-
10°C/min. The calcined bovine bones were position of the samples.
kept in desiccator. 2.5 Photocatalytic activity
2.3 Photocatalysts synthesis The photocatalytic hydrogen
The synthesis method involves a production experiments were carried out in
modified procedure reported earlier, the an air-tight three-necks round bottom flask
Ag2O-ZnO/HAp photocatalysts were under atmospheric pressure, at room
synthesized by hydrothermal method temperature and daylight irradiation using
(Bechambi et al. 2015).12 First, zinc acetate 20W LED lamp. First, the dried powder
dihydrate was dissolved by deionized water photocatalyst (50 mg) was suspended in 50
and stirred for 30 min to obtain different mL of glycerol-water mixture solution
solution containing zinc content at 3.0, 5.0 (5vol%). The reactor was degassed and
and 7.0wt%. Then acetic acid was added flowed with pure nitrogen gas (99.999%) to
into zinc acetate solution to avoid the ensure completely removal of oxygen from
formation of the hydroxides, and silver the reactor. Before irradiation process, the
nitrate (1.0wt% of Ag2O) was mixed with suspensions were shaken in the dark for 30
the above mixture. After that, HAp powder min to reach glycerol adsorption/desorption
(96, 94 and 92wt% respectively) was added equilibrium on surface of photocatalyst.
Then, the reaction mixture was stirred and
irradiated with a 20W LED daylight lamp on Ag2O-ZnO(7%)/HAp
a magnetic stirrer for 4 h. Then, the

conversion of glycerol was determined by
Transmittance (a.u.)
Ag2O-ZnO(5%)/HAp
UV-Visible spectrophotometer (Biochrom

Libra S11) and gaseous products were Ag2O-ZnO(3%)/HAp
analyzed with a gas chromatograph HAp

(Shimadzu GC-14A, TCD detector, using Ar
as carrier gas).
470
603
Bone
961
1458
430
3571
1413
563
1045
3. Results and Discussion 4000 3500 3000 2500 2000 1500 1000 500
3.1 Photocatalyst characterization studies Wavenumber (cm-1)
The crystalline structure, Figure 2. The FTIR spectra of HAp and
morphology and chemical composition of Ag2O-ZnO/HAp photocatalysts
as-synthesized HAp and Ag2O-ZnO/ HAp
samples were analyzed in order to ensure the In addition, the crystallite size of
photo-catalytic reforming of the proposed HAp can be calculated by using Scherrer's
precursors. equation. The average HAp crystal sizes
X-ray diffraction patterns of the HAp along with the (002) plane of the XRD
and Ag2O-ZnO/HAp samples were shown in results 47 nm, while all of Ag2O-ZnO/HAp
Figure 1. All the XRD patterns were closely photocatalysts showed similar crystallite
matched to stoichiometric HAp (corres- size which were smaller than HAp powder
ponding to JCPDS 09-0432) suggesting that (Table 2).
the thermal process did not change the The raw bovine bones and Ag2O-
structure of natural HAp. The XRD peaks of ZnO/HAp photocatalysts showed FTIR
HAp were observed at 25.9° (002), 31.8° absorption band (Figure 2) at 3571 cm-1
(211), 32.2° (112) and 32.9° (300). Typical represented hydroxyl groups, adsorption
XRD patterns of ZnO were also assigned at band at 1413 and 1458 cm-1 was carbonate
2θ values of 34.4° (002) and 36.2° (101), groups, which from the adsorption of carbon
which was agreement with JCPDS No. 36- dioxide (CO2) on the Ag2O-ZnO/ HAp. The
1451 with hexagonal wurtzite structure of characteristic bands at 470, 563, 603, 961
ZnO.13 However, the XRD pattern of doped and 1045 cm-1 were corresponding to
Ag2O on photocatalysts could not be clearly phosphate groups. The band at 430 cm-1 was
observed in this work (2θ=44.2°), due to assigned of ZnO. However, the band of Zn-
small amount of Ag content and may be O-P bond for ZnO-HAp could not be clearly
good dispersion of Ag2O on catalyst surface. observed except for Ag2O-ZnO (7.0wt%)/
HAp indicating the interaction between HAp
(211)
HAp
(112)(300)
and ZnO.14
ZnO
(002)
(101)
(002)
Ag2O-ZnO(7%)/HAp
The chemical analysis of HAp
analysed by SEM-EDX showed the weight
Intensity (a.u.)
Ag2O-ZnO(5%)/HAp content of calcium, phosphate and others in

Table 1. The HAp synthesized from natural
Ag2O-ZnO(3%)/HAp bovine bones has the molar ratio of Ca/P
1.56, slightly less than the Ca/P molar ratio
HAp of stoichiometric HAp (1.67). It was noted
that biological HAp are the nonstoichio-
10 20 30 40 50 60
2 Theta (Degree)
70 80 90
metric HAp structure with cation (Ca2+) and
anion (OH-) vacancies, due to the partial
Figure 1. XRD patterns of HAp and Ag2O-
replacement of some ion with Ca2+ ions
ZnO/HAp photocatalysts
a b
c d e
Figure 3. SEM images of (a) bones, (b) HAp, (c) Ag2O-ZnO (3wt%)/HAp, (d) Ag2O-ZnO
(5wt%)/HAp, (e) Ag2O-ZnO (7wt%)/HAp
Table 1. SEM-EDX quantitative results of for Ag2O-ZnO photocatalysts (Figure 3c-e)

HAp with different ZnO content (3.0, 5.0 and
Element line wt% (SD) 7.0wt%). It was observed that Ag2O-
Ca K 30.25 (3.60) ZnO(5.0wt%)/HAp was well dis-persed onto
PK 15.65 (0.76)
HAp surface, while Ag2O-ZnO (7.0wt%)/
Mg K 1.14 (0.06)
Na K 1.14 (0.12) HAp was nearly covered all HAp surface.
CK 5.82 (0.74) The specific surface area of the HAp
OK 46.12 (3.54) and Ag2O-ZnO/HAp samples was calculated
from N2 sorption measurements and
in the HAp lattice such as magnesium, application of the BET method shown in
sodium and non-metals (CO32-) which can Table 2. All specific surface area (SSA)
enhance the biological activities of HAp values of Ag2O-ZnO/HAp samples were
synthesized from organic source.15 higher than that of pure HAp indicating the
Figure 3a-e shows morphology of the good interaction of ZnO and HAp was
synthesized HAp and Ag2O-ZnO/HAp occurred during the synthesized process, thus
samples. All samples have particle size the agglomeration of HAp hardly to occur.
around 0.5-1.2 µm. The surface of bovine 3.2 Photo-activity studies
bones (Figure 3a) was dense and form larger The HAp, ZnO, and Ag2O/ZnO
particles than HAp, ZnO/HAp and Ag2O- photocatalysts supported on HAp with
ZnO/HAp samples (Figure 3b). The surface different ZnO content (3, 5 and 7wt%) were
of HAp powder shows porous and high used for photo-reforming of 5vol% glycerol
roughness surface because the organic solution under N2 atmosphere. The results
substances such as proteins and collagen were shown in Figure 4, demonstrating that
already decomposed from bovine bones.16 It the Ag2O-ZnO (5.0wt%)/HAp photocatalyst
can be clearly seen from Figure 3c-e that the gave higher percentage glycerol conversion
HAp particles were in the form of hexagonal than the other photocatalysts because of
shape. After using HAp as support material well dispersion of Ag2O and ZnO on HAp
Table 2. Structural properties of HAp and Ag2O-ZnO/HAp photocatalysts determined by N2
adsorption and XRD measurements
D (nm)a
Samples SSA (m2 g-1)
002 plane
HAp 1.1226 47.14
Ag2O-ZnO/HAp 3.0%b 2.7469 46.39
Ag2O-ZnO/HAp 5.0% b 2.6939 46.57
Ag2OZnO/HAp 7.0% b 2.2504 45.19
a
Calculated using Scherrer's equation (Dp=0.94λ/βCosθ) where Dp, λ, β, and θ are average crystalline size, X-ray
wavelength, line broadening (Peak half-width) in radians, and diffraction angle, respectively.
b
ZnO content (wt%).
glycerol.17 The mechanism pathways in

Scheme 1, show the conversion of glycerol
to other chemicals rather than hydrogen gas
and carbon dioxide, some organic acids can
be formed by dehydrogenation reactions
catalyzed by the metal, followed by
rearrangement reactions that take place in
solution or on the photocatalyst. These
organic acids lead to the formation of
alkanes from carbon atoms that are not
bonded to oxygen atoms.18 Therefore, the
product gasses produced during the photo-
reforming were analyzed to confirm the
hydrogen gas by using GC-TCD. The result
Figure 4. The performance of material type showed the GC peak of hydrogen and other
on the conversion (%) of glycerol for 4 h. peaks (may be methane, carbon dioxide, and
using 5vol% glycerol, 50 mg of materials at alkanes).
room temperature
4. Conclusion
surface. The Ag2O-ZnO (3.0wt%)/Hap con- The HAp and Ag2O/ZnO supported
tained low amount of ZnO, while the Ag2O- on HAp were successfully prepared by
ZnO (7.0wt%)/HAp particles were more calcination and hydrothermal method,
agglomerated on HAp surfaces and nearly respectively. These photocatalysts were
covered the surface. active for photocatalytic reforming reaction
of 5vol% glycerol solution under 20W LED
daylight at room temperature for 4 h. The
maximum reforming of glycerol was 36.6%
using Ag2O-ZnO (5.0wt%)/HAp photo-
catalyst.
Scheme 1. Reaction pathways for the Acknowledgements

generation of different products during the This work is a part of the project
conversion of glycerol granted by Research Fund of Prince of
Songkla University, Pattani campus
The highest percentage of glycerol (SAT600800S).
conversion in this work was 36.6% using
Ag2O-ZnO (5.0wt%)/HAp photocatalyst this References
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J. Photochem. Photobiol. A Chem. 2004, Mater. Lett. 2017, 205, 233-235.
162, 317-322. 15. Ofudje, E. A.; Rajendran, A.; Adeogun,
6. Di Paola, A.; Garcia-Lopez, E.; Marci, A. I.; Idowu, M. A.; Kareem, S.O.;
G.; Palmisano, L. J. Hazard. Mater. Pattanayak, D. K. J. Adv. Powder
2012, 211-212, 3-29. Technol. 2018, 29, 1-8.
7. Türkylmaz, S. S.; Güy, N.; Özacar, M. J. 16. Khoo, W.; Nor, F. M.; Ardhyananta, H.;
Photochem. Photobiol. A Chem. 2017, Kurniawan, D. J. Proc. Manuf. 2015, 2,
341, 39-50. 196-201.
8. Jaramillo-Páez, C.; Navio, J. A.; Hidalgo, 17. Strataki, N.; Bekiari, V.; Kondarides, D.
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R.L. J. Ind. Eng. Chem. Res. 2004, 43,
7683-7696.
A new solid state sensor using rhodamine derivative for copper ion
detection in aqueous solution
Nilobol Tungsombatvisit, Thitirat Inprasit, Penwisa Pisitsak*
Department of Materials and Textile Technology, Faculty of Science and Technology,
Thammasat University, Rangsit, Patumthani 12121, Thailand
*E-mail: penwisa@tu.ac.th
Abstract:
A novel colorimetric rhodamine-based sensor (Sensor-2) was synthesized from 2,5-
dihydroxybenzaldehyde rhodamine B hydrazine (DRBH) and ethylenediaminetetraacetic acid
dianhydride (EDTAD). FTIR analysis confirmed an esterification reaction between the
anhydride group of the EDTAD and the hydroxyl group of DRBH. In solution, Sensor-2
changed from colorless to pink when exposed to Cu(II) ions. To produce a solid-state sensor,
the anhydride groups were reacted with the hydroxyl groups in cellulose acetate, using N,N-
dimethylformamide as the reaction medium. The resulting material was cast into a sheet. This
was immersed in aqueous solutions containing five metal ions: Cu(II), Mn(II), Pb(II), Cd(II)
and Ni(II). When exposed to solutions containing 1-100 μM concentrations of Cu(II) ions,
the sensor visibly changed from colorless to pink. The color parameters were identified using
the CIELAB color space. Immersion in the other solutions produced negligible color change,
confirming the selectivity of the sensor.
1. Introduction molecules change in the presence of an ion.5

Industries such as dye The intrinsic sensitivity, portability and
manufacturing or electroplating release ionic capacity for rapid real-time monitoring
pollution to water resources. Inorganic makes this a useful tool for metal ion
effluent from these sources contains toxic detection.6 Zhihui et al. developed
metals such as Cu2+, Ni2+, Pb2+, Co2+, Cr3+, fluorescein hydrazide molecule as a
Zn2+, which may contaminate the food colorimetric sensor for mercury ions.7 Yong
chain, creating a potential health risk.1 et al. designed and synthesized a
Copper is a common transition metal colorimetric and fluorescent probe based on
element and one of the most abundant a resorufin molecule for hydrazine detection
critical trace element in the human body. It in rocket fuels. Detection was indicated by a
plays a fundamental role in a variety of color change from colorless to red.8
physiological processes.2 However, Rhodamine derivatives are of interest
exposure to high concentrations of copper as chemosensors for copper ions because of
ions is dangerous, as it can cause Wilson's their visible color change. They have been
disease, Menkes disease, Parkinson's used in solution and solid form.3 When used
disease, or Alzheimer's disease.3 in solution, the rhodamine powder can be
Techniques for detection of heavy used only once. In contrast, a solid state
metal ions include inductively coupled sensor is portable and can be used
plasma mass spectrometry, atomic repeatedly.9
absorption spectroscopy and electrochemical Ethylenediaminetetraacetic acid
methods. Although these techniques provide dianhydride (EDTAD) is a biodegradable
accurate measurement, they are expensive to and active agent containing two anhydride
use.4 A colorimetric chemosensor can be groups per molecule, each of which can act
easily prepared by designing molecules as a bridge between pairs of molecules
that change color in solution when their through an esterification reaction.10 Rathna
et al. prepared hydrogel from egg white
albumin (EWA) and then used EDTAD triethylamine as a catalyst, provided a novel
before and after chemical modification of its rhodamine derivative (Sensor-2), as shown
lysyl residues to incorporate the carboxylic in Figure 1.
group. The functional groups on the EWA O O
can adsorb Cu2+ ions.11

HO
OHC OH
N NH2 N N
OH
Cellulose acetate (CA) is an HO
abundant renewable biopolymer with N O N N O N
excellent properties12, being soluble in

-
Rhodamine B hydrazine Sensor 1
common solvents and easy to process. It O N

N
O
O
, NEt
3
contains hydroxyl groups that are capable of O

O
reacting with a wide range of functional O HO

O
OH
O
groups, e.g. carboxylic acid and anhydride. N
N
O N
N O
In this study, a novel colorimetric N O N

O
O
sensor based on a rhodamine derivative, Sensor 2
Sensor-2, was synthesized from 2,5-

dihydroxybenzaldehyde rhodamine B Figure1. Synthesis routes of Sensor-2
hydrazine (DRBH) and EDTAD. The sensor 2.3 Preparation of a solid sensor modified
was designed to contain an available with cellulose acetate
anhydride group which, due to its high Sensor-1 (0.01 mmol) and EDTAD
reactivity with other compounds, allows (10 mmol) were separately dissolved in 12.5
further chemical modification. In the ml of DMF. The two solutions were mixed
solution state, this sensor was found to be to obtain a clear solution. The mixture was
selective for Cu2+ ions. The incorporation of stirred for 3 h. CA (2 g) was dissolved in
Sensor-2 into CA produced a solid-state acetone (50 ml) and mixed with the Sensor-
sensor that could detect Cu2+ in water. The 1–EDTAD solution. Triethylamine (0.5 ml)
sensitivity and selectivity of the Sensor-2- was added to the mixture at room
treated CA was investigated using visual temperature. The reaction mixture became
detection and color measurement. increasingly viscous. The solvent volume
ratio was fixed at 1:2 (acetone:DMF). The
2. Materials and Methods mixture was stirred overnight, then cast into
2.1 Materials a sheet and dried at room temperature for 7
EDTAD (98%) and CA with a d. The obtained solid-state material was
number-averaged molecular weight of washed with ethanol three times. Finally, the
30,000 (Mn) were purchased from Sigma sample was cut into small pieces with
Aldrich. DBRH was prepared following the dimensions of 3x3x3 cm.
method of Milindanuch et al. (2016).13 2.4 Spectroscopic measurement
Acetone and N,N-dimethylformamide The FTIR spectra of Sensor-1,
(DMF) of analytical grade were purchased Sensor-2 and EDTAD in the range 500-4000
from Fisher Scientific. Triethylamine cm-1 were recorded on a Perkin Elmer
(99.5%) was obtained from LOBA Chemie. Spectrum GX. The color parameters were
Solutions of five metal ions were prepared measured using a spectrophotometer and
in ultrapure (type 1) water: CuCl2, MnSO4, expressed as CIE L*a*b*; L* (lightness), a*
Cd(Ac)2, NiSO4 and Pb(NO3)2. All (red to green), b* (yellow to blue). ΔE*
chemicals were used as received, without represents the total color difference between
further purification. the original color of the solid-state sensor
2.2 Synthesis of Sensor-2 and the color after exposure to metal ions.
A rhodamine B derivative, DBRH ΔE* was calculated as follows:
(Sensor-1), was synthesized by two-step
reaction, using the same method. Treatment ΔE* = [(ΔL*)2+(Δa*)2+(Δb*)2]1/2
of Sensor-1 with EDTAD, in the presence of
The solid-state sensor was immersed that Sensor-2 could be used to detect Cu2+
in solutions containing 100 µM of four ions.
metal ions: Mn2+, Cd2+, Ni2+ and Pb2+. To
evaluate the sensitivity of the sensor to Cu2+,
solutions with concentrations ranging from 1
µM to 100 µM were used.

The FTIR spectra of Sensor-1,
EDTAD and Sensor-2 are shown in Figure
2. Sensor-1 showed a characteristic peak Figure 3. Sensor-2 dissolved in a) water, b)
centered at 3396 cm-1, related to alcohol O- DMF and c) DMF in the presence of Cu2+
H stretching. EDTAD showed two bands at
1809 cm-1 and 1760 cm-1, associated with
C=O stretching of an anhydride moiety.
Sensor-2 showed a very broad band in the
region 3142 to 3645 cm-1, characteristic of
carboxylic acid–OH stretching. The band Figure 4. Color variation of the colorimetric
located at 1610 cm-1 was ascribed to C=O solid-state sensor before and after 1 h
stretching of the carboxylic acid group. This exposure to aqueous solutions of copper ions
implied that one of the hydroxyl groups of (1μM-100μM)
Sensor-1 was esterified with the anhydride Cu2+
group of EDTAD, giving rise to a carboxylic O
O
acid band in the spectrum of Sensor-2. The

N N
esterification between DBRH and EDTAD O
was confirmed by absorption at 1698 cm-1, O

N
corresponding to C=O stretching of ester.14

N O N O
N
O
O
HO
Figure 5. A proposed complex between

Sensor-2 and Cu2+
As the Sensor-2 was synthesized at a

1:1 mole ratio of DBRH to EDTAD, it was
assumed that one anhydride group would be
available to react with the CA. We therefore
prepared a solid-state colorimetric sensor by
incorporating Sensor-2 into CA. It was
found that the Sensor-2-treated CA also
changed from white to pink upon immersion
Figure 2. FTIR spectra of Sensor-1, Sensor- in an aqueous solution containing copper
2 and EDTAD ions (1μM-100μM). Increasing the copper
ion concentration resulted in a stronger color
The dissolution and sensitivity of shade, as shown in Figure 4. At 100μM of
Sensor-2 were investigated. As shown in copper ions concentrations the color change
Figure 3, Sensor-2 was insoluble in water of the sensor became stable after 1 h, as
but soluble in DMF. When the aqueous confirmed by comparison with the color
solution of Cu2+ (38 μM) was added to the after 24 h. We propose that Sensor-2 formed
DMF solution containing Sensor-2, the a 1:1 complex with Cu2+ (see Figure 5).
solution became pink. The result suggested
Table 1. Color parameters (L*, a*, b*, ΔE* ) of the solid-state sensor after 1 h immersion in
100 μM solutions of Cu2+, Mn2+, Cd2+, Ni2+ and Pb2+ (The “blank” sample was not immersed
in metal ion solution and was used as the reference when determining ΔE*.)
Metal ion L* a* b* ΔE* Color
Blank 80.15 -2.48 -6.49 -
2+
Cu 54.68 33.14 -23.09 46.83
Mn2+ 81.02 -1.09 -7.7 2.04
Pb2+ 82.13 -0.15 -7.66 3.27
Cd2+ 81.24 -1.62 -7.08 1.50
Ni2+ 82.42 0.86 -7.75 4.23
The selectivity towards Cu2+ was an available anhydride group that was able
studied by immersing the sensor in solutions to bond chemically with the hydroxyl group
of metal ions (Cu2+, Mn2+, Cd2+, Ni2+ and of cellulose acetate, producing a solid-state
Pb2+) at 100 μM. The color values based on sensor. A visible color change was observed
CIE L*a*b* are reported in Table 1. The in the presence of Cu2+, indicating good
sensor changed to pink when exposed to selectivity for copper ions in both solution
Cu2+ ions only, suggesting strong selectivity and in the solid state sensor. The pink color
for Cu2+. The L* values declined from 81.15 became stronger as the concentration of
to 54.68 as the color shade became darker copper ions increased from 1 μM to 100 μM.
following immersion of the sensor in the The selectivity of the solid-state sensor was
Cu2+ solution. The a* values increased from confirmed by immersion in solutions of
-2.48 to 33.14 and the b* values decreased other metal ions, which produced a
from -6.49 to -23.09, reflecting color negligible change in color.
changes towards red and blue, respectively.
The highest ΔE* was obtained from Cu2+, Acknowledgements
confirming the selectivity and sensitivity of The authors gratefully acknowledge
the sensor. The change in color parameters the financial support provided by National
when exposed to other metal ions was Research Council of Thailand (NRCT) and
marginal and undetectable with the naked the partial support provided by Central
eye. Scientific Instrument Center (CSIC), Faculty
UV-visible spectroscopy demonstrated of Science and Technology, Thammasat
no leaching of the sensor from the CA University. The authors also thank Dr.
substrate, confirming the strong affinity Tienthong Thongpanchang, Mahidol
between Sensor 2 and CA. Senna et al. University, for his support throughout this
report that EDTAD contains two anhydride work.
groups that act as crosslinkers between the
hydroxyl groups of CA.15 This suggests that References
a reaction between the available anhydride 1. Pereira, F. V.; Gurgel, L. V. A.; Gil, L.
group from the EDTAD-modified Sensor-2 F., J. Hazard. Mater. 2010, 176 (1),
and the hydroxyl group of the CA may 856-863.
prevent leaching of the sensor. 2. Sun, Z.; Li, H.; Guo, D.; Liu, Y.; Tian,
Z.; Yan, S., J. Lumines. 2015, 167
4. Conclusion (Supplement C), 156-162.
A novel rhodamine-based colorime- 3. Zhang, C.; Wan, L. Y.; Wu, S.; Wu, D.;
tric sensor for Cu2+ detection was Qin, X.; Ko, F., Dyes Pigm. 2015, 123
synthesized. The sensor molecule contained (Supplement C), 380-385.
4. Zhang, L.-F.; Zhao, J. L.; Zeng, X.; Mu, 10. Karnitz, O.; Gurgel, L. V. A.; Gil, L. F.,
L.; Jiang, X. K.; Deng, M.; Zhang, X.; Carbohydr. Polym. 2010, 79 (1), 184-
Wei, G., Sen. Actuators B: Chem. 2011, 191.
160 (1), 662-669. 11. Rathna, G. V. N.; Li, J.; Gunasekaran,
5. Sikdar, A.; Panja, S. S.; Biswas, P.; S., Polym. Inter. 2004, 53 (12), 1994-
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450. 12. Gan, S.; Zakaria, S.; Chia, C. H.; Chen,
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Dong, Y.-M.; Wang, Y.-W.; Peng, Y., 2017, 12 (3), 1-13.
Dyes Pigm. 2010, 87 (2), 164-172. 13. Milindanuth, P. Thammasat Master 's
7. Xie, Z.; Huo, F.; Su, J.; Yang, Y.; Yin, Degree Thesis, university Rangsit
C.; Yan, X.; Jin, S., Open J. Appl. branch, 2016.
Biosen. 2012, 1 (03), 9. 14. Kono, H.; Fujita, S., Carbohydr. Polym.
8. Qian, Y.; Lin, J.; Han, L.; Lin, L.; Zhu, 2012, 87 (4), 2582-2588.
H., Biosen. Bioelectron. 2014, 58 15. Senna, A. M.; Novack, K. M.; Botaro,
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9. Ozay, H.; Ozay, O., Chem. Eng. J. (Supplement C), 260-268.
2013, 232 (Supplement C), 364-371.
Amino-functionalized porphyrinic catalysts for electrochemical reduction
of carbon dioxide
Piyawat Sangpittayatorn, Patchanita Thamyongkit*
Department of Chemistry, Faculty of science, Chulalongkorn University, Bangkok 10330, Thailand
*E-mail: patchanita.v@chula.ac.th
Abstract:
In this work, preparation and investigation on catalytic activities for electro-
chemical reduction of carbon dioxide (CO2) of 5,10,15,20-tetra(4-aminophenyl)
porphyrin and its polymer were described. Electropolymerization of the amino-functionalized
monomer was performed on an indium tin oxide (ITO)-coated glass and carbon paper by
cyclic voltammetry in dimethylformamide containing a 0.1 M nBu4NPF6 supporting
electrolyte with a Pt plate as a counter electrode and a Ag/AgCl quasi-reference electrode
(QRE). The electrochemical reduction was carried out under CO2 atmosphere at potential
between 0 mV and –1700 mV vs. Ag/AgCl QRE with a scan rate of 50 mV·s–1 by using a
glassy carbon and a polymer-coated ITO-glass or carbon paper as working electrodes,
respectively. Results showed that current increase of 9% (at –1645 mV) and up to 73% (at –
1521 mV) was observed in a CO2-saturated condition when the target monomer and polymer
films were used as the catalysts, respectively. Moreover, due to higher surface area of the
carbon paper, the current of the reduction peak was found to be even higher, compared with
the case of the ITO-glass substrate. To summarize, the target monomer and its polymer
should be able to work as electrocatalysts for the reduction of CO2.
1. Introduction their properties by modifying their

Carbon dioxide (CO2) is known to be molecular structures.9 One of the most
one of major causes for greenhouse effect, commonly-used organic materials for this
associated with rising global surface application is porphyrin derivatives because
temperature, which is responsible for of their high thermal and photostability, and
atmospheric phenomena called global excellent photophysical and electrochemical
warming.1,2 Over the past century, properties.10 Moreover, these compounds
atmospheric concentration of CO2 has can form complexes with many transition
steadily increased as a consequence of metals to obtain a wide range of the
anthropogenic activities, mainly combustion photophysical and electrochemical
processes and deforestation.3 Therefore, properties.11 In addition, polyanilines had
conversion of this greenhouse gas into been widely used as conducting polymers in
useful products, such as liquid fuels or several electronic applications, including
organic compounds, has become of great electrocatalysis.12,13 In this work, we
interest.4 However, CO2, as the final product are interested in the electrocatalytic
of combustion of carbon-based compounds, performance of zinc-porphyrin derivatives
is a very stable molecule with ΔGf° of contains aniline groups at meso positions
394.39 kJ·M−1 and E° of −1.9 V vs. NHE.5 and its polymer toward the electrochemical
Electrocatalysis using coordination reduction of CO2 to produce useful products,
compounds as catalysts is a feasible way to such as carbon monoxide, formic acid and
overcome overpotential required to reduce methane, with hypothesis that the porphyrin
CO2.6,8 Organic and polymeric catalysts core will provide catalytic activity, while the
were proven to be potentials materials and oligoaniline linkages will facilitate
widely studied, owing to possibility to turn
electronic communication within the 2.3 Synthesis of 5,10,15,20-tetra(4-amino
polymeric system. phenyl)porphyrin (H2TAPP)
Following a published procedure,14 a
2. Materials and Methods solution of H2TAcPP (0.100 g, 0.118 mmol)
2.1 General in ethanol (30 mL) was treated with
All chemicals were analytical grade, concentrated HCl (20 mL) and refluxed for
purchased from commercial sources and 24 h. The reaction mixture was cooled down
used as received unless noted otherwise. and neutralized by a 2 M NaOH aqueous
1
H-NMR (400 MHz) spectra was recorded solution and extracted with CHCl3 (3×20
in (CD3)2SO. Chemical shifts (δ) mL). The organic phase was washed with
are reported in parts per million (ppm) water (20 mL) and brine (20 mL). Then, the
relative to the residual (CH3)2SO solution was dried over Na2SO4 and
signal at 2.50 ppm. Mass spectra were concentrated to dryness. The resulting crude
obtained using high resolution electrospray was purified by a silica column using 2−5%
ionization (HR-ESI) and matrix-assisted ethanol in CH2Cl2 as an eluent, leading to
laser desorption ionization (MALDI) H2TAPP as a purple solid (0.061g, 75%).
mass spectrometry with α-Cyano-4- 2.4 Synthesis of [5,10,15,20-tetra(4-ami
hydroxycinnamid acid (α-CCA) as a matrix. nophenyl)porphyrinato]Zinc(ІІ)
Absorption and emission spectra of (ZnTAPP)
all compounds were measured in dimethyl Following a standard method,15 a
formamide (DMF) at room temperature. solution of H2TAPP (0.050 g, 0.074 mmol)
2.2 Synthesis of 5,10,15,20-tetra(4-aceta in a mixture of DMF and CHCl3 (1:3, 30
midophenyl)porphyrin (H2TAcPP) mL) was reacted with a solution of
According to a known method,14 a Zn(OAc)2·2H2O (0.081 g, 0.37 mmol) in
solution of ρ-acetamidobenzaldehyde (0.820 methanol (3.7 mL) under reflux for 24 h.
g, 5.00 mmol) in CH2Cl2 (500 mL) in a 1 L After cooling down, the resulting mixture
round bottom flask covered with aluminum was washed with a 10% aqueous solution of
foil was purged with N2 for 15 min before NaHCO3 (20 mL), water (20 mL) and then
pyrrole (350 μL, 5.00 mmol) was injected. brine (20 mL). After removal of the solvent,
Then, the solution was treated with the crude was purified by a silica column
trifluoroacetic acid (TFA) (700 μL, 10.0 using 2−5% ethanol in CH2Cl2 as an eluent.
mmol) at room temperature for 1.5 h under The resulting dark purple solid was washed
N2 atmosphere. After that, 2,3-dichloro-5,6- with methanol and hexane under sonication
dicyano-1,4 benzoquinone (DDQ) (0.850 g, to yield ZnTAPP as a purple solid (0.036 g,
3.75 mmol) was added and the reaction was 78%).
continued for an additional hour. 2.5 Spectroscopic measurement of
Subsequently, trimethylamine (1.4 mL) was H2TAcPP
added and a volume of the solution was 1
H-NMR δ –2.91 (s, 2H, inner-H),
reduced to approximately 10% by 2.23 (s, 12H, CH3), 8.04 (d, 8H, J = 8.0 Hz,
evaporation. The resulting mixture was then Ph-H ortho to NHCOCH3), 8.14 (d, 8H, J =
passed through a silica pad using 1:1, 8.0 Hz, Ph-H meta to NHCOCH3), 8.87 (s,
ethanol:CH2Cl2 as an eluent. The purple 8H, ß-pyrrole H), 10.40 (s, 4H, NHCOCH3).
fraction was collected and concentrated to MALDI-TOF-MS 843.444 [M+], calcd
dryness. The crude product was purified 842.960 ([M+]; M = C52 H42N8 O4);
by a silica column eluted with 8−15% λabs 423, 518, 556, 652 nm; λem (λex = 423
ethanol in CH2Cl2 affording H2TAcPP as nm) 659, 723 nm.
a dark purple solid (0.160 g, 15%).
2.6 Spectroscopic measurement of of the Pt plate as the CE and the Ag/AgCl
H2TAPP QRE and a glassy carbon and the polymer-
1
H-NMR δ –2.75 (s, 2H, inner-H), coated substrate as the WEs for the
5.57 (s, 8H, NH2), 7.00 (d, 8H, J = 7.2 Hz, homogeneous and heterogeneous conditions,
Ph-H ortho to NH2), 7.84 (d, 8H, J = 7.2 respectively. The measurements were
Hz, Ph-H meta to NH2), 8.88 (s, 8H, ß- performed at the scan rate of 50 mV·s–1 and
pyrrole H). MALDI-TOF-MS 675.172 [M+], the number of the scanning cycles of 3.
calcd 674.812 ([M+]; M = C44H34N8); λabs
435, 529, 575, 664 nm; λem (λex = 435 nm) 3. Results and Discussion
693 nm. 3.1 Synthesis and characterization
2.7 Spectroscopic measurement of The synthesis of the target
ZnTAPP compounds as shown in Scheme 1 started
1
H-NMR δ 5.46 (s, 8H, NH2), 6.96 from condensation between pyrrole and
(d, 8H, J = 8.0 Hz, Ph-H ortho to NH2), acetamido benzaldehyde under acid catalysis
7.80 (d, 8H, J = 8.0 Hz, Ph-H meta to NH2), and subsequent oxidation, following a
8.83 (s, 8H, ß-pyrrole H). HR-ESI-MS literature procedure.14 Upon deprotection of
737.2112 [M+H]+, calcd 737.2114 ([M+H]+; acetyl groups of resulting porphyrin
M = C44H32N8Zn); λabs 438, 568, 614 nm; H2TAcPP by hydrolysis under acidic
λem (λex = 438 nm) 637 nm. condition to obtain H2TAPP which, after
2.8 Electropolymerization of ZnTAPP that, was subject to zinc-metalation by a
Electropolymerization of ZnTAPP standard method,15 ZnTAPP was yielded in
was performed by cyclic voltammetry in 78%. Spectroscopic data of all porphyrins
DMF containing 1 mM ZnTAPP and 0.1 M were consistent with those described in
nBu4NPF6 as a supporting electrolyte in a published literatures.15,16
three-electrode one-compartment system
using an ITO-coated glass or a carbon paper
as a working electrode (WE) and a substrate
for the polymer films. A Pt plate and silver
chloride coated on a silver wire (Ag/AgCl)
were used as a counter electrode (CE)
and a quasi-reference electrode (QRE),
respectively. Polymerization was
electrochemically carried out under N2
atmosphere at a potential ranging from 0
mV to 1400 mV vs. Ag/AgCl QRE with a
scan rate of 50 mV·s–1 and the number
of scanning cycles of 50.
2.9 Electrocatalytic activity determination
For a homogeneous catalytic system,
the electrochemical reduction of CO2 was
studied in DMF containing 0.5 mM
ZnTAPP and 0.1 M nBu4NPF6 under CO2
atmosphere by cyclic voltammetry at a
potential ranging from 0 mV and –2000 mV
vs. Ag/AgCl QRE, while the potential
between 0 mV and –1700 mV vs. Ag/AgCl
QRE was applied to DMF containing 0.1 M
nBu4NPF6 in case of a heterogeneous
system. Using the three-electrode one- Scheme 1. Synthesis of porphyrin
compartment electrochemical cell consisting derivatives
3.2 Electropolymerization studies that the spectra of ZnTAPP exhibited a
Cyclic voltammograms for the characteristic intense Soret band at 438 and
polymerization of ZnTAPP on the ITO- weak Q-bands at 568 and 614 nm.
coated glass as shown in Figure 1A
exhibited two oxidation peaks at 760 mV
and 1018 mV vs. Ag/AgCl QRE
corresponding to the oxidation of aniline
units and a porphyrin cores, respectively.17,18
However, with the increasing number of
scan, the aniline oxidation peak current was
found to decrease. This behavior is
attributed to increase in resistivity of the
film, which prevents the progress of
electrochemical polymerization process.19
Figure 2. Normalized absorption spectra of
a ZnTAPP solution in DMF (solid line) and
a poly(ZnTAPP) film (dashed line)
These features were also observed

for its polymer film with very slight red shift
as a result of the extended π-conjugation
system and possible macrocyclic-
aggregation in the polymer chains. A new
band at 369 nm observed in case of the
polymer film is consistent with the
Figure 1. Cyclic voltammograms of previously reported absorption of the
electropolymerization of ZnTAPP on (A) polyaniline,20 confirming the successful
an ITO-coated glass and (B) a carbon paper polymerization of ZnTAPP.
in a 0.1 M nBu4NPF6 solution in DMF 3.3 Investigations on the electrochemical
reduction of CO2
The electrochemical polymerization
of ZnTAPP on the carbon paper showed
two oxidation peaks at the similar potentials
as those observed for the one on the ITO-
coated glass (699 mV and 1167 mV vs.
Ag/AgCl QRE, Figure 1B). The results also
showed that the polymerization on the
carbon paper was found to have the current
enhancement of the aniline oxidation peaks
when the scanning cycles was increased and
required lower potential, compared with that
used for the polymerization on the ITO- Figure 3. Cyclic voltammograms of the
coated glass, indicating more efficient blank 0.1 M nBu4NPF6 solution in DMF
polymerization process on this substrate. under N2- (black dotted line) and CO2-(black
3.2 Photophysical properties dashed line) saturated conditions, and the
UV-visible absorption spectra of 0. 1 M nBu4NPF6 solution in DMF
ZnTAPP in DMF and its polymer film containing 0.5 mM ZnTAPP under N2-
(poly(ZnTAPP)) on the ITO-coated glass (blue solid line) and CO2- (red solid line)
are shown in Figure 2. The results showed saturated conditions
A cyclic voltammogram under the N2
atmosphere of the 0.1 M nBu4NPF6
electrolyte solution containing 0.5 mM
ZnTAPP showed a reversible reduction
peak at a peak potential (Epeak) of –1645 mV
vs Ag/AgCl QRE, corresponding to the
reduction of the common zinc porphyrin
complexes (Figure 3).21 Under CO2
saturation, the cyclic voltammogram showed
a similar reversible reduction peak at the
same Epeak as that observed in the N2-
saturated condition with slight increase in
current from 63 to 69 μA. However, when Figure 4. Cyclic voltammograms of the
the applied potential reached –1868 mV vs. blank 0.1 M nBu4NPF6 solution in DMF
Ag/AgCl QRE, which was an onset potential under N2- (black dotted line), CO2- (black
(Eonset) of this cyclic voltammogram, the dashed line) saturated conditions, and the
current was continuously increased by 47%, poly(ZnTAPP) film on the ITO-coated
compared with that observed under glass under N2- (blue solid line) and CO2-
the N2-saturated condition. This observation (red solid line) saturated conditions
indicated that ZnTAPP could promote the
electrochemical reduction of CO2 at this
potential. In case of the heterogeneous
condition, where the poly(ZnTAPP) film on
the ITO-coated glass was used as WE, a
cyclic voltammogram of the poly(ZnTAPP)
film under the N2-saturated condition did not
clearly showed the reduction peak, while
that observed under the CO2-saturated
condition showed the first reversible
reduction peak at Epeak of –900 mV vs.
Ag/AgCl QRE, corresponding to reduction
processes of the porphyrin film studied by
Closs G. L. et al (Figure 4).22 Figure 5. Cyclic voltammograms of the
In addition, the peak current was blank 0.1 M nBu4NPF6 solution in DMF
found to increase by 73% its Eonset (–1521 under N2- (black dotted line), CO2- (black
mV vs. Ag/AgCl QRE), compared with that dashed line) saturated conditions and the
observed under the N2-saturated condition, poly(ZnTAPP)-coated carbon paper under
suggesting that the poly(ZnTAPP) film may N2- (blue solid line) and CO2- (red solid
be able to serve as the catalyst for the line) saturated conditions
electrochemical reduction of CO2. Furthermore, due to large surface
The similar reduction peak pattern with area of the carbon paper electrode, the peak
less resolution was observed for current of the electrochemical reduction of
the poly(ZnTAPP) film on the carbon CO2 in this case was found to be
paper as shown in Figure 5. The results approximately 22% higher that than
showed that at –1506 mV vs. Ag/AgCl observed for the case when the ITO-coated
QRE, which is the Eonset of this process, the glass was used, suggesting that it might be a
current enhancement observed under suitable substrate for the heterogeneous
the CO2-saturated condition in comparison electrochemical reduction of CO2.
with the N2-saturated one was found
to be 8% (–0.99 mA vs. –0.91 mA). 4. Conclusion
A zinc complex of porphyrin bearing 6. Panda, M. K.; Ladomenou, K.;
the aniline meso-substituents and its Coutsolelos, A. G. Coord. Chem. Rev.
polymer film were successfully prepared 2012, 256 (21), 2601−2627.
and characterized by spectroscopic 7. Windle, C. D.; Perutz, R. N. Coord.
techniques. The electrocatalytic activities of Chem. Rev. 2012, 256 (21), 2562−2570.
these materials on the homogeneous and 8. Inglis, J. L.; MacLean, B. J.; Pryce, M.
heterogeneous reduction of CO2 were T.; Vos, J. G. Coord. Chem. Rev. 2012,
investigated by means of cyclic 256 (21), 2571−2600.
voltammetry. The results showed that the 9. Oh, Y.; Hu, X. Chem. Soc. Rev. 2013,
target porphyrin monomer exhibited 42 (6), 2253−2261.
significant catalytic activity for the 10. Jurow, M.; Schuckman, A. E.; Batteas,
homogeneous electrochemical reduction of J. D.; Drain, C. M. Coord. Chem. Rev.
CO2 in terms of the reduction current 2010, 254 (19–20), 2297−2310.
enhancement. In the case of the polymer- 11. Huang, X.; Nakanishi, K.; Berova, N.
coated carbon paper, the current of the Chirality 2000, 12 (4), 237−255.
reduction peak was found to be increased, 12. MacDiarmid, A. G.; Epstein, A. J.
compared with the case when the ITO- Faraday Discuss. Chem. Soc. 1989, 88
coated glass was used as the substrate, (0), 317−332.
possibly due to its higher surface area. 13. Hursan, D.; Kormanyos, A.; Rajeshwar,
Furthermore, studies on product formation K.; Janaky, C. Chem. Commun. 2016,
of their catalytic systems are under 52 (57), 8858−8861.
progresses and will be published elsewhere. 14. de Miguel, G.; Wielopolski, M.;
Schuster, D. I.; Fazio, M. A.; Lee, O. P.;
Acknowledgements Haley, C. K.; Ortiz, A. L.; Echegoyen,
We gratefully acknowledge the L.; Clark, T.; Guldi, D. M. J. Am.
financial support of the research from the Chem. Soc. 2011, 133 (33),
Graduate School of Chulalongkorn 13036−13054.
University (The 90th Anniversary of 15. Meng, W.; Breiner, B.; Rissanen, K.;
Chulalongkorn University Fund, Thoburn, J. D.; Clegg, J. K.; Nitschke,
Ratchadaphiseksomphot Endowment Fund). J. R. Angew. Chem. Int. Ed. 2011, 50
(15), 3479−3483.
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The preparation and characterization Ag and Ag alloy catalysts supported
on treated carbon
Jennarong Jaikang1, Anukorn Phuruangrat2, Thapanee Sarakonsri3, Aphiruk Chaisena1,
Siwat Thungprasert1*
1
Lampang Rajabhat University, Lampang 52100, Thailand
2
Department of Materials Science and Technology, Faculty of Science, Prince of Songkla University,
Songkla 90110, Thailand,
3
Department of Chemistry Center of Excellence for Innovation in Chemistry,
Faculty of Science Chiang Mai University, Chiang Mai 50200, Thailand
*E-mail: siwattawis@hotmail.com
Abstract:
Ag catalysts have been proposed as cathode material for direct ethanol fuel cell
(DEFC). In this research, 20% and 40% wt. Ag and AgNi were prepared to support on treated
carbon (Carbon Vulcan XC-72 by hydrogen peroxide) by NaBH4 reduction method in
ethylene glycol at room temperature. The XRD patterns confirmed the presence of Ag and
AgNi-alloy on carbon. The EDS indicated that metals (Ag and Ni) including and carbon are
major elements in the prepared catalysts. The SEM and TEM images showed good dispersion
of metals on treated carbon support. Particles size of Ag and AgNi-alloy on carbon support
were 24.21±7.26 and 19.14±5.30 nm.
1. Introduction AgCo/C13 has high electro-activity than

The direct ethanol fuel cell (DEFC) standard catalysts. The catalyst for cathode
is a green technology developed as a source fuel cell should have nano-size particles
of continuous clean energy. In general, with a good dispersion on carbon supporter.
platinum supported on carbon catalyst was In order to obtain nano-sized product, many
used as anode and cathode materials. At processes such as heat treatment in hydrogen
anode, platinum will catalyze the hydrogen gas and nitrogen gas.14,15 However, this
dissociation into protons and electrons. At method requires high energy and
cathode, platinum will catalyze the oxygen complicated procedure. This research use
dissociation. By using platinum catalyst at NaBH4 reduction method to reduce metal
cathode, the oxygen reduction reaction ion in solvent to form metal or metal alloy
(ORR) was reported to be two electrons because sodium borohydride (NaBH4) as a
mechanism, which produced hydrogen reducing agent due to its strong reductive
peroxide and destroyed electrode surface. ability.16,17 Moreover, good dispersion of
Moreover, platinum catalyst is an expensive catalysts on carbon support will be obtained
reagent.1,2 Presently, the cathode catalysts by using modified surface carbon with
such as platinum alloy and non-platinum hydrogen-peroxide.18,19
alloy has replaced the pure platinum In this research, the functional
catalyst. This research focuses on non- groups on surface of carbon were modified
platinum and metal alloy because catalysts by treated with hydrogen peroxide. After
do not agglomerate after long time operation that, the Ag and AgNi catalyst supported on
and use low platinum.3,4 The silver and treated carbon was synthesized by NaBH4
silver alloy with first row transition provide method using AgNO3 and Ni(NO3)2.6H2O as
good electrostatic force and low cost.5-7 The metal sources and ethylene glycol as a
silver alloy catalyst such as AgNi/C,8,9 solvent.
AgNi/PtAgNi,10 Cu@Ag/C,11 AgCu/C12 and
Carbon Vulcan XC-72 was treated
by hydrogen peroxide.20 The functional
groups of the treated and untreated carbon
were investigated by Fourier transform
infrared spectroscopy (FTIR, FTIR-8900,
Shimadzu). The experiment from 20% and
40% wt. Ag/C and AgNi/C catalysts were
started by adding treated carbons into
Figure 1. FT-IR spectrum of untreated
ethylene glycol (99.9%, J.T. Baker) After
carbon and treated carbon
that, stoichiometric amounts of AgNO3 (RCI
Labscan Limited 99.8%) precursors from
shown in Figure 2. They show the board
Ag/C and AgNO3 and Ni(NO3)3.6H2O (Loba
chemie Rvt 99.0%) from AgNi/C catalyst peak at 2θ ≈ 25.04o with corresponding to
(weight ratio of Ag:Ni as 1:1) were added. (002) plane of carbon (JCPDS No. 26-1079)
The stoichiometric amount of NaBH4 for all samples. The 20% and 40% wt. Ag/C
powder (96%, Merck) was added for a samples show peak at 2θ = 38.06o (111),
reduction process and the reaction was 44.22o (200), 64.39o (220) and 77.30o (311)
incubated with instantly stir for 12 hours. with corresponding to plane of Ag (JCPDS
The final powder was filtered, washed with No. 4-783). The 20% and 40% wt. AgNi/C
methanol and dried in oven for overnight. samples show peak at 2θ = 38.11o (111),
The prepared catalysts were characterized 44.31o (200), 64.41o (220) and 77.38o (311)
by X-ray diffraction (XRD, with corresponding to plane of AgNi.
Panalytical/Expert, Cu Kα (λ1.54)), Because the unit cell from these Ag and Ag-
alloy catalysts were cubic and space group
Scanning Electron Microscopy (SEM, JEOL
JSM-5910FE) equipped with Energy as Fm3m. The 2θ position of (111plane of
Dispersive Spectroscopy (EDS, INCA, The sample was compared in Table 1. From all
Microanalysis Suite Issue-16) and AgNi/C, the position of (111) plane was
Transmission Electron Microscopy (TEM) closer to Ag-alloy than Ag metal because
(JEOL JEM-2010) techniques. the unit cell from silver alloy was smaller
than silver metal. This alloy was counted as
3. Results and Discussion substitutional alloy; metal was replaced by
The modified functional groups on other metals at the same position .In this
the treated carbon surface were analyzed sample, nickel with the smaller atomic
compared with untreated carbon. They were radius than silver had replaced the silver
characterized by FTIR as shown in Figure 1. position. After the metals were mixed to
It can be seen that the treated carbon shows - form alloy, the acquired alloy phase
O-H stretch at 3500-3400cm-1, -C=O at provided the different unit cell parameters
1650 cm-1 and -C-O stretch at 1020 cm-1 from the metal. In case of the atomic
conform carboxylic group -)C(=O)-O-H). distance, the distance between the same
The appearance of this functional group was atomic types appeared further than the
due to hydrogen peroxide which was a distance from heteroatom by the effect of
strong oxidizing agent. This carboxylic the atomic force .For the same reason, the
group, which is a negatively charged position of (111) plane of AgNi catalysts
functional group, induce the formation of also appeared closer to silver alloy than
metal alloy catalysts on carbon due to silver metal.22 Reduction reaction take place
electrostatic interaction between a metal ion in a solution will allow metal ions to
and carboxylic group.21 combine with electrons to form alloy metal.
XRD patterns of Ag and AgNi The standard reduction potential of NaBH4
catalysts supported on treated carbon are in basic solution equals to – 1.65 V
(equation 1)23, which is low enough to
reduce all metal ions into metals (equation
2-3)24 with relatively high reaction rate.
BH4-+4OH-→BO2-+2H2O+4e- E0=-1.65V (1)
Ag+ + e- → Ag E0=+0.80V(2)
Ni2+ + 2e- → Ni E0=-0.25V (3)
Figure 3. SEM images of a) 20%Ag/C b)

40%Ag/C c) 20%AgNi/C (1:1) d) 40%
AgNi/C (1:1) catalyst
Table 2. Percentage weights of silver, nickel

and carbon on samples from EDS
Element (%weight)
Samples
C Ag Ni
Figure 2. XRD patterns of Ag and AgNi
catalysts on treated carbon 20%Ag/C 77.29 22.71 -
40%Ag/C 62.82 37.18 -
Table 1. Compared peak position of (111) 20%AgNi/C 86.65 11.11 2.24
between Ag/C and AgNi/C sample with unit 40%AgNi/C 81.35 14.39 4.26
cell parameters.
Samples 2θ (111) a (Å)
20%Ag/C 38.06° 4.09
40%Ag/C 38.06° 4.09
20%AgNi/C 38.11° 4.08
40%AgNi/C 38.11° 4.08
The SEM technique was show in

Figure 3. The picture was show particle size
and shape for carbon Vulcan XC-72
supported. The metals supporting was
smaller particle size which this technique
was detected. However, the qualitative and
quantitative elements for both samples were
detected by EDS spectrum as shown in Figure 4. TEM images and their corres-
Table 2. It shown that the intensities of EDS ponding SAD patterns images of a)
peaks of C, Ag and Ni. For XRD technique, 20%Ag/C b) 20%AgNi/C (1:1) catalyst
AgNi/C catalyst didn’t shown nickel or
nickel oxide peak. It conform that peak for from Ag and AgNi catalysts were
XRD technique as AgNi alloy. The amount 24.21±7.26 and 19.14±5.30 nm. In general,
of the observed metals was less than the catalyst with smallest nanoscales will
theoretical metals ratio because of the improve the fuel cell performance due to
limitation of this technique.19 high surface area. The selected area electron
The TEM technique was show in diffraction (SAD) patterns were inserted in
Figure 4. It was found that the catalysts TEM images. The 20% wt. Ag/C catalysts
were nanoparticles. Particles size was showed the ring pattern of (002) planes for
measured from several TEM images using carbon and (111) (200) and (220) planes of
image J program. The average particle size Ag. The 20% wt. AgNi/C catalysts showed
the ring pattern of (002) and (101) planes for 8. Feng, R. X.; Dong, H.; Cao, Y. L.; Ai,
carbon and (111) (200) and (220) of AgNi. X. P.; Yang, H. X. Int. J. Hydrog.
This technique was same result with XRD Energy 2007, 32, 4544–4549
technique and EDS technique. 9. Song, Xi.; Zhang, D. Energy 2014, 70,
223-230
4. Conclusion 10. Wua, D.; Cheng, D; Electrochimica
This research, Ag and AgNi alloy Acta 2015, 180, 316–322
supported on treated carbon Vulcan XC-72 11. Duan, D.; Liu, H.; You, Xi.; Wei, H.;
were successfully synthesized by NaBH4 Liu, S. J. Power Sources 2015, 293,
reduction method at room temperature. For 292-300
XRD and SAD were shown the catalysts as 12. Duan, D.; Liu, H.; You, Xi.; Wei, H.;
Ag and AgNi alloy on treated carbon for all Liu, S. Electrochimica Acta 2016, 198,
samples. Size of alloy particles was 212–219
measured from TEM images to be in 13. Joo, Y.; Ahmed, M. S.; Han, H. S.;
between 19 – 25 nm. Jeon, S. Int. J. Hydrog. Energy 2017,
42, 21751-21761
Acknowledgement 14. Luo, J.; Kariuki, N.; Han, L.; Wang, L.;
This work was granted from the Zhong, C. J.; He, T. Electrochimica
Center of Excellence for Innovation in Acta 2006, 51, 4821-4827.
Chemistry (PERCH-CIC), Office of the 15. Osmieri, L.; Escudero-Cid, R.;
Higher Education Commission, Ministry of Alessandro H. A.; Videlaa, M.; Ocón,
Education and Department of Chemistry, P.; SpecchiL, S. Appl. Catal. B:
Faculty of Science, Lampang Rajabhat Environ. 2017, 201, 253–265
University are gratefully acknowledged. 16. Wu, C.; Bai, Y.; Liu, D. X.; Wu, F.;
Pang, M.-L.; Yi, B. L. Catal. Today
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Technology and Application Vol. 2, 3, 29-33.
Wiley, 2003; 503-506. 18. Moreno-Castilla, C.; López-Ramón, M.
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Handbook of Fuel Cell Fundamentals 38, 1995-2001.
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K.S., J. Appl. Electrochem. 2001, 31, Klysubun, W.; Vilaithong, T. J. Alloy
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B. N.; Black, T.; Chen, D. A. J. Power Pro. Res. 2014, 15, 320-324
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The study of using nanostructured lipid carrier for turmeric encapsulation
in skincare application
Sukanya Thepwatee1*, Warunya Tienpratan1, Nuanlawong Sumanojitraporn1,
Jayanant Iemsam-arng2*
1
Department of Industrial Chemistry, King Mongkut’s University of Technology North Bangkok,
1518 Pracharat 1 Rd., Wongsawang, Bangsue, Bangkok 10800, Thailand
2
National Nanotechnology Center, National Science and Technology Development Agency,
*E-mail: sukanya.t@sci.kmutnb.ac.th, jayanant@nanotec.or.th
Abstract:
Turmeric is well-known for its antioxidant, anti-inflammatory, and antimicrobial
properties. However, its color staining, water insolubility, sensitivity to light, and low skin
permeability are the major problems for skincare applications. In this study, a nanostructured
lipid carrier (NLC) was used to overcome these problems. The stabilities of the turmeric
nanostructured lipid carriers (T-NLCs) was tested under different storage conditions, 4 °C,
25 °C , and 45 °C for a month, and under 6 cycles of heating-cooling condition between 4 °C
and 45 °C. Average particle sizes, size distributions, and zeta-potentials of the T-NLCs were
compared. The results demonstrated that the encapsulated turmeric had a good stability at 4
°C and under the heating-cooling condition. The release kinetic of curcumin from T-NLCs
was studied using a Franz diffusion cell with a polyethersulfone as a membrane. The
encapsulated turmeric showed good controlled release of over 8 hr, while the non-NLC
turmeric showed a poor skin permeability. This confirms the successful delivery and
controlled release system. Moreover, the T-NLCs have shown good anti-oxidant properties.
For the lotion formulation using T-NLC, it is clearly seen that the color of T-NLC lotion was
strongly destained and revealed satisfactory results compared to non-NLC turmeric.
1. Introduction without stability test. Zamarioli and

Curcumin, a chemical compound coworkers13 found that using solid lipid
found in turmeric plant (Curcuma longa), nanoparticle (SLN) could improve zeta
offers anti-oxidant and anti-inflammatory potential of curcumin particles into a range
properties.1-5 However, its poor dermal of −30.40 ± 4.16 mV which provides a
permeability, high sensitivity to light, low better resistance to particle aggregation.
water solubility (11 ng/ml) and strong However, encapsulation efficiency (52.92 ±
pigment need to be solved to apply into 5.41%) was much lower and most of
skincare application.6,7 curcumin was released within 3 hr. Fang et
Nanoencapsulation technology has al.20 demonstrated that using nanostructured
been widely used in food, cosmetics and lipid carrier (NLC) offers a greater dermal
pharmaceutical applications to overcome permeation and controlled release of
these problems.4,5,8-18 Due to a wide range of psoralens over SLN. Karimi et al.21 reported
potential application of curcumin, a lot of that NLC could prolong curcumin release of
works on encapsulation of curcumin have 42% for 20 h with enhanced shelf-life, anti-
been carried out. Sari et al.19 studied oxidant and anti-microbial activities. Similar
encapsulation of curcumin using to the report from Madan and coworkers22,
nanoemulsion. Small nanoparticles (~142 90.86% of encapsulation efficiency (EE)
nm) with an encapsulation efficiency more with a sustained release for over 10 hr was
than 90% were obtained. However, small achieved.
zeta potential of 6.9 ± 0.2 mV was reported Thus, this work aims at using nano-
structured lipid carrier (NLC) to improve 2.3 Preparation of T-NLCs lotion
dermal permeability and controlled release T-NLC lotion was formulated using
of curcumin while maintaining antioxidant ingredients shown in Table 2. Stearic acid,
activity of curcumin for skincare meadow lactone and simulgel NS were pre-
application. Moreover, it was expected that mixed in a beaker placed in a 50 °C water
dark yellow color of curcumin could be bath. The T-NLC formulation NW1 was
diminished after encapsulation. then added into the beaker and mixed using
a high-speed homogenizer with a speed of
2. Materials and Methods 5,000 rpm at room temperature for 5
2.1 Materials minutes.
Turmeric extract (85%
curcuminoids) was purchased from Table 2. T-NLC skincare formulation
Specialty Natural Products Co., Ltd. Percentage (%V/V)
Polyethylene glycol derivative (PEGdev), Component Base Non- T-NLC
NLC
non-ionic surfactant, stearic acid, sodium Turmeric extract - 1 -
benzoate, co-surfactant and natural wax T-NLC (NW1) - - 1
used as a solid lipid (SL) were purchased Stearic acid 1 1 1
from S. Tong Chemicals Co., Ltd. Meadow lactone 2 2 2
(Thailand). Synthetic oil or liquid lipid (LL) Simulgel NS 5 5 5
Titanium dioxide 1 1 1
was received from Sigma Aldrich DI water qs. 100 100 100
(Singapore). Meadow lactone and titanium
dioxide were received from Forecus Co., 2.4 Characterizations of T-NLCs
Ltd. (Thailand). Simulgel NS was purchased Mean particle size, polydispersity
from Chemico (Thailand). All chemicals index (PDI) and zeta potential were
and materials were used as received. recorded using Malvern Zetasizer Nano ZS.
2.2 Preparation of turmeric NLCs (T- 2.5 Stability of T-NLC
NLCs) After storage under different
T-NLCs were prepared with three conditions, phase separation of the samples
different lipid contents: without natural wax was visually observed. Change in mean
(NW0), with 0.1% natural wax (NW1) and particle size, polydispersity index (PDI) and
with 0.3% natural wax (NW3). zeta potential was evaluated by the Zetasizer
Compositions of each T-NLC are shown in Nano ZS.
Table 1. Clear solution of phase I (solid 2.6 In vitro release studies of T-NLCs
phase), phase II (water phase) and phase III Skin permeation and release kinetics
(lipid phase), were mixed together using a of curcumin from T-NLCs and turmeric in
probe sonicator (Q700, Qsonica, USA) with methanolic solution (0.1%) were performed
a set amplitude of 30% for 10 minutes and in a Franz diffusion cell using
cycles of 30-seconds pulse on/off to polyethersulfone membrane as a simulated
generate T-NLCs. The prepared T-NLCs human skin. The accumulated concentration
were stored in amber glass container. of turmeric in the receptor chamber was
colorimetrically analyzed by a fluorescent
Table 1. Composition of T-NLCs method (R2 = 0.9996) with the excitation
Percentage (%V/V)
Component
NW0 NW1 NW3
wavelength of 397 nm and emission at 508
Phase I PEGdev 0.5 0.5 0.5 nm using a microplate reader (Spectramax®
Turmeric extract 0.1 0.1 0.1 M2 Multi-detection Reader).
Phase II Surfactant 5.0 5.0 5.0 2.7 Anti-oxidant properties of T-NLCs
DI water 93.0 93.0 93.0 Antioxidant activities of T-NLCs
Sodium benzoate 0.1 0.1 0.1
Phase III Natural wax - 0.1 0.3
were evaluated by 1,1-diphenyl-2-
Synthetic oil 1 0.9 0.7 picrylhydrazyl (DPPH) assay. Briefly, the
Co-surfactant 0.5 0.5 0.5 DPPH in methanolic solution (2 µM) was
prepared and incubated in the dark for 30 negative surface charge shown in Figure
min. After the addition of DPPH methanolic 1(b). The strong surface charge helps
solutions to the samples, the absorbance of preventing particle aggregation and leads to
the solution was measured at 517 nm and the system stabilization.
expressed in term of percent inhibition
calculated as following equation.
%inhibition =
where Abs C is the absorbance of control

without sample addition and Abs S is the
absorbance of analytical sample. The anti-
oxidant activity of the samples was also
extrapolated to the anti-oxidant activity of
vitamin C (2-10 mg/mL, R2=0.9912).
2.8 Stability of lotion formulation
Stabilities of lotion formulations
were followed by the change of pH and
viscosity of the lotions after storage at
different temperature conditions using
Mettler Toledo MP 220 pH meter, Figure 1. (a) Mean particle size with
Switzerland and a viscometer from polydispersity index (PDI) (b) Zeta potential
Brookfield DV-II, USA. of T-NLCs
3. Results and Discussion 3.3 In vitro release of T-NLCs

3.1 Physical properties of T-NLCs
Various natural wax/synthetic oil
ratios or solid lipid/liquid lipid ratios
(SL/LL = 0:10, 1:9 and 3:7) was studied.
The results revealed that increase in solid
lipid (SL) content resulted in bigger particle
sizes (Figure 1, before storage). For, PDI
values, NW1 showed the smallest PDI of
0.31 indicating the most uniform
nanoparticle sizes obtained. These results
are accordant with the work reported by
Bahari et al.23
3.2 Stabilities of T-NLCs
To evaluate the impact of SL/LL
ratio to stability of the T-NLCs, samples
were stored at 4 °C, 25 °C and 45 °C for a
month and under 6 cycles of heating-cooling
condition between 4 °C and 45 °C Figure 2. Release profiles of T-NLCs
(abbreviated as 4/45 °C). The results (Figure compared to non-NLC turmeric in methanol
1(a)) demonstrated that NW1 was the most under (a) simulated human skin and (b)
stable colloidal system. Small particles with simulated human body conditions
low PDI changes were observed at the
storage temperatures of 4 °C, 45 °C and 4/45 Release profiles of T-NLCs were
°C. The results are accordant with strong determined under two different conditions: a
simulated human skin condition (pH of 5.5 vitamin C/mL) and NW3 (43% anti-oxidant
at 32 °C) and a simulated human body equivalents to 3.3 mg vitamin-C/mL). It
condition (pH of 7.4 at 37 °C). Figure 2 provided excellent antioxidant activity as
shows that the NLC system promoted skin turmeric in methanol but with much greater
permeation and controlled release of skin pemeation and controlled release. So,
curcumin for over 8 hr in both cases. Under this comfirms the successful delivery of
simulated human skin condition, release curcumin using the NLC system.
rates of curcumin was in the following order 3.5 Physical appearance and stability of
NW3>MW1>NW0. In the case of simulated lotion containing T-NLC
human body condition, similar release Figure 4 presents physical
profiles of T-NLCs were observed. The appearance of lotion with NW1 and lotion
risen pH and temperature resulted in the with non-NLC turmeric. The color of T-
fasten release kinetics. NLC lotion was strongly destrained
3.4 Antioxidant properties of T-NLCs compared to that of non-NLC turmeric. The
DPPH assay was carried out to T-NLC lotion can be applied on skin
examine the antioxidant properties of without leaving dark orange color stain. So,
T-NLCs (0.1%) compared to non-NLC this results clearly demonstrated the success
turmeric in methanolic solution (0.1%) as of using NLC system for color destraining
well as curcumin suspension in DI water of curcumin for skincare application.
(0.1%). Figure 3 shows that the anti-oxidant However, alternative experiment on
activity of turmeric in water was the worst quantitative analysis was currently focused.
due to its insolubility, while, turmeric
solution in methanol provided the best anti-
oxidant activity (90% equivalents to 8.9 ng
vitamin-C/mL).
Figure 4. (a) Lotion containing T-NLC

(NW1) and non-NLC turmeric (pure
curcumin powder) and color of the lotions
Figure 3. Antioxidant properties of T-NLCs (b) before and (c) after applying on human
comparing to non-NLC turmeric in DI water skin
and in methanol
To determine stability of the lotions,
Eventhough turmeric in methanol pH and viscosity of the lotion after storage
showed superior antioxidant perperties, were followed. As seen in Figure 5, pH and
curcumin could not be carried through viscosity of both T-NLC lotion and non-
human skin without the NLC system.Our NLC turmeric were not significantly
work demonstrated that NW1 (70% anti- changed in every case. The lotions have
oxidant equivalents to 8.4 mg vitamin- good stability enough to be stored at room
C/mL) was the formulation that provided the temperature or even higher temperatures
best antioxidant activity comparing to NW0 without dramatic degrading.
(39% anti-oxidant equivalents to 3.5 mg-
medicinal spice crops; Elsevier Science:
Burlington, 2013; pp. 47-59.
5. Shrotriya, S.; Ranpise, N.; Satpute, P.;
Vidhate, B. Artif. Cells, Nanomed.,
Biotechnol. 2017, 1-12.
6. Tønnesen, H. H.; Másson, M.; Loftsson,
T. Int. J. Pharm. 2002, 244 (1), 127-135.
7. Arct, J.; Ratz-Łyko, A.; Mieloch, M.;
Witulska, M. Indian J. Pharm. Sci.
2014, 76 (4), 374-378.
8. Guri, A.; Gulseren, I.; Corredig, M.
Food Funct. 2013, 4 (9), 1410-1419.
9. Gonçalves, G. M. S.; Silva, G. H. d.;
Barros, P. P.; Srebernich, S. M.;
Shiraishi, C. T. C.; Camargos, V. R. d.;
Figure 5. Comparison of (a) viscosity and
Lasca, T. B. Braz. J. Pharm. Sci. 2014,
(b) pH of lotion base, lotion with non-NLC
50 (4), 885-893.
turmeric and lotion with T-NLC (NW1)
10. Prasad, S.; Tyagi, A. K.; Aggarwal, B.
after a month of storage
B. Cancer Res. Treat. 2014, 46 (1), 2-18.
11. Ahmad, M. Z.; Alkahtani, S. A.; Akhter,
4. Conclusion
S.; Ahmad, F. J.; Ahmad, J.; Akhtar, M.
Nanostructured lipid carrier (NLC)
S.; Mohsin, N.; Abdel-Wahab, B. A. J.
of turmeric was developed and determined
Drug Targeting 2016, 24 (4), 273-293.
for its physicochemical properties as well as
12. Liu, W.; Zhai, Y.; Heng, X.; Che, F. Y.;
the stability. The release profile was also
Chen, W.; Sun, D.; Zhai, G. J. Drug
enhanced compared to the methanolic
Targeting 2016, 24 (8), 694-702.
solution. Then, the optimal nanocarrier was
13. Zamarioli, C. M.; Martins, R. M.;
further used in the topical preparation. It
Carvalho, E. C.; Freitas, L. A. P. Rev.
was found that the T-NLC preparation was
Bras. Farmacogn. 2015, 25 (1), 53-60.
stable and destained when applied on the
14. Esmaili, M.; Ghaffari, S. M.; Moosavi-
skin compared to the unencapsulated
Movahedi, Z.; Atri, M. S.; Sharifizadeh,
preparation.
A.; Farhadi, M.; Yousefi, R.; Chobert,
J.-M.; Haertlé, T.; Moosavi-Movahedi,
Acknowledgements
A. A. LWT - Food Sci. Technol. 2011,
The authors would like to thank
44 (10), 2166-2172.
supports and facilities from the Department
15. Paramera, E. I.; Konteles, S. J.;
of Industrial Chemistry, Faculty of Applied
Karathanos, V. T. Food Chem. 2011,
Science, King Mongkut’s University of
125 (3), 913-922.
Technology North Bangkok and the
16. Wang, Y.; Lu, Z.; Lv, F.; Bie, X. Eur.
National Nanotechnology Center of
Food Res. Technol. 2009, 229 (3), 391-
Thailand (NANOTEC).
396.
17. Gibbs, F. S. K. I. A. C. N. M. B. Int. J.
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19. Sari, T. P.; Mann, B.; Kumar, R.; Singh,
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2018, 22, 18-24.
Crosslinking of Thai silk fibroin and bioactive calcium silicate glass
for development of bone scaffolds
Pimnara Wattanachai, Peerapat Thongnuek*
Biomedical Engineering Program, Faculty of Engineering, Chulalongkorn University,
*E-mail: peerapat.t@chula.ac.th
Abstract:
Reinforcement of bioactive calcium silicate glass (BCSG) with Thai silk fibroin (TSF)
polymers is challenging, as TSF is prone to gel and thus undergoes phase-separation from the
solution. Our study using (3-glycidyloxypropyl)trimethoxysilane (GPTMS) as a crosslinker
discovered the ratio between BCSG and TSF that allowed both BCSG and TSF to
homogeneously form into porous scaffolds. Scanning electron microscopy showed the
existence of desirable interconnected pores in our hybrid materials. Fourier transform infrared
spectroscopy confirmed the crosslinks of BCSG and TSF by GPTMS. Analysis of the
dissolubility in water and in vitro biodegradation revealed the significantly enhanced stability
of the crosslinked hybrid scaffolds. The resulting hybrid scaffolds presents the admixture of
soft and elastic TSF fibres with stronger but brittle glass, mimicking bone organic and
inorganic composition to simultaneously replace and regenerate bone tissue in fractures.
1. Introduction interconnected pores to achieve more

As the world population is becoming specific surface area, and support the
aged, an increase in the demand for bone- transport of important molecules. They are
graft materials is predicted as bone has been also designed to be biodegradable such that
one of the most frequently grafted organs. bioactive molecules can be released during
Bone grafts can be derived from a second degradation. Most importantly, they must be
anatomic location of the same patient biocompatible.2
(autografts) or from another patient Silk fibroin (SF) from cocoons of
(allografts). Despite being a gold standard silkworms Bombyx mori is a biocompatible
for bone grafting, autografts may lead to and biodegradable polymeric protein.3 It has
donor site morbidity, and allografts raise been used for making biomaterials because
concerns over disease transmission.1 These of impressive mechanical properties,
limitations provide for finding alternative biocompatibility, biodegradability and low
methods. immunogenicity.4 SF can be used for various
Bone tissue engineering has widely tissue engineering applications such as bone,
been researched as a promising alternative to cartilage and skin. However, there are still a
the autografts and the allografts. In tissue number of problems regarding the use of SF
engineering, scaffolds are important because in bone tissue engineering. The most
they support fractures in tissue, provide 3D important problem is the lack of bioactivity,
framework for reconstructing functional as the regenerated tissue does not bond very
tissues, release bioactive compounds for well to the surface of SF.
regeneration, and attract regenerative cells Bioactive calcium silicate glass
such as stem cells. Therefore, bone scaffolds (BCSG) has been developed as a material
are designed to have mechanical for bone tissue engineering.5 The advantages
performance comparable to that of the native of BCSG over other materials are its
bone. The scaffolds are created to have 3D excellent bioactivity and degradability.
structure with suitable porosity and BCSG was shown to bond with living bone
through the formation of an apatite interface et al.10 40 grams of silk cocoon were
layer.6 Furthermore, properties of BCSG can degummed with 1 L of 0.02 M Na2CO3 and
be fine-tuned through compositional and rinsed thoroughly with de-ionised water to
structural variation.7 However, its remove sericin. The degummed TSF was
disadvantages are the brittleness and solubilized in 9.3 M LiBr solution for 4 h at
unstability.7 60 °C. The solution was then dialyzed in de-
To overcome the mentioned ionised water for 3 days.
problems, a crosslinking technique has been Preparation of BCSG solution
applied to covalently bond organic polymers BCSG solution was prepared by
and the silicate network of BCSG. In 2001, hydrolysis of TEOS in de-ionised water
Ren and his colleague were the first to use containing 1 N hydrochloric acid (volume
3-glycidoxypropyltrimethoxysilane ratio DI water:HCl = 3:1 and molar ratio DI
(GPTMS) (Figure 1) to crosslink gelatin water:TEOS = 4:1). The same volume of DI
chains.8 Later, Mahony et al. produced water was used for dissolving calcium
silica-gelatin hybrid scaffolds with GPTMS chloride and the two solutions were mixed
acting as a crosslinking agent between the together to obtain a sol with 70:30 molar
silica network and the gelatin.9 It was ratio between SiO2 and CaO.6
established that the epoxy ring on the Scaffold fabrication
GPTMS reacts with an amine group on a The ratios of the TSF, BCSG and
gelatin chain, whilst the methoxysilane on GPTMS for the fabrication of hybrids are
another end of the GPTMS get hydrolyzed presented in Table 1.
becoming silanol groups. Some of which
then undergo polycondensation to integrate Table 1. Composition of TSF-BCSG
into the silicate network.9 hybrids
Name Hybrid Crosslinked
composition with
(%wt) 10 wt%
GPTMS
TSF BCSG
TSF-BCSG 0 50 50 NO
Figure 1. Molecular structure of GPTMS TSF-BCSG 10 50 50 YES
In this work, the fabrication of For synthesis of the hybrid scaffolds,

hybrid scaffolds based on TSF and BCSG TSF was either functionalized with 10 wt%
was investigated. The stability and pore GPTMS or not. After that, the resulting TSF
characteristics of the hybrid scaffolds were was added to the BCSG solution, and the
then examined. mixture was then foamed by vigorous
agitation for 20 min. The foamed gels were
2. Materials and Methods aged for 3 days at 60 °C in closed beakers.
2.1 Materials The beakers were then left open to dry for
The cocoons of Thai silkworm another 24 h at 60 °C.
“Nangnoi Srisaket 1” were obtained from 2.3 Scaffold characterization
Queen Sirikit Sericulture Center, Nakhon- Chemical structures and crosslinking
ratchasima province, Thailand. Other The structures and chemical bonds
chemicals used in this study were of analy- were evaluated with an Attenuated Total
tical grade. Reflectance Fourier Transform Infrared
2.2 Scaffold fabrication Spectrometer (ATR-FTIR). The spectra
Preparation of TSF solution were collected in the range of 4000-510
TSF solution was prepared according cm-1.
to the method previously described by Kim Morphology and porous structure
The morphology and pore structure FTIR spectra of GPTMS alone, TSF
of TSF-BCSG 0 and TSF-BCSG 10 were alone, 10% GPTMS-functionalized TSF and
characterized by scanning electron TSF-BCSG 10 confirmed the incorporation
microscopy (SEM) at an accelerating of TSF with BCSG, and they also gave
voltage of 12-15 kV after sputter-coating evidence for the successful crosslinking by
with gold. The samples were imaged at GPTMS (Figure 2). The spectrum of the
100X. The SEM images were analyzed pure GPTMS showed a band at 1072 cm-1
using ImageJ to determine the average pore assigned to Si-O-C in the methoxysilane end
size. (Figure 1 and Figure 2, bottom line). In
Stability in water addition, the three bands at 1254.17, 909.37
The TSF-BCSG scaffolds (10±1 mg) and 854.06 cm-1 assigned to bonds in the
were immersed in 1 ml of de-ionised water epoxy ring on another end of the GPTMS
for 24 h at 37 °C. molecule were evident. Scaffolds made
The scaffolds were then retrieved solely of TSF exhibited amide I (C=O
from the de-ionised water and dried over stretching vibrations), amide II (C-N
night at 60 °C. The weight loss was used stretching vibrations) and amide III (N-H
as an indicator for the instability of the deformation vibrations) bands at 1623.20,
scaffolds in water. To determine the weight 1514.92 and 1233.79 cm-1, respectively
loss, the following equation was used: (Figure 2, 2nd line from bottom). Both
GPTMS-functionalized TSF and TSF-BCSG
Weight loss (%)= W0 - W1 x 100 10 showed the conservation of the amide I,
II and III bands (Figure 2, 3rd line from
W0 bottom and top line), confirming the
presence of TSF within the structures. In
where W0 is the dry weight before GPTMS functionalized TSF, a band at
immersion in de-ionised water. W1 is the dry 1100.65 cm-1 and a weak band at 850.67 cm-
weight after immersion in de-ionised water. 1
assigned to Si-O-Si and Si-OH respectively
In vitro biodegradation test were seen (Figure 2, 3rd line from bottom
50 mg of the scaffolds were and top line). This indicates that some of the
degraded at 37 ºC in PBS containing 1 methoxysilane groups in GPTMS were
unit/ml of protease XIV (Sigma) and 0.01 already hydrolyzed to form silanol groups,
wt% sodium azide as an antibacterial agent. and some of these groups further condensed
The enzyme solution was changed every 2 to become Si-O-Si. On the epoxy end of
days to ensure continuous enzyme activity. GPTMS, the spectrum of the GPTMS-
At 1, 3, 5, 7 and 10 days after degradation, functionalized TSF showed disappearance of
the remaining scaffolds were washed with the bands assigned to the bonds in the epoxy
DI water and freeze-dried. The dried ring (Figure 2, 3rd line from bottom). These
scaffolds were weighed and the percentage results confirmed that the epoxy ring was
of remaining weight was calculated as opened and used for bonding with amine
follows: groups in TSF. Furthermore, the more
prominent Si-O-Si and Si-OH bands were
Remaining weight (%) = (Wt/W0) x 100 observed in TSF-BCSG hybrids. That was
the result of the addition of, BCSG to the
where W0 and Wt represent the weight of system (Figure 2, top line).
scaffolds before and after degradation at 3.2 Morphology and pore structure
different time. The morphology of the scaffolds was
observed under SEM (Figure 3). Both TSF-
3. Results and Discussion BCSG 0 and TSF-BCSG 10 had pores with
3.1 Chemical structures and crosslinking
TSF-BCSG 0
Figure 2. The FTIR spectra of GPTMS, SF,

GPTMS-functionalized TSF and TSF-BCSG
10
Pore size: 128±36 µm
interconnection. The crosslinked scaffolds TSF-BCSG 10
had a larger pore diameter of 177± 74 µm
and the pores were more evenly distributed,
while the uncrosslinked scaffolds had a
smaller pore diameter of 128±36 µm. This
suggests that GPTMS affects the
morphology of the scaffolds. The greater
size of the pores in TSF-BCSG 10 could be
explained by the fact that GPTMS is a
foaming agent.11
3.3 Stability in water
The stability of the scaffolds was
determined by its insolubility in water
(Figure 4). The stability is hence inversely
proportional to weight loss. Apparently, Pore size: 177± 74 µm
TSF-BCSG 0 lost their weight twice as
much did the TSF-BCSG 10. That was Figure 3. The electron micrographs of TSF-
because the crosslinking stabilized the BCSG 0 and TSF-BCSG 10
scaffolds by forming covalent linkages
between the silicate network and the fibroin
network. In contrast, the two networks in
TSF-BCSG 0 had not been crosslinked, and
thus were hold together only by physical
entanglement and weak intermolecular
interactions such as van der Waals forces.
3.4 In vitro biodegradation
Degradation of the scaffolds in
protease XIV reaffirmed the success of
crosslinking. Both types of the scaffolds lost
their weight overtime in the protease XIV
solution (Figure 5). However, TSF-BCSG 0
always lost the weight more than TSF-
BCSG 10 over 10 days, reconfirming that Figure 4. Percentage of weight loss of TSF-
BCSG 0 and TSF-BCSG 10 after immersion
in de-ionised water for 24 h at 37 °C
the crosslinking by GPTMS stabilized the Examination of bioactivity and
scaffolds. Interestingly, degradation of the biocompatibility is needed in the future.
two types of the scaffolds during the first
day appeared to be most different, as TSF- Acknowledgements
BCSG 0 lost more than half of its weight The authors would like to express
while TSF-BCSG 10 lost less than 20% of our gratitude to all members of the
its weight. This agrees with our previously Biomaterial Laboratory, Faculty of
described experiment. After that, the rate of ngineering, Chulalongkorn University.
weight lost progressively decreased. This
experiment provides a starting point for References
tuning the degradation rate. 1. Khan, Y.; Yaszemski, M. J.; Mikos, A.
G.; Laurencin, C. T. J. Bone Jt. Surg.
2008, 90, 36-42.
2. Guenaelle, B.; David, M.; Magali, C.;
Luc, M.; Laurence, V. Tissue Eng. 2012,
21, 133-156.
3. Li, G.; Liu, H.; Wang, J. Mater. Sci.
Eng. C 2012, 32, 627-636.
4. Kundu, B.; Rajkhowa, R.; Kundu, S.;
Wang, X. Adv. Drug Deliv. Rev. 2013,
65, 457-470.
5. Fu, Q.; Saiz, E.; Rahaman, M.; Tomsia,
Figure 5. Percentage of remaining weight of A. Mater. Sci. Eng. C 2011, 31, 1245-
TSF-BCSG 0 and TSF-BCSG 10 after in 1256.
vitro degradation in protease XIV solution 6. Aza, P. N.; Guitan, F.; Aza, S. Scr.
for 10 days at 37 °C Metall. Mater. 1994, 31, 1001–1005.
7. Pereira, M. M.; Jones, J. R.; Orefice, R.
4. Conclusion L.; Hench, L. L. J. Mater. Sci. 2005, 16,
The TSF-BCSG hybrid scaffolds 1045-1050.
were successfully obtained using the 8. Ren, L.; Tsuru, K.; Hayakava, S.; Osaka,
foaming techniques. The FTIR results A. J. Sol-gel Sci. Technol. 2001, 21,
confirmed the crosslinking of TSF and 115-121.
BCSG through GPTMS. The resulting 9. Mahony, O.; Tsigkou, O.; Ionescu, C.;
GPTMS-crosslinked scaffolds showed the Minelli, C.; Hanly, R.; Ling, L.; Smith,
desirable interconnected pores. The M. E.; Stevens, M. M.; Jones, J.R. Adv.
crosslinked scaffolds also showed enhanced Funct. Mater. 2010, 20, 3835-3845.
stability in water, which is necessary for 10. Kim, U. J.; Park, J.; Kim, H. J.; Wada,
implantation. The work herein provides a M., Kaplan, D. L. Biomater. 2005, 26,
foundation for reinforcing brittle bio- 2775-2785.
activeglass with proteinaceous polymers. 11. Lei, B.; Wang, L.; Chen, X.; Chae, S. J.
Mater. Chem. B 2013, 1, 5153-5162.
The effect of nucleating agent (paraffin) on the supercooling in
encapsulated microcapsule for building application
Piyalak Ngernchuklin1*, Priyagorn Pholsrimuang2, Nestchanok Yongpraderm1,
Arjin Boonrung1, Preeyaporn Chaiyasat2, Amorn Chaiyasat2
1
Expert Center of Innovative Materials, Thailand Institute Scientific and Technological Research (TISTR),
Khlong 5, Khlong Luang, Pathumthani 12120, Thailand
2
Department of Chemistry, Faculty of Science and Technology, Rajamangala University of Technology
Thanyaburi, Klong 6, Thanyaburi, Pathumthani 12110, Thailand
*E-mail: piyalak@tistr.or.th
Abstract:
Phase change materials (PCMs) are attractive for many industrial applications such as
solar heat storages, textiles and building applications. PCM like paraffin wax was
encapsulated by polymer shell to increase heat transfer area, control the volume change of the
storage materials as phase change occur and protect the influence from the outside
environment. In this research, paraffin wax (Rubitherm®28:RT28) was encapsulated by
polymer shell from poly(methyl methacrylate-co-divinylbenzene) (P(MMA-co-DVB)) by a
microsuspension polymerization technique. In order to prevent supercooling effect, the
amount of paraffin: Tc 55 °C (0, 5, 10 and 15 wt% of RT28) as a nucleating agent was
studied. The microcapsules were characterized by using differential scanning calorimetry
(DSC) to observe the crystallization temperature (Tc), thermogravimetric analysis (TGA) for
degradation temperature, scanning electron microscope (SEM) for microstructure of
microcapsules and fourier transform infrared spectrometer (FT-IR) to compare the functional
group between bulk RT28 and encapsulated RT28. From the results, we obtained that when
adding the amount of paraffin (Tc 55 °C) at least 5 wt%, the crystallization temperatures of
encapsulated RT28 (18 and 24°C) was almost the same as that of bulk RT28 (25°C). In
addition, the latent heat of crystallization (∆Hc 182 J/g-core) of encapsulated RT28 was close
to that of bulk RT28 (∆Hc 195 J/g-RT28). In summary, the amount of paraffin (Tc 55 °C) as
nucleating agent can reduce supercooling in microcapsules. Therefore, P(MMA-co-DVB)
shell encapsulated Rubitherm®28 (RT28) can be used as thermal energy storage material for
building application.
1. Introduction when the temperature is decreased by

Recently, heat storage energy cooling, PCMs release energy and change
materials or phase change materials (PCMs) from liquid to solid phase.4 Among of
have been applied to textile technology,1 PCMs, the group of paraffin wax including
building applications2 and air condition.3 octadecane, hexadecane and tetradecane has
The PCM principle is the conversion of drawn attentions due to its high heat energy
solid to liquid, and vice versa, according to capacity, non-toxicity, non-corrosion, high
the change of temperature. At low stability and low cost but low thermal
temperature, PCMs are in the solid state. conductivity. To improve thermal
When the temperature is increased by conductivity, the encapsulation of paraffin
heating, PCMs absorb energy until reach to wax has been intensely developed to
the transition temperature. The phase increase surface area and heat transfer. The
changes from solid to liquid. In contrast, various encapsulation methods are solvent
evaporation, spray drying interfacial Divinylbenzene; DVB (Aldrich, Wisconsin,
polymerization and suspension USA; purity 80%) was washed with 1 M
polymerization (SP). The most attractive sodium hydroxide solution (NaOH; BDH
method is SP which uses internal phase Prolabo, Leuven, Belgium) to remove the
separation mechanism to generate polymerization inhibitors. RT28 (Rubitherm
microcapsule with high encapsulation Technologies GmbH, Berlin, Germany;
efficiency. However, the drawback of Commercial grade) was used as received.
encapsulation is supercooling effect of the Reagent-grade benzoyl peroxide; BPO
crystallization temperature (Tc) of which is (Merck, Munich, Germany) was purified by
lower than the pure paraffin wax.5 Such recrystallization. Paraffin (Tc 55 °C)
effect limits performance (absorbing- (Commercial grade, China) was used as the
releasing heat energy) of encapsulated nucleating agents as received. Poly(vinyl
microcapsules. Therefore, a method used to alcohol); PVA (Aldrich, Wisconsin, USA;
reduce supercooling effect is adding degree of saponification 87-90% molecular
nucleating agents. The purpose is to induce weight 3-7x104 g mol-1) was used as
crystallization in PCM and release heat at received.
normal temperature. To decrease 2.2 Synthesis of polymer microcapsule
6
supercooling, types and the amount of The microcapsules P(MMA-co-
nucleating agents (1-tetredecanol, paraffin DVB)/RT28 were synthesized from RT28
and 1-octadecanal) were studied in the encapsulated by P(MMA-co-DVB) using
system of co-polymer urea-melamine- microsuspension polymerization technique.
formaldehyde (shell) and n-octadecane, n- Firstly, two monomers (MMA and DVB)
nonadecane, and n-eicosane (core material) were homogenously mixed with BPO acted
by in-situ synthesization. It was found that as initiator. Secondly, RT28 and paraffin (Tc
the capsules from octadecane (core) with 1- 55 °C) as oil phase were mixed at 50-60 oC.
octadecanal (nucleating agent) could reduce Then, the two mixers were poured into the
supercooling. But such nucleating agent surfactant (1wt% PVA) and continuously
affected to broader size distribution and homogenized at 5000 rpm for 5 min until
larger capsules. obtaining the monomer droplets. After that,
Supercooling effect was found in our monomer droplet suspension was poured
encapsulation system. So, to reduce into round bottom flask sealed with silicone
supercooling in microcapsules of P(MMA- rubber septum. To keep such suspension in
co-DVB) shell encapsulated RT28 core, the vacuum system, air was pumped out
amount of nucleating agent (paraffin: Tc 55 alternatively nitrogen gas was purged in five
°C) to RT28 was studied by times. Finally, suspension in flask was
microsuspension polymerization (CRP) placed in silicone oil bath at 80 oC together
technique. with stirring for 8 hr to complete
polymerization process presented as
2. Materials and Methods microcapsules. The preparation of polymer
2.1 Materials microcapsules is shown in Figure 1. The
Methyl methacrylate; MMA study conditions for synthesization of
(Aldrich, Wisconsin, USA; purity 99%) was polymer microcapsules are displayed in
purified by passing through a column Table 1.
packed with basic aluminium oxide.
Figure 1. Schematic diagram for the preparation of copolymer/RT28 microcapsules by
microsuspension polymerization
Table 1. Conditions of microcapsule preparation at various paraffin concentrations

Concentration of paraffin (wt%)
Ingredients
5 10 15
MMA (g) 41.0 41.0 41.0
DVB (g) 21.0 21.0 21.0
RT 28 (g) 28.5 27.0 25.5
Paraffin (g) 1.5 3.0 4.5
BPO (g) 5.6 5.6 5.6
PVA aqueous
(g) 450.0 450.0 450.0
solution (1wt%)
Remarks
- Monomer/RT 28 ratio is 70:30 wt.%
- MMA/DVB ratio is 70:30 wt.%
2.3 Polymer capsules characterization by thermogravimetric analyzer (TGA, STA

Morphology of microcapsules was 449F3, Netzsch, German) at 30-600 oC with
observed by scanning electron microscope scanning rate 10 oC/min. Functional groups
(SEM, JEOL-JSM-5410LV, Japan) after of microcapsules were characterized by
washing by 2-propanol and drying. To fourier transform infrared spectroscopy
investigate the amount of polymer particle in (FTIR, IR Prestige 21 Shimadzu, Japan)
water, emulsion was set until polymer Heat of crystallization (ΔHc) was calculated
microcapsules were separated and followed from the equation (1)
by measuring of free polymer particle in
water. Thermal properties such as heat of A = (B/C) x 100 (1)
crystallization (ΔHc) and crystallization where
temperature (Tc) of poly(MMA-co-DVB) A = ΔHc of the encapsulated RT28 in joule
encapsulated RT28 were examined by per gram of encapsulated RT28 (J/g-RT28)
differential scanning calorimeter (DSC, 200 B = ΔHc of RT28 of dried microcapsules
F3, Netzsch, German) at 0-70 oC with from DSC graph (joule/gram of capsule)
scanning rate of 5 oC/min after 2-propanol C = %RT28 of dried microcapsules obtained
washing and drying in vacuum. The total from TGA thermogram.
amount of RT28 and paraffin were analyzed
3.1 Effect of paraffin to microcapsules
morphology
In this study, encapsulated
microcapsules were synthesized by CRP
technique. Paraffin (RT28) used as a thermal
energy storage material and BPO as initiator
were homogenously mixed and stirred to
form many monomer droplets distributed in
surfactant solution (PVA solution). During
polymerization, such monomers reacted Figure 2. SEM micrographs (a, b, c and d)
each other and then form long-chain of P(MMA-DVB)/RT28 microcapsules add
polymers until they reached to critical chain paraffin (wt%): 0 (a); 5 (b); 10 (c) and 15 (d)
length. After this reaction, polymer chain prepared by microsuspension CRP
could not dissolve into thermal energy
storage material within the droplets resulting As a result, RT28 had high density
in phase separation occurred. Excess together with low shell strength. Moreover,
polymer was moved to the surface and some monomers could dissolve into water
changed to the shell encapsulated thermal phase and then form as free polymers
energy material as a core.7 However, resulting in indented microcapsules, as
supercooling effect of encapsulated shown in Figure 2.
microcapsules had occurred. To solve this
problem, adding of nucleating agent to
increase Tc of microcapsules close to Tc of
pure thermal energy material was
performed. From previous research,6 the
nucleating agent (1-octadecanal) was not
only decreasing of cooling effect but also
affecting to particle size morphology and Figure 3. Suspensions after 1 week of
latent heat of microcapsules. Therefore, P(MMA-DVB)/RT28 microcapsules added
effect of the amount of nucleating agent paraffin (wt%): 0 (a); 5 (b); 10 (c) and 15 (d)
(paraffin Tc 55 oC) on morphology and by microsuspension (CRP)
thermal properties of microcapsules was
intensively investigated in this study. The behavior of microcapsule
The experimental conditions suspension was examined and found that
followed by Table 1 were investigated by suspensions after 7 days setting were
adding paraffin at 0, 5, 10 and 15wt%. The completely separated into two phases.
percent change of monomer to polymer at Lower part was the creamy microcapsules
various paraffin concentrations was found with higher density compared to water. The
that obtained microcapsules were smooth, upper part was the turbidity water from
the round shape and partially indented dissolved monomer as the free polymer
surface. This was because the decreasing of particles and polymerization took place by
temperatures between synthesis (80 oC) and homogeneous nucleation in emulsion
storage (25 oC) temperatures, resulting in the polymerization of water phase as displayed
transformation RT28 from liquid to solid in Figure 3.
phase.
reduce supercooling (Tc of microcapsules
with 5wt% paraffin = 18 and 24.5 oC and Tc
of pure RT28= 16.2 and 18 oC) as shown in
Table 3. This was because paraffin (Tc 55
°C) firstly generated crystals and then
induced to crystallization of RT28. As a
result, the crystallization temperatures (Tc)
of microcapsules were higher and close to
pure RT28.
Figure 4. TGA thermogram of copolymer
microcapsule using paraffin for RT28 pure
(a), Paraffin pure (Tc 55) (b), 5wt% (c),
10wt% (d) and 15wt% (e) at ratio of
monomer:RT28 = 70:30w/w% and
MMA:DVB = 70:30 w/w%
3.2 Thermal properties of RT 28 in

microcapsules
Figure 4 shows the TGA
thermograms of pure RT28, paraffin and
copolymer microcapsules (after the removal
of the free PMMA particles). The Figure 5. DSC thermograms of copolymer
information was used to calculate % loading microcapsules using paraffin for Paraffin
of encapsulated RT28. The results showed pure (a), RT28 pure (b), 5wt% (c), 10wt%
that all concentrations of paraffin provided (d) and 15wt% (e) at ratio of
experimental loading value were comparable monomer:RT28 = 70:30w/w% and
to the calculated one shown in Table 2. MMA:DVB = 70:30w/w%
From the result, it could conclude that high
encapsulation efficiency was achieved in Table 3. Latent heat (ΔH c ) and
this study system. crystallization temperatures (Tc) of RT28 in
microcapsule
Table 2. % Loading and encapsulation
efficiency of RT28 in microcapsules at Thermal properties
paraffin
monomer:RT28 of 70:30wt% (wt%) ΔH (J/g-core) T (oC)
c c
Loading (%) 0 186.17 16.20 21.30

paraffin Encapsulation
(wt%) (wt%) 5 182.21 18.00 24.50
Experiment Calculation
5 29.96 31.69 94.54 10 166.25 14.50 19.50
10 36.45 32.14 100.00 15 183.35 13.50 20.30
15 29.25 32.25 90.69 RT28 195.20 22.30 23.80

paraffin 151.50 55.10 -
Thermal property showed that heat
of crystallization values (ΔHc) of The FTIR spectra of RT28 pure,
microcapsules added paraffin at 5, 10 and paraffin pure, 0wt%, 5wt%, 10wt% and
15wt% were close to ΔHc of pure RT28 15wt% of paraffin in capsules show in
presented in Figure 5 and Table 3. In case of Figure 6. The absorbance peaks of pure
the crystallization temperature (Tc), it was RT28 at 2852 and 2922 cm-1 were sample
found that at least 5wt% of paraffin could alkane (C-H) asymmetric stretching
vibration. The peak at 719 cm-1 represented amount of nucleating agent (paraffin Tc 55)
the in-plane rocking vibration of the CH2 on the properties of microcapsules (RT28-
group and the peaks at 1377 and 1167 cm-1 P(MMA-co-DVB)) prepared by
were characteristic of RT28. The FTIR microsuspension polymerization technique
transmission spectra of pure paraffin at was conducted. It was found that the amount
2846, 2916 and 719 cm-1 were associated of paraffin at least 5wt%, microcapsules
with the aliphatic C-H stretching vibration provided smooth and round shape, 90-100
and the in-plane rocking vibration of the percent of loading with high encapsulation
CH2 group. The peaks at 1377 and 1162 efficiency. Importantly, the nucleating
cm-1 were associated with C-H bending and agent could reduce supercooling effect while
were characteristic of paraffin. The intense keeping of the same properties of ΔHc of
peaks of polymer shell at 1726 and 1145 pure RT28 in microcapsules. Finally,
cm-1 appeared due to the presence of ester Rubitherm®28 (RT28) encapsulated by
carbonyl group (C-O) and C-O of ester bond P(MMA-co-DVB) shell can be used as
stretching vibration, respectively. thermal energy storage material for building
applications.
Acknowledgements
This work was partially supported by
The National Research Council, Thailand
(given to P.N.) and Thailand Institute of
Scientific and Technological Research.
References
1. Azizi, N.; Ladhari, N.; Majdoub, M.
Application in Textile. Asian J. Text
2011, 1 (3), 130-137.
Figure. 6 FTIR spectra of copolymer
2. Sánchez, S. L.; Rodríguez, J. F.; Romero,
microcapsule with various amount of
A.; Sánchez, P. J. Appl. Polym. Sci. 2012,
paraffin:RT28 pure (a), paraffin pure (b),
124 (6), 4809-4818.
0wt% (c), 5wt% (d), 10wt% (e) and
3. Agyenim, F.; Hewitt, N.; Eames, P.;
15wt% (f) at ratio of monomer:RT28
Smyth, M. Renew. Sust. Energy Rev.
70:30w/w% and MMA:DVB 70:30w/w%
2010, 14, 615-628.
4. Qiu, X.; Song, G.; Chu, X.; Li, X.; Tang,
The FT-IR peaks of 0wt%, 5wt%,
G. Solal Energy 2013, 91, 212-220.
10wt% and 15wt% paraffin in capsules were
5. Sánchez, S. L.; Romero, J. F. R.;
matched with those peaks observed in the
Borreguero, A. M.; Carmona, M.;
pure RT28, pure paraffin and polymer shell.
Sánchez, P. Chem. Eng. J. 2010, 157,
These indicated that RT28 has been
216-222.
successfully encapsulated by polymer shell.
6. Zhang, X.-X.; Fan, Y.-F.; Tao, X.-M.;
The nucleating agent (paraffin: Tc 55 °C) has
Yick, K.-L. J. Colloid Interf. Sci. 2005,
been completely incorporated into the core
281, 299–306.
of the PCM microcapsules in this test.
7. Chaiyasat, P.; Ogino, Y.; Suzuki, T.;
Minami, H.; Okubo, M. Colloid Polym.
4. Conclusion
Sci. 2008, 286, 217-223.
In this research, the effect of the
Development of natural rubber based composite foam containing magnetite
nanoparticles for oil spill removal
Arisa Jaiyu*, Julaluk Pannoi, Passakorn Sueprasit, Siriporn Thongon
Expert Centre of Innovative Materials, Thailand Institute of Scientific and Technological Research,
Khlong Luang, Pathumthani 12120, Thailand
*E-mail: arisa@tistr.or.th
Abstract:
Marine oil spill is considered to be a serious pollution problem because of the
frequent occurrence in the last few decades, resulting in the acute and long-term effect on
human and marine animal health, environment and marine ecosystem. The development of
cost-effective and appropriate sorbent material for the oil spill removal seems necessary. In
this study, natural rubber latex, biodegradable and biocompatible polymer, was chosen as a
starting material for the oil absorbing foam. The magnetite nanoparticle was synthesized from
ferrous and ferric ions in the molar ratio 1:2. The natural rubber based composite foam
containing magnetite nanoparticles (NRF-MN) was successfully prepared via the Dunlop
process. The oil absorption properties and reusability of the composite foams were studied. It
was found that NRF-MN showed high oil absorption properties up to 12 g of crude oil/g and
could be utilized at least 5 times by a simple squeezing method. This study reveals that NRF-
MN from natural rubber latex is an efficient, eco-friendly, economic material for oil spill
removal.
1. Introduction the major drawback is that it is not

According to the statistics of the oil biodegradable and impossible to reuse.
spill in Thailand during 1973-2014, more Natural Rubber (NR) is the one of
than 215 times of oil spill incidences major economic plant in Thailand but 90%
occurred including ten of severe marine oil of NR is exported in raw rubber form which
spills (more than 20,000 L) resulting in the has low value-added. The Development of
acute and long-term effect on human and high value-added products from NR has
marine animal health, environment and gained more attention in Thailand.9
marine ecosystem.1,2 There are different Recently, modified NR was developed to
techniques to clean up the oil spill from use as sorbent material for oil spill
marine environment such as chemical removal.10,11 In 2013, Venkatanarasimhan
treatment using dispersants, in situ burning, developed ferromagnetic NR sorbent
bioremediation using microorganism and attracted to the magnet for oil spill treatment
physical adsorption using boom, skimmers by using dry NR mix with magnetite
or oil absorbent.3 Among these techniques, nanoparticle in chloroform solution.4
the use of sorbent materials for oil spill However to best of our knowledge, the
removal is considered to be one of the most ferromagnetic sorbent prepared directly
effective technique. A variety of sorbents from NR latex has not been reported before.
have been used for oil spill removal such as In this study, natural rubber latex,
natural materials (milkweed, cotton fiber, biodegradable and biocompatible polymer,
straw etc.), synthetic materials (polypropy was chosen as a starting materials for the oil
lene, polyurethane, polystyrene) and inor absorbing foam. For preparation of NR
ganic materials (organoclay, silica, fly ash based composite foam containing magnetite
etc.).4-7 Polypropylene is the one of nanoparticles (NRF-MN), the magnetite
commercial sorbent for oil spill removal8 but nanoparticle (MN) was synthesized and
incorporated in NR matrix foam The oil The ingredients for preparation of
absorption properties and reusability of the NRF-MN are shown in Table 2. Firstly, the
NRF-MN were studied. NR latex was stirred for 1 hour to remove
ammonia. Then, K-oleate, ZDEC, ZMBT,
2. Materials and Methods Sulfur, CPL were added into the NR latex
2.1 Materials and the NR compounding mixture was
10% Potassium Oleate (K-Oleate), stirred for 3 hours and then matured at room
50% zinc dietyl dithiocarbamate (ZDEC), temperature at least 24 hours. After
50% zinc-2-mercaptobenzotiazole (ZMBT), maturation, the NR compounding mixture
60% Sulfur (S), 50% butylated reaction was whipped into foam using a batch
product of p-cresol and dicyclo pentadiene foaming machine (mixer) via Dunlop
(CPL), 30% diphenyl guanidine (DPG), process until the volume was increased up to
50% zinc oxide (ZnO) 12.5% sodium three times. The MN, DPG, ZnO and SSF
silicofluoride (SSF) were all commercial were added and the foam was transferred
grades and used as received. Natural rubber into the mould (5 mm thickness). The foam
latex was purchased from Lucky Four was allowed to gel for 3-5 minutes at room
Company and characterized before using temperature and then cured in a hot air oven
(Table 1). at 100 ºC for 3 hours. After curing process,
the foam was stripped from the mould and
Table 1. Properties of natural rubber latex washed with water to remove the un-reacted
Properties Value elements. Then, the foam was dried in a hot
Total Solids Content (TSC) 62 wt% air oven at 80 Cº for 6-8 hours.13,14
Dry Rubber Content (DRC) 60 wt%
Ammonia Content 0.65 wt% Table 2. Formulation of natural rubber
Volatile Fatty Acid number based composite foam containing magnetite
(VFA number) 0.02 nanoparticles
KOH number 0.7 Ingredients Dry weight (pphr)
pH 10 60% NR latex 100
Mechanical Stability (MST) 1183 s
10% K-Oleate 1
Sludge Content 0.008 wt%
50% ZDEC 1.5
Coagulum Content 0.021 wt%
50% ZMBT 1
60% Sulfur 2.5
2.2 Preparation of magnetite nanoparticle
50% CPL 2.5
(MN)
MN was prepared by mixing 1:2 of 30% DPG 1.5
ferrous sulfate heptahydrate and ferric 50% ZnO 0.6
chloride hexahydrate in aqueous solution. 12.5% SSF 5
The solution mixture was sonicated for 1 MN 4
hour. After that, the MN was precipitated by
pouring this solution mixture to an ammonia 2.4 Characterization
solution (pH = 10). The solution mixture The MN, NRF-M and the original
was stirred at room temperature for 30 natural rubber foam (NRF) were identified
minutes and allowed to precipitate. The MN and characterization by X-ray diffraction
was separated by centrifuge at 9,000 rpm (XRD; Rigaku SmartLab). The crystallite
and rinsed with DI water at least 2 times. size of magnetite was determined from the
Then, it was dried at 50 ºC under vacuum full width at half maximum of XRD peak
for overnight.4,12 and Sherrer’s formula. The morphologies of
2.3 Preparation of natural rubber based the NRF-MN was observed by Field
composite foam containing magnetite Emission Scanning Electron Microscope
nanoparticles (NRF-MN) (FE-SEM; JEOL JSM-6304F). Photographic
images of the MN, NRF and NRF-MN were
captured with the digital camera.
2.5 Oil absorption Measurements MN
Two different petroleum oils (Crude
oil and diesel fuel) were used in this study.
NRF-MN (2x2x0.5 cm3) was weighted
accurately using a digital balance of the
resolution of 0.01 g. Then, NRF-MN was
immersed into petroleum oil at selected time Magnetite Fe3O4 from XRD library
and weighted again after that. The weight of
oil absorbed per gram of NRF-MN was
calculated by the following equation;
Woil absorbed /1g= ( Wa-Wi)/Wi
in which, Woil absorbed /1g is the weight of oil

absorbed per 1 g of NRF-MN, Wi represents
the initial weight of NRF-MN (gram), Wa Figure 1. The X-ray diffraction pattern of
represents the weight of the NRF-MN after MN and Manetite Fe3O4 from XRD library
immersed in oil.

3.1 Characterization of MN and NRF-MN
MN was successfully synthesized by
mixing 1:2 ratio of Fe2+/Fe3+ in the aqueous
solution. After precipitation and drying
process, black powder was obtained and
identified by XRD. It was found that MN
powder was corresponding to the reference
Magnetite Fe3O4 from XRD library (Figure
1). MN was ferromagnetic attracted to the
Figure 2. Photograph of MN and NR-MN
magnet (Figure 2). The crystallite size of
attracted to the magnet
MN was deduced to be 19 ± 0.5 nm from the
full width at half maximum of XRD peak
and Sherrer’s formula (calculated from 3
most intense XRD peak).
The NRF-MN was successfully
prepared using a batch foaming machine
(mixer) via the Dunlop process. The color of
200 µm 30x 200 µm 30x
NRF-MN was grey because the MN was
dispersed in NR matrix and then the NRF-
MN was strongly attracted to the magnet
(Figure 2). Density of NRF-MN was 0.17
±0.02 g/cm3 which calculated from the mass
and the volume of the NRF-MN. The
200 µm 45x
morphologies of NRF-MN (Figure 3) clearly 200 µm 130x
revealed the porous and the open cell Figure 3. SEM image of a) surface, b) cross
structure of the foam and the MN particles section, c) inner and d) inner with higher
seemed to bind well with the NR matrix. magnification of NRF-MN
3.2 Oil absorption properties and repeated for 5 cycles. It was found that
reusability of NRF-MN NRF-MN could be utilized at least 5 times
The oil absorption properties of and the oil absorption capacity of NRF-MN
NRF-MN were determined in this study. slightly decreased with an increase in the
The NRF-MN exhibited high oil absorption number of cycles. The relationship between
properties up to 12.1 g of crude oil/gNRF-MN the number of cycles and weight of oil
and 10.6 g of diesel oil/gNRF-MN. The absorbed is shown is Figure 5.
relationship between the contact time and
weight of oil absorbed at 0.17, 0.33, 0.67, 1, 12
5, 10, 20 and 30 minute of the absorption
process was presented. The oil absorption 10
rate throughout the first minute was rapid.
(Per Gram of Absorbent)

8
Weight of Oil Absorbed

The oil absorption capacity is almost
completed in 5 minutes for diesel fuel and 6
20 minutes for crude oil. This indicated that 4

the NRF-MN had a higher removal rate for
diesel fuel compared to the crude oil 2
because less viscous oil (diesel fuel) 0

penetrates by capillary action very fast when 1 2 3 4 5
Cycles
compared to highly viscous oil. On the
other hand, the NRF-MN had higher Figure 5. The relationship between the
maximum oil absorption capacity for crude number of cycles and weight of oil absorbed
oil compared to the diesel oil after 10
minutes of contact time (Figure 4). This The preliminary study of oil spill
result was may be due to the greater removal from water was studied in this
cohesion properties between NRF-MN and experiment. Crude oil was dispersed in the
crude oil.11 water surface in petri dish (Figure 6a) and
then the NRF-MN is dipped into the mixture
14.0 of oil/water (Figure 6b). After 10 minutes,
12.0
most of the crude oil was removed from the
water. The NRF-MN after oil absorbed
could be pulled off from the mixture of
(Per Gram of Absorbent)
10.0
Weight of Oil Absorbed
8.0
oil/water by using the magnet (Figure 6c,6d)
crude oil
6.0 diesel fuel a) b)

4.0
2.0
0.0
0 5 10 15 20 25 30
Contact time (min)
Figure 4. The relationship between the

contact time and weight of oil absorbed c) d)
The reusability of NRF-MN using

diesel fuel was determined at the contact
time of 5 min for 5 cycles. After the oil
absorption process, the NRF-MN was
simply squeezed to remove the diesel fuel
Figure 6. Photograph of the removal of
and then the NRF-MN was immersed into
crude oil from the mixture of oil/water by
diesel fuel again. These processes were
NRF-MN
Moreover, the NRF-MN after References
absorbed crude oil or diesel fuel was able to 1. http://www.md.go.th (Accessed 2018-
float on the water surface as shown in Figure 01-02)
7. This indicated that the NRF-MN could be 2. http://www.nationmultimedia.com/natio
utilized as oil absorbent for oil spill from the nat/Recent-oil-spill-10-times-larger-
water, river or sea. than-one-in-2013-30272382.html
a) b) c) (Accessed 2018-01-02)
3. Ornitz, B. E.; Champ, M. A. 2002, Oil
Spills First Principles: Prevention and
Best Response; Elsevier Science Ltd.
4. Venkatanarasimhani, S.; Raghavachari
Figure 7. Photograph of NRF-MN after D. J. Mater. Chem. A, 2013, 1, 868-876.
crude oil and diesel fuel absorbed 5. Amin, J. S.; Abkenar, M. V.;
Zendehboud, S. Ind. Eng. Chem. Res.
4. Conclusion 2015, 54, 10615−10621.
NRF-MN was successfully prepared 6. Zhou, X.; Zhang, Z.; Xu, X.; Men, X.;
via Dunlop process. The oil absorption Zhu, X. Ind. Eng. Chem. Res. 2013, 52,
properties and reusability of the composite 9411−9416.
foams were studied. It was found that NRF- 7. Zhu, Q.; Pan, Q.; Liu, F. J. Phys. Chem.
MN showed high oil absorption properties 2011, 115, 17464–17470.
up to 12 g of crude oil/g and could be 8. Wei, Q. F.; Mather, R. R.;
utilized at least 5 times by a simple Fotheringham, A. F.; Yang, R. D. Mar.
squeezing method. The NRF-MN before and Pollut. Bull. 2003, 46, 780–783.
after oil absorbed were attracted to the 9. https://www.ukessays.com/essays/econo
magnet because of the magnetite econ/rubber-export-industry-in-thailand-
nanoparticle in NR matrix. This procedure economics-essay.php (Accessed 2018-
could be applied in the real field usage by 01-02)
using the powerful magnet to pull off the 10. Danwanichakul, P.; Jaroenkarn, S.; Jum-
NRF-MN sheet from the water surface after pathi, P.; Dechojarassri, D. Bachelor’s
oil absorbed. The combinations of high oil degree Thesis, Thammasat University,
absorption properties, buoyancy and 2006.
ferromagnetic of NRF-MN make it a 11. Aronu, U. E. Master of Science Thesis,
promising sorbent material for oil spill University College of Boras, 2007.
removal from water. 12. Ratcha A.; Yoosak B.; Kongparakul, S.
Adv. Mat. Res. 2014, 844, 385-390.
Acknowledgements 13. Mei, L. M. Rubber Technology
The authors gratefully acknowledge Development 2010, 10, 23-26.
Thailand Institute of Scientific and 14. Roslim, R.; Amir, M. Y.; Augurio, P. T.
Technological Research (TISTR) for J. Eng. Sci. 2012, 8, 15–27.
financial, material and instruments support.
Fabrication of superhydrophobic gold microstructure
on polymer substrates
Siriwan Boonmeewiriya1, Supeera Nootchanat2, Sanong Ekgasit2, Kanet Wongravee2*
1
Program in Petrochemistry and Polymer Science, Faculty of Science,
2
Sensor Research Unit, Department of Chemistry, Faculty of Science,
*E-mail: kanet.w@chula.ac.th
Abstract:
Superhydrophobic surface has been used in the wide potential applications including
textiles, medical instruments, etc. In this study, the superhydrophobic gold films were
fabricated by reduction reaction between solution of sodium formate (HCOONa) and
chloroauric acid (HAuCl4) on polymer substrates. The effects of surface tension and
functional groups of the substrates such as glass slide, acrylic, and polydimethylsiloxane
(PDMS) on the hydrophobicity of the generated gold film were deeply investigated. Contact
angle was measured by in-house goniometer and the aggregated gold microstructures were
monitored by SEM. It was found that the superhydrophobic surface was created by roughness
from the aggregated gold microparticles (AuMPs). In the case, the gold growth solution can
be easily formed as a droplet on the surface with low γc (PDMS in the case). Therefore, the
deposition of AgMPs on the substrate was dense and later formed porous structure. Water
contact angle of 153⁰ was observed using these fabricated superhydrophobic gold films on
the PDMS substrates.
1. Introduction superhydrophobic surface. There are several

Superhydrophobicity is an researches focusing on fabrication of Au
exceptional property of non-wettable particles on the substrate to become a
surface.1 In the nature, Lotus leaf is a good surperhydrophobic material. In 2011, Zhu, et
example representing the superhydrophobic al.2 synthesized gold film with dendrite
surface. The superhydrophobic surfaces nanostructure on porous Si surface. It
have attracted great interest in recent years presented a superhydrophobic surface that
as they are self-cleaning surface and can be exhibited WCA near to 180⁰. Ishida, et al.3
applied in several applications such as developed the deposition process of gold
satellite dishes, solar cell, outer architectural nanoparticles (AuNPs) on different polymer
glass, textiles with waterproof and blood surfaces in 2008. Recently, the researcher
vessel replacement. To define as the developed Au on polymers using different
superhydrophobic surface, these surfaces synthesis protocol on many types of polymer
should provide an apparent water contract substrates. The fabricated Au-polymer
angle (WCA) of a water droplet more than substrates were used in many applications4-7
150o. such as electronic, sensor, catalyst or
Two ways to fabricate surface with Surface enhance raman spectroscopy
high WCA are to use low surface energy (SERS) substrate. However, superhydro-
materials or to modify the roughness of the phobic properties of Au film on polymer
material surface. Modified roughness substrates was rarely studied.
surface for superhydrophobicity receives In this research, we propose the rapid
more attention because various types of and simple process to synthesize Au film on
material can be chosen and modify surface polymer substrates.
from non-low surface material to be
Figure 1. The fabrication process of Au film on substrates (glass slide, acrylic and PDMS)
In this study, Au film was was successfully cured. PDMS sheet was cut
synthesized from reduction reaction between in 2x2 cm2 by cutter on cutting mat. All of
chloroauric acid (HAuCl4) and sodium substrates were used without any
formate (HCOONa) at room temperature. pretreatment.
Figure 1 shows overall scheme of the 2.3 Fabrication of Au film on substrates
synthesize process. Glass slide, acrylic and The Au growth solution was
polydimethylsiloxane (PDMS) were used as prepared in 10 mL by mixing of 100,000
substrates. In addition, volume of Au growth ppm HAuCl4∙3H2O 240 µL with the solution
solution was varied in order to investigate an of HCOONa pH 7 (1 M, 9,750 µL). We
aggregation level of Au particles which is used NaOH to control pH of HCOONa and
related to hydrophobicity of surface. added 10 µL of DI water (please re-write
Superhydrophobic was examined by WCA this sentence). The mixed solution was
measurement. Scanning electron microscope vigorously stirred for 10 seconds at room
(SEM) was used to characterize morphology temperature. The mixed solution was
of Au film on substrate. dropped in a desired volume varied from
100 to 200 µL on substrates (glass slide,
2. Materials and Methods acrylic and PDMS). The solution was leave
2.1 Chemicals and materials undisturbed until Au particles were formed
Gold(III) chloride trihydrate (HAu- and were deposited on the surface of
Cl4∙3H2O), ≥99.9% and sodium formate substrate. When color of solution changed
(HCOONa), ≥99.0% were purchased from from yellow to colorless, the reaction was
Sigma-Aldrich (Singapore). Acrylic was stopped by cleaning the Au films with DI
purchased from local market. Poly(methyl water several times to eliminate an excess
methacrylate) (PMMA) was purchased from reagent. The Au film on the substrate was
Sigma-Aldrich (Singapore). Sylgard 184 dried at room temperature. The morphology
silicone elastomer base and curing agent of Au film on the surface of substrate was
were purchased from DOW Corning characterized by SEM and hydrophobicity
Corporation (Midland, USA). All reagents was measured by water contact angle. To
were analytical grade and used without fabricate Au film on the different substrates,
further purification. All glassware was the Au growth solutions were freshly
cleaned up with detergent and DI water. prepared.
2.2 Substrate preparation 2.4 Water contact angle measurement
Glass slide and acrylic were cut in Nikon D90 digital camera with
2.5x2.5 cm2 by glass cutter and laser cutter Macro lens was used for capturing a picture
(T-BROS Laser), respectively. PDMS were of a water droplet on Au film substrate. The
prepared by mixing of Sylgard 184 silicone camera exposure was set to ISO 800, f/32
elastomer base and curing agent with a ratio and speed shutter 200 with YN600 Pro LED
of 10:1. Silicone solution was filled in and filter used as a light source. Sample
plastic petri dish and waited until polymer holding state was placed between camera
and light source. Bubble level was used to
control the parallel line between the camera
and objects. The positions of camera and
light source were fixed all times during the
experiment. A drop of 10 µL water was
injected on the Au film. Water contact angle
was calculated by ImageJ program with
Dropsnake plug-in (using 7 interface points).

3.1 Fabrication of Au film on substrates
Au films were prepared by the
chemical reduction between Au3+ (from
chloroauric acid) and formic acid used as the
gold ion source and reducing agent,
respectively. The chemical reduction Figure 2. Virgin substrates of glass slide
reaction was 2Au3+ + 3HCOOH → 2Au0 + (A1), acrylic (B1) and PDMS (C1). The Au
3CO2 + 6H+ (Ecell = +1.697 V) which is films on the substrates using the Au growth
spontaneous reaction. The Au films were solution of 100 and 200 µL, respectively:
fabricated on several substrates which are glass slide (A1-A2), acrylic (B2-B3) and
glass slide, acrylic and PDMS. The physical PDMS (C2-C3)
appearance of the film was a rounded shape
containing more condensed film in the 3.2 Water contact angle of the fabricated
middle rather than the edges as shown in Au film
Figure 2. The phenomenon is easily After Au films were successfully
observed on the polymer substrates. From fabricated on substrates, hydrophobicity of
Figure 2, it was found that the size of the Au the Au films was investigated and monitored
film strongly depends on the volume of Au by water contact angle. WCA measurement
growth solution and the substrates. was performed by dropping 10 µL of DI
Size of the fabricated Au films on the water on substrate and then immediately
different substrates was summarized in captured the water droplet images. The
Table 1. It was noticed that the substrate captured images were further analyzed by
with the lower critical surface tension ImageJ program with Dropsnake plug-in.
provides the smaller of Au film diameter. In The images of water droplets on the
our case, the smallest size of the Au film substrates which are glass slide (Figure 3:
was obtained by using PDMS which has the A1-A3), acrylic (Figure 3: B1-B3) and
lowest critical surface tension (20.1 PDMS (Figure 3: C1-C3) were presented. In
dynes/cm) compared to acrylic (37.5 all cases, the water droplet was easily
dynes/cm) and glass side (290 dynes/cm) formed on the Au films substrates rather
The value of critical surface tension than the virgin substrates. This observation
represents hydrophilicity or hydrophobicity suggests that the generated Au films
of the surface. A surface with high significantly enhance the hydrophobicity of
hydrophobicity gives a low critical surface the surface. The WCA was increased from
tension which affects the dispersion 27.89±4.06o to 132.98±14.48o, 79.73±4.24o
efficiency of liquid on the substrate. to 138.46±0.80o and 115.95±2.54o
Therefore, the small dispersive area was o
to153.69±4.56 for glass slide, acrylic and
obtained from the surface with the low PDMS, respectively. The influences of the
surface tension (high hydrophobicity) which
is PDMS in our case.
Table 1. Critical surface tension (dynes/cm) and diameter (cm) of the generated Au films
using the Au growth solution of 100 µL and 200 µL on the substrates which are glass slide,
acrylic and PDMS
Substrates Glass slide Acrylic PDMS
Critical surface tension 290.0 37.5 20.1
(dynes/cm)
Au growth solution (µL) Diameter of the fabricated Au films (cm)
100 1.2 0.8 0.7
200 1.8 1.2 0.9
volume of Au growth solution was also section, the morphological structures of the
investigated. It can be seen that the higher generated Au films on the substrate were
volume of Au growth solution, the higher investigated. The structures of the Au films
WCA was observed. generated by using volume of Au growth
This reveals that the sufficient solution at 50, 200 and 400 µL on PDMS
amount of the generated Au particles might substrate were monitored by SEM as shown
be required in order to provide the in Figure 4. It can be seen that the WCA
appropriate roughness on the substrates. In dramatically increases when the volume of
our case, the Au films using 200 µL of Au Au growth solution increases from 50 µL
growth solution on PDMS substrate give the (WCA = 112.68±0.70o) to 200 µL (WCA =
highest WCA (~153o) representing the 153.69±4.56o). However, the WCA slightly
superhydrophobic surface. increases when using the Au growth solution
3.3 Morphology of the fabricated Au film from 200 µL to 400 µL (WCA =
From the previous section, we found 163.32±2.02o) as shown in inset of Figure 4:
that the Au films enhance the A2, B2, and C2. From SEM images, it can
hydrophobicity of substrates. Amount of the be noticed that the Au films were uniformly
Au growth solution strongly affects to the distributed on the substrate. Then, the excess
hydrophobic property of the surface. In this Au particles in the system aggregated on
Figure 3. Comparison of virgin substrates (glass slide, acrylic and PDMS) and substrates
with Au film generated using 100 and 200 µL of Au growth solution (Inset figures show
droplet of 10 µL DI water on virgin substrates (A1-A3), Au film from 100 µL(B1-B3) and
200 µL (C1-C3) of Au growth solution.)
Figure 4. Au films from different volume of Au growth solution on PDMS: 50 µL(A1), 200
µL(B1) and 400 µL(C1). Droplet and water contact angle of 10 µL DI water on those Au
films (A2, B2, C2) were measured and morphology of Au films are exhibited by SEM images
with x1000 and x5000 respectively(A3-A4, B3-B4, C3-C4).
with irregular shape. This aggregation Acknowledgement

creates the roughness of the surface which This research was supported by
enhances the hydrophobicity of the surface. Grant for International Research Integration
The density of the Au particles increased Chula Research scholar, Ratchadaphisek-
when the volume of Au growth solution was somphot Endowment Fund.
used (please correct this sentence). In the
study, it examines that the sufficient amount References
of the generated Au particles is crucial 1. Ma, M.; Hill, R. M. Curr. Opin. Colliod.
parameter in order to generate the In. 2006, 11, 193-202.
superhydrophobic Au film. 2. Zhu, J.; Zangari, G.; Reed, M. L. J. Phys.
Chem. C. 2011, 115, 17097–17101.
4. Conclusion 3. Ishida, T.; Kuroda, K.; Kinoshita, N.;
In this study, the Au film with Minagawa, W.; Harata, M. J. Colloid.
superhydrophobicity was successfully Interface Sci. 2008, 323, 105-111.
developed on different substrates (glass 4. Tan, K. H.; Tajuddin, H. A.; Ahmad, R.;
slide, acrylic and PDMS). The Au film was Shuhaimi, A.; Johan, M. R. Optik. 2017,
synthesized using the simple chemical 50, 54-61.
reduction reaction between HAuCl4 and 5. Li, H.; Men, D.; Sun, Y.; Liu, D.; Li, X.;
HCOONa. The Au film fabricated on the Li, L.; Li, C.; Cai, W.; Li, Y. J. Colloid.
PDMS substrate provides the highest water Interface Sci. 2017, 505, 467-475.
contact angle (~153o) compared to the other 6. Budy, S. M.; Hamilton, D. J.; Cai, Y.;
substrates. Au film can be prepared by using Knowles, M. K.; Reed, S.M. Adv. Colloid
an appropriate substrate which presents the Interface Sci. 2017, 487, 337-347.
low critical surface energy and the sufficient 7. Ahmed, S. R.; Kim, J.; Tran, V. T.;
Au growth solution to create excess Au Suzuki, T.; Neethirajan, S.; Lee, J.; Park,
particles in order to promote the roughness E. Y. Sci. Rep. 2017, 7, 44495.
of the substrate surface. This protocol offers
a great opportunity to fabricate the
superhydrophobic film using the other
precise metals (e.g. Ag, Pt, Pd).
MXene/polymer based electrode for enhanced sensor performances in the
detection of H2O2
Supawat Neampet1, Jiaqian Qin2, Nadnudda Rodthongkum2*, Orawon Chailapakul3*
1
Program in Petrochemistry and Polymer Science, Faculty of Science, Chulalongkorn University,
Pathumwan, Bangkok 10330, Thailand
2
Metallurgy and Materials Science Research Institute, Chulalongkorn University, Soi Chula 12,
Phayathai Road, Pathumwan, Bangkok 10330, Thailand
3
Electrochemistry and optical spectroscopy Center of Excellent (EOSCE), Department of Chemistry,
Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
*E-mail: corawon@chula.ac.th, nadnudda.r@chula.ac.th
Abstract:
MXene-Ti3C2 was readily synthesized by etching method and used to form the
composite with conductive polymer for screen printed carbon electrode modification. The as
prepared composites were characterized by Fourier transform infrared spectroscopy (FTIR),
scanning electron microscopy (SEM) with energy dispersive X-rays spectroscopy (EDX) to
verify the presence of nanocomposites and illustrate their morphologies. Subsequently, the
modified electrodes were electrochemically characterized by using cyclic voltammetry (CV)
to examine the increased electrochemical conductivity compared with the unmodified screen-
printed carbon electrodes. Due to the substantially enhanced current response signal of a
standard ferrocyanide/ferrocyanide redox couple measured on the modified electrodes, it has
a high potential to use as new working electrodes in electrochemical sensor. Thus, these
electrodes were applied for the detection of hydrogen peroxide (H2O2), which is a common
electroactive product generating from enzymatic reactions in electrochemical biosensors. The
preliminary results verify that this system significantly increase the current response signal
towards H2O2 detection compared with the unmodified electrode system. Eventually, this
platform will be further utilized for enzymatic based electrochemical biosensor. These results
will be presented.
1. Introduction storage. Recently, MXene was applied as the

MXene is an interesting 2D material electrochemical transducer and used for
consisting of transition metal carbide and hydrogen peroxide measurement.1 Also,
nitride, which has attracted significant MXene presents great application prospects
research interest in recent years because of as electrochemical transducer in sensing
its unique properties such as conductivity applications.1,2 However, the modification of
and hydrophilicity leading to high stability MXene on electrode requires significant
in aqueous environment.1-3 MXene is effort for the well dispersion of MXene.
synthesized by selective etching of “A” Conducting polymers, such as polyaniline
element layer from MAX phase, resulting in (PANI), polypyrrole (PPY), and their
a morphology similar to the exfoliated composites, have also been widely used in
graphite. The potential uses of MXene as electrode surface modification.5 Among the
efficient electrode material for conducting polymers, PANI has attracted
electrochemical energy storage applications much attention because of its unique
have been demonstrated.4 However, the uses properties, such as high electrochemical
of MXene in sensing applications have been conductivity, environmental stability, and
given much less attention than energy biocompatibility.6
Hydrogen peroxide (H2O2) is an (Millipore, USA, R ≥ 18.2 MΩ cm-1) and
important mediator producing in biological used throughout the experiments.
metabolism and cell of many regulating 2.2 Apparatus
biological processes. Electrochemical The morphology and elemental
detection of H2O2 has become an interesting composition of Ti3C2 (MXene) were
method to meet application on-demand in characterized via scanning electron
clinical diagnosis. In addition, H2O2 is a microscopy (SEM) and energy dispersive X-
major reactive oxygen specy (ROS) of many rays spectroscopy (EDX) (JSM-6400; Japan
oxidative metabolic pathways (e.g. glucose Electron Optics Laboratory Co., Ltd.,
oxidase, cholesterol oxidase, lactate oxidase, Japan), respectively. All electrochemical
etc.) that mainly regulates the oxidative measurements were performed using cyclic
stress-related states. The unstable level of voltammetry (CV) via 910 PSTAT mini
ROS in the human body disturbs the (Metrohm Siam Company Ltd.) controlled
oxidative stress-related states, leading to with a 910 PSTAT software.
various diseases, such as cancer, diabetes, 2.3 Synthesis of MXene
cardiovascular disorders, and aging. Thus, MAX powders were pounded,
successful design of H2O2 sensors associated sieved, and dispersed in 20 mL of HF. Then,
with accurate, rapid, selective, and low-cost the dispersed MAX solution was stirred for
monitoring methods are extremely required 3 hours by using sonicator, and followed by
to analyze H2O2 levels. washing with deionized water until pH 7.0.
Here, the nanocomposite of After that, the solution was centrifuged at
MXene/PANI is prepared and used for 3000 rpm with an ultracentrifuge for 10 min,
applying as a hydrogen peroxide sensor. The and the supernatants were kept for freeze
as synthesized MXene was characterized dry. The obtained powder was used as
and then used for the preparation of MXene for all experiments.
nanocomposite with PANI. Then, 2.4 Modification of MXene on screen-
MXene/PANI modified screen-printed printed carbon electrode
electrode was used for hydrogen peroxide MXene-aniline nanocomposite
and lactate detection via cyclic voltammetry solution was prepared by adding 1 mg of
(CV). MXene in 1 mL of 0.1 M aniline monomer
in 0.5 M sulfuric acids, and then dispersed
2. Materials and Methods by sonication for 3 hours to obtain well
2.1 Chemicals and materials dispersed MXene-aniline nanocomposite
MAX (Ti3AlC2) powder was solution. For electrode modification, 100 µL
synthesized according to the previous report, MXene-aniline nanocomposite solution was
with a slight modification.7 Hydrogen dropped onto screen-printed carbon
fluoride (HF 50%) was obtained from Ajax electrode and then electropolymerized via
Finechem Pty Ltd. Hydrogen peroxide CV with scanning potential in a range from -
(H2O2 30%) was purchased from Merck 0.5 to +1.0 V for 4 cycles using a scan rate
Schuchardt OHG 85662 Hohenbrunn of 100 mV/s.
Germany. Aniline monomer, potassium 2.5 Electroanalytical measurement
ferricyanide (K3[Fe(CN6)]), potassium All electrochemical measurements
ferrocyanide (K4[Fe(CN6)]), phosphate were performed on potentiostat (910 PSTAT
buffer (PB, pH 7.0), lactate standard, and 50 mini, Metrohm Siam Company Ltd.) using
U/mL lactate oxidase were purchased from cyclic voltammetric technique. CV was
Sigma–Aldrich (St. Louis, MO, USA). All used, and the potential potential was
solutions were prepared in deionized water scanned from -0.5 V to +1.0 V with 100
Figure 1. SEM images of MXene with the magnification of (A) 5kx and (B) 20kx, and (C)
EDX spectrum of MXene
mV/s as scan rate for all measurements. The electrochemical characterization

MXene/PANI modified electrode was of MXene/PANI modified electrodes were
electrochemically characterized by CV using investigated by CV using a three-electrode
a standard solution of 1.0 mM [Fe(CN)6]3-/4- system in 1.0 mM [Fe(CN)6]3-/4- solution and
prior to H2O2 and lactate detection. compared to an unmodified electrode, PANI
modified electrode and MXene modified
3. Results and Discussion electrode as shown in Figure 2. The use of
3.1. Characterization of MXene MXene/PANI nanocomposite material
The morphology of MXene was showed the increase of anodic and cathodic
characterized by scanning electron peak currents compared with using PANI
microscopy (SEM) as shown in Figure 1. and MXene alone indicating the potentially
The SEM images of the obtained etched high sensitivity of this system, which
MXene using HF (Figure 1A-B) expose the approximately 5.6, 2.1 and 1.8 times higher
typical MXene morphology, which are than an unmodified electrode, MXene
similar to exfoliated graphite that can be modified and PANI modified electrodes,
attributed to the exfoliation of Al atom respectively. Compared to PANI modified
layers from the original MAX phase. electrode, the incorporation of MXene into
Moreover, the exfoliation of Al atoms layers nanocomposite improves the conduction
was confirmed by EDX pattern as shown in pathway on the electrode surface lead to
Figure 1C. The high intensity of Ti and C increase of both anodic and cathodic peak
atoms with low intensity of Al atom were currents of 1.0 mM [Fe(CN)6]3-/4-.
observed on the as prepared MXene Moreover, the presence of MXene and
confirmed that MXene was successfully PANI on electrode surface substantially
prepared in this work. From the EDX enhance the sensitivity of modified electrode
pattern, the peak of O and F peaks in EDX because of the high surface area measured
result indicate the present of water and HF by confocal microscopy, which corresponds
from synthesis process. well with the previous report8 and promoting
3.2. Electrochemical characterization of of electron transfer rate. Thus, MXene/PANI
MXene/PANI modified electrode modified electrode is selected as a potential
material for electrode surface modification
for further experiments.
Figure 3. Anodic peak currents integrated

from cyclic voltammetry of 1.0 mM
[Fe(CN)6]3-/4- using MXene /PANI modified
electrodes with different repeating cycles (2,
4 and 8 cycles) for MXene/PANI
electropolymerization of nanocomposite
3.3 Detection of H2O2 and lactate

For an electrochemical biosensor, the
determination of the H2O2 product obtained
from the oxidation of target analyte such as
lactate can be used for the indirect
Figure 2. (A) cyclic voltammograms of 1.0 electrochemical quantification of analyte.
mM [Fe(CN)6]3-/4- on unmodified, MXene The cyclic voltammograms of H2O2 using
modified, PANI modified and MXene/PANI MXene/PANI modified electrode is
modified electrodes at scan rate of 100 illustrated in Figure 4A. In the presence of
mV/s, and (B) anodic peak currents obtained H2O2, the anodic current was significantly
from cyclic voltammograms of Figure 2A increased confirming that MXene/PANI
modified electrode could be used as a novel
electrode for H2O2 sensor.
The effect of repeating cycles for
MXene/PANI modification on electrode
surface was also investigated and optimized
by CV using 1.0 mM [Fe(CN)6]3-/4- (Figure
3). Anodic peak current increased when
repeated electropolymerization from 2 to 4
cycles, indicating that incorporating
MXene/PANI significantly improved
electrical conductivity. However, the anodic Scheme 1. The enzymatic reaction between
lactate and LOX on MXene/PANI modified
peak current subsequently decreased when
electrode
the repeating cycle was higher than 8 cycles.
The decreasing in anodic current was from To specifically detect lactate, the
the high thickness of PANI at higher lactate oxidase enzyme (LOX) was
repeating cycles. Thus, 4 cycles was selected immobilized on MXene/PANI modified
as an optimal condition for all further electrode for specifical lactate detection. The
experiments. small amount of LOX (1 µL) was dropped
on MXene/PANI electrode and dried prepared and used for screen-printed
overnight at 4°C prior to detect lactate. The electrode modification for H2O2 detection.
detection of lactate is performed by The high conductivity and large surface area
measuring the H2O2 product that is of the MXene/PANI modified electrode
generated from an enzymatic reaction significantly improves the electrochemical
between lactate and LOX as shown in sensitivity for H2O2 detection. Under
Scheme 1. In the presence of lactate, the optimum conditions, MXene/PANI modified
anodic current signal increased indicated electrode can be applied for both H2O2 and
that this developed system could be applied lactate detection using enzymatic based
to detect lactate (Figure 4B). However, the reaction. This developed system might be
peak potential obtained from LOX used as an alternative device for H2O2
immobilized MXene/PANI modified screening in health monitoring.
electrode slightly shifts from 0.1 V to 0.3 V
due to the insulating properties of enzyme. Acknowledgements
Thus, this developed system will be further Ratchadaphisek Somphot
developed and applied for enzymatic based Endownment Fund, Chulalongkorn
electrochemical biosensor. University
References
1. Rakhi, R. B.; Nayak, P.; Xia, C.;
Alshareef, H. N. Sci. Rep. 2016, 6,
36422.
2. Liu, H.; Duan, C.; Yang, C.; Shen, W.;
Wang, F.; Zhu, Z. Sen. Actuators B:
Chem. 2015, 218, 60-66.
3. Romer, F. M.; Wiedwald, U.; Strusch, T.;
Halim, J.; Mayerberger, E.; Barsoum, M.
W.; Farle, M. RSC Adv. 2017, 7 (22),
13097-13103.
4. Zhang, X.; Zhang, Z.; Zhou, Z. J. Energy
Chem. 2018, 27 (1), 73-85.
5. Yukird, J.; Wongtangprasert, T.;
Rangkupan, R.; Chailapakul, O.;
Pisitkun, T.; Rodthongkum, N. Biosen.
Bioelectronics 2017, 87, 249-255.
6. Rodthongkum, N.; Ruecha, N.;
Rangkupan, R.; Vachet, R. W.;
Chailapakul, O. Anal. Chimi. Acta 2013,
Figure 4. Cyclic voltammograms of (A) 3 804, 84-91.
mM H2O2 and (B) 5 mM lactate compared 7. Qin, J.; Wang, Z.; Fang, L.; Wang, F.;
to 0.1 M PB pH 7.0 measured on Lei, L.; Li, Y.; Wang, J.; Kou, Z.; He, D.
MXene/PANI modified electrodes at a scan Solid State Commun. 2008, 148 (9), 431-
rate of 100 mV/s, insets show the anodic 434.
peak current from CV. 8. Wu, L.; Lu, X.; Dhanjai; Wu, Z. S.;
Dong, Y.; Wang, X.; Zheng, S.; Chen, J.
4. Conclusion Biosen. Bioelectronics 2018, 107, 69-75.
A nanocomposite based on
MXene/PANI has been successfully
Effects of natural fibers on morphology and acoustic properties of natural
rubber foam composites
Julaluk Phunnoi*, Pracha Lao-auyporn, Laksana Wangmooklang, Borwon Narupai,
Arisa Jaiyu, Passakorn Sueprasit
Expert Centre of Innovative Materials, Thailand Institute of Scientific and Technological Research,
Khlong Luang, Pathum Thani 12120, Thailand
* E-mail: julaluk@tistr.or.th
Abstract:
Acoustic materials play an important role in noise control solutions for many
situations, where it is necessary to protect health and environment quality. This research has
been carried out in order to develop natural rubber latex (NRL) for acoustic materials.
Dunlop process was used to prepare the natural rubber foam (NRF) containing natural fibers
such as rice husk (RH), coconut fiber (CF) and bagasse fiber (BF). The surface of natural
fibers was modified by using sodium hydroxide treatment. The effects of natural fibers on the
foam density, cell morphology and sound absorption coefficient were investigated. It was
found that the NRF with 10wt.% of bagasse fiber showed high sound transmission loss. The
open-cell foam was observed in cell morphology of all NRF composites. The foam density
was significantly affected by natural fibers addition. Natural fibers significantly enhanced
acoustic properties of NRF composites.
1. Introduction close proximity to a busy road or highway,

Natural rubber latex (NRL) is one of we are very likely exposed to traffic noise
the green polymer materials. The unique pollution around 85 dB.3 The solution of
characteristics of NRL are high strength, noise is an important issue. The noise
flexibility and elasticity.1 Most NRL have reduction is alternative choice to solve this
been used to prepare latex compounding problem, which used the process of
rubber for many applications and products. removing noise from a signal. Noise
Especially, it has been made by natural pollution was controlled using sound
rubber foam (NRF), which has light weight, absorption materials or acoustic materials
flexibility and elasticity. NRF have been which have been used to absorb a wide
used to produce pillows, mattress and sound frequency range of noises by effectively
proofing panels. dispersing sound energy on the process of
Nowadays, noise pollution effects on propagation of the sound wave.4
the environment, human health and In the past, sound absorption
economy, which caused by industrial materials or acoustic materials were
sources, transportation, household, public produced from synthetic fiber, which is
address system, and agricultural machines, harmful to human health and environment.5
etc. There are generally harmful and serious Therefore, natural fibers have been studied
health hazard. It has far-reaching as absorber materials instead of synthetic
consequences including physiological and fiber. Natural fibers can be obtained from
psychological effects on human beings.2 flora and fauna that are naturally bio-
According to the World Health Organization degradable, non-harmful to human health
(WHO) report that sound levels less than 70 and environment including low safety rick in
dB are not damaging to living life. The their process. Many research have been
exposure more than 8 hours of constant investigated the capability on the
noise beyond 85 dB may be hazardous. performance of natural fibers for use as
Consequently, we work for 8 hours daily in acoustic absorber. Several studies have been
focused on a similar group of natural fibers, Table 1. The properties of NRL
which have been discovered and suggested Properties of NRL Value
to be used as acoustical panel.6 Total solids content, TSC (wt.%) 61.71
This research focuses on the effects Dry rubber content, DRC (wt.%) 59.69
of natural fibers on NRF composite for Ammonia content (wt.%) 0.65
Volatile Fatty Acid number 0.023
acoustic material. The chosen natural fibers KOH number 0.74
are rice husk (RH), coconut fiber (CF) and pH of latex 10.44
bagasse fiber (BF). The natural fibers were Non-rubber solids (wt.%) 2.02
modified by using 10wt.% sodium Mechanical stability time, MST (s) 1,183
hydroxide treatment. The modified- natural Sludge content (wt.% rubber) 0.008
Coagulum content (wt.% rubber) 0.021
fibers were used as additives. Sulfur Mg2+ on solids (ppm) 19
vulcanization system was used to produce Viscosity (cPs) 125
vulcanized NRF composite. The suitable
fibers on vulcanized NRF composite were 2.3 Natural rubber foaming
determined. The density, average pore size, NRL samples were stirred to remove
cell morphology and sound transmission ammonia and adjusted pH value by using
were investigated. KOH. The blowing agent, accelerators,
sulfer and Vulcanox CPL were added to
2. Materials and Methods NRL. NRL compounds were stirred for 2 h
2.1 Materials and soaked overnight. After that, NRL
The properties of NRL, which was compounds were mixed with high speed to
used as raw materials in this study, are be foam. Natural fibers, activator and gelling
shown in Table 1. Solution and solid agent were added and dispersed for 4 min.
materials were added in NRL. 1) Solution; NRL foam was poured into the mold and
Zinc diethyldithiocarbamate (ZEDC), Zinc then heated for vulcanization.
mercaptobenzothiazole (ZMBT) and 2.4 Characterizations
Diphenlguanidine (DPG) were used as 2.4.1 Density and average pore sizes
accelerators. Sulphur was added to be a Bulk density of NRF was calculated
cross-linking agent. Zinc oxide (ZnO) was from mass and volume. The average pore
mixed as activator. Sodium silicofluoride sizes were determined using automated gas
(SSF) was added as gelling agent. Potassium sorption analyzer (Quantachrome, Autosorb
Oleate (K-Oleate) was used as blowing iQ, US) followed as The Brunnauer-Emmett
agent. Potassium hydroxide (KOH) was Teller (BET) method.
filled to adjust pH value. Vulcanox CPL was 2.4.2 Cell morphology
also used as protective agent. 2) Solid are The cell morphology for NRF
three kinds of natural fibers that are rice composites and natural fibers were
husk (RH), coconut fiber (CF) and bagasse investigated using Optical Inverted
fiber (BF). Besides these materials, sodium Microscope (OM, Rushmore, Axiovert 405,
hydroxide (NaOH) was used to treat the Germany) and Field Emission Scanning
natural fibers. Electron Microscope (FE-SEM, Jeol, JSM-
2.2 Natural fibers treatment 6340F, Japan), respectively. The NRF
The raw natural fibers were dried composites were cut in cross section. The
and sieved to size less than 850 µm. natural fibers were dried. Their surfaces
Then small fibers were soaked in were observed by FE-SEM.
the alkaline treatment with 10wt.% 2.4.3 Acoustic property
of NaOH solution for 10 min to remove Acoustic property of NRF was
dirt. After that, they were washed and heated investigated in the terms of sound
up to 80 oC for 2 h to remove the excess transmission loss. The diagram for sound
NaOH.
transmission testing was set as shown in The densities of NRF were slightly
Figure 1. NRF were attached at the hole of increased with increasing of RH content
the close container, which was installed a while, the densities of NRF with increasing
sound generator. The sound frequencies of CF and BF contents were evidently
125-8000 Hz were generated and were then increased. From standard deviation (SD)
adsorbed by NRF. The intensities of showed that small SD value effect to high
transmitted sound were measured in terms of dispersion ability of natural fibers in NRF.
decibel (dB). According to the results, the SD were small
with the addition of CF>BF>RH. It
indicated that RH and BF are lighter than CF
resulting in more dispersion of RH and BF
during mixed with NRF.
The pore size values of NRF with
different natural fiber contents are given in
Figure 3. The average pore diameters were
increased with increasing the natural fiber
contents. Considering the average pore
diameters with RH, CF and BF contents, the
pore diameter of NRF with BF and CF were
Figure 1. The schematic diagram of sound slightly different. But BF addition NRF
transmission testing setup showed smaller pore diameter than that of
RH. These results indicated that BF was
3. Results and Discussion more compatible in mixing with NRF than
3.1 Effect of natural fibers on density and those of RH and CF. The two properties
Average pore size including density and pore diameter of foam
The influences of natural fibers are significant factors to the acoustic
including RH, CF and BF on the densities properties which will be discussed in the
of NRF were summarized in Figure 2. next section.
Figure 2. The effect of natural fiber contents on density property of NRF
3.2 Effect of natural fibers on cell maximum sound transmission loss was 31
morphology dB which was tested at 4,000 Hz. Thus, it
The cell morphologies were might be implied that NRF containing BF
investigated by OM. Figure 4 showed cell has a good sound insulation capability.
morphology of NRF composites with RH, 3.4 Effect of treated-natural fiber on
CF and BF contents. The open-cell foams acoustic property
were observed in cell morphology of all The surface morphology of natural
NRF composites. The foam with open-cell fibers before and after treated with 10wt.%
has improved sound absorption abilities over NaOH was investigated by FE-SEM as
close-cell foam.4 Therefore, these NRF given in Figure 6. The morphology of BF
composites are suitable for acoustic material after treatment occurred higher tortuosity
application. and surface area than those of samples
3.3 Effect of natural fibers on acoustic without NaOH treatment.
property Figure 7 shows the influence of
The result of the sound transmission NaOH treatment with 5wt.% BF addition on
loss for NRF with RH, CF and BF contents sound transmission loss of NRF. At above
are shown in Figure 5. The sound 2000 Hz, The transmission loss values of
transmission loss of NRF containing natural treated sample were higher than that of
fibers (RH, CF and BF) was higher than untreated sample. The NaOH treatment
those of pure NRF. At the same loading of removed moisture absorbents of natural
natural fibers, NRF with BF content gave fiber such as oil and wax and increased the
more sound transmission loss than CF and roughness on fiber surface as well as
RH, respectively. The NRF with 10wt.% improved interfacial bonding between NRF
BF exhibited highest sound transmission and natural rubber, which led to enhance the
loss value in all of frequencies. The acoustic property.
Figure 3. The effect of natural fiber contents on average pore diameter
Figure 4. The cell morphology of NRF composites: NRF+5%RH (a), NRF+10%RH (b),
NRF+12%RH (c), NRF+5%CF (d), NRF+10%CF (e), NRF+12%CF (f), NRF+5%BF (g),
NRF+10%BF (h) and NRF+12%BF (i)
Figure 5. The effect of different natural rubber on acoustic property
Figure 6. The image of BF morphology: before treatment (a) and after treatment (b)
Figure 7. The effect of natural fiber treatment with 10 wt.% NaOH on acoustic property
4. Conclusion
The acoustic properties increased References
with adding the natural fiber in NRF. The 1. Roslim, R.; Amir Hashim, M. Y.;
NRF with 10 wt.% BF contents showed high Augurio, P. T. J. Eng. Sci. 2012, 8, 15–
sound transmission loss value in all of the 27.
testing frequencies; the exhibited maximum 2. Wei, D., Y.; Yan, L. J. Sci. China Tech.
sound transmission loss was 31 dB at 4,000 Sci. 2012, 55, 2278–2283.
Hz. It was indicated that NRF with 10 wt.% 3. https://www.environmentalpollutioncent
BF addition have a good acoustic insulation ers.org
property. The NRF with BF content and 4. Mamtaz, H.; Fouladi, M. H.; Al-Attabi,
treated by 10 wt.% NaOH for 10 min M.; Namasivayam, S. N. J. Eng. 2016,
enhanced the transmission loss value at high 2016, 1-11.
frequency. NaOH treatment increased the 5. Najib, N. N.; Ariff, Z. M.; Bakar, A. A;
roughness on surface fiber and improved the Sipaut, C. S. Mater. Des. 2011, 32, 505–
interfacial bonding between the matrix and 511.
natural rubbers. 6. Yahya, M. N.; Sambu, M.; Latif, H. A.;
Junaid, T. M. IOP Conf. Ser.: Mater. Sci.
Acknowledgements Eng. 2017, 226. 012013.
The financial support for this
research was provided by National Research
Council of Thailand (NRCT) and Thailand
Institute of Scientific and Technological
Research (TISTR).
Nitrogen-doped porous carbon derived from cattail flower waste via
hydrothermal treatment with chemical activation
Napat Kaewtrakulchai1, Gasidit Panomsuwan,2 Kajornsak Faungnawakij3, Apiluck Eiad-Ua1*
1
College of Nanotechnology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
2
Department of Materials Engineering, Faculty of Engineering, Kasetsart University, Bangkok, Thailand
3
National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency
(NSTDA), 111 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Nueng, Amphoe Khlong Luang,
Abstract:
Nitrogen doped porous carbon (NPC) have been performed by hydrothermal
treatment with chemical activation from cattail flower waste. The effects of varied
concentrations of ammonium hydroxide solution of 0.5, 1, 1.5, 2 M in hydrothermal
treatment were extensively investigated to develop a nitrogen content of the samples. The
pore formation and carbon percentage of NPC were successfully developed by using a
carbonization at 700 °C since ammonium hydroxide serves as both activating agent and
nitrogen containing source. NPC physicochemical properties are completely examined by
CHN elemental analysis, FTIR, and SEM to analyze a specific property such as elemental
composition, surface functional bonding, and surface morphology. Additionally, the
incorporation of nitrogen dopant into the carbon was intended to further enhance the
electrical performance.
1. Introduction substances have been applied for its

The utilization of biomass materials production.3
such as agricultural wastes, organic Recently, porous carbon was widely
substances derived a carbon materials has a utilized as a high surface area electrode in
much desirable attention in the fields of energy storage device. Unfortunately, their
environmental technologies (i.e. adsorbent, electrochemical performance has been limit
soil amender) and energy applications (i.e. because of their charge storage mechanism.
electronic device, solid biofuel) due to their Furthermore, porous carbon doped nitrogen
potentials as eco-friendly, low cost, and reveals an enhancement of electrical
carbon sequestration. As a result, several properties. Nitrogen dopes porous carbon is
technologies have been applied for the commonly synthesized via processing of
carbon material manufacturing.1 carbon with nitrogen containing precursor
However, the major process is a under a severe condition such as arc
thermochemical conversion such as discharge, chemical vapor deposition and
chemical vapor deposition, hydrothermal carbonization.4
treatment, pyrolysis or known as Consequently, this study aims to
carbonization.2 As we known that, porous prepared a nitrogen dope porous carbon by
carbon materials have some significances using cattail flower (Typha Angustifolia)
such as adsorptive, environmental, which is an abundant weed waste with a
mechanical, thermal, and electrical fibrous structure in Thailand as a carbon
characteristics for novel applications. source. The process is completely achieved
Conventionally, porous carbon could be in 2 simple steps; hydrothermal treatment
processed by using natural coal (i.e. with a various concentration of ammonium
bituminous, lignite, peat), coconut and palm hydroxide solutions for nitrogen doping and
shell. Till date, vast carbonaceous followed by carbonization to obtain a pore
formation during the reaction at high However, the yield of NPC was
temperature since ammonium molecule also obtained from the carbonization of prepared
serves both nitrogen source and activating hydrothermal char. The weight of NPC was
agent. reduced approximately 74 to 78% compared
to the initial weight of raw cattail flower
2. Materials and Methods which used in its manufacturing due to the
2.1 Preparation of NPC high temperature during carbonization7.
Cattail flower (Typha Angustifolia) Consequently, high ammonium
was collected from the local in Thailand. It concentration significantly causes a low
was firstly dried in an electrical oven at 90 NPC yield, as seen in Table 1.
°C for overnight. 20 g of prepared cattail
flower was mixed with 40 g of several Table 1. Production yield during NPC
concentrations of ammonium solution (0.5, processing
1, 1.5, 2 M). The mixture was subsequently Samples Yield (%)a Yield (%)b
moved into a stainless-steel reactor, and it CF 64.23 28.39
was heated to 200 °C for 24 h to serve a CF-0.5 59.37 26.17
nitrogen doping. Then, the solid product was CF-1 58.71 25.22
quenched via water and dried at 100 °C until CF-1.5 56.69 23.16
dried. Finally, NPC were obtained from the CF-2 54.92 22.54
carbonization at 700 °C for 2 h. a
Yield of Hydrothermal
2.2 Characterization b
Yield of NPC after carbonization.
The elemental composition of
produced NPC was determined by ultimate 3.3 Fourier transform infrared
analysis using CHN elemental analyzer spectroscopy (FTIR) analyses
(Leco truespec chns-628). The identification FTIR spectroscopy was employed in
of molecular bonding to samples structures order to investigated an overview on the
was analyzed through FTIR transmittance difference of chemical structure of both
spectra using a PERKIN ELMER UATR hydrothermal treated and NPC samples,
Two FTIR spectrophotometer in the Figure 1 and 2. The band at 3680-3000 cm-1
wavenumber range of 4000-400 cm-1. is assigned to the characteristic of - OH
Moreover, the surface morphology of stretching of hydroxyl and carbonyl groups
produced NPCs was observed by SEM on for lignin. However, lignin regions are also
Zeiss EVO50, Germany operating at 20 eV. found at 1380-1240 cm-1and 1460 cm-1.
Moreover, the broad bands centered 2925
3. Results and Discussion and 2850 cm-1 is identified C-H stretching of
3.1 Production yield of NPC polysaccharide that structured in cellulose
The production yield of NPC was and hemicellulose. The band between 1200-
evaluated by the calculation followed the 950 cm-1 also described to -C-O for cellulose
study of Martar et al. (2014).2,5 and hemicellulose. These band also displays
Hydrothermal treatment of cattail flower a chemical structure of a biomass polymer
with various concentrations of ammonium (i.e. cellulose, hemicellulose, lignin).2-4
solution for nitrogen doping has resulted the FTIR spectra indicated that the increase of
yield between 54.9 to 64.2% depending on ammonium concentration during
the different of the presented ammonium hydrothermal treatment gradually reduced
concentrations. Specifically, the degradation the functional structure resulted a pure
of cattail flower was elevated as increased carbon sample.
ammonium hydroxide concentration since Beside FTIR spectra of hydrothermal
ammonium molecule plays a role of treated samples, the bands which revealed a
catalytic degradation as alkaline
activation.3,6
Figure 1. FTIR spectra of hydrothermal
treated product compared with raw CF
Figure 2. FTIR spectra of NPC products
chemical structure of NPC samples also

determined. The carbonization at 700 °C is
effective for the purity of samples. As seen
in Figure 2, very chemical bands were not
observed due to the degradation by thermal
treatment combined a chemical activation.
According to the study of Zhen et al.
(2016), the bands at 1590 cm-1 can be Figure 3. SEM micrograph of a) NPC-0.5,
assigned to the presence of N-H in-plane b) NPC-1, c) NPC-1.5 and d) NPC-2
deformation vibration or C=C stretching
from aromatic rings. The peaks between 3.4 Scanning electron microscopy (SEM)
1150–900 cm-1 are attributed to the C-N analysis
stretching. The broad weak bands between The surface morphology of NCP
950 and 650 cm-1 correspond to out-of-plane was scanned by using SEM technique at
N-H deformation vibration.9 Consequently, magnifications of 300x and 500x as shown
nitrogen doping on a carbon surface can be in Figure 2. The prepared NPC has a fibrous
tentatively confirmed by using FTIR characteristic with size approximately 5
analysis. However, it should be analyzed microns. Smooth and compact surface
with a high qualitative technique as morphology has been observed from NPC-
elemental analysis and XPS scan.2,8 0.5 (Figure 3a) which is represented the
lowest concentration of ammonium solution
used in its production. However, the surface
roughness was increased in Figure 3b-c Ladkrabang for their assistance for facility
which is significantly elevated with supports.
concentration of ammonium solution
because of the chemical activation affected References
by ammonium at high temperature.9 1. Ana, K.; Jelena, D.; Tatjana, T. P.;
Marija, V.; Maja, P.; Vesna, M.; Zlatko,
4. Conclusion R.; Zoran, L. Carbon 2015, 95, 42-50.
Hydrothermal treatment using 2. Martar, S.; Linghui, Y.; Li, Z.; Conchi,
ammonium solution as nitrogen doping O. A. ACS Sustainable Chem. Eng. 2014,
followed by carbonization for nitrogen 2, 1049−1055.
doped porous carbon is an efficient method
3. Hasan, S.; Fuat, G. J. Cleaner
to develop a novel carbon material. The
Production 2016, 113, 995-1004.
nitrogen percentage of produced NPCs are
4. Imran, J. R.; Rajalakshmi, N.;
significantly increased with the
Dhathathreyan, K. S.; Ramaprabhu, S.
concentration of ammonium hydroxide
Inter. J. Hydrogen Energy 2015, 1-12
solutions. Besides the nitrogen contained in
5. Melike, D. O.; Uday, K. V.; Claudiu, T.
carbon structure, their porosity and carbon
L. Ind. Crops Prod. 2017, 95, 583–590.
content was also developed since
6. Thomas, E. R.; Denisa, H. J.; Kiran, K.;
ammonium molecules on carbon surfaces
plays a role of activation under a high Zhonghua, Z.; Gao, Q. L. J. Power
temperature condition during carbonization, Sources 2010, 195, 912–918.
simultaneously. Consequently, produced 7. Won-Hee, L.; Jun, H. M. ACS Appl.
NPC can be further applied in various areas Mater. Interfaces 2014, 13968−13976.
of electronic devices. 8. Latham, K. G.; Jambu, G.; Joseph, S. D.;
Donne, S. W. ACS Sustainable Chem.
Acknowledgements Eng. 2014, 2, 755−764.
The authors would like to thank to 9. Zhen, G.; Qiangfeng, X.; Hong, L.; Bing,
the College of Nanotechnology King L.; Haobin, W.; Yunfeng, L.; Cumman,
M o n gk u t ’ s In s t i t u t e o f T e c h n o l o gy Z. Sci. Rep. 2016.
Improving the efficiency of dye-sensitized solar cells with spray-coated
TiO2 electrode modified by CuO
Sakun Preedavijitkul*, Akawat Sirisuk
Faculty of Engineering, Chulalongkorn university, Pathumwan, Bangkok 10330, Thailand
*E-mail: sakun_moo@hotmail.com
Abstract:
This research investigated the application of the CuO/TiO2thin film electrode for dye-
sensitized solar cells (DSSCs). CuO and TiO2sols were synthesized separately via sol-gel
methods. The sols were mixed and sprayed onto the fluorine-doped tin oxide glass substrates.
The amount of CuO added to TiO2was varied at 0, 0.1, 0.5, 1.0 and 3.0%wt. The electrode
layer was sintered at 400 ˚C for two hours. The prepared DSSC electrodes were immersed
ineither a 0.3 mM solution of ruthenium-based N719or N3 dyes in ethanol. The electrolyte
solution was a mixture of 0.5 M lithium iodide, 0.05 M iodine,and 0.5 M 4-tert-butylpyridine
in acetonitrile.The current-voltage characterization of DSSCs was conducted under simulated
sun illumination. The presence of CuO in TiO2 electrode caused the red shift in UV-visible
light absorption. The DSSCs with 0.1% CuO/TiO2electrode coated with N3 dye possessed the
highest power conversion efficiency at4.03±0.58%, compared with that of DSSCs with pure
TiO2 (3.40±0.62%) electrode.
1. Introduction (FTO) glass; a dye sensitizer (e.g.

Electrical energyis an important factor ruthenium-based dye N719 or N3); an
for the development of the country. It can be electrolyte (I3-/I- redox couples) injected
either renewable or non-renewable, depending between the sensitizer and counter electrode;
on the resource that creates it. Renewable and a counter electrode (platinum and
energy is energy from renewable energy graphite) deposited on FTO glass. When the
sources such as sunlight, geothermal heat dye is excited by sunlight, it rapidly injects
and others. Solar cells convert the sunlight's an electron into photoanode electrode.
energy into electricity.1 Electrons are subsequently injected into the
Dye-sensitized solar cells (DSSCs), conduction band of the semiconductor and
the third generation solar cells, has gained quickly shuttled to the external circuit
attention because of their inexpensive through the conductive glass, producing an
production, highly efficient conversion of electric current. The electrolyte can restore
solar energy into electricity, and simple the dye to its original state by electron
manufacturing processes. DSSC possesses donation.7–9
conversion efficiency that is comparable to Metal oxide doping on TiO2
that of amorphous Si-based solar cells.2-5 nanostructure has been found to enhance the
In 1991, Grätzel et al. reported a photosensitivity of the TiO2 nanostructure.
DSSC, which is based on TiO2 nanoparticle, The metal oxide doping may enhance the
has highphotovoltaic conversion efficiency photosensitivity, promote surface reaction,
so this type of solar cell has attracted much and improve the efficiency of DSSC.10
commercial interest.6 Copper oxides (CuO) ischeap and
DSSC typically consists of a possesses low toxicity. With a narrow band
semiconductor (TiO2, ZnO,and others) thin gap of 1.2-1.85 eV), CuO can be coupled
film - served as a photoanode electrode - with a wide band gap semiconductor such as
that is coated on fluorine-doped tin oxide TiO2.11,12 The coupled TiO2and CuO can be
employed in photocatalysis, solar cells, and the solution was then added to TiO2 sol
others.9,13 Therefore, we studied the according to desire amount of CuO at 0, 0.1,
utilization of CuO/TiO2 hybrid nanoparticles 0.5, 1.0 and 3.0 %wt. while being stirred for
as an electrode in the DSSC.14 30 minutes.Homogeneous sol was obtained
at room temperature until further usein the
2. Materials and Methods spray coating process.
2.1 Materials 2.3 Preparation of ruthenium dyes
Nitric acid 65% (HNO3), Titanium To prepare the solutionof N3 or
tetraisopropoxide (Ti[OCH(CH3)2]4, TTIP), N719 dye, ruthenium-based N719or N3 dye
Lithium iodide (LiI), Iodine (I2), 4-tert- was weighed and was dissolvedin ethanol to
butylpyridine (4-TBP) and Sodium obtained 0.3 mM.The mixture was sonicated
hydroxide (NaOH) were purchased from inan ultrasonic bathfor 30 minutes until a
Sigma-Aldrich Inc. Copper(II) nitrate homogeneous solution was obtained.
trihydrate (Cu(NO3)2·3H2O) was purchased 2.4 Preparation of CuO/TiO2 electrode
from Honeywell Fluka. Acetonitrile FTO glass was cut into pieces with a
(C2H3N) and Acetone (C3H6O) were dimension of 2.0 × 2.5 cm2. The glass pieces
purchased from Labscan Ltd. Fluorine- cleaned three times with detergent solution,
doped tin oxide (FTO) conducting glass with acetone, and ethanol, respectively, using
a thickness of 2 mm and a resistance of 15 ultrasonic bathfor 15 minutes in each step.
Ω/cm2fluorine doped tin oxide coating on Fingerprints were wiped off using a tissue
one side, Surlyn film (25 µm thick sealing wettedwith ethanol and dried with blow
film), cis-bis(isothiocyanato)bis(2,2′-bipyri- dryer. Then the glass was covered with
dyl-4,4′-dicarboxylato)ruthenium(II) dye aluminum foil that was cut in a circular
(also known as N3) and ditetrabutyl- shape with a diameter of 0.5 cm. After that,
ammoniumcis-bis(isothiocyanato)-bis(2,2′- TiO2or CuO/TiO2 sol was deposited on the
bipyridyl-4,4′-dicarboxylato) ruthenium(II) FTO glass using an ultrasonic spray coater.
dye (also known as N719) were purchased The liquid feed rate to ultrasonic nozzle for
from Solaronix SA (Switzerland). TiO2 or CuO/TiO2 sol was set at 1 mL/min.
2.2 Preparation of CuO/TiO2 sol The power for frequency generator of
TiO2 was synthesized via a sol-gel ultrasonic nozzle was set at 3.5-3.6 W.
method by mixing the starting titanium In this research, the number of layers
isopropoxide (TTIP) in deionized water that of CuO/TiO2was 500 coats, which produced
contained 65% nitric acid as a catalyst.The a film with a thickness of approximately 10
volume ratio between TTIP, deionized µm. An electrode was sintered at 400 ˚C for
water, and nitric acid was 1:12:0.087. After two hours and left to be cooled at room
adding TTIP a white precipitate was temperature. The schematic diagram of the
instantaneously formed. The mixture was electrode is displayed in Figure 1.
vigorously stirred for three days at room
temperature until clear sol was obtained.Then
the clear sol was dialyzed in cellulose
membrane with a molecular weight cutoff of
3500. The distilled water used for dialysis
was changed daily until a pH of sol reached
to 3.5. Finally, TiO2 sol was kept into Figure 1. The schematic diagram of
refrigerator until further used. CuO/TiO2 electrode
In this research, Copper(II) nitrate
trihydrate was chosen as a precursor for Prior to dye immersion, an electrode
CuO. Copper(II) nitrate trihydrate was was gradually heated to 110 ˚C and was held
dissolved in deionized water. After its pH for 10 minutes to remove water.Then the
was adjusted to 3 using 5 M of nitric acid, electrode was slowly slid into a solution
ofeitherruthenium-based N719or N3 dyefor was made in the middle of the film, leaving
20-24 hours in the dark. Finally, the a square frame.
electrode was washed with ethanol to The two electrodes with sealing sheet
remove excess dye and was dried with a were clipped together with 2 clips on the
blow dryer. opposite end of the glass.Thesurlyn film was
2.5 Preparation of the platinum film softened by a heat gun and the sealing effect
counter electrode was obtained. To add the electrolyte solution,
The counter electrode was the solution was added dropwise until both
preparedon FTO glasses, which was cut into holes were completely filled. The
pieces with a dimension of 2.0 × 2.5 cm2. arrangement of the DSSCs component is
Then two small holes with a diameter of 1 shown in Figure 3.
mm was carefully drilled through the FTO After that both holes were sealed off
glass for subsequent filling of electrolyte by a clear adhesive tape to prevent the
solution. Next, the FTO glass was cleaned in electrolyte solution from leaking. The
the same manner as described in Section 2.4. resulting cell was ready for a measurement.
The platinum film was deposited on the
FTO glass using ion sputtering machine
(JEOL: JFC-1100E)at 10 mA of ion current
for six minutes. Sputtering area was 2.0 ×
2.0 cm2 in size. The schematic diagram is
displayed in Figure 2.
Figure 3. Fabrication of DSSC assembly for

testing
2.8 Physical and electrochemical

Figure 2. The schematic diagram of platinum characterization
film counter electrode The crystalline phase and crystallite
size of TiO2 and CuO/TiO2 was determined
2.6 Preparation of electrolyte using a SIEMENS D5000 X-ray
The electrolyte solution was a diffractometer with CuKα radiation (λ =
mixture of 0.5 M lithium iodide, 0.05 M 1.54439 Å) and Ni filter. The spectrum was
iodine, and 0.5 M 4-tert-butylpyridine in scanned at a rate of 0.04 min-1in the 2Ɵ
acetonitrile. The electrolyte solution was range of 20-80̊.
prepared by mixing 2 g of lithium iodide N2 physisorption was performed
(LiI), 0.38 g of iodine (I2) and 2.20 mL of 4- using a Micromeritics ASAP 2010 to
tert-butylpyridine (4-TBP) in 30 mL of determine the specific area from the BET
acetonitrile (Molar ratio LiI:I2:4-TBP = isotherm. The cumulative volume of pores
0.1:0.01:0.1). The solution was stirred for 30 and average pore diameter were estimated
minutes until homogeneity was obtained. from the total amount of N2 desorption by
2.7 Fabrication of dye-sensitized solar using BJH method.
cells The peak light absorption wavelengths
The DSSC was configured in a of ruthenium-based N719or N3 dyeswere
typical sandwiched cell by placing a TiO2 or determined by UV-Visible spectrophotometer
CuO/TiO2 electrode on the top of platinum (Perkin Elmer Lambda 650
film counter electrode with a 25 µm thick spectrophotometer) to be 314 nm for N3 dye
surlyn film inserted in the middle.Thesealing and 312 nm for N719 dye. To determine the
material was cut into a square with a amount of dye adsorbed on the CuO/TiO2
dimension of 2.0 × 2.0 cm2 and square hole film, the dye was dissolved in a solution of 0.1
M of NaOH in water and ethanol (1:1 volume The XRD peaksdetected at 2Ɵ of
fraction). The concentration of dye in the 25.3˚, 37.2˚, 48.2˚ and 62.8˚ were assigned
solution was later determined using UV- to anatase TiO2, whereas the peak at 27.4˚,
visible spectroscopy. 41.3˚ and 54.4˚ corresponded to be rutile
The light absorption of the catalysts phase and the peak at 30.9˚ was associated
was evaluated by UV-Visible spectroscopy. to brookite.15 A mixture of anatase and rutile
The diffuse reflectance spectrum of the TiO2 usually producesthe DSSC that
catalysts was obtained in the wavelength exhibits higher photovoltaic efficiency than
range of 200 – 800 nm with a step size of 1 pure anatase.16 XRD patterns of CuO/TiO2
nm.The band gap energy of the catalyst were shown in Figure 4. No XRD peak
wasestimated by equation (2-1); associated with CuO was detected.The
major phase in all samples was anatase with
1240
Eg = (2-1) some rutile and very small amount of
λ brookite. Table 1 lists the crystallite size of
TiO2 phase in CuO/TiO2. The crystallite size
when Eg is the band gap energy (eV) of the
became smaller when the amount of
catalyst and λis the wavelength at which the
CuO/TiO2was increased.
absorption begins (nm).
The electrochemical properties of
DSSC were determined by an IV-tester
under air mass (AM) 1.5 condition
(Keithley’s 2420 source meter). The current-
voltage characterization of DSSCs was
conductedunder simulated sun illumination.
The short current density (JSC), open circuit
voltage (VOC), and fill factor (FF) were
determined and this information was then
converted to the efficiency of a DSSC (η).
An area of our solar cell was 0.196 cm2. The
efficiency of DSSC (η) was determined by Figure 4. XRD patterns of CuO/TiO2
equation (2-2); powders at various percentages of CuO
VOC J SC FF Table 1. Crystallite size of anatase TiO2in

η= × 100 % (2-2) CuO/TiO2 calcined at 400˚C for two hours
Pin CuO/TiO2 Crystallite
(%wt.) size
when VOC is the open circuit voltage (V), (nm)
JSCis short current density (mA/cm2), FF is 0 8.70
0.1 8.00
the fill factor and Pin is the incident light
0.5 7.47
power (W/cm2). 1.0 7.35
3.0 6.68
3.1 Modification of TiO2 electrode layer Specific surface area and pore
by CuO properties of CuO/TiO2 were determined by
CuO/TiO2 sols were prepared by N2 physisorption.(as shown in Table 2.)
mixing different amounts of CuO and TiO2 Specific surface area, pore volume, and pore
sols to obtain the CuO amounts of 0.1, 0.5, diameter of 0.1% and 1.0% CuO/TiO2
1.0 and 3.0%wt. The CuO/TiO2 sol was differed only slightly when compared to pure
sprayed on the FTO glass substrates 500 TiO2.
times and was calcined at 400˚C for two The band gap energy of pure anatase
hours. TiO2 was 3.20 eV.17 The band gap energies
of various CuO/TiO2 were determined from to higher conversion efficiencies for the
Figure 5 and were reported in Table 3. An DSSCs with pure TiO2 and 0.1% CuO/TiO2.
increase in CuO/TiO2 content reduced its
band gap energy, resulting in ability to absorb
light in the visible region. With a newly
combined state because of the incorporation
of CuO into the lattice, an absorption peak in
the visible-light range occurred, and this
absorption increased with increasing CuO
content.18 In case of DSSC, the band gap
under 3.10 eV or absorption wavelength over
400 nm might not be suitable because it may
prevent some photons from reaching the dye
layer.
Figure 5. UV-visible adsorption characteristic
The amount of N719 or N3 dyes
of CuO/TiO2
adsorbed on CuO/TiO2 electrode was lower,
compared to pure TiO2 possibly because of
Table 4. The concentration of dyes contained
smaller specific surface area (see Table 4).
in the CuO/TiO2 electrode at various content
Table 2. Specific surface area, pore volume, of CuO
CuO/TiO2 Concentration of dyes
and average pore diameter of TiO2 and 0.1%
(%wt.) (×107mol/cm2)
CuO/TiO2 that were calcined at 400 ˚C for 2 N3 N719
hours 0 1.39 1.52
CuO/TiO2 BET BJH BJH 0.1 1.29 1.31
(%wt.) Surface area Porevolume Pore diameter 0.5 1.06 0.74
(m2/g) (cm3/g) (nm) 1.0 1.27 0.98
0 112.78 0.196 4.90 3.0 1.20 1.07
0.1 113.29 0.178 4.05
1.0 109.40 0.186 4.71
Table 5. Electrochemical properties of
Table 3. Band gap energy of TiO2 and DSSCs with CuO/TiO2 electrode coated with
CuO/TiO2 that were calcined at 400 ˚C for 2 N3 dye and were calcined at 400 ˚C for 2
hours hours
Sample VOC JSC FF η
CuO/TiO2 Wavelength Band gap energy
(%wt.) (V) (mA/cm2) (%)
(%wt.) (nm) (eV)
0 0.68 6.89 0.72 3.40±0.62
0 390 3.18
0.1 0.67 8.19 0.73 4.03±0.58
0.1 397 3.12
0.5 0.67 4.95 0.63 2.08±0.41
0.5 407 3.05
1.0 0.68 3.50 0.65 1.55±0.38
1.0 412 3.01
3.0 0.68 2.30 0.68 1.06±0.06
3.0 416 2.98
The I-V characteristics and Table 6. Electrochemical properties of

efficiencies of DSSC with TiO2 and DSSCs with CuO/TiO2 electrode coated
CuO/TiO2 electrodes were listed in Table 5. with N719 dye and were calcined at 400 ˚C
Five replicates of DSSC with each CuO for 2 hours
Sample VOC JSC FF η
content were prepared and they were (%wt.) (V) (mA/cm2) (%)
measured individually. The number 0 0.70 6.96 0.69 3.31±0.30
displayed was the average value of five 0.1 0.69 6.99 0.71 3.41±0.31
DSSCs value and the corresponding 0.5 0.66 3.69 0.67 1.64±0.68
standard deviation. 1.0 0.67 2.21 0.62 0.91±0.17
3.0 0.63 2.29 0.64 0.93±0.15
As seen in Table 4, the amount of
adsorbed dye in pure TiO2 and 0.1%
CuO/TiO2 were higher than the rest, leading
When the CuO content exceeded 0.1%, the Phillips, D. L. ACS Appl. Mater. Interf.
amount of adsorbed dye decreased and 2012, 4, 1254–1261.
consequently lowered the current density 5. Sauvage, F.; Decoppet, J. D.; Zhang, M.;
(JSC).19 The low efficiencies for DSSCs with Zakeeruddin, S. M.; Comte, P.;
CuO/TiO2 electrode that contained CuO Nazeeruddin, M.; Wang, P.; Grätzel, M. J.
greater than 0.1%wt. may be attributed to Am. Chem. Soc. 2011, 133, 9304–9310.
this low current density. Due to the high 6. O'Regan, B.; Grätzel, M. J. Nature 1991,
amount of adsorbed dye, the highest cell 353, 737–740.
efficiency of 4.03±0.58% was obtained with 7. Anders, H.; Gerrit, B.; Licheng, S.; Lars,
0.1% CuO/TiO2 electrode coated by K.; Henrik, P. J. Chem. Rev. 2010, 110
ruthenium-based N3 dye. (11), 6595–6663.
8. Justin, W. Y.; Seung,-H. A. L.; Yoji, K.;
4. Conclusion Emil, A. H.; Paul, G. H.; Thomas, A. M.;
CuO/TiO2 was successfully Ana, L. M.; Devens, G.; Thomas, E. M. J.
synthesized using sol-gel methods. Am. Chem. Soc. 2009, 131, 926–927.
Introduction of CuO caused the band gap to 9. Grätzel, M.; J. Photochem. Photobiol. C:
become narrower. When the CuO content Photochem. Rev. 2003, 4,145–153.
was increased beyond 0.1%wt., the amount 10. Dahyunir, D.; Siti, K. M. S.; Ade, U. B.;
of dye adsorbed on the electrode decreased Abdil, B.; Akrajas, A. U. J. Physica E
even though the specific surface area was 2017, 91, 185–189.
essentially unchanged. The highest power 11. Sun, Z.; Zhang, R. K.; Xie H. H.; Wang,
conversion efficiency was obtained with the H.; Liang, M.; Xue, S. J. Phys. Chem. C.
DSSC assembled with 0.1% CuO/TiO2 2013, 117 (9), 4364–4373.
electrode that was coated with N3 dye. 12. Zhao, W.; Sun, Y. L.; Castellano, F. N. J.
Am. Chem. Soc. 2008, 130 (38), 12566–
This research was supported by 13. Vaseem, M.; Umar, A.; Kim, S. H.; Hahn,
Center of Excellence on Catalysis, Faculty Y. B.; J. Phys. Chem. C. 2008, 112 (15),
of engineering, Chulalongkorn University. 5729–5735.
We would like to thank the Polymers for 14. Guo, E.; Yin, L. J. Phys. Chem. 2015, 17,
Energy Environment and Technology 563–574.
Research & Development Group 15. Porkodi, K.; Arokiamary, S. D. J. Mater.
(PENTEC), KMUTT for the I-V Sci. Technol. 2014, 30 (4), 328–334.
characteristic measurement. 16. Wang, H. H. J. Nanometer. 2011, 2011, 1–
7.
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www
Effect of chemical pretreatment on surface modification of carbon supports

derived from Cattail flower
Parncheewa Udomsap1,2, Nuwong Chollacoop2, Nilubon Jong-anurakkun3,
Yuji Yoshimura4, Apiluck Eiad-Ua1*
1
College of Nanotechnology,King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520,Thailand
2
National Metal and Materials Technology Center, National Science and Technology Development Agency,
3
Global Green Chemicals Public Company Limited, Bangkok 10900, Thailand
4
National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8565, Japan
Abstract:
Carbon supportsderived from Cattail flower as renewable biomass were prepared by
hydrothermal carbonization process at 180 °C for 8 hr then pyrolysis at 900 °C for 2 hr under
flow of N2. The carbon supports were pretreated separately with various acids as activated
chemicals including H2SO4, H3PO4, HNO3 and HCl. The effect of chemical pretreatments on
physical property of carbon was investigated by N2 adsorption and desorption isotherm. The
results found that the acid pretreatment can improve the surface areas of carbon. Especially
H3PO4, it can increase the surface area up to 860 m2/g with combination of micropore and
mesopore, which is close to value of commercial activated carbon (926 m2/g). The
improvement of the surface area of the carbon supports lead to high metal dispersion and
increase the active site, which enhance the catalytic activity of the catalyst. Furthermore, the
mesopore was more developed, which more facilitate diffusion of molecule.
1. Introduction decarboxylation, aromatization and re-

Currently, biomass has received condensation of biomass occur during the
attention as a precursor for carbonaceous hydrothermal carbonization process and be
materials production due to it is rich in stows the hydrochar with oxygenated
carbon in terms of carbohydrate, cellulose, functional group (OFG) which make it an
hemi-cellulose and lignin. Carbonnano- effective precursor for the production of
materials are widely used for a variety of chemically activated carbon. Reactivity of
application such as separation and precursors or the OFG content in the
purification of liquids and gases, solvent precursor is an important indicator of the
recovery and decolorizing, catalyst support, effectiveness of chemical activation and can
electrode and gas storage.1 In recent year, predict the extent to which porosity will be
hydrothermal carbonization has received developed in the activated carbon. By the
considered as an alternative method for way, many studied reported the BET surface
transformation of biomass into valuable area of hydrochars obtained from different
carbon materials. During hydrothermal starting raw materials were not over 100
carbonization process, biomass is heated in m2/g which is limitation of hydrochar.3
an oxygen free environment in the presence The activation of hydrochar with
of subcritical water and self-generated dehydrating chemical is widely used to
pressure.2 Carbonization temperature depend develop surface area of carbon.In chemical
on the type of starting materials and is activation, the biomass precursor is
typically employed in the range of 150-300 impregnated with a chemical agent and then
°C. The obtained solid product is called heated for carbonization in an inert gas
hydrochar. Hydrolysis, dehydration, atmosphere. The chemical agents help to
develop the activated carbon porosity by
means of dehydration and degradation. 6 hours. The obtained hydrochar products
Moreover, the advantages of the chemical were used as a precursor for chemical
activation process are lower pyrolysis activation. 5 g of precursor was pretreated
activation temperature, higher activated separately with various acids as activated
carbon yields, a large surface area, and well- chemicals, including H2SO4, H3PO4, HNO3
developed micro porosity.4 However, and HCl,at the same concentration 4
several chemical agents were studied for mol/Lfor a soaking time 3 hours. After the
activation of lignocellulosic materials. treatment, the mixtures were filtered and the
Potassium hydroxide (KOH) are used for the resulting solids were driedat 110 ºC for 12
activation of coal precursors or chars which hours. The carbonization and activation
can be develops large microporosity. On the ofthe impregnated sample was carried out in
other hand, the yield of activated carbon a horizon tubular reactor. Approximately 4 g
impregnated by basic agent is lower than of the impregnated sample was placed in the
those activated with zinc chloride or acid reactor and heated up to 900 ºC under
agents. The presence of metallic potassium nitrogen flow (200 cm3/min) at heating rate
and sodium will contaminate to the carbon of 10 ºC/min and held for 2 hours. After
matrix.5,6 The carbons obtained using zinc carbonization, the solid product was washed
chloride activation cannot be used in sequentially several times with hot distilled
pharmaceutical and food industries as it may water until neutral pH of the washing
contaminate the product. Moreover, it is solution. The produced samples were further
corrosive which is disadvantage to the dried at 110 ºC for 12 hours. The carbon
environment if inefficient chemical without chemical activation was carbonized
recovery.6 Therefore, the acid including at 900ºC under nitrogen atmosphere, which
H2SO4, H3PO4, HNO3 and HCl were was denoted CF. The activated carbons
attractive to use as activated chemical in this prepared with HNO3, HCl,H2SO4andH3PO4
research. Hydrochar derived from cattail were respectively CF-HN, CF-HC, CF-HS
flower as biomass were impregnated by and CF-HP.
separately acid. The change of textural and 2.3 Characterization
morphology of activated carbon were The textural properties of activated
investigated. carbons were characterized by determining
the nitrogen adsorption/desorption isotherm
2. Materials and Methods at -196 ºC using an automatic adsorption
2.1 Materials instrument (Micromeritic, ASAP2460). The
The biomass used for the preparation samples were degassed at 300 ºC for 12
of activated carbons was cattail flower hours before gas adsorption measurement.
which are abundant weed. Analytical grade The N2adsorption isotherm was obtained
of 98%w/w H2SO4, 85%w/w H3PO4, over a relative pressure, P/P0, ranging from
65%w/w HNO3 and 37%w/w HCl as approximately 10-6 to 1. The surface area of
chemical activating agents were purchased the activated carbons were determined by
from Sigma Aldrich. application of the Brunauer-Emmett-Teller
2.2 Preparation of activated carbon (BET). The BET surface areas were
Cattail flower as biomass starting assessed by applying relative pressure
materialswere converted to hydrochar via ranging from 0.01 to 0.3. The total pore
hydrothermal carbonization process. 30 g of volumes (Vtotal, cm3/g) were estimated to be
dried Cattail Flowers were mixed with 60 the liquid volumes of N2 at a high relative
mL of deionized water then transfer the pressure of 0.95. The micropore volume,
mixture to a 500 mL Glass-lined autoclave. Vmicro, was determined by the density
The autoclave reactor was heated to 180 ºC functional theory (DFT) method, and the
hold for 8 hours. After hydrothermal mesopore volume, Vmeso, was obtained by
treatment, the product was dried at 80 ºC for substraction of micropore volume and total
pore volume. The pore diameter distribution carbon without chemical activation as
of the activated carbon was obtained by reference carbon (CF) and activated carbon
applying the DFT method. Scanning by HNO3 (CF-HN) possess combination of
electron microscopy (SEM) analysis was isotherm type I and Type II, which arises
carried out on the activated carbon prepared from non-porous or macroporous surface.
from cattail flower under different However, it is indicative of presence of
conditions, to investigate its textural some microporous surface.
morphology, structure properties and the
development of porosity. (Zeiss EVO
MA10)

3.1 Characterizing pore structure of the
carbons
Pore structure of carbon can be
identified by adsorption of inert gases is
essential before liquid sorption. The shape
of the adsorption isotherm can provide
qualitative information on the adsorption
process and the extent of the surface area
available to the adsorbate. Figure 1 presents
the N2 adsorption-desorption isotherm at -
196 ºC of activated carbon from cattail
flower with different type of acid Figure 1. N2 adsorption-desorption isotherm
impregnation compared with carbon without of carbon without chemical activation (CF)
activation. It is apparent that isotherms of and activated carbon by HNO3 (CF-HN),
carbon activated by H2SO4 (CF-HS), H3PO4 HCl(CF-HC), H2SO4 (CF-HS) and
(CF-HP) and HCl (CF-HC) belonging to H3PO4(CF-HP)
type I and type IV according to the IUPAC
classification. A type I isotherm is The effect of chemical activation on
associated with microporous structures and a the BET surface area, micropore volume,
type IV isotherm indicated a mixture of and total pore volume are given in Table 1.
microporous and mesoporous materials. It The BET surface area and total pore volume
was clearly demonstrated that the amount of increase follow the order: CF < CF-HN <
adsorbed nitrogen was found to increase CF-HC < CF-HS < CF-HP. It can be seen
rapidly even at relative pressures lower than that the acid chemical activation can
0.1. At the beginning of each isotherm, improve the porosity of carbon. The
isotherms are type I, with a significant hydrochar precursors are subjected to acidic
uptake at low relative pressure, the conditions, both the acid-catalyzed
characteristics of microporous materials. hydrolysis of glycosidic linkages in
Furthermore, as the relative pressure polysaccharides and the cleavage of α- and
increases to 0.4, the isotherms exhibit β-aryl ether bonds in lignin occur, which
apparent hysteresis loops. The slope of the leads to the formation of gases, volatile
plateaus in these isotherms gradually fragments, and water soluble products. In
increases with increasing relative pressure. addition, acid accelerates the dehydration of
These results indicate that the pore structure the precursors, promoting the pore
is mainly mesoporous. Moreover, isotherm formation.8 Moreover, the ratio of
type IV can be classified to hysteresis loop micropore/mesopore volume of all samples
type IV (H4) which is character of narrow are consistent with N2 adsorption-desorption
slit-like pores and hollow sphere.7As for, isotherm results.
Considering the effect of acid, activated carbons mostly lies between 1 and
phosphoric acid reveals the best 5 nm, which obviously shows that the pore
performance due to it can increase the BET diameter are in the range of micropore.
surface area and total pore volume to 860 Moreover, activated carbon produced by
m2/g and 0.560 cm3/g, respectively, which is H3PO4 can be seen some pore diameter in
similar to the commercial activated carbon the mesopore range.
(SBET 926 m2/g and Vtotal 0.447cm3/g).
Table 1. Characteristics of activated carbons

prepared from cattail flower
Sample SBET Vmicro Vmeso Vtotal
(m2/g) (cm3/g) (cm3/g) (cm3/g)
CF 24 0.010 0.011 0.021
CF-HN 93 0.048 0.007 0.055
CF-HC 217 0.101 0.036 0.137
CF-HS 402 0.189 0.052 0.241
CF-HP 860 0.343 0.217 0.560
Commercial 926 0.320 0.127 0.447
AC
Furthermore, phosphoric acid can

develop the highest mesoporosity
proportion. Generally, activated carbon is
mainly microporous but it also contains
Figure 2. Pore diameter distribution
mesopores which is very important in
ofcarbon without chemical activation (CF)
facilitating the access of the adsorbate
and activated carbon by HNO3 (CF-HN),
molecules to the interior of the carbon
HCl(CF-HC), H2SO4 (CF-HS) and
particles.9 It is very essential and useful for
H3PO4(CF-HP)
catalyst support application. Hydrochloric
acid, nitric acid and sulfuric acid are
3.2 Comparison of the surfaces
classified the strong acid, the great of acid
morphology of activated carbon using
ionization constant (Ka). Whereas,
SEM
phosphoric acid is categorized of weak acid.
Scanning electron micrographs of the
The strong acid reacted with the hydrochar
surface morphology of the activated carbon
and volatile matter and diffused quickly out
samples are given in Figure 3. It is clear that
of the surfaces of particles during the
the carbon without chemical activation are
activation process. Furthermore, the
very low porous carbons, while the activated
gasification of surface carbon atoms became
carbon show the break-down of cellulose on
predominant, leading to an increase in the
the surface. Cavities were observed on their
weight loss and a low carbon yield.10Thus,
external surface. It seems that the cavities on
BET surface area and pore volume of
the surfaces of carbons resulted from the
activated carbon by strong acid series are
evaporation of the activating agent.11In the
lower than phosphoric acid. These findings
case of H2SO4 activation, the rough surface
are consistent with the literature; Activation
of the activated carbons with small cavities
soybean oil cake with strong base KOH
was formed. While, pore with a tunnel shape
presents BET surface area 618 m2/g, while
can be seen in carbon activated by H3PO4
weak base K2CO3shows higher surface area
which conform to N2 adsorption-desorption
(1353 m2/g) and carbon yield.5
isotherm result.
The DFT pore size distribution of
However, the others parameters,
activated carbons studied is shown in Figure
such as impregnation ratio of activation
2. The pore diameter distribution for the
20 µm 20 µm
20 µm 20 µm
20 µm
Figure 3. SEM images of activated carbons prepared with various acid activation (a) CF
(without chemical activation); (b) HNO3 (CF-HN); (c) HCl (CF-HC); (d) H2SO4 (CF-HS) and
(e) H3PO4 (CF-HP) at the same carbonization temperature 900 ºC
reagent, period of soaking and carbonization 4. Conclusion

temperature also effect to the occurrence of Cattail flower was utilized for the
porosity and the micropore/mesopore ratio production of activated carbon by acid
of activated carbon which optimum chemical activation. The maximum surface
condition are related to application of areas of the activated carbons prepared by
activated carbon. H3PO4 activation observed at 900 ºC, was as
large as values for commercial activated
carbons. The micropores and mesopores 3. Nizamuddin, S.; Baloch, H. A.; Griffin,
were well developed, which is advantages G. J.; Mubarak, N. M.; Bhutto, A.W.;
for metal loading as catalyst and molecule Abro, R.; Mazari, S. A.; Ali, B. S.
diffusion. Beside type of acid, the Renew. Sust. Energ. Rev. 2017, 73,
concentration of reagent, the period of 1289-1299.
impregnation and carbonization temperature 4. Yahya, M. A.; Al-Qodah, Z.; Zanariah
should be further studied. Moreover, the Ngah, C. W. Renew. Sust. Energ. Rev.
suitable chemical reagent should not be 2015, 46, 218-235.
toxic to environment. 5. Tay, T.; Ucar, S.; Karagoz, S. J. Hazard.
Mater. 2009, 165, 481-485.
Acknowledgements 6. Kumar, A.; Jena, H. M. Appl. Surf. Sci.
This research was financially 2015, 356, 753-761.
supported by Research and Researchers for 7. Prahas, D.; Kartika, Y.; Indraswati, N.;
Industries (RRi) project under The Thailand Ismadji, S. Chem. Eng. J. 2008, 140, 32-
Research Fund (TRF) and Global Green 42.
Chemicals Public Company Limited 8. Yorgun, S.; Yildiz, D. J. Taiwan Inst.
(GGC).And thankful to Nanoporous Chem. Eng. 2015, 53, 122-131.
Laboratory at College of Nanotechnology, 9. Su, J.; Chen, J. S. Microporous
King Mongkut’s Institute of Technology Mesoporous Mater. 2017, 237, 246-259.
Ladkrabang for their supporting. 10. Guo, Y.; Rockstraw, D. A. Carbon 2006,
44, 1464-1475.
References 11. Hayashi, J.; Kazehaya, A.; Muroyama,
1. Gonzalez-Garcia, P. Renew. Sust. Energ. K.; Watkinson, A. P. Carbon 2000, 38,
Rev. 2018, 82, 1393-1414. 1873-1878.
2. Jain, A.; Balasubramanian, R.;
Srinivasan, M. P. Chem. Eng. J. 2016,
283, 789-805.
Synthesis and photophysical properties of tetra-substituted pyrene
derivatives as blue emitting materials for electroluminescent devices
Patchareepond Panoy, Taweesak Sudyoadsak, Pichaya Pattanasattayavong,
Vinich Promarak*
Vidyasirimedhi Institute of Science and Technology, Wangchan, Rayong 21210, Thailand
*E-mail: vinich.p@vistec.ac.th
Abstract:
A new series of 1,3,6,8-substituted pyrene derivatives having 9,9-bis(4-
diphenylaminophenyl)fluorene and 7,9,9-tris(4-diphenylaminophenyl)fluorene as
substituents, namely Py-BisTPA and Py-TrisTPA were synthesized and characterized for
application as blue light-emitting materials for electroluminescent devices. Blue chromophore
pyrene is used as the core molecule due to its good charge carrier mobility and high thermal
stability while triphenylamine (TPA) substituted fluorene is used as substituents to enhance
hole-transporting abilities, suppress intermolecular interaction, and shorten fluorescence
lifetime. The structures of the synthesized molecules were confirmed by 1H-NMR, 13C-NMR,
and mass spectrometry. Optical properties were characterized by UV-Vis,
fluorescence/phosphorescence steady state and lifetime spectrometer (FLS). The emission
spectra of all compounds showed in the blue region of 460-470 nm in solution with an
approximately 15 nm red shift in thin film. Fluorescence lifetime of pyrene derivatives were
also found to be 0.5 to 3 ns.
1. Introduction properties for device fabrications, and those

Nowadays, organic light-emitting are solution processability, high thermal
diodes (OLEDs) are a field of interest in stability, intense luminescence efficiency,
both academic and industrial communities and enhanced charge carrier mobility. 7,8
because of their promising applications in Fluorene has also been widely used as a
full-color flat-panel displays and solid-state building block to form many emissive
lighting. 1,2 The search for stable and primary materials due to its high blue
color (red, green, and blue) emitters are photoluminescence efficiency, high carrier
crucial for the commercial application of mobility, and improved hole-injection
OLEDs. 3 Among the three, the development ability compared to other blue
of blue-emitting materials are still in chromophores. 9,10 To enhance the color
demand due to its imbalance of carrier purity and stability of fluorene-based
transporting, low efficiency and especially materials, large bulky groups or multi-
poor color purity. 4 Therefore, further efforts substituted rigid moieties is incorporated to
in this field with respect to increasing suppress aggregates and excimer. 11
efficiency and color purity of blue-emitters Triphenylamine (TPA) possesses a highly
have to be made. steric structure with hole-transporting
Due to its excellent emission properties and high thermal stability. In this
properties, strong absorption cross section work, we reported the synthesis and
and a long excited state lifetime, pyrene has characterization of 1,3,6,8-substituted
proved to be a promising chromophore for pyrene derivatives with novel TPA
making emitting materials for OLED substituted fluorene, namely Py-BisTPA
devices. 5,6 Additionally, pyrene and its and Py-TrisTPA. The chemical structure of
derivatives have shown several important these molecules was shown in Figure 1.
2. Materials and Methods N
2.1 General
All reagents were purchased from N
Aldrich, Acros, or TCI and used without

further purification. Tetrahydrofuran (THF) N
N -
was supplied by Burdick&Jackson and N
Py BisTPA
distilled by heating to reflux with sodium
N
and benzophenone under N2 atmosphere.
N
Dichloromethane for cyclic voltammetry
(CV) measurements was distilled twice from N
calcium hydride. Column chromatography

was performed with silica gel 60.
N N
2.2 Synthesis of Py-BisTPA and Py-
TrisTPA N N
2.2.1 Py-BisTPA
A mixture of 4 (150 mg, 0.19 mmol), N
1,3,6,8-tetrabromopyrene (20 mg, 0.03 N N

-
mmol), and Pd(PPh3)4 (8 mg, 0.007 mmol) Py TrisTPA
N
in THF (15 ml) and 2M aqueous Na2CO3
solution (1.54ml, 3.09 mmol) was refluxed N
at 80 °C for 48 h under N2 atmosphere. After N

N N
the reaction mixture was cooled to room

temperature, water (20ml) was added and
extracted with CH2Cl2 for 3 times. The Figure 1. Structures of Py-BisTPA and Py-
combined organic layer was separated and TrisTPA
washed with water and brine solution, then
dried over anhydrous Na2SO4. The solvent 4.63 mmol). The crude product was purified
was removed under vacuum and the residue by column chromatography over silica gel
was purified by column chromatography with using CH2Cl2/hexane (1:2) as the eluent
over silica gel with CH2Cl2/hexane (1:3) as to give Py-TrisTPA as a light yellow solid
the eluent to give Py-BisTPA as a light (170 mg, 81% yield). 1H NMR (600 MHz,
yellow solid (60 mg, 60% yield). 1H NMR CDCl3) δ (ppm): 8.01 (s, 4H), 7.93 (s, 2H),
(600 MHz, CDCl3) δ (ppm): 8.10 (s, 4H), 7.69 (d, J = 7.4 Hz, 8H), 7.63 (s, 4H), 7.59
8.01 (s, 2H), 7.81 – 7.75 (m, 8H), 7.73 (s, (s, 4H), 7.56 (d, J = 7.2 Hz, 4H), 7.53 (d, J =
4H), 7.65 (d, J = 7.6 Hz, 4H), 7.51 (d, J = 7.6 Hz, 4H), 7.42 (d, J = 8.0 Hz, 8H), 7.22 –
7.5 Hz, 4H), 7.42 (t, J = 7.3 Hz, 4H), 7.37 (t, 7.16 (m, 20H), 7.06 (d, J = 7.2 Hz, 28H),
J = 7.4 Hz, 4H), 7.20 (d, J = 7.3 Hz, 2H), 6.96 (s, 40H), 6.85 (d, J = 6.2 Hz, 36H),
7.12 (d, J = 8.4 Hz, 16H), 7.06 (s, 32H), 6.74 (d, J = 6.9 Hz, 36H). HRMS calculated
6.95 (d, J = 7.4 Hz, 32H), 6.84 (d, J = 8.1 for C284H198N12: 3778.5963; found
+
Hz, 32H); HRMS calculated for C212H146N8: 3778.3741 (M ).
2803.1670; found 2803.5489 (M+). 2.3 Methods
1
2.2.2 Py-TrisTPA H and 13C NMR spectra were
Py-TrisTPA was prepared according recorded with a Bruker AVANCE III HD
to the method used for Py-BisTPA by using (600 MHz) spectrometer. The mass spectra
3 (296 mg, 0.29 mmol), 1,3,6,8-tetrabromo- were recorded on Bruker Autoflex speedTM
pyrene (30 mg, 0.05 mmol) and Pd(PPh3)4 MALD-TOF. UV-Vis-NIR spectra were
(10 mg, 0.001 mmol) in THF (15 mL) and measured as a dilute solution in distilled
2M aqueous Na2CO3 solution (2.31 mL dichloromethane on a Perkin-Elmer Lambda
1050 spectrometer.
O
R Br
a) N
Br Br
N N
N N
b) 94% Br Br
Br
-
R O Py BisTPA
B
O d) 60%
1 R = H 60%
2 R = Br 94%
4
HO
c) 75% B N
HO
Br Br
N N N N
Br Br
b) 89% -
N O Py TrisTPA
N B
Br O d) 81%
3 5
Figure 2. Synthesis of the target molecules. Reagents and conditions: (a) CH3SO3H, 160 °C;
(b) Bis(pinacolato)diboron, Pd(PPh3)2Cl2, KOAc, toluene, 110 °C; (c) [Pd(PPh3)4], 2M
Na2CO3, THF, heat; (d) Pd(PPh3)4, 2M Na2CO3, THF, heat
Photoluminescence (PL) spectra and lifetime Py-TrisTPA were synthesized by Suzuki

were measured on Edinburgh instruments cross coupling of 1,3,6,8-tetrabromopyrene
fluorescence/ phosphorescence steady state with 4 or 5 in the presence of
and lifetime spectrometer (FLS 980). Cyclic Pd(PPh3)4/Na2CO3 as catalyst system in
voltammetry (CV) measurements were THF/water at reflux and obtained as light
performed at room temperature under inert yellow solids in good yields. Structures of
argon atmosphere in three electrode cells all compounds were confirmed by 1H-NMR,
13
with an Autolab potentiostat PGSTAT 101. C-NMR and mass spectrometry (MALDI-
The counter electrode was platinum. The TOF MS).
working and reference electrode were a 3.2 Optical properties
glassy carbon and Ag/AgCl, respectively. The optical properties of Py-BisTPA
and Py-TrisTPA were investigated in dilute
3. Results and Discussion CH2Cl2 solution and a thin film obtained by
3.1 Synthesis spin coating on a fused silica substrate
The synthetic route of the target (Figure 3a, Table 1). The absorption spectra
molecules was shown in Figure 2. The in solution of Py-BisTPA exhibited two
intermediates 1-2,12 313 and 4-514 were absorption peaks: one at approximately 310
synthesized according to the literature nm corresponding to the π-π* local electron
methods in the good yields. Py-BisTPA and transition of the pendant TPA moieties, and
Table 1. Optical and electrochemical properties of the synthesized molecules
λabs (nm) λem (nm) Lifetime (ns) Band
HOMO LUMO
Compound gap
(eV)b (eV)b
Soln Film Soln Film Soln Film (eV)a
Py-BisTPA 311, 410 315, 412 460 472 3.5 0.5 2.77 -5.37 -2.60
Py-TrisTPA 308, 372, 312, 374, 474 480 1.8 0.3 2.70 -5.31 -2.51
427 429
a
Estimated from the optical absorption edge, Eg = 1240/λonset
b
Calculated by HOMO = –(4.44 + Eonset), and LUMO = HOMO – Eg, where Eonset is the onset potential of
the oxidation.
a second absorption band about 410 nm

assigned to the π-π* electron transition of
the fluorene-pyrene conjugated backbone.
For the absorption spectra of Py-TrisTPA,
there are three absorption peaks. Two of
them are assigned similar to the absorption
of Py-BisTPA. In addition, the absorption
peak at 427 nm would be referred to the π-
π* electron transition of TPA-fluorene. In
the solid state, similar absorption features
with slight red-shifted compared to their
corresponding spectra in solution. In
addition, the emission spectra of Py-BisTPA
and Py-TrisTPA in solution showed an
emission peak in the blue region at 460 and
474, respectively. The emission of Py-
TrisTPA exhibited in the longer wavelength
more than Py-BisTPA due to more donor
effect of triphenylamine (TPA) in Py-
TrisTPA compound. The emission in thin
film of both compounds also appeared
slightly red-shifted because of the stacking
of the molecules as shown in Figure 3b.
3.3 Electrochemical properties
Electrochemical behaviors of all
compounds were investigated by cyclic
voltammetry (CV) and reported in Table 1.
From Figure 3c, Py-BisTPA and Py-
TrisTPA showed a quasi-reversible
oxidation processes with Eonset 0.93 and 0.87
V, respectively. The highest occupied
molecular orbital (HOMO) and lowest
unoccupied molecular orbital (LUMO)
energy levels were estimated according to
the electrochemical properties and UV–Vis
Figure 3. (a) UV/Vis absorption spectra, (b) absorption spectra. The calculated HOMO
photoluminescence spectra measured in of Py-BisTPA and Py-TrisTPA were -5.37
CH2Cl2 solution ( ̶ ̶ ̶ ) and thin film (----) and and -5.31, respectively.
(c) CV curves of the synthesized compounds
The HOMO level of Py-TrisTPA 4. Wu, C. H., Chien, C. H., Hsu, F. M.,
revealed upper than Py-BisTPA due to more Shih, P. I.; Shu C. F. J. Mater. Chem.
donor ability of triphenylamine in this 2009, 19, 1464.
molecule. The band gap of Py-TrisTPA 5. Zhao, Z. J.; Li, J. H.; Chen, X. P.; Wang,
(2.70 eV) was narrower than that of Py- X. M.; Lu, P.; Yang, Y. J. Org. Chem.
BisTPA (2.77 eV) which was corresponding 2009, 74, 383.
to the red-shifted of Py-TrisTPA compared 6. Xia, R. D.; Lai, W. Y.; Levermore, P. A.;
with Py-BisTPA in the emission spectra. Huang, W.; Bradley, D. D. C. Adv. Funct.
Mater. 2009, 19, 2844.
4. Conclusion 7. Zhang, H. J.; Wang, Y.; Shao, K. Z.; Liu,
Novel 1,3,6,8-substituted pyrene Y. Q.; Chen, S. Y.; Qiu, W. F.; Sun, X.
derivatives (Py-BisTPA and Py-TrisTPA) B.; Qi, T.; Ma Y. Q.; Yu, G.; Su, Z. M.;
were synthesized and investigated. All Zhu, D. B. Chem. Commun. 2006, 755.
compounds showed the emission spectra in 8. Sienkowska, M. J.; Farrar, J. M.; Zhang,
the blue region of 460-470 nm with strong F.;Kusuma, S.; Heiney, P. A.; Kaszynski,
luminescence efficiency. The donor ability P. J. Mater. Chem. 2007, 17, 1399.
of triphenylamine in Py-TrisTPA had an 9. Huang, H.; Fu, Q.; Zhuang, S.; Liu, Y.;
effect to the red-shifted of emission spectra Wang, L.; Chen, J.; Ma, D.; Yang, C. J.
and shorten luminescence to 1.8 ns while Phys
fluorescence lifetime of Py-BisTPA was 3.5 10. Wu, F.I.; Reddy, D. S.; Shu, C. F.; Liu,
ns in solution. M. S.; Jen, A. K. Y. Chem. Mater. 2003,
15, 269.
Acknowledgements 11. Gu, J. F.; Xie, G. H.; Zhang, L.; Chen, S.
This project was financially F.; Lin, Z. Q.; Zhang, Z.S.; Zhao, J.F.;
supported by the research grant of Xie, L. H.; Tang, C.; Zhao, Y.; Liu, S. Y.;
Vidyasirimedhi Institute of Science and Huang, W. J. Phys. Chem. Lett. 2010, 1,
Technology (VISTEC). Analytical 2849.
instruments were provided by the Frontier 12. Du, X.; Zhao, J.; Liu, W.; Wang, K.;
Research Center (FRC), VISTEC. Yuan, S.; Zheng, C.; Lin, H.; Tao, S.;
Zhang, X. H. J. Mater. Chem. C 2016, 4,
References 10301.
1. Tang, C. W.; Vanslyke, S. A. Appl. Phys. 13. Thangthong, A.; Prachumrak, N.;
Lett. 1987, 51, 913. Namuangruk, S.; Jungsuttiwong, S.;
2. Hung, L. S.; Chen, C. H. Mater. Sci. Kaewin, T.; Sudyoadsuk, T.; Promarak,
Eng., R 2002, 39, 143. V. Eur. J. Org. Chem. 2012, 5263.
3. Shih, P. I.; Tseng, Y. H.; Wu, F. I.; Dixit, 14. Jo, J.; Chi, C.; Hoger, S.; Wegner, G.;
A. K.; Shu, C. F. Adv. Funct. Mater. Yoon, D. Y. Chem. Eur. J. 2004, 10,
2006, 16, 1582. 2681.
Preparation and phase characterization of composites based on dispersion
of gold(III) chloride in dipolar discotic liquid crystal
Sawitree Juabram, Chanaiporn Danvirutai, Suwat Nanan*
Materials Chemistry Research Center, Department of Chemistry and Center for Innovation in Chemistry,
Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
*E-mail: suwatna@kku.ac.th
Abstract:
Composites based on dispersion of gold(III) chloride (AuCl3) in 1-nitro-2, 3, 6, 7, 10,
11-hexakis (hexyloxy) triphenylene (HAT6-NO2) have been prepared by using chemical
solution method. The thermal properties and phase transition temperatures of the composites
were determined from differential scanning calorimetric (DSC) method. All DSC curves of
the Au-doped HAT6-NO2 composites show clearing temperature (columnar-to-isotropic-
liquid phase transition) close to those of pure HAT6-NO2 matrix. Incorporation of 0.5-
1.0wt% AuCl3 in HAT6-NO2 does not destroy liquid crystal behavior of the matrix. The
photoluminescence (PL) emission spectra (λexcitation = 360 nm) of the composites were taken
in dichloromethane medium. The PL spectra show two emission peaks around 411 nm and
443 nm. Improvement of PL intensity is also observed with addition of gold(III) chloride up
to 1.0wt%. In addition, the UV–visible spectra of the composites have been monitored in
dichloromethane medium. After AuCl3 doping, a broad absorption throughout the visible
region was observed. The main peaks were at about 320 nm and 350 nm due to π→π∗
transition. This wide absorption over the visible region after AuCl3 doping makes the
composites as a potential candidate for photo applications and solar absorber.
1. Introduction electrical conductivity in DLC systems have

Discotic liquid crystals (DLCs) are been proposed for electronic and
fascinating nanomaterials with disc diameter optoelectronic devices, such as photovoltaic
2-6 nm. DLCs are emerging as one- cells,4 solar cells,5 organic light emitting
dimensional organic semiconducting diodes,6 and gas sensors.7 The efficiency of
materials. Due to the strong π-π interaction
1
such devices depends on the band gap of the
between the aromatic cores and the weak materials, which is typically about 4 eV for
interaction between the flexible aliphatic the DLC which therefore behave as
chains, the molecules can stack one over the insulators at normal conditions.8 On the
other, forming columns. These columns can other hand, they may be converted to useful
arrange in several ways forming columnar semiconductors by doping with either
hexagonal, columnar rectangular, columnar electron rich or electron deficient molecules
helical or columnar plastic phases.2 Typical into the supramolecular order of the liquid
column-column distance in the columnar crystalline phase. This of course means that
hexagonal phase is around 2–4 nm the doping should be maintained at an
depending on the aliphatic chain length and optimum concentration in order to retain the
the core-core distance within the column is liquid crystalline phase, while also be
around 0.35 nm with a length of a few tens introducing sufficient electron or hole
of nanometers.3 This strong intracolumnar concentration into the liquid crystalline
interaction and weak intercolumnar medium to increase the conductivity.9
interaction contribute to the quasi-one- Recently, dispersion of various
dimensional electrical conductivity along the metallic and semiconducting nanoparticles
columns. The quasi-one-dimensional such as titanium dioxide (TiO2),10 zinc
sulfide (ZnS),11 cadmium sulfide (CdS),12 identified by the immediate color change of
gold nanoparticles (AuNPs)13 and copper the solution from pale yellow to dark green.
nanoparticles (CuNPs)14 in the DLC matrix The composites based on addition of AuCl3 up
have been proposed. This is expected to to 1wt% have been prepared.
modify their physical and optical properties 2.3 Characterization of HAT6-NO2 and
of the composites for broad prospects of their AuCl3/HAT6-NO2 nanocomposites
applications. By doping with AuNPs , the The thermal properties and phase
dopants at low concentrations retain the transition temperatures of the HAT6-NO2
columnar phase with enhancement in the were determined by using differential
electrical conductivity by several orders of scanning calorimetric (DSC) and polarized
magnitude.15 For the CuNPs dopants, it has optical microscopic (POM) method. The
been shown that the presence of Cu NPs DSC experiment (Perkin Elmer Pyris One)
introduces surface plasmon resonance (SPR) was performed in N2 gas (100 mL/min)
and reduces the optical band gap of DLC.14 using a temperature range of 50 to 150 ºC
In this work, we focused on with a heating rate of 10 ºC/min. The
preparation of conducting nanocomposites polarized optical micrographs were
based on dispersion of gold(III) chloride determined using an Olympus BX51
(AuCl3) in DLC matrix, namely 1-nitro-2, 3, instrument, coupled with a Mettler heater
6, 7, 10, 11-hexakis (hexyloxy) triphenylene with scanning rate of 10 oC/min. The FT-IR
(HAT6-NO2) via simple chemical solution spectra were determined on a FT-IR
method. The optical properties, thermal spectrophotometer (Perkin Elmer Spectrum
properties and phase transition temperatures One) at room temperature in the
of the composites were also studied. wavenumber range of 4000-500 cm-1 with
10 scan and 4 cm-1 resolution. The UV–
2. Materials and Methods visible spectra were investigated on a
2.1 Preparation of HAT6-NO2 Spectrum One; Perkin Elmer
1-Nitro-2, 3, 6, 7, 10, 11-hexakis spectrophotometer over the wavelength range
(hexyloxy) triphenylene (HAT6-NO2) was of 200-800 nm. The room temperature
prepared according to the method described photoluminescence (PL) spectra were
elsewhere16 and the molecular structure is determined on Shimadzu RF-5301PC
shown in Figure 1. spectrofluorometer by taken the sample in
dichloromethane medium at a concentration
C6H13O OC6H13
of 1 mg/ 4 mL and were measured in the
NO2
wavelength range of 300-600 nm with the
excitation wavelength (λexcitation) of 360 nm.
C6H13O OC6H13 3. Results and Discussion

3.1 HAT6-NO2
C6H13O OC6H13 3.1.1 Vibrational spectroscopy
The vibrational spectrum was
Figure 1. Chemical structure of HAT6-NO2 recorded by Fourier transforms infrared
spectrophotometer (FT-IR) using KBr pellet
2.2 Preparation of AuCl3/HAT6-NO2 technique. The FT-IR spectrum of HAT6-
composites by chemical solution method NO2 is shown in Figure 2. The structure of
The conducting nanocomposites HAT6-NO2 consists of triphenylene ring and
based on dispersion of AuCl3 in discotic the substitutions group from ether. The
liquid crystal matrix have been prepared by peaks at 2995 and 2850 cm-1 are due to the
dissolving the discotic molecules in C–H stretching vibration of alkane. Peak
dichloromethane and then adding AuCl3. A arounds 1450-1550 cm-1 is attributed to C=C
slow evaporation of the solvent results in stretching from aromatic ring of
formation of a complex, which is easily triphenylene. The peak at 1070 cm-1 is
attributed to C-O stretching of ether. The
peak at 1290 cm-1 indicates NO2 stretching
vibration. The observed vibrational band
Heat Flow Endo Up (mW)

position and vibrational mode of HAT6-NO2
was summarized in Table 1.
Colh I
∆H = 7.09 J/g
HAT6-NO2
60 70 80 90 100 110 120 130 140 150

Temparature (oC)
Figure 3. DSC thermogram of HAT6-NO2

liquid crystal
Figure 2. FT-IR spectrum of HAT6-NO2 3.1.3 Polarized optical microscopy

The phase transition temperatures of
Table 1. FT-IR peak assignment of HAT6- discotic liquid crystal were also studied by
NO2 liquid crystal polarized optical microscopy (POM) with
Wavenumber Intensity Assignment scanning rate of 10oC/min. The polarized
-1
(cm ) optical micrographs were determined using
an Olympus BX51 instrument, coupled with
2950, 2850 Strong C-H (CH3)
a Mettler heater. The sample was
stretching sandwiched between a glass slide and a
1550, 1450 Strong C=C stretching of cover slip. The sample was heated to the
aromatic ring isotropic phases and each texture was
observed during heating. HAT6-NO2
1290 strong NO2 stretching
exhibited two phases namely hexagonal
1070 strong C-O stretching columnar liquid crystal phase (Colh) and
isotropic liquid phase (I). The clearing
3.1.2 Differential scanning calorimetry (Colh-to-I) temperature is 138 oC. The focal
The phase transition temperatures conic fan texture of hexagonal columnar
and associated enthalpy values of discotic liquid crystal phase is shown in Figure 4a-c.
liquid crystal were determined by 3.2 AuCl3/HAT6-NO2 composites
differential scanning calorimetry (DSC) with 3.2.1 Differential scaning calorimetry
a scanning rate of 10oC/min form both The phase transition temperatures and
heating and cooling processes. The DSC associated enthalpy values of discotic liquid
thermogram of pure HAT6-NO2 (Figure 3) crystal were determined by differential
showed one peak due to transition from scanning calorimetry (DSC) with a scanning
hexagonal columnar (Colh) phase to rate of 10 oC/min form both heating and
isotropic (I) phase known as clearing cooling processes. The DSC thermogram of
temperatures (Colh-to-I) at about 138 oC. It AuCl3/HAT6-NO2 in Figure 5 displayed one
is expected that at the melting point peak due to the clearing temperatures (Colh-
(transition from crystal to mesophase), the to-I) of 138 oC. It should be noted that the
alkyl chains of molecular crystals melt and clearing temperature form both heating and
provide fluidity to the mesophase while at cooling processes are comparable. Figure 5c
the clearing point (transition from shows the effect of AuCl3 content on
mesophase to the isotropic liquid) the transition temperatures of the composites.
unstacking of the central aromatic cores is
converted into an isotropic liquid state. 14
(a)
(b)
(c)
Figure 4. Polarized optical micrographs of

HAT6-NO2; (a) hexagonal columnar liquid
crystal phase at 85 °C, (b) 120 °C and (c)
124 °C
It is clearly seen that the clearing

temperature of the composites is not
significantly changed even addition of Figure 5. DSC thermograms of pure HAT6-
AuCl3 up to 1wt%. The slightly decrease of NO2 and AuCl3/HAT6-NO2 composites from
enthalpy with increasing dopant (a) first heating, (b) first cooling and (c) the
concentration may due to dilution effect. effect AuCl3 content on transition
3.2.2 UV-visible spectroscopy temperatures of the composites
The UV–visible spectra of all
composites have been taken in The spectra of composites showed a
dichloromethane. Pure dichloromethane has broad absorption band at 320 nm and 350
been taken as a reference sample. UV- nm corresponding to band gap energy of
visible spectra of AuCl3/HAT6-NO2 3.41 and 3.73 eV, respectively. This is due
composites in comparison to that of pure to π→π∗ transition in the discotic
HAT6-NO2 are shown in Figure 6. molecules.2 This wide absorption over the
14
(a)
(a)
12
10
(αhν)2
8
2 Eg = 3.41 eV
0
3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.60
hν (eV)
200
(b)
(b)
Figure 6. UV-Vis spectra of HAT6-NO2 150
and AuCl3/HAT6-NO2 composites
(αhν)2
100
Table 2. The optical band gap of HAT6-
NO2 and AuCl3/HAT6-NO2 composites 50
Optical band gap (eV)
Eg = 3.70 eV
Material
S0→S1 S0→S2 0
3.50 3.55 3.60 3.65 3.70 3.75 3.80 3.85 3.90
HAT6-NO2 3.41 3.70 hν (eV)
0.5 wt% AuCl3/ HAT6-NO2 3.41 3.73

Figure 7. Determination of optical band gap
1.0 wt% AuCl3/ HAT6-NO2 3.41 3.66 of pure HAT6-NO2 from absorption spectra:
(a) 320-360 nm and (b) 360-390 nm
UV region after doping AuCl3 makes the
composites as potential candidates for photo HAT6-NO2
applications. (a) 0.5% AuCl3/HAT6-NO2

1.0% AuCl3/HAT6-NO2
The optical band gaps (Eg) of the
PL emission (a.u.)
composites are related with absorption

coefficient (α) as follows14:
αhυ = A (hυ−Eg)n (1) (4.1)
where hv is incident photon energy with v

being the frequency of the incident light, A 400 420 440 460 480 500 520 540
is a constant, α represents absorption Wavelength (nm)
coefficient, α = 4πk/λ, (k is the absorption

index), λ is the wavelength, and n = ½ for (b)
the allowed direct band gap. By using HAT6-NO2
Equation 1, plot of (αhυ)2 vs hυ in Figure 7

0.5% AuCl3/HAT6-NO2
1.0% AuCl3/HAT6-NO2
PL excitation (a.u.)
provided optical energy band gap of the

samples and these are also summarized in
Table 2.
3.2.3 Photoluminescence study
Photoluminescence (PL) emission and
excitation spectra of pure HAT6-NO2 and
composites were taken in dichloromethane 310 320 330 340
Wavelength (nm)
350 360 370
medium at a concentration of 1 mg/4 mL.

Figure 8. PL emission spectra (a) and PL
excitation spectra (b)
Figure 8a shows the PL emission Kaen University for partial financial support
spectra of AuCl3/HAT6-NO2 composites in under Incubation Researcher Project.
comparison to that of pure HAT6-NO2. using
λexcitation of 360 nm. The composites with References
0.5wt% and 1wt% of AuCl3 showed peak at 1. Kumar, S. NPG Asia Mater. 2014, 6, 1-13.
about 431 and 443 nm, respectively while the 2. Nanan, S. KKU Sci. J. 2013, 41 (2), 320-
pure HAT6-NO2 liquid crystal matrix showed 329. Thai.
PL peak at about 423 nm. This indicates that 3. Kumar, P. S.; Kumar, S.; Lakshminarayan,
the interaction of AuCl3 with HAT6-NO2 V. J. Appl. Phys. 2009, 106, 093701.
discotic liquid crystal strongly influences the 4. Iino, H.; Takayashiki, Y.; Hanna, J. I.;
shift in PL peak position toward higher Bushby, R. J. Jpn. J. Appl. Phys. 2005, 44,
wavelength (red shift). L1310-2.
5. Yamanaka, N.; Kawano, R.; Kubo, W.;
4. Conclusion Masaki, N.; Kitamura, T.; Wada, Y.;
The composites based on dispersion Watanabe, M.; Yanagida. J. Phys. Chem.
of AuCl3 in discotic liquid crystal (HAT6- B. 2007, 111 (18), 4763–4769.
NO2) matrix have be prepared successfully. 6. An, Z.; Yu, J.; Jones, S. C.; Barlow, S.; Yoo,
The clearing temperatures of the composites S.; Domercq, B. Adv. Mater. 2005, 17,
are comparable to those of pure HAT6-NO2 2580-2583.
liquid crystal matrix. Incorporation of 0.5- 7. Sergeyev, S.; Pisula, W.; Geerts, Y. H.
1wt% AuCl3 in HAT6-NO2 does not destroy Chem. Soc. Rev. 2007, 36, 1902-29.
liquid crystal behavior of the matrix. After 8. Boden, N.; Bushby, R.; Movaghar, B.; Ency.
AuCl3 doping, there is a broad absorption Mater. Sci. Tech. 2001, 2, 2194-2203.
throughout the UV region showing the peaks 9. Kamaliya, B.; Vijay, K. M.; Yelamaggad,
at about 320 nm and 350 nm due to π→π∗ C. V.; Krishna Prasad, S. Appl. Phys. Lett.
transition. This wide absorption over the UV 2015, 106, 083110.
region after doping AuCl3 makes the 10. Roy, J. S.; Pal Majumder, T.; Dabrowski, R.
composites as potential candidates for photo J.Mol. Struc. 2015, 1098, 351-354.
applications. The interaction of AuCl3 with 11. Roy, J. S.; Majumder, T. P.; Dabrowski, R.;
HAT6-NO2 discotic liquid crystal strongly Dey, A.; Ray, P. P. Opt. Mat. 2015, 46,
influences the shift in PL peak position 467–471.
toward higher wavelength (red shift). 12. Yuan, K.; Chen, L.; Chen, Y. Chem. Eur. J.
Interestingly, improvement of PL intensity is 2014, 20, 11488 – 11495.
also observed with addition of AuCl3 up to 13. Basova, T. V.; Parkhomenko, R. G.;
1wt%. Polyakov, M.; Gürek, A. G.; Atilla, D.;
Yuksel, F.; Ryabchikova, E. I.; Kadem, B. Y.
Acknowledgements Dyes Pigm. 2016, 125, 266-273.
We are grateful to Materials 14. Yaduvanshi, P.; Mishra, A.; Kumar, S.;
Chemistry Research Center (MCRC), Dhar, R. J. Mol. Liq. 2015, 208, 160-164.
Department of Chemistry, Faculty of 15. Holt, L. A.; Bushby, R. J.; Evans, S. D.;
Science, Khon Kaen University for providing Burgess, A.; Seeley, G. J. Appl. Phys. 2008,
research facilities. S. Juabrum wishes to 103, 063712.
thank partial fund from MCRC as well. C. S. 16. Kumar, S. J. Phys. Chem. B. 1998, 102,
Nanan also would like to acknowledge Khon 4697-4710.
One step synthesis of PtCo/TiO2 catalysts by flame spray pyrolysis for
selective hydrogenation of furfural to furfuryl alcohol
Kitima Kruachao1, Okorn Mekasuwandumrong2, Piyasan Praserthdam1, Joongjai Panpranot1*
1
Center of Excellence on Catalysis and Catalytic Reaction,Department of Chemical Engineering,
2
Silpakorn University, Nakorn Pathom 73000, Thailand
*E-mail: joongjai.p@chula.ac.th
Abstract:
Furfural is a biochemical derived from lignocellulosic biomass that can be used to
produce furfuryl alcohol (FA), the raw material of several products such as lysine, furan
resins, lubricant oils, and ascorbic acid. The liquid-phase selective hydrogenation of furfural
to FA was carried out in a batch reactor at 323 K and 20 bar H2 using Pt/TiO2 (0.5wt% Pt)
and PtCo/TiO2 (0.5wt% Pt and 0-0.2wt.% Co) synthesized by one-step flame spray pyrolysis
(F) and impregnation (I) methods. The monometallic (F)-Pt/TiO2 and (I)-Pt/TiO2 exhibited
similar furfural conversion at ~83-85% after 2 h reaction time but the FA selectivity of the
(F)-Pt/TiO2 was much higher (95%) than the (I)-Pt/TiO2 (71.5%). As revealed by various
characterization results, the furfural hydrogenation activity and FA selectivity were found to
be correlated to %anatase of the TiO2 supports and the presence of the strong Pt-TiOx
interaction on the catalysts, respectively regardless of the preparation methods used. The
highest yield of FA at 97.5% can be obtained using (I)-Pt-0.2Co/TiO2 catalysts after 2 h
reaction time under the reaction conditions used.
1. Introduction towards C=O bond hydrogenation than the

Presently, fossil fuels are mainly corresponding monometallic Pt catalysts in
used as precursors in the production of many cinnamaldehyde hydrogenation reaction.3
petrochemical products but they are The addition of Co to Pt may be useful for
unsustainable and caused global warming. improving the hydrogenation of C=O to
Thus, it is interesting to use renewable produce FA in the selective hydrogenation
biomass as feedstock instead of fossil fuels. of furfural.
Furfural is a biomass-derived chemicals, Flame spray pyrolysis technique
received by acid-catalyzed dehydration of (FSP) is known as a one-step method for the
xylose, the main building-block of synthesis of nanoparticles. The advantages
hemicellulose constituent of lignocelluloses.1 of FSP are that the nanoparticles made from
The selective hydrogenation of FSP have high purity and relatively narrow
furfural to furfuryl alcohol (FA) is an size distribution and their properties can be
interesting reaction for producing lysine, tailored by controlling the synthesis
furan resins, lubricant oils, and ascorbic conditions such as precursor concentration
acid. Platinum (Pt) catalysts are commonly and dispersion gas flow rate.4,5 The FSP-
used in the selective hydrogenation of made catalysts have shown high catalytic
furfural because of their high catalytic performances in hydrogenation reactions
activity, however, they usually give medium than the catalysts prepared by the
selectivity of FA.2 conventional impregnation method.6
Cobalt (Co) is an interesting In this study, the Pt/TiO2 and
promoter for platinum catalysts. It has PtCo/TiO2 were prepared by FSP method
been reported that Pt-Co showed higher and tested in the selective hydrogenation of
hydrogenation activity and selectivity furfural to FA. The catalysts were also
characterized by N2 physisorption, X-ray naphthenate in xylene. The catalysts were
diffraction (XRD), CO pulse chemisorption dried in an oven at 100 °C overnight in air.
and H2-temperature-programmedreduction Afterwards, the catalysts were calcined in
(H2-TPR). oxygen at 500 °C for 2 h and reduced in H2
flow (25 cm3/min) at 500 ◦C for 2 h. The
2. Materials and Methods monometallic and bimetallic catalysts
2.1 General prepared by impregnation method are called
Platinum acetylacetonate, cobalt as (I)- Pt/TiO2 and (I)-PtCo/TiO2 .
naphthenate 6wt.% in mineral spirits, and 2.2.3 Catalytic hydrogenation
titanium(IV) butoxide reagent grade from The catalytic performances of the
Aldrich were used as platinum, cobalt, and prepared catalysts were tested in the liquid
titanium precursors, respectively. phase selective hydrogenation of furfural to
2.2 Catalyst preparation FA. Approximately 0.05 g of catalyst, 50 µL
2.2.1 FSP-synthesized catalysts of furfural and 10.0 mL methanol of were
The monometallic Pt/TiO2 (0.5wt% loaded into in a 100 mL stainless steel
Pt) and bimetallic PtCo/TiO2 (0.5wt% Pt autoclave reactor (JASCO, Tokyo, Japan).
and 0-0.2 wt.% Co) catalysts were prepared The reactor was purged with hydrogen to
by FSP method according to the procedure remove the air for three times. The reaction
reported in Ref.7. The designed amounts of was executed at 50 ◦C, 2 MPa of H2 for 120
metal precursor in xylene (MERCK; min. The reaction was controlled while
99.8vol.%) were prepared with total metal stirring the reaction mixture with a magnetic
concentration at 0.3 M. Liquid precursor stirrer. After the reaction, the reactor was
solution was then fed into flame reactor by a cooled to below room temperature with an
syringe pump at 5 mL/min and dispersed ice-water and carefully depressurized. The
with oxygen 5 L/min forming a fine droplets liquid product was then analyzed by a gas
by a gas- assisted nozzle fed by 5 L/min of chromatograph attached with a flame
oxygen (Thai Industrial Gas Limited; purity ionization detector.
>99%). Pressure drop (capillary tip) was 2.3 Catalyst characterization
maintained at 1.5 bar, the orifice gap area at The XRD patterns were collected
nozzle was adjusted. The spray was burned using a Siemens D5000 diffractometer with
by supporting flame lets fed with oxygen (3 a Ni filter and CuKα radiation. The BET
L/min) and methane (1.5 L/min) which were surface area, pore volume and pore diameter
positioned in a ring around the nozzle outlet. were determined by physisorption of N2
A sintered metal plate ring (8 mm wide, using a Micrometrics ASAP 2020
starting at a radius of 8 mm) provided an instrument. The percentages of platinum
additional 10 L/min of oxygen as sheath for dispersion were measured from CO-pulse
the supporting flame. The product particles chemisorption technique using a
were produced by passing through the flame Micromeritics ChemiSorb 2750 (pulse
at a temperature of approximately 2000◦C in chemisorption system). About 0.05 g. of
milliseconds and collected on a glass fiber catalyst was reduced under hydrogen flow
filter (Whatman GF/C, 15 cm in diameter) (25 cm3/min) at 500 °C for 2 h with a
with the aid of a vacuum pump. The heating rate of 10 ºC/min and cooled down
monometallic and bimetallic catalysts to the room temperature, and then helium
prepared by FSP method are called as gas was inserted into the sample cell (25
(F)- Pt/TiO2 and (F)-PtCo/TiO2. cm3/min) for remove air. The CO was
2.2.2 Impregnation-synthesized catalysts pulsed over the reduced catalyst until the
For comparison, the catalysts were TCD signal from the pulse was constant.
prepared by co-impregnation method. Next, 20 μL of carbon monoxide was
Titanium (P25) support was impregnated injected into catalysts and repeated until the
with platinum actetylacetonate and cobalt desorption peak were unchanged. The H2-
TPR experiments were performed to flame-made catalysts and impregnation
determine reducibility and reduction catalyst led to higher BET surface area and
temperature of platinum catalysts. The pore volume, and larger pore diameter of the
demonstration were carried out in a quartz catalysts.
U-tube reactor. For each measurements, all The reduction behaviors of the
the catalyst samples were pretreated with a monometallic and bimetallic catalysts
N2 flow (25 cm3/min, 1 h, 150 °C). The TPR obtained by different preparation methods
profiles were obtained by passing carrier gas were studied by H2-TPR technique. The
(10% H2 in nitrogen) through the catalyst results are shown in Figure 2. Both
samples (30 mL/min) with a temperature synthesized catalysts showed three reduction
ramp from room temperature to 600 °C at 10 peaks at 99-124 ◦C, 312-397 ◦C and 509-687
ºC/min. ◦C. The first reduction peak was correlated
to the reduction of PtOx to metallic Pt0.11-12
3. Results and Discussion The second peak at around 340 ◦C was
3.1 Catalyst characteristics associated to reduction of Pt species
The XRD patterns of the catalysts interacting with the TiO2 support to form Pt-
prepared by FSP and conventional TiOx interface site13,14 and the hydrogen
impregnation method are shown in Figure consumption above 500 ºC was due to the
1. The measurements were carried out at the reduction of surface capping oxygen of
diffraction angles (2θ) between 20º and TiO2.13 In this study, the second reduction
80°. All the synthesized samples showed the peaks were shifted to higher reduction
characteristic peaks of the crystalline phases temperatures when Co was added to the
of TiO2 consisting of anatase phase at Pt/TiO2 catalysts, suggesting stronger
2θ=25° (major), 37°, 48°, 55°, 56°, 62°, 71°, interaction between Pt and TiO2 support. It
and 75° and rutile phase at 2θ = 28° (major), is also possible that some cobalt particles
36°, 42°, and 57°8 The characteristic peaks migrated onto the Pt surface.15
of Pt and Co cannot be detected in all the
XRD patterns due to the low metal loading
and/or high dispersion of these metals . The
percentage of anatase phase was estimated
by the equation given in Ref.9 Table 1 shows
%anatase, BET surface area, pore volume,
pore diameter and average pore diameter of
the catalysts. It was found that adding Co
resulted in a drastically decrease of
%anatase content of the flame-made
catalysts from 89 to 57%. It has been
suggested that addition of a second element
during FSP synthesis of TiO2 established
higher number of defects, most likely Figure 1. The XRD patterns of the Pt/TiO2
oxygen vacancies, inside the TiO2 crystals and PtCo/TiO2
and affected to accelerating the anatase to
rutile transition.10 On the other hand, Comparing between the FSP and the
%anatase content of the impregnation impregnation catalysts, the second and the
catalysts was not much altered ranging third reduction peaks of the impregnation
between 85 to 92% as Co loading content catalysts appeared at higher temperature
increased from 0 to 0.2wt.%. The BET than the FSP-synthesized ones, indicating
surface area of the monometallic (F)-Pt/TiO2 higher degrees of interaction of the Pt-TiOx
and (I)-Pt/TiO2 were 42.6 and 52.2 m2/g, species on the impregnation catalysts. The
respectively. Increasing Co content for both peaks corresponding to PtOx species were
also detected on the (F)-Pt-0.1/TiO2 and phase of TiO2 as illustrated in Figure 3,
(F)-Pt-0.2Co/TiO2. regardless of the preparation methods used.
The Pt active sites were measured by
using CO pulse chemisorption results based
on the assumption that one CO molecule
adsorbed on one Pt site. The percentages of
platinum dispersion are shown in Table 2. It
was found that addition of Co into the
Pt/TiO2 catalysts resulted in higher
percentages of platinum dispersion for both
the flame-made and the impregnation
catalysts. The higher Pt dispersion
corresponded well to the stronger interaction
Pt-TiOx sites from the H2-TPR results. In
addition, it was suggested that addition of
Co-promoted the formation of Pt terrace
atoms.15
3.2 The selective hydrogenation of
furfural
The catalytic performances of the
prepared catalysts were investigated in the
selective hydrogenation of furfural at 50 ◦C
and 2 MPa of H2 with methanol as the
solvent. In this reaction, the desired selective
hydrogenation product FA was observed as
well as the side reaction product which
occurred from the methanol reaction Figure 2. H2-TPR profile of (a) FSP
forming solvent product (SP) as shown in catalysts and (b) impregnation catalysts
Figure 4.2 The conversion of furfural and
the selectivity to FA are shown in Table 2. It Similar results trend has been
appeared that the activities for furfural reported for supported Pd catalysts in
hydrogenation of both Pt/TiO2 and Pt- the acetylene hydrogenation that anatase
Co/TiO2 catalysts depended on %anatase TiO2 support gave higher hydrogenation
activity of the active metal than rutile one.16
Table 1. Physicochemical and catalytic properties of the Pt/TiO2 and PtCo/TiO2 catalysts
Entries Catalyst %Anatasea BET surface area Pore volumeb Pore diameterb
(m2/g) (cm3/g) (nm)
1 (F)-Pt/TiO2 89.6 42.6 0.12 14.0
2 (F)-Pt-0.04Co/TiO2 82.4 - - -
3 (F)-Pt-0.1 Co /TiO2 69.0 49.8 0.10 10.6
4 (F)-Pt-0.2 Co /TiO2 57.0 82.9 0.27 10.8
5 (I)-Pt/TiO2 85.8 52.2 0.28 16.5
6 (I)-Pt -0.04Co /TiO2 88.1 - - -
7 (I)-Pt -0.1Co /TiO2 89.7 59.5 0.35 18.8
8 (I)-Pt -0.2Co /TiO2 92.4 60.0 0.44 25.1
a
Base on the XRD results
b
Determined from the Barret-Joyner-Halenda (BJH) desorption method
Table 2. CO chemisorption and reaction results of the Pt/TiO2 and PtCo/TiO2 catalysts
Entries Catalyst Pt Conversion Selectivity (%)
dispersion (%)
(%)
FA SP
1 (F)-Pt/TiO2 23.5 83.4 95.1 4.9
2 (F)-Pt-0.04Co/TiO2 26.1 71.1 95.5 4.5
3 (F)-Pt-0.1 Co /TiO2 62.7 68.3 97.2 2.8
4 (F)-Pt-0.2 Co /TiO2 71.7 47.3 100 0
5 (I)-Pt/TiO2 34.1 84.6 71.5 28.5
6 (I)-Pt -0.04Co /TiO2 37.5 85.8 88.9 11.1
7 (I)-Pt -0.1Co /TiO2 43.2 86.4 93.5 6.5
8 (I)-Pt -0.2Co /TiO2 46.2 100 97.5 2.5
Reaction (50 µL furfural in 10 ml methanol) at 50 ºC with a 50 mg catalyst under 20 bar H2 after 120 min
showed higher FA selectivity than those

prepared by impregnation method, the
conversion of furfural was lower due to the
lower %anatase phase of the TiO2 supports.
Co addition resulted in the improved catalyst
performances in terms of FA selectivity for
both flame-made and impregnated catalysts.
The higher FA selectivity was correlated
well to the stronger Pt-TiOx species as
revealed by the H2-TPR results.
Figure 3. Correlation between furfural

conversion and %anatase content of the
TiO 2 supports
For a similar metal composition, the

FSP-made catalysts exhibited higher FA
selectivities than the impregnation ones. The
Figure 4. Simplified reaction scheme of the
FA selectivities, however, increased with
hydrogenation of furfural
increasing Co loading for both catalysts. It is
suggested that the stronger Pt-TiOx
Acknowledgements
interaction induced by Co addition promoted
The financial supports from the
the formation of FA and reduced the solvent
Thailand Research Fund (TRF) and the
product being formed. For the other
Ratchadaphiseksomphot Endowment Fund
catalyst system such as Ir/TiO2, the strong
for International Research Integration: Chula
Ir-TiOx interaction was also found to favor
Research Scholar are gratefully
FA selectivity in furfural hydrogenation.17
acknowledged.
4. Conclusion
References
The effects of preparation method
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Fundamental study of carbon materials from oil male palm flowers via
hydrothermal carbonization
Kanogpan Guntagerng1, Gasidit Panomsuwan2, Apiluck Eiad-ua1*
1
College of Nanotechnology, King Mongkut's Institute of Technology, Ladkrabang, Bangkok, Thailand
2
Department of Materials Engineering, Faculty of Engineering, Kasetsart University, Bangkok, Thailand
Abstract:
The application of biomass-derived energy is gaining in importance due to growing
environmental concerns. Oil male palm flowers are one of agricultural waste materials
produced from palm oil processing. This research aims to study the morphology of hydrochar
obtained via hydrothermal treatments of oil male palm flowers at 160–200 ℃ for 4–12 h,
followed by carbonization at the temperature range from 300 to 900 ℃ under a nitrogen
atmosphere for 2 h. The samples were characterized by scanning electron microscopy (SEM),
Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. The results
showed that the hydrochar obtained from carbonization at 900 ℃ for 2 hour time possessed
rough and rupturing surface, implying the development of porous structure. The FTIR results
revealed that the functional groups were disappeared at high carbonization temperature,
indicating the successful conversion of oil male palm flowers into carbon materials.
1. Introduction solid fuel).6 The HTC is usually performed

Lignocellulosic biomass materials at temperature range of 180–280 ℃ and
are derived from a variety of plants, pressures higher than water saturation
agricultural crop residues and animals, such pressure to against water into liquid state,
as corn,1 husk,2 wheat straw,3 and bagasse.4 under an inert atmosphere. Reaction time
Moreover, it is enormous and renewable has been reported in a broad range from 1
energy source that can help to reduce the min to several hours.7 Additives, such as
greenhouse gas emission as well as improve acids or bases, can affect the carbon
economy and energy security.5 Thailand is formation, which is then physically or
an agricultural country that produces a large chemically activated to obtain a porous
amount of biomass residues every day. carbon structure.8 Carbonization of biomass
Among several agricultural products, oil has a number of advantages when compared
palm is one of the major economic crops in with common biological treatment
Thailand. Oil male palm flower (OMPF) is processes. It generally takes only hours,
one of the major waste products in oil palm instead of the days or months required for
industry. However, no report is available biological processes, permitting more
concerning the hydrochar derived oil male compact reactor design.9
palm flower by applying thermochemical In this work, OMPF was employed
methods till now. Therefore, it would be as a precursor in the synthesis of carbon
worth if we can develope the effective materials via HTC process. The temperature
strategy to increase the value of oil palm and time used in hydrothermal process were
wastes by converting them to carbon varied in the range of 160–180 ℃ and 4–12
materials. h, respectively. The effects of hydrothermal
Hydrothermal carbonization (HTC) carbonization condition on the properties of
is a thermochemical conversion process carbons were characterized by scanning
from biomass into smokeless, high carbon electron microscopy (SEM), Fourier
content, and more valuable products (i.e.,
transform infrared spectroscopy (FTIR), and Hydrochars via HTC at 160, 180, and 200 ℃
Raman spectroscopy. in Figure 1 (b–j) and Figure 2 (b–j) suggest
that preparation at higher temperature and
2. Materials and Methods longer time causes a rough and rupture
The OMPFs were obtained from surface. Hydrothermal treatment of OMPF
Krabi province, Southern Thailand. They (fiber and pigment) at 200 ℃ 12 h was
were dried at 90 ℃ in air overnight. Their found to be the best conditions owing to a
size was then reduced by using a blender dominance of rupture surface and
machine, followed by sieving with a 700-μm degradation of the cellulose, hemicellulose
sieve. After that, OMPF-P (pigment) and and lignin component during HTC11 in
OMPF-F (fiber) were obtained. 10 g of comparison with raw OMPF (fiber and
OMPF-P and OMPF-F were mixed with 100 pigment).
mL of DI water and then transferred into a To identify the presence of
Teflon mold and stainless-steel reactor. The functional groups, the FTIR spectra of
reaction temperature was varied from 160 to OMPF (pigment and fiber) and hydrochars
180 ℃, while reaction time was varied from after HTC were collected as shown in Figure
4 to 12 h. The hydrochar was quenched via 3(a, b) and 4(a, b), respectively. As shown in
water and dried at 90 ℃ overnight. Figure 3(a, b), it can be observed broad
Hydrochar samples were carbonized at 300– absorption bands at 3000 and 3600 cm–1
900 ℃ for 2 h under N2 flow. The obtained and a small sharp peak at 2800−2950 cm–1,
carbon samples were characterized to corresponding to stretching vibration of O–
investigate their physical and chemical H (hydroxyl) and C–H stretching vibrations,
properties based on various techniques, respectively. The absorption bands at 1650
including SEM, FTIR, and Raman and 1720 cm–1 are attributed to C=O and
spectroscopy. C=C functional groups, reflecting their
aromatic nature, respectively.12 The bands in
3. Results and Discussion range of 1150–1250 cm–1 suggests the
The experiments were conducted presence of C–O and O–H bending
separately to investigate the effect of vibrations. In addition, small bands emerged
processing temperature and time. The at 950–1000 cm–1 is assigned to the C–H
experimental conditions are summarized in vibration in aromatic structure.13 – 16
Table 1. However, after carbonization
treatment, the absorption bands were
Table 1. Experimental conditions of disappeared and the FTIR spectra became
hydrothermal carbonization (HTC) featureless as depicted in Figure 4(a) and (b)
Condition Temperature (°C) Time (h) The absence of absorption bands at 1000–
1 160 4 1600 cm–1 and 3000–3600 cm–1 suggests the
2 160 8 occurrence of dehydration and
3 160 12 decarboxylation during carbonization.17
4 180 4 According to the FTIR results, it indicates
5 180 8
6 180 12
that carbonization at high temperatures
7 200 4 under inert atmosphere could lead to strong
8 200 8 reduction of the oxygen content through
9 200 12 decomposition of functional groups.
Furthermore, to evaluate the
The SEM images of hydrochar aromatic structures in hydrochar products,
surface obtained at different times and Raman spectroscopy analysis was
temperatures reveal the rupturing of the performed, as shown in Figure 5(a) and (b).
lignocelluosic structure in HTC process.10
Figure 1. SEM images of (a) (500)
OMPF-P untreated HTC, (b) (1000) HTC Figure 2. SEM images of (a) (500)
at 160 ℃ for 4 h, (c) HTC at 160 ℃ for 8 h, OMPF-F untreated HTC, (b) (1000) HTC
(d) HTC at 160 ℃ for 12 h, (e) HTC at 180 at 160 ℃ for 4 h, (c) HTC at 160 ℃ for 8 h,
℃ for 4 h, (f) HTC at 180 ℃ for 8 h (g) (d) HTC at 160 ℃ for 12 h, (e) HTC at 180
HTC at 180 ℃ for 12 h (h) HTC at 200 ℃ ℃ for 4 h, (f) HTC at 180 ℃ for 8 h, (g)
for 4 h, (i) HTC at 200 ℃ for 8 h, (j) HTC at HTC at 180 ℃ for 12 h, (h) HTC at 200 ℃
200 ℃ for 12 h for 4 h, (i) HTC at 200 ℃ for 8 h, (j) HTC at
200 ℃ for 12 h
Figure 3. FTIR spectra of OMPF (pigment and fiber) hydrochars synthesized from HTC
process (Ia) OMPF-P, (IIa) OMPF-P HTC at 200 ℃ for 4 h, (IIIa) OMPF-P HTC at 200 ℃
for 8 h, (IVa) OMPF-P HTC at 200 ℃ for 12 h, (Ib) OMPF-F, (IIb) OMPF-F HTC at 200
℃ for 4 h, (IIIb) OMPF-F HTC at 200 ℃ for 8 h, (IVb) OMPF-F HTC at 200 ℃ for 12 h
Figure 4. FTIR spectra of OMPF (pigment and fiber) (Ia) OMPF-P carbonized at 300 ℃ for 2
h, (IIa) OMPF-P carbonized at 500 ℃ for 2 h, (IIIa) OMPF-P carbonized at 700 ℃ for 2 h,
and (IVa) OMPF-P carbonized at 900 ℃ for 2 h, (Ib) OMPF-F carbonized at 300 ℃ for 2 h,
(IIb) OMPF-F carbonized at 500 ℃ for 2 h, (IIIb) OMPF-F carbonized at 700 ℃ for 2 h, and
(IVb) OMPF-F carbonized at 900 ℃ for 2 h
Figure 5. Raman spectra of OMPF (pigment and fiber) (Ia) OMPF-P 300 ℃ for 2 h, (IIa)
OMPF-P 500 ℃ for 2 h, (IIIa) OMPF-P 700 ℃ for 2 h, and (IVa) OMPF-P 900 ℃ for 2 h,
(Ib) OMPF-F 300 ℃ for 2 h (IIb) OMPF-F 500 ℃ for 2 h, (IIIb) OMPF-F 700 ℃for 2 h,
and (IVb) OMPF-F 900 ℃ for 2 h
The Raman spectra of carbonized samples 4. Conclusion

had two broad overlapping bands at about HTC of OMPF was performed by
1350 cm−1 (D peak) and 1600 cm−1 (G varying processing time (4–12 h) and
peak), which are related to benzene or temperature (160–200 °C). The reaction
condensed benzene rings of amorphous temperature and time in HTC process play
carbons.18,19 This result confirms the the significant roles in controlling the
existence of small aromatic structure, which properties of hydrochar products. Among
is consistent with the abovementioned FTIR several conditions investigated, we found
results. that the hydrochar products prepared by
hydrothermal process at 200 ℃ for 12 h had 7. Toufiq, M.; Janet, A.; Bejamin W.;
rough and rupturing surface as compared Daniela, B.; Judith, P.; Joan, L.; Jan, M.
with raw materials. Further carbonization at Bioenerg. Res. 2014, 1, 11–29.
900 ℃ for 2 h resulted in a complete 8. Nizamuddin, S.; Shrestha, S.; Athar, S.;
degradation of function groups through both Ali, B.; Siddiqui, M. Adv. Eng. Res.
dehydration and decarboxylation. We can 2015, 32, 489–505.
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derived from OMPF (pigment and fiber) via Biores. Technol. 2013, 142,
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Photocatalytic degradation of methylene blue over polydopamine-Ag/TiO2
Kamonthip Tammarakwattana*, Akawat Sirisuk
Faculty of Engineering, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand
*E-mail: kat.kamonthip@gmail.com
Abstract:
This research investigated the photocatalytic degradation of methylene blue in
aqueous phase over Ag/TiO2 catalyst that was modified by polydopamine (PDA). Titanium
dioxide was synthesized via a sol-gel method and silver (2%wt) was loaded onto TiO2 by an
incipient wetness impregnation method. Then polydopamine-Ag/TiO2 catalyst was prepared
by an impregnation method, using dopamine as a precursor. The amounts of dopamine added
were 5 and 10%wt, respectively. The photocatalytic degradation of methylene blue in
aqueous phase was conducted under both UV and visible light irradiations over pure TiO2,
2% Ag/TiO2, 5% PDA-2% Ag/TiO2 and 10% PDA-2% Ag/TiO2 catalysts. In each
experiment the catalyst was dispersed in an aqueous solution of 10 ppm methylene blue. The
suspension was stirred in the absence of light for 60 minutes and was then irradiated at an
ambient temperature by either UV or visible light bulbs for 120 minutes. The concentration
of methylene blue was measured at every 15 minutes using a UV/Visible spectrophotometer.
Addition of polydopamine to Ag/TiO2 photocatalyst improved photocatalytic performance in
methylene blue degradation. The improvement was more pronounced under visible light
irradiation.
1. Introduction More recently, bioinspired

Photocatalytic water treatment polydopamine (PDA) has gained a wide
process using semiconductor photocatalysts range of interest in functionalizing the
has been considered as an eco-friendly and surface of biomaterials as well as in
clean technology.1-3 The most widely-used different fields such as biosensing, catalysis,
photocatalyst is TiO2 because of its low and energy storage.11-17 PDA can be
manufacturing cost, low toxicity, and good synthesized easily through the self-
photochemical stability, and high activity.4,5 polymerization of dopamine, which is cheap
However, its relatively large bandgap of 3.2 and widely available.18,19 PDA absorbs a
eV gives rise to the catalyst being active in wide spectrum of light in both UV and
the ultraviolet region only.6 Several visible region and exhibits good
techniques have been developed to extend photoconductivity under visible light
its use under visible light to take advantage irradiation, leading to an increase of
of solar energy (e.g., doping, dye photogenerated charge carriers due to its
sensitization, and coupling with other polymeric π-conjugated structure leading to
semiconductors).7,8 electrical conduction capability.20,22
In recent years, various metals have In this study we investigated the
been investigated for the modification of photocatalytic degradation of methylene
titanium dioxide so as to improve its blue in aqueous phase over Ag/ TiO2
photocatalytic efficiency.9 Silver-modified catalysts that were modified by PDA at the
TiO2 catalyst has shown improvement in amounts of 5 and 10 %wt, respectively. The
photocatalytic activity of dye degradation experiments were conducted under both UV
under both UV and visible light and visible light irradiations.
irradiations.10
2. Materials and Methods was rinsed with DI water three times and the
2.1 Materials collected solid was dried at 110 °C.
The chemicals used in the 2.5 Photocatalytic experiments
preparation of the catalyst include silver The photocatalytic activity of the
nitrate (AgNO3, Aldrich), titanium catalyst was measured for the
tetraisoproproxide (Ti[OCH(CH3)2]4 97%, photodegradation of methylene blue under
Aldrich), nitric acid (HNO3 65%, Aldrich), UV and visible light irradiations at an
dopamine hydrochloride (C8H11NO2⋅HCl ambient temperature. Initially, 0.4 g of
Aldrich), sodium hydroxide (NaOH, catalyst was added to 400 mL of an aqueous
Aldrich) and Tris(hydroxymethyl)- solution of 10 ppm methylene blue. The
aminomethane (tris-buffer solution, mixture was stirred in the absence of light
Aldrich). for one hour to obtain adsorption
2.2 Synthesis of titanium dioxide equilibrium. After that the mixture was
Titanium dioxide was synthesized exposed to either UV irradiation using 75W
via a sol-gel method. Titanium isopropoxide UV-C lamp (Philips), the wavelength of
(TTIP) was poured into deionized water (DI) UV-C is 250 nm which the shortest
containing a measure amount of 65% nitric wavelength irradiation (UV-C) of UV
acid. The mole ratio of DI:HNO3:TTIP is irradiation was proved that the most
55.55:0.1064:0.271. After the addition of effective degradation of methylene blue23, or
TTIP, white precipitate was formed visible light irradiation using 18W TL-D
instantaneously. The mixture was stirred for Standard Colors lamp (Philips). During 120
3 days until clear sol was obtained and then minutes of irradiation, 4 mL of the solution
clear sol was dialyzed in cellulose was extracted every 15 minutes to measure
membrane with MW cutoff of 3500. The the concentration of methylene blue using
distilled water used in dialysis was changed UV-visible spectrophotometer.
daily until a pH of sol reached 3.5. Finally, 2.6 Spectroscopic measurement
the sol was dried and calcined at 400 °C for The BET surface area of the catalysts
two hours. was measured, using Micromeritics ASAP
2.3 Preparation of Ag/TiO2 2020 surface area analyzer. Fourier
The Ag/TiO2 catalyst was prepared transform infrared (FTIR) spectra of the
by an incipient wetness impregnation PDA-Ag/TiO2 catalyst were obtained from
method. A desired amount of AgNO3 was an ATR-FTIR spectrometer in wavelength
dissolved in DI water and was loaded onto 400-4000 nm, using Thermo Scientific
TiO2 in order to obtain silver at 2 %wt. The Nicolet 6700 FT-IR spectrometer. The X-ray
catalyst was dried and then was calcined at diffraction (XRD) analysis was conducted
400 °C for two hours. using a SIEMENS D5000 X-ray
2.4 Synthesis of PDA-Ag/TiO2 catalyst diffractometer with a Cu Kα radiation
First, desired amounts of Ag/TiO2 source at a scan rate of 0.04 min−1 in the 2Ɵ
and dopamine hydrochloride (i.e., to obtain range of 20-80o. The UV−visible diffuse
5 and 10%wt of PDA) were added to 100 reflectance spectra of the sample in the
mL of DI water. The mixture was stirred at a wavelength range of 200-800 nm were
room temperature for 30 minutes. After that, obtained using Perkin Elmer Lambda 650
100 mL of tris-buffer solution (10 mM) was spectrophotometer. The concentration of
added and the pH of the mixture solution methylene blue was measured by Perkin
was adjusted to 8.5 using NaOH solution. Elmer Lambda 650 spectrophotometer at the
The mixture was stirred at 60 °C for 12 wavelength of 664 nm.
hours. Then the mixture was cooled down to
a room temperature and was centrifuged 3. Results and Discussion
under 2000 rpm for 20 minutes. The solid 3.1 XRD analysis
XRD spectra of pure TiO2, Ag/TiO2, bands observed at 2000 cm-1 and around
5% PDA-Ag/TiO2, and 10% PDA-Ag/TiO2 2200-2400 cm-1 were characteristic of C-H
catalysts are presented in Figure 1. From the and O=C=O bonds, respectively.27-30 When
spectra of the four samples diffraction peaks Ag/TiO2 was modified with PDA, the peaks
at 2Ɵ values of 25.37°, 37.86°, 38.61°, at 1150, 1200, 1350 and 1750 cm-1,
48.12° and 75.0°, which were associated corresponding to C-O, C-NH2, C-O-H, and
with the anatase phase, were observed, while aromatic C-C bonds, respectively, were
diffraction peaks at 27.48°, 56.65°, 64.2° more pronounced due to the presence of
and 68.79° corresponded to the rutile PDA.31
phase.24 A small peak at 2Ɵ of 27.52° 3.3 Specific surface area measurement
represented PDA in the catalyst.25,26 The The specific surface areas of the
crystallite sizes of the catalyst were catalysts are listed in Table 1. The specific
calculated using Debye-Scherer equation surface area of Ag/TiO2 was smaller than
and listed in Table 1. The crystallite size of that of pure TiO2 because of the second
pure TiO2 was 9.8 nm and modification of calcination. When PDA was loaded onto
the catalyst with Ag and PDA decreased the Ag/TiO2, the specific surface area decreased
crystallite size of the catalyst. even further, suggesting that the coverage of
PDA prevented N2 probe molecule from
entering the pores.
Figure 1. XRD result of the four catalysts
Table 1. Surface area and crystallite size of

TiO2, Ag/TiO2, 5%PDA-Ag/TiO2 and Figure 2. FTIR spectra of (a) TiO2, (b)
10%PDA-Ag/TiO2 calcined at 400 °C for 2 Ag/TiO2, (c) 5% PDA-Ag/TiO2 and (d)
hours 10%PDA-Ag/TiO2
Sample SBET Crystallite
(m2 g-1) size (nm) 3.4 UV-visible absorption spectra
Pure TiO2 131.47 9.8
To assess the optical absorption
Ag/TiO2 107.75 9.1 properties of the catalyst, UV visible
5%PDA-Ag/TiO2 103.22 8.3 spectroscopy was conducted in the range of
10%PDA-Ag/TiO2 87.89 7.6 200 to 800 nm. As shown in Figure 3(a),
TiO2 does not absorb any light in the visible
3.2 FTIR analysis region (greater than 400 nm). When Ag was
The FT-IR spectra of TiO2, Ag/TiO2, doped into the TiO2, the visible light
5% PDA-Ag/TiO2, 10% PDA-Ag/TiO2 response improved slightly (see Figure
catalysts are shown in Figure 2. The band at 3(b)). Moreover, PDA modification has a
wavenumbers of 600-700 cm-1 was considerable influence on the optical
attributed to Ti-O-Ti stretching vibration, property of PDA-Ag/TiO2. An increase in
which was observed in every sample. The PDA in the catalyst gave rise to a drastic
increase in the absorption of light in the
visible-light region, suggesting that the
PDA-Ag/TiO2 possesses a potential to
harvest more visible light than pure TiO2 and
Ag/TiO2. The band-gap energy of the
catalyst is estimated from and is displayed in
Figure 4. The band-gap energies of TiO2,
Ag/TiO2, 5% PDA-Ag/TiO2, and 10% PDA-
Ag/TiO2 were approximately 3.2, 3.02, 2.4
and 2.0 eV, respectively. As the amount of
PDA increased, the band gap of the catalyst
became narrower. The narrowing of the
band gap suggested that PDA-Ag/TiO2 Figure 4. Plots of the Kubelka–Munk
composite was able to absorb light with function versus the energy of the incident
longer wavelength, compared to TiO2. light, as derived from the diffuse reflectance
Consequently, the composite would exhibit UV–Vis spectra for (a) TiO2, (b) Ag/TiO2,
a better performance in photocatalytic (c) 5% PDA-Ag/TiO2 and (d) 10%PDA-
degradation of dye. Ag/TiO2
3.5 Photocatalytic degradation of
methylene blue
The photocatalytic degradation of
methylene blue was chosen as a model
reaction to evaluate the photocatalytic
activities of the four catalysts under UV and
visible light irradiations. As shown in Figure
5(a) and 5(b), the concentration of
methylene blue decreased in first 60 minutes
in the absence of light because of adsorption
on the surface of the catalyst. Under UV
irradiation the conversion of methylene blue
in the system containing pure TiO2 is about
48.4% after 180 minutes. Doping TiO2 with
Ag raised the conversion slightly. However,
when the Ag/TiO2 was modified by PDA,
Figure 5. Photocatalytic degradation of

methylene blue result under (a) UV and (b)
visible light irradiations
Figure 3. UV-vis absorption spectra of (a)
TiO2, (b) Ag/TiO2, (c) 5% PDA-Ag/TiO2 the conversion of methylene blue was
and (d) 10%PDA-Ag/TiO2 increased to 69.3% and 85.4% for the
system containing 5% and 10% PDA, 1. Hoffmann, R. M.; Martin, T. S.; Choi,
respectively. W.; Bahnemann, W. D. Chem. Rev.
As seen in Figure 5(b), the 1995, 95, 69-96.
photocatalytic conversions of methylene 2. Cho, Y. J.; Kim, I. H.; Lee, S.; Choi, W.
blue under visible light irradiation were J. Catal. 2015, 330, 387-395.
lower than that of the system under UV 3. Hamdy, M. S.; Saputera, W. H.;
irradiation for all four catalysts. The Groenen, E. J.; Mul, G. J. Catal. 2014,
addition of Ag prevented the recombination 310, 75-83.
of photogenerated electron-hole pair and 4. Pathak, P.; Meziani, J. M.; Li, Y.;
facilitated the transfer of electrons to oxygen Cureton, T.; Sun, Y. P. Chem. Commun.
adsorbed on the surface of the catalyst.32,33 2004, 10, 1234-1235.
As discussed above the presence of PDA 5. Pathak, P.; Meziani, J. M.; Castillo, L.;
enhanced the light absorption ability of the Sun, Y. P. Green Chem. 2005, 7, 667-
catalyst in the visible region, leading to 670.
more photogeneration of electrons and holes 6. Fujishima, A.; Rao, T. N.; Tryk, D. A. J.
Consequently, the conversion of methylene Photochem. Photobiol. 2000, 1, 1-21.
blue was increased when the amount of 7. Dong, S.; Feng, M.; Fan, M.; Pi, Y.; Hu,
PDA added to the catalyst was increased L.; Han, X.; Liu, M.; Sun, J. RSC Adv.
from 5% to 10%. Furthermore, PDA could 2015, 5, 14610-14630.
act like a gripper with a high adsorption 8. Lim, J.; Murugan, P.;
efficiency for organic matter. Lakshminarasimhan, N.; Kim, Y. J.;
Lee, S. J.; Lee, H.S.; Choi, W. J. Catal.
4. Conclusion 2014, 310, 91-99.
In summary, PDA-Ag/TiO2 catalysts 9. Cozzoli, D. P.; Comparelli, R.; Fanizza,
were successfully prepared by a sol-gel E.; Curri, L. M.; Agostiano, A.; Laub, D.
method, followed by impregnation methods. J. Am. Chem. Soc. 2004, 126, 3868-
The addition of PDA caused the band gap of 3879.
the catalyst to become narrower. Therefore, 10. Ko, S.; Banerjee, K. C.; Sankar, J.
the PDA-Ag/TiO2 composite was able to Composites Part B. 2011, 42, 579-583.
absorb light in the visible region. The results 11. Zhou, H. W.; Tang, F. S.; Yao, H. Q.;
of the photodegradation of methylene blue Chen, R. F.; Yang, H. H.; Wang, X. R.
under both UV and visible light confirms Biosens. Bioelectron. 2010, 26, 585-589.
that the PDA-Ag/TiO2 catalyst exhibit high 12. Liu, R.; Mahurin, S. M.; Li, C.; Unocic,
photocatalytic activity. As the amount of R. R.; Idrobo, J. C.; Gao, H.; Pennycook,
PDA was increased, the efficiency of the S. J.; Dai, S. Angew. Chem. 2011, 50,
photocatalytic activity was higher. The 6799–6802.
PDA-Ag/TiO2 catalyst not only exhibited 13. Kong, J.; Yee, A. W.; Yang, L.; Wei, Y.;
superior photocatalytic degradation Phua, S. L.; Ong, H. G.; Ang, M. J.; Li,
properties but also possessed excellent X. Lu, X. Chem. Commun. 2012, 48,
adsorption affinity for organic chemicals. 10316-10318.
14. Sureshkumar, M.; Lee, P. N.; Lee, C. K.
Acknowledgements J. Mater. Chem. 2011, 21, 12316-12320.
This research was supported by 15. Li, F. Y.; Wang, F. S.; Wu, H.; Wang, T.
Center of Excellence on Catalysis and J.; Jiang, J. J. Mater. Chem. 2012, 22,
Catalytic Reaction Engineering, Department 19617-19620.
of Chemical Engineering, Faculty of 16. Zhang, L.; Shi, F. J.; Jiang, Y. Z.; Jiang,
Engineering, Chulalongkorn University. J. Y.; Meng, J. R.; Zhu, Y. Y.; Liang Y.
P.; Zheng, Y. ACS Appl. Mater.
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Synthesis and photophysical properties of carbazole and triphenylamine
dendrons end capped-phenanthrocarbazole for light-emitting applications
Jirat Chatsirisupachai, Taweesak Sudyoadsuk, Vinich Promarak*
Abstract:
Herein we reported on the design, synthesis and photophysical properties of carbazole
and triphenylamine dendrons end capped-phenanthrocarbazole namely TPA1-PCZ-TPA1 (1),
CBZ1-PCZ-CBZ1 (2), TPA2-PCZ-TPA2 (3) and CBZ2-PCZ-CBZ2 (4) as shown below.
Phenanthrocarbazole as core was end capped with different dendrons using Suzuki cross-
coupling reaction to yield target molecules 1 to 4 in moderate to good yields. Absorption and
emission spectra of phenanthrocarbazoles 1 to 4 were analyzed both in toluene solution and
thin film coated on fused silica. All of them showed excitation peaks of the
phenanthrocarbazole core at around 485 nm with red-shift on thin film. For emission spectra,
all compounds showed an emission in green region at around 500 nm with red-shift on thin
film. Higher generation of dendrons significantly enhances quantum yield of thin films from
less than 10% to higher than 20%. Energy transfer from end capped-dendron to
phenanthrocarbazole core was observed and evaluated. Fluorescence lifetime and
chromaticity of phenanthrocarbazole 1 to 4 were also studied.
1. Introduction dendrimers based on phenanthrocarbazole

Recently, development of organic cores and end-cap carbazole dendrons to
semiconducting (OSC) materials as investigate their properties to be used as
versatile, lower cost electronic devices are lighting materials for OLEDs. The target
of interest. Among them, molecules are shown in Figure 1.
phenanthrocarbazole is one of the most
widely used in many fields such as organic
light emitting diodes (OLEDs),
photodetectors, organic photovoltaic (OPV)
cells, organic field effect transistors
(OFETs) since it has appropriate properties
such as planarity, strong electron conducting
ability, thermal stability and easy synthesis
method.1-10 However, since its structure is
planar, it suffers from aggregation and
quenching. In 2014, Promarak group has
reported that carbazole dendrons can help
improve performance of emissive core by
reducing interaction between them for
OLEDs.9-10 So far, there are a lot of studies
on phenanthrocarbazole but to the best of
our knowledge, there is not much study on
the properties of phenanthrocarbazole as
small molecule dendrimers yet. Therefore, it Figure 1. Structure of target molecules 1 – 4
is of great interest to synthesize a series of
2. Materials and Methods 2.2.2 G1Ph-PCZ-PhG1 (2)
2.1 General Following the same synthetic
Unless stated otherwise, all reagents approach for compound 1, the reaction of
used in the experiments were purchased compound 5 (150 mg, 0.28 mmol),
from commercial sources without further compound 7 (340 mg, 0.70 mmol), kalium
purification. Tetrahydrofuran (THF) was carbonate (387 mg, 2.80 mmol) and
dried by sodium through distillation, with tetrakis(triphenylphosphine) palladium(0)
benzophenone as chromogenic reagent. For (33 mg, 0.03mmol) was conducted to
flash column chromatography, silica gel produce G1Ph-PCZ-PhG1 (110 mg, 36%)
with 200 ~ 300 mesh was used. as a yellow solid. 1H-NMR (600 MHz,
2.2 Synthesis of compounds 1 – 4 CDCl3) δ (ppm): 8.79 (d, J = 6.8 Hz, 2H),
2.2.1 TPA1-PCZ-TPA1 (1) 8.34 (d, J = 7.2 Hz, 2H), 8.28 (s, 4H), 7.98
A mixture of compound 5 (150 mg, (d, J = 7.7 Hz, 4H), 7.92 (s, 2H), 7.89 (d, J =
0.28 mmol), compound 6 (202 mg, 0.70 7.8 Hz, 2H), 7.83 (d, J = 7.9, 4H), 7.62 (d, J
mmol), kalium carbonate (387 mg, 2.80 = 8.5, 4H), 7.59 (d, J = 8.5, 4H), 4.70–4.61
mmol), and tetrakis(triphenylphosphine)- (m, 2H), 2.43–2.34 (m, 1H), 1.57 (s, 36H)
palladium(0) (33 mg, 0.03 mmol) in THF 1.55–1.30 (m, 8H), 1.04 (t, J = 7.4 Hz, 3H),
(15 ml)/H2O (1.5 ml) was refluxed at 65 oC 0.92 (t, J = 7.2 Hz, 3H); HRMS calculated
for 24 hr under N2 atmosphere. After for C80H81N3: 1083.643. Found: 1083.616.
cooling down to room temperature, water 2.2.3 TPA2-PCZ-TPA2 (3)
was added and then extracted with Following the same synthetic
dichloromethane. The organic extract was approach for compound 1, the reaction of
dried over natrium sulfate and concentrated compound 5 (50 mg, 0.09 mmol), compound
in vacuum. The crude product was purified 8 (200 mg, 0.23 mmol), kalium carbonate
by chromatography using hexane/ (130 mg, 0.93 mmol) and tetrakis-
dichloromethane (3:1) as an eluent to give a (triphenylphosphine)palladium(0) (11 mg,
yellow solid of TPA1-PCZ-TPA1 (163 mg, 0.01 mmol) was conducted to produce
67%). 1H-NMR (600 MHz, CDCl3) δ (ppm): TPA2-PCZ-TPA2 (125 mg, 68%) as a
8.69 (br, 2H), 8.29 (br, 2H), 7.82 (t, J = 7.8 yellow solid. 1H-NMR (600 MHz, CDCl3) δ
Hz, 2H), 7.78 (s, 2H), 7.66 (d, J = 6.6 Hz, (ppm): 8.77 (br, 2H), 8.42 (s, 4H), 8.29 (br,
2H), 7.39 (t, J = 7.7 Hz, 8H), 7.36 (d, J = 2H), 8.01 (d, J = 6.5 Hz, 4H), 7.94 (s, 2H),
8.3, 4H), 7.14 (t, J = 7.2, 4H), 4.52 (d, J = 7.91 (t, J = 7.7 Hz, 2H), 7.84 (d, J = 7.4 Hz,
5.7, 2H), 2.35–2.24 (m, 1H), 1.51–1.23 (m, 4H), 7.73 (d, J = 8.0 Hz, 4H), 7.69 (d, J =
8H), 0.98 (t, J = 7.4 Hz, 3H), 0.90 (t, J = 7.2 7.9, 4H), 7.66 (d, J = 8.1, 8H), 7.30 (t, J =
Hz, 3H); HRMS calculated for C64H53N3: 7.5, 16H), 7.21 (br, 8H), 7.18 (d, J = 7.4,
863.424. Found: 863.383. 16H), 7.04 (t, J = 6.1, 8H), 4.70 (br, 2H),
Figure 2. Synthetic scheme of compounds 1 – 4
2.39 (br, 1H), 1.56–1.36 (m, 8H), 1.02 (t, J = 3. Results and Discussion
7.2 Hz, 3H), 0.88 (t, J = 7.2 Hz, 3H); HRMS 3.1 Synthesis of compounds 1 – 4
calculated for C136H101N7: 1831.812. Found: The synthetic routes of compound 1
1831.783. to 4 are presented in Figure 2.
2.2.4 G2Ph-PCZ-PhG2 (4) Phenanthrocarbazole core 5 was synthesized
Following the same synthetic following a reported procedure.2 Palladium-
approach for compound 1, the reaction of catalyzed Suzuki-coupling reaction between
compound 5 (75 mg, 0.14 mmol), compound dibromo phenanthrocarbazole core 5 and
9 (324 mg, 0.35 mmol), kalium carbonate dendrons precursor 6 to 9 were conducted to
(194 mg, 1.40 mmol), and yield target products 1 to 4 in moderate to
tetrakis(triphenylphosphine) palladium(0) good yields as shown in figure 2. The
(16 mg, 0.01 mmol) was conducted to structures and purities of compound 1–4
produce G2Ph-PCZ-PhG2 (130 mg, 47%) as were confirmed by 1H NMR, 13C NMR and
a yellow solid. 1H-NMR (600 MHz, CDCl3) high resolution MALDI-TOF MS.
δ (ppm): 8.87 (d, J = 7.4 Hz, 2H), 8.44 (d, J 3.1 Optical properties
= 8.2 Hz, 2H), 8.40 (s, 4H), 8.28 (s, 8H), The UV-Vis absorption spectra of
8.17 (d, J = 7.4 Hz, 4H), 8.04 (br, 4H), 7.99 compound 1 to 4 in toluene solution and thin
(t, J = 7.4 Hz, 4H), 7.94 (d, J = 8.5 Hz, 4H), film are shown in Figure 3, and the
7.78 (d, J = 8.6 Hz, 4H), 7.56 (d, J = 8.5, correlated optical parameters are
8H), 7.48 (d, J = 8.6, 8H), 4.79 (d, J = 4.8 summarized in table 1. All of them showed
Hz, 2H), 2.48 (br, 1H), 1.46–1.36 (m, 8H), similar absorption spectra in toluene
1.11 (t, J = 7.2 Hz, 3H), 0.96 (t, J = 7.1 Hz, solution covering from 250 to 500 nm. The
3H); HRMS calculated for C144H141N7: absorption spectra exhibited two major
1968.125. Found: 1968.218. absorption peaks at around 300 – 325 nm
2.3 Methods and 450 – 475 nm referred to the end-
Nuclear magnetic resonance (NMR) capping dendrons and phenanthrocarbazole
spectra were conducted on a Bruker core, respectively. The spectral absorptions
AVANCE III HD (600 MHz) spectrometer. of the thin films of the four compounds were
MALDI-TOF mass spectrometric red shifted and broadened. For emission
measurements were performed on Bruker spectra, all four compounds showed similar
Autoflex speedTM MALDI-TOF. UV–vis- emission bands both in toluene solution and
NIR absorption spectra were measured on a thin film with emission maximum at around
PerkinElmer Lambda 1050 spectrophoto- 500 nm in solution and red shifted in thin
meter. Photoluminescence (PL) spectra, film as shown in Figure 4. Noted that they
fluorescence quantum yield, and life time
were measured on Edinburgh Instruments
FLS 980. Cyclic voltammetry (CV)
measurements were recorded on an
electrochemistry workstation (Autolab
potentiostat PGSTAT 101) under argon
atmosphere to determine electrochemical
redox potentials. The working electrode was
a glassy carbon. Platinum wire was used as
the counter electrode. Ag/AgCl (Ag in a
0.01 mol/L KCl) electrode was used as the
reference electrode. A 0.1 M anhydrous
solution tetrabutylammonium hexyl
Figure 3. UV-Vis absorption spectra of
fluorophosphate (Bu4NPF6) in dichloro
compounds 1 – 4
methane was used as the electrolyte.
Table 1. Optical, electrochemical, and photophysical properties of compounds 1 to 4
Compoun λabs (nm)a λemis (nm)a Eg (eV)b HOMO LUMO Quantum Lifetime
(eV)c (eV)d yield (%)a (ns)a
1 302, 461/305, 466 514/529 2.52 -5.00 -2.50 92/8 3.5/~1
2 298, 454/299, 458 508/518 2.58 -5.13 -2.57 100/12 4.2/1.1
3 334, 454/330, 462 494/514 2.60 -5.17 -2.58 - 2.3/~1
4 298, 453/299, 457 504/520 2.60 -5.18 -2.59 100/20 2.8/1.6
a
In toluene solution/In thin film.
b
Calculated from the absorption edge wavelength.
c
Estimated from the onset oxidation potentials.
d
Calculated from (HOMO level and Eg).
are independent to excitation wavelength as The CV curves of all compound

excitation either at the dendron (around 300 shows multi quasi-reversible oxidation
nm) or at the core (around 450 nm), the processes with no reduction process. The
emission spectra obtained are mostly HOMO and LUMO energy levels of
identical. This proves that energy can be compound 1 to 4 were calculated from their
efficiently transferred from the end-capping oxidation onset potentials (Eoxonset) and
dendrons to the emissive core. energy gaps (Eoptg) and the results are listed
in table 1.
3.3 Photophysical properties
Chromaticity of all compounds is
shown in figure 6, labeling as 1 – 4 for
compound 1 to 4 in solution and 5 – 8 for
compound 1 to 4 in thin film respectively.
Label C and D are standard green CIE 1974
and 1931, respectively. Fluorescence
quantum yield and life time of compound 1
to 4 were also studied and summarized in
Table 1.
Figure 4. PL emission spectra of

compounds 1 – 4
3.2 Electrochemical properties

Cyclic voltammetry measurements
were carried out to investigate
electrochemical behaviors of compound 1 to
4. and the resulting data are shown in Figure
5.
Figure 6. Chromaticity CIE coordinates for

compounds 1 – 4
All of them have fluorescence life

Figure 5. CV curves of compounds 1 – 4 time in nanosecond scale. As for the
quantum yields, second generation dendrons 3. Zeng, Z.; Lee, S.; Zafra, J. L.; Ishida,
can help reduce aggregation and results in M.; Bao, N.; Webster, R. D.; Navarrete,
higher quantum yield up to 20% in thin film. J. T. L.; Ding, J.; Casado, J. Kim, D.;
Energy transfer was measured to be up to Wu, J. Chem. Sci. 2014, 5, 3072–3080.
75%. 4. Park, K. H.; Yun, H.; Lu, W.; Chung, D.
S.; Kwona, S.; Kim, Y. Dyes Pigm.
4. Conclusion 2014, 103, 214–221.
In summary, carbazole and 5. Deng, Z.; Chen, L.; Wu, F.; Chen, Y. J.
triphenylamine dendrons end capped- Phys. Chem. C 2014, 118, 6038–6045.
phenanthrocarbazole 1 to 4 were synthesized 6. Li, X.; Zheng, Z.; Jiang, W.; Wu, W.;
in 36% – 68% yield. They are soluble in Wang, Z.; Tian, H. Chem. Commun.
most common organic solvents. All 2015, 51, 3590−3592.
compounds show emission in green region 7. Yang, L.; Yao, Z.; Liu, J.; Wang, J.;
with λmax around 500 nm in toluene solution Wang, P. ACS Appl. Mater. Interfaces
and a little red-shifted in thin film. They can 2016, 8, 9839−9848.
have high quantum yield in thin film up to 8. Sung, M. J.; Yoon, S.; Kwon, S.; Kim,
20%. CV measurements also reveal the Y.; Chung, D. S. ACS Appl. Mater.
HOMO and the LUMO energy levels to be Interfaces 2016, 8, 31172−31178.
around -5.0 to -5.2 eV and -2.5 to -2.6 eV. 9. Atakan, G.; Gunbas, G. RSC Adv. 2016,
6, 25620–25623.
Acknowledgements 10. Zhang, X.; Tan, H.; Yan, Y.; Hang, Y.;
This project was supported by the Yu, F.; Qub, X.; Hua, J. J. Mater. Chem.
PhD research grant of Vidyasirimedhi B, 2017, 5, 2172−2180
Institute of Science and Technology 11. Moonsin, P.; Prachumrak, N.;
(VISTEC). Analytical instruments were Rattanawan, R.; Keawin, T.;
provided by the Frontier Research Center Jungsuttiwong, S.; Sudyoadsuk, T.;
(FRC), VISTEC. We would like to express Promarak, V. Chem. Commun. 2012, 48,
our gratitude to the Pure and Applied 3382−3384.
Chemistry International Conference 2018 12. Prachumrak, N.; Pansay, S.;
(PACCON2018) for giving us the Namuangruk, S.; Kaewin, T.;
opportunity to participate in the poster Jungsuttiwong, S.; Sudyoadsuk, T.;
session. Promarak, V. Eur. J. Org. Chem. 2013,
29, 6619−6628.
References 13. Sudyoadsuk T.; Moonsin, P.;
1. Chen, H.; He, C.; Yu, G.; Zhao, Y.; Prachumrak, N.; Namuangruk, S.;
Huang, J.; Zhu, M.; Liu, H.; Guo, Y.; Jungsuttiwong, S.; Keawina, T.;
Lia, Y.; Liu, Y. J. Mater. Chem. 2012, Promarak, V. Polym. Chem. 2014, 5,
22, 3696−3698. 3982–3993.
2. Chen, H.; Guo, Y.; Sun, X.; Gao, D.;
Liu, Y.; Yu, G. J. Polym. Sci. A Polym.
Chem. 2013, 51, 2208–2215.
Simple hole-transporting materials based on
bis(2,4-dimethoxyphenyl)carbazole for perovskite solar cell
Thanyarat Chawanpunyawat, Duangratchaneekorn Muenmart, Taweesak Sudyoadsuk,
Pichaya Pattanasattayavong, Vinich Promarak*
Vidyasirimedhi Institute of Science and Technology, Payupnai, Rayong 21210, Thailand
*E-mail: vinich.p@vistec.ac.th,
Abstract:
Hole-transporting materials (HTMs) are important in perovskite solar cell (PSC)
because they enhance the device efficiency by extracting the holes and transporting these
charges to the electrode. Commonly used spiro-OMeTAD is expensive and required a
complex synthesis. Here we report simple small organic molecule HTMs based on
triphenylamine and triazine cores substituted with (2,4-dimethoxyphenyl)carbazole. C3 and
C6 active sites of carbazole unit were protected with an electron donor 2,4-dimethoxyphenyl
group which would improve the charges transport, electrical properties and electrochemical
stability. Tris(4-(3,6-bis(2,4-dimethoxyphenyl)-9H-carbazol-9-yl)phenyl)amine (diOMeCbz-
TPA) and 2,4,6-tris(4-(3,6-bis(2,4-dimethoxyphenyl)-9H-carbazol-9-yl)phenyl)-1,3,5-triazine
(diOMeCbz-TAZ) were synthesized via Suzuki and Ullmann coupling reactions. The band
gap of 3.34 eV for diOMeCbz-TPA and 2.74 eV for diOMeCbz-TAZ were obtained from
UV-Vis analysis. HOMO energy level in solution measured by cyclic voltammeter was found
at -5.25 eV and –5.33 eV for diOMeCbz-TPA and diOMeCbz-TAZ, respectively, which
higher than that of perovskite (-5.43eV), hence favoring the hole injection and transport.
Moreover, both of materials showed high glass transition temperature at 233 ˚C and 235 ˚C
for diOMeCbz-TPA and diOMeCbz-TAZ, respectively in TGA-DSC measurement. So, these
new materials could be comparable to spiro-OMeTAD for high efficiency PSC.
1. Introduction to enhance the donor ability2 and increase

Perovskite solar cell (PSC) is one of more charge injection from 2,4-
the promising technology these days, as it dimethoxyphenyl moiety.3
has high efficiency that can be competed
with the conventional silicon based solar 2. Materials and Methods
cell.1 One challenge in this field is the 2.1 General
expensive hole transporting material All reagents and catalyst were
(HTM); 2,2',7,7'-tetrakis(N,N-di-p-methoxy- purchased from TCI and used without
phenyl-amine) 9,9'-spirobifluorene (spiro- further purification. All bases were
OMeTAD), the cost can decrease further by purchased from Carlo. (±)-trans
changing spiro-OMeTAD to the simple Diaminocyclohexane were purchased from
synthesized organic HTM. This aim can be Acros. Solvents were purchased from Carlo
done by designed a star-shaped molecule and used without further distillation.
that the moieties are twisted and have less Tetrahydrofuran (THF) purchased from
planar stacking, so it will enhance the Burdick&Jackson and was distilled with
charge transport property. Here, we sodium/benzophenone under N2 atmosphere
designed and synthesized a star-shape before used.
compound based on triphenylamine and 2.2 Synthesis of diOMeCbz
triazine as the core molecules, using A mixture of 3,6-diiodocarbazole4 (1
carbazole substituted at C3 and C6 position g, 2.39 mmol), 2,4-dimethoxyphenylboronic
acid (0.96 g, 5.27 mmol), Pd(PPh3)4 (0.11 g, column chromatography using a mixture of
2.39 mmol), 2,4-dimethoxyphenylboronic CH2Cl2 and hexane (60% v/v CH2Cl2-
acid (0.96 g, 5.27 mmol), Pd(PPh3)4 (0.11 g, hexane) as eluent and followed by
0.09 mmol) and 2M sodium carbonate recrystallization with a mixture of CH2Cl2
(Na2CO3) (7.36 g, 69.46 mmol) aqueous and MeOH.
solution in THF was degassed with N2 for 5 diOMeCbz-TPA, 37%, cream
times, the reaction mixture was stirred at solids, H NMR (600 MHz, CDCl3) δ 8.25
1
reflux under N2 for 24 hours. After being (6H, s), 7.63 (6H, d, J = 8.8 Hz), 7.60 (6H,
cooled to room temperature, water (30 mL) dd, J = 8.5, 1.6 Hz), 7.55 (6H, d, J = 3.4
was added and extracted with CH2Cl2 (60 Hz), 7.53 (6H, d, J = 3.6 Hz), 7.38 (6H, d, J
mLx3) the combined organic phases were = 7.1 Hz), 6.62 (6H, d, J = 7.3 Hz), 6.61
washed with water (30 mL) and brine (6H, s), 3.88 (18H, s), 3.83 (18H, s) ppm;
solution (30 mL). Dry over anhydrous 13
C NMR (151 MHz, CDCl3) δ 159.99,
Na2SO4, filtered and the solvent was 157.58, 131.58, 128.11, 127.75, 124.45,
removed under pressure. The pure 123.58, 121.08, 104.71, 99.17, 55.68, 55.48
compound was obtained by silica gel ppm. MADI-TOF (m/z) calcd for
column chromatography using a mixture of C102H84N4O12: 1556.6086; found 1556.9772
CH2Cl2 and hexane (60%v/v CH2Cl2- (M+).and
hexane) as eluent and followed by diOMeCbz-TAZ, 45%, yellowish
recrystallization with a mixture of CH2Cl2 solids, 1H NMR (600 MHz, CDCl3) δ 9.11
and MeOH. The pure compounds were (6H, d, J = 8.4 Hz), 8.27 (6H, s), 7.92 (6H,
obtained in white solid with 60% yield. 1H d, J = 8.3 Hz), 7.62 (12H, s), 7.38 (6H, d, J
NMR (600 MHz, CDCl3) δ 8.16 (2H, s), = 8.9 Hz), 6.62 (6H, d, J = 5.5 Hz), 6.62
8.02 (1H, s), 7.55 (2H, dd, J = 6.6, 1.8 Hz), (6H, s), 3.88 (18H, s), 3.84 (18H, s) ppm;
7.41 (2H, d, J = 8.4 Hz), 7.34 (2H, d, J = 9 13
C NMR (151 MHz, CDCl3) δ 171.23,
Hz), 6.60 (2H, d, J = 4.2 Hz), 6.60 (2H, s), 160.07, 157.59, 142.20, 139.60, 134.52,
3.87 (6H, s), 3.81 (6H, s) ppm. MALDI- 131.57, 130.87, 130.75, 127.97, 126.57,
TOF (m/z) calcd for C28H25NO4: 439.1784; 124.28, 124.09, 121.31, 109.43, 104.74,
found 439.2309 (M+). 99.18, 55.70, 55.48 ppm. MADI-TOF (m/z)
2.3 Synthesis of diOMeCbz-TPA and calcd for C105H84N6O12: 1620.6147; found
diOMeCbz-TAZ 1621.6087 (M+H+).
Triiodo-triphenylamine (100 mg, 2.4 Methods
1
0.161 mmol) or tri-(4-bromo-phenyl)- H and 13C NMR spectra were
triazine (100 mg, 0.184 mmol) were mixed recorded on a Bruker AVANCE III HD
with diOMeCbz (282.27 mg, 0.642 mmol), (600MHz) spectrometer using CDCl3 as
K3PO4 (136.34 mg, 0.642 mmol) and CuI solvent in all cases. Molecular masses were
(24.48 mg, 0.129 mmol). Toluene was measured with a Bruker Autoflex speed
added and stirred at room temperature until mass spectrometer. UV–vis spectra and
it dissolved. The mixture was degassed were recorded as a dilute solution in distilled
under N2 for 5 times followed by addition of reagent grade CH2Cl2 on a Perkin-Elmer
(±)-trans-diaminocyclohexane (14.68 mg, Lambda 1050 spectrometer. Differential
0.128 mmol). The reaction mixture was scanning calorimetry (DSC) measurement
stirred at reflux under N2 for 48 hours. After was used a PerkinElmer DSC-8500 thermal
being cooled to room temperature, water (20 analyzer at a heating rate of 10 ˚C/min from
mL) was added and extracted with CH2Cl2. room temperature to 400 ˚C under N2
The combined organic phase was washed atmosphere. Cyclic voltammetry (CV)
with water and brine solution. Dry over measurements were carried out under inert
anhydrous Na2SO4, filtered, and the solvent argon atmosphere with an Autolab
was removed under pressure. The pure potentiostat PGSTAT 101 using a three-
compound was obtained by silica gel electrode system fitted with a platinum rod
O
O O O
I I B O
CH3COOH HO OH
N KIO3, KI N Pd(PPh3)4, 2M Na2CO3 O

H H THF N
H
diICbz I
diOMeCbz
60% yield
N Br
I I
O O
O
- -
1,2 t rans daminocyclohexane, N N
O u , , o uene, heat
C I K 3 PO 4 t l N
N Br Br
- -
1,2 t rans daminocyclohexane,
O CuI, K3PO4, toluene, heat
O N O
O O
N N
O
O
O O
O
O
N N
O O
O N O
-
diOMeCbz TPA
N N O
37% yield
-
diOMeCbz TAZ
N e
45% yi ld
O
O
O O
Scheme 1. Synthetic route to diOMeCbz-TPA and diOMeCbz-TAZ
counter electrode, a glassy carbon working column chromatography using mixed

electrode and a Ag/Ag+ reference electrode. CH2Cl2 and hexane as eluent and were
attained as solids in moderate yields. All
3. Results and Discussion compounds were chemically characterized
3.1 Synthesis by standard method including H- and C-
As depicted in Scheme 1, diOMeCbz NMR and mass spectrometry.
was first synthesized via iodination of 3.2 Optical properties
carbazole with KI and KIO3 in acetic acid, The optical properties of the target
then Suzuki cross-coupling with 2,4- molecules were measured in a dilute CH2Cl2
dimethoxyphenyl boronic acid at C3 and C6 solution (3 μM) and film (1 mg/mL at spin
of iodinated carbazole catalyzed with rate 1,000 rpm), the data were collected in
Pd(PPh3)4/Na2CO3 in THF/water. Figure 1 and Table 1. The absorption spectra
DiOMeCbz-TPA and diOMeCbz-TAZ were of diOMeCbz-TPA showed a strong
then synthesized via Ullmann coupling absorption band at 301 nm which would be
reaction between core molecules of triiodo- corresponded to the -* transition of
triphenylamine or tri-(4-bromo-phenyl)- carbazole.7 In addition, this molecule had an
triazine and diOMeCbz in the presence of intramolecular charge transfer (ICT) band at
K3PO4/CuI/(±)-trans-diaminocyclohexane in 320-380 nm that would assigned to the
toluene at refluxing temperature. The
reaction mixtures were purified by silica gel
Table 1. Optical and electrical properties of diOMeCbz-TPA and diOMeCbz-TAZ
Eonset Band gapa Tg Tm
Absorption Eonset HOMOb LUMOb
(solution/film) (solution/film)
diOMeCbz-TPA 301, 308 371/382 3.34/3.26 0.81 -5.25 -1.91 233 282
diOMeCbz-TAZ 386, 403 434/453 2.85/2.74 0.89 -5.33 -2.48 235 -
5 125 248
Spiro-OMeTAD 305, 384 -/- 3.17/3.05 - -5.22 -2.05
a
Band gap calculated from (eV)
b, c
HOMO = -(Eonset + 4.44), LUMO = HOMO + Eg6
charge transfer between carbazole donor and

TPA acceptor moiety. The band gap was
found at the onset of 371 nm (3.34 eV).
Moreover, when measured the absorption as
the thin film, UV-vis spectra were slightly
red-shifted due to the stacking of the
molecules in solid state and band gap got
narrower to3.26 eV. The same result was
found with diOMeCbz-TAZ, the band gap in
solution was 2.85 eV and it got narrower to
2.74 eV in solid state.
3.3 Electrical properties
HOMO and LUMO of the molecules
were investigated using cyclic voltammetry
(CV) from 0 to 2.0 eV for oxidation process
and 0 to -2.0 eV for reduction process.
Tetrabutylammonium hexafluorophosphate
was used as a supporting electrolyte in
CH2Cl2 solution under argon flow. All data
were plotted in Figure 1C and listed in
Table 1. From the CV curve, diOMeCbz-
TPA showed an irreversible oxidation
process with the onset at 0.81 eV and a
quasi-reversible process for diOMeCbz-
TAZ with the onset at 0.89 eV. At this
measurement condition, we had not found
the reduction process of all compounds. The
calculated HOMO showed -5.25 eV and
-5.33 eV and LUMO showed -1.91 eV and -
2.48 eV for diOMeCbz-TPA and
diOMeCbz-TAZ, respectively. The deeper
HOMO level of diOMeCbz-TAZ might
because of the acceptor ability of the triazine
core that decreased the electron density at
the occupied molecular orbital, in contrast to
Figure 1. The absorption spectrum of (a) the triphenylamine core that of increased the
diOMeCbz-TPA and (b) diOMeCbz-TAZ in electron density result in a higher HOMO
solution (solid line) and film (dot line). energy level. HOMO level both compounds
(c) CV of diOMeCbz-TPA (black) and were in the suitable level to perovskite
diOMeCbz-TAZ (red) in CH2Cl2 material within +0.2 eV from valence band
of perovskite (-5.43 eV) as described in were in the suitable level to perovskite
Figure 2.5b material. Also, the amorphous characteristic
of them made these two compounds suitable
to use as HTM for the perovskite solar cell.
Acknowledgements
Vidyasirimedhi Instituted of Science and
Technology (VISTEC). All instruments
were supported from Frontier Research
Center (FRC).
References
1. Yang, Y.; You, J. Nature 2017, 544, 155-
156.
2. Zassowski, P.; Ledwon, P.; Kurowska,
A.; Herman, A. P.; Jarosz, T.;
Figure 2. Summary of energy level of Lapkowski, M.; Cherpak, V.; Stakhira,
synthesized compounds comparing to spiro- P.; Peciulyte, L.; Volyniuk, D.;
OMeTAD and perovskite layer Grazulevicius, J. V. Synthetic Met. 2017,
223 (Supplement C), 1-11.
3.4 Thermal properties 3. Degbia, M.; Schmaltz, B.; Bouclé, J.;
The thermal properties were Grazulevicius, J. V.; Tran-Van, F. Polym.
important to study for materials that used in Int. 2014, 63 (8), 1387-1393.
the solar cell and both compounds were 4. Albrecht, K.; Yamamoto, K. J. Am.
characterized using DSC measurement. For Chem. Soc. 2009, 131 (6), 2244-2251.
diOMeCbz-TPA, it showed a semi- 5. (a) Bailie, C. D.; Unger, E. L.;
crystalline material as it had both glass Zakeeruddin, S. M.; Gratzel, M.;
transition temperature (Tg) at 233 ˚C and McGehee, M. D. Phys. Chem. Chem.
melting point (Tm) at 282 ˚C. For Phys. 2014, 16 (10), 4864-70; (b) Yu, Z.;
diOMeCbz-TAZ, it showed an amorphous Sun, L. Adv. Energy Mate. 2015, 5 (12),
material with Tg at 235 ˚C and there was no 1500213. (c) Shao, J. Y.; Li, D.; Tang,
trace of Tm. Both compounds showed a good K.; Zhong, Y. W.; Meng, Q. RSC Adv.
thermal property to use as the HTM for PSC. 2016, 6 (95), 92213-92217.
6. Leonat, L.; Sbarcea, G.; Branzoi, I. V.
4. Conclusion U.P.B. Sci. Bull. Series B 2013, 75 (3),
Two new HTMs were successfully 111-118.
synthesized. The absorption spectra of both 7. Promarak, V.; Ichikawa, M.;
compounds showed the wide band gap Sudyoadsuk, T.; Saengsuwan, S.;
materials, cyclic voltammetry showed the Jungsuttiwong, S.; Keawin, T. Synthetic
calculated HOMO level of both compound Met. 2007, 157 (1), 17-22.
TADF materials based on carbazole substituted on triphenyl-triazines; the
effect of acceptor strength
Pattarawadee Therdkatanyuphong, Taweesak Sudyoadsuk, Vinich promarak*
Vidyasirimedhi Institute of Science and Technology, Pa Yup Nai, Wangchan, Rayong 21210, Thailand
Abstract:
Thermally activated delayed fluorescence (TADF) materials are recently attractive as
new class of fluorescence emitter for high efficiency and low-cost organic light emitting
diodes (OLED). A donor-acceptor characteristic molecule with small singlet-triplet energy
gap (∆EST) is the key for TADF materials to harvest both singlet and triplet excitons via
reverse intersystem crossing (RISC) which directly impact to their properties and
performances. Herein, new TADF materials consisting of a carbazole dendrimer as donor unit
and 2,4,6-triphenyl-1,3,5-triazine as acceptor unit were designed and synthesized. To study
the effect of acceptor strength on TADF properties, different electron withdrawing
substituents on triphenyl-triazine unit were introduced including fluoro and cyano groups.
The structure of all target molecules was identified by NMR spectroscopy, and mass
spectrometry. The optical properties were investigated by UV-visible and photoluminescence
techniques. Moreover, their TADF characteristics were investigated by fluorescence lifetime
spectrometer (FLS).
1. Introduction electrochemical stability. In this work,

Since 2009, Adachi et al. reported donor-acceptor structures consisting of
the first thermally activated delayed 3,3'',6,6''-tetra-tert-butyl-9'H-9,3':6',9''-
fluorescence (TADF) emitters in OLEDs,1 tercarbazole (G2) as donor moiety and
which can provide the ultimate 2,4,6-triphenyl-1,3,5-triazine (TRZ)
electroluminescent efficiency by efficient derivatives as acceptor moieties were
up-conversion of exciton from the lowest designed and synthesized for study the
triplet excited state (T1) to lowest singlet effect of acceptor strength to the TADF
excited state (S1) via reverse intersystem properties.
crossing (RISC), the research area of TADF
materials was rapidly developed. The basic 2. Materials and Methods
principle of TADF is minimizing the energy 2.1 General
difference between the singlet and triplet All reagents and solvents were
excited states (∆EST), which enables the purchased from commercial sources and
thermally activated RISC. Usually, small used without further purification. All the
∆EST can be achieved by separation of the reactions were carried out under nitrogen
highest occupied molecular orbital (HOMO) atmospheres and Schlenk techniques unless
and the lowest unoccupied molecular orbital otherwise noted. Solvents in nitrogen
(LUMO),2 leading to twisted donor-acceptor atmospheres reactions were dried using
structure of TADF materials. standard procedures. Column
Derivatives of triazine acceptor and chromatography was carried out using
carbazole donor are versatile TADF gravity feed chromatography with Merck
emitters,3-5 due to their thermal and silica gel mesh, 60 Å.
Figure 1. Synthesis route of carbazole substituted on triphenyl-triazine (TRZ-G2)
derivatives
2.2 General method for synthesis of TRZ- 119.40, 116.24, 111.29, 109.09, 34.74,
G2 derivatives 32.05; MADI-TOF (m/z) calcd for
NaH (3 eq) was added to 3,3'',6,6''- C73H68N6: 1028.5505; found 1029.1497
tetra-tert-butyl-9'H-9,3':6',9''-tercarbazole (M+H+).
(G2) (1.2 eq) in mixture of DMF/THF. The 4,4'-(6-(4-(3,3'',6,6''-Tetra-tert-butyl-
mixture was stirred at 70oC for 30 minutes. 9'H-[9,3':6',9''-tercarbazol]-9'-yl)phenyl)-
After that, core structure (1-3) (1 eq) was 1,3,5-triazine-2,4-diyl)dibenzonitrile (CN-
added to the mixture, and then stirred at TRZ-G2): Obtained as orange solid. Yield
100oC for 18 hours. The mixture was diluted 34%. 1H NMR (600 MHz, CDCl3) δ 9.110
with dichloromethane and washed with (2H, d), 8.938 (4H, d), 8.280 (2H, s), 8.169
distilled water. The combined organic (4H, s), 8.028 (2H, d), 7.935 (4H, d), 7.789
phases were dried over anhydrous Na2SO4 (2H, d), 7.663 (2H, d), 7.470 (4H, d), 7.352
and dried in vacuo to give the crude product. (4H, d), 1.471 (36H, s); 13C NMR (150
The pure compound was purified by column MHz, CDCl3) δ 171.65, 170.76, 142.73,
chromatography using DCM/n-hexane as 140.08, 139.79, 139.64, 134.40, 132.63,
eluent and followed by recrystallization with 131.59, 131.16, 129.53, 126.98, 126.21,
a mixture of CH2Cl2 and MeOH. 124.57, 123.61, 123.22, 119.48, 118.30,
3,3'',6,6''-Tetra-tert-butyl-9'-(4-(4,6- 116.35, 116.29, 111.20, 109.03, 34.75,
diphenyl-1,3,5-triazin-2-yl)phenyl)-9'H- 32.04; MADI-TOF (m/z) calcd for
9,3':6',9''-tercarbazole (TRZ-G2): Obtained C75H66N8: 1078.5410; found 1078.4587
as yellow solid. Yield 40%. 1H NMR (600 (M+).
MHz, CDCl3) δ 9.131 (2H, d), 8.848 (4H, 9'-(4-(4,6-Bis(4-fluorophenyl)-1,3,5-
d), 8.272 (2H, s), 8.165 (4H, s), 7.987 (2H, triazin-2-yl)phenyl)-3,3'',6,6''-tetra-tert-
d), 7.784 (2H, d), 7.637 (8H, m), 7.470 (4H, butyl-9'H-9,3':6',9''-tercarbazole (F-TRZ-
d), 7.358 (4H, d), 1.462 (36H, s); 13C NMR G2): Obtained as yellow solid. Yield 33%.
(150 MHz, CDCl3) δ 171.94, 142.64, 1
H NMR (600 MHz, CDCl3) δ 9.096 (2H,
141.09, 140.13, 140.00, 136.2, 135.72, d), 8.848 (4H, dd), 8.283 (2H, s), 8.174 (4H,
132.75, 131.35, 130.95, 129.06, 128.76, s), 7.991 (2H, d), 7.785 (2H, d), 7.663 (2H,
126.87, 126.15, 124.41, 123.60, 123.18,
d), 7.476 (4H, d), 7.363 (4H, d),7.295 (4H, Then, the three target compounds were
t), 1.477 (36H, s); 13C NMR (150 MHz, synthesized from the alkylation reaction
CDCl3) δ 170.95, 170.85, 166.85, 165.17, between intermediates 1-3 and G2 in the
142.67, 141.26, 140.12, 139.95, 135.42, presence of base KOH in DMF/THF. They
132.16, 131.44, 131.42, 131.38, 130.93, were attained as yellow-orange solids. The
126.87, 126.17, 124.45, 123.61, 123.20, structures of TRZ-G2 derivatives were
119.43, 116.26, 115.96, 115.81, 111.25, characterized by NMR spectroscopy and
109.08, 34.75, 32.05, 29.71; MADI-TOF MALDI-TOF mass spectrometry.
(m/z) calcd for C73H66F2N6: 1064.5317; 3.2 Photophysical properties
found 1065.1128 (M+H+). The photophysical properties of
2.3 Methods TRZ-G2 derivatives were analyzed in
NMR spectra were recorded on toluene solution by UV-Vis and emission
Bruker Ascend (600 MHz for 1H and 150.9 spectra as shown in Figure 2. Three major
MHz for 13C) spectrometer. Chemical shift absorption patterns located in the range of
(δ) are reported relative to the residual in 280-310, 326-360, and 360-450 nm
deuterated solvent peak in part per million corresponding to the π-π* transition of
(ppm). MALDI-TOF mass spectra of all carbazole, n-π* transition of carbazole, and
target products were recorded on Bruker charge-transfer (CT) band, respectively. The
Autoflex speed, using α-cyano-4- absorption spectra show the longer CT band
hydroxycinnamic acid as a matrix. UV- where the acceptor strength is increased.
Visible spectra were recorded using toluene Moreover, the estimation of band gap
as solvent on Perkin Elmer Lambda 1050 energy (Eg) was calculated by using
UV/Vis/NIR spectrometer. Fluorescence wavelength onset (λonset) from absorption
emission spectra and photoluminescence spectra. From the optical band gap and
decay profiles were recorded using HOMO energy level, which calculated from
Edinburgh Instruments FLS980 the onset of oxidation, the LUMO energy
spectrometer. Electrochemical analysis was level was estimated. These corresponding
performed on Autolab PGSTA101 by using results are show in Table 1.
tetrabutylammonium hexafluorophosphate The maximum emission peaks of
as electrolyte with dichloromethane as TRZ-G2, F-TRZ-G2, and CN-TRZ-G2
solvent and Ag/AgCl as reference electrode. were 460, 472, and 512 nm, respectively.
Changing the acceptor strength from H
3. Results and Discussion group (poor electron withdrawing unit) to
3.1 Synthesis and characterization CN group (high electron withdrawing unit)
Synthesis of TRZ-G2 derivatives are induces the shift of emission maximum to
presented in Figure 1. All target molecules, longer wavelength. Because the acceptor
TRZ-G2, F-TRZ-G2 and CN-TRZ-G2,
TRZ-G2 1.0
were synthesized by two coupling reactions
Molar absorptivity (x105 M-1cm-1)
F-TRZ-G2
Normalized PL intensity (a.u.)
1.2 CN-TRZ-G2
including Suzuki coupling reaction and 0.8
alkylation reaction. The sequential Suzuki
coupling, between cyanuric chloride and (4- 0.8 0.6
fluorophenyl)boronic acid, and then with

0.4
phenylboronic acid or (4-
0.4
cyanophenyl)boronic acid, were used to 0.2
synthesis intermediate 1 and intermediate 2,
respectively. While intermediate 3 was 0.0
300 350 400 450 500 550 600 650 700
0.0
prepared by the single step of Suzuki Wavelength (nm)

coupling reaction, between cyanuric Figure 2. UV-Vis and normal emission
chloride and (4-fluorophenyl)boronic acid. spectra of TRZ-G2 derivatives
Figure 3. The emission spectra (Top), and fluorescence decay time (Bottom) of TRZ-G2, F-
TRZ-G2, and CN-TRZ-G2 in absence and presence of oxygen
Table 1. Optical and electronic properties of all compounds

λemmax Eg[a] HOMO[b] LUMO[c] τ air sat. [d] τ oxygen-free[e] CIE (x, y)
(nm) (eV) (eV) (eV) (ns) (ns)
TRZ-G2 460 3.02 -5.41 -2.39 7 9 (0.156, 0.175)
F-TRZ-G2 472 2.97 -5.40 -2.43 8 11 (0.164, 0.237)
CN-TRZ-G2 512 2.76 -5.44 -2.68 20 95 (0.272, 0.530)
[a]
Measured in toluene at room temperature. [b] Calculated from the absorption edge of the UV-Vis spectrum; Eg
= 1240/λonset. [c] Calculated by HOMO = -(4.44 + Eoxonset); LUMO = HOMO - Eg, where Eoxonset is the onset
potential of oxidation. [d] Measured lifetime in air saturated condition. [e] Measured lifetime in oxygen-free
condition.
strength was effect to LUMO energy level. (Table 1). To further confirm the delayed
At the higher strength of acceptor unit, the fluorescence, the emission spectra show that
lower LUMO energy level was obtained, the elimination of oxygen from TRZ-G2
leading to the decreasing of energy gap of derivatives compounds in toluene can lead
stronger acceptor strength compound as to a great increase in the steady-state
shown in Table 1. fluorescence intensity.
3.3 TADF analysis
To get insight into the possible 4. Conclusion
TADF mechanism, the emission spectra and A new TADF series of carbazole
fluorescence lifetime of TRZ-G2 substituted on triphenyl-triazines was
derivatives were primarily investigated in designed and synthesized for study the
absence and presence of oxygen (Figure 3). effect of acceptor strength on the TADF
In both conditions, TRZ-G2 and F-TRZ- compound by varying the substitution unit
G2 only display the prompt fluorescence, from hydrogen to fluoro and cyano group.
while CN-TRZ-G2 exhibits both prompt Their emission spectra extend in wide range
and delayed fluorescence. However, the from blue to green based on the substitution
increasing of lifetimes was observed for all unit. Moreover, this study demonstrated that
compound in the oxygen-free environment these molecules improved ICT
characteristics, and increased the References
fluorescence lifetime, by increasing the 1. Endo, A.; Ogasawara, M.; Takahashi, A.;
strength of acceptor unit. Yokoyama, D.; Kato, Y.; Adachi, C. Adv.
Mater. 2009, 21 (47), 4802-4806.
Acknowledgements 2. Adachi, C. Jpn. J. Appl. Phys. 2014, 53
This work was financially supported (6), 060101.
by the research grant of Vidyasirimedhi 3. Wex, B.; Kaafarani, B. R. J. Mater.
Institute of Science and Technology Chem. C 2017, 5 (34), 8622-8653.
(VISTEC). The analytical instruments were 4. Cha, J. R.; Lee, C. W.; Lee, J. Y.; Gong,
provided by Frontier Research Center M. S. Dyes Pigm. 2016, 134, 562-568.
(FRC), VISTEC. 5. Matulaitis, T.; Imbrasas, P.; Kukhta, N.
A.; Baronas, P.; Buciunas, T.;
Banevičius, D.; Kazlauskas, K.;
Gražulevičius, J. V.; Juršėnas, S. J. Phys.
Chem.C 2017, 121 (42), 23618-23625.
CO2 methanation over NiO/NiAl2O4 spinel catalysts
Chanya Thamma, Piyasan Praserthdam, Joongjai Panpranot*
Center of Excellence on Catalysis and Catalytic Reaction, Department of Chemical Engineering,
*E-mail: joongjai.p@chula.ac.th
Abstract:
Methanation of carbon dioxide is a practical process to recycle CO2 emission into
alternative fuels. In the present work, a series of NiO/NiAl2O4 catalysts were prepared by co-
precipitation method with a stoichiometric mixture of nickel nitrate and aluminium nitrate
solutions (Ni/Al=1/2) at different precipitation temperatures (25 and 80 oC) and calcined at
500, 700, and 900 oC. The XRD results confirmed the NiAl2O4 spinel phase for those
calcined at 700 oC and higher, regardless of the precipitation temperature used. The presence
of spinel phase was responsible for the decrease of BET surface area and high temperature
reduction peaks shown in the H2-temperature program reduction results due to the strong
interaction between nickel and alumina in the form of NiAl2O4. However, it was found that
all the NiO/NiAl2O4 catalysts exhibited higher activity for CO2 methanation at 350 oC
atmospheric pressure than the impregnation NiO/Al2O3 catalyst. The best catalyst
performances (91% conversion of CO2 and 100% of selectivity of methane) were obtained
over the NiAl2O4 catalysts prepared using precipitation and calcination temperatures at 80 oC
and 900 oC, respectively.
Introduction under atmospheric pressure at a not so high

Nowadays renewable energy is temperature but the conversion is still low.2
important to displace fossil fuels and the CO2 methanation is highly exothermic
demand is increasing continuously. Green reaction so that thermally stable catalysts
energy such as biogas is attractive and with high activities are preferred.3 Alumina
environmentally friendly alternative to supported nickel catalyst is one of the most
reduce the extravagant fuels being used. commonly used catalyst for CO2
However, biogas consists mostly methane methanation due to its good catalytic
and carbon dioxide. At present, CO2 performance and very low price. In most
methanation is a practical way to upgrade studies, Ni/γ-Al2O3 prepared by
biogas into high methane concentration and conventional methods such as impregnation,
lower the amount of CO2 emission. The CO2 sol-gel, co-precipitation, and mechanical
hydrogenation to methane also called the mixing showed low catalytic activity at low
Sabatier reaction is an important catalytic temperature and were easily to deactivate at
process; high temperature.4 In hydrogenation of
acetylene reaction, the formation of NiAl2O4
CO2 + 4H2 CH4+2H2O can provide a stabilizing effect and decrease
H298K = -252.9 kJ/mol coking rate of catalysts.5 Furthermore, in dry
stream reforming reaction, a comparison of
and has a wide range of applications MgO- -Al 2 O 3 and MgAl 2 O 4 showed that
including the purification of synthesis gas superior activity and stability of nickel
for the production of ammonia and syngas.1 catalysts can be obtained in the
In addition, the formation of methane from Ni/MgAl2O4.6
CO2 is a simple reaction that can operate
In the present work, a series of Table 1. Co-precipitation and calcination
NiO/NiAl2O4 catalysts were prepared by co- conditions of the prepared catalyst
precipitation method and tested in the CO2 Co-
Calcination Catalysts
methanation at 350 ⁰C. The effects of No precipitation
temp (⁰C) nomenclature
temp (⁰C)
precipitation temperature and calcination 1 25 500 Ni-Al1 500
temperatures on the characteristics and 2 25 700 Ni-Al1 700
catalytic performances of the NiAl2O4 spinel 3 25 900 Ni-Al1 900
were focused. 4 80 500 Ni-Al2 500
5 80 700 Ni-Al2 700
6 80 900 Ni-Al2 900
2. Experimental
NiAl2O4 catalysts were prepared by TPR measurements were carried in quartz u-
co-precipitation method with molar ratio of tube reactor to determine the behavior of
Ni/Al was 1:2. Nickel nitrate (Ni (NO3)2 • reduction temperature of the catalysts. The
6H2O) and aluminium nitrate (Al (NO3)3 • samples were pretreated by N2 with flow
9H2O) were used as the metal precursors. rate 30 cm3/min at 200 ⁰C for 1 h then
Firstly, nickel nitrate and aluminium nitrate cooled down to the room temperature. After
were diluted in DI water with concentration that, the TPR profiles were obtained by
of solution 0.5 molar and then adjust pH of flowing the carrier gas (10%H2/Ar) through
solution by adding NH4OH from pH 4 to 8.5 the catalysts samples at 30 cm3/min and
and stirring until precipitated. The temperature ramping from 30 ºC to 800 °C
temperature of co-precipitation were varied with the temperature rating 10 ºC/min.
at 25 ⁰C and 80 ⁰C. After that the precipitate 2.3 Catalytic activity
The catalytic activity of the prepared
powder were filtrated and dried at 110 ⁰C
catalysts were evaluated in CO2 methanation
for 12 h in air and calcined at temperature
reaction at 350 ⁰C for 3 h under atmospheric
500,700, or 900 ⁰C for 5 h. The preparation
and the molar gas mix ratio of CO2:H2:N2
conditions and the nomenclature of the
1:10:4 with the total flow rate is 40 cm3/min.
prepared catalysts are shown in Table 1. For
Approximately 0.1 g catalyst was packed in
comparison purposes, NiO/Al2O3 was also
9 mm diameter of the tubular quartz reactor.
prepared by impregnation method with a
Prior to the reaction test, the samples were
similar molar ratio of Ni/Al 1:2 using
nickel nitrate (Ni (NO3)2 • 6H2O) and Al2O3 pretreated in H2 at 450 ⁰C for 2.30 h with
commercial (Fluka) as precursor of metal flow rate is 30 cm3/min. The composition of
and support, respectively. The catalyst was outlet gas was analyzed by pulse inlet to
GC-TCD with molecular sieve and Porapack
dried at 110 ⁰C overnight and calcined at
Q columns.
550 ⁰C for 2 h.
2.2 Catalytic characterization
The BET surface area, pore volume,
3.1 Catalytic characterization
and average pore size diameter were
The BET surface area, pore volume,
calculated from the nitrogen adsorption
and average pore size diameter are shown in
isotherms at 77 K using a BEL-SORP
Table 2. The catalyst prepared at
automated system. The XRD pattern were
precipitation temperature 80 ⁰C had higher
obtained by using a SIEMENS XRD D5000
BET surface area (59-105 m2/g) than the
X-ray diffractometer with CuKα radiation
catalysts prepared at room temperature (81-
the patterns were recorded over a range of
118 m2/g) except those calcined at 500 ⁰C.
2 angles from 10° to 80°. The average
The Ni-Al1 500 had higher BET surface area
crystallite size of NiAl2O4 were calculated
(190 m 2 /g) than Ni-Al 2 500 (161 m 2 /g).
by using the Scherrer’s equation. The H2-
Table 2. Characterization of NiAl2O4 spinel catalysts
Average Average crystallite size
Catalysts BET surface Pore volume pore size (nm)
area (m2/g) (cm3/g) Diameter (nm) NiO phase NiAl2O4 phase
Ni-Al1 500 190 0.27 4.4 4.84 -

Ni-Al1 700 105 0.21 4.6 6.2 8.24
Ni-Al1 900 59 0.23 9.7 12.2 8.1
Ni-Al2 500 161 0.31 4.5 7.6 -
Ni-Al2 700 118 0.32 6.5 13.44 9.1
Ni-Al2 900 81 0.31 10.8 16.04 9.0
The BET surface area decreased 8.1 when the calcination increased from 500
drastically with increasing calcination to 900oC. A similar trend was also observed
temperature. On the contrary, the average for the NiAl2 catalysts. The average
pore diameter of the catalysts calcined at crystallite size of NiO phase increased form
higher calcination temperature was larger 7.6 nm to 16.0 nm as well as a slightly
than those calcined at lower temperature, decreased average crystallite size of
especially for the Ni-Al2 series in which NiAl2O4 from 9.1 to 9.0 nm when the
larger pore diameter (4.5-10.8 nm) were calcination temperature increased from 500
obtained, comparing to the Ni-Al1 series to 900 oC. It is noticed that the average
(4.4-9.7 nm). The lower surface area of the crystallite size of NiO of the NiAl2 catalysts
samples was probably due to the formation was larger than the NiAl1 catalysts whereas
of NiAl2O4 spinel phase by calcination at there was no significant difference in the
high temperature.7 The XRD patterns of the average crystallite size of NiAl2O4 for both
catalysts are shown in Figure 1 and the NiAl1 and NiAl2 catalysts.
calculated average crystallite size of each
nickel phase are given in Table 2. All the
catalysts exhibited the characteristic peaks
corresponding to both NiO and NiAl2O4
phases8. The XRD patterns were quite
similar for both Ni-Al1 and Ni-Al2 catalysts.
The samples calcined at 500 ºC (Ni-Al1 500
and Ni-Al2 500) revealed only the NiO
peaks at 2θ degrees 37º , 43º and 62.5º.7,9
The NiAl2O4 peak were not observed. The
characteristic peaks corresponding to the
spinel NiAl2O4 appeared as small shoulder
peaks at 2θ degrees 37º, 47.5º and 65.2º
when the samples were calcined at 700ºC.
Furthermore, the XRD patterns of NiAl2O4
were clearly observed for the samples
calcined at 900 ºC in which high intensity of Figure 1. The XRD patterns of NiAl2O4
the NiAl2O4 were presented at 2θ degrees spinel catalysts: (a) Ni-Al1 500, (b) Ni-Al1
37º, 47.5º, 59.1º, and 65.2º. For the NiAl1 700, (c) Ni-Al1 900, (d) Ni-Al2 500, (e) Ni-
catalysts, the average crystallite size of the Al2 700 and (f) Ni-Al2 900 ; • NiAl2O4 ; o
NiO phase increased from 4.8 nm to 12.2 NiO
nm while the average crystallite size of
NiAl2O4 slightly decreased from 8.24 nm to
In other words, precipitation and activity due to decrease the sintering
calcination temperatures strongly affected deactivation of catalyst at high temperature.
the sintering of NiO phase but had little
effect on the thermally stable NiAl2O4. The Table 3. The H2 – TPR results
results from the H2-TPR profiles of the The reduction peak (⁰C)
samples are shown in Figure 2 and Catalysts
1st peak 2nd peak
summarize in Table 3. Two reduction peaks
Ni-Al1 500 518 705
were observed in the TPR profiles at the
Ni-Al1 700 565 741
temperature range 500-600 oC and > 700 oC, Ni-Al1 900 580 >800
corresponding to the reduction of NiO to Ni-Al2 500 554 745
metallic Ni0 and the reduction of Ni2+ Ni-Al2 700 576 771
incorporated in the nickel aluminate structure, Ni-Al2 900 593 >800
respectively10 . The reduction peaks shifted
towards higher temperatures when the 3.2 Catalytic activity
calcination temperature was increased for The catalytic activity tests for CO2
both Ni-Al1 and Ni-Al2 with the Ni-Al2 series methanation were investigated at 350 ⁰C for
exhibited higher reduction temperature than the 3 h reaction time. The catalyst performances
Ni-Al1. in terms of CO2 conversion and CH4 yield
are shown in Figure 3. All the catalysts
showed high stability during the 3 h time on
stream. The Ni-Al2 900 catalyst exhibited
f the highest conversion of CO2 among the
catalysts studied at 91% with 100% methane
e selectivity without CO formation. The Ni-
Al1 900 catalyst also showed high CO2
conversion at 88% conversion which was
d slightly less than the Ni-Al2 900 catalyst.
Interestingly, The CO2 conversion of Ni-Al1
c 500, Ni-Al2 500, Ni-Al1 700, and Ni-Al2 700
were essentially similar at around 82%
b conversion. The CO2 conversion of the
Ni/Al2O3 catalyst prepared by impregnation
a with similar amount of Ni loading was
found to be at 74%, which was lower than
all the precipitated NiO/NiAl2O4 catalysts.
Temperature (⁰C) In this work, the formation of NiAl2O4
Figure 2. The H2-TPR profiles of NiAl2O4 spinel phase could enhance the conversion
spinel catalysts: (a) Ni-Al1 500, (b) Ni-Al1 of CO2 and CH4 yield in CO2 methanation
700, (c) Ni-Al1 900, (d) Ni-Al2 500, (e) Ni- reaction (no changes in CH4 selectivity) and
Al2 700 and (f) Ni-Al2 900 the catalysts calcined at 900 oC showed the
best catalyst performances, despite the larger
The reduction temperature may be a crystallite size of the NiO and NiAl2O4
little effect in the conversion of CO2 and phases. Nevertheless, in a previous study by
CH4 yield because the highly reduction Sahli. L et al.,11 larger crystallite size of the
temperature and the reduction time had been bulk NiAl2O4 also showed high activity in
used that optimized potential to reduce NiO the steam reforming of methane reaction.
that a easily reducible material to Ni metal Furthermore, the presence of NiAl2O4 could
active site. Conversely, the formation of exert a positive effect on Ni sintering.
NiAl2O4 exert the positive effect to catalytic
and high dispersion of Ni on NiAl2O4 upon
100
reduction.
95
90 Conclusion
%CO2 conversion, % CH4 yield
The Ni-Al2 900 catalysts which

85
prepared by co-precipitation of nickel nitrate
80 and aluminium nitrate at 80 oC and calcined
75 at 900 oC showed the highest CO2
conversion and CH4 yield at 91% at
70 relatively low reaction temperature (350 oC).
65 Ni-Al1 700 The precipitation and calcination
60
Ni-Al1 900 temperatures strongly influenced the
Ni-Al2 700 crystallite size of the NiO phase but had
55 Ni-Al2 900 little effect on the NiAl2O4 phase. There
Ni-Al1 500
50 Ni-Al2 500
were no significant differences in the
Ni/Al2O3 catalyst activities for those calcined at 500
45
and 700 oC. All the NiO/NiAl2O4 catalysts
40 showed higher activity than the NiO/Al2O3
0 20 40 60 80 100 120 140 160 180 200
catalyst prepared by conventional
Time (min) impregnation under similar reaction
Figure 3. CO2 conversion and CH4 yield of conditions used. The enhancement of both
NiAl2O4 spinel catalysts during CO2 CO2 conversion and CH4 yield may be due
methanation at 350 ⁰C for 3 h (180 min) to NiAl2O4 exerted a positive effect on Ni
metal crystallite size after reduced resulting
The particle size of Ni metal changed in higher dispersion on NiAl2O4 support and
to smaller size after reduced in pure H2 at the stabilizing effect of the NiAl2O4 spinel
high temperature and resulted in high phase.
disposition on the NiAl2O4 catalyst
support.11,12 In the CO2 reforming of Acknowledgements
methane, an enhancement of CO2 Financial Supports from the Thailand
conversion and CH4 yield over NiAl2O4 was Research Fund, the National Research
attributed to the presence of spinel structure Council of Thailand (NRCT), and the
that was active and highly stable, high Ratchadaphiseksomphot Endowment Fund
dispersion of NiO on the spinel, and the for International Research Integration: Chula
interaction of nickel metal and the support.5 Research Scholar are gratefully
Moreover, in the hydrogenation of acetylene acknowledged.
over Ni/NiAl2O4 catalyst, the formation of
NiAl2O4 can provide a stabilizing effect References
resulting in the inhibition of metal 1. Wei, W.; Jinlong, G. Frontiers. Chem.
aggregation and lowering coking rate Sci. Eng. 2010, 5 (1), 2-10.
formation.9 Furthermore, the formation of 2. Aziz, M. A. A.; Jalil, A. A.; Triwahyono,
NiAl2O4 can improve of coke resistance, and S.; Ahmad, A. Green. Chem. 2015, 17
enhance stability of catalyst for dry (5), 2647-2663.
reforming of methane reaction.13 In the 3. Stangeland, K.; Kalai, D.; Li, H.; Yu, Z
present work, the presence of both NiO and Energy Procedia 2017, 105, 2022-2027.
NiAl2O4 led to high activity in CO2 4. Zeng, Y.; Ma, H.; Zhang, H.; Ying, W.;
methanation due probably to the formation Fang, D. Fuel 2014, 137, 155-163.
of active substoichiometric spinel structure
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Santanaria, J. J. Catal. 1995, 159, 313– Rivas, B.; González-Velasco, J. R.;
322, 159. Gutiérrez-Ortiz, J. I.; López-Fonseca, R.
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New J. Chem. 2016, 40 (5), 4049-4060. 190-201.
7. Yolanda Cesteros, P. S., Francisco 11. Sahli, N.; Petit, C.; Roger, A. C.;
Medina Sueiras, J. S. E. Chem. Mater Kiennemann, A.; Libs, S.; Bettahar, M.
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8. Li, C. Y.; Zhang, H. J.; Chen, Z. Q. 193.
Appl. Surface Sci. 2013, 266, 17-21. 12. Su, X.; Xu, J.; Liang, B.; Duan, H.;
9. Jiménez-González, C.; Boukha, Z.; de Hou, B.; Huang, Y. J. Energy. Chem.
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Catal. A: General 2013, 466, 9-20. 2508-2516.
Hole-transporting thermally activated delayed fluorescence (TADF)
materials based on carbazole dendronized anthraquinone derivatives for
organic light-emitting diodes
Namthip Khammultri, Rossatorn Muangpaisal, Taweesak Sudyoadsuk, Vinich Promarak*
Vidyasirimedhi Institute of Science and Technology, Wang Chan, Rayong 21210, Thailand
*E-mail: vinich.p@vistec.ac.th, s15_namthip.k@vistec.ac.th
Abstract:
Thermally activated delayed fluorescence (TADF) is alternative luminescence
material. Because of it can harvest energy from triplet state by thermal activation to obtain
100% internal quantum efficiency. In this work, novel hole-transporting TADF materials has
been developed from highly efficient red-emitted TADF molecule AQTPA containing
anthraquinone as an acceptor unit and triphenylamine as donor unit. Two anthraquinone
derivatives namely AQTPA-G1 and AQTPA-G2 having carbazole dendrimers as hole-
transporting moiety substituted on triphenylamine of AQTPA were synthesized. All target
molecules were confirmed structure by NMR and mass spectrometry. Their photophysical
properties and TADF characteristics were investigated using UV-VIS, steady state,
fluorescence lifetime and phosphorescence lifetime (FLS), and photoelectron (AC-2)
spectrometers. AQTPA-G2 exhibited outstanding life time in neat film demonstrating a
promising for highly OLEDs performance.
1. Introduction In 2014, Zhang and his colleagues reported

In recent years, organic light- highly efficient red TDAF materials based
emitting diodes (OLEDs) devices are the on anthraquinone molecules.9 In device
interesting luminescence devices which fabrication process, OLEDs materials are
having many advantages such as fast being often fabricated using host materials
response time, high resolution, low power which improve charge transport in emitting
consumption, brightness, flexible display, materials to improve the device
thin-film devices, large fabrication area and efficiency.10,11 The error of optimum doping
wide viewing angle.1-3 Thermally activated concentration is problems of fabricating
delayed fluorescence (TADF) has been process. In this work, we focus on develop
wildly known as new generation of emitting hole-transporting TADF materials from
materials which converts electron from AQTPA by adding carbazole dendrimers at
triplet state to singlet state lead to internal the terminal ends of the molecule as
quantum efficiency up to 100% by thermal AQTPA-G1 and AQTPA-G2. The target
energy.4-6 The design of TADF material molecules were investigated photophysical
should separate HOMO and LUMO energy and TADF properties.
level by creating donor-acceptor structure.7
The energy level of singlet and triplet state 2. Materials and Methods
should be lower than 0.5 eV. Anthraquinone 2.1 General
is one of the attractive materials for All reagents were used without other
developing emitting materials because of purification from TCI or Aldich companies.
their good chemical and physical properties All solvents were provided by Thai
such as thermal stability, electron-accepting companies and used without other
property and photoluminescence property.8 distillation.
2.2 Synthesis of 2,6-bis(4-(bis(4-bromo (4:6) followed by recrystallization with a
phenyl)amino)phenyl)anthracene-9,10- mixture of CH2Cl2 and methanol.The
dione (AQTPA-Br) product was obtained as orange solid (0.19g,
2,6-Bis(4-diphenylamino) phenyl) 55%). 1H NMR (600 MHz, CDCl3) δ 8.61
anthracene-9,10-dione (AQTPA)9 (0.50 g, (1H, s), 8.43 (1H, d), 8.15 (4H, d), 8.07 (1H,
0.72 mmol) was dissolved in THF 40 ml. dd), 7.78 (2H, d), 7.55 (4H, d), 7.50 (4H,
NBS (0.61 g, 3.60 mmol) was added in dd) 7.46-7.42 (12H, m), 1.50 (42H, d) ppm;
small portions at 0 °C. The mixture was 13
C NMR (150 MHz, CDCl3) δ 183.09,
stirred at room temperature for 2.5 hours. 148.48, 146.30, 145.87, 143.03, 139.47,
The solvent was removed by rotary 134.35, 133.77, 133.33, 132.05, 131.66,
evaporator. CH2Cl2 (40 ml) and water (40 128.60, 128.34, 127.95, 125.76, 125.05,
ml) was added for extraction. The organic 124.13, 123.75, 123.51, 116.41, 109.38,
phase was washed in water (50 ml x 2), 34.88, 32.17, 29.83 ppm; MALDI-TOF
brine solution (50 ml) and dried over (m/z)calcd for C130H126N6O2: 1803.9976;
anhydrous Na2SO4. The solvent was found 1804.3665 (M+).
removed until dry out by rotary evaporator. 2.4 Synthesis of 2,6-bis(4-(bis(4-
Then recrystallization with a mixture of (3,3'',6,6''-tetra-tert-butyl-9'H-[9,3': 6',9''
hexane and CH2Cl2. The product was -tercarbazol]-9'-yl) phenyl) amino)
obtained as yellow solid (0.64 g, 89%) 1H phenyl)anthracene-9,10-dione (AQTPA-
NMR (600 MHz, CDCl3) δ 8.53 (1H, s), G2)
8.38 (1H, d), 7.99 (1H, dd), 7.65 (2H, d), AQTPA-G2 was prepared in similar
7.41 (4H, d), 7.16 (2H, d), 7.10 (4H, t) ppm; AQTPA-G1 by adding AQTPA-Br (0.18 g,
13
C NMR (150 MHz, CDCl3) δ 183.01, 0.17 mmol), 3,3'',6,6''-tetra-tert-butyl-9'H-
147.86, 146.14, 146.09, 134.24, 133.36, 9,3':6',9''-tercarbazole (G2)12 (0.64 g, 0.89
132.70, 131.99, 131.62, 128.44, 128.25, mmol). The product was purified by column
126.23, 124.97, 123.79, 116.53 ppm; chromatography. The system for eluting is a
MALDI-TOF (m/z)calcd for C50H30 mixture of CH2Cl2 and hexane (3:7)
Br4N2O2: 1009.9000; found 1010.1352 followed by recrystallization with a mixture
(M+). of DCM and methanol. The product was
2.3 Synthesis of 2,6-bis(4-(bis(4-(3,6-di- obtained a yellow solid (0.23g, 36%). 1H
tert-butyl-9H-carbazol-9-yl)phenyl) NMR (600 MHz, CDCl3) δ 8.65 (1H, s),
amino)phenyl)anthracene-9,10-dione 8.45 (1H, d), 8.26 (4H, s), 8.16 (8H, s), 8.10
(AQTPA-G1) (1H, d) 7.86 (2H, d), 7.50 (8H, t), 7.64 (8H,
A mixture of AQTPA-Br (0.20 g, dd) 7.54 (2H, d), 7.46 (8H, d), 7.34 (8H, d)
0.20 mmol, 3,6-di-tert-butyl-9H-carbazole 1.47 (72H, s) ppm; 13C NMR (150 MHz,
(G1)12 (0.33 g, 1.19 mmol), K3PO4 (0.42 g, CDCl3) δ 147.99, 146.75, 142.60, 140.51,
1.98 mmol) and CuI (0.08 g, 0.40 mmol) 140.16, 134.25, 132.52, 131.70, 130.98,
was degassed with N2 3 times. Then, toluene 128.75, 128.42, 128.29, 126.00, 125.67,
30 ml was added in the reaction followed by 125.11, 124.89, 123.99, 123.55, 123.15,
(±)-trans-1,2-diaminocyclohexane (0.04 119.38, 116.23, 111.14, 109.06, 34.72,
ml). The reaction mixture was heated to 120 32.03, 31.57; MALDI-TOF (m/z) calcd for
°C for 3 days. The crude reaction was added C258H246N14O2: 3573.9645; found 3575.7251
with water (100 ml) and extracted with (M+H+).
CH2Cl2 (40 ml x 2). The combined organic 2.5 Methods
1
phase was washed in water (40 ml), brine H and 13C NMR spectra were
solution (40 ml) and dried over anhydrous recorded on a Bruker AVANCE III HD
Na2SO4.The solvent was removed until dry (600MHz) spectrometer using CDCl3 as
out by rotary evaporator. Purification by solvent. Molecular masses were measured
column chromatography over silica gel with a Bruker Autoflex speed mass
eluting with a mixture of CH2Cl2 and hexane spectrometer. UV–vis spectra, fluorescence
Br
Br
O O
N N
NBS
o
N THF, 0 C N
O O
Br
AQTPA AQTPA-Br
Br
NH2
NH2
G1
or
G2
CuI, K3PO4,
toluene, reflux
X
X
N N O
N
X=
N N N
O
X
AQTPA-G1 AQTPA-G2
X
Figure 1. Synthetic pathway of AQTPA-G1 and AQTPA-G2
spectra and life time were investigated as a confirmed four bromine substituents in the
dilute solution in spectroscopy grade air- structure. AQTPA-G1 and AQTPA-G2
saturated toluene and degassed toluene on a were then synthesized using Ullmann
Perkin-Elmer Lambda 1050 spectrometer coupling reaction of AQTPA-Br with G1
and an Edinburgh Instruments FLS 980 and G2 carbazoles catalyzed by
spectrometer, respectively. Photoelectron K3PO4/CuI/(±)-trans-1,2-diaminocyclohexa-
(AC-2) were recorded on Riken ne in toluene at reflux. AQTPA-G1 and
photoelectron spectrometer. AQTPA-G2 were attained as orange and
yellow solids respectively. Chemical
3. Results and Discussion structures of all compounds were
3.1 Synthesis of AQTPA-G1 and AQTPA- investigated by 1H NMR, 13C NMR and
G2 MALDI-TOF.
Target molecules were designed to 3.2 Photophysical properties
improve hole transporting capability by Absorption spectra of AQTPA-G1
adding carbazole dendrimers on to the and AQTPA-G2 were determined by UV-
AQTPA core molecule as AQTPA-G1 and vis and photoluminescence (PL)
AQTPA-G2. The synthesis pathway is spectroscopy in toluene solvent as shown in
shown in Figure 1. The intermediate Figure 2(a). The electronic transition
AQTPA-Br was synthesized by demonstrates π-π* transition of
bromination reaction of AQTPA with NBS anthraquinone core molecule and carbazole
in THF at low temperature. Mass spectrum dendrons at 335 and 298 nm respectively.
data of the obtained isolated product showed
molecular weight at 1010.1352 m/z that
Table 1. Photophysical data of target molecules at room temperature

Compound λabs,max sol λem,max sol λem,max film Ega HOMOb LUMOc τd
(nm) (nm) (nm) (eV) (eV) (eV) (ns)
AQTPA 456 613 663 2.20 -5.57 -3.37 5.61
AQTPA-G1 462 680 653 2.12 -5.60 -3.48 4.76
AQTPA-G2 447 613 624 2.21 -5.78 -3.57 16.02
a
Onset emission of neat films Eg = λonset/1240
b
HOMO investigated by Photoelectron spectrometer (AC-2) where gold at -4.7 eV.
c
LUMO calculated by HOMO-Eg
d
An Average lifetime in neat film
Figure 2. Absorption and PL spectra of the molecules in a) toluene and b) neat film. c)
Fluorescence decay time of the molecules in neat film investigated by FLS
The onset absorption of neat film using steady state, fluorescence lifetime and
corresponding to energy bandgap for phosphorescence lifetime (FLS). AQTPA-
AQTPA-G1 and AQTPA-G2 are shown in G2 in neat film indicate outstanding life
Table 1. AQTPA-G1 and AQTPA-G2 time at 16.02 ns shown in Figure 2(c).
exhibit slightly blue-shifted emission in neat 3.3 TADF Properties
film especially AQTPA-G2 which have TADF properties were investigated
higher generation carbazole dendrons units briefly by study effect of oxygen in PL
(Figure 2(b)). HOMOs of AQTPA-G1 and spectra and life time according to oxygen
AQTPA-G2 exhibit at 5.60 and 5.78 eV, can quench triplet state of compounds.
respectively by AC-2 spectrometer. C-N AQTPA-G1 and AQTPA-G2 indicated
bond of carbazole dendrons affect electron higher intensity emission and increase life
density in molecules and improve hole- time of molecules when degassed solvent
transporting property. Furthermore, they shown in Figure 3.
result in reducing the aggregation and life
time of materials. Life time was investigated
Figure 3. Effect of oxygen investigated by PL spectra (left) and fluorescence decay time
(right) of AQTPA-G1 (a and b) and AQTPA-G2 (c and d)
4. Conclusion
Novel hole-transporting TADF 4. Uoyama, H.; Goushi, K.; Shizu, K.;
materials have been synthesized. Carbazole Nomura, H.; Adachi, C. Nature 2012,
dendrons can improve hole transport 492, 234-238.
capability especially AQTPA-G2. AQTPA- 5. Dias, F. B.; Bourdakos, K. N.; Jankus,
G2 exhibits band gap 2.21 eV and emission V.; Moss, K. C.; Kamtekar, K.
at 624 nm. Furthermore, AQTPA-G2 T.;Bhalla, V.; Santos, J.; Bryce, M. R.;
indicates outstanding lifetime at 16.02 ns. Monkman, A. P. Adv. Mater. 2013, 25,
The hole-transporting TADF materials are 3707-14.
promising to demonstrate highly OLEDs 6. Tao, Y.; Yuan, K.; Chen, T.; Xu, P.; Li,
performance. H.; Chen, R.; Zheng, C.; Zhang, L.;
Huang, W. Adv. Mater. 2014, 26, 7931-
This work was supported by 7. Adachi, C. Jpn. J. Appl. Phys. 2014, 53,
Vidyasirimedhi Institute of Science and 060101.
Technology (VISTEC). We thank Frontier 8. Itoh, T. Chem. Rev. 1995, 95, 2351.
Research Center (FRC) at VISTEC provided 9. Zhang, Q.; Kuwabara, H.; Potscavage,
instrument for characterization and analysis. W. J. J.; Huang, S.; Hatae, Y.; Shibata,
T.; Adachi, C. J. Am. Chem. Soc. 2014,
References 136, 18070-81.
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Brown, A. R.; Marks, R. N.; Mackay, 11. Jung, B.; Yoon, C.; Shim, H.; Do L.;
K.; Friend, R. H.; Burns, P. L.; Holmes, Zyung, T. Adv. Funct. Mater. 2001, 11,
A.B. Nature 1990, 347, 539. 430.
2. Braun, D.; Heeger, A. J. Appl. Phys. Lett. 12. Moonsin, P.; Prachumrak, N.;
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3. Komoda, T.; Ide, N.; Varutt, K.; Yamae, Jungsuttiwong, S.; Sudyoadsuk, T.;
K.; Tsuji, H.; Matsuhisa, Y. J. Soc. Inf. Promarak, V. Chem. Commun. 2012, 48,
Disp. 2011, 19, 838. 3382–3384.
Thermally activated delayed fluorescence (TADF) materials based on
triphenylamine end capped benzothiadiazole for OLEDs
Jakkapan Kumsampao, A-monrat Thangthong, Taweesak Sudyoadsuk, Vinich Promarak*
Abstract:
Thermally activated delayed fluorescence (TADF) materials enable a new opportunity
for the development of highly efficient fluorescent OLEDs because of achieving 100%
internal quantum efficiency and do not require expensive metallic elements. In this work,
three novel TADF molecules, TPA-BTZ-H, TPA-BTZ-F and TPA-BTZ-CN were
synthesized by using Suzuki coupling reaction. Unsubstituted benzothiadiazole (BTZ-H),
difluoro-benzothiadiazole (BTZ-F), and dicyano-benzothiadiazole (BTZ-CN) as the acceptor
were end capped with triphenylamine (TPA) as the donor to form D-A-D structure TADF.
Their physical, electrochemical properties and TADF characteristics in toluene were studied
by UV-Vis, cyclic voltammetry (CV) and steady State, fluorescence and phosphorescence
lifetime spectrometer (FLS). The PL spectra of the three compounds showed large Stoke
shifts where TPA-BTZ-H emitted yellow-orange light with emission peak at 595 nm, TPA-
BTZ-F blue-shift to yellow light with an emission peak at 578 nm and TPA-BTZ-CN red-
shifted to red light with an emission peak at 674 nm. The fluorescence color of these
compounds can be controlled by using different electron withdrawing groups which represent
the most method to tune the band gap that cover the range from yellow to red color emission,
which can be beneficial for the improvement of the OLEDs.
1. Introduction delayed emission that are involved in the

Organic light-emitting diodes TADF process, which is easily disturbed by
(OLEDs) have received remarkable interest external conditions, such as heat and oxygen
in organic materials field due to their or triplet-quenching by O2.4 The
potential applications in full color-flat-panel photophysical properties of these materials
displays and solid-state lighting with high could be tuned by a suitable combination of
electroluminescence (EL) efficiency, the donor and acceptor structures.5,6 As the
flexibility, high contrast, lightweight, and mention above, that is our challenges to
good color range.1 Generally, the emitting synthesize the novel TADF materials for
materials are focus as main point study to OLEDs. In this work, new TADF materials
enhance the performances of OLEDs.2 based on benzothiadiazole (BTZ) as the
Recently, metal-free thermally activated acceptor were end capped with
delayed fluorescent (TADF) emitters triphenylamine (TPA) as the donor were
become a viable alternative for development designed and characterized.
of high efficient OLEDs with 100% internal
quantum efficiency (IQE) achieving from 2. Materials and Methods
both singlet (25%) and triplet (75%) 2.1 General
excitons and easily obtained in the pure All chemicals were purchased from
form.3 Sigma-Aldrich, Tokyo Chemical Industry
Interestingly, TADF is a special Co., Ltd (TCI) and used without further
radiative transition of excitons base on purification. THF used as the solvent for the
reversible intersystem crossing (RISC) from synthesis was purified by distillation from
triplet to singlet excited state generates the Na/benzophenone. All final products were
purified by column chromatography with purified by column chromatography (1/3
Merck silica gel mesh, 60 Å before the CH2Cl2/hexane). The resulting product was
measurements. further purified by recrystallizing with
2.2 Synthesis of 4,7-dibromo-2,1,3- CH2Cl2/MeOH to give an orange powder of
benzothiadiazole (2) TPA-BTZ-H in 92% (0.39 g, 0.63 mmol).
A mixture of 2,1,3-benzothiadiazole 1
H NMR (600 MHz, CDCl3) δ (ppm): 7.07
1 (4.00 g, 29.34 mmol) and HBr (50 mL) in (t, J = 7.8 Hz, 4H), 7.15-7.25 (m, 12H), 7.30
a two-necked round-bottomed flask was (t, J = 7.8 Hz, 8H), 7.74 (s, 2H), 7.89 (d, J =
stirred at room temperature. A solution of 8.4 Hz, 4H). 13C NMR (151 MHz, CDCl3) δ
Br2 (15.51 g, 97.05 mmol) in HBr (30 mL) (ppm): 123.0, 123.3, 124.9, 127.4, 129.4,
was added dropwise very slowly to the 129.9, 132.2, 154.2. MALDI-TOF-MS: m/z
mixture. After total addition of Br2, the [M]+ calcd for C42H30N4S: 622.2191, found:
reaction was heated at reflux for 6 hours. 622.2844.
Then, the reaction mixture was cooled to 2.4 Synthesis of 5,6-difluoro-4,7-bis
room temperature, and a sufficient amount (triphenylamine)-2,1,3-benzothiadiazole
of a saturated solution of NaHSO3 was (TPA-BTZ-F)
added to completely dispose any excess Br2. A mixture of 4,7-dibromo-5,6-
The mixture was extracted CH2Cl2 (3 × 100 difluoro-2,1-3-benzothiadiazole 3 (0.20 g,
mL) and water (100 mL). The combine 0.61 mmol), 4-(diphenylamino)
organic layer was dried with anhydrous phenylboronic acid 4 (0.37 g, 1.28 mmol),
Na2SO4, filtrated and concentrated using Pd(PPh3)4 (0.035 g, 0.03 mmol) was
rotary evaporator. The crude residue was dissolved in dry THF (25 mL). Then, 2M
purified by column chromatography (1/4 Na2CO3 (6 mL) was added. The mixture was
CH2Cl2/hexane) on silica gel and degassed under nitrogen atmosphere (3
recrystallized by CH2Cl2/MeOH to afford a times) at room temperature follow by
white solid of dibrominated product 2 in heating at reflux for 48 hours. After the
80% (6.90 g, 23.47 mmol). 1H NMR (500 reaction completed, the reaction was cooled
MHz, CDCl3) δ (ppm): 7.73 (s, 2H), 13C to room temperature and water (50 mL) was
NMR (125 MHz, CDCl3) δ (ppm): 113.9, added to quench the reaction. The product
132.4. CL-QTOF-MS: m/z [M]+ calcd for was extracted with CH2Cl2 (3 × 50 mL). The
C6H2Br2N2S: 291.8305, found: 294.8294. organic phase was separated and dried over
2.3 Synthesis of 4,7-bis(triphenylamine)- anhydrous Na2SO4, filtered and concentrated
2,1,3-benzothiadiazole (TPA-BTZ-H) under reduced pressure. The crude product
A mixture of 4,7-dibromo-2,1-3- was purified by column chromatography
benzothiadiazole 2 (0.20 g, 0.68 mmol), 4- (1/4 CH2Cl2/hexane) on silica gel and
(diphenylamino) phenylboronic acid 4 (0.43 recrystallizing by CH2Cl2/MeOH to obtain a
g, 1.49 mmol), Pd(PPh3)4 (0.04 g, 0.03 yellow powder of TPA-BTZ-F in 68%
mmol) was dissolved in dry THF (25 mL). (0.27 g, 0.41 mmol). 1H NMR (600 MHz,
Then, 2M Na2CO3 (6 mL) was added. The CDCl3) δ (ppm): 7.08 (t, J = 3.7 Hz, 4H),
reaction mixture was degassed under 7.17-7.22 (m, 12H), 7.30 (t, J = 7.8 Hz, 8H),
nitrogen atmosphere at room temperature 7.72 (d, J = 9.0 Hz, 4H). 13C NMR (151
follow by heating at reflux for 48 hours. MHz, CDCl3) δ (ppm): 117.8, 121.8, 123.3,
After the reaction completed, the mixture 123.7, 125.3, 129.4, 131.4, 147.3, 149.6 (J =
was cooled to room temperature and water 324.9). MALDI-TOF-MS: m/z [M]+ calcd
(50 mL) was added to quench the reaction. for C42H28F2N4S: 658.2003; found:
The product was extracted with CH2Cl2 (3 × 658.2449.
50 mL). The organic layer was collected, 2.5 Synthesis of 5,6-dicyano-4,7-
dried over anhydrous Na2SO4 and bis(triphenylamine)-2,1,3-benzothia-
evaporated under vacuum. The crude diazole (TPA-BTZ-CN)
product was adsorbed on silica gel and
H H H H
Br2, HBr
Br Br
reflux, 6 h
N N N N
S S
1 2, 80%
X X X X
HO Pd(PPh3)4, 2M Na2CO3
Br Br + B N N N
HO THF, reflux, N2, 48 h
N N N N
S S
2, X = H 4 TPA-BTZ-H, X = H, 92%
3, X = F TPA-BTZ-F, X = F, 68%
F F NC CN
KCN, 18-Crown-6,
N N N N
THF, reflux, Ar, 24 h
N N N N
S S
TPA-BTZ-F TPA-BTZ-CN, 87%
Scheme 1. Synthetic route of TPA-BTZ-H, TPA-BTZ-F and TPA-BTZ-CN
A mixture of TPA-BTZ-F (0.10g, calcd for C44H28N6S: 672.2096, found:

0.15 mmol) KCN (0.059 g, 0.91 mmol) and 672.2561.
18- crown-6 (0.004 g, 0.015 mmol) was 2.6 Methods
1
dissolved in anhydrous THF (15 mL) and H NMR spectra were recorded using
degassed under argon atmosphere at room a Bruker AVANCE 500 MHz spectrometer
temperature. The reaction was then heated at or a Bruker AVANCE III HD 600 MHz
refluxed for 24 hours. After the reaction spectrometer. 13C NMR spectra were
completed, the mixture was cooled to room recorded on a Bruker AVANCE 125 MHz
temperature and water (50 mL) was added to spectrometer or a Bruker AVANCE III HD
quench the reaction. The product was 151 MHz spectrometer. All spectra were
extracted with CH2Cl2 (3 × 50 mL). The recorded in CDCl3 solvent and chemical
organic layer was collected, dried over shifts are reported as δ values in part per
anhydrous Na2SO4 and evaporated under million (ppm) relative to tetramethylsilane
vacuum. The crude product was purified by (δ 0.00), CDCl3 (δ 7.26) as internal standard.
column chromatography (1/2 Molecular masses were determined by
CH2Cl2/hexane) on silica gel and matrix-assisted laser desorption-ionization
recrystallization with CH2Cl2/MeOH to time-of-flight mass spectrometry (MALDI-
afford a red powder of TPA-BTZ-CN in TOF-MS) using Bruker Autoflex speed
87% (0.09 g, 0.13 mmol). 1H NMR (600 instrument and liquid chromatography-time-
MHz, CDCl3) δ (ppm): 7.14 (t, J = 7.4 Hz, of-flight tandem mass spectrometer (LC-
4H), 7.18 (d, J = 8.6 Hz, 4H), 7.24 (d, J = QTOF-MS) using a Bruker compact QTOF
7.9 Hz, 8H), 7.34 (t, J = 7.7 Hz, 8H), 7.69 instrument. Ultraviolet-visible (UV-vis)
(d, J = 8.6 Hz, 4H). 13C NMR (151 MHz, absorption and photoluminescence emission
CDCl3) δ (ppm): 111.6, 116.1, 120.4, 124.5, spectra of the target compound were
124.6, 126.0, 129.6, 131.5, 140.5, 146.7, measured using a PerkinElmer LAMBDA
150.1, 154.6. MALDI-TOF-MS: m/z [M]+ 1050 UV/Vis/NIR and Edinburgh
Table 1. Photophysical and electrochemical properties of TPA-BTZ-H, TPA-BTZ-F and
TPA-BTZ-CN
Compound λabs [nm] sola λPL [nm] sola Egb [eV] HOMO/LUMOc [eV]
TPA-BTZ-H 314, 460 595 2.31 -5.26/-2.95
TPA-BTZ-F 310, 448 578 2.44 -5.38/-2.94
TPA-BTZ-CN 324, 537 674 2.03 -5.44/-3.41
a
Measured in toluene (10-5 M) at room temperature. b Estimated from the edge of the long wavelength
absorption, Eg = 1240/λonset. c HOMO level was calculated by HOMO = -(4.44 + E(OX)onset) eV, and LUMO level
was determined by LUMO = HOMO + Eg eV, where E(OX)onset is the onset potential of the oxidation curve.
instruments FLS 980 spectrophotometer, 3. Results and Discussion

respectively. The electrochemical properties 3.1 Synthesis
were investigated by cyclic voltammetry Scheme 1 describes the synthetic
using an Autolab potentiostat PGSTAT 101. route to all three target molecules. 4,7-
Cyclic voltammetry (CV) was carried out in Dibromosubstituted benzothiadiazole (2)
argon-purged CH2Cl2 at room temperature was prepared in good yield by the reaction
with a three-electrode system consisted of of 2,1,3-benzothiazole (1) with molecular
Ag/AgCl as the reference electrode, a glassy bromine in hydrobromic acid. TPA-BTZ-H
carbon as the working electrode, and a and TPA-BTZ-F were the synthesized via
platinum rod as the counter electrode. The Suzuki coupling reaction of
supporting electrolyte n-Bu4NPF6 was used benzothiadiazole cores 2 and 3 with 4-
as a supporting electrolyte. (diphenylamino) phenylboronic acid (4)
using a catalytic system of
Pd(PPh3)4/Na2CO3, respectively. They were
obtained in good yields as yellow to orange
solids. TPA-BTZ-CN was generated by
nucleophilic aromatic substitution of TPA-
BTZ-F with potassium cyanide in the
presence of 18-crown-6 and obtained as a
red solid in 87%. All target molecules were
chemically characterized by H- and C- NMR
and mass spectrometry.
3.2 Photophysical properties
The photophysical properties of three
compounds were measured by UV-vis
absorption and photoluminescence (PL)
using as toluene solvent. The results show in
Figure 1 and summarized in Table 1. The
normalized absorption spectra of TPA-BTZ-
H, TPA-BTZ-F and TPA-BTZ-CN exhibit
two major absorption bands of π-π*
transition at 285-360 nm, and the visible
broad absorption centred on 460, 448 and
537 nm can be recognized to be the
intramolecular charge transfer (ICT)
transition from the diphenylamine (TPA) as
Figure 1. (a) Normalized UV-Vis spectra of a donor moiety to the benzothiadiazole
TPA-BTZ-H, TPA-BTZ-F and TPA-BTZ- (BTZ) as an acceptor moiety, respectively
CN in toluene (10-5 M), (b) Normalized PL (Figure 1a). The optical bandgap (Eg) can be
spectra of TPA-BTZ-H, TPA-BTZ-F and estimated from the edge of the UV-vis
TPA-BTZ-CN in toluene (10-6 M)
Figure 2. (a) PL spectra of TPA-BTZ-H in toluene (10-6 M) under air and argon. (b) PL
spectra of TPA-BTZ-F in toluene (10-6 M) under air and argon. (c) PL spectra of TPA-BTZ-
CN in toluene (10-6 M) under air and argon. Inset of (a), (b) and (c) are PL images of a
solution in toluene under 365 nm UV light
absorption, giving the values of 2.31, 2.44 from 6 × 106 to 1.8 × 107. These results
and 2.03 eV for TPA-BTZ-H, TPA-BTZ-F indicate that the three emitting materials
and TPA-BTZ-CN, respectively. These have sufficiently small ΔEST values, the
results in the calculated Eg value of TPA- energy up-conversion from T1 to S1 state by
BTZ-F is 0.13 eV larger than that of TPA- reverse intersystem crossing (RISC) due to
BTZ-H. While, the Eg value of TPA-BTZ- O2 free triplet-quenching that is involved in
CN is 0.28 eV lower than that of TPA- the TADF feature which compose of prompt
BTZ-H. and delayed fluorescence.
The PL spectra show the maximum 3.4 Electrochemical properties
emission wavelength at 595, 578 and 674 The cyclic voltammograms (CV) of
nm corresponded to TPA-BTZ-H, TPA- the compounds are shown in Figure 3.
BTZ-F and TPA-BTZ-CN, respectively During the anodic scan in dichloromethane,
(Figure 1b). The results demonstrate large TPA-BTZ-H, TPA-BTZ-F and TPA-BTZ-
Stokes shift (130 nm to 137 nm) of three CN exhibited reversible oxidation behavior,
compounds, and also reflect the feature of which can be attributed to the oxidation of
ICT transitions from the TPA donor to BTZ electron donating TPA moiety. The HOMO
acceptor. TPA-BTZ-H emitted yellow- energy level of TPA-BTZ-H, TPA-BTZ-F
orange light (595 nm), while adding fluoro and TPA-BTZ-CN were estimated to be
group (TPA-BTZ-F) made the compound -5.26 eV, -5.38 eV, and -5.44 eV,
blue-shift to yellow light (578 nm). In case respectively, from the onset of oxidation
of adding cyano group (TPA-BTZ-CN) curves. CV curve of TPA-BTZ-CN (Figure
made red-shifted to red light (674 nm). 3a) revealed additional peak at lower
3.3 TADF properties potential on the cathodic scan (Epc) at 0.88
To investigate the anticipated TADF V, which corresponded to electrochemical
feature of three target molecules, they were coupling reactions of the TPA radical cation
measured in the presence and absence of (TPA⋅+). Repeated CV scans of this
triplet-quenching oxygen in toluene (Figure compound also showed an increasing
2). The results show that excluding of air change in their CV curves (Figure 3b).
from toluene solution, increasing of the Demonstrating a series of electrochemical
fluorescence intensity. In case of TPA- reactions leading to electro-polymerization
BTZ-H, going from 7 × 105 to 8 × 105 M. of those radical cation species on the glassy
While TPA-BTZ-F increasing from 5 × 106 carbon electrode surface. The HOMO level
to 6 × 106 M, and TPA-BTZ-CN rising was reduced upon addition of electron
BTZ, fluoro and cyano groups, compared
with unsubstituted BTZ were successfully
synthesized. The different deactivating
groups demonstrate different electron
withdrawing characters which control the
photoluminescence (PL) color. Compared
with yellow-orange emission color of TPA-
BTZ-H, TPA-BTZ-F exhibits blue-shifted
to yellow emission color. On the other hand,
TPA-BTZ-CN displays red-shifted to red
emission color. This report provides a
simple and effective approach to obtain
TADF emitting materials, and significant for
the efficient application in OLEDs.
Acknowledgements
Vidyasirimedhi Institute of Science and
Technology (VISTEC).
References
1. Reineke, S.; Lindner, F.; Schwartz, G.;
Seidler, N.; Walzer, K.; Lüssem, B.,
Leo, K. Nature 2009, 459, 234–238.
2. Baldo, M. A.; O'Brien, D. F.; You, Y.;
Shoustikov, A.; Sibley, S.; Thompson,
Figure 3. (a) Oxidation part of the CV M. E.; Forrest, S. R. Nature 1998, 395,
curves of TPA-BTZ-H, TPA-BTZ-F and 151–154.
TPA-BTZ-CN and (b) multiple CV scans of 3. Uoyama, H.; Goushi, K.; Nomura, H.;
TPA-BTZ-CN in dichloromethane Adachi, C. Nature 2012, 492, 234–238.
4. Zhang, J.; Chen, W.; Chen, R.; Liu, X.
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BTZ-CN, respectively (Table 1). Yasuda Y.; Adv. Func. Mat. 2016, 26,
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4. Conclusion 6. Ni, F.; Wu, Z.; Zhu, Z.; Chen, T.; Wu,
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with two different deactivating groups on
Liquid-phase selective hydrogenation of furfural to furfuryl alcohol on
RuCo/TiO2 catalysts
Natdanai Nanthasanti*, Piyasan Praserthdam, Joongjai Panpranot
Faculty of Engineering, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
*E-mail: natdanaibest13@hotmail.com
Abstract:
Furfuryl alcohol (FA), an important intermediate for the production of many fine
chemicals, is typically produced by the selective hydrogenation of lignocellulosic biomass-
derived furfural using supported metal catalysts. In this study, the addition of a small amount
of Co in the range of 0.2-0.8wt% was found to improve the catalytic activity and FA
selectivity of 1.5wt.% Ru/TiO2 catalysts in the liquid-phase selective hydrogenation of
furfural to FA at 50 °C and 2.0 MPa H2. The unpromoted Ru/TiO2 showed furfural
conversion and FA selectivity 32% and 90%, respectively after 2 h reaction time. Under the
similar conditions, the 0.6Co1.5Ru/TiO2 exhibited the best catalytic performances with
91.7% conversion of furfural and 97.5% FA selectivity. It is suggested that Co addition
favored the C=O hydrogenation. The catalysts were also characterized by X-ray diffraction,
N2-physisorption, H2-temperature program reduction, and CO chemisorption.
1. Introduction Although the catalyst is active for furfural

Currently, the global trend of using hydrogenation but it has some adverse
renewable energy is increasing progressively effects such as the poisonous catalyst that
due to global warming, the limited fossil can cause pollution and ultimately affects
fuel resources, and other pollution problems. the environment.4,6
Biomass is considered as an important The use of noble metals such as Pt,
feedstock for the production of fuels and Pd, and Ru supported on titania has received
chemicals.1 Furfural is a renewable chemical considerable interest in recent catalyst
that can be produced from xylose conversion development in liquid-phase selective
of lignocellulosic biomass. It can also be hydrogenation reactions.7,8 Titania structure
found in the decomposition of agricultural was found to significantly affect the physio-
waste such as wood, grass, and corncobs.2,3 chemical properties of catalysts. For
Furfuryl alcohol (FA) is a substance widely example, platinum on anatase titania showed
used in chemical industry for the production higher activity than rutile phase supported
of fine chemicals, polymers, and other ones in the p-nitrophenol hydrogenation.9 Pt
intermediates such as tetrahydrofurfuryl nanoparticles supported on SiO2 exhibited
alcohol, 2,3-dihydropyran, resin, fibers, high conversion and selectivity to FA under
lysine, vitamin C, and lubricants.1,4 A mild conditions with methanol polar solvent
variety of products for instance FA, furan, 2- while the use of non-polar solvents resulted
methylfuran, 2-methyl tetrahydrofuran, and in poor furfural conversion.10
tetrahydrofurfuryl alcohol can be obtained in Ru/C and Pd/C catalysts have been
furfural hydrogenation but the mostly studied in the aqueous-phase hydrogenation
aspiring product is furfuryl alcohol.5 of furfural at a temperature of 50 °C and
Industrially, selective hydrogenation hydrogen pressure of 0.5 MPa. The Ru
of furfural is carried out in liquid or vapor catalyst showed low activity whereas Pd
phase using copper chromite catalysts at catalyst had high conversion and FA
high temperature and high pressure. selectivity.6 However, for the bimetallic Pd-
Ru/TiO2, increasing the amount of Ru in the texture characteristics of the catalysts (BET
PdTiO2 raised the selectivity of 2- surface area, pore volume, average pore size
methylfuran and FA but decreased the diameter) were obtained on a BEL-SORP
conversion of furfural.7 The addition of non- automated system. The reducibility and
nobel metal such as tin to Ru catalysts at reduction temperature of Ru and Ru-Co
Sn/Ru ratio of 0.4 facilitated the C=O catalysts were determined by a quartz u-tube
hydrogenation, improving the FA reactor. Firstly, 0.1 g catalyst sample was
selectivity.11 pretreated for 1 h with a N2 flow 25 cm3/min
In this study, bimetallic Ru-Co at 200°C and the samples were cooled down
catalysts supported on anatase phase titania to room temperature. Then, the TPR
were prepared with different molar ratios of experiment was performed using 10%
Ru/Co and tested in the hydrogenation of H2/Ar with a flow 25 cm3/min and a ramp
furfural to FA. rate 10°C/min from 30°C to 800°C. The
XRD patterns were collected on a
2. Materials and Methods SIEMENS XRD D5000 x-ray diffractometer
2.1 Materials with Cu Kα radiation from 10° to 80° 2θ and
Furfural (99%), methanol (99.9%), the crystallite size was determined by the
product, and by-products of reaction were Scherrer’s equation.
purchased from Sigma Aldrich and used as 2.4 Catalytic activity measurements
received. Ruthenium(III) nitrosylnitrate Prior to the reaction tests, the
solution metal precursor was purchased Ru/TiO2, Co/TiO2, and Ru-Co/TiO2
from Alfa Aesar and cobalt naphthenate Catalysts were reduced with H2 flow 25
metal precursor was purchased from Sigma cm3/min for 2 h at 300 °C. The liquid phase
Aldrich. TiO2 anatase phase was purchased hydrogenation reaction of furfural was
from Alfa Aesar. Pure hydrogen (99.9%) carried out using a 100 cm3 stainless steel
were obtained from Linde Gas. autoclave reactor (JASCO, Tokyo, Japan).
2.2 Catalyst preparation After reduction 5 mg of catalyst was loaded
The Ru/TiO2 and Ru/TiO2 with 1.5 into reactor with methanol solvent 10 cm3
wt% content of metal was prepared by and furfural 50 uL. The reactor was purged
incipient wetness impregnation method of with H2 for three times and heated in a water
the TiO2 anatase phase support with solution bath. The furfural hydrogenation reaction
of ruthenium (III) nitrosylnitrate and cobalt was heated to 50 °C and 20 bar H2 pressure
naphthenate. All the bimetallic catalysts under stirring speed to 1000 rpm for 2 h.
supported on TiO2 anatase phase were After completion of the reaction, the
obtained by incipient wetness impregnation reaction was cooled to under 25 °C with
method with 1.5wt% content of cooling water and the mixing product was
ruthenium(III) nitrosylnitrate solution metal centrifuged. The sample product
preursor and 0.2, 0.4, 0.6, 0.8wt% content of identification was analyzed by GC with FID
cobalt naphthenate. After impregnation, the detector.
catalysts were dried in air at room
temperature for 6 h and dried in oven at 110 3. Results and Discussion
°C for 12h then calcined in air at 550 °C for 3.1 Characterization of Ru/TiO2, Co/TiO2
4 h at a heating rate of 10 °C min−1. and Ru-Co/TiO2 Catalysts
2.3 Catalyst characterization The XRD patterns of the
The metal dispersion of Ru on the monometallic (Ru/TiO2) and the bimetallic
Ru/TiO2 and RuCo/TiO2 catalysts were of ruthenium with different amounts of
determined by CO pulse chemisorption cobalt (Ru-0.2Co/TiO2, Ru-0.4Co/TiO2, Ru-
using Micromeritics ChemiSorb 2750 pulse 0.6Co/TiO2, and Ru-0.8Co/TiO2) are shown
chemisorption system assuming a in Figure 1. All the samples displayed the
stoichiometry factor Co/Ru atom = 1. The crystalline diffraction peaks 2θ between 10°
and 80° showing the characteristic peaks of reduction peak at 440°C. The first shoulder
titania in both anatase and rutile phases. The and the main reduction peak were attributed
anatase phases were exposed at 25.3° to the two-step reduction of cobalt oxide:
(major), 37.9°, 48.1°, 54.1°, 55.2°, and 75.5° Co3O4 → CoO → Co0. The last shoulder
and the rutile phases occurred at 27.7° peak could be related to the interaction
(major), 37.1°, 38.7°, 62.9°, 69.2°, and between Co and Ti.19, 20 The H2-TPR profile
70.4°12. The XRD peaks corresponding to of Ru/TiO2 showed the reduction peak of
Ru and Co species were not detected for all Ru(III) to Ru(0) at 203 °C while the
the samples due probably to the low bimetallic Ru-Co/TiO2 catalysts exhibited
amounts of metal loading.7 Table 1 shows the shift of the reduction peaks of Ru(III) to
the N2 physisorption results in terms of BET Ru(0) towards higher temperature,
surface area, pore volume, and average pore suggesting an interaction between Ru and
diameter. The surface areas of the Co. The degree of Ru-Co interaction was
monometallic Ru/TiO2 catalyst was 50 m2/g maximized at 0.2wt% Co content as the
whereas those of the bimetallic Ru-Co/TiO2 highest reduction temperature shift was
were ranging between 41-48 m2/g. The observed at 233 oC. When Co content was
addition of cobalt second metal resulted in increased to 0.4, 0.6, and 0.8wt% the H2-
further decrease of surface area of the TPR profile showed the shift back to lower
monometallic Ru/TiO2. However, the pore temperature which may be due to the
volume and average pore diameter remained increasing size of the metal. There were no
unchanged. significant differences for the broad peaks at
406-408 °C among the catalysts studied
except for the Ru-0.8Co/TiO2 in which the
peak was shifted to lower temperature at 393
o
C. It is suggested that addition higher
amount of Co at 0.8wt% led to weaker
interaction between Ru and TiO2. Moreover,
the adding of Co content resulted in higher
peak area of the Ru reduction peaks due
probably to the incorporation of Co species
in the Ru reduction peaks. The reduction
peaks of cobalt oxides were not detected in
the TPR profiles of Ru-Co/TiO2.
Figure 1. The XRD patterns of the Ru/TiO2
and Ru-Co/TiO2 catalysts
The H2-TPR experiments were

carried out to study the reducibility and the
metal-metal and metal-support interaction of
the Ru/TiO2 and Ru with different amounts
of Co on TiO2 catalysts and the results are
shown in Figure 2. The catalysts presented
two reduction peaks. The sharp peak
between 200 °C and 250 °C was assigned to
the reduction of Ru(III) to Ru(0) and the
metal-metal interaction17 and the broad peak
at 408 °C can be assigned to the reduction of Figure 2. H2-TPR profile of the Ru/TiO2,
Ru interacting with TiO2.18 The H2-TPR Co/TiO2 and Ru-Co/TiO2 catalysts
profile of Co/TiO2 shows two shoulder
peaks at 371 °C and 492 °C and a main
3.2 Liquid phase hydrogenation of lowest CO chemisorption, corresponding to
furfural the strongest degrees of Ru-Co as shown by
Furfural hydrogenation to FA was the highest shift of Ru reduction peak in the
carried out as a test reaction to evaluate the H2-TPR profiles. The amount of CO
performances of Ru/TiO2 and Ru-Co/TiO2 chemisorption then gradually increased from
catalysts. Table 2 shows the catalytic results 5.0 to 9.0 when Co content was increased
of 1.5 wt% Ru catalyst with different Co from 0.2 to 0.6wt%. The highest furfural
contents from 0.2 to 0.8wt% in the liquid- conversion was also observed on the Ru-
phase selective hydrogenation of furfural at 0.6Co/TiO2. Thus, there should be an
50 °C, 20 bar of H2 after 2 h reaction time optimum interaction between Ru and Co to
and the CO chemisorption results. The maximize the furfural conversion. Further
addition of Co to Ru/TiO2 catalyst increased increase of Co content from 0.6 to 0.8 wt%
the conversion of furfural from 31.8% to resulted in a drop of CO chemisorption as
56.9-91.7% with the Ru-0.6Co/TiO2 showed well as furfural conversion and the shift of
the highest furfural conversion. The FA Ru reduction peak in the H2-TPR to lower
selectivity also increased from 90% of the temperature, compared to the other
monometallic Ru/TiO2 to 96-98% for the RuCo/TiO2. Nevertheless, the selectivity of
bimetallic Ru-Co/TiO2. Despite the lowest FA was improved for all the bimetallic
catalytic activity, the Ru/TiO2 exhibited the catalysts, compared to the monometallic
highest CO uptake because the size of metal Ru/TiO2. It is suggested a strong interaction
on support was small but the adding of Co between Ru-Co facilitated the C=O
contents were increased the size of metal hydrogenation in the selective
while decreased CO uptake.11 The Co/TiO2 hydrogenation of furfural to FA. In the
showed negligible conversion in furfural present work, the highest yield of FA at
hydrogenation and the lowest amount of Co ∼88% was obtained over the Ru-0.6Co/TiO2
uptake. Among the bimetallic RuCO/TiO2 after 2 h reaction time under the reaction
catalysts, the Ru-0.2Co/TiO2 exhibited the conditions used.
Table 1. N2 physisorption results of the monometallic Ru, and bimetallic Ru-Co catalysts
BET surface area Pore volume Avg. Pore diameter
Entries Catalysts
(m2/g) (cm3/g) (nm)
1 Ru/TiO2 50 0.29 25
2 Ru-0.2Co/TiO2 42 0.30 32
3 Ru-0.4Co/TiO2 42 0.29 28
4 Ru-0.6Co/TiO2 48 0.30 25
5 Ru-0.8Co/TiO2 41 0.30 30
Table 2. Effect of addition of Co into the monometallic Ru catalysts and Co chemisorption

Conversion FA Yield CO chemisorption
Entries Catalysts Selectivity (%)
(%) (%) (CO uptake × 1017)
FAa Othersb
1 Ru/TiO2 31.8 90.0 10.0 28.6 13.3
2 Ru-0.2Co/TiO2 56.9 95.7 4.3 54.5 5.0
3 Ru-0.4Co/TiO2 81.2 95.7 4.3 77.7 7.2
4 Ru-0.6Co/TiO2 91.7 97.5 2.5 87.8 9.0
5 Ru-0.8Co/TiO2 86.4 97.1 2.9 83.9 7.8
6 Co/TiO2 0 - - - 4.1
Reaction furfural 50 uL in 10ml methanol at 50 °C with a 0.05 g catalyst under 20 bar H2 after 120 min
a
FA = furfuryl alcohol.
b
Others = solvent products (2-furaldehyde dimethyl acetal).
4. Conclusion
The effect of Co addition on the 7. Aldosari, O.; Iqbal, S.; Miedziak, P.;
Ru/TiO2 catalysts prepared by impregnation Brett, G.; Jones, D.; Liu, X.; Edwards,
method was investigated in the liquid phase J.; Morgan, D.; Knight, D.; Hutchings,
hydrogenation of furfural to FA at 20 bar of G. Catal. Sci. Technol. 2016, 6,
H2 and 50 oC. A small amount of Co (0.2- 234−242.
0.8 wt%) resulted in an interaction between 8. Ordomsky, V. V.; Schouten, J. C.;
Ru and Co as revealed by H2-TPR and CO Schaaf, J.; Nijhuis, T. A. Appl. Catal. A:
chemisorption results. The conversion and Gen. 2013, 451, 6−13.
selectivity of FA for all the bimetallic were 9. Rizhi, C.; Yan, D.; Weihong, X.;
higher that the monometallic Ru/TiO2 while Nanping, X. J. Chem. Eng. 2006, 14,
the Co/TiO2 did not exhibit activity in 665−669.
furfural hydrogenaton. The 1.5%Ru- 10. Taylor, M.; Durndell, L.; Lsaacs, M.;
0.6%Co/TiO2 catalyst was the optimum Parlett, C.; Wilson, K.; Lee, A.;
composition that gave the highest FA yield Kyriakou, G. Appl. Catal. B: Environ.
(88%) under the conditions used. 2016, 180, 580−585.
11. Musci, J.; Merlo, A.; Casella, M. Catal.
Acknowledgements Today. 2017, 296, 43−50.
Financial Supports from the Thailand 12. Elsalamoy, R.; Mahmoud, S. J. Chem.
Research Fund (TRF) and the 2017, 10, 194−205.
Ratchadaphiseksomphot Endowment Fund 13. Cui, H.; Park, J.; Park, J. Appl. Surface.
for International Research Integration: Chula Sci. 2013, 282, 844−850.
Research Scholar are gratefully 14. Elmasides, C.; Kondarides, D. I.;
acknowledged. Grunert, W.; Verykios, X. E. J. Phys.
Chem. B. 1999, 103, 5227−5239.
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Synthesis of boron carbide powder from cellulose and boric acid using
freezing-thawing technique
Wiphawi Jansuda, Sujarinee Sinchai*
Department of Materials science, Faculty of science, Chulalongkorn University, Pathumwan,
*E-mail: sujarinee.k@chula.ac.th
Abstract:
Boron carbide is well known for its high hardness and low density. However,
synthesis of high purity boron carbide powder is complicated. Studies on boron carbide
powder synthesis have been widely reported in order to reduce process temperature and
obtain fine microstructure. In this study, low temperature synthesis of boron carbide powder
from the highly homogeneous precursor improved by freezing-thawing technique was
attempted. Precursors prepared from boric acid (BA) and cellulose microcrystalline powder
(CL) were heated under inert gas atmosphere to promote the reaction to form boron carbide.
BA:CL mole ratios were varied as the 4:1 to 5.6:1. Phase, morphology and chemical structure
of the synthesized powder were identified by X-ray diffraction (XRD), scanning electron
microscope (SEM) and Fourier-transform infrared spectroscopy (FTIR), respectively. Mole
ratio between BA and CL had an effect on the purity of synthesized powder where the
composition with boron excess was preferred.
1. Introduction reducing agent. The powder synthesized by

Boron carbide (B4C) has attracted this technique has desirable characteristics
increasing attention from many researchers for the further forming and sintering
due to its extremely high hardness with low processes.
density (∼2.52 g/cm3), high melting point Carbothermic reduction of boric acid
(∼2540 °C), good chemical inertness, high using petroleum coke or carbon black as a
capture cross-section for neutrons and high carbon source is typically employed to
elastic modulus (∼445 Gpa).1-4 This produce B4C powder. The reaction proceeds
excellent combination of properties make in 3 steps which can be presented as the
B4C a leading candidate material for following:
numerous advanced applications including 4H3BO3 → 2B2O3 + 6H2O (1)
abrasive powder, neutron moderator in B2O3 + 3CO → 2B + 3CO2 (2)
nuclear reactor, wear resistant equipment 4B + C → B4C (3)
and lightweight armor.5 The overall reaction:
Various synthetic techniques to H3BO3 + 7C→ B4C + 6CO + 6H2O (4)
produce variety forms of B4C powder have However, this process is strongly
been reported. The most widely used endothermic and carried out at a high
technique for synthesizing B4C powder is temperature of approximately 2000°C
carbothermic reduction because the process resulting in the agglomeration of the
is inexpensive and feasible to upscale to synthesized powder. Furthermore, free-
commercial production. Carbothermic carbon as a second phase caused by volatile
reduction involves the reduction reaction to boron oxide species during the reduction
form B4C powder using carbon as a process also persists after synthesis,
indicating that the mole ratio between freezing-thawing cycles were investigated.
carbon and boron in the starting materials is FTIR, XRD and SEM analyses were utilized
an important process parameter. to characterize the precursor bonding, phase
During the recent years, low present and morphology of the synthesized
temperature synthesis of B4C powder by powder, respectively.
using organic precursors has been reported.
Isao et al. 6 successfully synthesized B4C 2. Materials and Methods
powder at 1500°C by condensation reaction 2.1 Materials
of boric acid with 2-nydroxy benzyl alcohol Boric acid (BA; Fisher Chemical,
(HBA). Hiroaki et al.7 used boric acid and 99.5%) and cellulose (CL; Sigma-Aldrich)
1,2,3 propanetriol as precursors and were used as boron and carbon sources,
obtained B4C powder at 1400 °C. Ikuo et al.8 respectively. These materials were used as
received.
also produced B4C powder at 1300 °C from
2.2 Preparation of precursor
boric acid and polyvinyl alcohol.
10wt% aqueous solution of BA was
A promising alternative to produce
B4C powder by using polymer precursor prepared at 80 °C and then CL was slowly
route provides the chemical homogeneity in added into BA solution. The BA:CL mole
molecular scale and facilitates the diffusion ratio of all recipes is shown in table 1. The
distance for reaction so that synthesis at low mixture was frozen in the refrigerator at
temperature can be achieved. Saccharides, -60 °C for 12 h. After freezing step, the
mixture was kept at room temperature so
e.g. glucose, sucrose, cellulose, have been
that it turned back into liquid (12 h).
employed to produce B4C powder. 9-12 After freezing-thawing process has been
Muhammad et al.13 synthesized crystalline conducted for 1 or 3 cycles, the mixture was
B4C powder using glucose, starch and dehydrated in an oven at 80 °C for
cellulose as sources of carbon. In cellulose 24 h.
precursor, crystalline B4C powder with high
surface area and low residual carbon could Table 1. Recipe name, mole ratio of BA:CL
be synthesized at 1200 °C. They concluded and number of freezing-thawing cycle.
that the dispersibility as well as the Recipe BA:CL Freezing-thawing
homogeneity of B2O3 and carbon fibers in mole cycle
cellulose precursor rendered the low ratio
F4-1 4:1 1
temperature synthesis of crystalline B4C F4-3 4:1 3
powder. F5.6-1 5.6:1 1
Homogeneity of dispersion of boron F5.6-3 5.6:1 3
and carbon sources was expected to be
enhanced by using freezing-thawing 2.3 Synthesis of B4C powder
technique. The swollen structure of cellulose The obtained precursor was
after freezing process is supposed to synthesized under heat treatment at 1300 °C
increase degree of dispersion of boron for 3 h in Argon flow with a flow rate of 200
species in such structure during the thawing ml/min, in tube furnace. The synthesized
step. powder was stored in desiccator, ready for
In this work, low temperature characterization step. Process diagram for
synthesis of boron carbide powder from the this study is shown in Figure 1.
highly homogeneous precursor improved by 2.4 Characterizations
freezing-thawing technique was attempted. The Fourier transformation infrared
The effects of synthesis parameters (FTIR; Nicolet, Model Impact 400) spectra
including boric acid:cellulose mole ratio and
of the precursor were measured. Phase revealed the appearance of bands at 3000-
present of the synthesized powder was 3500, 2900 and 1000-1500 cm-1 those were
determined using X-ray diffraction (XRD; attributed to the O-H, C-H and C-O
Bruker, D8 Advance). The morphology of stretching vibration, respectively. Presence
B4C powder was observed by scanning of –OH and –CO functional groups in CL
electron microscope (SEM; JOEL, JSM- facilitated the homogeneous condensation
6480LV). with BA to produce appropriate precursor
for B4C synthesis. Compared to the starting
materials, FT-IR spectra of the precursors
showed some different features. The bands at
1430 and 1650 cm-1 corresponding to
symmetric and anti-symmetrical stretching
of COO-14 were observed in the precursor
spectra. The bands at 1000-1200 cm-1 were
assigned to B-O-C stretching vibration15
indicating condensation of BA and CL. The
higher intensity of O-H vibration and smaller
B-OH bending vibration peaks those found
in precursors spectra further verified the
condensation reaction. In addition, the O-H
vibration bands in 3-cycle-freezing-thawing
precursors at 3000-3500 cm-1 were found to
be smaller than those of F4-1 and F5.6-1,
implying the smaller absorption caused by
the freezing-thawing process.
Figure 1. Process diagram of B4C powder

synthesis

3.1 FT-IR spectra of precursors
The FT-IR spectra of the starting
materials (BA and CL) were studied in order
to be compared with the precursors those Figure 2. FT-IR spectra of BA, CL and
having different BA:CL mole ratios and precursors
freezing-thawing cycles, as shown in Figure
3.2 Phase analysis of synthesized powder
2. For BA spectrum, the broad bands
Phase formation of the synthesized
between 3000-3500 and 1350-1550 cm-1
powder was analyzed by XRD as shown in
corresponded to the O-H and B-O stretching Figure 3. It was found that the peaks
vibration, respectively, while the sharper corresponding to rhombohedral B4C crystal
peak appeared at 1200 cm-1 was assigned to (JCPDS # 35-0798) were observed in the
the bending vibration of B-OH. CL spectrum XRD pattern of F5.6-1 and F5.6-3, whereas
F5.6-3 gained the higher peak intensity. The mixture after freezing process. Two layers
fact that powder synthesized from the of gel and the frozen solution were
precursor having BA:CL mole ratio of 5.6:1 observed.
facilitated the formation of B4C phase
implied that boron-rich precursor was
preferred in this system. The unreacted
carbon peaks were observed in all 4
powders. The peaks corresponding to B2O3
which found in F4-1 and F5.6-1 disappeared
in the powders those experienced 3 cycles of
freezing-thawing process, indicating the
improved homogeneity of boron species
dispersed in CL structure during the thawing
Figure 4. FTIR spectra of (i) F5.6-3
step.
precursor, (ii) F5.6-3 and (iii) F5.6-1
FT-IR spectra of the F5.6-1 and F5.6-
synthesized powders.
3 synthesized powders shown in Figure 4
were studied to confirm the XRD results.
The band noticed ~800 cm-1 in both
synthesized powders was assigned to the B-
B-C bond16. In addition, the presence of
bands at 1650 and 2900 cm-1 in the
precursors which corresponding to
antisymmetrical stretching of C=O and
stretching of C-H vibration, respectively,
disappeared in the synthesized powders.
Figure 5. BA-CL mixture (F4-3) after

freezing at -60 °C for 12 h
A series of SEM images of CL,

precursors and synthesized powders are
shown in Figure 6 and 7. Compared to CL,
smoother surfaces were observed in the BA-
CL precursors, where the surfaces of F5.6-1
and F5.6-3 were smoother than those of F4-
Figure 3. XRD patterns of synthesized 1 and F4-3. Such surface erosion caused by
powders the acid treatment, affirming the reaction
between BA and CL. Figure 7 (ii) shows the
3.3 Morphology microstructure of synthesized powder. In
The B4C powder was synthesized by F4-1, some B2O3 particles were found on the
fiber surface of CL relict while there was no
heating BA-CL precursor at 1300 °C for 3 h
evidence of B4C. The B2O3 particles those
in Ar flow. Freezing-thawing technique was
observed in F4-1 and F5.6-1 were not
applied during the precursor preparing step
detected in 3-cycle-freezing-thawing
to enhance the reaction between BA and CL,
powders, supporting the XRD results and
thus improving the dispersion homogeneity.
confirming an improvement in dispersion
Figure 5 shows the feature of BA-CL
degree by freezing-thawing process. B4C
particles with the size of 3-7 micron were precursors indicated the condensation
found in F4-3, F5.6-1 and F5.6-3. In reaction between BA and CL. The
addition, no fibrous structure of previous CL synthesized powder from boron rich
left in F5.6-3. precursor that experienced 3 cycles of
freezing-thawing process; F5.6-3, gained the
highest B4C peak intensity from XRD
analysis. In addition, B2O3 and fibrous
structure of CL relict were not observed in
the microstructure of F5.6-3 synthesized
powder. These results verified the
enhancement of B4C crystallization by
freezing-thawing process.
Acknowledgements
Figure 6. SEM image showing CL rough
The author would like to gratefully
surface
acknowledge Department of Materials
Science, Faculty of Science, Chulalongkorn
University for research support as well as
Assoc.Prof.Dr. Vimolwan Pimpan and
Asst.Prof Dr. Wanpen Tachaboonyakiat for
the helpful discussion.
References
1. Mondal, S.; Banthia, A. K. J. Eur. Ceram.
Soc. 2005, 25, 287-291.
2. Hadian, A. M.; Bigdeloo, J. A. J. Mater.
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5. Jianxin, D. Mater. Sci. Eng. 2005, 408,
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6. Hasegawa, I.; Fuji, Y.; Takayama, T.;
Figure 7. SEM images of (i) BA-CL Yamada, K. J. Mater. Sci. Lett. 1999, 18,
precursors and (ii) synthesized powders 1629-1631.
of (a) F4-1, (b) F4-3, (c) F5.6-1 and 7. Wada, H.; Ito, Sh.; Kuroda, K.; Kato Ch.
(d) F5.6-3
Chem. Lett. 1985, 14, 691-692.
4. Conclusion 8. Yanase, I.; Ogawara, R.; Kobayashi, H.
In this study, synthesis of B4C Mater. Lett. 2009, 63, 91-93.
powder was attempted by heat treatment of 9. Pilladi, T. R.; Ananthanivan, K.;
BA-CL precursors at 1300 °C for 3 h in Ar Antonysamy, S. Powder Technol. 2013,
flow. Freezing-thawing technique was also 246, 247-251.
applied on the precursors in order to
facilitate the dispersion of boron species in
CL structure. FT-IR analysis of the
10. Pilladi, T. R.; Ananthanivan, K.; 14. Tomasik, P.; CRC Press 2004b 93-128.
Antonysamy, S. J. Mater. Sci. 2012, 47, 15. Rafi-ud-din; Zahid, G. H.; Ahmad, E.;
1710-1718. Hussain, S. Z.; Maqbool, M.; Subhani,
11. Sudoh, A.; Konno, H.; Habazaki, H.; T.; Syed, W. A. J. Inorg. Organomet.
Kiyono, H. Tanso. 2007, 226, 8-12. Polym. 2015, 25, 995–999.
12. Konno, H.; Sudoh, A.; Aoki Y.; 16. Ahmed, Y.; El-Sheikh, S. M.; Ewais, E.
Habazaki, H. Mol. Cryst. Liq. Cryst., M. M.; Abd-Allah, A. A.; Sayed, S. A. J.
2002, 386, 15-20. Mater. Eng. Perform. 2017, 26, 1444-
13. Maqbool, M.; Din, R. U.; Zahid, G. H.; 1454.
Ahmad, E.; Asghar, Z.; Subhani, T.;
Shahzad, M.; Kaleem, I. Mater. Express
2015, 5, 390-400.
Styrene/stearyl methacrylate foams as oil absorbent
Voratida Chuensukum1,2, Orathai Boondamnoen1,2, Pakorn Opaprakasit3, Atitsa Petchsuk4,
Mantana Opaprakasit1,2*
1
Department of Materials Science, Faculty of Science, Chulalongkorn University, Phayathai,
2
Center of Excellence on Petrochemical and Materials Technology, Chulalongkorn University,
3
School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology (SIIT),
4
National Metal and Materials Technology Center (MTEC), National Science and Technology Development
Agency (NSTDA), Pathum Thani 12120, Thailand
*E-mail: mantana.o@chula.ac.th
Abstract:
Styrene/stearyl methacrylate foams have been developed for use as oil absorbents
using a high internal phase emulsion (HIPE) method. Effects of monomer compositions and
contents of surfactant and cross-linking agent on the foam’s microstructures and oil
absorption efficiency are investigated. A good foam structure derived from styrene (S) and
stearyl methacrylates (SMA) monomers were successfully prepared with 0.75 and 1 g of
surfactant per 50 mL of aqueous phase. However, S/SMA foams with inhomogeneous foam
structure, investigated by scanning electron microscope, were obtained when the S/SMA
weight ratios of lower than 1 was employed. The oil absorption capacity of the S/SMA foams
tends to increase with the S/SMA ratios. The results from 1:1 S/SMA foams prepared with
20-50% w/w of divinylbenzene (DVB) per monomers contents revealed that their oil
absorption efficiency is possibly improved by lowering the contents of the cross-linking
agent. The absorption capacity of these oil absorbents is ranging from 6 to 19 g/g. Although
high oil absorbency can be achieved with S/SMA foams, their oil recovery capacity by a
pressing method is challenging, as the sample cracks upon pressing. Further improvement of
this property is on-going.
1. Introduction morphology and physical properties are

Oil absorbent polymers have gain controlled by oil/water ratio and the contents
vast attention because they are possibly of surfactant and crosslinking agent.7, 9
applied as clean-up materials for spilled oils However, the study on preparation of HIPE
and industrial or household waste oils. Their foams using alkyl acrylates is much
sorption capacity can be controlled by scarce.11-13
chemical structures and morphology, in This research is aimed to develop oil
which the selected polymers should have absorbent foams from alkyl acrylate and
good mutual solubility with the intended styrene (S) monomer. Among many alkyl
oils. Therefore, alkyl acrylates and styrene acrylates, stearyl methacrylate (SMA) is of
have been widely used as oil absorbent interest, as its long alkyl chain provides
materials.1-5 In addition, crosslinked benefit for oil absorption applications.5, 6, 14
structure is typically designed for improving A HIPE method is adopted for preparing the
their absorbency.5,6 Moreover, porous S/SMA foams. Effects of the S/SMA ratios
structures and void spaces are required. and the contents of the surfactant (span80)
Styrene foams with high porosity have been on morphology and oil absorption capacity
widely developed using a high internal of the S/SMA foams are investigated.
phase emulsion polymerization (HIPE) Moreover, S/SMA foams with various
approach.7-11 It was reported that the foam contents of crosslinking agent (divinyl
benzene; DVB) are developed in an attempt received after drying at 80 °C, and a
to enhance their oil absorbency and oil constant weight were reached.
recovery ability. 2.3 Oil absorptivity evaluation
Oil absorption capacity of the HIPE
2.Materials and Methods foams was evaluated according to ASTM
2.1 Materials F726-06 by using gasoline and palm oils.
SMA (purity > 95%, Tokyo Dried cubic specimens (∼0.1 g) were soaked
Chemical Industry Co. Ltd) and S (purity ≥ in the oil (10 mL). After 24 hours, the
99%, Aldrich Chemical Co.) were used as samples were picked up with tweezers and
monomers for preparing HIPE foams. DVB laid on a filter paper to remove excess oil.
(purity > 80%, technical grade, Aldrich The oil absorption capacity (Q) was
Chemical Co.) was used as a cross-linking calculated, according to the following
agent. All reactants were used as received. equation1:
Nonionic surfactant Span80 was supplied by
Sigma-Aldrich. Potassium persulfate Q = M2/M1 (1)
(K2S2O8,) (ACS reagent, purity ≥ 99%,
Sigma-Aldrich) was used as an initiator. where M2 is the weight of oil absorbed
2.2 Preparation of poly HIPE foams sample, and M1 is the original dry weight
Poly HIPE foams were prepared by 2.4 Foam morphology
varying S/SMA weight ratios and the Thin specimens of the HIPE foams
contents of DVB and Span80, as were characterized by a JSM-6480LV
summarized in Table 1. The total weight of Scanning Electron Microscope (SEM)
monomers and crosslinking agent was kept (JOEL, Japan) at 5kV. An Image-J software
at 3.15 g. An oil-phase solution consisting of was applied to the SEM images taken at
monomers, crosslinking agent and surfactant 2500X and 1000X to measure cell size of
was firstly prepared. Then, an aqueous- the samples. At least 50 points of cells were
phase solution containing 1.5 g K2S2O8 in measured, and the average value was
1000 mL de-ionized water was prepared. reported.
The oil-phase solution was continuously 2.5 Compressive property of HIPE foams
stirred in a 100 mL beaker for 5 minutes, Specimens, with a dimension of 15 x
before 50 mL of the aqueous solution was 15 x 15 mm3, were cut from dry and oil-
added drop-wise over a period of 15 absorbed S/SMA foams. Their
minutes. The mixture was vigorously stirred compressibility was examined by pressing in
for 5 minutes by a mechanical stirrer at 500 a SUMO® press at 10 bar. The compression
rpm. The mixture was, then, poured into a depth of the samples was set at 33% and
silicone mold and placed in an oven 66% of their original height. Physical
overnight at 60 °C. HIPE foams were appearance of the specimens after pressing
for 10 seconds was reported.
Table 1. Preparation conditions of poly HIPE foams, in terms of S/SMA monomers ratios,
DVB contents, and surfactant compositions in the oil-phase solution
DVB content Concentration of span80 for preparing S/SMA foams
(% wt) 100:0 80:20 50:50 30:70 0:100
50 1 and 0.75 g/50ml
30 - 0.75 g/50ml -
20 - - 0.75 g/50ml - -
3. Results and Discussion seen in Figure 3 that a reduction of the
3.1 Morphology and oil absorbencies of surfactant content dramatically affects the
HIPE foams cell sizes of the 100:0 S/SMA foams, in
Physical appearance and SEM which an increase from ∼8 to 18 µm is
images of the prepared S/SMA foams are observed. However, less effect is observed
strongly dependent on the S/SMA feed in other S/SMA foams, whose cell size
ratios and the span80 contents. At low difference is only 1-2 µm.
contents of span80, good foam structure is
obtained when the contents of SMA are
varied from 0 to 100%, as shown in Figure
1(a) - (c). However, the good foam structure
cannot be obtained, when 100% SMA and
the content of span80 of 1g/50 mL are used, Figure 2. SEM micrographs of foams
as shown in Figure 1(d). This implies that prepared with 50%DVB and 1 g/50 mL of
emulsion stability is interfered by the span80, using S/SMA ratios of: (a) 100:0,
presence of SMA and the surfactant. As the (b) 50:50, and (c) 0:100
polarity of SMA is higher than styrene
monomer, diffusion rate of monomer
molecules in the oil phase into the aqueous
phase is increased with an increase in the
SMA content. Therefore, the network
structure generated by this water-in-oil
emulsion can collapse easier. Moreover, an
increase in the surface area of aqueous
phase, induced by high surfactant content,
leads to higher degree of instability.
Figure 3. Average cell sizes of S/SMA

foams characterized from SEM micrographs
Figure 1. Physical appearance of HIPE
foams prepared at 0.75 g/50 mL of span80 Figure 4 illustrates density of the
and S/SMA ratios of: (a) 100:0, (b) 50:50 S/SMA foams, calculated from bulk weight
and (c) 0:100 and (d) 0:100 S/SMA foam and volume measurements. The density is
prepared with span80 of 1 g/50 mL ranging from ∼0.04 – 0.08 g/cm3, or ∼2.5 –
5 lb/ft3. Given that commercial polyurethane
SEM images, as shown in Figure 2, foams available in the market for soft to
reveal that the received S/SMA foams are luxury foams have density in the range of
composed of a large number of closed-cells 0.8 to 3.5 lb/ft3, the resulting S/SMA foams
interconnecting through small windows prepared in this study can be classified as
located all over the cell wall. Figure 3 high density foams. In addition, the density
summarizes average cell sizes of the foam of the foams tends to decrease with
structures. The results indicate that sizes of decreasing contents of SMA, span80 and
the cells and windows tend to increase with DVB. The oil absorbency of the S/SMA
increasing of the S/SMA ratio, and a foams was tested with gasoline and palm
decrease of the span80 contents. Cells of oils. Results from Figure 4-5 reflect that the
S/SMA foams prepared with 1 and 0.75g/50 foam absorption capacity (Q) and the
ml have average size ranging from 4-8 µm density are affected by the contents of SMA
and 5-18 µm, respectively. It is obviously monomer span80 and DVB. Higher contents
of these substances produce denser foams, chemicals are required. Therefore, feasibility
with lower Q values. A linear relationship of applying this oil recovery approach to the
between the foam density and the Q values S/SMA foams is evaluated. The tests were
implies that the number of voids containing conducted by placing absorbed foam sample
in the S/SMA foams plays a major role in with original height of 15 mm between 2
controlling their oil absorption properties. compressive plates. The samples were
Figure 5 shows that the S/SMA foams can compressed by travelling the upper plate to
be used as oil-absorbent materials for both 5 and 10 mm below the top surface of the
ester oils and hydrocarbon oils. Effects of specimens. Figures 6(a)-(b) show that S-
the foam morphology on the oil absorbency foams and SMA-foams are completely
of S/SMA foams are similar for gasoline and broken after holding at both selected
palm oils. positions, with a pressure of 10 bar for 10
sec. In contrast, physical appearances of the
foams, as shown in Figures 6(c) and Figure
7, reveal that the 50:50 S/SMA foams can
withstand the compression force better than
pure S-foams and pure SMA- foams. While
the foam, produced at 50:50 S/SMA with a
50%wt DVB content, can hold their shape
only at 33% compressive depth (Figure 6(c)
and 7(a)), the 50:50 S/SMA foams, with
lower cross-linking agent, can retain their
shape up to 66% compressive depth.
Figure 4. Palm oil absorption efficiency
(bar chart) and density () of S/SMA foams
as a function of the S/SMA ratio and the
contents of span80 and DVB; (■) 1 g/50mL
of span80 with 50%DVB and (■)( )( ) Figure 6. Physical appearance, after
0.75 g/50mL of span80 with 50%wt, 30%wt compressive testing at 5 mm (33%), of
and 20%wt DVB, respectively S/SMA foams: (a) 100:0, (b) 0:100, and (c)
50:50 S/SMA foams prepared from
0.75g/50mL of span80 with 50%wt DVB
Figure 7. Physical appearance, after

compressive testing at 10 mm (66%), of
50:50 S/SMA foams prepared with DVB at:
(a) 50%wt, (b) 30%wt, and (c) 20%wt
Figure 5. Oil absorption efficiency (Q) of
S/SMA foams prepared with 1 g/50mL of 4. Conclusion
span80 and 50%wt DVB The S/SMA foams prepared in this
study have a density ranging from ∼0.04 –
3.2 Compressive property of HIPE foams 0.08 g/cm3 and exhibit oil absorption
Mechanical pressing is effective capacity (Q) for gasoline and palm oils in
method for recovering oils from the the range of 6 - 19 g/g. These properties are
absorbent foams. This is because no harmful strongly dependent on the S/SMA ratio and
the contents of the surfactant (span80) and
crosslinking agent (DVB). The foams 3. Chen, J.; Fang, P.; Du, Y.; Hou, X.
prepared with high S/SMA ratios and low Colloid. Polym. Sci. 2016, 294, 119–
amounts of span80 tend to have larger cell 125.
size and lower density. Therefore, they 4. Yuan, X.; Chung, M. Energy Fuels
possess much higher Q values. Although 2012, 26, 4896–4902.
pure styrene foams have good oil 5. Duan, Y.; Bian, F.; Huang, H. Polym.
absorbency, they are fragile to compressive Adv. Technol. 2015, 27, 228–234.
forces. Pressing or squeezing to recover the 6. Jang, J.; Kim B. S. J. Appl. Polym. Sci.
absorbed oil is not possibly applied to S- 2000, 77, 903–913.
foams. In contrast, 50:50 S/SMA foams 7. Williams, J. M.; Wrobleski, D. A.
prepared with 0.75g/50ml of span80 and 20- Langmuir. 1988, 4, 656–662.
30%wt DVB possess high Q and squeezable 8. Cameron, N. R.; Sherrington, D. C. Adv.
potency. Polym. Sci. 1996, 126, 163–214.
9. Williams, J. M.; Gray, A. J.; Wilkerson,
Acknowledgements M.H. Langmuir. 1990, 6, 437–444.
The authors thank the Center of 10. Menner, A.; Bismarck, A. Macromol.
Excellence on Petrochemical and Materials Symp. 2006, 242, 19–24.
Technology (PETROMAT) and Chula 11. Lin, Y.; Li, T.; Liu, Y. J. Macromol.
Unisearch (Grant no: CU-58-041-AM) for Sci., Part B: Physics. 2015, 54, 102–111.
financial supports. 12. Tai, H.; Sergienko, A.; Silverstein, M. S.
Polym. Eng. Sci. 2001, 41, 1540–1552.
References 13. Sergienko, Y. A.; Tai, H.; Narkis, M.;
1. Ji, N. Y.; Chen, H.; Yu, M. M.; Qu, R. Silverstein, M. S. J. Appl. Polym. Sci.
J.; Wang, C. H. Polym. Adv. Technol. 2002, 84, 2018–2027.
2011, 22, 1898–1904. 14. Sun, J.; Xu, Y.; Chen, H.; Tan, Z.; Fan,
2. Jang, J.; Kim, B. J. Appl. Polym. Sci. L. Sep. Sci. Technol. 2014, 49, 2518–
2000, 77, 914–920. 2524.
Preparation of silica supported zirconium-based heterogeneous catalysts by
surface organometallic chemistry strategy for the conversion of CO2 to
cyclic carbonates under mild conditions
Ounjit Sodpiban, Silvano Del Gobbo, Valerio D’Elia*
Department of MaterialsSscience and Engineering, School of Molecular Science and Engineering,
Vidyasirimedhi Institute, Payupnai, Wangchan, Rayong 21210, Thailand
*E-mail: Valerio.delia@vistec.ac.th
Abstract:
Surface organometallic chemistry (SOMC) is a powerful approach to prepare well-
defined catalytically active sites on the support surface. In this work, organo-zirconium
compounds were prepared and grafted on silica by a facile method. Several materials
differing by the podality of the resulting complexes and by the zirconium surface density with
zirconium-loading between 0.21 and 0.52%wt on silica which has specific surface area 200
m2g-1 were produced. These materials were applied as reusable heterogeneous catalysts to the
conversion of CO2 to cyclic organic carbonates displaying high activity and selectivity under
mild conditions. The catalysts could be reused several times and the correlation between
zirconium-leaching and catalytic activity was carefully investigated. This readily available
family of catalysts constitutes an excellent candidate for application using impure CO2 as a
feedstock.
1. Introduction The development of heterogeneous

The increasing CO2 levels in the catalysts able to operate under such
atmosphere and the unavoidably related challenging reaction conditions (low partial
environmental concerns have been in the CO2 pressure, presence of pollutants) would
spotlight of international debates, legislation be required in the industrial applications.
and research in the last few decades. It is Surface Organometallic Chemistry (SOMC)
becoming clearer that a major reduction of is a powerful approach for the preparation of
amount of CO2 released into the atmosphere heterogeneous catalysts.7 It can produce the
will have to come from a decreased immobilization organometallic on the
dependence on fossil fuels in favour of support surface of catalysts and reaction
renewable energies and from higher energy intermediates leading to a better insight into
efficiency.1 In parallel, chemistry-based the mechanism of alkane,8 olefin and imine
strategies must be developed for the metathesis reactions,9 non-oxidative
treatment of CO2 in flue gases.2 The coupling of methane to ethane and
conversion of CO2 to epoxides is a well- hydrogen,10 oxidative dehydrogenation of
known process to produce value-added propane,11 etc. Herein, we prepared
cyclic carbonates that are employed as green ZrCl4(OEt2)2 grafting on surface of
solvents and as chemical intermediates in dehydroxylated silica at 200 °C and 700 °C
industry.3 In the homogeneous phase, the by SOMC approach to generate various
cycloaddition reaction can be carried out structural surface complexes, which
under mild conditions using organometallic determined by FT-IR, elemental analysis
complexes4 or early transition metal halides5 and SS-NMR. These materials can use as
as molecular catalysts. Metal halides of catalysts for the conversion of CO2 to cyclic
zirconium, yttrium and scandium are organic carbonates under mild conditions
excellent catalysts in the cycloaddition of and by using flue gas as a source of CO2.
CO2 from diluted flows to epoxides.6
2. Materials and Methods for 5 hours. The solid was separated by
2.1 General filtration and washed by diethyl ether (5 x
All reactions were carried out under 25 mL). All volatile solvent was removed by
an atmosphere of nitrogen in glovebox or high vacuum pump (10-5 mbar) for 12 hours
using standard Schlenk techniques. All to provide white powder.
materials were purchased from commercial 2.3 Characterization of catalysts
suppliers. Zirconium(IV) chloride (ZrCl4, ≥ FT-IR spectra were recorded under a
99.5%) was purchased from Merck. For nitrogen atmosphere on a Thermoscientific
epoxide, propylene oxide (≥ 99.0%) was Nicolet iS10 FT-IR spectrometer. Each
purchased from Merck, epichlorohydrin (≥ spectrum was collected by 32 scans. The
99.0%) and styrene oxide (97%) were zirconium content was determined on a
purchased from TCI. The epoxides were Varian 720-ES AXIAL ICP-OES. Elemental
generally used as received except propylene analyses were provided by Mikro-
oxide was distilled before use. All solvents analytisches Labor Pascher. Combustion
were dried before use by pure process analyses (CHN) were performed with on a
technology solvent purification system and Thermoscientific Flash 2000 combustion
degassed by freeze-pump-thaw cycles on a analysis machine. 1H solid state NMR
high vacuum (10-5 mbar) line. spectra were collected on a Bruker 400 WB
Dehydroxylated silica was prepared from Plus spectrometer. All solution state NMR
Aerosil-200 (Degussa, specific area 200 spectra were collected on either a Bruker
m2/g), dehydroxylated under dynamic high AVANCE 400 MHz or Bruker AVANCE
vacuum for 18 hours at 200 °C (SiO2-200) or 600 MHz spectrometer and all signals were
700 °C (SiO2-700). Carbon dioxide (CO2, ≥ referenced to residual solvent resonances
99.999%) and Nitrogen (N2, ≥99.999%) (1H and 13C).
from Bangkok Industrial Gas Co., Ltd. were 2.4 Catalysis experiments
used as supplied. Flue gas contained various In each experiment, catalyst 0.3
impurities derived by the combustion of mol%Zr, TBAB 1 mol% (0.43 mmol) and
heavy fuel oil containing 2.6wt.% sulfur for epoxide (43mmol) were mixed in a 100 mL
the thermal treatment of CaCO3, the stainless steel autoclave inside a glovebox.
subsequent grinding process and moisture The reactor was pressurized with pure CO2
(remove by condensation). Flue gas act as a (initial pressure: 10 bar) or flue gas (initial
source of CO2. The concentration of CO2 in pressure: 7 bar; partial CO2 pressure 1.05
the flue gas was measured as 15% CO2 (v/v). bar corresponding to ca. 4.3 mmol CO2).
2.2 Preparation of catalysts The system was heated at 60 °C for 18 h.
Three catalysts were prepared by After this period the autoclave was allowed
reacting of ZrCl4(OEt2)2 precursor (mixing to cool to room temperature and was
of ZrCl4 and 4 equiv. diethyl ether) with the transferred into a glovebox. The liquid phase
silanol groups (≡SiOH) of differently was taken for 1H NMR analysis of the
dehydroxylated silica supports (SiO2-700, reaction products and taken for ICP-OES
[≡SiOH]: 0.30 mmol/g, and SiO2-200, analysis of zirconium-leaching. The solid
[≡SiOH]: 0.78 mmol/g).12 Catalyst 1 was material was isolated by decanting the liquid
produced by grafting of 1.1 equiv. and was washed 5 times with diethyl ether
ZrCl4(OEt2)2 on SiO2-700. While catalyst 2 (5 mL). The catalyst was then dried by high
and 3 were generated by grafting of 1.33 and vacuum pump (10-5 mbar) for 12 hours
0.66 equiv. ZrCl4(OEt2)2 on SiO2-200 before use in subsequent reactions.
respectively. Both ZrCl4 (OEt2)2 precursor
and dehydroxylated silica support were 3. Results and Discussion
mixed with diethyl ether (25 mL) in one side 3.1 Characterization of catalysts
of a double Schlenk to afford a white slurry. Silica was treated at different
The slurry was stirred at room temperature temperatures 200 °C and 700 °C (SiO2-200
and SiO2-700) under high vacuum system (10- of material) for catalyst 1, 4.8wt% (0.52
5
mbar) before grafting by ZrCl4(OEt2)2 mmol Zr/g of material) for catalyst 2 and 2.6
precursor. Two kinds of absorption bands wt% (0.28 mmol Zr/g of material) for
arising from ν(OH) stretching vibrations can catalyst 3 (Table 1). Base on the presence of
be observed from the IR spectra of the SiO2- 0.3 and 0.78 mmol silanol/g of SiO2-700 and
200 and SiO2-700 (Figure 1). The sharp band at SiO2-200 respectively,12 these values allowed
around 3747 cm-1 is ascribed to isolated the calculation of 0.70, 0.67 and 0.36
silanols, whereas the broad band at 3500– Zirconium atom grafted per silanol. The
3750 cm−1 is attributed to silanols Zirconium/silanol ratios of catalyst 2 and 3
interacting between each other.13 The SiO2 are lower than catalyst 1, however, grafted
treated at different temperatures SiO2-200 is more heterogeneous than grafted
subsequently reacts with the ZrCl4(OEt2)2 SiO2-700 and the grafting of precursors
precursor in the H2O-free and O2-free normally leads to a mixture of bipodal and
system, which results in producing the monopodal surface species.14 Elemental
catalyst 1-3. The reaction between SiO2 and analysis allowed for the determination of a
ZrCl4(OEt2)2 leads to a rapid and remarkable molar Cl/Zr ratio is 3 for catalyst 1 which
decrease of the sharp band at 3747 cm−1 of agree with expectation that ZrCl4(OEt2)2
the isolated silanol group (≡SiOH) in the IR precursor will react with only isolated
spectra. On the contrary, the broad band silanol of SiO2-700 to generate a monopodal
attributed to the interacting silanols dropped species [(≡SiO)-ZrCl3Ln]. For both catalyst
slightly. Therefore, the isolated silanol 2 and 3 the molar Cl/Zr ratio is 2.5 that
(≡SiOH) at the surface of SiO2 can be means at the presence surface complexes
considered as the active site for bonding the bearing only two chlorine atoms which can
ZrCl4(OEt2)2 precursor. Moreover, after be expected to a bipodal species[(≡SiO)2-
grafting new peaks of organic molecule are ZrCl2Ln]. The presence of carbon in
present in 2800-3000 cm-1 (νs(C-H)) and complexes was determined by combustion
1390-1480 cm-1 (δ(C-H)) regions which can analysis, which corresponding to molar C/Zr
be indicated to zirconium coordinated with ratio of 6.5 for catalyst 1. This value is
diethyl ether occur on the surface support. slightly lower than expected (C/Zr = 8).
Based on the structure of the precursor the
structure of catalyst 1 can be considered as
the presence of two surface species with
differing in the number of coordinated
diethyl ether, which is one or two. For the
catalyst 2 and 3 the C/Zr molar ratios were
determined to 4.0 and 5.1 respectively,
which predicted structure for this both
catalyst should be a single molecule of
diethyl ether per zirconium center.
Moreover, 1H SS-NMR was determined to
understand deeper information about
hydrocarbon structure of three catalysts. The
1
Figure 1. FT-IR spectra of surface support H NMR spectra of all catalysts show in the
before and after grafting by ZrCl4⸱(OEt2)2 similar pattern that consist of chemical shift
precursor to provide catalyst 1-3 at 4.3, 3.8, 1.4 and 1.1 ppm (Figure 2). From
the spectra of all catalyst occur two peaks of
The zirconium loading was methylene group (4.3 and 3.8 ppm) and two
determined by ICP-OES spectroscopy signals of methyl group (1.4 and 1.1 ppm).
corresponding to 1.9wt% (0.21 mmol Zr/g This can be suggested that all catalysts
Table 1. Elemental analysis values for catalyst 1-3
Entries Catalyst [Zr] wt%a [Zr]/[ [Cl] wt% [C] wt%
(Silica support) ([Zr] (mmol/g)) ≡SiOH]b ([Cl]/[Zr])c ([C]/[Zr])c
1 1 (SiO2-700) 1.9 (0.21) 0.70 2.3 (3.1) 1.63 (6.5)
2 2 (SiO2-200) 4.8 (0.52) 0.67 4.6 (2.5) 2.49 (4.0)
3 3 (SiO2-200) 2.6 (0.28) 0.36 2.5 (2.5) 1.71 (5.1)
a
Determined by ICP-OES
b
Molar ratio between zirconium and surface silanol
c
Molar ratio between chlorine or carbon and zirconium
consist of different types of diethyl ether

molecules, being shifted downfield because
of the coordination to the Lewis acidic
zirconium center.15
Figure 3. Proposed structure of catalyst 1

(top), of catalyst 2 and 3 (bottom)
Catalyst 1 and 3 enhanced similar

Figure 2. 1H MAS solid-state NMR catalytic activity and they can be reused for
spectrum of catalyst 1 further three reactions but catalyst 2
displayed similar catalyst 1 and 3 for only
According to all information form first run, then conversion dropped to 48% in
elemental analysis and 1H SS-NMR spectra. second run. Even though structure of
We can propose structure of catalyst 1 as catalyst 1 and 3 are different, they show
coordination of one diethyl ether molecule very similar catalytic performance. From
of ZrCl4·(OEt2)2 precursor and one siloxane these results suggest that podality of the
of surface support to generate [(≡SiO- surface did not effect on catalytic activity.
)ZrCl3·OEt2 (Os(-Si≡)2))] and [(≡SiO- We also studied catalytic performance with
)ZrCl3·(OEt2)2] (Figure 3). For catalyst 2 various conditions by changing substituent
and 3 can purpose structure as mixture of group of epoxides, pressure of CO2, amount
one diethyl ether with two or three chlorines of Zr-loading and using flue gas. From
([(≡SiO)2ZrCl2·OEt2·(Os(-Si≡)2)]; bipodal results all catalysts can be used as
complex or [(≡SiO)ZrCl3·OEt2·(Os(-Si≡)2)]; heterogeneous catalyst for coupling between
monopodal complex). CO2 and epoxide and afforded high catalytic
3.2 Catalysis experiments activity and high selectivity under mild
Catalyst 1-3 were used as conditions. Moreover, the catalysts can be
heterogeneous catalysts for cycloaddition reused several times with slightly dropped
reaction of CO2 to epoxides to generate conversion in each run because of
cyclic organic carbonates (Table 2.). zirconium-leaching.
Table 2. Catalysis performance of catalyst 1-3 for the cycloaddition of CO2 to epoxidesa
Entries Catalyst R CO2 source/ Run 1 Run 2 Run 3 Run 4

[CO2] (bar) Yield (%)c Yield (%)c Yield (%)c Yield (%)c
1 1 CH3 Pure CO2/10 96 84 76 68
2 2 CH3 Pure CO2/10 90 48 46
3 3 CH3 Pure CO2/10 93 83 67 56
4 3 C6H5 Pure CO2/10 93 87 83 80
5 3 CH2Cl Pure CO2/10 98 91 87 84
6 3 CH3 Pure CO2/5 85
7 3 CH2Cl Pure CO2/1 75 70
8b 3 CH2Cl Pure CO2/10 98
9b 3 CH2Cl Pure CO2/1 96
10 1 CH3 Flue gas/1.05 8d (80)e 7.8d (78)e 7.7d (77)e
d e d e
11 2 CH3 Flue gas/1.05 9.9 (99) 8.7 (87) 7.9d (79)e
a
Mixing propylene oxide (43mmol), TBAB (1 mol%) and catalyst (Zr 0.3 mol%) in autoclave with 10 bar CO2
at 60°C for 18 hours.
b
Reaction for 24 hours.
c
Calculated by 1H-NMR spectroscopy.
d
Isolated yield.
e
Percent conversion of initial CO2.
4. Conclusion be a represent of important progress

The ZrCl4(OEt2)2 precursor was innovation in single-site heterogeneous
grafted on surface of the dehydroxylated catalyst field.
silica at 200 °C and 700 °C by SOMC
approach to generate catalyst 1-3. These Acknowledgements
catalysts were characterized be FT-TR, This work has been supported by the
elemental analysis and 1H SS-NMR to get Department of Molecular Science and
the structural information of the surface Engineering, Vidyasirimedhi Institute of
complexes. Catalyst 1-3 displayed as Science and Technology.
heterogeneous catalyst for the cycloaddition
reaction of CO2 to epoxide to provide cyclic References
organic carbonates under mild conditions. 1. Hoffert, M. I. Science 2010, 329 (5997),
The catalysts were recoverable and reusable 1292-1294.
several times for further catalytic cycles. 2. Song, C. Catal. Today 2006, 115 (1–4),
The catalytic performance of these catalyst 2-32.
did not depend on podality of the surface 3. Schäffner, B.; Schäffner, F.; Verevkin,
and degree of isolated zirconium center. S. P.; Börner, A. Chem. Rev. 2010, 110
Moreover, catalyst 1 and 2 were tested by (8), 4554-4581.
using flue gas as a source of CO2. Under this 4. Ma, R.; He, L.-N.; Zhou, Y. B. Green
challenging condition that consist of various Chem. 2016, 18 (1), 226-231.
pollutions, these catalysts still were able to 5. Monassier, A.; D'Elia, V.; Cokoja, M.;
convert CO2 and could be reused. Therefore, Dong, H.; Pelletier, J. D. A.; Basset, J.
in this work we present example M.; Kühn, F. E. ChemCatChem 2013, 5
heterogeneous catalysts for the conversion (6), 1321-1324.
of actual flue gas. It is clear that the 6. Barthel, A.; Saih, Y.; Gimenez, M.;
development of heterogeneous catalysts and Pelletier, J. D. A.; Kuhn, F. E.; D'Elia,
processes as that reported in this study can
V.; Basset, J. M. Green Chem. 2016, 18 K.; D’ Elia, V.; Basset, J.M. ACS
(10), 3116-3123. Catalysis 2016, 6 (9), 5908-5921.
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Chem. Res. 2016, 49 (4), 664-677. Basset, J. M. Comptes Rendus Chimie
8. Basset, J. M.; Coperet, C.; Soulivong, 2004, 7 (8), 725-736.
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Chem. Res. 2010, 43 (2), 323-334. C.; Basset, J. M. J. Am. Chem. Soc.
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L.; Paul, J. F.; Nicholas, C. P.; Gauvin, Merle, N.; Szeto, K. C.; De Mallmann,
R.M.; Taoufik, M. Chem. Commun. A.; Del Rosal, I.; Maron, L.; Girard, G.;
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10. Soulivong, D.; Norsic, S.; Taoufik, M.; M. ACS Catalysis 2014, 4 (11), 4232-
Coperet, C.; Thivolle-Cazat, J.; Chakka, 4241.
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Llorens, I.; Hazemann, J. L.; Köhler, Chem. Soc. 2015, 137 (24), 7728-7739.
Process optimization and characterization of activated carbon from cattail
weed waste prepared by chemical activation for efficient CO2 removal
Nardnutda Katunyoo1, Araya Smuthkochorn1, Napat Kaewtrakulchai1, Duangduen Atong2,
Kanit Soongprasit2, Apiluck Eiad-ua1*
1
College of Nanotechnology, King Mongkut’s institute of Technology Ladkrabang, Thailand
2
Nation Metal and Materials Technology Center (MTEC), Thailand
Abstract:
The major reason of rapid CO2 emission is resulted from human activities such as
burning of fossil fuels, transportation and air polluted from industrial process causing the
increase of average global temperature at a faster rate. Several technologies (e.g. CO2
conversion and carbon storage technology) have been developed to reduce a carbon dioxide
from the atmosphere. Reducing CO2 emissions is of great importance to theglobalcommunity,
and carbon capture and storage (CCS) is one of effective options for reducing these harmful
CO2 emissions. Recently, development of activated carbon from waste and biomass for
carbon dioxide capture has been reported with high separation performance and offers a cost-
effective solution. Thus, this study explores the activated carbon manufactured from cattail
leaves (CL) by using KOH activation, the activated carbon was processed by hydrothermal at
200 °C for 24 hours and substituted to chemical activation at 500 to 900 °C for 1 h. Then, the
characteristics of cattail leaves-based activated carbon were analyzed by SEM, FTIR, N2
adsorption isotherm, and Raman spectroscopy. From this investigation, the prepared activated
carbon will be an efficient adsorbent for the CO2 emission capture.
1. Introduction useful adsorbent for CO2 capture due to their

Global warming is a current large surface areas, suitable pore size
temperature on earth have increased, severe distribution,6 high mechanical strength and
impact on humans and environment. This stable adsorption-desorption cycles.
main reason came from greenhouse effect Basically, activated carbon is produced via
related to greenhouse gases (i.e. ozone, physical or chemical activation, physical
methane, nitrous oxide, chlorofluorocarbon activation is using a carbonization process
and carbon dioxide), that can trap heat and with high temperature to prepare char under
infrared radiation. carbon dioxide has the activating gas atmosphere such as steam
sourced from electricity production, and carbon dioxide,7 Whereas, chemical
transportation, agriculture activities, activation is treated with chemical solution,
commercial, industry and residential sectors. for example sodium hydroxide and
The principal methods used to capture CO2 potassium hydroxide. Activated carbon has
are adsorption and membrane separation.1,2 been classified according to raw materials
The methods of CO2 adsorption by amines used in its preparation such as natural coal,
are commonly used and highly effective. peat, industrial wastes and biomass.
However, there are some important technical Activated carbons are usually prepared from
but economic drawbacks.3 Adsorbents have agricultural waste because of its inexpensive
a high adsorption performance and high cost, it can change lignocellulose to carbon
thermal stability. Activated carbon4 and become an interesting material for
zeolite5 has very high surface areas resulted adsorbents.
from porosity, size of pores larger than In this study, we prepared activated
adsorbate molecules.Activated carbon is a carbon from the cattail leaves, which are a
weed waste found in all areas throughout of activating at 500, 700 and 900 °C was noted
Thailand. The preparation was performed in as CL1, CL2 and CL3, respectively. The
2 step processes (hydrothermal- activated carbon from chemical activation
carbonization and chemical activation using with KOH at 500, 600, 700, 800 and 900 °C
KOH as activating agent). Produced was noted as CAC1, CAC2, CAC3, CAC4
activated carbon was characterization by and CAC5, respectively. After the
scanning electron microscopy, Fourier- preparation, prepared samples were
transform infrared spectroscopy. The aim of characterized by scanning electron
this work is to study the effects of microscope (SEM) used for the surface
carbonization temperatures and chemical morphology analysis, Fourier transform
properties of the cattail flower derived infrared spectroscopy (FT-IR, Perkin Elmer
activated carbon. Spectrum 2) for the surface function
bonding analysis.
2.1 Raw materials preparations 3. Results and Discussion
Waste biomass (cattail leaves, CL) 3.1 Production of activated carbon
was obtained from weed in all areas The carbon yield (%) obtained by the
throughout Thailand. The chemical chemical activation are approximately 60%
component analysers of cattail leaves are excepted the sample which was burned off
shown in Table 1. The collected CL were at 500 °C showing high yield off 63.43 %.
dried at 110 °C overnight to remove The yield from physical activation around
moisture and then crushing to be particle 42-17% are lower than chemical activation.
sizes below 1 mm. Figure 1 is observation for the increase of
carbonization temperature led to the
Table 1. The chemical component analyses reduction of carbon yield due to the
of cattail leaves disappearance of organic substances.
Structural Cattail leaves Production yield results are not really high
Cellulose 63.2 % compared to the one obtained by chemical
Hemicellulose 8.7 %
Lignin 9.6 % activation the sample prepared by the
physical process reveals a lower yield of the
2.2 Preparation and characterization of order 42-17%.
activated carbon 3.2 Fourier transform infrared
Activated carbon were preparation spectroscopy (FT-IR)
from cattail leaves by Hydrothermal- These analyses were present of
Carbonization process, prepared CF was functional group on the activated carbons
heated at 200 °C for 12 h in a stainless-steel surface prepared from cattail leaves. The
chamber. and then carbonized in nitrogen band observed in Figure 2a activated
carbons after hydrothermal-carbonization
flow at 500, 600, 700, 800 and 900 °C for 1
without chemical activated, the functional
h. by a ramp rate for 10 o/min. The biochar
group was decrease when the temperature
was chemical activating with a saturated
solution of potassium hydroxide (KOH) 4 increased from 500 °C to 900 °C the final
molar for 24 h at room temperature. After product was only pure carbon, the band at
that was washed with 0.1 M of HCl and 1514 and 1078 cm-1 was related to the C-C
distilled water several times until pH of and C-O stretching indicated the functional
filtrate became neutral. The washed sample group of lignin. At 874 cm-1, C-C stretching
indicated group of hemicellulose, 796 cm-1
were dried at 90 °C overnight to prepare the
C-H rocking was the groups of
activated carbon.
hemicellulose, cellulose and lignin. In
On the other hand, biochar obtained
Figure 2b, the effect of impregnated with
from cattail leaves without chemical
KOH function group of the activated
carbons surface was appeared the band at
1563 cm-1, 1078 cm-1 was related to the C-O
bonding indicated to cellulose and lignin, at
1370 cm-1 was related to the C-H bending
indicated to lignin, at 879 cm-1 and 768
cm-1 was related to the C-C stretching and
C-H rocking indicated to hemicellulose,
cellulose and lignin. The increased
temperatures are melting burn off only pure
carbons was the final product.
Figure 2b. FTIR-ATR spectra of the

activated carbons impregnated with KOH at
different temperature (a) 500 °C, (b) 600 °C,
(c) 700 °C, (d) 800 °C and (e) 900 °C
morphology of the activated carbons from

cattail leaves. In Figure 3a-e shows SEM
image of activated carbons without
impregnated at different temperature of 500
°C, 600 °C, 700 °C, 800 °C and 900 °C,
respectively. However, the high dense and
Figure 1. Carbon yield with/without low roughness surface without the cavities
impregnation with KOH at different on the surface is show in Figure 3a-b which
temperatures is carbon products performed at
carbonization temperature of 500 °C and
600 °C. Generally, the pore and surface
roughness begins to form by the release of
volatile substances at the temperature
greater 700 °C without activation process5,
as show in Figure 3a-e. Whereas, various
pores apparently found with the increasing
of carbonization temperature as seen in
Figure 3a-c. A wide cavity on their surface
is resulted from the thermal degradation of
hemicellulose, cellulose and lignin. On the
other hand Figure 4a-e illustrates various
Figure 2a. FTIR-ATR spectra of the pores of the carbon product performed by
activated carbons without impregnated at using KOH as an activating agent.
different temperature (a) 500 °C, (b) 600 °C, Furthermore, Figure 4e displayed the
(c) 700 °C, (d) 800 °C and (e) 900 °C degradation of pore at the highest
temperature in carbonization due to the
effect of extreme temperature.
3.3 Scanning electron microscopy
Scanning electron microscope
(SEM) measurements were performed using 4. Conclusion
SEM Zeiss EVO MA10 operated at 10 kV This study represents of activated
applied to observe the surface physical carbons prepare from cattail leaves by
hydrot herm al carboni z at i on proces s
Figure 3. SEM images (300x and 500x) of Figure 4. SEM images (300x and 500x) of
activated carbons without impregnated at activated carbons activate with KOH at
different temperature (a) 500 °C, (b) 600 °C, different temperature (a) 500 °C, (b) 600 °C,
(c) 700 °C, (d) 800 °C and (e) 900 °C (c) 700 °C, (d) 800 °C and (e) 900 °C
followed by chemical activation, the main References
parameter is temperature which significantly 1. Deng, S.; Wei, H.; Chen, T.; Wang, B.;
affected on the porosity of produced Huang, J.; Yu, G. Chem. Eng. J. 2014,
activated carbon. The carbon yield achieved 253, 46–54.
by chemical activation are higher than 2. Kacem, M.; Pellerano, M.; Delebarre, A.
carbonization. According to this study, we Fuel Process. Technol. 2015, 138, 271–
expected to prepare a porous carbon from 283.
waste biomass resource for direct reduction 3. Sneddon, G.; Greenaway, A.; Yiu, H. H.
of greenhouse gases which is the major P. Adv. Energy Mater. 2014, 4, 1–19.
cause of global warming in further 4. Jime’nez, V.; Ramírez-Lucas, A.; Díaz,
investigation. P.; Sa’nchez, J. A.; Romero, A. Environ.
Sci. Technol. 2012, 46, 7407–7414.
Acknowledgements 5. Kamimura, Y.; Shimomura, M.; Endo,
The authors would like to thank the Microporous Mesoporous Mater. 2016,
College of Nanotechnology, King 219, 125–133.
Mongkut's institute of Technology 6. Fiuza-Jr, R. A.; Neto, R. J. M.; Correia,
Ladkrabang (KMITL), Bangkok Thailand L. B.; Andrade, H. M. C. J. Environ.
and the financial support of the Thailand Manage. 2015, 16, 198–205.
Research Fund to the National Metal and 7. Chao, C.; Wha-Seung, A. Chem. Eng. J.
Materials Technology Center (MTEC) via 2011, 166, 646–651.
the YSTP program of National Science and 8. Jarosław, S.; Urszula. N.; Antoni, W. M.;
Technology Development Agency Rafał, J. W.; Beata, M. J. CO2 Utilization
(NSTDA), Pathumthani Thailand (Grant 2017, 18, 73–79.
GNA-CO-2560-4662-TH/2560) for support
this study.
Conversion of CO2 to cyclic carbonates catalyzed by lanthanide salen
complexes
Vatcharaporn Aomchad1, Valerio D’Elia2*
Department of materials science and engineering, school of molecular science and engineering,
Vidyasirimedhi institute, Payupnai, Wangchan, Rayong 21210, Thailand
*E-mail: valerio.delia@vistec.ac.th
Abstract:
Salen ligands have been widely applied in coordination chemistry of transition metals
because of their excellent advantages such as ease of preparation, low cost and high tunability
of the steric and electronic effects by modifications of the organic scaffolds. In
organolanthanide chemistry, lanthanide salen complexes are considered as efficient catalysts
able to promote several chemical transformations, however, they have not yet been employed
for the conversion of CO2 to value added products. In this work, scandium and yttrium salen
complexes were synthesized and used as homogeneous catalysts in the synthesis of value-
added cyclic carbonate products by the coupling reaction of CO2 and epoxides. This work
shows the potential of lanthanide salen complexes to catalyze the conversion of CO2 into
cyclic carbonates under mild reaction conditions and with a low catalyst loading.
1. Introduction the carbonation reaction catalyzed by metal

Carbon dioxide (CO2) is one of complex YCl3/TBAB (TBAB:
greenhouse gases responsible for global tetrabutylammonium bromide) could be take
warming mainly caused by the thermal used place under ambient conditions even when
of fossil fuels from human activities.1 flue gas was used as not pure source of
However, CO2 is considered as a C1 CO2.5 And moreover, readily available
building block and an inexhaustible, cheap, ascorbic acid was discovered as an
nontoxic, non-flammable green source of environmental catalyst for this
carbon for the synthesis of more than 20 cycloaddition.6 The binary system of
known value-added products.2 Among them, ascorbic acid/TBAI (TBAI:
the coupling of epoxides and CO2 to tetrabutylammonium iodide) could be
generate cyclic carbonates have gained a lot catalyzed the cycloaddition of CO2 to
of attention because of their usefulness various epoxides under mild conditions.
applications such as solvents, electrolytes, These routes can avoid the emission of CO2
monomers for polymer synthesis, synthetic to the atmosphere resulting in a greener
building blocks, fine chemicals, industrial process.
lubricants, and industrial scale for the Salen ligands have been widely
synthesis of polycarbonates.3 The utilization applied in coordination chemistry of
of CO2 as a C1 feedstock to produce value- transition metals because of their excellent
added cyclic carbonate products is an advantages such as ease of preparation, low
alternative pathway to reduce the amount of cost and high tunability of the steric and
CO2 emission. However, to find out suitable electronic effects by modifications of the
catalysts for CO2 conversion is still a organic scaffolds.7 Binary catalytic system
challenging task.4 consisting of metal salen complexes and
Recently, our group has quaternary ammonium halides co-catalysts
demonstrated the cycloaddition reactions have been successfully applied to catalyze
that can be proceeded even under the coupling reaction of epoxides with CO2.8
atmospheric CO2 conditions. For example, In organolanthanide chemistry, lanthanide
salen complexes are considered as efficient were obtained on a Bruker Avance III HD
catalysts able to promote several chemical (1H, 600 MHz; 13C, 125 MHz).
transformations, however, they have not yet 2.2 Synthesis of lanthanide salen
been employed for the conversion of CO2 to complexes
value added products.9 In this work, The complex 1 was prepared
scandium and yttrium salen complexes were according to a slightly modified literature
synthesized (see Figure 1) and used as procedure, as described for
homogeneous catalysts in the synthesis of [Salen]Y[N(SiHMe2)2][thf] (see Scheme
value-added cyclic carbonate products by 1).11 The mixture of N,N’-Bis(3,5-di-tert-
the coupling reaction of CO2 and epoxides. butylsalicylidene)-1,2-cyclohexanediamine
This work shows the potential of lanthanide (L1-H2) 1 mmol and Tris[N,N-
salen complexes to catalyze the conversion bis(trimethylsilyl)amide]yttrium 1 mmol in
of CO2 into cyclic carbonates under mild hexane (5 mL) and thf (5 mL) was stirred at
reaction conditions and with a low catalyst room temperature for 5 days. The solution
loading. was evaporated. Then, trituration with
pentane to give the complex as yellow solid.
General Procedure for Complexes 2
(Scheme 1). According to a slightly
modified literature procedure, as described
for (Salen)Sc(Cl)(thf) (2a)9b,12 N,N’-Bis(3,5-
di-tert-butylsalicylidene)ethylenediamine
(L2-H2) was dissolved in thf, and added to
the flask contained 2 equiv. of KH. The
bubbling of hydrogen gas was occurred, and
Scheme 1. Synthesis of lanthanide salen the solution color changed from pale yellow
complexes used in this study to dark golden yellow. After gas evolution
disappeared, YCl3(thf)3 was added and the
2. Materials and Methods white insoluble material began to form. The
2.1 General reaction was stirred at room temperature
All of the manipulations of metal overnight. Afterwards, the mixture was
complexes and CO2 conversion were filtered and extracted with thf. The
performed under a nitrogen-filled glovebox combined solvent was evaporated. Then,
(MBRAUN MB-200B-MOD; <0.1 ppm O2, trituration with hexane generated
<0.1 ppm H2O) or standard Schlenk (Salen)Y(Cl)(thf) (2b) as a pale-yellow
techniques. Solvent purification was solid.
performed by solvent purification system 2.3 The conversion of CO2 to cyclic
(MBRAUN MB-SPS). Metal halides were carbonate
purchased from Alfa Aesar (YCl3 ultra dry, The activity of lanthanide salen
99.99%; ScCl3 99.9%). Tris[N,N- complexes was studied toward a benchmark
bis(trimethylsilyl)amide]yttrium was reaction of the coupling of epichlorohydrin
purchased from Sigma-Aldrich. Salen with CO2. In glove box, starting with 0.1-0.5
ligands were prepared according to well mol% of lanthanide salen complexes and
established procedures in the literature.10 0.2-1 mol% of nucleophilic co-catalysts
CDCl3 and C6D6 were obtained from were charged with 50-mL Schlenk flask
Cambridge Isotope Laboratories, inc. C6D6 equipped with a magnetic stirrer bar. Next,
was dried over molecular sieve for 48 h the Schlenk flask was took out and equipped
before used and stored in a glovebox. with Schlenk line to add 25.5 mmol of dry-
Epichlorohydrin, TBAB, and TBAI were epichlorohydrin. Afterward, a rubber
purchased from Merck. CO2 (>99.999%) balloon containing CO2 was connected and
was purchased from Praxair. NMR data flushed the flask with a part of CO2 in the
balloon for a few seconds to remove the air
inside the flask. The reaction was carried out
at room temperature for 1 day. After this
period, an aliquot of the reaction mixture
was determined a substrate conversion by
1
H NMR spectroscopy.

3.1 Synthesis of lanthanide salen
complexes
The condensation of salicylaldehydes Figure 1. 1H NMR spectra of salen L1-H2
with the corresponding diamines afforded (a) and complex 1 (b) in C6D6
the desired salen ligands. To prepare
lanthanide salen complexes, most of them The comparing NMR spectrum of
were prepared based on silylamide and salen ligand L1-H2 and complex 1 was
metal halide routes. Therefore, complex 1 shown in the Figure 1. The -OH peak at
was prepared as the representative of 14.05 ppm of the salen ligand disappeared to
silylamide route and complexes 2a and 2b ensure the complexation might take place to
were prepared as the representatives of form the complex 1. For complex 2a and 2b
metal halide route. Complex 1 was were characterized by NMR spectroscopy
characterized by NMR spectroscopy using using C6D6 as a solvent. In general, the
C6D6 as a solvent. The NMR spectrum of NMR spectra of 2a and 2b were similar due
complex 1 were matched the previously to the same group of lanthanide metals. The
reported data however we have proposed the comparing NMR spectrum of L2-H2 and
different synthetic pathway.13 complex 2a and 2b was shown in the Figure
2.
Figure 2. 1H NMR spectra of salen L2-H2 (a) complex 2a (b) and complex 2b (c) in C6D6
The complexation brought about the lanthanide salen complexes toward the
absence of -OH peak around 14 ppm of the cycloaddition of epoxides and CO2.
salen ligand observed in both 2a and 2b
NMR spectra to form lanthanide salen
complexes. Moreover, when employed
ethylenediamine-backbone to synthesize
complex 2a and 2b the spectrum around the
Scheme 2. A generally accepted mechanism
backbone regions (2-4 ppm) were split due
for the metal-catalyzed coupling of CO2 and
to their isotropic NMR shifted phenomenal
epoxides.
observed in transition metal complexes
Therefore, 2D NMR was performed to study
The result of a study using
the relationship of the spectrum. The COSY
lanthanide salen complexes as Lewis acid
spectrum of complex 2a in C6D6 was shown
catalysts in the combination with
in the Figure 3. The result suggested that the
nucleophilic co-catalysts was shown in the
splitting peaks around ethylenediamine-
Table 1. We first examined the effect of
backbone have correlated to each other.
nucleophilic co-catalysts toward the
complex 1 catalyzed the conversion of CO2
and epichlorohydrin into 3-chloropropylene
carbonate carried out under room
temperature for 1 day. The result showed
that using either TBAB or TBAI could
promote this reaction and the conversions
were not significant different (entries 1 and
2). Therefore, TBAI was chose instead of
TBAB to be a suitable nucleophilic co-
Figure 3. COSY spectra of complex 2a catalyst due to their leaving ability of iodide
anions. Increasing an amount of catalyst
3.2 The conversion of CO2 to cyclic loadings could slightly promote the CO2
carbonate conversion catalyzed by complex 1 (entries
The catalyst system that can catalyze 3).
the coupling reaction of epoxides with CO2
consisting of a metal Lewis acid together Table 1. Coupling of CO2 with
with a suitable nucleophilic co-catalyst so epichlorohydrin catalyzed by lanthanide
called the binary catalyst system. A typical salen complexes in combination with
acceptance mechanism for this coupling is nucleophilic co-catalystsa
shown in the Scheme 2.14 The activation of
epoxides begins by a metal of Lewis acid
coordinated to the oxygen atom of epoxides
(M–O coordination) resulting the weakened
of C-O bond of epoxides to ease for the ring Catalyst Co-catalyst
Entries %Conversion
(mol%) (mol%)
opening step. Next, the nucleophilic attacks
1 1 (0.1) TBAB (0.2) 13
on the less hindered methylene carbon of 2 TBAI (0.2) 16
epoxides generated intermediate I. Then, 3 1 (0.5) TBAI (1.0) 38
CO2 is inserted through M-O bond to 4 2a (0.1) TBAI (0.2) 11
generate hemi-carbonate intermediate II. 5 2b (0.1) TBAI (0.2) 8
Finally, the ring closing step affords the 6 2b (0.5) TBAI (0.2) 38
a
desire cyclic carbonates and regenerates Reaction conversions determined by NMR after 24
Lewis acid. In view of a high Lewis acidity h. Experimental conditions: CO2 1 atmospheric
pressure, epichlorohydrin 25.5 mmol, room
nature of scandium and yttrium we decide to temperature, under solvent free condition.
investigate the activity of the synthesized
For complexes 2a and 2b the 3. Büttner, H.; Longwitz, L.; Steinbauer, J.;
conversions were quite similar due to the Wulf, C.; Werner, T. Top. Curr. Chem.
same group of scandium and yttrium (entries 2017, 375 (3), 50.
4 and 5). Both lanthanide salen complexes 4. (a) Comerford, J. W.; Ingram, I. D. V.;
bearing trimethylsilylamide (TMS) axial North, M.; Wu, X. Green Chem. 2015, 17
ligand of complex 1 and the halide axial (4), 1966-1987; (b) Song, Q. W.; Zhou,
ligand of complex 2b caused the formation Z. H.; He, L. N. Green Chem. 2017, 19
of 3-chloropropylene carbonate in the (16), 3707-3728; (c) Shaikh, R. R.;
similar percentage conversions even when Pornpraprom, S.; D’Elia, V. ACS Catal.
increasing the amount of catalyst loadings 2018, 8 (1), 419-450.
(entries 3 and 6) was not enhanced the 5. Barthel, A.; Saih, Y.; Gimenez, M.;
activity. Pelletier, J. D. A.; Kuhn, F. E.; D'Elia,
V.; Basset, J. M. Green Chem. 2016, 18
4. Conclusion (10), 3116-3123.
In summary, lanthanide salen 6. Arayachukiat, S.; Kongtes, C.; Barthel,
complexes were synthesized and employed A.; Vummaleti, S. V. C.; Poater, A.;
for the first time in combination with Wannakao, S.; Cavallo, L.; D’Elia, V.
nucleophilic co-catalysts to catalyze the ACS Sustainable Chem. Eng. 2017, 5 (8),
coupling of CO2 and terminal epoxide. This 6392-6397.
work displays the potential of lanthanide 7. (a) Cozzi, P. G. Chem. Soc. Rev. 2004, 33
salen complexes to catalyze the coupling of (7), 410-421; (b) Bellemin-Laponnaz, S.;
CO2 with epichlorohydrin to afford the Dagorne, S., Coordination Chemistry and
desired cyclic carbonate under mild reaction Applications of Salen, Salan and Salalen
conditions however the results with these Metal Complexes. PATAI'S Chemistry of
compounds are likely to be improved. Functional Groups; John Wiley & Sons,
Therefore, further optimization of the Ltd: 2009.
catalyst structures by the synthesis of 8. (a) North, M.; Pasquale, R.; Young, C.
bimetallic complexes that is currently in Green Chem. 2010, 12 (9), 1514-1539;
progress in our laboratory. (b) Clegg, W.; Harrington, R.W.; North,
M.; Pasquale, R. Chem. Eur. J. 2010, 16
Acknowledgements (23), 6828-6843; (c) Decortes, A.;
This work has been supported by the Castilla, A. M.; Kleij, A. W. Angew.
Department of Molecular Science and Chem. Int. Ed. 2010, 49 (51), 9822-9837.
Engineering, Vidyasirimedhi Institute of 9. (a) Wu, B.; Gallucci, J. C.; Parquette,
Science and Technology. J.R.; RajanBabu, T. V. Chem. Sci. 2014,
5 (3), 1102-1117; (b) Meermann, C.;
References Sirsch, P.; Tornroos, K. W.; Anwander,
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Valuable Products under Homogeneous Shen, Q. Organometallics 2013, 32 (9),
Catalysis A2 - Reedijk, Jan. 2608-2617; (d) Gu, W.; Xu, P.; Wang,
Comprehensive Inorganic Chemistry II Y.; Yao, Y.; Yuan, D.; Shen, Q.
(Second Edition), Poeppelmeier, K., Ed. Organometallics 2015, 34 (12), 2907-
Elsevier: Amsterdam, 2013; pp 563-586. 2916.
2. Alves, M.; Grignard, B.; Mereau, R.; 10. (a) Larrow, J. F.; Jacobsen, E. N.;
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Catal. Sci. Technol. 2017, 7 (13), 2651- J. Org. Chem. 1994, 59 (7), 1939-1942;
2684. (b) Früh, N.; Togni, A. Angew. Chem.
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11. (a) Lin, M. H.; RajanBabu, T. V., Org. 13. Li, G.; Wang, L.; Yao, Z.; Xu, F.
Lett. 2002, 4 (9), 1607-1610; (b) Runte, Tetrahedron: Asymmetry 2014, 25 (13),
O.; Priermeier, T.; Anwander, R. Chem. 989-996.
Commun. 1996, (11), 1385-1386. 14. Zhou, F.; Xie, S.-L.; Gao, X. T.; Zhang,
12. Meermann, C.; Törnroos, K. W.; R.; Wang, C. H.; Yin, G. Q.; Zhou, J.
Anwander, R. Inorg. Chem. 2009, 48 Green Chem. 2017, 19 (16), 3908-3915.
(6), 2561-2570.
Preparation and characterization of Pb(Mg1/3Nb2/3)1-xTixO3 ceramics to
fabricate flexible piezoelectric composites
Sukanya Samadoloh1, Napatporn Promsawat2, Ekwipoo Kalkornsurapranee1,
Kowit Lertwittayanon1, Soodkhet Pojprapai3, Methee Promsawat1*
1
Department of Materials Science and Technology, Faculty of Science,
2
Synchrotron Light Research Institute, Muang, Nakhon Ratchasima 30000, Thailand
3
School of Ceramic Engineering, Institute of Engineering, Suranaree University of Technology,
Muang, Nakhon Ratchasima 30000, Thailand
*E-mail: methee.p@psu.ac.th
Abstract:
Pb(Mg1/2Nb2/3)1-xTixO3 ceramics were prepared which possessed the density of 7.8
g/cm3 and the grain size ranged from 3-5 µm. Dielectric constant reached a maximum value
at x = 0.34. Remanent polarization tended to decrease while coercive field and tetragonality
tended to increase with increasing x. The highest piezoelectric properties, i.e. d33 = 410 pC/N
and g33 = 19.8×10-3 V⋅m/N were found in the ceramic with x = 0.34. This composition
powder was used as dispersed particles in natural rubber matrix for further producing flexible
piezoelectric composite. The composite with 100 phr powder addition showed the highest
output voltage.

Flexible piezoelectric composites Pb(Mg1/3Nb2/3)1-xTixO3 ceramics,
have been received much attention for where x = 0.30, 0.32, 0.34, 0.36, 0.38 and
applications as piezoelectric sensors and 0.40 (designed as PMNT30, PMNT32,
generators.1,2 Recently, both lead-based and PMNT34, PMNT36, PMNT38 and
lead-free piezoelectric materials have been PMNT40, respectively), were prepared from
used as dispersed phases3,4 while various commercial starting powders (TOPLUS,
kinds of polymers were used as matrices.5,6 China) by a solid state sintering at 1240 °C
Pb(Mg1/3Nb2/3)1-xTixO3 (PMNT) is a for 2 h. Sintered density, phase analysis,
complex perovskite piezoelectric exhibiting microstructure, dielectric, ferroelectric and
high piezoelectric responses, in particular at piezoelectric properties of prepared PMNT
x = 0.30-0.40.7,8 For mass production ceramics were characterized by the
purpose of the material, the use of laboratory Archimedes’ method, X-ray diffraction
grade precursors is quite inconvenient due to (XRD), a scanning electron microscope
their expensive costs. This may be solved by (SEM), an LCR meter, a Sawyer-Tower
using of lower-grade precursors with much circuit and a piezo-d33 meter, respectively.
lower prices. The PMNT powders showing the best
Therefore, in this work, PMNT piezoelectric were prepared for using as a
ceramics were prepared. The effects of Ti dispersed phase of flexible piezoelectric
content (x) on properties of the ceramics composites with natural rubber matrix. The
were studied. The PMNT composition with various contents (0, 60, 80, 100, 120 and
the best piezoelectric response was then 150 phr) of the powders were added into
selected to prepare PMNT powders for piezoelectric composites. Output voltage
further forming flexible piezoelectric generated during applying compressive
composites with natural rubber matrix. stress of composites was monitored and
Output voltage of the prepared piezoelectric recorded.
composites was also measured.
Figure 1. SEM images of fractured surfaces of (a) PMNT30, (b) PMNT32, (c) PMNT34,
(d) PMNT36, (e) PMNT38 and PMNT40 ceramics
3. Results and Discussion phase, corresponded to JCPDS file no. 27-

All the prepared ceramics possessed 1199, with perovskite structure was
the density of ∼7.8 g/cm3, i.e. a relative observed in PMNT30 sample. A coexistence
density of ∼97%. The mixed mode of inter- of rhombohedral and tetragonal phases was
and transgranular fractures was observed in found in PMNT32 and PMNT34 samples. A
all the ceramics, as shown Figure 1. Grain pure tetragonal phase, corresponded to ICSD
size was found to be ranged from 3-5 µm. file no. 78-0298, was belonged to the
No impurity or unwanted phase was samples with x = 0.36-0.40. The observed
observed in all the samples, as the XRD result was consistent with the previous result
patterns shown in Figure 2. A rhombohedral observed by Noheda et al.8 Tetragonality
(c/a) tended to increase with increasing x.
Dielectric constant (εr) and dielectric
loss (tanδ) measured at a frequency of 100
kHz of ceramics were given in Table 1. εr
slightly increased when x increased from
0.30 to 0.34. However, εr tended to increase
with further increasing x. tanδ did not
change with x. It was believed that the
domain configuration in PMNT34 ceramics
was suitable to facilitate the domain wall
motion under a low signal electric field,
leading to a greater dielectric response.
Polarization-electric field (P-E)
hysteresis loops measured at 27 °C and at 25
Hz of ceramics were illustrated in Figure 3.
Symmetric and square P-E loops, indicating
normal ferroelectric behavior9, were
observed in all ceramics. Remanent
polarization (Pr) and coercive field (Ec)
Figure 2. XRD patterns of PMNT ceramics values were listed in Table 1. Pr did not
contributed to a lower number of switchable
domains. The increase in g33 of PMNT40
ceramic was due to the decrease in dielectric
constant. This means that the generated
charge due to an applied force can be fully
transferred to the sample surface, resulting
in the higher generated voltage. Because
PMNT34 ceramics showed the best
piezoelectric properties, therefore, the
ceramic powder of this composition was
selected to be used as the dispersed phase of
piezoelectric composites with natural rubber
matrix. The properties of the samples in this
Figure 3. P-E hysteresis measured at 25 Hz work were compared to those of the sample
of PMNT ceramics from our previous works, as shown in Table
change with x = 0.03-0.34. However, Pr 1.
significantly decreased as x increased to Plots of output voltage as a function
0.36. Pr did not decrease further when x was of measurement time of NR/PMNT34
above 0.36. Ec tended to increase with composites were shown in Figure 4.
increasing x. In PMNT system, the size of Average output voltage tended to increase
domains increases with increasing x.8 This and reached a maximum value of about 2.0
causes a stronger interaction strength V with increasing PMNT34 powder content
between polar-macrodomains, leading to a up to 100 phr. The output voltage was
lower number of switchable domains. reduced when PMNT34 powder content was
Moreover, the higher interaction strength above 100 phr. This may be due to an
between domains also caused an increase in agglomeration of PMNT powders. The
the energy required for domains to switch, result suggested that the composite
resulting in an increase in coercive field.10 incorporated with 100 phr PMNT34 power
Piezoelectric coefficient (d33) and was a promising material used for
piezoelectric voltage coefficient (g33) of applications as piezoelectric sensors with
PMNT ceramics were shown in Table 1. g33 high mechanical flexible endurance.
of samples was calculated from the equation
proposed in the previous work reported by 4. Conclusion
Haertling.9 d33 and g33 of PMNT30 ceramics In this work, PMNT ceramics with
tended to increase and reached the Ti contents ranged from 0.30-0.40 mole
maximum values at x = 0.34. Both d33 and fraction were successfully prepared. The
g33 tended to decrease with increasing x up prepared ceramics possessed the density and
to 0.38. At a higher x content, the increased the grain size of ∼7.8 g/cm3 and 3-5 µm,
tetragonality and number of 90° domains respectively. Tetragonality and Ec tended to
Table 1. Dielectric, ferroelectric and piezoelectric properties of PMNT ceramics

Sample ε r* tan δ* Pr(µC/cm2) Ec (kV/cm) d33(pC/N) g33(x10-3 V•m/N)
PMNT30 2129 0.063 33 5 310 16.4
PMNT32 2198 0.049 31 6 364 18.7
PMNT34 2311 0.043 31 6 406 19.8
PMNT36 2096 0.046 27 7 348 18.7
PMNT38 1986 0.036 27 8 321 18.2
PMNT40 1578 0.042 27 10 271 19.4
PMNT35** 2600 0.040 26 7 530 16.0
*The shown dielectric property parameters were measured at 27 ºC at a frequency of 100 kHz.
**The sample was prepared from the laboratory11,12
Figure 4. Plots of output voltage as a function of measurement time of piezoelectric
composites
increase while εr decreased with increasing Actuators A. doi:10.1016/j.sna.2016.06.

x. Pr decreased when x was above 0.34. d33 011.
and g33 values were maximized at x = 0.34. 5. Zeng, Z.; Gai, L.; Petitpas, A.; Li, Y.;
Flexible piezoelectric composites with Luo, H.; Wang, D.; Zhao, X. Sens.
PMNT34 powders as dispersed phase and Actuators A doi:
natural rubber as matrix phase were 10.1016/j.sna.2017.07.059.
prepared. The composite with 100 phr 6. Jung, I.; Shin, Y. H.; Kim, S.; Choi, J.
PMNT34 powders showed a high output Y.; Kang, C. Y. Appl. Energy. 2017,
voltage which was a promising material for 197, 222−229.
applications as flexible piezoelectric 7. Alguero, M.; Moure, A.; Pardo, L.;
sensors. Holc, J.; Kosec, M. Acta. Mater. 2006,
54, 501−511.
Acknowledgements 8. Noheda, B.; Cox, D. E.; Shirane, G.;
This research was supported by Gao, J.; Ye, Z.-G. Phys. Rev. B 2002, 66,
Natural Rubber Innovation Research 054104/1−7.
Institute, Prince of Songkla University 9. Haertling, G. H. J. Am. Ceram. Soc.
(Grant No. SCI601093S). 1999, 82, 797−818.
10. Moulson, A. J.; Herbert, J. M.
References Electroceramics; 2nd Eds. John Wiley &
1. Guggilla, P.; Batra, A. K.; Edwards, M. Sons; Chichester, 2003.
E. J. Mater. Sci. 2009, 44, 5469−5474. 11. Promsawat, M.; Watcharapasorn, A.;
2. Huang, X.; Xie, L.; Jiang, P.; Wang, G.; Ye, Z. G.; Jiansirisomboon, S. J. Am.
Liu, F. J. Phys. D: Appl Phys. 2009, 42, Ceram. Soc. 2015, 98, 848−854.12.
245407/1–10. 12. Promsawat, M.; Ye, Z. G.;
3. Babu, I.; de With, G. Compos. Sci. Watcharapasorn, A. Mater. Lett. 2017,
Technol. doi:10.1016/j.compscitech. 205, 126−129.
2013.11.027.
4. Ji, S. H.; Cho, J. H.; Jeong, Y. H.; Paik,
J. H.; Yun, J. D.; Yun, J. S. Sens.
Preparation of complex hydrogels composed of chitosan, sodium
tripolyphosphate and κ-carrageenan as xanthone encapsulating material
Apisara Yimprayul1, Nataporn Sowasod1*,
Wiwut Tanthapanichakoon2,3
1
Division of Chemical Process Engineering Technology, Faculty of Engineering and Technology,
King Mongkut's University of Technology North Bangkok (Rayong Campus), Rayong 21120, Thailand
2
SCG Chemicals Co. Ltd., Rayong 21150, Thailand
3
Royal Society of Thailand 10300, Thailand
*E-mail: nataporn.s@eat.kmutnb.ac.th
Abstract:
Xanthone is a bioactive compound extracted from the peel of Garcinia mangostana
fruit (or mangosteen). However, its antioxidant and antibacterial properties could suffer rapid
deterioration when exposed to strong light and atmospheric oxygen. The objective of this
study is to obtain complex hydrogels composed of chitosan, sodium tripolyphosphate (TPP)
and κ-carrageenan as encapsulation material in order to promote long-term stability of
xanthone, which is effective for topical treatment of skin problems such as acne. In order to
obtain the most promising formulation that endows the highest stability of xanthone stored up
to 45 days at 4 °C and 25 °C, observations and simple characterizations were conducted on 24
formulations consisting of chitosan (0.4 and 3%), TPP (0.1 and 0.2%), κ-carrageenan (0.2,
0.5 and 1%), and xanthone extract (0.1 and 0.3%), in which the ratios of the complex
hydrogels to xanthone were kept at 7:1 and 9:1. It was found that the smallest average size of
nanoparticles of 690.5 nm was observed in Specimen C which contained 3% chitosan, 0.3%
xanthone, 0.5% κ-carrageenan and 0.2% TPP while the ratio of hydrogel to xanthone was 7:1.
In addition, Specimen C exhibited very good and fairly good particle size stability at 4◦C and
25◦C, respectively. However, its zeta potential increased from 24.1 to 28.2 and 32.5 mV,
respectively, after 30- and 45-day storage at 4 °C.
1. Introduction antioxidant, antibacterial, anti-inflammatory

Recently, the consumption of fruits and anticancer effects.1
that contain high amount of antioxidants has The encapsulation modes could be
gained popularity due to increasing public classified as reservoir type, core-shell type,
awareness of their health. Pericarps of matrix type, etc. Researchers are interested
various fruits have been used in folk in preparing and applying hydrogel
medicine for the treatment of many human encapsulation to drug delivery systems
illnesses.1 Mangosteen (Garcinia because hydrogels possess three-
Mangostana Linn) is one of the fruits often dimensional network of hydrophilic
used as an ingredient in commercial polymers that are insoluble and swell in
products, including nutritional supplements, water. A complex hydrogel system made
herbal cosmetics, and pharmaceuticals. The from non-toxic biopolymer materials should
major bioactive compounds found in be able to protect functional properties of
mangosteen are phenolic acid, prenylated bioactive xanthone. More specifically, a
xanthone derivatives, anthocyanins, and complex xanthone-carrying hydrogel
procyanidins. Extracted from the peel of composed of chitosan, sodium
mangosteen, xanthone has a variety of tripolyphosphate (TPP) and κ-carrageenan
biological activities, for instance, could be anticipated to protect, stabilize, or
control the release of the xanthone core
because chitosan as hydrophilic polymer can resulting complex gel (c) was ultrasonically
effectively cross-link with counter poly- mixed for 2 min with a xanthone-containing
anions like TPP and κ-carrageenan to ethanol solution (oil phase) (d). The ratios of
control the release of xanthone. complex hydrogel to xanthone (RHX) were
In this study, a technique to 7:1 and 9:1 (v/v). Next the resulting 24
encapsulate oil-soluble xanthone in a colloidal specimens were screened down to
complex hydrogel system was investigated. 5 promising specimens A, B and C (RAX
The present objective is to obtain a most 7:1) and specimens D and E (RAX 9:1)
promising formulation to produce a mixture before subsequent characterization and
of encapsulated xanthone nanoparticles with storage up to 45 days at 4 °C and 25 °C,
storage stability of up to 45 days. Further respectively. The screened and selected
investigation into its effectiveness for specimens A to E are listed in Table 1 and
topical treatment of skin problems will be their photos shown in Figure 2.
carried out in subsequent work. 2.3 Characterizations
2.3.1 Average nanoparticle size of
2. Materials and Methods xanthone-containing complex hydrogels
2.1 Materials and zeta potential
Commercial grade chitosan was The average nanoparticle sizes and
obtained from Seafresh Chitosan (Lab) Co. zeta potentials were measured using
Ltd., Thailand. Tween 80 was purchased Zetasizer model Nano-ZS, Malvern
from Ajax Finechem. Sodium Instruments, England. Our preliminary
tripolyphosphate (TPP), xanthone, acetic experimental results revealed that the
acid, κ-carrageenan and ethanol were optimal ratios of complex hydrogel to
purchased from Sigma Aldrich. xanthone were 7:1 and 9:1, so the effect of
2.2 Preparation of xanthone-containing chitosan concentration in mixture (a) and κ-
complex hydrogels carrageenan concentration in mixture (b) on
A total of 24 formulations consisting the nanoparticle size and zeta potential was
of chitosan (0.4 and 3%), TPP (0.1 and investigated with 3% chitosan and 0.5 to 1%
0.2%), κ-carrageenan (0.2, 0.5 and 1%), and κ-carrageenan suspensions, respectively.
xanthone extract (0.1 and 0.3%) were 2.3.2 Physical stability of xanthone-
prepared, in which the ratios of the complex containing complex hydrogels
hydrogels to xanthone were kept at 7:1 and The physical stability was
9:1. The preparation procedure of xanthone- determined by measuring the average
containing complex hydrogel was particle sizes and zeta potentials after the
schematized in Figure 1. First, an aqueous specimens were stored at 4◦C and 25◦C over
chitosan suspension containing xanthone in a period of 45 days.
acetic acid was mixed with sodium 2.3.3 Microstructures
tripolyphosphate (TPP) for 2 min using an The morphology of the complex
ultrasonic homogenizer (Sonics & Materials. hydrogels was observed through scanning
Co. Ltd., USA.). The obtained chitosan electron microscopy (SEM) (Model 1450
suspension (a) was ultrasonically mixed for VP, LEO). Gold coating was applied on the
2 min with a polymer suspension of κ- fractured surface of a stub of the specimen
carrageenan (b) at a ratio of 1:1. Then this of interest. SEM micrographs were taken
mixture was mixed and emulsified and examined in terms of morphology, size
ultrasonically for 2 min with Tween80. The and distribution of the nanoparticles.
Figure 1. Preparation procedure of xanthone-containing complex hydrogel
Table 1. Selected formulations of xanthone-containing complex hydrogel

Xanthone Chitosan κ-carrageenan TPP Ratio of complex
Specimen
Solution solution suspension solution hydrogel to xanthone
A 0.1% 0.4% 0.2% 0.1% 7:1
B 0.1% 0.4% 0.5% 0.1% 7:1
C 0.3% 3% 0.5% 0.2% 7:1
D 0.3% 3% 0.5% 0.2% 9:1
E 0.3% 3% 1% 0.2% 9:1
Figure 2. Photos of selected xanthone-containing complex hydrogel
3. Results and Discussion Therefore, it was decided to select specimen
3.1 Effects of κ-carrageenan solution C with the smallest particle size for storage
concentration and ratio of complex gel to stability study at 4 °C and 25 °C.
xanthone (RHX) 3.2 Physical stability
Table 2 shows the effect of κ- Table 3 shows the effect of storage
carrageenan solution concentration and temperature (4 °C and 25 °C) and time on
RHX on the average particle size and zeta the average size and zeta potential of
potential of specimens C, D and E. specimen C on the physical stability of
Obviously, specimen C with κ-carrageenan xanthone-containing complex hydrogels.
solution concentration of 0.5% and RHX of The average size of the capsules was
7:1 had the smallest particle size (690.5 nm) essentially unchanged after storage at 4 °C
compared to specimen D with κ-carrageenan for 45 days but at 25 °C the size increased
solution concentration of 1% and RHX of by about 16%. In contrast, the zeta potential
9:1, which had slightly larger particle size increased by about 50% over 45 days of
(1643.3 nm). Specimen E with κ- storage at 4 °C and slightly more at 25 °C.
carrageenan solution concentration of 1% The preferred temperature for storage of the
and RHX of 9:1 had the largest particle size complex hydrogels was 4 °C since
of 1839.3 nm. aggregation was reported to occur more
quickly at 25 °C.3
Table 2. Effect of κ-carrageenan solution
concentration and ratio of complex hydrogel Table 3. Effect of storage temperature and
to xanthone on its average particle size and time on average size and zeta potential of
zeta potential specimen C
Specimen Average size Zeta potential
code (nm) (mV) Time Average size (nm) Zeta potential (mV)
C 690.5 24.1± 1.7 (days) 4 °C 25 °C 4 °C 25 °C
D 1643.3 40.7± 7.5
E 1839.3 28.9± 6.4 0 690.5 690.5 24.1± 1.7 24.1± 1.7
30 693.6 772.2 28.2± 2.3 34.3± 3.4
45 686.8 802.4 32.5± 2.5 38.2± 15
On the other hand, the κ-carrageenan
solution concentration (0.5 vs. 1%) was
found to have more effect on the zeta
potential than that of RHX (7:1 vs. 9:1).
Prego et al.2 have suggested that an increase
in particle size indicates attachment of
polymer molecules of κ-carrageenan to the
surface of the oil core. Therefore, it may be
considered that the increase in average size
of the complex hydrogels with κ-
carrageenan indicated that κ-carrageenan
was preferably located on the surface of the
chitosan polymer mainly due to their
electrostatic attraction, which as a resut may
be useful for stabilization of the xanthone oil
core.3
It was also found in our preliminary
screening that, in the case of specimens with
Figure 3. SEM micrographs of xanthone-
chitosan solution concentration of 0.4%, a
containing complex hydrogels in Specimens
higher TPP concentration resulted a bigger
A, B and C
average particle size, which is consistent
with the finding of Dhudani et al.5,6
3.3 Microstructure Acknowledgement
Figure 3 (A, B and C) shows the The authors are grateful to King
general shape of the complex hydrogel Mongkut's University of Technology North
network for specimens A, B and C, Bangkok (Rayong Campus) and Center of
respectively. It could be confirmed from the Excellence in Particle Technology,
SEM images that droplets of about 1 μm in Chulalongkorn University, for use of their
diameter appeared in the stub specimens. lab facilities.
Obviously, the most interesting specimen C
had a high concentration of fine droplets of References
xanthone-containing hydrogels. 1. Machmudah, S.; Yasa, Q.; Shiddiqi, A.;
Kharisma, A. D.; Kanda, H.; Winardi, S.;
4. Conclusion Goto, M. J. Adv. Chem. Eng. 2015, 5,
Complex hydrogels composed of 117.
chitosan, sodium tripolyphosphate and κ- 2. Prego, C.; Torres, D.; Fernandez, M. E.;
carrageenan as xanthone-encapsulating Novoa-Carballal, R.; Quiñoá, E.; Alonso,
material were prepared. The smallest M. J. J. Control. Release. 2006, 111, 299-
average size of 690.5 nm was observed in 308.
Specimen C which contained 3% chitosan, 3. Lertsutthiwong, P.; Noomun, K.;
0.3% xanthone, 0.5% κ-carrageenan and Jongaroonngamsang, N.; Rojsitthisak, P.;
0.2% TPP while the ratio of hydrogel to Nimmannit, U. Carbohydr. Polym. 2008,
xanthone was 7:1. In addition, Specimen C 74, 209-214.
exhibited very good and fairly good particle 4. Kailakua, S. I.; Mulyawantia, I.;
size stability at 4 °C and 25 °C, respectively. Alamsyah, A. N. Procedia Chem. 2014,
However, its zeta potential increased from 9, 235 – 241.
24.1 to 28.2 and 32.5 mV, respectively, after 5. Dhudani, A. R.; Kosaraju, S. L.
30- and 45-day storage at 4 °C. Carbohydr. Polym. 2010, 81, 243-251.
6. Tanthapanichakoon, W.; Sowasod, N.;
Charinpanitkul, T. Ceram. Trans. 2007,
198, 185–192.
Perylene diimide functionalized with fused-thiophenes for organic field
effect transistors: synthesis and electronic properties
Anna Pachariyangkun, Wiyada Saennawa, Taweesak Sudyoadsuk,
Pichaya Pattanasattayavong, Vinich Promarak*
Vidyasirimedhi Institute of Science and Technology, Wang Chan, Rayong 21210, Thailand
*E-mail: vinich.p@vistec.ac.th, s15_anna.p@vistec.ac.th
Abstract:
Perylene diimides (PDIs) have emerged as an interesting material in organic
electronics due to their excellent chemical and physical properties such as its high electron
affinity, charge mobility, as well as absorptivity. Two new perylene diimide derivatives: 2,9-
bis(2-ethylhexyl)-6,13-bis(thieno[3,2-b]thiophen-2-yl)-5a,5a1-dihydroanthra[2,1,9-def:6,5,10
-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetraone (PDI-T) and 5,12-bis(dithieno[3,2-b:2',3'-
d]thiophen-2-yl)-2,9-bis(2-ethylhexyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-
1,3,8,10- (2H, 9H)-tetraone (PDI-DT), achieved by functionalizing the basic perylene core
at the bay positions with fused-thiophenes, have been synthesized. Both compounds were
investigated for a potential application as n-type semiconductor in organic field effect
transistors (OFETs).
1. Introduction deficient (n-type) material. Excellent

Development of organic chemical and physical properties of PDI
semiconductors, specifically organic field make it a promising class of electron
effect transistors (OFETs), has been accepting and electron transporting materials
researched intensively due to their great which are used in various applications such
potential in the field of “plastic electronics” as light harvesting arrays,6 field effect
in terms of production cost, light-weight transistors,7 light-emitting diodes,8 and
substrates, and large area production.1,2 The photovoltaics.9
key to high performance OFETs is to design In this study, two perylene diimide
and synthesize molecules with high derivatives PDI-T and PDI-DT were
planarity, good stability, high charge carrier investigated as n-type semiconductor. Their
mobility, large on/off ratio, and low optical, electrochemical, thermal, structural,
threshold voltage.3,4 A field effect transistor and morphological properties were studied
is commonly used as a signal amplifier or as using different measurements.
a switch. In an OFET, current flows from
one electrode, called source, to another 2. Materials and Methods
electrode, called drain, along the organic 2.1 Materials
semiconductor layer called channel by All reagents were purchased from
controlling the gate electrode.1 Both p-type Aldrich, Acros, or Tokyo Chemical Industry
and n-type organic semiconductors are and used without further purification. All
needed in the industrial applications, solvents were supplied by Thai companies
however, the development of n-type organic and used without further distillation.
semiconductor material falls far behind p- Tetrahydrofuran (THF) was distilled from
type organic semiconductor.5 sodium/benzophenone under nitrogen
Perylene diimide (PDI), with five atmosphere prior to use. Dichloromethane
conjugated benzene rings as its framework for cyclic voltammetry (CV) experiment
and carboxylic imide, is an ideal electron
was washed with conc. H2SO4 and distilled 2.4 Synthesis of PDI-T
twice from calcium hydride. PDI-di-Br (200 mg, 0.26 mmol),
2.2 Synthesis of PDI thieno[3,2-b]thiophen-2-ylboronic acid (104
Perylene-3,4,9,10-tetracarboxylic mg, 0.57 mmol), Pd(PPh3)4 (15 mg, 0.01
dianhydride (PTCDA) (5.00 g, 12.75 mmol), mmol), and 2M Na2CO3 (2.58 mL) were
2-ethyl hexylamine (8.32 mL, 51.00 mmol), added into a 100 mL round-bottom flask.
and zinc acetate (2.79 g, 12.75 mmol) were THF (40 mL) was added as a solvent then
added into a 250 mL round-bottom flask. the mixture was stirred and degassed with
Quinoline (150 mL) was added as solvent N2 for 5 min. The mixture was stirred at
and the mixture was stirred and refluxed reflux under N2 for 24 h. After cooling, the
under N2 for 3 h at 180 °C. The reaction mixture was extracted with CH2Cl2 (70 mL
mixture was cooled and poured into 1 M x 2). The combined organic phase was
HCl (20 mL) to form precipitate. The washed with water (70 mL), brine solution
precipitate was collected by filtration and (70 mL), dried over anhydrous Na2SO4,
washed with water and MeOH. The obtained filtered, and evaporated to dryness. The
solid product was dried in vacuum. The residue was further purified by silica gel
crude was further purified by silica gel column chromatography eluting with
column chromatography eluting with mixture of CH2Cl2 and hexane (3:1) to
CH2Cl2 to obtain PDI as dark red solids obtain PDI-T as dark purple solids (22%).
(93%). 1H NMR (600 MHz, CDCl3) δ ppm: 1
H NMR (600 MHz, CDCl3) δ ppm: 8.72 (s,
8.57 (d, J = 7.8 Hz, 2H), 8.46 (d, J = 7.8 2H), 8.24 (q, 4H), 7.55 (s, 2H), 7.51 (d, J =
Hz, 2H), 4.08-4.17 (m, 2H), 1.95-1.97 (m, 5.28 Hz, 2H), 7.30 (d, J = 5.22 Hz, 2H),
1H), 1.32-1.41 (m, 8H), 0.95 (t, J = 7.8 Hz, 4.08-4.16 (m, 4H), 1.92-1.95 (m, 2H), 1.30-
2H), 0.89 (t, J = 7.2 Hz, 3H); MALDI-TOF 1.42 (m, 16H), 0.93 (t, J = 7.38 Hz, 6H),
(m/z) (M+) calcd. for C40H42N2O4: 614.3145, 0.88 (t, J = 7.02 Hz, 6H); 13C NMR (151
found 615.3441. MHz, CDCl3) δ 163.55, 163.44, 145.25,
2.3 Synthesis of PDI-di-Br 140.92, 140.14, 135.68, 134.26, 133.21,
PDI (7.30 g, 11.88 mmol) and FeCl3 132.90, 129.89, 129.84, 129.02, 128.80,
(9.67 g, 59.40 mmol) were added into a 250 127.79, 122.31, 122.18, 119.88, 44.40,
mL round-bottom flask. CHCl3 (70 mL) was 37.97, 30.77, 28.70, 24.05, 23.08, 14.10,
added as a solvent. Br2 (11.64 mL) diluted 10.64; MALDI-TOF (m/z) (M+) calcd. for
with CHCl3 (50 mL) was added dropwise C52H48N2O4S4: 892.2497, found 892.3911.
via additional funnel. The mixture was 2.5 Synthesis of PDI-DT
stirred at reflux for 24 h. After cooling, PDI-di-Br (200 mg, 0.26 mmol),
sodium thiosulphate was added to the dithieno[3,2-b:2',3'-d]thiophen-2-ylboronic
mixture to remove excess bromine. The acid (136 mg, 0.57 mmol), Pd(PPh3)4 (15
mixture was stirred for another 24 h at room mg, 0.01 mmol), and 2M Na2CO3 (2.58 mL)
temperature. The mixture was evaporated to were added into a 100 mL round-bottom
dryness and the residue was further purified flask. THF (40 mL) was added as a solvent
by silica gel column chromatography eluting then the mixture was stirred and degassed
with mixture of CH2Cl2 and hexane (2:1) to with N2 for 5 min. The mixture was stirred
obtain PDI-di-Br as red solids (68%). 1H at reflux under N2 for 24 h. After cooling,
NMR (600 MHz, CDCl3) δ ppm: 9.44 (d, J the mixture was extracted with CH2Cl2 (70
= 8.16 Hz, 2H), 8.89 (s, 2H), 8.66 (d, J = mL x 2). The combined organic phase was
8.16 Hz, 2H), 4.09-4.20 (m, 4H), 1.94-1.99 washed with water (70 mL), brine solution
(m, 2H), 1.31-1.41 (m, 16H), 0.95 (t, J = (70 mL), dried over anhydrous Na2SO4,
7.38 Hz, 6H), 0.90 (t, J = 7.02 Hz, 6H); filtered, and evaporated to dryness. The
MALDI-TOF (m/z) (M+) calcd. for residue was further purified by silica gel
C40H40Br2N2O4: 770.1355, found 770.8993. column chromatograph y eluting with
mixture of CH2Cl2 and hexane (1:1) to carried out under inert argon atmosphere
obtain PDI-DT as dark green solids (35%). with an Autolab potentiostat PGSTAT 101
1
H NMR (600 MHz, CDCl3) δ ppm: 8.76 (s, using platinum counter electrode, glassy
2H), 8.34 (d, J = 8.16 Hz, 2H), 8.26 (d, J = carbon working electrode, and Ag/AgCl
8.16 Hz, 2H), 7.60 (s, 2H), 7.46 (d, J = 5.28, reference electrode in distilled CH2Cl2 with
2H), 7.36 (d, J = 5.28, 2H), 4.07-4.16 (m, tetra-n-butylammoniumhexafluorophosphate
4H), 1.93-1.95 (m, 2H), 1.28-1.40 (m, 16H), (n-Bu4NPF6) as a supporting electrode at a
0.93 (t, J = 7.32 Hz, 6H), 0.88 (t, J = 7.02 scan rate of 50 mV s-1. High resolution
Hz, 6H); 13C NMR (151 MHz, CDCl3) δ MALDI-TOF mass spectra were recorded
163.51, 143.73, 142.28, 142.25, 135.74, with a Bruker Autoflex speed mass
134.40, 133.04, 132.95, 132.68, 131.04, spectrometer. X-ray diffraction (XRD) of
130.03, 129.84, 129.21, 127.98, 127.26, organic semiconductor powder were
122.45, 122.30, 121.30, 120.86, 53.41, measured on a Bruker New D8 Advance
44.39, 37.96, 30.75, 29.70, 28.69, 24.04, diffractometer. The detector was moved by
23.08, 14.11, 10.64; MALDI-TOF (m/z) 2θ steps of 0.02°. Morphologies of PDI-T
(M+) calcd. for C56H48N2O4S6: 1004.1938, and PDI-DT films were investigated using
found 1004.0329. Park Systems NX-10 atomic force
2.6 Measurements microscope (AFM) using true non-contact
1
H and 13C spectra were recorded on mode and NCHR cantilever. Differential
a Bruker Avance III HD 600 MHz scanning calorimetry (DSC) measurements
spectrometer using CDCl3 as solvent in all were carried under nitrogen atmosphere
cases. UV-Vis spectra were recorded as a using Perkin Elmer DSC-8500 thermal
dilute solution in a spectroscopic grade analyzer at a heating rate of 10 °C/min.
dichloromethane on a Perkin-Elmer Lambda
1050 spectrometer. CV measurements were
O N O
S
S S
S
O O O O N O (iii) O N O
(i) R PDI-T
R
O O O O N O O N O
(iv)
S
PTCDA
S
S S
S
PDI : R = H
S
(ii)
O N O
PDI-di-Br : R = Br
PDI-DT
Figure 1. Synthesis route to PDI-T and PDI-DT. Reagents and conditions: (i) Zn(OAc)2, 2-
ethyl hexylamine, quinoline, 180 °C, 3 h. (ii) FeCl3, Br2, CHCl3, 60 °C, 24 h. (iii) thieno[3,2-
b]thiophen-2-ylboronic acid, Pd(PPh3)4, 2M Na2CO3, THF, 65 °C, 24 h. (iv) dithieno[3,2-
b:2',3'-d]thiophen-2-ylboronic acid, Pd(PPh3)4, 2M Na2CO3, THF, 65 °C, 24 h
3. Results and Discussion is shown in Figure 3. 10 mg of the
3.1 Synthesis of PDI-T and PDI-DT synthesized compounds and 350 mg of n-
The desired PDI derivatives PDI-T Bu4NPF6 of supporting electrode were
and PDI-DT were synthesized in three steps dissolved in distilled dichloromethane. The
as illustrated in Figure 1. Imidization of energies of the HOMO and LUMO levels of
PTCDA with 2-ethyl hexylamine at the both compounds were deduced from these
presence of zinc acetate as catalyst gave PDI measurements and are reported in Table 1.
dark red solids with improved solubility due
to alkyl chains. Bromination of the resultant
with bromine in chloroform produced 1,7
dibrominated PDI regioisomers which are
widely used as starting materials for a broad
variety of PDI derivatives.10,11 PDI-di-Br
was then coupled with thieno[3,2,-b]
thiophene-2-boronic acid and dithieno[3,2-
b:2’,3’-d]thiophene-2-boronic acid via
Suzuki-Miyaura and Sonogashira cross-
coupling to afford PDI-T and PDI-DT
respectively. Both PDI-T and PDI-DT are
soluble in all common organic solvents at
room temperature and its structure was Figure 2. UV-vis absorption spectra of PDI-
confirmed by 1H and 13C NMR and mass T and PDI-DT solutions (CH2Cl2) and thin
spectrometry. films (CHCl3: toluene)
3.2 Optical and electrochemical studies
The optical properties of PDI-T and
PDI-DT were investigated UV-vis
spectroscopy in a dilute CH2Cl2 solution and
thin film shown in Figure 2. Both PDI-T
and PDI-DT showed similar absorption
pattern, but PDI-DT showed more red-
shifted absorption due to the extended π-
conjugation.12 In the solid state (thin films
spin-coated from CHCl3: toluene solutions),
both PDI-T and PDI-DT showed a
significant red shift in its absorption onset,
which can be attributed to solid-state
packing effects. The electrochemical
properties of PDI-T and PDI-DT were Figure 3. Cyclic voltammograms measured
investigated using cyclic and differential in dichloromethane with n-Bu4NPF6 as a
pulse voltammetry (DPV) in supporting electrolyte at a scan rate of 50
dichloromethane and the voltammogram mV s-1
Table 1. Optical and electrochemical properties of PDI-T and PDI-DT

Compound HOMOa (eV) LUMOb (eV) Egc (eV) λabs sol/filmd (nm) Tme (°C)
PDI-T -5.7 -3.84 1.86 284, 444, 584/ 287, 463, 594 340
PDI-DT -5.6 -3.84 1.76 324, 467, 611/ 327, 486, 633 375
a, b
HOMO = -(4.44 + Eonset (oxidation)), LUMO = Eg + HOMO13
c
Band gap calculated from Eg = 1240/λonset
d
Measured in CHCl2 solutions; thin film spin coated from CHCl3: toluene (1:1) solutions
e
Determined from DSC calculations 10 °C/min under N2
3.3 Structural and morphological studies square (RMS) of 0.97 nm and 3.8 nm for
XRD measurements of PDI-T and PDI-T and PDI-DT respectively. The
PDI-DT powder performed by Bruker New pinholes could be attributed to the volatility
D8 Advance diffractometer is shown in at ambient conditions of chloroform
Figure 4. Both compounds showed resulting in too rapid solvent evaporation.
crystallinity which could induce ideal carrier Therefore, it is desirable to use higher
transport as charge transport in organic boiling point solvents which allows a better
semiconductors is dependent in control of the processing conditions.
the intermolecular dynamics of charge Chlorobenzene was selected for film spin
carriers. A degree of perfect molecular order coating solvent. Fig. 5c showed PDI-DT
is crucial for electrical conductivity. film with reduced pinholes and RMS of 0.60
nm. Still, the optimized condition for spin
coating must be investigated further.
4. Conclusion
In summary, we have successfully
synthesized two n-type organic
semiconductors, PDI-T and PDI-DT via
Suzuki coupling reactions. PDI-T and PDI-
DT showed similar optical and
electrochemical properties. Crystallinity of
both compounds make it an ideal n-type
semiconductor for OFETs. Morphologies of
the thin film spin coated from chlorobenzene
showed lower pinholes than thin film spin
Figure 4. XRD patterns of PDI-T and PDI-
coated from CHCl3: toluene (1:1) solution
DT powder
due to the slower evaporation rate allowing
the molecules to rearrange and form
AFM images of PDI-T and PDI-DT
smoother films. Thus, appropriate spin
thin films spin coated from different
coating and annealing conditions is essential
conditions are shown in Figure 5. Fig. 5a
for enhancing the crystallinity of the films.
and 5b showed thin films spin coated from
The optimization of evaporation and
chloroform: toluene (1:1) under ambient
solution techniques will be investigated
condition with a typical spinning rate of
further to obtain the ideal film morphology
1000 rpm and then annealed at 100 °C. Both
of the organic semiconducting layer.
AFM images showed rough surface
morphology with pinholes and root mean
Figure 5. AFM images of thin films (a) PDI-T from chloroform: toluene (b) PDI-DT from
chloroform: toluene (c) PDI-DT from chlorobenzene
References 8. Ego, C.; Marsitzky, D.; Becker, S.;
1. Wu, W.; Liu, Y.; Zhu, D. Chem. Soc. Zhang, J.; Grimsdale, A. C.; Müllen, K.;
Rev. 2010, 39 (5), 1489-1502. MacKenzie, J. D.; Silva, C.; Friend, R. H.
2. Forrest, S. R. Nature 2004, 428 (6986), J. Am. Chem. Soc. 2003, 125 (2), 437-
911-918. 443.
3. Wang, M.; Ford, M.; Phan, H.; Coughlin, 9. Schmidt-Mende, L.; Fechtenkötter, A.;
J.; Nguyen, T. Q.; Bazan, G.C. Chem. Müllen, K.; Moons, E.; Friend, R. H.;
Commun. 2016, 52 (15), 3207-3210. MacKenzie, J. Sciences 2001, 293
4. Cheng, H.; Huai, J.; Cao, L.; Li, Z. Appl. (5532), 1119-1122.
Surf. Sci. 2016, 378, 545-551. 10. Würthner, F., Chem. Commun. 2004,
5. Zhao, Y.; Guo, Y.; Liu, Y. Adv. Mat. (14), 1564-1579.
2013, 25 (38), 5372-5391. 11. Wasielewski, M. R., J. Org. Chem.
6. Kelley, R. F.; Shin, W. S.; Rybtchinski, 2006, 71 (14), 5051-5066.
B.; Sinks, L. E.; Wasielewski, M.R. J. 12. Sugiyasu, K.; Fujita, N.; Shinkai, S.
Am. Chem. Soc. 2007, 129 (11), 3173- Angew. Chem. 2004, 116 (10), 1249-
3181. 1253.
7. Schmidt, R.; Ling, M. M.; Oh, J. H.; 13. Muenmart, D.; Prachumrak, N.;
Winkler, M.; Könemann, M.; Bao, Z.; Tarsang, R.; Namungruk, S.;
Würthner, F. Adv. Mat. 2007, 19 (21), Jungsuttiwong, S.; Sudyoadsuk, T.;
3692-3695. Pattanasattayavong, P.; Promarak, V.
RSC Adv. 2016, 6 (44), 38481-38493.
Removal of Cd2+ and Pb2+ ions by aluminium oxide and the
prepared aluminium oxide nanoparticles
Saytanar Tun1*, Nay Thwe Kyi2, Kyaw Naing3
1
Department of Chemistry, University of Yangon, Myanmar, 2Pro Rector, Dagon University,
3
Deputy Director General, Department of Higher Education, Myanmar
*E-mail: decembersaytanar@gmail.com
Abstract:
Aluminium oxide (Al2O3) is one of the most versatile ceramic oxides. In this
study, aluminium oxide nanoparticles were synthesized by sol-gel assisted auto-combustion
method and were characterized by using TG-DTA, XRD, FT IR and FE-SEM
techniques. The resulted γAl2O3 nanoparticles were used to remove of Cd2+ and Pb2+
ions from model solution and contaminants in wastewater at different contact time and
dosage. Then the concentration of Cd2+ and Pb2+ ions in the solution after
adsorption were determined by atomic absorption spectrophotometer (AAS). These
observations clearly indicated that the removal of metal ions purely depends on the
amount of adsorbent and contact time. The comparison of adsorption properties of
Al2O3 and prepared γ Al2O3 nanoparticles were studied. Al2O3 and the prepared Al2O3
nanoparticles were be compared in removal of Cd2+ and Pb2+ ions under identical
conditions from wastewater, 96.31 % for Cd2+ ion and 56.81% for Pb2+ ion were
removed by Al2O3. The prepared γ Al2O3 nanoparticles can completely remove Cd2+
and Pb2+ ions contaminants in 100 mL wastewater. Therefore, the prepared γAl2O3
nanoparticles were more powerful than Al2O3.

Nano is one billionth of a meter. In All the chemicals used were obtained
Greek, nano means dwarf. Particles of from reagent (BDH).The apparatus used
dimension of about 1 to 100 nanometers are consist of TG-DTA (Rigaku Thermoplus TG
treated as particles. Nanotechnology is the 8120), XRD (Regaku, D/max 2200, Japan),
application of scientific knowledge for the FT IR (Perkin–Elmer (Spectrum GX) fourier
purpose of producing such particle and transform infrared spectrophotometer), FE-
system using them.1 Nano-alumina is one of SEM (EVO 60, Brand: Cards ZEISS,
the most important and extensively explored Germany) and AAS (Perkin-Elmer Analyst
ceramic materials widely used as catalysts or 800). Aluminium oxide nanoparticles were
catalyst supports for chemical reactions, prepared by sol-gel synthesis using
electrical insulators, structural composites aluminium nitrate and citric acid in alkaline
for spacecraft, abrasive and thermal wear solution. Figure 1 show flow chart for sol-
coatings, membrane applications as well as gel assisted auto-combustion synthesis of
adsorbent for water and wastewater Al2O3 particles.
treatment.2
Table 1. TG-DTA data of alumina gel
Temperature Weight TGA DTA
Range (ºC) loss(%) Remark Remark
29.86– 225.13 17.22 Dehydration Endo peak

related to
99 ºC
loss of
hydrated
water and
solvent
225.13– 71.30 Decomposi- Exo peak
597.73 tion related to
273 ºC decomposi
tion of
organic
matter
29.86– 596.88 88.52 Combustion Exo peak
Figure 1. Flow chart for sol-gel assisted 385 ºC due to
auto-combustion synthesis of Al2O3 particles combustio
n of
residual
3. Results and Discussion organic
3.1 TG-DTA thermogram of Al2O3 gel matter
Figure 2 shows TG-DTA
thermogram of alumina gel. Table 1 show 3.2 XRD studies on calcinated products of
TG-DTA data of alumina gel. Al2O3 nanoparticles
Figure 3 shows changes of XRD
patterns of alumina with different
calcination temperatures. According to XRD
data, phase changes were observed in
alumina products calcinated at different
temperatures. Up to 800°C, the alumina was
amorphous nature. After calcination at 900
°C, γAl2O3 phase was appeared. After
calcination at 1000 and 1100 °C, the peaks
related to γ-Al2O3 phase are become smaller.
After calcination at 1150°C, the residual
γAl2O3 phase is completely disappeared and
pure αAl2O3 phase was obtained. XRD data
of the γAl2O3 nanoparticles were showed
characteristic peaks related to miller indices
of 111, 311, 222, 400, 331, 422, 511 and
440, these peaks are well matched with
standard library data of (JCPDS 79-1558>
Al2O3). By using Scherrer equation,
crystalline sizes of the Al2O3 nanoparticles
obtained at calcination temperature of 900,
Figure 2. TG-DTA thermogram of alumina 1000, 1100 and 1150 °C were 35.15, 37.52,
gel 45.77 and 48.10 nm, respectively. Therefore
crystalline size increased with calcination
temperature.
3.3 FTIR spectrum of Al2O3 nanoparticles
In this Figure 4 shows FT IR
spectrum of Al2O3 nanoparticle obtained
after calcinations at 900 °C. FT IR data of
Al2O3 nanoparticles was clearly indicated
the presence of strong peaks at 594, 462 and
432 cm-1. These peaks were related with
symmetric and asymmetric vibration of Al-
O bond in Al2O3 nanoparticle.
Figure 4. FTIR spectrum of γAl2O3

nanoparticles (400-2000 cm-1) calcinated at
900 °C
3.4 FE-SEM microphotograph of γAl2O3

nanoparticles
Figure 5 shows FE-SEM micro-
photograph of γAl2O3 nanoparticles. In FE-
SEM microphotograph, flake-like structures
of Al2O3 nanoparticles were observed.
Figure 5. FE-SEM microphotograph of

Figure 3. Changes of XRD patterns of
γAl2O3 nanoparticles calcinated at 900 °C
Al2O3 nanoparticles with different
calcinations temperatures
3.5 Effect of contact time on adsorption of of γAl2O3 nanoparticles and the best removal
cadmium ion percent was found to be 74.20 %.
Nanoalumina has several crystalline
form and mainly used as γ-alumina form. γ- Table 3. Effect of dosage of Cd2+ Ion
Al2O3 nanoparticles is a suitable material as removal on γAl2O3 nanoparticles
an adsorbent because of its large specific Weight of [Cd2+] at
Removal
surface area, high adsorption capacity, No. γAl2O3 equilibrium
percent (%)
mechanical strength, and low-temperature NPs (g) (ppm)
modification. In this research, adsorption of 1 0.1 5.35 46.45
cadmium ion on aluminium oxide 2 0.2 5.26 47.35
nanoparticles was studied using 10 ppm 3 0.3 4.92 50.79
model cadmium sulphate solution at
4 0.4 4.00 59.91
different contact time. The changes of
5 0.5 2.57 74.20
removal percent of cadmium were shown in 2+
Vol. of Cd ion (10 ppm) solution = 20 mL
Table 2. From these observations, it was Time = 180 min
found that cadmium removal percent
increased with an increase in contact time, 3.7 Removal of cadmium ion in
but removal percent was not evident at 210 wastewater by using Al2O3
min. Therefore the optimum contact time In this research, cadmium ion
was 180 min for Cd2+ ion. removal in wastewater was carried out with
two dosage of Al2O3. It was found that
Table 2. Effect of contact time of Cd2+ ion cadmium ion removal percents increased
removal on γAl2O3 nanoparticles with weight of Al2O3. Table 4 show
[Cd2+] at cadmium removal percents in wastewater by
Contact time Removal
No. equilibrium using Al2O3.
(min) percent (%)
(ppm)
1 30 6.83 31.64 Table 4. Cadmium removal percents in
2 60 6.25 37.50 wastewater by using Al2O3
3 90 5.63 43.64 Weight Initial [Cd2+] Removal
4 120 5.35 46.42 No. of Al2O3 [Cd2+] after Percent
(g ) (ppm) removal (%)
5 150 4.73 52.62
byAl2O3
6 180 4.03 59.62 (ppm)
7 210 4.00 59.98
2+ 1 0.5 3.26 0.97 70.25
Vol. of Cd ion (10 ppm) solution = 20 mL
γAl2O3 NPs = 0.1 g 2 1.0 3.26 0.12 96.31
Wastewater = 100 mL
3.6 Effect of dosage on adsorption of Contact time = 180 min
cadmium ion
The adsorption capacities of 3.8 Removal of cadmium ion in
prepared γAl2O3 nanoparticles were studied wastewater by using γAl2O3 nanoparticles
by using model cadmium sulphate solution. In this research, cadmium ion
Different dosages of γAl2O3 nanoparticles removal in wastewater was carried out with
were stirred in 10 ppm model solution for two dosages of γAl2O3 nanoparticles. It was
180 min and determined the final found that cadmium ion removal percents
concentration of cadmium using atomic increased with weight of γAl2O3
absorption spectrophotometer (AAS). Table nanoparticles. Table 5 shows cadmium
3 shows the effect of dosage of Cd2+ ion removal percents in wastewater by using
removal on γAl2O3 nanoparticles. It can be γAl2O3 nanoparticles. The higher dosage of
seen that the maximum cadmium removal γAl2O3 nanoparticles can remove completely
percent increased with an increasing dosage cadmium ion contaminant in wastewater.
Table 5. Cadmium removal percents in absorption spectrophotometer (AAS). Table
wastewater by using γAl2O3 nanoparticles 7 shows effect of dosage of Pb2+ ion
Weight Initial [Cd2+] Removal removal on γAl2O3 nanoparticles. It can be
No. of [Cd2+] after Percent seen that the maximum lead removal percent
γAl2O3 (ppm) removal (%)
increased with an increasing dosage of
NPs (g) by γAl2O3
NPs (ppm) γAl2O3 nanoparticles and the best removal
percent was found to be 96.91%.
1 0.5 3.26 0.04 82.40
2 1.0 3.26 0.00 100.00 Table 7. Effect of dosage of Pb2+ ion
removal on γAl2O3 nanoparticles
Wastewater = 100 mL
Contact time = 180 min Weight of [Pb2+] at
Removal
No. γAl2O3 NPS equilibrium
percent (%)
3.9 Effect of contact time on adsorption of (g) (ppm)
lead ion
1 0.1 0.87 91.28
In this research, lead ion removal by
γAl2O3 nanoparticles using 10 ppm model 2 0.2 0.72 92.72
lead nitrate solution at different contact 3 0.3 0.57 94.26
time. The change of removal percent of 4 0.4 0.44 95.58
cadmium were shown in Table 6. From
these observations, it was found that lead ion 5 0.5 0.30 96.91
2+
removal percent increased with an increase Vol. of Pb ion (10 ppm) solution = 20 mL
in contact time, but removal percent was not Time = 150 min
evident at 150 to 210 min. Therefore the
3.11 Lead ion removal in wastewater by
optimum contact time was 150 min for Pb2+
using Al2O3
ion.
In this experiment, lead ion removal
in wastewater was carried out with two
Table 6. Effect of contact time of Pb2+ ion
dosage of active Al2O3. Tables 8 shows lead
removal on γAl2O3 nanoparticles
removal by using Al2O3. It was found that
[Pb2+] at lead removal percents increased with weight
Contact time Removal
No. equilibrium of Al2O3.
(min) percent (%)
(ppm)
3.12 Lead ion removal in wastewater by
1 30 0.96 90.37 using γAl2O3 nanoparticles
2 60 0.83 91.67 In this experiment, lead ion removal
3 90 0.73 92.64
in wastewater was carried out with two
4 120 0.50 94.99 dosages of Al2O3 nanoparticles. Tables 9
5 150 0.41 95.90 shows lead ion removal percent in
6 180 0.40 95.92 wastewater by using Al 2 O3 nanoparticles.
7 210 0.40 95.94
2+
Vol. of Pb ion (10 ppm) solution = 20 mL
Table 8. Lead removal by using Al2O3
γAl2O3 = 0.1 g Weight Initial [Pb2+] Removal
3.10 Effect of dosage on adsorption of No. of [Pb2+] after Percent
Al2O3 (ppm) removal (%)
lead ion
(g) by Al2O3
In the present work, adsorption (ppm)
capacity of prepared γAl2O3 nanoparticles
was studied by using model lead nitrate 1 0.5 4.33 2.95 31.79
solution. Different dosages of γAl2O3 2 1.0 4.33 1.87 56.81
nanoparticles were stirred in 10 ppm model Wastewater = 100 mL
solution for 180 min and determined the Contact time = 150 min
final concentration of lead using atomic
It was found that lead ion removal percents removed by one gram Al2O3 and the
increased with weight of Al2O3 prepared one gram γAl2O3 nanoparticles
nanoparticles. The higher dosage of Al2O3 were completely removed Cd2+ ion and Pb2+
nanoparticles can remove completely lead ion contaminants in 100 mL wastewater.
ion contaminant in wastewater. The outcome of these researches is that the
prepared γAl2O3 nanoparticles can be used
Table 9. Lead removal percents by using for heavy metal remediation in wastewater
γAl2O3 nanoparticles and water treatment for domestic purposes
Weigh Initial [Pb2+] Removal as powerful adsorbents.
No. t of [Pb2+] after Percent
γAl2O3 (ppm) removal (%) Acknowledgements
NPs by Al2O3 I would like to express my deep
(g) NPs
(ppm)
appreciation to Professor Dr. Hinn Hinn
Aye, Head of Department of Chemistry,
1 0.5 4.33 1.76 98.77 University of Yangon for her kind and
2 1.0 4.33 0.00 100.00 constant encouragement. I wish to express
Wastewater = 100 mL my profound gratitude to my research
Contact time = 150 supervisor, Dr. Kyaw Naing, Deputy
Director General, Department of Higher
4. Conclusion Education, Myanmar for his kind guidance,
In this study, aluminium oxide helpful advice, comments, encouragement,
nanoparticles were synthesized by sol-gel and numerous invaluable suggestions. I wish
assisted auto-combustion method. The to convey my sincere thanks to Dr. Nay
prepared γAl2O3 nanoparticles were Thwe Kyi, Pro-Rector, Dagon University
characterized by using TG-DTA, XRD, from the deep of my heart for her close
FT-IR and FE-SEM techniques. The resulted supervision, patient, invaluable suggestions,
γAl2O3 nanoparticles were used to remove guidance and encouragement.
of Cd2+ ion and Pb2+ ion from modal
solution and contaminants in wastewater and References
was studied the comparison of adsorption 1. Sheeparamatti, B. G.; Sheeparamatti, R.
properties of the prepared γAl2O3
B.; Kadadevaramath, J.S. IETE Tech.
nanoparticles and Al2O3. In this work, the
Al2O3 and the prepared γAl2O3 nanoparticles Rev. 2007, 24, 5-8.
can be compared removal of Cd2+ ion and 2. Banerjee, S.; Dubey, S.; Gautam, R. K.;
Pb2+ ion under identical conditions, 96.31% Chattopadhyaya, M. C.; Sharma, Y. C.
of Cd2+ ion and 56.81% of Pb2+ ion were Arabian J. Chem. 2017, 1-16.
Fabrication and characterization of fouling-resistant membrane based on
polyamide thin film composite incorporated with
functionalized graphene oxide
Nuntanat Worrakunpiset, Chavakorn Samthong, Anongnat Somwangthanaroj,
Chalida Klaysom*
*E-mail: chalida.kl@chula.ac.th
Abstract:
Fouling is one of the biggest challenges in membrane technology. Developing
advanced membranes that are resistant to foulants and easy to recover is thus essential. A
fouling-resistant membrane could be achieved by carefully controlled charge, roughness and
hydrophilicity of the membrane surface. In this work, a thin film nanocomposite membrane
was fabricated based on polyacrylonitrile (PAN) substrate. Polyamide thin film as selective
layer, was synthesized onto the substrate via an interfacial polymerization between m-
phenylenediamine (MPD) and trimesoyl chloride (TMC). In order to enhance membrane
surface hydrophilicity, the selective thin film was incorporated with graphene oxide (GO) and
amine-functionalized graphene oxide (GO-NH2). The effects of filler loadings on membrane
morphology, surface functionality and surface hydrophilicity were investigated by SEM,
ATR-FTIR, AFM, and contact angle goniometer. Results showed that GO addition had an
influence on the interfacial polymerization reaction and surface property of the final
membrane. There was no difference of surface morphology between membranes with and
without GO addition due to the few GO loading onto membrane surface. Nevertheless,
membranes with GO and GO-NH2 exhibited higher surface hydrophilicity, which was
attributed to the oxygen-containing functional groups of GO and GO-NH2 embedded in the
thin film layer. .
1. Introduction is one of the methods for an improvement of

Thin film composite (TFC) membrane fouling-resistant. Among various kinds of
has been mostly used in nanofiltration and nanoparticles, graphene oxide (GO)
reverse osmosis processes. Composing thin incorporation showed remarkable
polyamide selective layer on polymeric performance enhancement of the modified
substrate, TFC membrane presents superior membrane. Due to the oxygenated functional
permeate flux and salt rejection comparing to groups in the structure, GO embedded in the
other membrane types. Nevertheless TFC thin film layer induced more hydrophilicity
membrane contains hydrophobic surface and negative charges on the membrane
inducing foulant molecules to attach on the surface, resulting in flux improvement and
surface, this finally results in flux decline and anti-fouling property of membrane.8
lower separation efficiency.1 Hence, many Polyamide thin film is synthesized
researches have been focused on investigating from interfacial polymerization between two
the membrane surface modification in order reactive monomers that contain amine and
to obtain more hydrophilicity and negative acryl chloride groups1. Thus, adding amine
charges on the surface, making the membrane functionalized GO (GO-NH2) onto thin film
resist to foulant molecules. Incorporation of layer is expected to increase stability of the
nanoparticles such as silica nanoparticles,2-3 embedded GO due to more interaction
aluminium-contained nanoparticles4-5 and between the functionalized GO and the
carbon nanoparticles,6-7 onto thin film layers monomers.
In this study, polyamide thin film degree of polymerization. The substrate was
nanocomposite (TFN) membranes with dipped in the MPD aqueous solution for 30
embedded GO and GO-NH2 were fabricated minutes. After removing PAN substrate
based on polyacrylonitrile (PAN) substrate. from the solution and being air dried for 80
Influence of incorporated GO and GO-NH2 seconds, TMC solution was gently poured
loadings on membrane morphology, surface onto the substrate for interfacial
functionality, and surface hydrophilicity was polymerization of polyamide for further 80
investigated. seconds. The solution was drained off from
the thin film composite membrane following
2. Materials and Methods by rinsing with n-hexane to remove the
2.1 Materials unreacted monomers from the membrane.
Polyacrylonitrile (PAN, MW 150,000 The obtained membrane was kept in DI
g/mol), triethylamine (TEA, ≥99.5%) and water at room temperature.
sodium dodecyl sulfate (SDS, ≥99%), were For GO/GO-NH2 addition, the GO
purchased from Sigma Aldrich, USA. N, N- nanoparticles were added into DI water and
Dimethylformamide (DMF) was purchased ultrasonicated for 30 minutes to disperse the
from QRëC, New Zealand. The novatexx GO nanoparticles. Afterwards, the GO
2470 nonwoven fiber was obtained from dispersed solution was added into the MPD
Viledon – Freudenberg, German. Normal aqueous solution to obtain the final GO
hexane (n-hexane, ≥98.5%) was purchased concentration varied between 0-50 ppm. The
from Carlo Erba, Italy. Trimesoyl chloride preparation procedure of TFN membranes
(TMC, 99.8%) and 1,3-phenylenediamine containing GO/GO-NH2 was the same as
(MPD, 100%), were purchased from Merck mentioned above.
KGaA, German. Graphene oxide and amine 2.4 Membrane characterization
functionalized graphene oxide were kindly Morphology of membrane surface
provided by Prof. Armando T. Quitain, was analyzed by a scanning electron
Department of Applied Chemistry and microscope (SEM, Hitachi S-3400N). Prior
Biochemistry, Kumamoto University, Japan. to SEM sample preparation, membrane
All reagents were analytical grade and were samples were dried at room temperature.
used without further purification. Then, gold sputtering was applied on the
2.2 Preparation of PAN substrate samples to provide high electric conductivity
Polymer solution was prepared by before the investigation. Surface roughness
vigorously stirring 16 wt% of PAN in DMF of membranes was measured using an
at 60°C until the solution became atomic force microscope (AFM, Veeco
homogeneous. The polymer solution was Methodology group, Dimension 3100).
placed at room temperature until there was Chemical composition, the structure
no bubble in the solution. Then, PAN of membranes and the presence of
solution was casted onto the non-woven functionalized GO on membrane surface
backing support using 250 µm casting knife were investigated by attenuated total
before being immersed into a coagulation reflectance Fourier transform infared
bath of DI water to induce phase inversion spectroscopy (ATR-FTIR, Nicolet 6700).
of the solution. The substrate sheet was kept Membrane samples were dried at room
in DI water at 25°C until use. temperature and were scanned at
-1
2.3 Preparation of TFN membranes wavenumber from 400-4000 cm . Surface
2wt% of MPD aqueous solution and hydrophilicity of membrane was
0.15wt% of TMC in n-hexane were prepared characterized by a contact angle goniometer
as monomer solutions for polymerization. In (OCA – 40) by dropping 1 µl of distilled
the aqueous solution, 0.5wt% of TEA and water onto the membrane surface, at five
0.1wt% of SDS were added, to enhance
different positions, and the average contact is too small to have an effect on the structure
angles were examined. of the thin film.

3.1 Characteristics of membranes>>>>>
surface functionality
Presence of GO incorporated onto
thin film layer and the thin film composition
were proved by ATR-FTIR. As shown in
Figure 1, all membranes exhibit amide
characteristic of C=O stretching, aromatic
ring, C−N stretching and N−H bending at
the peaks of 1664, 1610 and 1541 cm-1,
respectively.9 For GO/GO-NH2 addition in
TFN membrane, FTIR shows higher peak
intensity at 3335 cm−1 indicating more -OH
stretching of carboxyl groups of embedded
GO/GO-NH2 on membrane surface.10 Peaks
at 1079–1251 cm−1 correspond to C-O
stretching of the C-OH/C-O-C groups.11 Figure 2. SEM images of PAN support (a)
Moreover, membrane with embedded GO- and polyamide selective layer of TFN
NH2 presents higher intensity of the C=O membrane with 0 ppm GO (b), 25 ppm of
and N−H peaks because of more amide GO (c), 25 ppm of GO-NH2 (d), 50 ppm of
formation between NH2 functionality of GO GO (e), and 50 ppm of GO-NH2 (f) in MPD
and acryl chloride group of TMC. solution
Figure 1. FTIR spectra of TFN membrane

with 0 ppm GO (a), 50 ppm of GO (b), and
50 ppm of GO-NH2 (c) in MPD solution
3.2 Surface morphology and hydrophilic-

city of membranes
Figure 2 illustrates SEM images of
membrane surface with GO/GO-NH2
incorporation, whereas the surface
morphology and roughness of the
membranes are presented in Figure 3. The
two figures show insignificant difference of Figure 3. AFM images of polyamide
membrane morphology and surface selective layer of TFN membrane with 0
roughness after GO addition. This might be ppm GO (a), 25 ppm of GO (b), 25 ppm of
because the amount of GO nanoparticles GO-NH2 (c), 50 ppm of GO (d), and 50 ppm
incorporated in the polyamide thin film layer of GO-NH2 (e) in MPD solution
Table 1. Water contact angle and surface angle goniometer. It was found that small
roughness of the TFN membranes number of GO and GO-NH2 addition had no
effect on the smoothness of membrane
Membrane code Contact angle (°) surface. However, the thin film
TFC 82.4±3.6 nanocomposite membranes with embedded
TFN-50 GO 48.5±1.3 GO-NH2 gave more hydrophilicity than the
TFN-50 GO-NH2 43.1±2.8 original membranes and those with the
pristine GO.
Membrane hydrophilicity was
studied by water contact angle measurement. Acknowledgements
As shown in Table 1, the water contact The authors are grateful to Prof.
angles of membranes with GO/GO-NH2 Armando T. Quitain, Department of Applied
incorporation greatly drop with increasing Chemistry and Biochemistry, Kumamoto
GO concentration implying more University, Japan for supporting GO and
hydrophilicity of the modified membranes GO-NH2 used in this work.
which is attributed to the oxygenated groups
of the GO.12 Furthermore, membranes References
containing GO-NH2 have slightly more 1. Baker, R. W. Membrane Technology
hydrophilic surface than those with pristine and Applications, 3rd ed.; John Wiley &
GO at the same loading. Hence, Sons, Ltd: 2012.
functionalization of amine group on GO 2. Yin, J.; Kim, E. S.; Yang, J.; Deng, B. J.
surface is believed to enhance the GO Membr. Sci. 2012, 423-424, 238-246.
attachment onto the thin film layer due the 3. Wu, H.; Tang, B.; Wu, P. J. Membr. Sci.
improved interaction between the GO 2013, 428, 341-348.
nanoparticles and reactive monomers. More 4. Li, H.; Shi, W.; Zhang, Y.; Du, Q.; Qin,
surface hydrophilicity would lessen foulants X.; Su, Y. Sep. Purif. Technol. 2016,
attachment onto the membrane surface 166, 240-251.
affecting the anti-fouling performance of 5. Saleh, T. A.; Gupta, V. K. Sep. Purif.
membrane. However, in order to confirm the Technol. 2012, 89, 245-251.
stability advancement of GO incorporated 6. Khan, A. U .H.; Khan, Z.; Aljundi, I.H.
onto the thin film layer by NH2 Desalination. 2017, 420, 125-135.
functionalization and the fouling-resistant 7. Zheng, J.; Li, M.; Yu, K.; Hu, J.; Zhang,
enhancement of membrane, more detailed X.; Wang, L. J. Membr. Sci. 2017, 524,
characterizations and membrane 344-353.
performance tests will be further conducted 8. Liu, Q.; Xu, G. R. Desalination. 2016,
and reported in the future work. 394, 162-175.
9. Klaysom, C.; Hermans, S.; Gahlaut, A.;
4. Conclusion Van Craenenbroeck, S.; Vankelecom, I.
Polyamide thin film composite F.J. J. Membr. Sci. 2013, 445, 25-33.
membranes incorporated with amine 10. Shen, L.; Xiong, S.; Wang, Y. Chem.
functionalized graphene oxide were Eng. Sci. 2016, 143, 194-205.
prepared via interfacial polymerization 11. Zhang, H.; Bin, L.; Pan, J.; Qi, Y.; Shen,
between MPD aqueous solution and TMC J.; Gao, C.; Van der Bruggen, B. J.
organic solution. Functionality, surface Membr. Sci. 2017, 539, 128-137.
morphology and hydrophilicity of 12. Hegab, H. M.; Zou, L. J. Membr. Sci.
synthesized membranes were characterized 2015, 484, 95-106.
by ATR-FTIR, SEM, AFM and contact
The morphology of zeolite 13X/PDMS mixed matrix membrane for
flue gas separation
Nampetch Padungpetch1, Chavakorn Samthong1, Kajornsak Faungnawakij2,
Anongnat Somwangthanaroj1, Chalida Klaysom1*
1
2
National Nanotechnology Center (NANOTEC),
National Science and Technology Development Agency, Thailand
*E-mail: chalida.kl@chula.ac.th
Abstract:
The increase of carbon dioxide has led to the climate change and global warming.
Hence, the removal of CO2 is required. The membrane technology is considered as an
efficient and environmentally friendly process for flue gas separation. However, there is still
a limitation for acquiring the promising membranes such as poor CO2 permeability and
CO2/N2 selectivity. Accordingly, this study aims to develop the composite membrane for CO2
separation. The synthesized composite membranes were composed of a support layer of
polyethersulfone (PES) and a thin selective layer of polydimethylsiloxane (PDMS). The
concentration of PDMS solution, coating time and zeolite 13X addition were investigated.
The chemical structure of PDMS coating was confirmed by an attenuated total reflectance
(ATR) - Fourier transform infrared spectroscopy (FTIR). The morphology of membrane was
characterized by scanning electron microscopy (SEM), which evaluated thickness of a
selective layer and dispersion of filler. The results showed that the thickness of a selective
layer increased with an increase concentration of coating solution and coating time. In
addition, 30 % of filler loading had a good distribution in the selective layer.
1. Introduction installation area. Moreover, advantages of

Since the rapid growth of population membrane separation are no hazardous
and industries, human nowadays has chemical usage, environmental friendly
enormously consumed energy resources process, easy to scale-up, and uncomplicated
(i.e., petroleum, natural gas, coal, oil shale) operation.2,3
for transportation, manufacturing process, The dense membrane structure is
and so on. Furthermore, the conversion of often used for the gas separation process due
these fossil fuels to energy always to very small molecular size of gases, which
concurrently emits carbon dioxide gas, separate gases based on the solution-
which lead to the climate change and global di1ffusion mechanism.4,5 The feed gas is
warming.1,2 Thus, the carbon dioxide must absorbed onto the surface of the membrane
be removed from flue gas (CO2/N2) before and diffuses through the membrane by
releasing into the atmosphere. concentration gradient as a driving force. To
There are several technologies for promote the gas transfer, membranes with a
CO2 separation such as water scrubber, thin selective layer are preferred. An
chemical absorption, pressure swing incorporation of inorganic filler that has an
adsorption, and membrane separation. The intrinsic property of superior gas selectivity
membrane separation is an interesting and into the continuous polymer phase forming
rapidly developed technology. This mixed matrix membrane could suppress the
technology uses low energy and small limitation of PDMS mentioned above.
In this work, polydimethylsiloxane 2. Materials and Methods
(PDMS) was used as a selective layer 2.1 Materials
because of its superior gas permeability, Polyethersulfone (PES, MW 58,000
which is due to high flexibility of siloxane g/mol) was purchased from Sigma Aldrich.
linkages whose the chemical structure is PDMS was prepared by mixing the
shown in Figure 1. In addition, the PDMS is precursors sylgard 184A/184B at a ratio of
a rubbery material that has given good 10:1 by volume. N-methyl-2-pyrrolidone
thermal, chemical and oxidative stability.3 (NMP) and n-hexane were purchased from
However, the CO2/N2 selectivity is very low Acros Organics and Carlo Erba,
in PDMS material. respectively. Deionized water was used as a
To suppress the limitations solvent for phase inversion step. Zeolite
mentioned above, fabrication of selective 13X, which is used as filler, was obtained
layer and a filler to combine the advantages from National Nanotechnology Center
of an organic polymer with inorganic fillers (NANOTEC).
would improve CO2 permeability and 2.2 Fabrication of support
CO2/N2 selectivity. Zeolite, the one of PES support was prepared via phase
promising fillers is widely used for inversion method. Initially, PES was
developing new mixed matrix membrane for dissolved in NMP solvent at 25wt%
gas separation applications. Zeolite 13X is a concentration under a stirring condition at
synthetic form of faujasite-Na (FAU) zeolite 75 ºC for 5 h until homogeneous solution
with high Al content (Si/Al ratio of 1–1.5) was obtained. Then, the solution was kept 3
preferable for an adsorption of polar h at room temperature for degassing the
molecules like CO2.6 Tutuk Djoko Kusworo trapped air bubbles. The prepared PES
and co-worker7 reported an increase in CO2 solution was casted by casting knife with
permeability of mixed matrix membrane 200 µm thickness and immersed in
made of PI/PES and zeolite 13X. Murat G. deionized water for precipitation. Finally,
Siier and co-worker8 incorporated zeolite the membrane support was dried at room
13X in polyethersulfone (PES) to temperature for 24 h.
significantly enhance the CO2/N2 selectivity. 2.3 Fabrication of composite membrane
The purpose of this study was to The mixed PDMS was dissolved in
fabricate mixed matrix thin membranes n-hexane at room temperature under stirring
composing of PDMS and zeolite 13X on a for 1 h to obtain a PDMS solution. Support
porous support layer. The effect of membranes were coated by PDMS solution
concentration of PDMS solution, coating via dip-coating method. For composite
time and zeolite 13X addition on the membranes, the filler was added in PDMS
membrane morphology was investigated. solution under ultrasonic for 40 min. After
that, the composite membrane was dried at
room temperature for 3 days.
2.4 Sample characterization
The cross-section and surface
morphology of the prepared membrane was
examined by a scanning electron
microscopy (SEM). Membranes were
fractured in liquid nitrogen in order to
Figure 1. The chemical structure of observe a cross-section of membrane. The
Polydimethylsiloxane (PDMS)9 sample was gold sputtered before the
measurement.
The chemical structure of PDMS
coating was confirmed by an attenuated total
reflectance (ATR) - Fourier transform
infrared spectroscopy (FTIR) (Nicolet
6700). The first step is to collect a
background spectrum that can be subtracted
from the test spectra. Next, the membrane is
analyzed by FTIR system. This generates
the absorbance spectra showing the unique
chemical bonds of sample material.

3.1 FTIR characterization
The coating of PDMS on PES (a)
support was confirmed by FTIR spectra in
Figure 2. For PDMS layer, the absorption
bands at 890 and 2961 cm-1 were assigned to
Si-C bond and C-H bond (methyl group),
respectively. The Si-O-Si stretching peaks
were detected at 1100-1000 cm-1.10 In PES
support layer, the absorption peaks were
attributed to benzene ring stretching at 1580
cm-1, C–C bond stretching at 1488 cm-1,
aromatic ether stretching at 1244 cm-1 and
(b)
C–O bond stretching at 1106 cm-1,
respectively.11 Additionally, the Si–O
stretching peak was observed at 974 cm-1
and appearing the stretching of Al–O
vibrations at 795 cm-1 for zeolite 13X.12
(c)
Figure 2. The FTIR spectra of (a) PES

support, (b) 1% PDMS coated on PES
support and (c) 10% zeolite 13X + 5%
PDMS coated on PES support
3.2 Effect of PDMS concentration and

coating time (d)
Three PDMS coating concentrations Figure 3. The SEM images of a cross-
(1, 5 and 10%) with similar coating time (5 section of (a) PES support (b) 1% PDMS,
min) and PES support were prepared and (c) 5% PDMS and (d) 10% PDMS
compared. SEM images of a cross-section of membrane
Table 1. The selective thickness of SEM images by SemAfore program. From
composite membrane at different PDMS Table 1, an increase in PDMS concentration
concentrations tends to increase a layer thickness due to an
PDMS Selective thickness increased PDMS polymer content.
concentration (%) (µm) PES supports were coated with 5%
1 1.39±0.26
of PDMS concentration at different coating
5 1.46±0.15
10 2.08±0.12 time (5, 10 and 15 min) and the morphology
of the resultant membranes are shown in
Figure 4. An increase in selective thickness
of membrane was attributed to an increase
coating time as summarized in Table 2.
Moreover, there is the infiltration of PDMS
solution into the support layer in Figure 4(c)
due to long coating time.
Table 2. The selective thickness of

composite membrane at different coating
times
Coating time Selective thickness
(min) (µm)
(a)
5 1.46±0.15
10 1.88±0.17
15 2.65±0.42
As the result, 5% of PDMS

concentration and 5 min of coating time
gave thin selective layer, which improves
the gas transfer resistance. Thus, this
condition was selected for studying the
effect of zeolite 13X addition in the next
section.
(b) 3.3 Effect of zeolite 13X addition on the
morphology of membrane
Morphology of membranes at 30 %
of zeolite 13X loadings on a selective layer
was observed by SEM micrographs in
Figure 5. It was observed that there is a good
Infiltration
distribution of filler. After this, fabricated
membranes with a thin selective layer were
used for CO2/N2 separation. The thin
selective layer will improve CO2
permeability and mixed matrix membrane of
(c)
PDMS and zeolite 13X will enhance CO2/N2
Figure 4. The SEM images of a cross-
selectivity.
section of membrane at different coating
times (a) 5 min, (b) 10 min, and (c) 15 min
4. Conclusion
The effects of PDMS concentration,
membrane are displayed in Figure 3. The
coating time and zeolite 13X addition on
selective layer thickness was measured from
Synchrotron Light Research Institute (SLRI)
to K.F. (BRG6080015).
References
1. Kentish, S. E.; Scholes, C. A.;
Stevens, G. W. Recent Pat. Chem.
Eng. 2010, 1, 52-66.
2. Liang, C. Z.; Yong, W. F.; Chung, T. S.
J. Membr. Sci. 2017, 541, 367-377.
(a) 3. Madaeni, S. S.; Badieh, M. M. S.;
Vatanpour, V. Polym. Eng. Sci. 2013,
53, 1878-1885.
4. Wijmans, J. G.; Baker, R. W. J. Membr.
Sci. 1995, 107, 1-21.
5. Liu, S. L.; Shao, L.; Chua, M. L.; Lau,
C. H.; Wang, H.; Quan, S. Prog. Polym.
Sci. 2013, 38, 1089-1120.
6. Wolińska-Grabczyk, A.; Kubica, P.;
Jankowski, A.; Wójtowicz, M.; Kansy,
J.; Wojtyniak, M. J. Membr. Sci. 2017,
(b) 526, 334-347.
Figure 5. The SEM images of surface 7. Kusworo, T. D.; Ismail, A. F.;
membrane at zeolite 13X loadings (a) 0% Mustafa, A.; Li, K. Reaktor. 2008,
and (b) 30% 12, 68-77.
8. Süer, M. G.; Baç, N.; Yilmaz, L. J.
thickness of selective layer in mixed matrix Membr. Sci. 1994, 91, 77-86.
membrane were investigated. As the result, 9. Shokri Doodeji, M.; Zerafat, M. M.;
the optimum coating condition was obtained Yousefi, M. H.; Sabbaghi, S.
from observed morphology of membrane as Desalination. 2018, 426, 60-68.
5% of PDMS concentration, 5 min of 10. Moradi, M. R.; Pourafshari Chenar, M.;
coating time and 30% of zeolite 13X Noie, S.H.; Hesampour, M.; Mänttäri,
loading. The optimum condition will be M. Polym. Test. 2017, 63, 101-109.
further examined by gas separation test. 11. Zhu, S.; Shi, M.; Zhao, S.; Wang, Z.;
Wang, J.; Wang, S. RSC Adv. 2015,
Acknowledgements 5, 27211-27223.
The authors acknowledge the 12. Boroglu, M. S.; Gurkaynak, M. A.
financial support from the Thailand Polym. Bull. 2011, 66, 463-478.
Research Fund (TRF), NANOTEC, and
Surface transformation of porous bioactive glass
Thanatcha Kanthamoon, Radchada Buntem*
*E-mail: radchadab@yahoo.com
Abstract:
The bioactive glass (BG) has a capability to bond with living bone by forming
hydroxyapatite on its surface. High porosity of the material increases the degree of surface
transformation. The porous bioactive glass in this research was prepared using sol-gel
technique. The BG sol, prepared by mixing deionized water, nitric acid, ethanol, tetraethyl-
orthosilicate, triethylphosphate and Ca(NO3)2, was added to 1% w/v carboxymethylcellulose
(CMC) solution. The wet gel was then dried and calcined at 600ºC for 3 hr to obtain
BG+CMC600. The disc with the diameter and thickness of 13 and 2 mm respectively was
prepared under high pressure using 0.4 g of BG+CMC600. The disc was kept in a simulated
body fluid (SBF) for three different periods: 7, 14 and 21 days. The SBF solution was
replaced every two days. The collected solution was further analyzed for Ca2+ concentration
using atomic absorption spectroscopy. The surface morphology of the BG+CMC600 after
soaking with SBF solution showed the globular crystals with nanoporous structure. The
presence of hydroxyapatite on the surface of BG+CMC600 (after 14 days in SBF solutions)
was evidenced by Raman analysis. The Ca2+ concentrations was fluctuated for 10 soaking
days due to the Ca2+ exchange between BG+CMC600 surface and SBF solution.
1. Introduction This is evidenced that the higher

Various bioactive materials like surface area of the bioactive glass helps
bioactive glasses, bioactive glass–ceramics, increase the dissolution rate. The increase in
and calcium phosphate ceramics, have been the porosity and/or a decrease in the particle
developed and some of them are now size lead to high specific surface area of the
applied to repair and reconstruct damaged materials.6 The porous materials like silica
bones. Among these materials, bioactive can be prepared by the pyrolysis of the
glasses initially discovered by Hench1 have silica/organic polymer hybrid materials.
many advantages like excellent Interactions between silica and polymeric
osteoconductivity and bioactivity, ability to chains occur through covalent or hydrogen
deliver cells, and controllable biodegrad- bonding which prevents particle
ability. The surfaces of the bioactive glasses aggregation. The thermal treatment and the
can be transformed into hydroxyapatite,2 the heat generated by the combustion can
main component in bone, via the dissolution promote structural relaxation and open up
mechanism of ions from the glass surface.3 the walls of the existing pores to form
This mechanism is enhanced by the SiO2 mesoporous silica.7 This can potentially
network in which surface Na+ and Ca2+ are overcome the drawback of thermal
first exchanged with H+ from the biological reorganization of the inorganic network.8
fluid, generating Si-OH bonds.4 The release Various polymers like chitosan,9 chitosan-
of phosphate ions results in the pH increase polyacrylic acid (CS-PAA),10 were used to
which helps promote the formation of Si-OH bind with silica colloidal particle to form the
bonds. The deposition of Ca2+ and PO43- silica/polymer hybrids. The calcination of
ions onto the surface an amorphous calcium organic/inorganic hybrid materials can be
phosphate layer, which then crystallizes into applied to prepare porous bioactive glass
biomimetic hydroxyapatite.3,5 materials. Carboxymethyl-cellulose, a
readily and thermally degradable BG sample was analyzed by FT-IR and
biopolymer, possesses carboxylic groups SEM/EDS.
which can effectively bind with silanol 2.2 Preparation of BG+CMC600
groups on bioactive glass surface. In this The BG sol, prepared as the method
research, the bioactive glass containing in 2.1 was slowly mixed with 1% w/v CMC
carboxymethylcellulose was prepared via sol solution and the mixture was then stirred for
gel process followed by calcination to obtain 24 hours. The mixture was left dry at room
porous bioactive glass. The in vitro temperature with subsequent oven-dried at
bioactivity was performed in a simulated 120 °C for 3 hours to obtain BG+CMC. It
body fluid (SBF). The surface was then calcined at 600 °C to obtain
transformation was characterized by Fourier BG+CMC600.
Transform Infrared Spectroscopy and 2.3 In vitro biomineralization studies
Scanning Electron Microscopy (SEM). The BG600 and BG+CMC600 were
released amount of Ca2+ was analyzed by fabricated in the disc form. 0.4 g of each
Atomic Absorption Spectroscopy (AAS). sample was pressed using 5 tons for 10 min
to obtain a circular disc. It was then
2. Materials and Methods immersed in a simulated body fluid (SBF)
Tetraethylorthosilicate (TEOS), prepared according to the previous method12
triethly-phosphate (TEP), Ca(NO3)2·4H2O by adding NaCl (7.995 g), KCl (0.224 g),
and car-boxymethylcellulose (CMC) were CaCl2·2H2O (0.368 g), MgCl2·6H2O (0.305
obtained from Sigma-Aldrich and used as g), K2HPO4·3H2O (0.174 g), NaHCO3
received. All solvents used were of (0.349 g) and Na2SO4·10H2O (0.161 g) to 1
analytical grade and dried over molecular L of distilled water. The pH of the solution
sieves. The functional groups of the samples was adjusted to 7.4 by the addition of
were analyzed using Spectrum 100 FT-IR Tris/HCl. The samples immersed in SBF
spectrophotometer (Perkin Elmer). While were kept for incubation at 37 ºC for 7, 14,
the surface morphology of the samples were and 21 days. The SBF solution was replaced
measured using Scanning Electron every 2 days to maintain the concentration
Microscopy (SEM : JSM-6480LV (JEOL) of the ionic species in the SBF due to the
with EDS (Oxford instruments). The nucleation of biominerals on the samples.
calcium ion concentrations in simulated The Ca2+ concentration in the collected SBF
body fluid were analyzed using atomic solution was analyzed by Atomic
absorption spectrometer (Perkin Elmer, Absorption Spectrophotometry (AAS). After
AAnalyst 800). The pure bioactive glass the precise time duration, the samples were
sample (BG) was adapted from previously removed, washed with deionized water to
published method.11 remove the adsorbed minerals, dried and
2.1 Preparation of BG analyzed using FT-IR and SEM/EDS for
Deionized water (0.1918 mol), mineralization.
HNO3 (4.8091 × 10-3 mol), ethanol (0.1920
mol) and TEOS (0.0480 mol) were mixed 3. Results and Discussion
and the mixture was stirred for 30 minutes. 3.1 Characterizations of the bioactive
Then TEP (4.798 × 10-3 mol) was added glass samples
while stirring. After 30 minute, BG was prepared by mixing silica
Ca(NO3)2·4H2O was added (0.0271 mol). sol with triethylphosphate and calcium
The mixture was stirred for another 3 hours nitrate with subsequent calcination at 600
to obtain BG sol. The sol was left dry at °C. While BG+CMC was prepared by
room temperature, subsequently dried in the mixing BG sol with CMC solution and dried
oven at 120 °C and eventually calcined at at 120 °C. BG+CMC was calcined at 600
600 °C for 3 hours to obtain BG600. The °C. BG+CMC was calcined at 600 °C to
obtain BG+CMC600. In BG sol, the silica stretching vibrations of the P–O groups in
particles were formed after mixing TEOS Q2 and Q1 structural units respectively19
with water, acid and ethanol. Calcium and (where Q is the PO43- ion and the superscript
phosphate ions were subsequently adsorbed designates the number of bridging oxygen
on the silica surface. After being calcined, bonds formed with other PO43- ions). While
ions like Ca2+ and PO43- were diffused into in the case of BG, the IR spectrum is similar
silica matrix of BG. Carboxymethylcellulose to BG+CMC600.
in BG samples containing CMC provides
carboxylic and hydroxyl active sites for the
attachment with silica, Ca2+ and PO43-. After
being calcined, the porous material,
BG+CMC600, was obtained. FTIR spectra
of CMC and BG+CMC are shown in Figure
(a)
1. The silica particles in BG+CMC exhibits
broad bands centering at 1100 cm-1 (b)
attributed to Si-O-Si symmetric stretching
and Si-O stretching vibrations of the silanol
groups. In the case of BG+CMC, Figure 1. IR spectra of (a) CMC and (b)
the characteristic bands at 1636, 2934 and BG+CMC600 before calcination
3434 cm-1 are related to carboxyl group,
asymmetric C-H stretching and O-H (a)
stretching vibration in CMC respectively.
The IR spectrum of BG+CMC exhibits
broadening band of O-H stretching (b)
indicating the formation of hydrogen bond
interactions between silanol groups (Si-OH)
from incomplete polycondensation of the
TEOS13 and hydroxyl groups of CMC.
Furthermore, the carboxylic groups of CMC
are capable of forming hydrogen bonds with Figure 2. IR spectra of (a) BG600 and (b)
the silanol groups (Si-OH) on the surface of BG+CMC600
the silica produced in the sol–gel process.14
The asymmetric and symmetric stretching of 3.2 In vitro biomineralization studies
nitrate ions are normally found at 1423, BG600 and BG+CMC600 were
1384 and 1357 cm-1.15-16 However they all mineralized through immersion in simulated
are obscured by the characteristic C-H body fluids with ion concentrations higher
bending signals. The two peaks at 823 and than those present in human blood serum for
735 cm-1 referring to the bending modes of 7, 14 and 21 days. The IR spectra of BG600
the nitrate ions15,17 was clearly observed. and BG+CMC600 are presented in Figure 3
The IR spectra of BG600 and BG+CMC600 and 4 respectively. In all immersed samples,
are shown in Figure 2. The BG+CMC600 the broad bands around 3430 and 1645 cm-1
exhibits the characteristic bands of O-H are consecutively assigned to the O-H
stretching and bending of silanol groups at stretching and O-H bending modes of water
3475 and 1646 cm-1. While in the case of and silanol (Si-OH) group. The formation of
BG600, those related to O-H sinals are not carbonatehydroxyapatite was evidenced
obviously found. The bands at 1043 cm-1 from the bands at 1423 and 874 cm-1
and at 786 cm-1, assigned to the asymmetric assigned to the C – O stretching and bending
and symmetric stretching modes of Si-O-Si of carbonate ion. The appearance of the
respectively.18 Moreover, the broad shoulder double band at 603 and 565 cm-1 is
at 1160 cm-1 are due to the asymmetric attributed to P-O bending of apatite
formation. The Si-O-Si stretching solution are shown in Figure 5. While those
overlapped with P-O asymmetric stretching of BG+CMC600 and mineralized
appears as a broad band around 1039 cm-1. BG+CMC600 are shown in Figure 6. Before
mineralization, BG and BG+CMC sample
show homogenous rough surface with
smoothness degree, which is an indication
for a homogenous distributions of CMC
fibers in silica-gel network. After immersing
BG600 or BG+CMC600 in SBF for 7, 14,
21 days, shows the formation of apatite layer
comprised of spherical globules as seen in
Figure 5 and 6. On the surface, the flake-like
apatite was formed with the long oval and
circular-shape holes on the surface. The
0 surface transformation in BG+CMC600
seems to be faster as compared to the crystal
7 growth on BG at the same exposure time.
14
(a) (b)
21
Figure 3. IR spectra in 4000-400 cm-1

(upper) and 2000-400 cm-1 (lower) of
BG600 before and after immersing in SBF
for 7, 14 and 21 days (c) (d)
Figure 5. BG600 (a) before and after SBF

for (b) 7 (c) 14 and (d) 21 days
(a) (b)
(c) (d)
Figure 4. IR spectra in 4000-400 cm-1

(upper) and 2000-400 cm-1 (lower) of
BG+CMC600 before and after immersing in
SBF for 7, 14 and 21 days Figure 6. BG+CMC600 (a) before and after
SBF for (b) 7 (c) 14 and (d) 21 days
The SEM micrographs of BG600 and
mineralized BG after the immersion in SBF
The EDS analyses were performed or H3O+ ions from the SBF solution forming
on the globular surfaces of all samples. The silanol groups (Si–OH) on the glass surface.
Ca/P molar ratios of all samples are The amount released from BG+CMC600
during first 4 days is higher than that from
summarized in Table 1. The Ca/P molar
BG. This may result in a faster rate of
ratios of apatite are lower than the surface transformation in BG+CMC600 in
theoretical Ca/P molar ratio (= 1.67) of pure accordance with the SEM data. The release
apatite (Ca5(PO4)3(OH)). Calcium deficient of calcium ion was then decreased to
hydroxyapatite is normally formed on the approach equilibrium at day 8.
bioactive glass surface.11 The carbonate
hydroxyapatite was initially formed due to
the presence of carbonate ion in SBF
solution.20-21 The immersed BG600 or
BG+CMC600 samples release Ca2+ ions (as
evidenced by AAS analysis) which can be
exchanged with H+ in the solution or can
react with OH- ions to form Ca(OH)2. This
results in the pH increase inducing a higher
Figure 7. Variation in concentration of
amount of carbonate ions. calcium ions in SBF at different exposure
time
Table 1. The Ca/P molar ratios in bioactive
glass samples 4. Conclusion
The apatite formation was observed
Sample Molar ratio of Ca/P on the surface of BG600 and BG+CMC600.
The faster rate was found in the
BG600 24.24 BG+CMC600 as evidenced from SEM
BG600_SBF7 1.39 analysis. The release of calcium ion from
BG+CMC600 immersed in SBF is higher
BG600_SBF14 1.38 than that from BG600.
BG600_SBF21 1.32
Acknowledgements
BG+CMC600 -
Many thanks to Faculty of Science,
BG+CMC600_SBF7 1.47 Silpakorn University for research facilities.
BG+CMC600_SBF14 1.45
References
BG+CMC600_SBF21 1.57 1. Hench, L. L.; Splinter, R. J.; Allen, W.
C.; Greenlee, T. K. J. Biomed. Mater.
3.3 The release of Ca2+ from bioactive Res. 1971, 5, 117-141.
glass samples 2. Kokubo, T. Biomaterials 1991, 12, 155-
The dissolution behavior of BG600 163.
and BG+CMC600 during bioactivity study 3. Hench, L. L. J. Am. Ceram. Soc. 1991,
was monitored by the amount of Ca2+ 74, 1487-1510.
released into SBF at different time. The 4. González, P.; Serra, J.; Liste, S.; Chiussi,
concentration of released calcium ions from S.; León, B.; Pérez-Amor, M. J. Non
both samples were increased until the Cryst. Solids. 2003, 320, 92-99.
maxima were reached at day 4 (see Figure 5. Gunawidjaja, P. N.; Lo, A. Y. H.;
7). This is attributed to the exchange of Izquierdo-Barba, I.; García, A.; Arcos,
calcium ions from the two samples with H+ D.; Stevensson, B.; Grins, J.; Vallet-
Regí, M.; Edén, M. J. Phys. Chem. C. 15. Irish, D. E.; Walrafen, G. E. J. Chem.
2010, 114, 19345-19356. Phys. 1967, 46, 378-384.
6. Vichery, C.; Nedelec, J. M. Materials 16. Piccirillo, A. M.; Ciarletta, M.;
2016, 9, 288-304. Borysenko, S. D. Atti Accad. Pelorit.
7. Raman, K. N.; Anderson, M. T.; Pericol., Cl. Sci. Fis. Mat. Nat. 2012, 90,
Brinker, C. J. Chem. Mater. 1996, 8, 1-8.
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8. Boury, B.; Chevalier, P.; Corriu, R. J. P.; Borysenko, S. D. Atti Accad. Pelorit.
Delord, P.; Moreau, J.J.E.; Chiman, Pericol., Cl. Sci. Fis. Mat. Nat. 2011, 89,
M.W. Chem. Mater. 1999, 11, 281-291. 5–11.
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Solids. 2001, 285, 123-127. Gallicchio, M.; Pacifico, S. J. Drug
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11. Catauro, M.; Bollino, F.; Renella, R. A.; Pandey, M.; Deb, S. K. BARC News
Papale, F. Ceram. Int. 2015, 41, 12578- Lett. 2007, 285, 167–173.
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12. Kokubo, T.; Takadama, H. Biomaterials J. Non-Cryst. Solids. 1992, 143, 84–92.
2006, 27, 2907-2915. 21. Hench, L. L. J. Am. Ceram. Soc. 1991,
13. Li, R.; Clark, A.E.; Hench, L. L. J. Appl. 74, 1487–1510.
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1328.
Development organic coolants for electronics industry
Rattikierati Tnuputdhanapun1, Duangkamon Jiraroj1, Sukkaneste Tungasmita2,
Sarapong Choumwong3, Chakkrit Supavasuthi3, Duangamol Tungasmita1*
1
Department of Chemistry, Faculty of Science, Chulalongkorn University,
Patumwan, Bangkok 10330, Thailand
2
Department of Physic, Faculty of Science, Chulalongkorn University,
3
Western Digital (Thailand) Co., Ltd., Bangpa-in, Ayutthaya 13170, Thailand
*E-mail: duangamol.n@chula.ac.th
Abstract:
Organic coolant is used in the metal cutting process in the electronics industry, to
reduce the temperature caused by cutting. Good coolant should have both cooling properties
and low side effects. The specific heat capacity of an amine triisopropanolamine (TIPA) is
higher than triethanolamine (TEA) which analyzed by differential scanning calorimetry
(DSC) and both amines were compared with commercial coolants. TIPA also has lower side
effects in corrosion rate than TEA measured by potentiodynamic polarization and low
swelling properties as increasing only 0.72%.
1. Introduction EAs generally have low vapor

The basic functions of a cutting fluid pressures, and this reduces the inhalation
are the cooling of the part tools, as well as hazard in industry.4 From previous research,
the lubrication of the interface of tool and the effect of hydroxyl on alkanolamine was
cutting chips, and the flow of materials from studied. Affect of energy when hydroxyl is
the cutting area. Especially in the cutting close to the nitrogen position, the more
area that present a high excess temperature, energy is used to break the bond.5 In this
the cooling property of the cutting fluids study investigates the enthalpy values used
prevents bending situations. to change the state of liquid to gas of
Cutting fluids are generally classified triethanolamine and triisopropanolamine and
into three types (neat cutting oils, water- then calculates the specific heat capacity.
soluble fluids and gases) according to the Figure 1, alkanolamines can absorb excess
amount and type of oil that they contain.1 heat generated by metal cutting. Boric acid
Alkanolamine or ethanolamines (EAs), is used to reduce corrosion of metals.6
triethanolamine (TEA), diethanolamine
(DEA) and monoethanolamine (MEA) have
been added as integral components to
cutting fluids, to stabilize the pH surfactant
or inhibit corrosion.2
Amino alcohols are derivatives of
ammonia in which one, two, or three alkanol Figure 1. Triethanolamine (left) and
groups are attached to one amino group; the triisopropanolamine (right)
respective EAs are MEA (HOCH2CH2NH2),
DEA (HOCH2CH2)2- NH) and TEA 2. Materials and Methods
((HOCH2CH2)3N). EAs are strong bases and 2.1 Materials
can reduce simultaneously both as aerosol The first functional organic coolant
and vapor in the workplace atmosphere (C1) was prepared from triethanolamine,
because of the broad range of vapor TEA, (95%-A.C.S. Xenon Limited
pressures.3 Partnerships) and boric acid (99%-Sigma-
Aldrich). The second functional organic 3. Results and Discussion
coolant (C2) was prepared from tri- 3.1 Corrosion test
isopropanolamine, TIPA, (95%-A.C.S. From potentiodynamic polarization,
Xenon Limited Partnerships). The copper the polarization curves obtained from both
strips were used to study the corrosion and types of coolant (C1&C2), in comparison
the nitrile (Buna-N, NBR) o-ring rubber
with boric acid, water and the standard
was used in swelling.
2.2 Corrosion test commercial coolant were illustrated in
The potentiodynamic polarization Figure 2. The corrosion rate of the solutions
was used to measure corrosion potential were calculated from this polarization
(Ecorr) and corrosion current (Icorr) in order to curves. A corrosion protection can be
study corrosion rate and corrosion behaviors indicated by the highest Ecorr and low
of the mixed coolants on copper. The corrosion rate. Among those solutions, boric
copper strips were cut to about 1.5 cm x 4
acid gave the highest Ecorr value at -25.60
cm Ag/AgCl was used as the reference
probe in the measurement. The counter mV. The corrosion rate was calculated at
electrode (cathode) was platinum and about 42.17 µm/month. As the boric acid
working electrode (anode) was copper strip. was mixed with each coolant, the corrosion
The C1-coolant for this measurement was rate of both coolants increased higher than
prepared from the mixture of TEA, boric pure boric acid. The corrosion rate for C1-
acid and water in ratio of 30:10:60 by and C2-coolants were at 100.23 µm/month
weight%. The C2-coolant was prepared
and 54.02 µm/month, respectively. However,
from TIPA instead of TEA at the same
amount. the standard commercial coolant also has a
2.3 Heat capacity analyses low corrosion rate, but it is easy to corrode
The specific heat capacity affects to with copper, compared to both mixed
the cooling and heat transfer properties of coolants. Compare the corrosion behavior
each coolant. The heat that used to change between C1 and C2 coolants, it was found
the state of a substance from a liquid into a
that the C2-coolant showed less copper
gas was obtained to calculate the value of
specific heat capacity for each coolant corrosion and lower corrosion rate than C1-
formula. The measurements were done by coolant. This indicated TIPA can protect
using the differential scanning calorimeter copper better than TEA.
(DSC)-DSC3 STAR® SYSTEM, Mettler
Toledo, at the temperature from 30oC to
250oC, at a heating rate of 10oC/min.
2.4 Swelling test
Another possibility side effect
caused by the coolant, is the swelling of the
rubber o-ring. The o-ring is used as a part of
the coolant flow system. The rate of
swelling of the rubber can be related to the
erosion of the o-ring and can lead to leak.
The o-rings were immerged in each coolants
at different concentrations, at about 30oC.
The swelling test was done by measuring Figure 2. Polarization curves of C1 and C2
weight, thickness, internal and external coolants, compared to boric acid, water and
diameters of the o-rings every 24 hours for a
the standard commercial coolant
week.
3.2 Heat capacity analyses amine has some influences on the specific
From the DSC measurement, the heat capacity, as mentioned above.
area under the graph between heat flow and 3.3 Swelling test
temperatures can be referred as the energy
of state changing, and the specific heat
capacity can be calculated.7 From Table 1, it
shows that the specific heat capacity of
TIPA is higher than the heat absorption rate
of TEA, at the same concentration. The
specific heat capacity increases with an
increasing of concentration of each
substance. Based on the structure of TEA
and TIPA, the distance between the Figure 3. Percentage swelling of rubber in
hydroxyl and nitrogen affects the heat different solutions after one week
capacity, due to the influence of hydrogen
bonding between nitrogen and hydrogen of The swelling test of the rubber,
hydroxyl within the structure.5 caused by the coolants and other solutions
for a week, is displayed in Figure 3. The
Table 1. Specific heat capacity values of result showed the C2-coolant 2 has the
TEA and TIPA at different concentration, lowest rubber swelling at about 0.72%. As
obtained from DSC. the rubber starts to swell, the swelling rate
Specific heat capacity was steady over time. Then there was a loss
Chemicals
(Jg-1oC-1) of elasticity and then it was eventually
TEA 10% 27.26 destroyed.8
TEA 20% 28.87
TEA30% 29.46 4. Conclusion
TIPA10% 29.72
The specific heat capacity of a
TIPA20% 31.90
special mixed coolant can be determined by
TIPA30% 36.11
the amount of heat absorption. This value
Table 2. Specific heat capacity of mixed
can be used to indicate among of heat that
coolants compared with commercial coolant.
can be taken during the cutting. Increasing
Specific heat capacity
Chemicals
(Jg-1oC-1)
the concentration of hydroxyl amine the
water 4.34 coolant can lead to an increasing of the
Boric acid 28.05 specific heat capacity. TIPA showed a better
Coolant 1 29.57 cooling performance than TEA with lower
Coolant 2 36.72 side effects, such as very low swelling rate
Commercial coolant 32.05 compared to TEA and low corrosion rate.
TIPA can be applied to use in coolant as
30%wt of TEA and 30%wt of TIPA alternative component for a better cutting
were used to prepare the C1- and C2-
performance.
coolants, respectively. The specific heat
capacity values of C1- and C2-coolants were
shown in Table 2, compared to boric acid, Acknowledgements
water and the standard commercial coolant. The authors would like to thank
The specific heat capacity of C1-coolant was Western Digital (Thailand) Co., Ltd.,,
found lower than C2-coolant. The specific Research and Researcher for Industry (RRi),
heat capacity values also increased with the
addition of TEA or TIPA. Thus, hydroxyl
Thailand Research Fund (TRF), for the 5. Golec, K.; Hill, E. C.; Kazemi, P.;
financial supports on this project. Skold, R. O. Tribology 1989, 22, 375-
382.
References 6. Chen, D.; Howe, K. J.; Dallman, J.;
1. Sullivan, P. A.; Eisen, E. A.; Woskie, S. Letellier, B. C. Corrosion Sci. 2008, 50,
R.; Kriebel, D.; Wegman, D. H.; 1046-1057.
Hallock, M. F. Am. J. Ind. Med. 1998, 7. Cerdeirina, C. A.; Miguez, J. A.
34, 36-48. Themochimica Acta. 2000, 347, 37-44.
2. Gillbert, G. P. Met. 2007, 41, 388–397. 8. Costa, H. M.; Nunes, C. R.; Visconte,
3. Wright, T. E.; Godard, H. P. Corrosion L.Y. Raw Mat. App. 2001, 5, 242-249.
1954, 10, 195–198.
4. Kim, S. B.; Yoon, C. S.; Park, D. Safety
and Health at Work 2010, 1, 175-182.
The effect of film-forming amines (FFAs) on flow-accelerated corrosion
(FAC) of carbon steels in single-phase water
Salinla Charintara1, Chutima Kongvarhodom1, Derek H. Lister2, Shintarou Mori3*
1
2
University of New Brunswick, Fredericton, New Brunswick, Canada
3
Kurita Water Industries, Nakano-ku, Tokyo, Japan
*E-mail: shintarou.mori@kurita.co.jp
Abstract:
Flow-accelerated corrosion (FAC) is a process in which the normally protective
magnetite layer on carbon steel dissolves in flowing water or wet steam. It causes wall
thinning of carbon steel piping, which may contribute to the risk of pipe ruptures. Additives
to the system, such as dissolved oxygen, ammonia and FFAs (film-forming amines) mitigate
FAC, and elements such as chromium in the carbon steel reduce the attack. To determine the
effectiveness of a new commercial FFA, experiments were done on a 1.0 mm-bore on-line
FAC probe machined from STPT 480 steel with 0.001% chromium and installed in a high-
temperature water loop. It was exposed to single-phase water at 140 ºC, which is
approximately the temperature of maximum FAC rate. The pH was adjusted with ammonia
and dissolved oxygen was a parameter; the FFA was added part-way through the experiment.
The results indicated that high pH with ammonia reduced FAC rate, as expected. The steel
responded to the FFA in a similar way to that of A106B steel with 0.02% chromium: an
addition of FFA to give initial concentration of ~1 ppm demonstrated reduction from neutral
condition in FAC up to ~90% in A106B (pH25 ºC 9.0) and ~82% in STPT480 (pH25 ºC 9.2).
1. Introduction cause local cracking in steam generators of

Flow-accelerated corrosion (FAC) dual-cycle plants such as pressurized-water
causes wall thinning of carbon steel piping reactors (PWRs) so oxygen is of the order of
exposed to flowing water (single-phase) or tens of parts per billion (ppb - µg·kg-1).2 In
wet steam (two-phase). The major impact FAC, as its name implies, the higher
for the FAC is the feedwater oxidizing- velocity of flowing fluid increases the
reducing potential (ORP) because when chance of erosion occurring on the metal
carbon steel is operated under reducing surface; this aggravates the removal rate of
feedwater chemistry, the protective oxide magnetite film. Over time, the pipe wall
film called magnetite will be formed on the may eventually become thin enough to let
metal surface and the film solubility is the pressure burst the container. The
mightily influenced by reduction condition.1 mechanism of FAC is classified into 6 steps
Nevertheless, if feedwater is changed to an (see Figure 1): corrosion, precipitation,
oxidizing treatment by eliminating the diffusion, dissolution, hydrogen production,
reducing agent or adding oxygen, this can and erosion.3 The other common mitigating
reduce the solubility of the oxide film; hence method that is applied frequently is
the FAC can be stifled. The addition of alkalizing agents may show protection
small amount of dissolved oxygen (DO), against the possible impact from acidic
depending on the pH, is regularly applied in species. The examples of alkalizing agent
fossil plants and boiling water reactors are ammonia, ethanolamine, and
(BWRs) in order to mitigate corrosion. cyclohexylamine (CHA).
However, only small amount of oxygen can
would add value to the product by providing
it with a verified qualification.

The experiment is conducted in a
high-temperature/pressure system (see
Figure 2) under single-phase conditions in
order to inspect the probe and evaluate the
water chemistry and FAC conditions. This
experimental loop, with wetted parts made
mainly of 316 stainless steel, has a
Figure 1. Corrosion of carbon steel under maximum operating temperature of 315 ºC
FAC conditions and pressure of 12.5 MPa with a maximum
flow rate of 0.56 L/min. The loop has been
Recently, film-forming amines used before for many projects researching
(FFAs) have been introduced into the market FAC. In this project, the loop was operated
and their use in utility steam-water cycle/ at 140ºC and pressures up to 6 MPa.
HRSGs (heat recovery steam generators) is In the loop, the water was pumped to
continuing to increase. FFAs are aliphatic the high-pressure test section, where the
organic compounds with an unbranched corrosion rate was measured, then to the
hydrocarbon chain. FFAs are known as full-flow condenser and pneumatic back-
corrosion mitigating agents because they are pressure regulator. Only one probe at a time
claimed to form hydrophobic film on the was installed in this project. After exiting
metal surface by attaching their amine heads the high-pressure section, the low-pressure
to the metal or metal oxide and pointing water passed through filters, ion-exchange
their hydrocarbon tails toward water. This columns, a flow-metering device, and an
hydrophobic film acts as a barrier against Orbisphere (oxygen analyzer) before re-
flowing fluid and can form multiple layers entering the tank. Sample fluid was
by the attractive force between amine-amine withdrawn from the system via the port
and aliphatic-aliphatic groups.4,5 The film is below the tank. The 1.0 mm-bore probe was
also believed to remain intact even after the machined from carbon steel STPT 480 with
dosage has been stopped; therefore, even the low chromium content as shown in table 1.
dosage falls below the required level for a The electrical resistance probe has a wall
short period of time, no immediate harm thickness which can withstand the loop
occurs.5 However, the FFA concentration pressure but it is thin enough to give a
must be kept constantly low because the sensitive measurement of electrical
chemical and physical properties of fluid resistance. It relies on the fact that the
may change due to the formation of micelles electrical resistance along the probe
after reaching the critical concentration. increases as the wall thickness decrease. An
Moreover, the excessive amount of FFA can electrical source meter supplies constant
also promote fouling in downstream current along the length of the probe’s
equipment. reduced section in the middle and voltage
The FFAs investigated in this drop is logged every 3-4 minutes. The
experiment is a commercial FFA product experimental tasks were conducted as shown
from Kurita Water Industries. The FFA in the Table 2.
product, New Product A, was tested in a
closed-test loop under well-controlled
condition in order to provide results that
Figure 2. Flow schematic diagram of the loop (hot section represented in red)3
Table 1. Composition of carbon steel for probe manufacture (weight %)

Material C Si Mn P S Cr Mo
STPT 480 0.25 0.24 0.80 0.017 0.010 0.001 <0.01
Table 2. Experimental matrix

Task Time pH25ºC Base FFA DO
# (day) (ppm) (ppb)
Single-phase 1 6 9.6 NH3 - ~0
water (140°C, 2 5 9.2 NH3 - ~0
feed flow rate 3 11 9.2 NH3 0.4 ~0
0.56 L/min) 4 10 9.2 NH3 0.4 20
5 14 9.2 NH3 - 20
3. Results and Discussion 9.6. However, with an excessive amount of

The result of the test is shown in ammonia, the pH25ºC went up to 9.8, which
Figure 3; the red line represents the average reduced the FAC rate to 0.47 mm/y. Fresh
temperature of the test section; the blue band H-OH ion-exchange was valved-in to reduce
represents the probe wall thickness; the pH25 ºC to 9.6, then the ammoniated resin
purple and orange dots represent the FFA was engaged to maintain the condition for
concentration and the pH at 25 ºC of the about 7 days; this increased the corrosion
feedwater, respectively. At the initial stage, rate to 1.06 mm/y. The H-OH ion-exchange
the probe was exposed to neutral conditions was valved in again to reduce pH25 ºC to 9.2;
with single-phase water at 140 ºC, which this required almost two days to remove
gave the FAC rate of 6.14 mm/y. After that, enough ammonia for the feedwater to reach
ammonia-saturated ion-exchange resin was desired pH.
valved in on day 6 to increase the pH25 ºC to
Figure 3. Probe wall thickness and conditions (single-phase, 140ºC, 0.001% Cr steel)
A further adjustment of pH25ºC to 9.2 it was close to zero for the rest of the run.
for six days gave an FAC rate of 1.77 mm/y. The removal of FFA from the loop on day
After that, New Product A was injected into 40 by ammoniated ion-exchange, while
the system on day 21. The FFA keeping the pH constant at 9.2, had no effect
concentration went up to 0.7 ppm but then on the FAC, which remained immeasurable
fell sharply to zero because the ammonia- until the end of the run at day 56.
saturated ion-exchange was unintentionally The probe was removed from the
left valved-in. Nevertheless, the FFA loop, sectioned longitudinally and labelled
injection reduced the FAC rate to 1.07 from A to G indicating its analysis locations
mm/y. A second injection, with the ion- as shown in Figure 4. A and G were the
exchange valved out, to an FFA inlet and outlet of the probe, respectively,
concentration of 0.6 ppm had no immediate while C, D, and E were in the FAC-
effect on the FAC rate, which fell gradually monitoring area (reduced section). A water-
over the next few days to reach 0.65 mm/y droplet test was done by applying droplets
by day 33. The FFA concentration declined of deionized water on the surface of the
apparently exponentially and reached an probe to observe its hydrophobicity. The
equilibrium value of about 0.1 ppm at day droplets were spherical throughout the
40. At day 33 an unexpected power surge length of the probe (see Figure 5), indicating
disturbed the electronics controlling the a hydrophobic property imparted by the
resistance measurements and caused an remaining FFA attached on the probe
apparent increase in the probe wall thickness surface. Furthermore, Raman spectra taken
over a day or so. On day 34 a special right after the sectioning indicated that the
mixture of nitrogen and oxygen was probe surface was covered by a highly
introduced to the loop by sparging into the Raman-absorbing substance such as an
tank to produce a dissolved oxygen (DO) organic compound, confirming that FFA
level of 20 ppb. The FAC rate appeared to remained on the probe surface (see Figure
fall to 0.09 mm/y; however, the resistance 6).
measurement was then rather unsteady, and SEM images of the probe surface at
the FAC rate apparently fluctuated, although locations A, D, and G are shown in Figure 7.
Scallops, characteristic of FAC in single- New Product A was added to ammoniated
phase water, were observed on the probe feedwater at pH 9.2 the FAC rate fell from
surface. Even in low magnification, the SEM 1.77 to 1.07 mm/y but the pH was
images show obvious signs of corrosion, unaffected. New Product A mitigated FAC
especially just downstream of the probe more effectively than using ammonia alone
entrance. At higher magnification, the SEM under deoxygenated conditions. The
images show lamellar structures of the oxide addition of dissolved oxygen to a
with small deposits all over the porous concentration of ~20 ppb stifled FAC and
surfaces. further chemistry adjustments had no effect;
the FAC remained stifled till the end of the
4. Conclusion run.
The effectiveness of the FFA New Furthermore, the STPT carbon steel
Product A for mitigating Flow-Accelerated responded to FFA addition in a similar way
Corrosion (FAC) under feedwater conditions to that of A106B steel with 0.02%
was done on a 1.0 mm-bore, on-line FAC chromium. Thus, the addition of FFA with
probe machined from STPT480 steel with CHA to neutral water to an initial
0.001% chromium. The probe was exposed concentration of ~1 ppm and pH25 °C 9.0
to single-phase water at 140°C with a flow previously reduced the FAC of A106B by
rate of 0.56 L/min and the dissolved oxygen ~90%;6 here, the addition of ammonia to
was controlled at ~0 ppb for a deoxygenated neutral water to pH25 °C 9.6-9.2, followed by
condition, then was raised to ~20 ppb for an the addition of FFA to 0.8 ppm reduced the
oxygenated condition. For the deoxygenated FAC of STPT 480 by ~98%.
condition, the experiments started at neutral
pH, which gave an FAC rate of 6.14 mm/y.
Then the pH25°C was adjusted with ammonia
to 9.8, 9.6, and 9.2, which gave values of
FAC rate of 0.47, 1.06, and 1.77 mm/y,
respectively. Decreasing pH level by only
Figure 4. Sectioned probe with analyzed
0.2 units from a value of 9.8 can
position
significantly increase corrosion rate. After
Figure 5. Water droplet on the probe surface shows hydrophobicity
Figure 6. Raman spectra of the sectioned probe analyzed immediately after sectioning (left to
right: inlet, center, and outlet)
Figure 7. SEM images of the sectioned probe at different magnifications
Acknowledgements References
I would like to express my sincere 1. EPRI, Guideline for Controlling Flow-
appreciation to Dr. Derek H. Lister, Dr. Accelerated Corrosion in Fossil and
Chutima Kongvarhodom and Dr. Carl W. Combined Cycle Plant. 2017.
Turner for their patient guidance and 2. Lister, D. PP. Chem. 2011, 13, 188-196.
enthusiastic encouragement throughout this 3. Lertsurasakda, C. PhD’s Degree Thesis,
project. I would also like to express my University of New Brunswick, 2013.
sincere gratitude to all UNB Nuclear group 4. Lister, D. Proc. Int. Conf. 2016.
members. Without their valuable assistance 5. IAPWS TGD8-16, Application of Film
and great support, this work would not have Forming Amines in Fossil, Combined
been possible. Cycle, and Biomass Power Plants. 2016.
6. EPRI, Further Investigation of Critical
Parameters of FAC in Two-Phase Flow.
2016.
The green synthesis of gold nanoparticles using Crinum moorei extract
Warinda Marujiwat, Nongnuj Muangsin*
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
*E-mail: nongnuj.j@chula.ac.th, nongnuj.ms@gmail.com
Abstract:
Crinum moorei as medical herb has different pharmacological activities such as anti-
inflammatory and antioxidant properties components. Gold nanoparticle (AuNP) is an
interesting anti-cancer drug carrier which can solve anti-cancer drug’s problems with good
biocompatibility, non-toxicity and suitable size for the intracellular uptake to treat cancer.
From the beginning, other reducing agents were used to synthesize AuNPs, however, they
lead to the contamination of chemicals on AuNP’s surfaces which limits the medical
application or long process and time consuming. Therefore, plant extracts were studied to
make a remarkable dual functional synthesized AuNPs agent and acted as anti-cancer drug
agent. In this research, AuNPs were prepared by using Crinum moorei extract as a reducing
and stabilizing agent in one-pot synthesis. The synthesized AuNPs, 1 mM HAuCl4 and
5 %(W/V) Crinum moorei extract was optimized by using various volume and was
characterized by Ultraviolet–visible spectroscopy, Fourier-transform infrared spectroscopy,
Dynamic light scattering and Transmission electron microscopy. The results from UV-Vis
spectroscopy show surface plasmon resonance band (SPR) around 530 nm and TEM image
show spherical shape with the particle size 65±10 nm.
1. Introduction process which taking advantage of the

Nowadays, the problems of anti- enhanced permeability and retention effect
cancer drug such as non-specific to target (EPR).2 Moreover, gold nanoparticles have
cells remain. This problem causes the suitable size, low toxicity, high drug loading
damaging of normal cells. Fortunately, nano capacity, and uncomplicated synthesize
drug carriers’ advantages such as non- method.3
toxicity, suitable size, and their good There are a few popular methods to
properties in biological system are the synthesize gold nanoparticles, for example,
factors to overcome anti-cancer drug’s issue. chemical method, physical method and
Thus, nano size drug carriers such as biosynthesis method (green synthesis).
polymeric nanoparticles, iron oxide However, the classical chemical and
nanoparticles and metal nanoparticles have physical methods may lead to the
been studied and developed.1 One of the environmental contaminations and
most interesting anti-cancer drug carrier damaging of normal cell due to the large
nowadays is metal nanoparticle, because of amount of hazardous chemical product and
its tunable synthesis and modification, impurity product of produced nanomaterials,
moreover, the metal nanoparticle is easy to respectively. The green synthesis uses plant
follow up after treat which is the advantage extract as both reducing agent and anti-
in medical treatment. cancer drug.
In recent times, gold nanoparticles Crinum moorei has been known as
(AuNPs) are the most outstanding metal traditional medical herb and widely
nanoparticles with 1 to 100 nm, this size is distributed in Thailand. Its chemical
similar to the biomolecules, such as components in the extract have phenolic
nucleotides or proteins which can pass compounds, containing hydroxy and
through the cancer cell by passive targeting carbonyl group that can reduce Au3+
(HAuCl4) to Au0 (gold nanoparticle), that 2.3 Synthesis of gold nanoparticle
means Crinum moorei extract can act as The synthesis of AuNPs was carried
reducing agent. Moreover, Makarov and co- out by mixing HAuCl4 solution with
worker found that the sugar in plants extract Crinum moorei extract (CM) and sodium
can also remain the shape of metal hydroxide. The 1 mL total volumn is
nanoparticle, because aldose and ketose operated by deionized water in this
sugar can act both as reducing agent and experiment.
antioxidants agent.4 For anti-cancer 2.3.1 Effect of Crinum moorei extract
property, Fawole and co-workers found that volume
Crinum moorei can be used as anti- The synthesized conditions were
inflammatory agent, because Crinum moorei evalutated at 60 °C by using 20 µL of 50
extract can inhibit against cyclooxygenase-1 mM HAuCl4, 15 µL of 5% (W/V) sodium
(COX-1) and cyclooxygenase-2 (COX-2) hydroxide solution. The volume of 5%
enzymes. These compounds lead to produce (W/V) Crinum moorei extract is presented in
the main symptom of inflammatory that may Table 1.
cause of cancer. They also found that
Crinum moorei has good antioxidant Table 1. The synthesized gold nanoparticles
activity5. From these investigations, Crinum by various volume of 5% (W/V) Crinum
moorei will be good alternative agent to use moorei extract
as both reducing agent and anti-cancer drug. Volume of
Phenolic Volume of Volume of
In this research will focus on synthesis of compound HAuCl4 NaOH
CM (µM)
(µg) (µL) (µL)
AuNPs, using Crinum moorei extract as a
10 0.884 20 15
dual-action reducing and stabilizing agent in 20 1.77 20 15
one-pot synthesis and was characterized by 30 2.65 20 15
Ultraviolet–visible spectroscopy, Fourier- 40 3.54 20 15
transform infrared spectroscopy, Dynamic 50 4.42 20 15
light scattering and Transmission electron
microscopy. 2.3.2 Effect of sodium hydroxide solution
volume
2. Materials and Methods The synthesized gold nanoparticles
2.1 Materials conditions at 60 °C by using 20 µL of 5%
Fresh bulb and root of Crinum (W/V) Crinum moorei extract, 20 µL of 50
moorei were obtained from Bangkok, mM HAuCl4 and various volume of 5%
Thailand. Methanol for extract was AR (W/V) NaOH solution such as 0, 15, 30, 45,
grade and purified according to standard and 60 (µL).
procedures Chloroauric acid (HAuCl4) was 2.3.3 Effect of temperature for synthesis
obtained from Aldrich Co., USA. of gold nanoparticles
2.2 Preparation of Crinum moorei The synthesized gold nanoparticles
Bulb and root of Crinum moorei conditions used 20 µL of 5% (W/V) Crinum
were into small pieces and dried in room moorei extract, 15 µL of 5% (W/V) NaOH
temperature. Then, 500 mL methanol was solution and 20 µL of 50 mM HAuCl4
used to extract 70 g dried Crinum moorei by solution at various temperature (40-80 °C).
refluxing for 12 hrs.6 Plant extract was 2.4 Characterization of gold nanoparticle
filtered through filter paper and concentrated The prepared AuNPs were
using rotary evaporator at 70 °C. The crude characterized with Ultraviolet–visible
extract was kept in refrigerator. The crude spectroscopy (HP 8435) to confirm surface
extract shows the total phenolic compound plasmon resonance property (SPR) of
(reducing agent) content as 88.4 µg/mL. AuNPs around 520-550 nm. The AuNPs
sample and Crinum moorei extract were
dropped and grounded with KBr, then
analyzed by using a Fourier-transform peak intensity at around 530 nm. Thus, the
infrared spectroscopy (Nicolet 6700) in the condition at 20 µL of 5% (W/V) crinum
range of 400–4000 cm-1 to confirm moorei extracts will be used as optimized
formation between AuNPs sample and condition to find optimized sodium
Crinum moorei extract. The morphological hydroxide solution volume in next section.
of the prepared AuNPs were illustrated by
Transmission electron microscopy (JEM-
1400) and equipped with an energy-
dispersive X-ray spectrometer and Dynamic
light scattering (Zetasizer Nanoseries model
S4700).

3.1 Effect of Crinum moorei extract
This experiment focuses to
synthesize the appropriate size and shape of
AuNPs which should have scale from 1 to
100 nm, these sizes have the similar size
with the biomolecules that can pass through Figure 1. UV-vis spectra and optical photos
the target cancer cell. Fortunately, AuNPs is of synthesized AuNPs prepared from
easy to follow reaction by observing color of solution of 10 to 50 µL of 5% (W/V)
the prepared solution which give purple-red Crinum moorei extracts
or red wine color solution (The size of
particles is around 60-80nm).7 The darker 3.2 Effect of sodium hydroxide solution
color solution gave the bigger particles
(Aggregation process). This phenomenal of
AuNPs can be explained by surface plasmon
resonance band (SPR) which not only found
in gold nanoparticles but also in the other
metal such as silver nanoparticle. Herein, the
optical photos of the synthesized AuNPs
samples in Figure 1 shows that only the
synthesized AuNPs at 20 µL Crinum moorei
extracts (1.77 µg phenolic compound)
condition gave red wine color while the
others such as 10 µL Crinum moorei
extracts gave light purple color and 30-50
µL Crinum moorei extracts gave dark
purple, mean that the aggregation occurred. Figure 2. UV-vis spectra and optical photos
The reason behind this result may cause by of synthesized AuNPs prepared from
there are less gold nanoparticles in solution 0 to 60 µL of 5% (W/V) sodium hydroxide
of the condition at 10 µL Crinum moorei solution
extracts. In contrast of condition at 30-50 µL
Crinum moorei extracts, there is a great The optical photos in Figure 2 shows
number of gold nanoparticles in solution the synthesized gold nanoparticles samples.
which can aggregate and form to be bigger Only the prepared AuNPs which prepared at
particles. In addition, the optical photos as condition 15 µL of 5% (W/V) NaOH gave
well as corresponding with UV-vis spectra red wine color which mean the appropriate
that solution of the condition at 20 µL size. In the other hand, at 0 µL NaOH gave
Crinum moorei extracts appeared the highest colorless that mean no AuNPs occur. Since
the pH, plays important role in AuNPs aromatic amines (1,405 cm-1) and aliphatic
synthesis, then the solution pH should amines (1,060 cm-1) functional groups.12,13
appropriate to form AuNPs.8 Okitsu et al. Analysis of the FT-IR spectrum of prepared
reported also that the changing of the pH AuNPs (Figure 4b), which obtained the
could result (i) the change in redox potential optimized condition as the previous
of Au ion, (ii) the change in the catalytic described.
activity on surface of gold seed, (iii) the
change in reactivity of Au ion on gold seeds,
(iv) the shape and size of AuNPS. They
suggested that the alkaline solution is the
key to control size and shape.9 Adding
NaOH may also help to increase ring
opening reaction in Crinum moorei’s
sugar.10 This transform sugar can also be the
one of reducing agent. The UV-vis spectra
show SPR peak at 530 nm which get along
with the optical photo. Therefore, the
appropriated condition to synthesize AuNPs
is 20 µL of 5% (W/V) Crinum moorei
extract, 15 µL of 5% (W/V) NaOH and 20 Figure 3. UV-vis spectra and optical photos
µL of 50 mM HAuCl4. of synthesized AuNPs prepared from
3.3 Effect of temperature for synthesizing temperature between 40 °C to 80 °C
gold nanoparticles
As show in Figure 3, the particle size
of gold nanoparticles decreases, when the
temperature was increase between 40 °C to
80 °C and AuNPs clearly increasing
productivity with increasing temperature.
Mountrichas et al. confirmed that the higher
is temperature reaction, the lower size of
AuNPs. They also explained that the smaller
AuNPs occur at higher temperature, because
at the higher temperature the hydrodynamic
radius is higher during the incubation Figure 4. FT-IR spectra of Crinum moorei
period.11 UV-vis spectra showed the highest extract (a) and prepared AuNPs obtained by
temperature gave the SPR peak around 530 optimized condition (b)
nm. According to the optimization by
observing the effect of Crinum moorei FT-IR bands of Crinum moorei
extract volume, NaOH solution volume and extract appeared differently from prepared
synthesizing temperature, UV-vis spectra AuNPs bands. The decreased band at 3,428
and optical photos show that the optimized cm-1 of ‒OH group and additional bands at
condition is 20 µL of 5% (W/V) Crinum 2,954 and 2,854 cm-1 of C‒H stretching in
moorei extract, 15 µL of 5% (W/V) NaOH carbonyl groups suggested that ‒OH group
and 20 µL of 50 mM HAuCl4 at 80 °C. of phytochemicals in Crinum moorei extract
3.4 Fourier-transform infrared spectro- reduced Au3+ form to Au0 form. Then, the
scopy (FT-IR) after interaction the original transmittance
FT-IR spectrum of Crinum moorei level of C‒H stretching in carbonyl
extract (Figure 4a) showed strong FT-IR compounds band changed considerably
band characteristic of hydroxyl (‒OH) group which indicated the oxidation of ‒OH
(3415 cm-1), C=C of benzene (1,637 cm-1), groups to aldehyde.
Figure 5. TEM images (left) and EDX spectrum (right) of prepared AuNPs obtained by
optimized condition
These results showed the strong The smallest detected particles,

interactions with the phytochemicals in starting from ~30 nm in size, had nearly
Crinum moorei extract during their spherical shapes, while the larger particles,
synthesis. ~100 nm in size. Mostly the particle size is
3.5 Transmission electron microscopy around 60 nm which showed in the
(TEM) and energy dispersive X-ray histogram of size distribution of AuNPs.
spectroscopy (EDX) The samples exhibit similar symmetric
Figure 5 (left) shows TEM images distribution curves with a very small slow
and histogram of prepared AuNPs showed decaying tails towards large particle
mostly same shapes and sizes of particles. It diameters, it still shows a good condition of
clearly seen the grey area around black size which is lower than the expectation size
center of gold nanoparticles which (100 nm) that can be allowed to pass
suggesting gold nanoparticles were through cancer cells. Moreover, these curve
surrounded by phytochemicals of Crinum shapes are similar to those obtained in the
moorei. UV-Vis part as showed in the previous
result. Moreover, the EDX spectrum (Figure
5 (right)) could confirm the existing of gold
nanoparticles by showing Au peaks.
Therefore, it could confirm that the black
sphere particles in TEM image is gold
nanoparticle. Size distribution of prepared
AuNPs from DLS particle size analysis. The
average size of synthesized gold
nanoparticles in solution around 94 nm
(Figure 6), Comparing the size between
TEM, it is bigger than TEM image (Figure
5) which had particle size around 60 nm.
Due to the reason that the stretching of
phytochemical compounds which covered
Figure 6. DLS particle size analysis of on AuNPs in that of solution14, while the
prepared AuNPs obtained by optimized gold nanoparticle sample for TEM was
condition dried. It can be suggested that the
phytochemicals of Crinum moorei is able to
act as stabilizer for gold nanoparticles.
4. Conclusion 4. Kobayashi, S.; Tokumoto, T.; Kihara,
The green synthesis of gold M.; Imakura, Y.; Shingu, T.; Taira, Z.
nanoparticle was successfully synthesized Chem. Pharm. Bull. 1984, 32 (8), 3015-
by using Crinum moorei extract which gave 3022.
mostly spherical shape with the particle size 5. Fawole, O. A.; Amoo, S. O.; Ndhlala, A.
around 65±10 nm. This method can be R.; Light, M. E.; Finnie, J. F.; Van
considered as easy, simple, eco-friendly, and Staden, J. J. Ethnopharmacol. 2010, 127
effective method to synthesis gold (2), 235-241.
nanoparticles. In addition, Crinum moorei 6. Jain, P. K.; EI-Sayed, I. H.; EI-Sayed,
extract has more efficient than just reducing M. A. Nano Today. 2007, 2 (1), 18-29.
agent, its interesting property which act as 7. Liu, J.; Qin, G.; Raveendran, P.;
anti-cancer drug would be evaluate in next Ikushima, Y. Chem. Eur. J. 2006, 12 (8),
work. 2131-2138.
8. Okitsu, K.; Sharyo, K.; Nishimura, R. J.
Acknowledgements Phys. Chem. A. 2014, 118, 22754−2275.
The authors gratefully acknowledge 9. Elgorashi, E. E.; Drewes, S. E.; Morris,
all the supports from Chulalongkorn C.; Van Staden, J. Biochem. Syst. Ecol.
University. 2003, 31 (6), 601-615.
10. Mountrichas, G.; Pispas, S.; Kamitsos,
References E. I. J. Phys. Chem. C. 2014, 118 (39),
1. Wang, E. C.; Wang, A. Z. Integr. Biol. 22754-22759.
2014, 6 (1), 9-26. 11. Kajani, A. A.; Bordbar, A. K.; Zarkesh
2. Ajnai, G.; Chiu, A.; Kan, T.; Cheng, C. Esfahani, S. H.; Razmjou, A. RSC Adv.
C.; Tsai, T. H.; Chang, J. J. Exp. Clin. 2016, 6 (68), 63973-63983.
2014, 6 (6), 172-178. 12. Das, R. K.; Gogoi, N.; Bora, U.
3. Yong Song, J.; Jang, H. K.; Kim, B. S. Bioprocess Biosyst. Eng. 2011, 34 (5),
Process Biochem. 2009, 44, 1133-1138. 615-619.
Makarov, V. V.; Love, A. J.; Sinitsyna, 13. Komenek, S.; Luesakul, U.; Ekgasit, S.;
O. V.; Makarova, S. S.; Yaminsky, I. V.; Vilaivan, T.; Praphairaksit, N.; Puthong,
Taliansky, M. E.; Kalinina, N. O. Acta S.; Muangsin, N. AAPS PharmSciTech.
Naturae 2014, 6 (1), 35-44. 2017, 18 (4), 1104-1115.
Effects of silver sulfadiazine on crosslinking and properties of Thai silk
fibroin/gelatin films
Patchara Umprasert1, Siriporn Damrongsakkul1,2, Juthamas Ratanavaraporn1*
1
Biomedical Engineering Program, Faculty of Engineering, Chulalongkorn University,
2
*E-mail: juthamas.r@chula.ac.th
Abstract:
Thai silk fibroin, a protein derived from Bombyx Mori. Silkworm cocoon, and gelatin,
a denatured collagen, were introduced to fabricate blended silk fibroin/gelatin (50/50) films
in this study. For this blend, the appropriate crosslinking technique to reduce solubility of
gelatin and to stabilize gelatin with silk fibroin is usually required. We found that silver
sulfadiazine (SSD) which is a cationic antibiotic for wound infection treatment had the
effects on chemical structure and crosslinking of the blended silk fibroin/gelatin films. The
effects of SSD at various concentrations on chemical structure, crosslinking, and solubility of
the blended silk fibroin/gelatin films were investigated. The results showed that incorporation
of SSD increased thickness and crosslinked (water in-soluble) fraction of the films while the
swelling ability was reduced possibly due to ionic crosslinking. The shift of amide peaks of
the blended films in the presence of SSD analyzed by Fourier Transform Infrared (FTIR)
spectroscopy supported that SSD changed chemical structure of proteins and may crosslink
silk fibroin and gelatin. The Thai silk fibroin/gelatin films incorporating SSD can be further
studied as controlled release dressing for the treatment of infected wound.
1. Introduction biocompatibility and Arg-Gly-Asp (RGD)

Nowadays, natural-derived and sequence to promote cell attachment and
biodegradable biomaterials have played proliferation.3 In our previous researches,
significant role in medical applications, such gelatin was introduced to blend with silk
as tissue engineering and drug delivery fibroin and fabricated into various forms e.g.
system. porous scaffold, microsphere, and
Silk is a protein produced from nanofiber.4-6 We showed that the blended
silkworms, spiders, or other insects. Silk silk fibroin/gelatin had good mechanical
protein from Bombyx Mori silkworms properties and extended degradation rate due
consists of two major protein components, to the silk fibroin component while the
which are sericin (25-30%) and fibroin (70- biological activities were improved by the
75%). Recently, many researches focused on gelatin component. However, gelatin is
using silk fibroin as scaffolding and drug water-soluble, the appropriate crosslinking
carrier because it has excellent mechanical is usually required to reduce the solubility of
properties and slow degradation rate gelatin and to stabilize gelatin with silk
comparing to other natural-derive fibroin. Chemical agents such as
biomaterials while generates minimal glutaraldehyde and carbodiimide are widely
inflammatory and immune responses.1-2 used to crosslink either gelatin or silk
However, silk fibroin itself shows poor fibroin.7 The disadvantage of these
biological activities, the blending of silk crosslinking agents is the residual toxicity.
fibroin with other biological active polymers Recently, there were reports about the
may be required in some applications. effects of cations on chemical structure and
Gelatin, a denatured collagen, possesses crosslinking of proteins.8-9 In this study, we
introduced silver sulfadiazine (SSD), which were weighed (W1) and the swollen
is a cationic antibiotic for wound infection thickness of the films (T1) was measured.
treatment, to incorporate into the blended The percentage of thickness increased and
silk fibroin/gelatin films. The effects of SSD swelling ratio of the films were calculated
at various concentrations on the crosslinking by the following equations:
and chemical structure of the silk
fibroin/gelatin films obtained were percentage of thickness increased (%) =
investigated. The silk fibroin/gelatin films x 100 (1)
incorporating SSD will be further studied as
controlled release dressing for the treatment
of infected wound. Swelling ratio = (2)
2. Materials and Methods 2.4 Evaluation of gel (water-insoluble)

2.1 Materials fraction of the films
The cocoons of Thai silkworms The films were cut into 5 mm x 10
“Nangnoi Srisaket 1” were kindly supplied mm and weighed (W0). Then, the films were
by Queen Sirikit Sericulture Center, immersed in 5 ml of deionized water at 37
Nakhonratchasima province, Thailand. The °C for 24 h. The films were then dried in an
regenerated silk fibroin solution (5- oven at 60°C, for 24 h. The dried films
6%wt/wt) was prepared following the remained were weighed (W1). The gel
method previously described by Lerdchai et (water insoluble) fraction of the films was
al.10 Type A gelatin (MW = 100,616 kDa) calculated by the following equation:
was obtained from Nitta Gelatin Co., Osaka,
Japan. Silver (I) sulfadiazine (MW = 357.14 gel fraction (%) = x 100 (3)
g/mol) was purchased from Sigma-Aldrich
laborchemikalien, Germany. Others 2.5 FTIR analysis
chemicals used in this study were of The chemical structure and
analytical grade. functional groups presented in the films
2.2 Preparation of silk fibroin/gelatin were analyzed using Fourier transform
films incorporating SSD infrared (FTIR) spectroscopy (Perkin-Elmer,
The regenerated silk fibroin solution USA). The FTIR analysis was based on the
was mixed with gelatin solution to prepare identification of absorption bands (ATR
the blended silk fibroin/gelatin (50/50) mode) concerned with the vibrations of
solution at 4%wt/wt. Various concentrations functional groups presented in the films. All
of SSD (0, 0.12, 0.62, 1.16, 1.69, and spectra were recorded in the wavenumber
2.16%wt/wt) was added to the blended silk range from 4000 to 500 cm-1 at the
fibroin/gelatin solution and stirred for 10 resolution of 4 cm-1.
min at 25°C. Then, the mixture was cast in a 2.6 Statistical analysis
mold and air-dried at ambient temperature All quantitative data were shown as
under darkness. After 2-3 days, the silk mean ± standard deviation. The statistical
fibroin/gelatin films incorporating SSD were significance was determined by one-way
obtained. analysis of variance (ANOVA). A value of
2.3 Measurement of thickness and water p<0.05 was considered to be significant.
swelling ability of the films
The dried films were cut into 5 mm x 3. Results and Discussion
10 mm and weighed (W0). The thickness of 3.1 Thickness and swelling ratio of the
dried films (T0) was measured using films
micrometer. Then, the films were immersed The thickness of the dried and
in 5 ml of phosphate buffer saline (PBS, pH swollen silk fibroin/gelatin films and their
7.4) at 37°C for 24 h. The swollen films percentages of increased thickness are
reported in Table 1. Both the dried (511-659 SSD would stabilize the silk fibroin/gelatin
µm) and swollen films incorporating SSD films possibly by ionic crosslinking,11-12
(684-921 µm) were significantly thicker therefore the water-soluble fraction of the
than the dried and swollen films without films was significantly reduced.
SSD (396 and 612 µm, respectively). The
thickness of the films seemed to be Table 2. Swelling ratio of silk
increased with increasing SSD fibroin/gelatin (50/50) films incorporating
concentration. On the other hand, the SSD (0, 0.12, 0.62, 1.16, 1.69, and
percentage of increased thickness of the film 2.16%wt/wt) (* represents the significant
without SSD (55%) was higher than the difference at p < 0.05 relative to the films
films incorporating SSD (33-42%). The without SSD)
weight swelling ratio of the silk SSD concentration Swelling ratio
fibroin/gelatin films presented in Table 2 (%wt/wt) (by weight)
showed the highest swelling ratio at 3.24 for 0 3.24 ± 0.08
the film without SSD. Incorporation of SSD 0.12 2.38 ± 0.02*
reduced swelling ratio of the films to 2.03- 0.62 2.12 ± 0.02*
2.38 in a concentration dependent manner. 1.16 2.1 ± 0.04*
The less percentage of thickness increased 1.69 2.08 ± 0.07*
and water swelling ratio of the films 2.16 2.03 ± 0.01*
incorporating SSD than the film without
SSD was possibly due to the ionic Table 3. Gel (water-insoluble) fraction of silk
crosslinking that denser the polymer
network.11-12 fibroin/gelatin (50/50) films incorporating
3.2 Gel (water-insoluble) fraction SSD (0, 0.12, 0.62, 1.16, 1.69, and
of the films 2.16%wt/wt) (* represents the significant
Gel (water-insoluble) fraction difference at p < 0.05 relative to the films
percentage of the silk fibroin/gelatin films is without SSD)
presented in Table 3. The gel fraction of the SSD concentration Gel fraction percentage
film without SSD was only 38% (water (%wt/wt) (%)
solubilized fraction ∼ 62%) while the gel 0 38.54 ± 3.79
fraction of the films incorporating SSD were 0.12 70.84 ± 2.12*
increased to 71-78% (water solubilized 0.62 73.61 ± 3.39*
fraction ∼22-29%). The gel fraction 1.16 72.87 ± 4.10*
percentage of the films incorporating SSD 1.69 76.62 ± 2.61*
tended to increase with the increasing SSD 2.16 78.26 ± 1.18*
concentration. The results supported that
Table 1. Thickness of the dried and the swollen silk fibroin/gelatin (50/50) films
incorporating SSD (0, 0.12, 0.62, 1.16, 1.69, and 2.16%wt/wt) and their percentages of
thickness increased
(* represents the significant difference at p < 0.05 relative to the films without SSD)
SSD concentration Thickness of the dried Thickness of the swollen Percentage of thickness
(%wt/wt) films (μm) films (μm) increased (%)
0 396.33 ± 13.42 612 ± 25.33 54.27 ± 0.96
0.12 511.33 ± 17.86 725.67 ± 31.90 41.30 ± 1.29*
0.62 513 ± 27.74 684.33 ± 17.44 35.41 ± 3.83*
1.16 592 ± 23.37 825 ± 24.67 40.09 ± 1.37*
1.69 662.67 ± 2.33 921.33 ± 16.05 38.10 ± 1.90*
2.16 659 ± 12.67 899 ± 14.67 36.62 ± 0.40*
Figure 1. ATR-FTIR spectra of silk fibroin/gelatin (50/50) films incorporating SSD (0, 0.12,
0.62, 1.16, 1.69, and 2.16%wt/wt)
3.3 FTIR spectra of the films (water in-soluble) fraction of the films are
The FTIR spectra of the film without increased while the swelling ability is
SSD and those incorporating SSD are shown reduced due to the ionic crosslinking by
in Fig. 1. The characteristic peaks of SSD. The shift in chemical structure
proteins including hydroxyl group (3270 cm- indicated that there might be some
1
), amide I (1650 cm-1), amide II (1550 cm- crosslinking occurred within the silk
1
), and amide III (1274 cm-1), are found in fibroin/gelatin films incorporating SSD.
all silk fibroin/gelatin films.13 The films Further investigation is necessary to explore
incorporating SSD at every concentration the mechanism of crosslinking of silk
showed peaks of pyrimidine groups (1415 fibroin/gelatin by SSD for clearer
cm-1), sulfur dioxide group (1100 cm-1), and understanding.
S-N group (1077, 977, 833 cm-1) of SSD.
These peaks were clearly seen in the films Acknowledgements
incorporating high concentration of SSD This research was supported by
(1.69 and 2.16%wt/wt). We also observed Innovation Hub – Ageing Society from
that the amide peaks of the films Council of University Presidents of Thailand
incorporating SSD were slightly shifted and Faculty of Engineering, Chulalongkorn
from those of the film without SSD, University.
indicating the change in chemical structure
or the crosslinking occurred in the films References
incorporating SSD. 1. Thurber, A. E.; Omenetto, F. G.; Kaplan,
D.L. Biomaterials 2015, 71, 145–157.
4. Conclusion 2. Tungtasana, H.; Shuangshoti, S.;
This study demonstrated that SSD Kanokpanont, S.; Kaplan, D. L.;
could be used as an ionic crosslinking agent Bunaprasert, T.; Damrongsakkul, S. J.
to reduce the solubility of gelatin and to Mater. Sci. Mater. Med. 2010, 21, 3151–
stabilize gelatin with silk fibroin. The SSD 3162.
incorporation influenced on chemical 3. Vepari, C.; Kaplan, D. L. Prog. Polym.
structure and crosslinking of the blended silk Sci. 2007, 32, 991–1007.
fibroin/gelatin films. The thickness and gel
4. Jetbumpenkul, P.; Amornsudthiwat, P.; 9. Zhou, L.; Chen, X.; Shao, Z. Z.; Huang,
Kanokpanont, S.; Damrongsakkul, S. Y. F.; Knight, D. P. J. Phys. Chem.
Int. J. Biol. Macromol. 2012, 50, 7-13 2005, 109, 16937–16945.
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Damrongsakkul, S. J. Mater. Sci: Mater. Ratanavaraporn, J.; Kanokpanont, S.;
Med. 2013, 25, 401-410 Damrongsakkul, S. J. Pharm. Sci. 2015,
6. Okhawilaia, M.; Rangkupanb, R.; 105, 221–230.
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Int. J. Biol. Macromol. 2010, 46, 544- Pharmaceutics 2015, 7, 305-319.
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Kanokpanont, S.; Damrongsakkul, S. 13. Kaewprasit, K.; Promboon, A.;
Int. J. Biol. Macromol. 2012, 50, 7-13. Kanokpanont, S.; Damrongsakkul, S. J.
8. Zhou, L.; Chen, X.; Shao, Z. Z.; Zhou, Biomed. Mater. Res. B. Appl. Biomater.
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FEBS Letters 2003, 554, 337-341.
Role of β-silver sulfide solid electrolyte on Ag nanofilament fabrication for
surface-enhanced Raman spectroscopy activity
Phichaya Fueaimi1, Pannee Leeladee1,2*, Thawatchai Tuntulani1, Annop Klamcheun3,
Alongkot Treetong3, Tuksadon Wutikhun3, Panita Kasamechonchung3,
Chaweewan Sapcharoenkun3*
1
2
Research group on Materials for Clean Energy Production STAR, Department of Chemistry,
Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
3
National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency
(NSTDA), Pathumthani 12120, Thailand
*E-mail: pannee.l@chula.ac.th, chaweewan@nanotec.or.th
Abstract:
In this work, we have developed a facile method to fabricate a high-density of silver
nanofilaments (Ag NFs) based on β-silver sulfide (β-Ag2S) solid electrolyte for using as a
surface-enhanced Raman spectroscopy (SERS) substrate. A chemical bath deposition was
chosen to prepare β-Ag2S solid electrolyte due to its low-cost and high-throughput deposition
method. Electron beam irradiation was used to construct Ag NFs due to its short reaction
time, high efficiency and the absence of chemical residues after the reaction.
The amount of β-Ag2S has a strong effect on the Ag NFs formation. The highest density of
Ag NFs as SERS substrate was found to be 1.03x109 filaments/cm2. The SERS performance
of methylene blue (MB) adsorbed on Ag NFs substrate has been investigated with green laser
(532 nm). The maximum SERS enhancement factor of 1.57x106 was achieved. The limit of
detection of MB obtained from Ag NFs substrate was found to be at 1 µM.
1. Introduction that α-Ag2S is stoichiometric whereas β-

Silver (Ag) nanomaterials have been Ag2S and γ-Ag2S are nonstoichiometric
increasingly used in many applications, for comprising of a small deficiency or small
example, antibacterial,1 bio-sensing,2 excess of silver.12 During the last two
3 4
electronic, photocatalyst due to their decades, the Ag/Ag2S heteromaterials
unique chemical and physical properties. In become the promising substance because of
the last decades, Ag nanomaterials have also the coupling of quantum confinement
been employed as the surface enhanced leading to the plasmon-enhanced emission
Raman spectroscopy (SERS) substrates.5,6 or absorption. An extensive number of
SERS provides greatly enhanced Raman methods have been reported for the
signal for Raman-active analyte molecules synthesis of Ag/Ag2S heteromaterials with
that have been adsorbed onto specially different particle sizes, shapes and surface
prepared noble metal surfaces such as properties.13 Nayark et al. reported the
gold,7 silver8 and copper.9 formation and annihilation of a Ag atomic
Silver sulfide (Ag2S) is one of the bridge using a solid-electrochemical
excellent metal chalcogenides as a reaction in a nanogap between a Ag2S solid-
semiconducting sulfide due to its unique electrolyte electrode and a counter platinum
physical and chemical properties with high electrode.14 However, Ag2S phases have an
ion conductivity and thermal stability.10,11 effect on the preparation of Ag
Ag2S solid electrolytes consist of 3 phases nanoprotrusion from solid electrochemical
in different structures including α-Ag2S reaction. In 2014, Tanaka et al. reported a
(monoclinic), β-Ag2S (body center cubic) chemical bath deposition (CBD) used for
and γ-Ag2S (face center cubic). It is thought low-cost fabrication of Ag2S thin films and
the Ag filaments were grown by electron solutions of Na2S for sulfidation for 6 h. The
irradiation on β-Ag2S (cubic) only.15 The different concentrations of Na2S were
different concentrations of initial sulfide prepared at 0.5, 2, 10, and 20 mM. XRD,
reactant play a key role on the number of UV-Vis spectrophotometer (Carry 5000,
sulfide nuclei and the rate of deposition.16 Agilent Technologies) and confocal Raman
The nanostructure of Ag2S has been studied spectrometer (NT-MDT NTEGRA
extensively in the past two decades, SPECTRA) were used for the compositional
however, there is currently no study on the analysis. The roughness of the surface was
phase transition of Ag2S. In this work, the measured using tapping mode AFM.
optimum condition for β-Ag2S synthesis 2.3 Fabrication of Ag NFs
was obtained by varying the Na2S The Ag NFs were fabricated on Ag2S
concentrations. The effect of β-Ag2S on Ag thin films by using electron irradiation in
NF fabrication was studied by employing Field emission scanning electron microscope
electron irradiation. A qualitative refinement (FE-SEM, HITACHI SU-8030) with
of Ag NFs and its performance as SERS accelerating voltage of 20 kV for 10 min.
substrate were investigated. The maximum 2.4 SERS measurement
enhancement factor of 1.57 x 106 was The Ag NFs on Ag2S thin films
achieved. (Ag/Ag2S) fabricated via electron irradiation
in FE-SEM were used as SERS substrate.
2. Materials and Methods Methylene blue (MB), Raman active
2.1 The preparation of Ag thin films molecules, was employed to observe SERS
Ag thin films were fabricated on activity. The 10-3 M aqueous solution of MB
silicon (Si) (100) by using radio frequency was dropped on the SERS substrates and
(RF) reactive magnetron sputtering dried for 15 min in the ambient air. The
deposition technique. A 3-inch diameter with confocal Raman spectroscopy with IX71
a 0.25-inch thickness Ag was used as a Olympus microscope with 100x objective
sputtering target. The Si (100) substrates lens was used to investigate the SERS
were cleaned with acetone, ethyl alcohol and spectra, using a laser beam with
deionized water, respectively for 15 min an excitation wavelength of 532 nm.
each and then dried with the nitrogen gas. A charge-couple device (CCD) with a
The samples were transferred to the high resolution of 4 cm-1 was employed to record
vacuum sputtering chamber for Ag thin film the SERS spectra. The SERS spectra were
deposition. When a base pressure was acquired with exposure time of 10 seconds
reached to 5.0 ×10-6 mbar, a constant flow of and 3-time accumulations. The scanning area
Ar gas was introduced into the chamber at of Raman imaging was 100 μm2. The
20 sccm. The RF power and working exposure time of scan speed and resolution
pressure were used at 100 watt and 7.5×10-3 of imaging were 0.3 s per pixel and 32x32
mbar, respectively. The thickness of Ag pixels, respectively.
thin films was about 100 nm, which was
measured by the thickness monitor 3. Results and Discussion
(INFICON SQM-160). The crystal 3.1 Ag2S thin films
orientation of samples was characterized by After the 100 nm Ag thin films were
X-ray diffractometer (XRD, Bruker D8 grown on Si (100) substrates, the Ag2S thin
ADVANCE). Atomic force microscope films were prepared by dipping the Ag thin
(AFM, SEIKO SPA400) was employed to films into 0.5, 2, 10 and 20 mM of Na2S for 6
investigate the morphology of Ag thin films. h. The surface morphology of Ag and Ag2S
2.2 Synthesis of Ag2S thin films thin films was studied by AFM as shown in
The Ag thin films were dipped into aqueous
Figure 1. The characterization of Ag and Ag2S thin films prepared from 2 mM for 6 h. a)
AFM images of Ag and Ag2S thin films, b) XRD patterns of Ag, Ag2S and Si with 2Ɵ = 30-
50๐, c) Raman spectrum of Ag2S and d) UV-Vis spectra of Ag2S and Ag thin films
Figure 1(a). Root mean square (RMS) concentration of Na2S increased from 0.5
roughness of Ag2S thin film with 16.9 nm is mM to 10 mM, the Iβ/Iα significantly
higher than that of Ag thin film with 3.6 nm. decreased from 10 to 1. This can be
This indicates that the morphology of Ag attributed to the higher concentration of
thin film was changed after the sulfidation Na2S, the faster the reaction rate of
reaction. The X-ray profiles analysis of Ag2S sulfidation leading to the lower
thin films compared with that of Ag and Si rearrangement of Ag2S unit cell. The sulphur
thin films (Figure 1(b)) reveals that both of ions were preferentially deposited on Ag
Ag and Ag2S thin films include Ag (111) and (111) surface. Since the sulphur deposition
Ag (200) peaks. Mixture-phase crystal rate is slower at lower concentration of Na2S,
structures of α-Ag2S, acanthite structure, and Ag2S was gradually rearranged to form β-
β-Ag2S, argentite structure were observed in Ag2S BCC unit cell which is denser unit cell
Ag2S thin films. Figure 1(c) demonstrates the than α-Ag2S monoclinic unit cell.12
Raman spectrum of Ag2S thin film that
consists of Ag-S stretching peak at Raman
shift of 243 cm-1. The optical transmittance
of Ag and Ag2S was studied by using UV-
Vis spectrophotometer. The results are
shown in Figure 1(d) indicating that
transmittance (%T) of Ag2S was higher than
that of Ag thin film. Various concentrations
of Na2S aqueous solutions were studied for
the synthesis of β-Ag2S. The relationship
between Na2S concentration and intensity
ratio of β-Ag2S and α-Ag2S (Iβ/Iα) from XRD Figure 2. Relationship between the XRD
spectra for 6 h dipping time is shown in intensity ratio of β-Ag2S to α-Ag2S and Na2S
Figure 2. The highest Iβ/Iα was found at 0.5 concentration of 0.5, 2, 10 and 20 mM for
mM Na2S. It can be seen that when the 6 h dipping time
3.2 Ag nano-filaments (Ag NFs) After the electron irradiation, we
After dipping Ag thin film substrate demonstrate the utilization of Ag NFs SERS
in 0.5, 2, 10 and 20 mM Na2S for substrate. MB at the concentration of
approximately 6 h, Ag2S was fabricated and 1x10-3 M was used to observe the
then irradiated by electron beam from SEM enhancement effect of the fabricated SERS
with 20 kV accelerated voltage for 10 mins substrate via Raman spectroscopy as shown
as shown in Figure 3. The density of Ag NFs in Figure 4. The peak at 521 cm-1
was calculated by the number of Ag NFs corresponds to the Raman scattering of the
divided by the area. The results show that the crystalline Si substrate. The Raman shift at
highest density of Ag NFs of 5.93x109 1623 cm-1 is the characteristic peak of MB.
NF/cm2 was obtained from 0.5 mM Na2S. The Raman spectra show that the peak
When the concentration of Na2S increased, intensity of MB at 1623 cm-1 on Ag NFs
the density of Ag NFs decreased. This result grown on Ag2S (SERS substrate) was
was in good agreement with the amount of enhanced when compared to those on the
β-Ag2S as shown in Figure 2 and agree well commercial SERS and Ag from sputtering.
with the study of Tanaka et al.15 According The enhancement factor (EF) was calculated
to the study of the electron irradiation, to compare the enhancement effect between
β-Ag2S argentite which has a superionic Si substrate and Ag NFs SERS substrate
conductivity can be transformed to Ag via (equation 1).
electron irradiation. Ag+ ion in β-Ag2S can
be diffused faster along the line and surface (1)
defects than that in α-Ag2S. This is because
Ag+ ions in β-Ag2S is at the center of unit ISERS: The enhance intensity of adsorbed MB
cell while Ag+ ion in α-Ag2S is in the lattice. molecules on SERS substrate
The linear diffusion is faster than the lattice IRef: The spontaneous Raman scattering
diffusion, they are also termed as high intensity from bulk MB molecules under the
diffusivity or easy diffusion paths.17,18 The laser spot on the blank Si substrate.
diffused Ag+ ion was reduced on the surface NSERS: The number of MB molecules
of the thin film to form Ag NFs. Our results uniformly spreading on the SERS substrate
confirm that β-Ag2S has a strong effect on under the laser spot.
Ag NF growth. NRef: The number of the bulk MB molecules
excited by the laser without Raman enhance
effect
Figure 3. SEM images of Ag NFs after

irradiation with 20 kV at 10 mins. The
scanning study area is 13.8x13.8 µm2. (The
sample was dipped into 0.5, 2, 10 and 20 Figure 4. Raman spectra of methylene blue
mM of Na2S for 6 h) (MB) at a concentration of 10-3 M on Si,
3.3 SERS activity test Ag2S, Ag from sputtering technique and
Ag NFs SERS substrates
The EF of AgNFs SERS substrate 4. Yang, W.; Zhang, L.; Hu, Y.; Zhong, Y.;
was achieved at 1.57x106 which is 2 orders Wu, H. Bin; Lou, X. W. Angew. Chemie -
of magnitude higher than that of Ag from Int. Ed. 2012, 51 (46), 11501–11504.
sputtering (2.05x104). The limit of detection 5. Yi, Z.; Li, X.; Luo, J.; Yi, Y.; Xu, X.; Wu,
of MB obtained from Ag NFs SERS P.; Jiang, X.; Wu, W.; Yi, Y.; Tang, Y.
substrate was found to be at 1 µM. Plasmonics 2014, 9 (2), 375–379.
This result indicates that the highest density 6. Fateixa, S.; Nogueira, H. I. S.; Trindade,
of Ag NFs plays an important role in surface T. Phys. Chem. Chem. Phys. 2015, 17
plasmon resonance (SPR). The greater (33), 21046–21071.
enhancement is observed when the SPR 7. Hatab, N. A.; Hsueh, C. H.; Gaddis, A.
wavelength of Ag NFs matches with the L.; Retterer, S. T.; Li, J. H.; Eres, G.;
wavelength of the excitation laser. Zhang, Z.; Gu, B. Nano Lett. 2010, 10
(12), 4952–4955.
4. Conclusion 8. Tian, C.; Ding, C.; Liu, S.; Yang, S.;
We successfully fabricated the Ag Song, X.; Ding, B.; Li, Z.; Fang, J. ACS
NFs as the high sensitivity SERS substrate Nano 2011, 5 (12), 9442–9449.
via the electron irradiation of β-Ag2S solid 9. Muniz-Miranda, M.; Gellini, C.;
electrolyte. The optimum condition for β- Giorgetti, E. J. Phys. Chem. C 2011, 115
Ag2S fabrication was found to be at 0.5 mM (12), 5021–5027.
Na2S for 6 h. β-Ag2S play a critical role and 10. Lim, W. P.; Zhang, Z.; Low, H. Y.; Chin,
directly affect the Ag NFs formation. The W. S. Angew. Chemie - Int. Ed. 2004, 43
highest density of Ag NFs was achieved at (42), 5685–5689.
5.93 x 109 filaments/cm2 with 0.5 mM Na2S. 11. Ivanov-Shitz, A. K. Crystallogr. Reports
The EF of Ag NFs SERS was achieved at 2007, 52 (2), 302–315.
1.57x106. The limit of detection obtained 12. Sadovnikov, S. I.; Gusev, A. I. J.
from highest density of Ag NFs SERS Nanoparticle Res. 2016, 18 (9), 1–12.
substrate was found to be at 1 µM. 13. Tran, Q. H.; Nguyen, V. Q.; Le, A. T.
Adv. Nat. Sci. Nanosci. Nanotechnol.
Acknowledgements 2013, 4 (3),1-20.
This work was financially supported 14. Nayak, A.; Tamura, T.; Tsuruoka, T.;
by National Nanotechnology Center Terabe, K.; Hosaka, S.; Hasegawa, T.;
(NANOTEC) from the National Science and Aono, M. J. Phys. Chem. Lett. 2010, 1
Technology Development Agency (NSTDA) (3), 604–608.
and the 90th Anniversary of Chulalongkorn 15. Tanaka, H.; Akai, T.; Tanaka, D.;
University Scholarship under contract Ogawa, T. e-Journal Surf. Sci.
number as GCUGR1125603062M (No. 58). Nanotechnol. 2014, 12 (April), 185–188.
We also acknowledge the scholarship 16. Tang, A.; Wang, Y.; Ye, H.; Zhou, C.;
supported by the Thailand Graduate Institute Yang, C.; Li, X.; Peng, H.; Zhang, F.;
of Science and Technology (TGIST) under Hou, Y.; Teng, F. Nanotechnology 2013,
contract number as SCA-CO-2559-2317-TH. 24 (35) , 1-10.
17. Gusev, A.I.; Sadovnikov, S.I.
References Semiconductors 2016, 50 (5), 682–687.
1. Sondi, I.; Salopek-Sondi, B. J. Colloid 18. Truls N. Defects Transp. Cryst. solids
Interface Sci. 2004, 275 (1), 177–182. 2005, 1-29.
2. Cho, W. J.; Kim, Y.; Kim, J. K. ACS
Nano 2012, 6 (1), 249–255.
3. Wei, H.; Ratchford, D.; Li, X. E.; Xu, H.;
Shih, C.K. Nano Lett. 2009, 9 (12), 4168–
4171.
Corrosion resistance properties of poly(acrylic acid-co-sodium styrene
sulfonate) inhibitors on low carbon steel AISI 1018
in recirculating cooling water
Parinya Jitreewas1,2, Siriporn Pranee3, Sukanya Thepwatee1, Narong Pungwiwat1,
Samitthichai Seeyangnok1,2*
1
2
3
Abstract:
The aim of this research is mainly focused on the synthesis and the ability to inhibit
corrosion of poly(acrylic acid-co-sodium styrene sulfonate) (PAA-co-NaSS copolymer) with
various weight ratios of monomers. The PAA-co-NaSS copolymers is synthesized via
solution polymerization and characterized by FT-IR, 1H-NMR, GPC, TGA and turbidity
tests. The performance of the synthesized polymer as a corrosion inhibitor is evaluated on
low carbon steel AISI 1018 working electrode by potentiodynamic polarization and
electrochemical impedance spectroscopy (EIS) techniques in a temperature range of 25-70
°C.
1. Introduction copper wire was soldered to the rear side of

The main problems faced in the the electrode and embedded in epoxy resin.
recirculating water cooling system are such The exposed surface of the electrode was
that the corrosion is very costly and wet-polished with abrasive paper up to 2000
dangerous. Thus, carboxylated polymers are grits, rinsed with acetone and air dried.
widely applied as corrosion inhibitors. They 2.2 Synthesis and characterization of
exhibit effective corrosion inhibition polymer
because of long chain carbon backbone and A series of polymers were
multiple carboxylate groups. The multiple synthesized by free radical polymerization.
carboxylate groups can effectively block Acrylic acid (AA) and sodium styrene
large area of the corroding metal.1-4 sulfonate (NaSS) are monomers and
Poly(acrylic acid) consists of carboxylate potassium persulfate (K2S2O8) is an initiator,
group which can also adsorb onto the metal respectively (Figure 1). The weight ratios of
surface. However, there are some poor monomer and an initiator were summarized
properties existed in this method such as in Table 1. The mixed solution was stirred
slightly soluble in water. To improve the for 5 minutes under N2 atmosphere and
solubility property, PAA-co-NaSS refluxed at 70 °C for 90 minutes. The 10%
copolymer is being emphasized. H2O2 in methanol was introduced into
reactor to stop polymerization process and
2. Materials and Methods solvent was then removed and dried in an
2.1 Materials oven at 70 °C overnight. After that, the
AISI 1018 type carbon steel was synthesized polymers were characterized by
used as working electrode with 1 cm2 FTIR, 1H-NMR, GPC, TGA and turbidity
surface area exposed to the corrosive test technique.
solution. For the electrical connection, a
O
HO S O H
O
O OH HO O m
K2S2O8 n
HO O
O S O Potassium persulfate
Acrylic acid O S O O S O
(AA) ONa ONa ONa
Sodium Styrene Sulfonate
(NaSS) Poly(acrylic acid-co-sodium styrene sulfonate)
(PAA-co-NaSS)
Figure 1. Synthesis of PAA-co-NaSS
Table 1. The weight ratio of AA : NaSS : K2S2O8 in each polymer

Polymer AA (g) NaSS (g) K2S2O8 (g) a
PAA 10 0 0.1666
PAA-co-NaSS-91 9 1 0.1455
PAA-co-NaSS-82 8 2 0.1260
PAA-co-NaSS-73 7 3 0.1079
PAA-co-NaSS-64 6 4 0.0912
PAA-co-NaSS-55 5 5 0.0758
a
gram of initiator to control a degree of polymerization approximately 50
2.3 Performance test of polymers as a solution with and without inhibitors,

corrosion resistance properties respectively.
Potentiodynamic polarization and
EIS measurements were tested by using 3. Results and Discussion
Potentiostat (Autolab ECO CHEMIE) 3.1 Synthesis and characterization of
controlled by a GPES 4.7 corrosion analysis polymer
and FRA 4.7 software model. The The FTIR spectrum of PAA and AA
experiment used a conventional three monomer is shown in Figure 2 Thus, it can
electrode with consist of a Ag/AgCl as the be found that the absorption peak at 1635
reference electrode, a platinum electrode as cm−1 is belonged to the C=C stretching of
the auxiliary electrode and the working AA monomer and disappear in FTIR spectra
electrode was 1 cm2 of low carbon steel of PAA, it can confirm that the
AISI 1018. The conditions were operated at polymerization of AA monomer is achieved.
a temperature of 25-70°C and a scan rate of The absorption peak at 3200-3550 cm−1 is
1 mV s−1 starting from −1.5V to 1V vs. assigned to the O-H stretching of acrylic
Ag/AgCl reference electrode. EIS acid group, the characteristic absorption
measurements were operated at frequency peaks at 1714 cm−1 correspond to the C=O
range from 100 kHz to 10 mHz using AC stretching of a carbonyl in acrylic acid
signal with 2 mV amplitude peak to peak. group. The FTIR spectrum of synthetic
After that, the inhibition efficiency (IE) of PAA-co-NaSS copolymers is shown in
the inhibitor of the steel corrosion was Figure 3 Thus, it can be seen that the main
calculated with the following equation 1. characteristic peaks previously in Figure 2
I 0 − I corr indicating the existence of acyclic and the
IE (%) = corr × 100 (1) apparent peak at 1475 cm-1 is
I corr 0
corresponded to C=C in the aromatic ring of
where Icorr and Icorr0 are the corrosion styrene group, it can be demonstrated that
current values of steel immersed in a test NaSS monomer can be synthesized as
copolymer with AA monomer.
O-H Stretching C=O Stretching C=C Stretching 24052016-CO-DI-BI.60.fid
1H PAA 2
PAA
PAA
PAA
AA 24052016-CO-DI-BI.130.fid
PNaSS
1H PNaSS 1
PNaSS
Wavenumber (cm-1)
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 -3
f1 (ppm)
1
Figure 2. FTIR spectra of PAA and AA Figure 4. H-NMR spectra of PAA and
monomer PNaSS
O-H Stretching C=O Stretching C=C Stretching 24052016-CO-DI-BI.150.fid
PAA-co-NaSS-291
1H 291 5
PAA-co-NaSS-91
24052016-CO-DI-BI.10.fid
PAA-co-NaSS-282
1H 282 4
PAA-co-NaSS-82
PAA-co-NaSS-91
24052016-CO-DI-BI.110.fid
PAA-co-NaSS-82 PAA-co-NaSS-273
1H 273 3
PAA-co-NaSS-73
PAA-co-NaSS-73 24052016-CO-DI-BI.20.fid
PAA-co-NaSS-264
1H 264 2
PAA-co-NaSS-64
PAA-co-NaSS-64
24052016-CO-DI-BI.30.fid
PAA-co-NaSS-55 PAA-co-NaSS-255
1H 255 1
PAA-co-NaSS-55
Wavenumber (cm-1) 16 15 14 13 12 11 10 9 8 7 6
f1 (ppm)
5 4 3 2 1 0 -1 -2 -3
1
Figure 3. FTIR spectra of PAA-co-NaSS Figure 5. H-NMR spectra of PAA-co-
copolymers NaSS copolymers
In order to confirm the structure of sulfonate monomer was incorporated to

the synthesized PAA, PNaSS and PAA-copolymer chain. In other words, The PAA-co-
NaSS copolymer, the 1H-NMR spectra in NaSS copolymer was successfully
D2O were carried out and shown in Figure 4 synthetized.
and Figure 5, it is seen that the proton The molecular weight of PAA-co-
resonance at 2.0-4.0 ppm is ascribed to NaSS copolymers was analyzed using GPC
methylene protons in the backbone of PAA technique. The results of molecular weight
polymer. The proton resonance at 7.5 and by number, average molecular weight, and
7.6 ppm are aromatic proton of PNaSS. The poly dispersity index (PDI) were
appearance of these 2 proton resonance summarized in Table 2.
signals indicate that sodium styrene
Table 2. Result obtained from GPC analysis

Polymer Mn (kDa) Mw (kDa) PDI
PAA-co-NaSS-91 1074 2272 2.1155
PAA-co-NaSS-82 1217 2139 1.7572
PAA-co-NaSS-73 804 1788 2.2239
PAA-co-NaSS-64 568 1619 2.8485
PAA-co-NaSS-55 900 1675 1.8619
The average molecular weight of structure. This suggests that the polymer
each copolymer is in a range of 1600-2300 samples are effectively thermal stability
kDa and the PDI of copolymer is shown in a since an operating temperature of cooling
range of 1.75-2.84. It pointed to the tower is around 70°C.
synthesized polymer are low dispersity of The result of turbidity study was
molecular weight. summarized in Table 4. It shows that
The thermal property of the PAA PAA homopolymer cannot be dissolved in
polymer and PAA–co-NaSS copolymers water. For PAA-co-NaSS copolymers, their
was analyzed and the result was summarized solubility can be enhanced by the presence
in Table 3. It is clear that there are three of sulfonate group. Even though copolymers
stages for degradation of each polymer are composed of PAA, they are soluble in
sample. The first decomposition stage (150– water. It can be explained that NaSS played
350 °C) was assigned to a removal of all an important role in solubility control of
volatile matter including entrapped moisture these copolymers.
or solvent. The second decomposition stage 3.2 Performance test of polymers as a
(350–550 °C) could be attributed to the corrosion resistance properties
decomposition of carboxyl group of PAA The corrosion rate of carbon steel
and sulfonate group of PNaSS. Furthermore, AISI 1018 in 0.01 M NaCl solution with
an increase in AA monomer results in absence and presence of polymer inhibitor
greater degradation percentage as listed in was investigated by potentiodynamic
Table 3. The last decomposition stage at measurements as shown in Figure 6 and the
high temperature (550–1100 °C) was result of inhibition efficiency (IE) of the
generally associated with a further progress inhibitor calculated with the equation 1 was
in the decomposition of polymer backbone displayed in Figure 7.
Table 3. Result obtained from thermogravimetric analysis

% Decomposition
Polymer
150-350 °C 350-550 °C 550-1100 °C Residue
PAA 32.64 47.81 12.77 10.26
PAA-co-NaSS-91 27.51 46.31 20.94 8.54
PAA-co-NaSS-82 24.89 42.22 26.94 8.94
PAA-co-NaSS-73 26.35 33.40 33.30 10.06
PAA-co-NaSS-64 21.33 24.32 42.02 15.29
PAA-co-NaSS-55 21.20 23.33 45.34 12.60
Table 4. Turbidity of polymer solution

Polymer concentration (ppm)
Polymer 20 30 40 50 60 80 100
Turbidity of polymer solution (NTU)
PAA 14.29 19.71 26.43 38.71 50.71 60.71 69.14
PAA-co-NaSS-91 0.14 0.71 0.71 1.29 1.57 2.14 2.57
PAA-co-NaSS-82 0.00 0.57 0.86 0.86 0.86 1.43 2.14
PAA-co-NaSS-73 0.00 0.00 0.57 0.71 0.86 1.00 1.57
PAA-co-NaSS-64 0.14 0.29 0.57 0.57 0.71 1.00 1.29
PAA-co-NaSS-55 0.00 0.00 0.14 0.43 0.57 0.71 0.71
The EIS measurements (Figure 8)
showed the Nyquist diagrams measured on
the carbon steel electrode in 0.01 M NaCl
solution at room temperature in the absence
and presence of various types of polymer
inhibitors at 100 ppm. All the obtained plots
showed one capacitive loop and the diameter
of the semicircle increased with decreasing
amount of acrylic acid segment in the
polymer which suggested that acrylic acid
Figure 6. Potentiodynamic polarization segment could promoted acidity to the steel
curves of carbon steel in 0.01M NaCl surface.
solutoin in the absence and presence of
polymer inhibitor 4. Conclusion
The PAA-co-NaSS copolymer with
weight ratio of monomer at 6:4 shows the
highest efficiency as corrosion inhibitor with
82.64% at 70 °C of 100 ppm dosage. The
observation from the tests clearly showed
that the copolymer with 6:4 ratio is an
optimum useful as scale and corrosion
inhibitor for recirculating water cooling
system in industrial.
Acknowledgements
The authors would like to extend
Figure 7. Inhibition efficiency (IE) of the their appreciations to Department of
polymer inhibitor Industrial Chemistry, Faculty of Applied
Science, Graduate College, King Mongkut’s
University of Technology North Bangkok
for their continual supports in providing
efficient information and operations.
References
1. Migahed, M. A.; Rashwan, S. M.;
Kamel, M. M.; Habib, R. E. J. Mol. Liq.
2016, 224, 849–858.
2. He, C.; Tian, Z.; Zhang, B.; Lin, Y.;
Chen, X.; Wang, M.; Li, F. Ind. Eng.
Chem. Res. 2015, 54, 1971−1981.
3. Lin, G.; Xue, M.; Yang, H. Desalination
Figure 8. Nyquist plot for carbon steel in 2017, 419, 133–140.
0.01M NaCl solution in the absence and 4. Rahmani, K.; Jadidian, R.; Haghtalab, S.
presence of polymer inhibitor Desalination 2016, 393, 174–185.
A top-down fabrication of Thai silk fibroin/gelatin composited
microparticles
Orathai Suphawannawiboon1, Jutarat Jamkratoke2, Sorada Kanokpanont1*
1
Pathumwan, Bangkok 10330, Thailand
2
Queen Sirikit Sericulture Center (Nakhon Ratchasima), Nakhon Ratchasima, Thailand
*E-mail: sorada.k@chula.ac.th
Abstract:
Several microparticles fabrication methods require the use of organic solvent.
Residues solvent may be toxic to cells or reduce the ability of biological cells if not fully
removed. This study aims to fabricate solvent-free porous microparticles using a simple
technique via top-down approach. Silk fibroin (SF), derived from Bombyx mori Thai silk
cocoons (Nangnoi Srisaket1), was blended with gelatin (GA) at different blending ratios
(SF100, SF80GA20, SF50GA50, SF20GA80 and GA100). The composite scaffolds were
fabricated by freeze-drying and glutaraldehyde crosslinking at vary glutaraldehyde
concentration 0.10, 0.15 and 0.20%v/v. The resulting sponges were grinded into
microparticles and were sieved to obtain microparticles in the range of 1000-2000 µm. The
gel fraction of microparticles showed that over 90% structure remained intact. In vitro
enzymatic biodegradation was investigated by incubating microparticles in 1 U/mL protease
type XIV solution (pH7.4) for 24 hours. The remaining weight percentage of SF100,
SF80GA20, SF50GA50, SF20GA80 and GA100 were around 61-64%, 37-48%, 15-23%, 4-
5% and 0.4-3% respectively. The result showed that silk fibroin extended degradability,
comparing to the pure gelatin microparticles. This top-down fabrication appears to be a
simple and safe choice for producing solvent-free porous microparticles which can be
candidate for drug delivery and tissue engineering applications.
1. Introduction Suitable materials for biomedical

Several methods and techniques for applications such as tissue engineering and
preparation of microparticles have been drug delivery, should be biocompatible,
developed in the past. The most well-known biodegradable and cause minimal
strategies for fabricating microparticles inflammatory reaction.7 Silk fibroin (SF) is a
include dispersion, precipitation, emulsion, fibrous protein which derived from Bombyx
coacervation, spray drying and rapid mori silkworm. It has been broadly used as
expansion of supercritical solutions.1-4 biomaterial in biomedical applications for
However, many of these methods involve decade due to its outstanding mechanical
the use of organic solvents which causes and and biological properties such as high
effects on health and environmental mechanical strength, biocompatibility,
pollution. Most of the organic solvents biodegradability and low immunogenicity.8
irritate skin and damage to brain, nervous However, SF does not promote cell adhesion
system and internal organs such as the liver as well as other bioactive materials.9 To
and kidneys.5 Moreover, the residual solvent enhance cell adhesion and proliferation,
in microparticles may be toxic to cells or gelatin was additionally used as bioactive
reduce the ability of biological cells if not material. Gelatin is a denatured polymer
completely removed.6 This concern of from collagen, containing RGD sequence
existence solvent effects led to developing which mediates cell attachment.10
solvent-free fabrication methods for In our previous work, we have
microparticles. reported the blending of SF/GA in various
forms such as films,11 electrospun fibers,12 crosslinked solution was frozen overnight
microspheres13 and scaffold.14-16 Here, we prior to lyophilization for 72 h. After freeze
focused on developing solvent-free drying, the SF/GA sponges were removed
fabrication methods for microparticles. The the residual toxic aldehyde groups of
porous microparticles with high surface glutaraldehyde by immersing in 0.1 M
were prepared by using a simple technique glycine solution for 3 h and extensive
via top-down approach. The preparation washing in DI water. Then, the sponge was
procedure starts from fabricating the freeze dried again and was grinded using
crosslinked SF/GA sponge, then grinding food processing blender (HR2117, PHILIPS,
into microparticles. The influences of the Japan) into microparticles. Finally, the
glutaraldehyde concentration and SF/GA microparticles were fractionated by using
composition was studied. sieves (Humboldt, USA) with aperture size
500, 1000 and 2000 µm. Only
2. Materials and Methods microparticles with size in the range of
2.1 Materials 1000-2000 µm were selected for further
Nangnoi Srisaket 1, Thai silk study.
cocoons, Bombyx mori were cultured and 2.4 Morphological observation
maintained by Queen Sirikit Sericulture The samples were sputter-coated
Center, Nakhon Ratchasima province, with gold. Physical appearances and surface
Thailand. Gelatin Type A (isoelectric point morphology of microparticles were observed
= 9) was kindly supplied by Nitta Gelatin on a scanning electron microscope (SEM,
Inc., Japan. All chemicals used in this study JSM 5400, JEOL, Japan).
were analytical grade. 2.5 Gel fraction of microparticles
2.2 Preparation of Thai SF aqueous The weight loss of microparticles in
solution water was used to evaluate the effectiveness
Thai SF aqueous solution was of crosslinking. The water solubility of
prepared with the modified method from microparticles was determined using gel
previously reported by Kim et al.17 Briefly, fraction technique.18 The known dry weight
Thai silk cocoons were degummed in microparticles were immersed in DI water
boiling 0.02 M Na2CO3 solution for 20 min, and incubated at 37 °C for 24 h. After that,
and then rinsed thoroughly with deionized the microparticles were removed from DI
(DI) water to remove sericin gum. After water and dried at 60 °C overnight. The
drying, the degummed silk fibers were percent of gel fraction was calculated using
dissolved in 9.3 M LiBr solution with the following equation:
ratio 1:4 (w/v) at 60 °C for 4 h. The fibroin
dissolution was dialyzed in DI water for 2
days. The final concentration of Thai silk where wi represents the initial weight of
fibroin aqueous solution was about 6–6.5 microparticles and wf represents the final
%w/v. weight of microparticles (dry basis).
2.3 Preparation of Thai SF/GA 2.6 In vitro enzymatic biodegradation test
composited microparticles In vitro enzymatic biodegradation
Thai SF solution was blended with was investigated by incubating the known
GA solution at different weight ratios dry weight microparticles (w1) in 1 U/mL
(SF100, SF80GA20, SF50GA50, SF20GA- protease type XIV solution (pH 7.4)
80 and GA100) to obtain a final containing 0.01%w/v sodium azide (NaN3)
concentration at 4% w/v. Glutaraldehyde as an antibiotic at 37 °C for 24 h.19
was gently added into the mixing solution at At the designated time, the remained
varying concentration of 0.10, 0.15 and microparticles were collected, rinsed
0.20%v/v to crosslink the proteins. The repeatedly with DI water and freeze dried.
20x 150x 1000x
SF100
SF50GA50
Figure 1. SEM images of crosslinked SF/GA microparticles at two different blending ratio
SF100 and SF50GA50
The dried microparticles were weighed (w2) of sponges before the grinding. The pore
and calculated to determine the percentage size and porosity of sponge were reported to
of remaining weight using following be dependent with the blending ratio, the
equation: sponges with higher GA content had
significantly larger pore size and more
porosity than the ones with lower GA
2.7 Statistical analysis content.16 The largest size of microparticles
The quantitative data were averaged (> 2000 µm) was found to have the highest
and expressed as the mean ± standard size distribution. While the population of
deviation. All statistical calculations were
determined by one-way analysis of variance
(ANOVA) using IBM SPSS Statistics 22 for
Windows. Significant level was considered
significant at P <0.05.

3.1 Morphology and size of the
microparticles
Morphology of Thai SF/GA
microparticles at blending ratio SF100 and
SF50GA50 were showed in Figure 1. The
microparticles with irregular structure and
interconnected pores were observed. The
microparticles were grinded using blender, Figure 2. The particle size distribution after
resulting in a random structure and a range grinding at different blending ratio. Particles
of size distributions. Figure 2 shows the size : < 500 µm (-35mesh), 500-1000
particle size and size distribution after µm (-18+35mesh), 1000-2000 µm
grinding at vary SF/GA mixing ratio. (-10+18mesh) and >2000 µm (+10mesh)
Increasing the amount of gelatin content led
to the larger size of the microparticle. This microparticles with the size between 1000
might depend on the pore size and porosity and 2000 µm which was selected for further
study was around 25-45%w/w. The efficient
production of desired microparticles seem to
be slightly low because the grinding time
and rotational speed were insufficient.
Therefore, the optimal condition will be
considered in the future study.
3.2 Gel fraction of microparticles
In this study, the SF/GA
microparticles at various blending ratios
were prepared by freeze drying and
glutaraldehyde crosslinking at vary
glutaraldehyde concentration 0.10, 0.15 and
0.20%v/v. The crosslink bonds were formed Figure 4. Percentages of weight remaining
between dialdehyde of glutaraldehyde and of SF/GA microparticles at different weight
two different amine groups of protein, thus blending ratios after incubated in 1 U/mL
the glutaraldehyde carbon chain was a protease type XIV solution (pH 7.4)
covalent bridge between the amine containing 0.01% (w/v) sodium azide at 37
moieties.20 The microparticles were °C for 24 h. Glutaraldehyde concentrations
crosslinked in order to reduce the water were used for crosslinking: 0.10%v/v,
solubility of gelatin and improve thermal, 0.15%v/v and 0.20%v/v.
mechanical and stability properties. To (⁎ and ⁎⁎ represented significant difference among
determine crosslink density, the weight sample at p<0.05 at same composition)
remained of microparticles in water was
evaluated using gel fraction technique. The concentration and SF/GA blend ratio on the
non-crosslinked fraction was supposed to be weight percentage of gel fraction of
dissolved in water. Figure 3 shows the microparticles. The percentages of gel
influences of the glutaraldehyde fraction of all crosslinked microparticles
were around 90–95%. The significant
difference among the crosslinked
microparticle was not observed. For non-
crosslink microparticles, the highest percent
gel fraction was found on the SF/GA
microparticles blended at 50/50 (14%) while
at another blending ratios were just around
2-6%. Comparing the gel fraction between
non-crosslinked and crosslinked
microparticle, there were highly significant
differences between both groups. This could
confirm the accomplishment of crosslinking
at any studied glutaraldehyde concentration.
Figure 3. Percentages of weight remaining 3.3 In vitro enzymatic biodegradation test
of SF/GA microparticles after immersion in In this work, in vitro enzymatic
water at 37 °C for 24 h. Glutaraldehyde biodegradation was investigated only at 24 h
concentrations were used for crosslinking: time period to observe the effect of SF/GA
non-crosslinking, 0.10%v/v, 0.15%v/v blending ratio and glutaraldehyde
and 0.20%v/v. concentration on the degradation behavior of
(⁎ and ⁎⁎ represented significant difference among
sample at p<0.05 at same composition)
microparticles. The percentage of remaining
weight of the microparticles after incubated
in 1 U/mL protease type XIV solution
(pH7.4) was shown in Figure 4. The
remaining weight percentage of SF100, 2. Jyothi, N. V.; Prasanna, P. M.; Sakarkar,
SF80GA20, SF50GA50, SF20GA80 and S. N.; Prabha, K. S.; Ramaiah, P. S.;
GA100 were around 61-64%, 37-48%, 15- Srawan, G. Y. J. Microencapsulation
23%, 4-5% and 0.4-3% respectively. The 2010, 27 (3), 187–197.
significantly slower degradation rate of SF- 3. Joye, I. J.; McClements, D. J. Curr.
based microparticles compare to GA-based Opin. Colloid Interface Sci. 2014, 19,
microparticles was clearly observed. This 417–427.
indicated that silk fibroin enhanced stability 4. Deshmukh, R. K.; Wagh, P.; Naik, J.
and extended degradability of the Drying Technol. 2016, 34 (13), 1593-
microparticles. Moreover, it was found that 1603.
concentrations of glutaraldehyde had 5. Sanni Babu, N.; Mutta Reddy, S. J.
significant effect on degradation at some Chem. Pharm. Sci. 2014, 3, 49-52.
blending ratio (SF80GA20 and SF50GA50). 6. Lees-Haley, P. R.; Williams, C. W. J.
Since each composition of crosslinked Clin. Psychol. 1997, 53, 699.
microparticles had similar water resistance 7. Meinel, L.; Fajardo, R.; Hofmann, S.;
and degradation behavior, we proposed to Langer, R.; Chen, J.; Snyder, B.;
maximum efficiency and minimum chemical Novakovic, G. V.; Kaplan, D. L. Bone.
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4. Conclusion 4, 74.
The preparation of solvent-free 9. Moy, R. L.; Lee, A.; Zalka, A. Am. Fam.
microparticles via top-down technique was Physician. 1991, 44, 2123–2128.
successfully. The morphology of 10. Wang, Y.; Kim, H. J.; Novakovic, G. V.;
microparticles were irregular structure with Kaplan D. L. Biomaterials 2006, 27,
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influences of the SF/GA blending ratio 11. Kaewprasit, K.; Promboon, A.;
affected pore size and porosity of Kanokpanont, S.; Damrongsakkul, S. J.
microparticle. The crosslinked Biomed. Mater. Res., Part B. 2014, 102
microparticles were more stable than those (8), 1639-1647.
of non-crosslinked microparticles, resulting 12. Somviparta, S.; Kanokpanonta, S.;
in a high percentage of insoluble gel Rangkupanb, R.; Ratanavaraporna, J.;
fraction. For the degradation test, this Damrongsakkula, S. Int. J. Biol.
indicated that the presence of silk fibroin in Macromol. 2013, 55, 176– 184.
microparticles could reduce the In Vitro 13. Ratanavaraporna, J.; Kanokpanonta, S.;
Enzymatic Biodegradability. In further Damrongsakkula, S. Mater. Sci. Mater.
study, these SF/GA microparticles will be Med. 2014, 25, 401–410.
introduced as micro-scaffolds for cell 14. Chamchongkaset, J.; Kanokpanont, S.;
culture. Kaplan, D. L.; Damrongsakkul, S. Adv.
Mater. Res. 2008, 55-57, 685-688.
Acknowledgements 15. Tungtasana, H.; Shuangshoti, S.;
The authors gratefully acknowledge Shuangshoti, S.; Kanokpanont, S.
support from the Ratchadaphiseksomphot Kaplan, D. L.; Bunaprasert, T.;
Endowment Fund of Chulalongkorn Damrongsakkul, S. J. Mater. Sci. Mater.
University (CU-58-002-HR). Med. 2010, 21, 3151–3162.
16. Lerdchai, K.; Kitsongsermthon, J.;
References Ratanavaraporn, J.; Kanokpanont, S.;
1. O'Donnell, P. B.; McGinity, J. W. Adv. Damrongsakkul, S. J. Pharm. Sci. 2016,
Drug Delivery Rev. 1997, 28, 25-42. 105, 221-230.
17. Kim, U. J.; Park, J.; Kim, H. J.; Wada, 19. Wongnarat, C.; Srihanam, P.
M.; Kaplan, D.L. Biomaterials, 2005, Engineering 2013, 5 (1), 61-66.
26, 2775–2785. 20. Kulkarni, A. R.; Soppimath, K. S.;
18. Yang, S.; Wu, Z.; Yang, W.; Yang, M. Aminabhavi, T. M. Pharm. Acta Helv.
Polym. Test. 2008, 27, 957-963. 1999, 74, 29–36.
Conjugated polycarbazole nanoparticles dispersion in aqueous solution for
printed organic light-emitting diodes
Patteera Funchien, Duangratchaneekorn Muenmart, Taweesak Sudyoadsuk,
Vinich Promarak*
Vidyasirimedhi Institute of Science and Technology, Payupnai, Rayong 21210, Thailand
Abstract:
Two conjugated polycarbazole nanoparticles (PCBZ-H and PCBZ-G1) as blue
emitting material for OLEDs were designed and synthesized through Suzuki cross-coupling
mini-emulsion polymerization of 2,7-dibromo-N-octylcarbazole and 2,7-dibromo-N-(7-(3,6-
di-tert-butyl-carbazol-N-yl)heptyl)carbazole monomers with N-(9-heptadecanyl)-2,7-bis
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)carbazole catalyzed by Pd(dppf)Cl2/Et4NOH in
aqueous solution of SDS surfactant. Both CPNs showed particle size around 29-50 nm with
molecular weight in range of 23,000-25,000 g/mol and polydispersity index of 2.0-3.0. They
emitted brightly in blue color with emission wavelengths of 420-450 nm. Moreover, the
synthesized CPNs can be fabricated on paper or any substrates by ink-jet printing, which is
interesting for further study because it is an important technology for electronic devices
manufacturing, owing to reducing the cost and environmental hazards.
1. Introduction stable aqueous dispersions of nanoparticles,

Recently, organic light-emitting thus making device fabrication easier.6
diodes (OLEDs) are interesting because of Therefore, in this work, we report the
their potential use in solid-state lightening synthesis and characterizations of new
technologies with high efficiency at 50-90% polycarbazole based blue-light emitting
range. Moreover, it could reduce global CPNs dispersed in water as ink for ink-jet
electricity use and power consumption1 and printing OLEDs, which will be a large
holds the promise for long life time, and potential for further study.
environmental friendly light sources.2
However, printed OLEDs have a large 2. Materials and Methods
attention because of higher efficiency and 2.1 Materials
reducing the process cost, so OLEDs are All reagents were obtained from
processed directly from solution by spin Tokyo Chemical Industry (TCI) Co., LTD or
coating, ink-jet printing and other Aldrich, or Acros and used without further
techniques.3 purification. All AR-grade solvents for
Conjugated polymer nanoparticles chemical reaction were supplied by Carlo, or
(CPNs) have been widely applied in various Burdick&Jackson and used without further
applications such as optoelectronic, distillation. Tetrahydrofuran (THF) was
photonics, bio-sensing and biomedical distilled from sodium/benzophenone under
imaging. Moreover, CPNs are prepared nitrogen atmosphere prior to use. The
easily including high brightness, good solvents for column chromatography were
photostability and high quantum yield.4 the commercial quality from Thai company.
There are two main methods to synthesize 2.2 Synthesis of 2,7-dibromo-9-octyl-9H-
CPNs: mini-emulsion and reprecipitation.5 carbazole (1)
The former has many advantages including 2,7-Dibromocarbazole (1 g, 3.08
stable CPNs, high concentration and short mmol) was mixed with DMF (12.31 mL)
processing time. CPNs can be prepared as and KOH (0.49 g, 12.31 mmol) at room
temperature. Then after 30 minutes of Carbazole (10 g, 59.81 mmol) was
stirring, bromooctane (2.38 g, 12.31 mmol) dissolved in the mixture solvent between
was added into the reaction. The mixture CH3NO2 (50 mL) and CH2Cl2 (25 mL) (1:2
was stirred for 6 hours. After the completion by volume) and ZnCl2 (24.46 g, 179.42
of reaction, the reaction was poured into mmol) was added into the mixture as well.
water and extracted with AcOEt. The Then 2-chloro-2-methylpropane (20 mL,
combined organic phases were washed with 179.42 mmol) was added dropwise via
water followed by dried with anhydrous additional funnel into the reaction. The
Na2SO4. The product (1) of 89% yield as a reaction was sonicated in ultrasonic bath for
white solid was obtained by recrystallization 60 minutes. The mixture was poured into
with a mixture of CH2Cl2 and MeOH. 1H water and extract with dichloromethane.
NMR (600 MHz, CDCl3) δ ppm: 7.89 (d, J The G1 (3) of 72% yield as a white solid
= 4.1 Hz, 2H), 7.53 (d, J = 1.4 Hz, 2H), 7.34 was obtained by recrystallization with a
(dd, J = 8.3 Hz, 2H), 4.19 (t, J = 7.4 Hz, mixture of CH2Cl2 and MeOH. 1H NMR
2H), 1.84 (quin, J = 7.3 Hz, 2H), 1.40-1.32 (600 MHz, CDCl3) δ ppm: 8.08 (s, 2H), 7.46
(m, 4H), 1.30-1.21 (m, 6H), 0.88 (t, J = 7.0 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H),
Hz, 3H). 13C NMR (151 MHz, CDCl3) δ 6.11 (s, 1H), 1.45 (s, 18H). APCI/Q-TOF
ppm: 141.39, 122.53, 121.47, 121.30, (m/z) calcd for (M+) C20H25N: 279.1987;
119.70, 112.02, 43.36, 31.77, 29.28, 29.14, found 280.2015.
28.76, 27.17, 22.60, 14.05. APCI/Q-TOF 2.5 Synthesis of 2,7-dibromo-9-(8-(3,6-di-
(m/z) calcd for (M+) C20H23Br2N: 437.0177; tert-butyl-9H-carbazol-9-yl)octyl)-9H-
found 438.0195. carbazole (4)
2.3 Synthesis of 2,7-dibromo-9-(8- G1 (3) (0.604 g, 2.16 mmol) and
bromooctyl)-9H-carbazole (2) KOH (0.323 g, 5.76 mmol) were stirred in
2,7-Dibromocarbazole (1 g, 3.08 DMF (10 mL) at room temperature for 30
mmol) was mixed with DMF (12.31 mL) minutes. Then 2 (0.74 g, 1.4 mmol) was
and base potassium hydroxide (0.49 g, 12.31 added in one portion, and mixture was
mmol) at room temperature. Then after 30 stirred overnight at room temperature. After
minutes of stirring, 1,8-dibromooctane (3.35 the completion of reaction, the reaction was
g, 12.31 mmol) was added into the reaction. poured into water and extracted with ethyl
The mixture was stirred for 6 hours. After acetate. The combined organic phase was
the completion of reaction, the reaction was washed with water followed by dried with
poured into water and extracted with ethyl sodium sulfate anhydrous. The target
acetate. The combined organic phase was molecule (4) was purified by silica gel
washed with water followed by dried with column chromatography eluting with
sodium sulfate anhydrous. The product (2) mixture of CH2Cl2 and hexane (1:4) to
was obtained by recrystallization with a obtain 4 as a white solid (63%). 1H NMR
mixture of CH2Cl2 and MeOH in order to (600 MHz, CDCl3) δ ppm: 8.09 (d, J = 1.8
give the desired compound as a white solid Hz, 2H), 7.86 (d, J = 8.2 Hz, 2H), 7.51 (d, J
in 66% yield. 1H NMR (600 MHz, CDCl3) = 1.5 Hz, 2H), 7.49 (dd, J = 8.5, 1.9 Hz,
δ ppm: 7.89 (d, J = 8.2 Hz, 2H), 7.53 (s, 2H), 7.32 (dd, J = 8.2, 1.6 Hz, 2H), 7.28 (d,
2H), 7.34 (d, J = 8.2 Hz, 2H), 4.20 (t, J = 7.3 J = 8.6 Hz, 2H), 4.21 (t, J = 7.2 Hz, 2H),
Hz, 2H), 3.39 (t, J = 6.8 Hz, 2H), 1.87-1.81 4.17 (t, J=7.3 Hz, 2H), 1.83-1.80 (m, 4H),
(m, 4H), 1.43-1.38 (m, 2H), 1.38-1.36 (m, 1.45 (s, 18H), 1.35-1.30 (m, 8H). 13C NMR
4H), 1.33-1.29 (m, 2H). APCI/Q-TOF (m/z) (151 MHz, CDCl3) δ ppm: 141.45, 141.38,
calcd for (M+) C20H22Br3N: 514.9282; found 138.99, 123.21, 122.69, 122.56, 121.49,
515.9300. 121.30, 119.71, 116.25, 112.01, 108.00,
2.4 Synthesis of 3,6-di-tert-butyl- 43.27, 43.11, 34.64, 32.07, 29.24, 29.07,
9H-carbazole or G1 (3) 29.04, 28.62, 27.19, 27.02. APCI/Q-TOF
(m/z) calcd for (M+) C40H46Br2N2: analyzer. The DLS results polymer quoted
714.2007; found 715.2014. are the average of three measurements.
2.6 Synthesis of polymers PCBZ-H and Molecular mass of compounds was analyzed
PCBZ-G1 by using Liquid Chromatography-
Sodium dodecyl sulfate (SDS) (0.5 Quadrupole-Time-of-Flight Mass Spectro-
g) and deionized water (50 mL) were meter of Bruker compact QTOF. UV–vis
transferred to a three-neck flask and added spectra were recorded on a Perkin-Elmer
with tetraethylammonium hydroxide Lambda 1050 spectrometer and fluorescence
(Et4NOH) (0.19 mL, 0.46 mmol) then the spectra were measured on an Edinburgh
solution was degassed by bubbling with Instruments FLS 980 spectrometer,
nitrogen for 30 minutes. Monomers 1 or 4 respectively.
(0.1 g, 0.23 mmol or 0.14 mmol,
respectively) and 9-(heptadecan-9-yl)-2,7- 3. Results and Discussion
bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 3.1 Synthesis and characterization
2-yl)-9H-carbazole (0.16 g, 0.23 mmol) The synthesis routes to the
were dissolved in toluene (2 mL), to which monomers and polymers are displayed in
hexadecane (78 µl) was also added, and this Scheme 1. Alkylation of 2,7-dibromo-
solution was degassed for 5 minutes in the carbazole with 1-bromooctane and 1,8-
same manner. Pd(dppf)Cl2 (6.7 mg, 9.13× dibromooctane in the presence of KOH as
10-3 mmol) was added to monomer solution, base gave monomer 1 and compound 2,
which then was transferred to the reaction respectively. Further alkylation of 2 with
vessel. The reaction mixture was emulsifier carbazole 3 under the same afforded
by ultrasonication for 15 minutes while monomer 4 in reasonable yield. The mini-
cooling with an ice bath. The three-neck emulsion polymerization of the monomers 1
flask was resealed and mini-emulsion was and 4 with 9-(heptadecan-9-yl)-2,7-
heated at 70 oC for overnight. bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2.7 Methods 2-yl)-9H-carbazole was carried out using
1
H and 13C NMR spectra were Suzuki Miyaura cross-coupling reaction
recorded on a Bruker AVANCE III HD with Pd(dppf)Cl2 as catalyst, Et4NOH as
(600MHz) spectrometer using CDCl3 as base, SDS as surfactant, and
solvent in all cases. The molar mass of the hexadecane/toluene/water as solvent system.
polymer produced in each reaction was
analyzed using gel permeation
15
Normalized Number Distribution
chromatography (GPC) that were recorded PCBZ-H

PCBZ-G1
from Malvern Omnisec software of Malvern
Viscotek TDAmax with RI detector and 10
using tetrahydrofuran (THF) as an eluent.
The system was calibrated with low
polydispersity polystyrene standards, in the 5
range 1,200 to 4.2 × 106 g/mol, from Agilent
Technologies. Particle size analysis of the
dispersions was completed using dynamic 0
10 100
light scattering (DLS). The particle size Diameter (nm)
distributions of diluted samples were
Figure 1. The number-average particle size
measured at 25 °C using a Particulate
distribution of CPNs from DLS
Systems Nanoplus zeta/nano particle
i
Br Br Br Br
N 89% N
H
ii
Br Br Br Br
N 66% N Br B
r
H
N
iv
63%
Br
N
iii
N 72%
N
H H
v N
O O
Br Br B B
N O N O N n
X PCBZ-H (X=H) X
PCBZ-G1 (X= )
N
H
Scheme 1. Synthetic pathway of monomers and polymers. Reagents and conditions: (i)
bromooctane, KOH, DMF, R.T., 6 h. (ii) 1,8-dibromooctane, KOH, DMF, R.T., 6 h. (iii)
tBuCl, ZnCl2, CH3NO2, DCM, sonicate, 1 h (iv) KOH, DMF, R.T., 12 h. (v) Pd(dppf)Cl2,
hexadecane, toluene, water, SDS, Et4NOH, 70 oC, 24 h
The mixture was emulsifier by ranging from 23,000–25,000 g/mol. PCBZ-
ultrasonication while cooling with an ice G1 containing bulky substituent displayed
bath and the heated at 70 oC for overnight. broader molecular weight distribution
Both of polymers PCBZ-H and PCBZ-G1 polydispersity (PD) of 3.33 compared to that
were obtained as white colloidal solutions. of PCBZ-H (PD = 2.52).7 Additionally, the
They were characterized using GPC and polymers exhibited low of PDI (0.2-0.3)
DLS technique as shown in Table 1. Both with particle size less than 50 nm as shown
polymers show high molecular weight in Figure 1.
Table 1. Characterization of conjugated polycarbazole PCBZ-H and PCBZ-G1
Concentration
Mna(g/mol) Mw b(g/mol) PDc dnd (nm) PDIe
(mg/mL)
PCBZ-H 3.1 23,313 58,352 2.523 14 0.30
PCBZ-G1 2.7 25,680 85,895 3.345 49 0.20
a,b
Determined by GPC in THF with PS standard
c
Polydispersity of polymers from GPC technique PD= Mw/Mn
d
The number average particle size of CPNs
e
Polydispersity index of particles
d,e
Measured by DLS technique
3.2 Optical properties In comparison maximum absorption

The optical properties of PCBZ-H peak between PCBZ-H and PCBZ-G1, the
and PCBZ-G1 were measured by UV-vis peak of PCBZ-G1 was slightly red-shifted
and photoluminescence spectroscopy in a because of donating group of G1. However,
diluted water solution, which their spectra the absorption band of PCBZ-H and PCBZ-
were collected in Figure 2. PCBZ-H had G1 were in region 350-400 nm which
maximum absorption peak at wavelength belong to carbazole ring had been reported
378 nm. While PCBZ-G1 exhibited a by Fu et al.8 The emission spectra of PCBZ-
broader absorption band with the absorption H and PCBZ-G1 were very similar. They
maximum at 384 nm. showed an emission peak at 428, 430 nm
(a) and a shoulder at 454, 455 nm respectively.
Both polymers exhibited blue color
fluorescence with quantum yield in range of
2-5 % as reported in Table 2. Moreover, our
primary study review that these polymer
nanoparticles can be printed on glass
substrate using a conversional ink-jet printer
and show a bright blue emission as shown in
Figure 3.
(b) (c)
Figure 3. Photograph of PCBZ-H film

printed on glass substrate under UV
irradiation
Table 2. Summarized optical properties of
Figure 2. (a) Absorption and PCBZ-H and PCBZ-G1
photoluminescence spectra of conjugated λabsa λemb CIEc
polycarbazole nanoparticles dispersion in Φd(%)
(nm) (nm) (x,y)
aqueous solution at room temperature. (b) PCBZ-H 378 428 0.16, 0.08 2.523
and (c) are PL image of CPNs solution PCBZ-G1 384 430 0.16, 0.07 3.345
a,b
under UV irradiation PCBZ-H and PCBZ- Wavelength of absorption and emission maxima
respectively
G1, respectively c
CIE diagram 1931
d
Photoluminescence quantum yield
T. D.; Scholz, B. J.; Mayr, C. Phys.
4. Conclusion
Status Solidi (a) 2013, 210 (1), 44-65.
This study reports the synthesis of
2. Thejokalyani, N.; Dhoble, S. Renew.
conjugated polycarbazole nanoparticles by
Sust. Energ. Rev. 2014, 32, 448-467.
the Suzuki−Miyaura reaction to obtain high
3. Al Attar, H. A.; Monkman, A. P.;
molecular weight polymers in range of
Tavasli, M.; Bettington, S.; Bryce, M.
23,000-25,000. Moreover, the polymers
R. Appl. Phys. Lett. 2005, 86 (12),
showed high concentration dispersed in
121101.
aqueous solution that are suitable for
4. Feng, L.; Zhu, C.; Yuan, H.; Liu, L.;
electronic devices fabrication. PCBZ-H and
Lv, F.; Wang, S. Chem. Soc. Rev. 2013,
PCBZ-G1 emitted in blue color.
42 (16), 6620-6633.
Importantly, they can be fabricated on paper
5. Behrendt, J. M.; Guzman, J. A. E.;
or any substrates by ink-jet printing, which
Purdie, L.; Willcock, H.; Morrison, J. J.;
is interesting for further study because it is
Foster, A. B.; O'Reilly, R. K.; McCairn,
an important technology for electronic
M. C.; Turner, M. L. Reac. Funct.
devices manufacturing, owing to reducing
Polym. 2016, 107, 69-77.
the cost and environmental hazards.
6. Muenmart, D.; Foster, A. B.; Harvey,
Acknowledgements A.; Chen, M. T.; Navarro, O.;
This research was supported by Promarak, V.; McCairn, M. C.;
Vidyasirimedhi Instituted of Science and Behrendt, J. M.; Turner, M. L.
Technology (VISTEC). We thank Frontier Macromolecules 2014, 47 (19), 6531-
Research Center (FRC) at VISTEC that 6539.
provided instruments for characterization 7. Qin, S.; Geng, Y.; Discher, D. E.; Yang,
and analysis. S. Adv. Mater. 2006, 18 (21), 2905-
2909.
References 8. Fu, Y.; Bo, Z. Macromol. Rapid Comm.
1. Brütting, W.; Frischeisen, J.; Schmidt, 2005, 26 (21), 1704-1710.
The effect of NaCl on SiC formation from sugarcane bagasse ash
via SHS method
Matthana Khangkhamano1*, Rungrote Kokoo2
1
Department of Mining and Materials Engineering, Faculty of engineering,
Prince of Songkla University, Hat Yai 90112, Thailand.
2
King Mongkut’s University of Technology North Bangkok, Bangsue, Bangkok 10800, Thailand
*E-mail: kmatthana@eng.psu.ac.th
Abstract:
SiC powder was synthesized via reaction of sugarcane bagasse ash (SiO2 source) with
metallic Mg and activated carbon using self-propagating high temperature synthesis (SHS)
method. Various amounts of NaCl were introduced into the reaction system to evaluate its
effect on SiC formation and morphology. Phase composition and microstructure of as-
synthesized powder were examined using X-ray diffraction (XRD) and scanning electron
microscopy (SEM). XRD curves revealed that SiC and MgO were major phases whereas Si
and Mg2SiO4 were minor phases. Undesired phases were further leached out using acid
solutions of HCl and CH3COOH.
1. Introduction this study. Two aspects of study are

In the last years, a large amount of communicated here including transfor-
sugarcane bagasse has been produced as mation of SBA into an important non-oxide
agricultural waste by the sugar/alcohol ceramic powder, SiC, using self-propagating
industry worldwide. This fibrous residue is high temperature synthesis (SHS) and
left over after sugarcane extraction is investigation of the effect of NaCl addition
normally reused in the sugarcane/alcohol on SiC formation and morphology. In recent
industries as biomass fuel in boilers for heat years, fabrication of SiC from agricultural
generation. In this process, a huge volume wastes has been widely studied to reduce the
of ash, known as sugarcane bagasse ash increased quantity of the wastes. For
(SBA), is generated as a solid waste by- example, Niyomwas11 fabricated agglo-
product.1 Such waste ash is as high as merated Si-SiC composite powders from
576,600 tons per year in Thailand2 and rice husk ash using SHS process. Li et al.12
mostly is disposed of as waste in landfills also used rice husk to produce SiC
causing a nuisance to the health and nanoparticles and nanowiskers via micro-
especially to environment.3 Up to date, wave heating method. Hongjie et al.13
although utilization of the ash is still synthesized sub-micrometer β-SiC powder
minimal compared to its huge production, a from the reaction of fly ash with carbon at
limited number of researches pointed out about 1400°C. However, the product powder
that the ash can be used as a replacement obtained from SHS process is normally in
material in cement concrete and tiles4-6 a the form of coarse grains due to rapid
reinforcement in polymers7 and adsorbents heating and cooling rate of SHS process.
for heavy metal removal from waste Although addition of NaCl into the reaction
water.8-10 system is advised to reduce particle size of
Because of the limited application of SHS product powder,14 excess adding
SBA and for minimizing the adverse amounts affect SHS reaction. Here, SBA
impacts of SBA, transforming the waste ash waste was used as SiO2 source to react with
into useful products has been presented in
metallic Mg and activated carbon in a SHS overnight in an oven prior to phase
reactor under an argon environment. NaCl identification and microstructure
was used as diluents to investigate its effects examination.
on SiC formation and morphology of the
product powder. Current Controller
Power
Argon Tank
2. Materials and Methods Source
2.1 Raw materials Pressure Guage Vacuum Pump
The raw materials used for this study

were sugarcane bagasse ash (82.93% SiO2), Tungsten Filament
Sample
activated carbon (Ajax Finechem, 99% C),
magnesium (Riedel-deHaen, 99% Mg) and
sodium chloride (Ajax Finechem, 99.9%
NaCl) powders.
2.2 Experimental procedure
A planetary ball mill (Fritsch GMBH. SHS Reactor
Pulverisette) at a rotational speed of 250 rpm Figure 1. Schematic of the experimental
for 1 h was used in this work to reduce the setup.15
particle size of sugarcane bagasse ash
(SBA). Reactant powders were weighed as 2.3 Characterization
stoichiometric molar ratio of SiO2: C: Mg: The chemical composition of as-
NaCl= 1:1:2:n (n = 0, 0.1, 0.2 and 0.3 mole) received SBA was identified by X-ray
of the reaction formula (2) and dry mixed in fluorescence energy dispersive spectrometer
a ball mill jar for 2 h.Then, a cylindrical (XRF, PW2400, Philips, Netherlands). The
graphite mold with a cavity of 25 mm phases and crystalline structures of the as-
diameter was filled with the milled reactant synthesized and leached products were
mixture where was then a mechanically examined by a powder X-ray diffraction
compressed to form a green compact sample. (XRD, XPert MPD, Philips Netherlands).
The sample was then loaded into a reaction JCPDS cards used were SiC (01-073-1708),
chamber of SHS reactor which MgO (01-089-4248), Si (01-075-0589), and
Mg2SiO4 (01-084-1402). Spectra were
schematically represented in Figure 1. The
recorded at 30 mA and 40 kV using Ni-
reaction chamber was evacuated and filled
filtered Cu-Kα radiation (λ=0.154178 nm).
with argon gas at the pressure of 0.5 MPa.
The scan rate (2θ) was 3°/min with a step
This operation was repeated at least twice in size of 0.05°. Morphologies of the leached
order to ensure an inert environment during powder were characterized by a scanning
reaction revolution. The combustion was electron microscope (SEM, Quanta 400, FEI,
then generated by ignition of a tungsten
Czech Republic), respectively.
filament. Then, under self-propagating
conditions, the reaction front travels until
reaches the opposite end of the sample. After
3.1 Thermodynamic analysis
reaction, the product was allowed to cool to
room temperature. The reacted sample was In combustion synthesis, adiabatic
temperature (Tad) is important for predicting
washed repeatedly with distilled water to
the reaction of the reactants and also the
remove NaCl and leached using acid
occurrence of the self-propagating reaction.
solutions of HCl:CH3COOH (v/v) at room
Therefore, theoretically, the adiabatic
temperature for 2 h to remove impurity
temperature (Tad), the amount of exothermic
phases. The leached powder was then dried
heat given off by a combustion reaction, was
calculated based on the assumption of the temperature, and Tad is the adiabatic
change in enthalpy (ΔH) of the reaction temperature.
which used to heat up the reactants and no Thermodynamic calculations for
energy was lost by convection to the equilibrium concentration of stable species
surrounding environment. In the case of SiC produced by SHS reaction were calculated
synthesis from elements, the reaction can be using HSC® program17 based on the Gibbs
written as follows: energy minimization method.18 The
calculating results showed that it is
Si + C = SiC (1) thermodynamically feasible to synthesize
composites via the reaction system of SiO2-
The reaction (1) has a moderate C-Mg due to a highly exothermic reaction at
enthalpy of product formation and thus has room temperature (ΔH = -655.15 kJ) and
relatively low adiabatic combustion thermodynamic instability at room temper-
temperature (Tad=1586.85ºC). Thus, it is not ature. As accepted that the reaction can be
easy to accomplish a self-sustained SHS self-sustained combustion when adiabatic
process in this system. However, the other temperature of the reaction higher than
way to produce SiC powder by using SHS 1800ºC.19 From calculation, adiabatic
method involves sequence of two reactions temperature of each sample with the
that take place in the combustion front: difference amount of NaCl (0, 0.1, 0.2 and
reduction of silica by a metal to produce 0.3 mole) of the reactions are (Tad) 2386.5,
pure silicon, followed by silicon reaction 2288.9, 2195.3 and 2108.9°C which are
with carbon. The SiO2 + (Mg, Al) + C feasible for SHS reaction via this system.
system is much more exothermic, as 3.2 Characterization of SHS product
compared to binary Si + C composition. Figure 2 and 3 show XRD curves of
Thus, it is relatively easy to initiate the SHS as-synthesized and leached powders,
mode in such reduction-type mixture respectively, with different mole ratios of
without using any special enhancing NaCl addition. SiC was found evidently
means.16 In this system, the overall along with by-product MgO in all samples
combustion reaction for reduction synthesis while SiO2, Si and Mg2SiO4 remained as
of SiC, when magnesium (Mg) is used as a minor phases. The resulting SiC were in both
reducing element and sodium chloride is α-SiC and β-SiC phases. It was noticed that,
used to decrease particle size of the SiC
with increasing NaCl contents, residual SiO2
powders, can be written as follows:
was noticeably decreased whereas Si and
MgO were increased indicating the increased
SiO2 + C + 2Mg + nNaCl
reduction rate of SiO2 by Mg. Increasing
= SiC+ 2MgO + nNaCl (2)
NaCl amount gives rise to a larger amount
The adiabatic temperature (Tad) of of liquid phase, caused by NaCl melting, in
the SHS process can be calculated from the the reaction system resulting in an increase
enthalpy of the reaction. This is the in contact opportunities between the
maximum theoretical temperature that the reactants during the reaction. In addition, it is
reactants can reach, and it is determined worth to note that addition of NaCl as an
from equation (3). This equation applies to inert diluent into SHS reactions results in
phase change occurring between the initial lower reaction temperature.14 This is in
temperature and Tad. accordance with the results presented here
(3)
that the more NaCl, the more Si, but the
higher degree in lower reaction temperature
Where, ΔH is the enthalpy of the reaction, and thus the higher degree in incomplete
ΔHf is the enthalpy of transformation, Cp is reaction within a short time (seconds). The
the specific heat capacity, Tm is the melting presence of an impurity Mg2SiO4 in all SHS
Figure 2. XRD patterns of as-synthesized Figure 3. XRD patterns of leached products
products of SiO2-C-Mg-nNaCl System in of SiO2-C-Mg-nNaCl System in different
different mole ratio of NaCl (a) 0, (b) 0.1, mole ratio of NaCl (a) 0, (b) 0.1, (c) 0.2 and
(c) 0.2 and (d) 0.3 mole (d) 0.3 mole
Figure 4. SEM images of leached products of SiO2-C-Mg-nNaCl System in different mole

ratio of NaCl (a) 0, (b) 0.1, (c) 0.2 and (d) 0.3 mole
products might be due to the reaction of gas Figure 4 shows morphologies of the
phase products, i.e., Mg(g) and SiO(g) at high leached powders containing various NaCl
temperature during an SHS reaction.20 contents. Agglomerated, spherical-like and
However, it was claimed that this fine particles were observed. With increasing
intermediate Mg2SiO4 phase can be leached NaCl amounts, particle size was
out using acid solution of HF: H2O (v/v) significantly decreased from 100 nm
while stirring at 40 °C for 1 h.21 (without NaCl addition) to 35 nm (with 0.3
mole of NaCl addition) indicating the effect of international RILEM conference,
of NaCl on morphologies of the product Barcelona, Spain, Nov 8-11, 2004.
powder which is in agreement with Çamurlu 3. Foo, K. Y.; Hameed, B. H. J. Hazard.
and Maglia22 and Khanra, et al.23 Two Mater. 2009, 172, 523-531.
reasons are given here for the reduction in 4. Ganesan, K.; Rajagopal, K.; Thangavel,
particle size when NaCl was increased. One K. Cem. Concr. Compos. 2007, 29, 515-
is that the lower reaction temperature, 524.
caused by NaCl diluents, and hence grain 5. Modani, P. O.; Vyawahare, M. R. Proc.
growth suppression. The other is evaporation Eng. 2013, 51, 25-29.
of NaCl during combustion process. NaCl 6. Alavéz-Ramírez, R.; Montes-García, P.;
might has partially melted and vaporized Martínez-Reyes, J.; Altamirano-Juárez,
giving rise to a coating layer on the surface
D. C.; Gochi-Ponce, Y. Constr. Build.
of SiC particles9 which might result in a
reduction in grain growth of the product Mater. 2012, 34, 296-305.
particles. 7. Santos, R. J.; Agostini, D. L. S.; Cabrera,
F.C.; Reis, E. A. P.; Ruiz, M. R.;
4. Conclusion Budemberg, E. R.; Teixeira, S. R.; Job,
Fine SiC particles were synthesized C. Polímeros. 2014, 24, 646-653.
from SBA waste via SHS process to 8. Rao, M.; Parwate, A. V.; Bhole, A. G.
investigate the effect of NaCl on SiC Waste Manage. 2002, 22, 821-830.
formation. The results clearly showed that
9. Gupta, V. K.; Ali, I. J. Colloid Interface
NaCl had a significant effect on both SiC
Sci. 2004, 271, 321-328.
formation and morphologies. The more NaCl
addition is, the lower residual SiO2 and the 10. Gupta, V. K.; Jain, C. K.; Ali, I.; Sharma,
finer particle size become. However, Si M.; Saini, V. K. Water Res. 2003, 37,
impurity increased with the increased NaCl 4038-4044.
due to incomplete reaction at lower 11. Niyomwas, S. J. Met. Mater. Miner.
combustion temperature caused by NaCl 2009, 19, 21-25.
diluents. 12. Li, J.; Shirai, T.; Fuji, M. Adv. Powder
Technol. 2013, 24, 838-843.
Acknowledgements 13. Hongjie, W.; Yonglan, W.; Zhihao, J. J.
Many thanks to the Faculty of
Engineering, PSU and KMUTNB for Mater. Process. Technol. 2001, 117, 52-
financial support. Also thanks to Assoc.Prof. 55.
Sutham Niyomwas and the center of 14. Khanra, A. K.; Pathak, L. C.; Godkhindi,
excellence in materials engineering M. M. J. Mater. Process. Technol. 2008,
(CEME), PSU, for facility provision, and Si 202, 386-390.
Thu Myint Maung for fruitful discussion. 15. Khangkhamano, M.; Singsarothai, S.;
Kokoo, R.; Niyomwas, S. Int. J. Self-
References
Propag. High-Temp Synth. 2018, 27,
1. Souza, A. E.; Teixeira, S. R.; Santos, G. (accepted for publication).
T. A.; Costa, F. B.; Longo, E. J. Environ. 16. Alexander, S. M. Combustion synthesis
Manage. 2011, 92, 2774-2780. of silicon carbide; properties and
2. Cordeiro, G. C.; Filho, R. D. T.; applications of silicon carbide; Gerhardt
Fairbairn, E. M. R.; Luis, M. M. R., Ed.; Intech: Croatia, 2011; pp. 389-
T.; Oliveira, C. H. Proceedings 409.
17. Outokumpu HSC Chemistry® for 21. Tawat, C. J. Aust. Ceram. Soc. 2017,
windows, version HSC 4.1. Finland: 53, 245–252.
Outokumpu Research Oy; 1999. 22. Çamurlu, H. E.; Maglia, F. J. Eur.
18. Gokcen, N. A.; Reddy, R. G. Ceram. Soc. 2009, 29, 1501-1506.
Thermodynamic; Plenum Press: New 23. Khanra, A. K.; Pathak, L. C.; Mishra, S.
York, 1996. K.; Godkhindi, M. M. Mater. Lett.
19. Moore, J. J.; Feng, H. J. Prog. Mater Sci. 2004, 58, 733-738.
1995, 39, 243-273.
20. Yermekova, Z.; Mansurov, Z.;
Mukasyan, A. Int. J. Self-Propag. High-
Temp Synth. 2010, 19, 91-96.
Preparation and application of poly(acrylamide) based polymers
as corrosion inhibitor
Phanita Tansiri1,3, Parinya Jitreewas1,3, Siriporn Pranee2, Narong Pungwiwat1,
Sukanya Thepwatee1, Samitthichai Seeyangnok1,3*
1
2
3
Abstract:
Stainless steels are one of the essential materials made for industrial use. Its physical
properties in term of durability and its chemical resistance are the vital factors to be
considered. Stainless steels are as such used to produce various kind of equipment such as
boilers, cooling towers and pipe systems. On the other hand, there are some poor properties
existed in the steels that can be corroded by exposing them to water. However, there are
many different methods that can be used to protect metals from corrosion. One such popular
way is by installing a corrosion inhibitor. Poly(acrylamide) based polymers are thus
employed in the water as corrosion inhibitor. This research is mainly focused on the synthesis
and the ability to inhibit the corrosion of stainless steel. Poly(acrylamide) based polymers are
synthesized by free radical polymerization using ammonium persulfate as an initiator and
characterized by FT-IR, TGA and acidity test. The efficiency of corrosion inhibitor is then
analyzed on 1018 carbon steel by potentiodynamic polarization measurement. The corrosion
behavior is investigated by electrochemical impedance spectroscopy technique.
1. Introduction Water-soluble poly(acrylamide) is

Carbon steel has important role in widely used in a variety of corrosion
industrial for producing various kinds of inhibitor1. The problem is not just corrosion
equipment such as boilers, cooling towers but there is a scale. The most popular
and pipe systems.1,5 Their properties in term method for this problem is to use a chemical
of durability and chemical resistance are the inhibitors such as poly(acrylic acid), because
vital factors to be considered. The most of their strong scale inhibition performance.4
problem of carbon steel is corrosion that Therefore, the researcher would like to
happed by ions in water, such as Ca2+, SO42- develop the properties to inhibit corrosion
and Cl-. However, there are many ways to and scale forming.
prevent corrosion such as galvanic In this work, the poly(acrylamide)
corrosion, crevice corrosion, pitting, (PAM) was synthesized via free radical
intergranular corrosion, selective leaching or polymerization using ammonium persulfate
dealloying, erosion corrosion and stress as an initiator and PAM-co-PAA copolymer
corrosion.2 One of the most popular ways is was synthesized in the similarly process by
to use a corrosion inhibitor. The organic adding acrylic acid (AA) monomer into the
compounds containing functional groups reaction. After that, the synthesized
with heteroatom because they can create polymers were characterized by FTIR and
coordinative interactions between lone pair TGA technique. The inhibition efficiency of
electrons in the molecule with the metal and the synthesized polymers on the carbon steel
the chemicals used must be environmentally AISI 1018 in 0.01M NaCl solution was
friendly.3,5 investigated using potentiodynamic and EIS
measurement by different concentrations of 20 mg of the polymer was ground and
inhibitor (25, 50, 100, 150 and 200 mg L-1) heated up from 40 to 800 °C under 50
at 293 K. mL.min-1 nitrogen flowing atmosphere.
Acidity of polymer (pH Test)
2. Materials and Methods The synthesized polymer was
2.1 General dissolved in 100 mL distilled water and then
Acrylamide monomer (AM), acrylic the acidity value was analyzed by pH meter.
acid monomer (AA) and ammonium
persulfate (NH4S2O8) are analytically pure 2.4 Inhibition efficiency of polymeric
grade. The corrosion solutions were inhibitor
prepared by dissolving specified amount of Potentiodynamic measurement
copolymer in 0.01 M NaCl. Distilled water The potentiodynamic measurement
was used in all the studies. Carbon steel was was performed on AutoLab PGSTAT 10
used in this work is AISI 1018 grade steel. using three electrode cell. The working
2.2 Synthesis of PAM and PAM-co-PAA electrode was used as a carbon steel AISI
PAM and PAM-co-PAA (DP25) 1018 with one face of the electrode of
were synthesized by free radical constant 1 cm2 exposed area was sanded
polymerization shown in Scheme 1. The with No. 600 and 2000 and then washed
AM, AA and NH4S2O8 in weight ratios as with distilled water before test. A silver
shown in Table 1. Monomers and initiator chloride electrode (Ag/AgCl2) was used as
were dissolved in 500 mL distilled water reference electrode. A platinum was used as
and the reaction mixture was stirred at 50 °C the counter electrode. The polarization study
for 5 hrs after that 10 mL of 10% H2O2 in was applied voltage from -1 to 1 V with the
methanol was added into the reactor to stop scanning rate at 5 mVs-1. The inhibition
the reaction. The final solution was efficiency was calculated according to the
evaporated under reduced pressure and dried following Eq. 1.4
overnight at 80 °C in air oven to obtain (Eq. 1)
colorless clear solid.
where icorr and icorr(inh) are the corrosion
current density of absence and presence of
Table 1. Weight ratio of monomers and polymeric inhibitor.
initiator on polymerization process
Electrochemical impedance measurement
Ratio AM AA NH4S2O8
monomer monomer (g)
(EIS)
(g) (g) The impedance spectrum was
PAM 20 - 0.115 measured at OCP in the frequency range of
8:2 8 2 0.116 10 kHz to 0.05 Hz with 5 mV amplitude sine
7:3 7 3 0.115 wave and impedance parameters were
6:4 6 4 0.113 analyzed using FRA software.
2.3 Measurement
Infrared spectroscopy (FTIR) 3. Results and Discussion
All samples were analyzed using a 3.1 Characterization of AM, PAM and
FTIR spectroscopy. The samples powder PAM-co-PAA by FTIR
was mixed with KBr powder. The wave The FTIR spectra of AM, PAM and
number of the IR absorption peaks of PAM-co-PAA copolymer are shown in
functional groups was determined in the Figure 1. The absorption peaks at 3353 and
range of 4000–400 cm− 1. 3438 cm-1 (a) are assigned to N-H stretching
Thermogravimetric analysis (TGA) of amide group. The absorption peak of C=O
stretching at 1674 cm-1 and the absorption
To analyze the thermal property, 10- peak of –C=C- stretching at 1613 cm -1
+
Acrylamid Acrylic Poly(acrylamide-co-acrylic
Scheme 1. Synthesis of PAM-co-PAA
completely disappear in (b) and (c) reveals

PAM-co-PAA 6:4
that free radical polymerization between 100 PAM-co-PAA 7:3
AM, PAM and PAM-co-PAA had been PAM-co-PAA 8:2
80
occurred.
Weight (%)
60
(a) (b) (c)
PAM-co-PAA 6:4
40
PAM-co-PAA 7:3
20
PAM-co-PAA 8:2 0 200 400 600 800
Temperature (°C)
PAM
Figure 2. TGA curves of PAM-co-PAA
AM
4000 3500 3000 2500 2000 1500 1000 500 Table 2. Acidity of polymeric initiator
Wavenumber (cm-1)
Figure 1. FTIR spectra of AM, PAM and Ratio pH
PAM 5.546
PAM-co-PAA 8:2 2.437
7:3 2.601
3.2 Characterization of PAM-co-PAA by 6:4 2.851
TGA
The thermogravimetric thermal 3.3 Inhibition efficiency of polymeric
stability obtained by TGA was show in inhibitor by Potentiodynamic measurements
Figure 2. The results of each PAM-co-PAA The performance of corrosion
copolymers were showed three stage of inhibition is shown in Figure 3. The
decompositions (Table 3). The first stage at inhibition efficiency followed with
201-300 oC is the evaporation of the solvent PAM:PAA ratios of 10:0 (PAM) > 8:2 > 7:3
and water and some decomposition of amide > 6:4 respectively. At 200 mgL-1 of PAM,
group to generate ammonia gas. The second the highest corrosion inhibition was 97.39%.
stage at 300-350 oC is a decomposition of The corrosion efficiency was decreased with
carboxylic acid to give CO2. The last stage the increasing of AA monomer because the
at high temperature, the decomposition of carboxylic acid group in AA part can
the carbon backbone of the polymer increase the acidity of the solution.
structure was considered. At high 3.4 Inhibition efficiency of polymeric
temperature, the thermal decomposition inhibitor by EIS measurement
behavior of copolymers shows that the Figure 4 shows Nyquist plots of
thermal stability, followed with ratio PAM for carbon steel AISI 1018 in 0.01 M
8:2 > 7:3 > 6:4 respectively. NaCl at 293 K with different inhibitor
From Table 2, acidity of polymer concentrations. The dimeters of semicircles
was increased with increasing AA monomer increase with increasing of concentration of
because AA monomer has a carboxyl group. PAM solution. The impedance plots are not
Table 3. % Decomposition of copolymer
Ratio % Decomposition
Temperature (oC) 200-300 300-350 350-400
8:2 95.83 77.48 25.06
7:3 81.42 76.54 23.83
6:4 95.69 78.09 21.89
–C-C- carbon backbone of PAM-co-PAA.

The TGA measurement indicated that the
thermal decomposition behavior of
copolymers with the thermal stability
followed by the weight ratios of monomer of
8:2 > 7:3 > 6:4, respectively. The acidity of
the copolymer increases with increasing of
AA monomer. The efficiency of corrosion
inhibition is found that the highest corrosion
inhibition of 97.39% is obtained at 200 ppm
Figure 3. Corrosion inhibition (%) of PAM
of PAM.
and PAM-co-PAA
Acknowledgements
acknowledge Department of Industrial
Chemistry, Faculty of Applied Science, and
partially supported by the Graduate Thesis
Research Grant (GTRG) from the Graduate
College, King Mongkut's University of
Technology North Bangkok.
References
1. Xueling, Y.; Changjun, Z.; Yibie, Q.
Starch/Stärke. 2014, 66, 968–975.
Figure 4. Nyquist plots of PAM
2. Maqsood, A. M.; Mohd, A. H.; Firdosa,
N.; Shaeel, A. A.; Zaheer, K.
perfect semicircles, and this variation has Electrochem. Sci. 2011, 6, 1927 – 1948.
been assigned to frequency dispersion. 3. Tianli, H.; Jinyun, L. Procedia Eng.
2010, 7,404-409.
4. Conclusion 4. Xiaorui, G.; Fengxian, Q.; Ke, D.;
PAMs and PAM-co-PAAs are Xinshan, R.; Kaichen, H.; Jicheng, X.;
synthesized via free radical polymerization Dongya, Y. Appl. Clay Sci. 2014, 99,
using ammonium persulfate as an initiator. 187-193.
The synthesized polymers were 5. Migahed, M. A,; Rushwan, S. M,;
characterized by FTIR measurement Kamel, M.M,; Habib, R.E.J. Mol. Liq.,
revealed that the –C=C- functional groups of 2016, 224, 849-858.
AM and AA are converted to
Synthesis of hydrophobically-modified poly(ethyleneimine) as surfactant
for cationic CdSe/ZnS quantum dots with high colloidal stability
Jamornpan Yangcharoenyuenyong1,2, Tirayut Vilaivan1, Numpon Insin1,2*
1
Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai, Bangkok 10330, Thailand
2
Phayathai, Bangkok 10330, Thailand
*E-mail: numpon@gmail.com
Abstract:
Semiconductor nanoparticles or quantum dots (QDs), especially CdSe/ZnS, are of
interest as bio-sensors due to their unique size-dependent optical properties and
photostability. To apply QDs in biological applications, the surface of QDs must be modified
to become charged or hydrophilic. Poly(ethyleneimine) or PEI has been used in the
modification of various nanoparticles to create water-dispersible cationic nanoparticles.
However, the colloidal and photo stability of the modified nanoparticles was limited. In this
study, we prepared a photo-stable colloidal cationic PEI/QDs using a new modified polymer
in a micelle formation process. To induce the micellar structure, PEI was partially acylated
with octanoic acid (Oct) via EDC/NHS coupling to introduce some hydrophobicity and
coated onto QDs surface via micelle formation in chloroform/water biphasic mixture. The
Oct-PEI/QDs were characterized using UV-vis and fluorescent spectroscopy to confirm that
the optical properties were not affected by the modification process, which is significantly
different from the PEI/QDs prepared from direct ligand exchange process. Moreover, the
zeta-potential of the Oct-PEI/QDs is 30-60 mV, which is similar to other PEI/nanoparticles
reported (20-30 mV) due to acylation of PEI, and the resulted Oct-PEI/QDs with the molar
ratio of 60:1 exhibit lower cytotoxicity for their less positive charges.
1. Introduction silanization, capping with thiol molecules,

Colloidal semiconductor nano- etc.9-11
particles or quantum dots (QDs) are Coating QDs surface with
inorganic nanoparticles whose size and band hydrophilic polymers is one of the most
gap are inversely related, resulting in employed methods to create water-soluble
tunability of emission.1,2 Their unique and biocompatible nanoparticles.4,12,13
optical properties make them potentially However, most of the polymers extensively
useful for applications in biological imaging, investigated for this purpose are nonionic
sensors, etc.3,4 and anionic polymers since most of cationic
One type of QDs that become widely polymers have shown some problems with
used is CdSe/ZnS because of their stability and cytotoxicity. The limitation of
photostability and wide range of band gap using cationic polymers hinders many
covering almost all the visible region.5,6 applications that require cationic
However, the conventional cadmium based nanoparticles. The cationic polymer which
QDs have intrinsic toxicity due to the has been studied to be suitable for
presence of cadmium, which remains the bioapplications is poly(ethylene)imine (PEI)
major hindrance for their use in in vivo because of its interesting proton-sponge
applications.7,8 Many recent studies on effect.14,15 However, there is still some
cadmium-based QDs revealed one approach limitation on using PEI since high molecular
to produce biocompatible CdSe/ZnS QDs is weight PEI exhibits cytotoxicity.16
the methods of surface modification such as
Scheme 1. Schematic of QDs surface modification via micelle formation
There are two major methods for phases of CdSe/ZnS QDs were characterized
coating nanoparticles with hydrophilic by a D/MAX 2200 X-ray diffractometer
polymers. One is replacing the original (Rigaku, Japan) with Cu-Kα radiation at an
surfactants on the nanoparticles with the accelerating voltage of 40 kV and an applied
polymers with a suitable functional group.17 current of 30 mA. Transmission Electron
The second method is by forming micellar Microscopy (TEM) (model TECNAI 20,
structures onto the hydrophobic Philips, US) at Burapha University was used
nanoparticles without replacing the original to estimate the average size of CdSe and
surfactants.18,19 Compared to the first CdSe/ZnS QDs. A Zetasizer Nano ZS
method, the second method is known to (Malvern, UK) was used to measure zeta-
increase the hydrodynamic radii of the potential and size of Oct-PEI/QDs and
nanoparticles, but the optical properties PEI/QDs.
could be better maintained.19,20 In the case of
QDs, both methods has been reported for 2.2 Synthesis of QDs
nonionic and anionic polymers,18,21,22 but CdSe/ZnS QDs were synthesized as
only the first method was reported for previously described.9 In brief, for CdSe
cationic polymers.23 In this study, we report core, cadmium-2,4-pentanedionate
the formation of PEI/QDs with micellar (Cd(C2H7O2)2, 0.09 g), 1,2-hexadecanediol
structure by modifying the PEI with (C16H34O2, 1.7 g) and 6 mL of 1.5 M of tri-
octanoic acid to form the amphiphilic n-octylphosphine selenide (TOPSe) solution
modified PEI (Oct-PEI) as shown in Scheme were dissolved in tri-n-octylphosphine
1. Low molecular weight PEI (MW = 800, (TOP, 4 mL) and rapidly injected into a
PDI = 1.33) is used in this work to minimize mixture of tri-n-octylphosphine oxide
the cytotoxicity. The stability and (TOPO, 6 g) and hexadecylamine (HDA, 6
cytotoxicity of the resulted QDs (Oct- g). The mixture was then heated to 360๐C
PEI/QDs) are compared with the PEI/QDs under N2 atmosphere and maintained at this
prepared using the direct surfactant temperature for 1 h. The solution was cooled
exchanging using unmodified PEI. down to 160๐C for further growth. To
synthesize CdSe/ZnS QDs, 10 mL of CdSe
core were precipitated by centrifuged with
2.1 Reagents and instruments
ethanol and redissolved in hexane. Then the
Unless specified, chemicals were
CdSe core were injected into a mixture of
purchased from Sigma-Aldrich (St.Louis,
zinc-acetate dihydrate (Zn(OAc)2·2H2O,
MO) and used without further purification.
0.21 g) and HDA (1.6 g) in 5 mL of
UV-vis spectrophotometer (HP8453,
trioctylamine under N2 gas. Then, freshly
Agilent, US) and fluorescence spectro-
prepared solution of sulfur powder (0.06 g)
fluorometer (Cary Eclipse, Agilent, CA)
in 5 mL of TOP was added dropwise to the
were used to characterize the absorption and
mixture at 150๐C to obtain the CdSe/ZnS
emission spectra of the QDs. The crystalline
core/shell QDs. UV absorption, fluorescence
emission spectra, XRD and TEM images the medium containing unreacted dye was
were used for characterization of particle removed, and 150 μL per well DMSO was
optical properties and sizes. added to dissolve the achieved purple
2.3 Modification of QDs surface formazan crystals. The absorbance was
For cationic surface modification, measured in a BioTek Elx80 at a wavelength
Branched poly(ethylene)imine-ethylene of 570 nm.
diamine (MW = 800) was 30% acylated
with octanoic acid through EDC/NHS 3. Results and Discussion
coupling (Oct-PEI) and coated onto QDs 3.1 Characterization of QDs
surface via micelle formation. The
CdSe/ZnS QDs and Oct-PEI with different
molar ratio between QDs and Oct-PEI (1:60,
1:70, 1:80 and 1:90) were dissolved in 5 mL
of chloroform and left overnight in the air at
room temperature. After the chloroform was
evaporated by rotary evaporator (Buchi,
Switzerland), the mixtures of QDs and Oct-
PEI were dissolved in DI water. The Oct-
PEI/QDs were thoroughly purified by Figure 1. XRD pattern of CdSe/ZnS QDs
dialysis (MWCO 30,000) to remove (black line), JCPDS no. 19-0191 CdSe
unreacted polymer, and their optical pattern (red line), JCPDS no. 77-2100 ZnS
properties were compared with PEI/QDs. pattern (blue line) and the inset is TEM
The PEI/QDs was obtained similar to a image
previous study.23 Briefly, QDs precipitate
(0.06 μmol) and PEI (0.2 g) were dissolved The CdSe/ZnS QDs was synthesized
in 1 mL chloroform and left overnight using hydrothermal method. From Figure 1,
before the QDs were precipitated in XRD pattern of as-synthesized QDs showed
cyclohexane and re-dissolved in DI water. the characteristic peaks of cadmium selenide
2.4 Cytotoxicity measurement in zinc blende cubic structure and zinc
Mouse fibroblast L929 cells were sulfide in sphalerite-zinc blende structure,
used as a cell model. Cells were grown in confirming that the synthesized QDs
Dulbecco’s Modified Eagles Medium contained both CdSe and ZnS. In addition,
(DMEM; Gibco, US) supplemented with 4 extra peaks around 22-23º is most likely
mM L-glutamax, 1% Na-pyruvate, 100 came from the mixture of excess surfactants
U·mL-1 penicillin and 10% fetal bovine on the surface of QDs, which has been
serum (FBS) (all from OBCR-laboratories, observed previously in the similar QD
Thailand), in a humid atmosphere at 37 °C. synthesis process.24 The sizes of the
For MTT assay, cells were cultured in 96- synthesized CdSe/ZnS QDs were measured
well plates (10,000 cells in 200 μL DMEM by TEM as shown in the inset of Figure 1.
per well). After 24 h of incubation to bring The average diameter of the QDs is 4.54 ±
the cells to confluence, the cell culture 0.61 nm.
medium was replaced with fresh culture Oct-PEI which was partially acylated
medium containing materials (Oct-PEI/QDs through EDC/NHS coupling. Its structure
in different molar ratio and PEI/QDs) at a was confirmed using infrared spectroscopy.
concentration 10 and 100 μg·mL-1. After 24 From the spectra in Figure 2, it was
h of incubation with different materials at observed that the peak of carbonyl of amide
37 °C, the 20 μL of 5 mg·mL-1 MTT assay group (O=C-N) appeared in the spectrum of
stock solution in PBS was added to each Oct-PEI (1648.08 cm-1, Figure 2b). This
well after 24 h incubation at 37 °C and peak was not presented in the spectrum of
further incubated for 1.5 h at 37 °C. Finally, PEI (Figure 2a). Moreover, the Oct-PEI also
exhibited the signal from the functional synthesized CdSe/ZnS QDs in hexane. From
group of amine (3300-3500 cm-1)25 similar the relative fluorescence spectra (Figure 4a
to PEI. The structure was also confirmed by and 4b), we observed that all the Oct-
high resolution ESI-QTOF mass PEI/QDs gave ~3 times weaker fluorescent
spectrometry. Oct-PEI showed series m/z intensity than the original QDs, but the
about 1288.89 which increased from series signals were enough to be observed (Figure
m/z of PEI (about 878.98). From these 4c). Freshly prepared Oct-PEI/QDs with the
results, it can be indicated that the Oct-PEI molar ratio of 80:1 exhibited strongest
was not completely acylated and should still fluorescent signals, but after 14 days, the
perform as a cationic polymer. signals from Oct-PEI/QDs with the molar
ratio of 60:1 was significantly increased but
the signals were slightly decreased in the
ones with molar ratios of 70:1 and 80:1.
Moreover, the emission wavelength of Oct-
PEI/QDs was slightly red-shifted. This
phenomenon might be due to the incomplete
micelle formation between Oct-PEI and
QDs at the molar ratio of 60:1 leading to
some aggregation of the QDs leading to an
increase in the size of QDs resulting in the
red-shifted emission and decrease in
fluorescence. After storing for 14 days, the
Figure 2. Infrared spectra of PEI (a) micelle formation and surface stabilization
compared with Oct-PEI (b) has become more complete and the
fluorescent signal restored.
After confirming its functional
groups and molecular weight, Oct-PEI was
coated onto the QDs surface via micelle
formation. Figure 3. showed that the
absorption spectra of Oct-PEI/QDs still
showed the broad absorption character with
the first absorption peak at ̴ 550 nm similar
to as-synthesized CdSe/ZnS QDs. In
contrast, the first absorption peak
disappeared in PEI/QDs, suggesting that the
QDs have lost their unique optical
properties. Moreover, the fluorescent signal
of PEI/QDs (Figure 4c) was significantly
less than Oct-PEI/QDs. These results are
most likely due to the surface disturbance
and dissolution of the QDs from the direct Figure 3. Absorption spectra of CdSe/ZnS
ligand exchange process. These observations QDs, PEI/QDs and different molar ratio of
demonstrated the benefits of using Oct-PEI Oct-PEI/QDs
over the PEI on the quality of the resulted
QDs. Size and zeta-potential data of Oct-
Moreover, we optimized the molar PEI/QDs according to Table 1 showed that
ratio between Oct-PEI and QDs by all the particles exhibit positive charge as
comparing the fluorescent intensity of the designed. Higher ratio of polymer to QDs,
Oct-PEI/QDs with different molar ratios at gave higher value of zeta-potential,26,27
the same QDs concentration with as- suggesting that the charge could be
controlled by adjusting the molar ratio.
Moreover, all the Oct-PEI/QDs are
significantly larger than CdSe/ZnS QDs
especially in Oct-PEI/QDs with the molar
ratio of 80:1 likely because the micelle
structure may contain several individual
particles.
Figure 5. Cytotoxicity of Oct-PEI/QDs in

different concentration and PEI/QDs against
L929 cells
3.2 Cytotoxicity Measurement

The cytotoxicity of the Oct-PEI/QDs
were affected by concentration, charge and
size of the particles. The results of MTT
assays in Figure 5. showed that at the QDs
concentration of 100 μg·mL-1, the level of
cell viability is lower than at the
concentration of 10 μg·mL-1. The Oct-
PEI/QDs with molar ratio of 60:1 showed
the highest level of cell viability when
compared to other ratios and PEI/QDs. The
reason may relate to the lower positively
charged due to less quantity of PEI on the
surfaces. Moreover, from the result showing
that level of cell viability of the Oct-
PEI/QDs with the molar ratio of 80:1 is
higher than 70:1, this might be from the
Figure 4. Relative fluorescence spectra of significantly larger size of the micelles with
different molar ratio of Oct-PEI/QDs in 1 the ratio of 80:1 leading to less surface area
day (a) and 14 days (b) compared with to interact with the cells and less
CdSe/ZnS QDs and images of QDs under a cytotoxicity.
backlight irradiation (c)
4. Conclusion
Table 1. Sizes and zeta-potentials of Oct- In conclusion, the cationic water-
PEI/QDs with different molar ratios soluble Oct-PEI/QDs were successfully
measured by zeta-sizer analysis produced via micelle formation resulting in
Materials Size (d, nm) Zeta-potential (mV) QDs with superior photo-properties of
Oct-PEI/QDs maintaining fluorescent intensity when
267.2±7.28 36.9 compared with PEI/QDs prepared from
60:1
Oct-PEI/QDs ligand exchange process. The molar ratio
252.8±24.9 38.1
70:1 between Oct-PEI and QDs of 60:1 is the
Oct-PEI/QDs most suitable for bio-applications as they
390.7±20.7 62.5
80:1 exhibit less cytotoxicity while still maintain
high fluorescence properties after storage.
Acknowledgement 12. Yang, W.; Ni, J.; Luo, F.; Weng, W.;
This work was supported by the Wei, Q.; Lin, Z.; Chen, G. Anal. Chem.
Grant for International Research Integration: 2017, 89, 8384-8390.
Chula Research Scholar, 13. Yezhelyev, M. V.; Qi, L.; O’Regan, R.
Ratchadaphiseksomphot Endowment Fund, M.; Nie, S.; Gao, X. J. Am. Chem. Soc.
CU Graduate School Thesis Grant and 2008, 130, 9006-9012.
Center of Excellence on Petrochemical and 14. Wen, S.; Zheng, F.; Shen, M.; Shi, X. J.
Materials Technology. The authors thank Appl. Polym. Sci. 2013, 3807-3813.
Oral Biology Research Center, Faculty of 15. Han, B.; Wang, W.; Wu, H.; Fang, F.;
Dentistry, Chulalongkorn University for Wang, N.; Zhang, X.; Xu, S. Colloids.
laboratory facilities and instruments and Surf. B Biointerfaces 2012, 100, 209-
Assist. Prof. Patcharee Ritprajak for the 214.
cytotoxicity studies. 16. Kim, S.; Choi, J. S.; Jang, H. S.; Suh,
H.; Park, J. Bull. Korean Chem. Soc.
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Preservation of nanodroplets by the colloid-electrospinning technique
Kusuma Thongchaivetcharat, Ratchapol Jenjob, Daniel Crespy*
*E-mail: daniel.crespy@vistec.ac.th
Abstract:
Nanodroplets of different hydrophobic liquids such as essential oils or monomers are
interesting in applications for biomedicine, food science, and self-healing materials.
However, emulsions of nanodroplets cannot be stored for long time due to their
destabilization by coalescence and Ostwald ripening. We present here a method for
preserving the structural integrity and size of nanodroplets. For this, we embed nanodroplets
in dextran nanofibers by colloid-electrospinning. Afterwards, the nanofibers can be easily
dissolved to form again the original nanodroplets. The proof-of-principle was shown for
droplets of essential oils such as peppermint oil which has a similar size between before
embedding in nanofibers (150 nm) and after re-dispersion from nanofibers (157 nm).
Moreover, the self-healing application was studied by encapsulated of the healing agents in
nanofibers and then dissolved the nanofibers to liberate the healing agents for generating self-
healing material which was investigated by dispersive Raman microscope.
1. Introduction of drugs and the permeation profiles of

Nanodroplets have applications in poorly soluble drugs can be improved
biomedicine, food science, and self-healing significantly.4-6
materials. Unfortunately, the applications of Herein, we develop a method to
nanodroplets were limited because of the protect and extend the lifetime of
very high Laplace pressure associated with nanodroplets by electrospinning them. Fiber
their sizes. Therefore, nanodroplets cannot matrixes serve as support for the embedded
usually be stored for a long time. One nanodroplets.7 The idea is to encapsulate
strategy to store nanodroplets is to nanodroplets into nanofibers and liberate the
encapsulate them in polymer or inorganic nanodroplets from nanofibers by dissolving
nanocapsules.1,2 Although nanocapsules can the fibers in water. Furthermore, we also
protect the liquid core, the transportation or investigated this concept for a potential
storage of nanocapsules is still a problem application for self-healing materials. For
because it requires additional storage this, we embedded nanodroplets of
capacity and more energy for transportation monomer and catalyst into nanofibers by
compared to pure nanodroplets. To avoid side-by-side electrospinning.
these issues, we propose a method to store
nanodroplets and subsequently release them 2. Materials and Methods
from nanofibers fabricated by 2.1 Materials
electrospinning. Dextran (Mr ~ 70,000), peppermint
Electrospinning is a simple and oil (PO), and Grubbs catalyst 2nd generation
popular technique to fabricate nanofibers were purchased from Sigma-Aldrich.
with unique characteristics such as high Dicyclopen tadiene (DCPD, 95%), sodium
surface area and high density of inter-fibrous dodecyl sulfate (SDS, 99%), and n-
pores.3 Many groups reported the hexadecane (99%) were obtained from
preparation of nanofibers to entrap and ACROS organics. Chloroform (CHCl3,
release drugs upon dissolution of the fibers ≥99%), cyclohexane (≥99.8%), and n-octane
in water. With this method, dissolution rate (99%) were supplied from Carlo Erba. The
water mentioned in this document is dyed DCPD emulsion (5 g) was added to an
deionized water. The bodipy dye (λabs = 520 aqueous solution of dextran (50wt%) and the
nm, λem = 551 nm) was synthesized as emulsion (5 g) of Grubbs catalyst in CHCl3
reported previously.8 was added an aqueous solution of dextran
2.2 Preparation of the miniemulsions (50wt%). Dextran (4 g) was slowly added in
A continuous phase containing SDS both solutions and mixed for 2 h.
(7.2 g) in water (24 g) was added into a Afterwards, the two emulsions were
dispersed phase which is composed of electrospun simultaneously as the same
different types of oil (6 g) (octane, PO, conditions. The collected nanofibers were
CHCl3 containing 45 mg Grubbs catalyst, dissolved in 100 µl water or 100 µl of an
pure DCPD, and DCPD containing 5, 10, aqueous solution of AlCl3. The solutions
and 20 mg of fluorescent dye), and were then dropped on glass slides to prepare
hexadecane (250 mg). The mixture was for the characterization.
stirred at 1000 rpm at room temperature for 2.5 Analytical methods
1 h. Then, the miniemulsions were formed Nanodroplets were measured their
after ultrasonicating with 3/4-inch tip at 70% sizes and observed their morphologies by
amplitude for 180 s. DLS (NanoPlus, Particulate systems) and a
2.3 Embedding emulsion droplets in scanning electron microscope (SEM) (JSM-
nanofibers 7610F, JEOL). Dextran nanofibers loaded
Initially, dextran (1 g) was dissolved with nanodroplets of self-healing agents
into water (1 g). Then, the dispersion (5 g) were taken the fluorescence images with a
of octane, PO, and DCPD miniemulsions confocal laser scanning microscopy
were added into the dextran solutions with (FLUOVIEW FV3000, Olympus
gently stirring. Afterwards, dextran (4 g) Corporation) at an excitation wavelength of
was charged into the mixture and stirred for 553 nm. Self-healing material was
2 h in order to prepare 50wt% dextran monitored by dispersive Raman microscope
solutions to be an electrospinning (Senterra II, R200-532, Bruker) with a 523
dispersion. The polymer solution was filled nm laser source and a 20x microscope
in a 10 mL syringe and pumped through a magnification.
needle with a diameter of 1.6 mm at a feed
rate of 0.3 mL.h-1 controlled by a syringe 3. Results and Discussion
pump (TL-F6, Tong Li Tech). The distance 3.1 Morphologies of the nanofibers loaded
between the needle and the collector was with oil nanodroplets
fixed to 10 cm. Then, electrospinning (TL- Octane (3wt% compared to dextran)
Pro-BM, Tong Li Tech) was performed for nanodroplets was first entrapped in dextran
20 min with electrical potentials of 40 kV nanofibers to verify that dextran was a
with opposite and symmetric polarity at the suitable matrix. The dispersion containing
needle and collector, the negative voltage octane nanodroplets and dextran were then
being at the collector. electrospun and yielded smooth nanofibers.
2.4 Re-dispersion of the nanodroplets We also prepared nanodroplets of other
The nanofibers (10 mg) containing hydrophobic compounds such as essential
nano droplets were dissolved in water (3 g). oil (peppermint oil, PO) and the monomer
The obtained dispersions after dissolving dicyclopentadiene (DCPD). PO and DCPD
were then measured by dynamic light (3wt% compared to dextran) nanodroplets
scattering (DLS). were mixed with dextran solution and then
2.5 Self-healing experiment electrospun. The nanodroplets were
The nanodroplets of 0.32%wt DCPD successfully embedded in the nanofibers and
and nanodroplets of 0.15%wt Grubbs yielded smooth nanofibers as shown in
catalyst in CHCl3 were prepared separately: Figure 1.
The concept of embedding
nanodroplets into dextran nanofibers was
further supported in self-healing materials.
We used dyed DCPD and a second
generation Grubbs catalyst to be a model of
starting materials for this application.
10wt% of dyed DCPD and 10wt% of a
catalyst were embedded separately in
nanofibers by side-by-side electrospinning.
Then, the nanofibers were collected and
observed the morphology by a confocal laser
scanning microscopy (CLSM) as shown in
Figure 1. SEM micrographs of dextran
Figure 2. The results indicated that the
nanofibers (a) containing the nanodroplets
droplets of DCPD were distributed along the
of octane (b), PO (c), and DCPD (d)
fibers.
3.2 Sizes of nanofibers and nanodroplets
After electrospinning, fibers were
observed by SEM and the resulting fibers
were then dissolved in water in order to
prove the stability of nanodroplets and
compare the diameters of nanodroplets with
their sizes before electrospinning. The
diameter of nanofibers was proportional to
the size of nanodroplets. Therefore, the
diameter of nanofibers loaded with DCPD
nanodroplets was largest. The hydrodynamic
diameters of the nanodroplets are shown in
Table 1. The hydrodynamic diameters of the
nanodroplets after dissolution were found to
be very close to the hydrodynamic diameter
of the nanodroplets before electrospinning. Figure 2. Schematics depicting nanofibers
Thus, nanodroplets could be successfully with embedded reactive droplets prepared
re-dispersed without noticeable coalescence. by side-by-side electrospinning (a). CLSM
images of the nanofibers taken via different
Table 1. Sizes of nanofibers loaded with modes: Differential interference contact
nanodroplets and average hydrodynamic (DIC) (b), fluorescence (c), and overlay of
diameters of nanodroplets before both modes (d)
electrospinning and after dissolution of the
In order to observe if a self-healing
fibers
reaction could be triggered, the nanofibers
Diameter Diameter of were dissolved with water. Subsequently,
Types of the the nanodroplets [nm] the dissolved fibers were characterized by
of oil nanofibers Electrospinning- After Raman spectroscopy. As shown in Fig. 3c,
[nm] dispersion dissolution
the self-healing reaction did not occur.
Octane 292 ± 97 313 ± 16 256 ± 6 Indeed, the nanodroplets of healing agents
PO 219 ± 74 150 ± 0 157 ± 2 remained colloidally stable and therefore,
DCPD 512 ± 122 314 ± 4 315 ± 3 the droplets kept their structural integrity
and did not coalesce to react. On the
3.3 Characterization of self-healing contrary, the addition of an aqueous solution
materials of AlCl3 triggered the self-healing reaction
by destabilizing the nanodroplets. The
formation of the product of the self-healing electrostatic interactions between nano-
reaction, polydicyclopentadiene (PDCPD), droplets, resulting in coalescence. As a
was monitored by Raman spectroscopy as result, a self-healing reaction occurred,
shown in Figure 3. The bands at 1653 cm-1 leading to the formation of a polymer.
and 1665 cm-1 displayed cis-conformation Moreover, the embedding and release of
and trans-conformation of vc=c-stretching nanodroplets can be further explored for
vibrations of the aliphatic double bonds of cosmetics, food products, and biomedicine.
PDCPD9 (Figure 3d).
Acknowledgements
This work was financially supported
by the Vidyasirimedhi Institute of Science
and Technology. Instrumental support from
the Frontier Research Center (FRC) at
VISTEC is also acknowledged.
Furthermore, the authors would like to thank
Ms. Yuvaret Viturawong from Spacemed
Ltd., Bangkok, Thailand, for operating the
confocal laser scanning microscope.
References
Figure 3. Raman spectra of self-healing and 1. Meier, W. Chem. Soc. Rev. 2000, 29,
reference samples; a) Dextran; b) poly 295-303.
dicyclopentadiene; c) dextran nanofibers 2. Zhao, Y.; Lv, L. P.; Jiang, S.;
with embedded nanodroplets dissolved in Landfester, K.; Crespy, D. Polym. Chem.
H2O; d) dextran nanofibers with embedded 2015, 6, 4197-4205.
nanodroplets of healing agents dissolved in 3. Ritcharoen, W.; Thaiying, Y.; Saejeng,
an aqueous solution of AlCl3 Y.; Jangchud, I.; Rangkupan, R.;
Meechaisue, C.; Supaphol, P. Cellulose
4. Conclusion 2008, 15, 435.
We presented a method to preserve 4. Chen, Y.; Chen, H. R.; Zeng, D. P.;
the structural integrity of nanodroplets. Tian, Y. B.; Chen, F.; Feng, J. W.; Shi,
Nanodroplets of octane, peppermint oil, and J. L. ACS Nano 2010, 4, 6001-6013.
dicyclopentadiene were incorporated within 5. Yu, D. G.; Gao, L.D .; White, K.;
dextran nanofibers via colloid- Branford-White, C.; Lu, W. Y.; Zhu,
electrospinning. The nanodroplets were re- L. M. Pharm. Res. 2010, 27, 2466-2477.
dispersed by dissolution of nanofibers and 6. Illangakoon, U. E.; Nazir, T.; Williams,
their initial size could be maintained. In G. R.; Chatterton, N. P. J. Pharm. Sci.
addition, this concept was not only used to 2014, 103, 283-292.
protect and transport nanodroplets but also 7. Jiang, S.; Lv, L. P.; Landfester, K.;
extended for self-healing applications. For Crespy, D. Acc. Chem. Res. 2016, 49,
this, we encapsulated nanodroplets of 816-823.
healing agents into dextran nanofibers 8. More, A. B.; Chakraborty, G.; Mula, S.;
matrix. The dextran nanofibers loaded with Ray, A. K.; Sekar, N. J Fluoresc 2017,
nanodroplets of healing agents were 1-12.
dissolved in an aqueous solution of AlCl3. 9. Jiang, S.; Ma, B. C.; Reinholz, J.; Li, Q.;
Droplets of catalyst and monomer were Wang, J.; Zhang, K. A.; Landfester, K.;
liberated from dextran nanofibers. The Crespy, D. ACS Appl. Mater. Interfaces
trivalent aluminium cation then screened the 2016, 8, 29915-29922.
Influence of monetite on photocatalytic activity of
titanium dioxide P–25
Pakpassagun Somwong1, Neeranut Kuanchertchoo2, Dujreutai Pongkao Kashima1,3*
1
Department of Materials Science, Faculty of Science, Chulalongkorn University, Phayathai Rd.,
Wangmai, Pathumwan, Bangkok 10330, Thailand.
2
Department of Materials Technology, Faculty of Science, Ramkamhaeng University,
Huamark, Bangkapi, Bangkok 10240, Thailand.
3
Pathumwan, Bangkok 10330, Thailand.
*E-mail: dujreutai@gmail.com
Abstract:
Monetite (Dicalcium phosphate anhydrous, CaHPO4) is one of a chemical compound
in calcium orthophosphate group. In nature, it is often found in mineral or hard skeleton of
animals e.g. shell, bone and teeth. Monetite powder was prepared from brushite (Dicalcium
phosphate dihydrate, CaHPO4.2H2O) by planetary milling for 20 min using water as a milling
media and subsequently drying in oven at 180 °C for 24 h. This research focused on using of
monetite as an adsorbent for adsorption of organic dye, methylene blue (MB) and methyl
orange (MO). It was found that decreasing the particle size of monetite resulted in an increase
the adsorption ability of MB, MO. After the adsorption of organic dye on monetite
completed, TiO2 P-25 was added in order to degrade adsorbed organic dye under UV
irradiation and investigated by UV-VIS spectrophotometer (UV-VIS). Optimization of
monetite to TiO2 P-25 ratio was performed by controlling the weight ratio at 1:1, 3:1, and 5:1.
Photocatalytic activity test by MO organic dye degradation indicated that photocatalytic
activity of TiO2 P-25 could be enhanced by monetite.
1. Introduction adsorb on the surface of monetite powder.

Monetite (Dicalcium phosphate We focus on investigation the influence of
anhydrous, DCPA, CaHPO4)1 is one of a monetite powder on photocatalytic activity
chemical compound in calcium of TiO2 P–25.10-12 Photocatalytic activity test
orthophosphate group. Its chemical formula was performed by monitoring the
is similar to brushite (Dicalcium phosphate degradation of two different types of organic
dihydrate, DCPD, CaHPO4.2H2O)2,3 dyes, cationic dye; methylene blue (MB)
however, monetite is an anhydrous form. and anionic dye; methyl orange (MO).
Due to their non-toxicity, they have widely Moreover, the weight ratio of monetite to
used in many applications such as drug TiO2 P–25 was controlled at 1:1, 3:1, and
delivery, dental and orthopedics.4-7 5:1 wt%, respectively in order to observe the
Furthermore monetite shows an adsorption influence of monetite powder on
efficiency.8,9 Its chemical structure includes photocatalytic activity of TiO2 P–25.
discrete PO43- tetrahedral and Ca2+ ion, the
latter coordinated by a shell of oxygen 2. Materials and Methods
atoms.1 Calcium in the structure can 2.1 Synthesis of monetite powder
dissociate to Ca2+ when exposed to weak Monetite powder was prepared from
acid. brushite (Fluka Chemika). The grinding
In this research, monetite was mixed method in planetary mill (FRITSCH,
with titanium dioxide P–25 (TiO2 P–25) for Pulverisette 6) using 35 g of brushite mixed
adsorption of organic dye. Cation from with 50 ml RO (reverse osmosis) water as a
calcium will attract anion and make it milling media. Brushite was ground with
speed of 400 rpm for 20 min subsequently
drying in oven at 180 °C for 24 h, finally
monetite powder was obtained. Monetite
powder was then ground in agate mortar and
sieved through 325 mesh.
2.2 Sample characterization
Phase characterization of monetite
was done by X-ray diffraction (XRD,
Bruker, D8 Advance) from 2θ 5°-80° with
0.5 sec/step. Morphology was characterized Figure 1. XRD patterns of monetite powder
by scanning electron microscopy (SEM, and brushite, starting material
JEOL, JSM–6480LV) and elemental
analysis was performed by energy dispersive
analysis (EDS) dot mapping technique.
2.3 Photocatalytic activity test
The photocatalytic activity of the
samples were evaluated through degradation
of 0.026 mM MB, 150 mL and 0.056 mM
MO, 150 mL with the same absorbance unit
at 1.4 under UV light irradiation: (Lamp:
Philips TL-D 18 w). The light intensity
between light source and samples was 2
mW/cm2 and for each result, an average
absorbance of 3 samples was taken. Weight Figure 2. SEM micrograph of monetite
ratio of monetite powder to TiO2 P–25 was powder
controlled at 1:1, 3:1, and 5:1 wt%. Prior to
add TiO2 P–25, monetite powder was added The morphology of monetite was
into MB and MO dye solution and let them analyzed by SEM at magnification 20,000X.
adsorbed each other in the dark environment The plate-like shape was clearly observed as
for 1 h. After the adsorption of MB and MO shown in Figure 2 with particle size in
on monetite powder was in equilibrium (at 1 submicron range at around 0.5-1 µm.
h.), TiO2 P–25 was then subsequently added EDS dot mapping in Figure 3a and
into MB and MO under UV irradiation. UV– 3b showed the distribution of Ca and Ti
VIS spectrophotometer (UV–VIS, Perkins element which is randomly distributed when
Elmer, Lambda 35) with wavelength 664 nm monetite and TiO2 P–25 at 1:1 wt% was
for MB and 466 nm for MO was used to well mixed. The results indicated that there
monitor the absorbance of each dyes to is a possibility that the well distribution
check whether the photocatalytic between monetite powder and TiO2 P–25
degradation was influenced or not. would influence the photocatalytic
efficiency of TiO2 P–25. Monetite acted as
3. Results and Discussion adsorbent of organic dye on the surface,
3.1 Sample characterization after that TiO2 P–25 would easily
Phase analyses from XRD in Figure photocatalytically degrade the adsorbed dye
1 showed that the pure monetite phase was under UV light irradiation.
synthesized from brushite, starting material, 3.2 The photocatalytic degradation of
after planetary milling for 20 min and oven organic dyes
drying at 180°C for 24 h. Brushite, a hydrous 3.2.1 Methylene blue
form, lose the crystallized water molecule In Figure 4, the results showed that
and converted to an anhydrous form, monetite powder did not influence the
monetite.
a
Figure 4. The absorbance of 0.026 mM

MB determined by UV-VIS spectrophoto-
b metry.
3.2.2 Methyl orange
c
Figure 5. The absorbance of 0.056 mM
MO determined by UV-VIS spectrophoto-
metry.
degrade 97% of MB under UV light

irradiation.
In Figure.5, the results showed that
monetite powder could influence the
photocatalytic activity of TiO2 P–25 on
Figure 3. SEM micrograph of monetite degradation of MO. By using the weight
mixed with TiO2 P–25. ratio of monetite:TiO2 P–25 = 3:1 and 5:1, it
a.) monetite mixed with TiO2 P–25 at was found that the photocatalytic
1:1wt%. degradation of MO was better than that
b.) EDS analysis of monetite mixed with obtained from pure TiO2 P–25. Pure TiO2 P–
TiO2 P–25 at 1:1wt% showing the dot 25 with adding of monetite at 3:1 and 5:1
mapping of CaKα. wt% could degrade MO with 34% and 38%
c.) EDS analysis of monetite mixed with efficiency under UV light. However, the
TiO2 P–25 at 1:1wt% showing the dot concentration of MO used in this
mapping of TiKα. photocatalytic test is twice of MB
concentration, prolonging the degradation
photocatalytic activity of TiO2 P–25 on time over than 2 h is necessary to be done.
degradation of MB. Even at the maximum We have also observed the photocatalytic
weight ratio of monetite:TiO2 P–25 = 5:1, activity of monetite:TiO2 P–25 = 1:1, 3:1,
the photocatalytic degradation of MB was and 5:1wt% on degradation of MO at lower
not better than that of pure TiO2 P–25. Pure concentration of 0.026 mM as shown in
TiO2 P–25 without adding monetite could Figure 6. The result showed that MO could
University and Department of Materials
Technology, Faculty of Science,
Ramkamhaeng University.
References
1. MacLennan, G.; Beevers, C. A. Acta
Cryst. 1995, 8, 579–583.
2. Faleh, T.; Zeeshan, S.; Jake, B. Acta
Figure 6. The absorbance of 0.026 mM Biomater. 2012, 8, 474–487.
MO determined by UV-VIS spectrophoto- 3. Sergey, V. D. Mater. Sci. Eng. C. 2015,
metry 55, 272–326.
4. Maria-Pau, G.; Cristina, C.; Montserrat,
completely degrade with 96% efficiency E.; David, P.; Edgar, B. M. Adv. Drug
within 5 h. The results implied that adding Deliv. Rev. 2012, 64, 1090–1110.
of monetite to high concentration 0.056 mM 5. Nathalie, L.; Ange, N.; Patrick, S.
of MO dye solution could influence the Procedia Eng. 2014, 83, 423–431.
photocatalytic activity of TiO2. MO dye 6. Medvecky, L.; Stulajterova, R.;
could adsorb on the plate surface of Giretova, M.; Mincik, J.; Vojtko, M.;
monetite powder and let TiO2-P25 degrade Balko, J.; Briancin, J. Dental materials
that dye under UV irradiation. However, 2017, 442–451.
monetite could not influence the 7. Jesús, T.; Iskandar, T.; Jatsue, C. A.;
photocatalytic activity of TiO2-P25 when Isabel, T.; Mohammad, A.; Enrique, L.
degradation of low concentration MO dye. C.; Gonzalo, H.; Faleh, T. Ann. Anat.
This is because the nanoparticle size of 2015, 200, 126–133.
TiO2-P25 could play a key role regarding 8. Chensi, S.; Liuxi, W.; Yanhong, C.; Su,
adsorption and degradation of MO dye. L.; Sadia, R.; Yan, G.; Jianshe, L. Chem.
Eng. J. 2016, 303, 391–400.
4. Conclusion 9. Mirkovi´, M. M.; Lazarevi´ Pasti, T. D.;
Monetite could adsorb MO, an Dosen, A. M.; Cebela, A. Z.; Rosic, A.
anionic dye, on the surface however, A.; Matovic, B. Z.; Babic, B. M. RSC
monetite could not adsorb MB which is Adv. 2016, 6, 12219–12225.
cationic dye. As a result, we have found that 10. Adawiya, J. H.; Riyad Hassan, AL–A.;
monetite could influence the photocatalytic Ghadah Rasim, K.; Chafic Touma, S.
activity of TiO2 P–25 only for anionic dye Energy Procedia. 2017, 119, 332–345.
degradation. 11. Shayan, M. A.; Donia, F.; Jason, S.;
Rose, A. J. Hazard. Mater. 2018, 341,
Acknowledgements 404–423.
The authors would like to 12. Shidong, J.; Setsuaki, M.; Masanobu, K.;
acknowledge the financial and research Koji, I. Mater. Res. Bull. 2009, 44, 768–
facility from Department of Materials 77.
Science, Faculty of Science, Chulalongkorn
z
Nano-zinc oxide-doped activated carbon for feed additive from popped rice
Duangporn Saramas1, Sanong Ekgasit2*
1
Technopreneurship and Innovation Management Program, Graduate School, Chulalongkorn University,
Phyathai Road, Pathumwan, Bangkok 10330, Thailand
2
Sensor Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University,
Phyathai Road, Pathumwan, Bangkok 10330, Thailand
*E-mail: sanong.e@chula.ac.th
Abstract:
Nano-zinc oxide-doped activated carbon from popped rice was prepared. Popped rice
with porous structure was done by hot air heating of un-milled rice. Ground popped rice was
chemically treated with zinc chloride solution. Pyrolysis was carried out under a constant
nitrogen flow (5 l/h). The temperature of the pyrolysis was raised at 10oC/min up to 800oC
and was maintained for 1 h. The adsorption properties of activated carbon were investigated
by ultraviolet-visible spectroscopy (UV-Vis). The results showed that the prepared activated
carbon with maximum particle size of 45 µm presented high adsorption capacity of
methylene blue comparable to commercial activated carbon. The structure and morphology
of zinc oxide nanoparticles were characterized by X-ray diffraction (XRD) and transmission
electron microscopy (TEM). Nanoparticles of zinc compounds with particle size in the range
of 10-20 nm were generated on the surface of activated carbon. High capacity of toxin
adsorption was exhibited by the produced activated carbon. The percent adsorption of
aflatoxin B1, zearalenone, and fumonisin B1 were 91.43, 90.54, and 90.30, respectively. The
outcome indicates that high surface area of activated carbon based on popped rice containing
zinc oxide nanoparticles could be used as animal feed additive.
1. Introduction effects of mycotoxins is the use of

Mycotoxins were considered as one mycotoxin binders defined as substances for
of the most dangerous contaminants of reduction of the contamination of feed. The
human food and animal feed.1 Exposure to use of inorganic adsorbent materials is one
mycotoxins could lead to disease or death in of the best known approaches to protect
both humans and animals. Food and animals against the harmful effects of
Agricultural Organization (FAO) of the mycotoxin contaminated feed. Activated
United Nations estimated that up to 25% of carbon is one of selected inorganic
world’s food crops are significantly adsorbent material presented the adsorption
contaminated with mycotoxins.2 Global properties for being used as mycotoxin
losses due to mycotoxins and their impact binder.5
on human and animal health have been Activated carbon is typically referred
linked with national economic implications as carbonaceous material with well-
which can occur at all levels of food and developed porosity, and large surface area. It
feed production including crop and animal also presents good physicochemical
production, processing, and distribution.3 stability, high adsorptive capacity, high
Among many different mycotoxins, mechanical strength, and high degree of
aflatoxin, fumonisin, and zearalenone are surface reactivity.6-8 Activated carbon has
well known for their toxicity and been widely used in various applications
occurrence, especially in animals.4 such as energy storage, environmental
Several approaches have been remediation, recovery of solvents. The
developed to counteract mycotoxins in adsorption properties of activated carbon
animal feeds. A recent and apparently rely on several parameters including source
promising approach to mitigate the harmful of materials, surface area, and pore size
distribution. Moreover, activating agents, ZnCl2 was dissolved in ethanol, then popped
processing time, impregnation condition, rice was well impregnated in ZnCl2 solution
carbonization temperature, and others play with ratio of ZnCl2 to popped rice at 1:1 and
an important role in the adsorption 1:2. Impregnated samples were kept for 24
properties of activated carbon.9-10 h. After aging, the mixture was subjected to
Apart from activated carbon, zinc filter and dry at 110 ◦C. Chemical activation
oxide (ZnO) nanoparticles are also used in was then carried out under constant nitrogen
animal feed industry. ZnO nanoparticles flow (5 l/h). The temperature of the
have been used as alternative to existing pyrolysis oven was raised at 10 oC /min up
antibiotic. The addition of ZnO to 800 oC, and held at 800 oC for 1 h before
nanoparticles in animal feed can increase cooling down to room temperature.
productive performance and effectively Thereafter, activated carbon samples were
reduce the occurrence of post-weaning ground by ring mill and sieved to a particle
diarrhea in piglets.11 size range of 20-45 µm. Upon completion
The aim of the study is to prepare of the chemical activation, the produced
activated carbon containing ZnO activated carbons were characterized
nanoparticles through carbonization process according to their surface properties. In this
by using zinc chloride (ZnCl2). In this study, commercial activated carbon, and
research, popped rice was selected as a raw popped rice carbon without chemical
material for preparation of activated carbon. activation were compared as indicated in
According to the pore size and open pore Table 1.
characteristic of popped rice,12 it is
interesting to find the alternative approach Table 1. Abbreviation of samples
for adding value of Thai rice. The properties Abbreviation Description of Sample
CAC Commercial activated carbon
of activated carbon based on popped rice
PRC Popped rice carbon
including structure and morphology of ZnO AZ1 Activated carbon at ZnCl2
nanoparticles were evaluated. The adsorp- impregnation ratio of 1:1
tion capacity of toxins by activated carbon AZ2 Activated carbon at ZnCl2
containing ZnO nanoparticles are also impregnation ratio of 1:2
measured to find the potential for
commercialization. 2.3 Characterization
The optical adsorption spectra of the
2. Materials and Methods prepared samples were done on a double
2.1 Materials beam UV-vis spectrophotometer. The
Un-milled rice was selected as formation of ZnO in activated carbon was
starting material. A local waxy rice variety, performed by PANalytical X’Pert PRO X-
called “Laow-taek” (Oryza sativa L.) was ray diffractometer (XRD) using CuKα (n =
obtained from Yasothon province, Thailand 1.5406 Å) radiation source operating under a
and used for the preparation of popped rice. voltage of 40 kV and a current of 30 mA.
Analytical grade ZnCl2 was obtained from The diffraction angle (2θ) was varied
Fisher Scientific. A commercial activated between 10o and 70o. Transmission electron
carbon was used for comparison. Other microscope (TEM) images were performed
reagents used were all analytical grade. on a model of JEOL JEM 2010F with a
2.2 Preparation working voltage of 200 kV.
Popped rice with porous structure 2.4 Toxin adsorption measurement
was prepared by hot air heating of un-milled An in vitro technique was adapted13-14
rice grains at 215 oC. Popped rice samples in order to simulate the digestion procedure
were ground and sieved in order to obtain a of pigs. Each sample consisted of 2.5 mL of
particle size of 1-2 mm. The ground samples phosphate buffer (0.1 M, pH 6.0) and 0.5%
were allowed to dried in an oven at 110 oC. suspension of activated carbon samples was
added to conical tube, and then followed by measuring the absorbance of the solution in
5 mL of diluted mycotoxin standard the cuvette where de-ionized water was used
solution. Regarding control treatment, 5 mL as reference. The adsorption spectra were
of phosphate buffer was added. The pH measured on UV–Vis spectrophotometer (at
value was adjusted to pH 2.0 by adding 1.0 666 nm). MB solution in the absence of
M HCl for simulation pH in the stomach. activated carbon was also included in the
Each sample was incubated for 2 h in figure as control sample. Figure 1 shows
shaking incubator at 39°C, then 1 mL of the adsorption activity of samples as a
phosphate buffer (0.2 M, pH 6.8) was added function of time. According to the result,
to the conical tube. For simulating the AZ1 presents the fastest MB adsorption
conditions in the small intestine, 300 μL of followed by AZ2, CAC, PRC, respectively.
1.0 M NaOH was added and incubated at pH It is observed that activated carbon with
6.8 for 4 h. After incubation, the mixture ZnCl2 activation can increase the
was centrifuged and the supernatant was disappearance of MB significantly.
employed for analysis of residual unbound 3.2 X-ray diffraction (XRD)
toxins. In this study, the enzyme-linked XRD technique is used to study
immunosorbent assay (ELISA) test kits were crystalline nature of materials. Figure 2
used to detect the unbound aflatoxin B1, shows the XRD patterns of the four samples.
zearalenone and fumonisin B1 content. Overall, there are 2 broad peaks at 2𝜃 =
25.1o and 45o related to (002) and (100)
3. Results and Discussion diffractions, respectively. This reveals a
3.1 UV-Vis adsorption predominantly amorphous nature of
Methylene blue (MB) was used as carbon15. In Figure 2(a), the sharp narrow
representative dye to evaluate the adsorption peak at 26.1oindicated the existence of
activity of activated carbon AZ1 and AZ2 graphitized crystallite in the activated
compared to CAC and PRC. carbon sample16. However, Figure 2(b)
shows the absence of a sharp peak compared
to Figure 2(a).
Figure 1. Adsorption activity as a function

of time by using MB
Figure 2. XRD patterns of (a) CAC, (b)
For each condition, 0.01 g of sample PRC, (c) AZ2, and (d) AZ1
was dispersed in 5 ppm MB aqueous
solution (50 ml). All samples were well Structural identification of activated
shaken in the syringe connected to Swinnex carbon containing ZnO nanoparticles of
filter holder. Solution was then passed AZ2 and AZ1 was reported in Figure 2(c)
through a 5 µm Millipore filter paper in and 2(d), respectively. XRD pattern confirm
order to measure the adsorption. Each the formation of ZnO nanoparticles, as can
experiment was conducted for 5 min with 3 be seen from crystallinity. The defined
ml solution extracted every 1 min. The peaks at 31.6, 34.2, 36.2, 47.4 and 56.6,
disappearance of MB was observed by which corresponds to (100), (002), (101),
(102), (110) crystal planes of ZnO, toxins were included aflatoxin B1,
respectively. The position of peak can zearalenone and fumonisin B1.
confirm the occurrence of hexagonal
wurtzite structure of ZnO which is
consistent with the values in the standard
JCPDS 36-145. Surprisingly, peaks
corresponding to zinc magnesium phosphate
are also observed by comparing the
diffraction patterns with standard JCPDS
31-1468. The Miller indices of reflection are
assigned as * for zinc oxide and # for zinc
magnesium phosphate.
3.3 Transmission electron microscope
(TEM)
Transmission electron microscope
(TEM) were carried out on the samples in
order to investigate nanoparticles of zinc
compounds combining both zinc oxide and
zinc magnesium phosphate. TEM images of Figure 4. TEM images of AZ2
zinc compounds nanoparticles of AZ1 and
AZ2 are shown in Figure 3 and Figure 4, ELISA test kits were used to check
respectively. Nanoparticles are nearly the amount of unbound mycotoxin. Initial
spherical and quite monodisperse. By TEM amount of toxin was tested with different
analysis, it is observed that average particles level due to the maximum
size is 10-20 nm. It means that permissible concentration of each mycotoxin
nanoparticles of zinc compounds are type. Initial amount of aflatoxinB1,
successfully obtained for AZ1 and AZ2. zearalenone, fumonisinB1 was 20 ppb, 200
ppb, and 1 ppm, respectively. The percent
adsorption of activated carbon was
calculated using the following equation:
Percent adsorption = (IMT-UMT) x 100
IMT
where IMT is the initial amount of
mycotoxin and UMT is referred to the
residual amount of unbound mycotoxin after
digestion procedure.
The percent adsorption of 3 different
types of toxin is presented in Table 2. The
produced activated carbon, AZ1, shows
significantly high capacity of toxin
adsorption greater than 90%. The adsorption
of aflatoxin B1, zearalenone and fumonisin
B1 by activated carbon is 91.43%, 90.54%,
Figure 3. TEM images of AZ1 and 90.30%, respectively. In general, the
binding efficacies of commercially available
3.4 Evaluation of toxin adsorption mycotoxin binders for various mycotoxins
Sample AZ1 was selected to evaluate are different, varying from 0-90% with some
percent adsorption of mycotoxin. Three of being more specific to some mycotoxins and
some not.17-18 According to the results, AZ1
shows great potential to further develop as 2. Adams, M.; Motarjemi, Y. Basic Food
mycotoxin binder. Safety for Health Workers; World
Health Organization, Geneva, 1999.
Table 2. In vitro present adsorption of 3. Galvano, F.; Piva, A.; Ritieni, A.;
toxins by AZ1 Galvano, G. J. Food Prot. 2001, 64 (1),
Toxin Initial amount Toxin 120-131.
of toxin adsorption 4. Pettersson, H. Controlling mycotoxins
(%)
in animal feed. In Mycotoxins in Food:
Aflatoxin B1 20 ppb 91.43
Zearalenone 200 ppb 90.54 Detection and Control; Magan, N.;
Fumonisin B1 1 ppm 90.30 Olsen, M., Eds.; CRC Press, Boca
Raton: Florida, 2004; pp. 262-304.
4. Conclusion 5. Kolosova, A.; Stroka, J. World
The results show that popped rice by Mycotoxin J. 2011, 4 (3), 225-256.
chemical activation of ZnCl2 can provide 6. Yahya, M. A.; Al-Qodah, Z.; Ngah, C.
activated carbon with the presence of ZnO Z. Renewable Sustainable Energy Rev.
nanoparticles. The prepared activated 2015, 46, 218-235.
carbon exhibits high adsorption capacity of 7. Cuhadaroglu, D.; Uygun, O. A. Afr. J.
MB compared to commercial activated Biotechnol. 2008, 7 (20).
carbon. 8. Sahu, J.; Acharya, J.; Meikap, B.
Moreover, the study based on the in Bioresour. Technol. 2010, 101 (6),
vitro experiment indicates the sufficient 1974-1982.
potential of activated carbon containing ZnO 9. Hirunpraditkoon, S.; Tunthong, N.;
nanoparticles to bind three main types of Ruangchai, A.; Nuithitiku, K. World
mycotoxin mostly found in Thailand Acad. Sci. Eng. Technol. 2011, 78, 711-
including aflatoxin, zearalenone, and 715.
fumonisin. High surface area of activated 10. Patil, B.; Kulkarni, K. Int. J. Adv. Eng.
carbon based on popped rice with ZnO Res. Stud. 2012, 1 (2), 109-13.
nanoparticles show the promising results to 11. Raguvaran, R.; Manuja, A.; Manuja, B.
be used as animal feed additive. K. Immunome Res. 2015, 11 (2), 1.
12. Konharn, L. Master's Thesis,
Acknowledgements Chulalongkorn University, 2016.
This research has been financially 13. Kong, C.; Shin, S. Y.; Kim, B. G.
supported by the 90th Anniversary of SpringerPlus. 2014, 3 (1), 346.
Chulalongkorn University scholarship, 14. Boisen, S.; Fernández, J. A. Anim. Feed
Chulalongkorn University. The authors Sci. Technol. 1997, 68 (3), 277-286.
would like to thank Klean Greentech Co., 15. Xu, J.; Chen, L.; Qu, H.; Jiao, Y.; Xie,
Ltd. for the financial support of toxin J.; Xing, G. Appl. Surf. Sci. 2014, 320,
adsorption experiment. 674-680
16. Dandekar, A.; Baker, R.; Vannice, M.
References Carbon 1998, 36 (12), 1821-1831.
1. Council for Agricultural Science and 17. Waseem, A.; Ahmad Shah, S.; Sajjad,
Technology. Mycotoxins: Economic and A.; Rauf Siddiqi, A.; Nafees, M. J.
Health Risks; Council for Agricultural Chem. Soc. Pak. 2014, 36 (6).
Science and Technology Task Force 18. Dawson, K. A.; Evans, J.; Kudupoje, M.
Report No 116. Aimes, IA, 1989. Science and technology in the feed
industry; Nottingham University Press:
Nottingham, UK, 2001; 169-181.
Magnetic nanoparticles for organic synthesis
Parintip Rattanaburi1* , Vachira Choommongkol2, Khemmaphat chudam1, Nussaba Juntorn1
1
General Science Program, Faculty of Education, Nakhon Si Thammarat Rajabhat University,
Meaung Nakhon Si Thammarat 80280, Thailand
2
*E-mail: parintip.joy@gmail.com
Abstract:
Nanoparticles are being expressed as fundamental building blocks of nanotechnology
which commonly used in solution phase organic synthesis to facilitate product purification.
This technique requires the attachment of catalyzed to magnetic nanoparticles to facilitate
their isolation and enable the chemist to monitor reaction using common analytical
techniques. Recently, Magnetic nanoparticles have been considered as the new alternative
material for used as nanoparticles. These nanoparticles become popular reagent due to the
advantages over other organic reagents e.g. easy magnetic separation, a short reaction time,
ease of the morphology with catalyst. Moreover, magnetic nanoparticles are not swell and
will not be dissolved in any organic solvent and commercially available. In this study,
magnetic nanoparticles were prepared from FeCl3 and FeCl2 to give Fe3O4 magnetics
nanoparticles (Fe3O4-MNPs). The morphology and elemental composition of the composites
were characterized by transmission electron microscopy (TEM) and Fourier transform
infrared spectroscopy (FT-IR). Fe3O4-MNPs were applied to synthesize amide compounds
and the products from the reaction can be obtained in excellent yield (90% yield).
1. Introduction In this study, magnetic nanoparticles were

Catalyst employed for solution phase synthesized and their applications as catalyst
organic synthesis, a very significant role in were evaluated. Nanotechnology has
modern science and technology as they experimented a tremendous rise in the last
improve reaction yields, short reaction decade. In particular, the design of
times. Different types of solids are used in nanoparticles has represented a very
heterogeneous catalysis. They may be promising strategy alternative to
metals, metal oxides, metal sulphides, clays conventional processes, especially of great
etc. these materials may be used in their application in environmental and biomedical
pure form or in the form of their mixtures. problems.3,4
Further catalyst may be crystalline, Nanoparticles are commonly used in
microcrystalline and amorphous. These solution phase organic synthesis to facilitate
catalysts are used in a state of file powder so product purification. This catalyst to
as to give a large surface area. The main facilitate their isolation and enable the
advantage of using nanoparticles particles chemist to monitor reaction using common
sizes is their high effective surface area and analytical techniques. These catalyst means
low sedimentation rates.1,2 that can be employed in order to drive the
Solution phase organic synthesis reaction to completion, while the catalyst
using nanoparticles has emerged as a can be easily removed from the reaction
powerful technique for the preparation of mixture simply by external magnetic.
diverse compounds. Reaction with Reaction with these catalyst does not suffer
nanoparticles does not suffer from from such disadvantages as difficult product
disadvantages concerning typical catalyst as isolation and low yield due to loss of
difficult product isolation and low yield due product in purification step. Spreading
to loss of product during purification step. catalyst on finely divided nanoparticles as
catalyst is also known to produce an 2.2 Synthesis of N-phenylacetamide
enhanced reaction rate, greater efficiency Acetyl chloride (25 mg) was added
and the lower mobility of catalyst makes into a stirred solution containing aniline (1.2
them safer to handle.5,6 eq) and Fe3O4-MNPs (cat.) in
In this study, the catalyst was dichloromethane (Scheme 2). The reaction
synthesized with FeCl3.6H2O and mixture was stirred at room temperature for
.
FeCl2 4H2O to give magnetic nanoparticles 30 minute. After the reaction was
(Fe3O4-MNPs). Its application was completed, Fe3O4-MNPs were separated via
investigated in the synthesis of N- external magnetic and washed with
phenylacetamide. The reactions gave high dichloromethane. The combined reaction
yields of products without need for further mixture was evaporated to give N-
purification. Fe3O4-MNPs offer several phenylacetamide as a yellow solid (90%
advantages including ease of product yield). (90% yield). 1H NMR (400 MHz,
isolation, short reaction times under mild CDCl3) δ 2.2 (1H, s) and 7.0-7.6 (5H, m).
and neutral conditions.

The reagents of analytic grade
.
(FeCl3 6H2O, FeCl2.4H2O, NH3, aniline and
acetyl chloride) were use as raw materials.
All other solvents and chemicals were Scheme 2. Synthesis of N-phenylacetamide
obtained from commercial sources and used
without prior purification. 3. Results and Discussion
2.1 Synthesis of Fe3O4-MNPs The obtained magnetic nanoparticles
FeCl2.4H2O 0.50 mg and (Fe3O4-MNPs) were further characterized by
FeCl3.6H2O were dissolved in 10 ml of Fourier transform infrared spectroscopy
water in round-bottomed flask. Whilst the (FT-IR). As shown in Figure 1, the
solution was stirred vigorously, 10 ml of concurrence of Fe3O4 infrared absorption
ammonia was slowly added. The solution peaks at about 580-650 cm-1, while that of
was stirred at 80 oC for 60 minutes then the –OH bond is at 3363 cm-1.
Fe3O4-MNPs was obtained. The morphology of Fe3O4-MNPs
were further characterized using
transmission electron microscopy (TEM) as
shown in Figure 2. These samples clearly
confirms that can be nanocomposites with a
sized of 15-20 nm at the beginning of the
process was obtained.
To demonstrate the applicability of
the obtained Fe3O4-MNPs, they were
applied as catalyst in the synthesis of N-
phenylacetamide. The reaction in
homogeneous solution was also performed
for comparison. 200, 400 and 600 mg of
Fe3O4-MNPs were use as catalyst in the
solution phase synthesis. The product was
isolated by concentrated the crude reaction
mixture was separated via external
Scheme 1. Synthesis of Fe3O4-MNPs magnetic. Table 1 showed the yield
obtained from both reactions. It was found
that the entry 3 can gave high yields of the
product (90% yield).
Table 1. The parcentage yields obtained

from the synthesis of N-phenylacetamide
Entries Fe3O4MNPs (mg) %yield
1 - 58.48
2 200 80.05
3 400 90.00
4 600 56.58
Figure 3. NMR spectra of the crude

reaction mixtures from N-phenylacetamide
synthesis using Fe3O4-MNPs as catalyst
4. Conclusion
Magnetic nanoparticles (Fe3O4-
MNPs) has been synthesized and can be
used as an effective catalyst in the synthesis
Figure 1. IR spectra of Magnetic of N-phenylacetamide. This procedure is
nanoparticles (Fe3O4-MNPs) fast, produces excellent yield, and allows
easy isolation of product without any
aqueous work up and/or column
chromatography.
Acknowledgements
The authors would like to thank the
Nakhon Si Thammarat Rajabhat University
and Department of Chemistry, Faculty of
Science, Meajo University for facilities
provision.
Figure 2. TEM of Fe3O4-MNPs
References
1. Wu W.; He Q.; Jiang C. Nanoscale Res.
Considering the purity of the N-
Lett. 2008, 3, 397–415.
phenylacetamide NMR was used to analyze
2. Wei Y.; Han B.; Hu X.; Lin Y.; Deng X.
the purity of the crude reaction mixtures
Procedia Eng. 2012, 27, 632–637.
obtained from the reaction using Fe3O4-
3. Gao J.; Gu H.; Xu B. Nanomaterials.
MNPs as catalyst (Figure 3). The chemical
2014, 4, 222–241.
shift at 2.2 ppm is corresponsed to the –CH3
4. Govan J.; Gun’ko Y. K. Acc. Chem. Res.
of the methyl group of product while at δ
2009, 42, 1097–1107.
7.0-7.6 ppm belong to the aromatic group.
5. Chen, X.; Arruebo, M.; Yeung, K. Catal.
Thus resulting in Table 1 was shown percent
Today 2013, 204, 140–147.
yield of product when using Fe3O4-MNPs as
6. Zhou, W.; Gao, X.; Liu, D.; Chen, X.
catalyst, it gave good yields in short reaction
Chem. Rev. 2015, 115, 10575–10636.
times and under mild and neutral conditions.
Physical properties of gelatin and silk fibroin microspheres
Aviga Soonmongkol1, Jutarat Jamkratoke2, Sorada Kanokpanont1*
1
Phaya Thai Road, Bangkok 10330, Thailand
2
Queen Sirikit Sericulture Center, Nakhon Ratchasima, Thailand
*E-mail: sorada.k@chula.ac.th
Abstract:
In this study, gelatin type A (G) and silk fibroin (SF) which is obtained from Thai silk
cocoons, Bombyxmori, Leuang Pi Roj were used to developmicrospheres for cell carrier
application. The incorporation of silk fibroin into gelatin microspheres is expected to enhance
the mechanical properties of the gelatin microspheres. The G/SF microspheres were
fabricated by water in oil emulsion technique. In the microspheres preparation, variables that
affected to the size of microspheres were studiedincluding water/oil ratio, stirring speed and
total solid weight of G and SF. To stabilize microspheres, 0.05%v/v glutaraldehyde (GA) was
used as a crosslinking agent. The physical and chemical properties of the microspheres were
investigated. The results showed that the obtained microspheres were round shape with
smooth surface. The sizes of microspheres were in the range of 32-100 microns at 62-83
%yield. The crosslinking degree evaluated by ninhydrin assay were in the range of 20-27%.
Crosslinking degree depends on the content of SF. Increasing content of SF from 0-20%wt
resulted in decreasing swelling ratio (1.6 to 1.3), gel fraction (86 to 71%) and increasing half-
life degradation under enzymatically condition (3 to 16 h).
1. Introduction are produced as a composite or blending

Gelatin (G) is a denatured collagen with materials.
which is water soluble material. G plays a Silk fibroin (SF) is a natural fibrous
role in major protein components of protein which is derived from domesticated
cartilage, bone, skin and various connective silkworm, Bombyxmori. The SF is reported
tissue.1,2 It is biodegradable and nontoxic to form random-coil and amorphous type
since it consists of only basic amino acids which is the main secondary structures of
and sugars which are favorable with human fibroin, antiparallel β-pleated sheet. SF has
body.3 Because of its extensive properties, G many excellent properties such as good
has been widely used in various medical mechanical strength, toughness, elasticity
application. G based electrospun fiber mats and biocompatibility.8,9 Moreover, due to its
were employed for controlled release of biodegradability, SF was exhibited as a
active agents.4 G-based composite scaffolds biomaterial in various forms such as films,
have been studied in field of tissue sponges, microspheres, gels and
regeneration.5,6 In addition, it also generally scaffolds.6,7,10-11 The SF fibers can be
promotes cell attachment and proliferation. formed by the simple technique of
For example, G based porous scaffold, electrospinning for use in vascular grating12
which have cell adhesive RGD sites in G in which it acts as an accelerated healing
molecules, could be used to improve cell membrane for the guided bone
adhesion.7 On the other hand, the major regeneration.13 Microspheres can also be
drawback are its weak mechanical strength fabricated from an aqueous SF solution and
and fast degradation rate, which limits load were investigated as aplatform for controlled
bearing applications. To achieve the drug delivery.14 In addition, SF based
optimum properties between cell interaction scaffolds have been explored in field of cell
and mechanical strength, G based scaffolds carriers. Some recently research revealed SF
based scaffold can support human bone- solution was dialyzed against DI water using
marrow derived mesenchymal stem cell dialysis tube (Cellulose tube, MWcutoff =
(hMSC) by promoting cell adhesion and 12,000-16,000 Da, width 40 mm, Viskase
growth.15 Companies Inc., Japan). The dialyzed water
The incorporation of SF into G was replaced until its conductivity was equal
scaffold exhibits impressive properties. SF to that of the DI water. The final
can enhance the strength while maintaining concentration of SF solution was around 6-
elasticity and controlled degradation rate of 7%wt.
the blended material. Meanwhile, G exhibits 2.3 Zeta potential measurement
hydrophilicity part which is suitable for cells Zeta potentials were analyzed by
adhesion. Microspheres is a novel type of Zeta-Sizer (650, Malvern, UK). All
scaffold that was employed for medical solutions including G solutions, SF solutions
applications for century. Since they are and G/SF solutions at different weight
small particles which provide high surface blending ratios, were prepared at 25 ºC and
area per volume that is suitable for cells to pH 7.4. The solutions were injected into a
grow in large amounts. quartz-measuring cell containing two
In this study, we aimed to prepare electrodes and repeated three runs per
G/SF microspheres by a simple technique, experiment.
water in oil emulsion. The stabilized G/SF 2.4 Preparation of the G/SF microspheres
microspheres were produced using The G/SF microspheres were
glutaraldehyde crosslinking technique. The fabricated by water in oil emulsion
effects of water to oil (W/O) ratios, stirring technique.17,18 For an aqueous phase, 2.5, 5,
speeds, weight blending ratio of G/SF and and 7.5%wt G/SF solution at different
total solid weight of blended solutions on weight blending ratios of G/SF (100/0,
the formation of microspheres were 90/10, and 80/20) solutions were prepared at
investigated and compared in this work. 40C. Soybean oil was preheated to 40 C.
The G/SF solution was gently dropped into
2. Materials and Methods oil phase under various homogenized speeds
2.1 Materials at 40 C for 10 min. The emulsion
Thai domestic silk cocoons, temperature was then decreased to 4 C and
Bombyxmori, Leuang Pi Roj (hybrid race of continuously homogenized. The excess oil
Thai Nanglai race and Japan (J108) race), on the droplets were washed out with cold
was cultured and maintained by Queen acetone and by centrifugation at 4 C. The
Sirikit Sericulture Center, Nakhon obtained microspheres were then air-dried
Ratchasima, Thailand. Type A gelatin overnight. These non-crosslinked
prepared by the acid pretreatment of porcine microspheres were immersed in 0.05 %vol.
skin collagen (isoelectric point = 9) was of GA in acetone:water (3:1) solution at 4C
purchased from by Nitta Gelatin Inc., Japan. for 20 h under darkness, followed by rinsing
All chemicals in analytical grade were used with 0.1 M glycine and DI water to remove
as received. unreacted aldehyde groups of GA.17 The
2.2 Preparation of Thai silk fibroin GA-crosslinked microspheres were then
solution freeze-dried for 72 h and size sorting was
The protocol of SF solution doneby sieving (32-75 m, 75-100 m and
preparation was modified from Kim et al.16
>100 m, IIDA Seisakusho co.,Ltd, Japan).
Degumming of cocoons to remove sericin,
2.5 Morphology and size analysis of
the cocoons were boiled in 0.02 M Na2CO3
microspheres
twice for 20 min and rinsed with deionized
Morphology of dried G/SF
(DI) water, followed by air-drying at 252 microspheres were observed under scanning
C. The degummed SF was dissolved in 9.3 electron microscope (SEM, SU3500,
M LiBr in an oven at 60 C for 4 h. The Hitachi,Japan) at the accelerating voltage of
3 kV after sputter-coated with gold. The where Gi and Gr represent initial and
averagesize of microspheres was evaluated remaining weight of G/SF microspheres,
from samples (n = 100) using Image J respectively (n = 4).
software (the US National Institutes of 2.8 In vitro biodegradation test
Health, Bethesda, MA, USA). Five mg of G/SF microspheres were
2.6 Determination of crosslinking degree incubated in 0.5 U/mL of Protease XIV
The content of free amino (NH2) solution14 (pH 7.4) containing an anti-
groups in non-crosslinked and crosslinked bacterial agent at 37 °C. The enzymatic
microspheres were estimatedandcrosslinking solution was changed every 2 days. The
degree was calculated by using ninhydrin remained microspheres after incubation
assay.19 Five mg of lyophilized were then freeze dried. The dried
microspheres were rehydrated in 100 L and microspheres were weighed to calculate the
1 mL of ninhydrin solution. They were half-life of degradation.
added to all samples, followed by incubation 2.9 Statistical analysis
at 100 C for 20 min in the dark. Then, their One way analysis of variance
temperature was reduced to room (ANOVA) was used to analyze individual
temperature and 5 mL of 50% isopropanol set of data. In all cases, the results were
was added to each sample. The absorbance considered to be significantly different when
of the solution was measured using a p-value < 0.05 (n = 4)
spectrophotometer at 570 nm. The
crosslinking degree of GA-crosslinked 3. Results and Discussion
microspheres was calculated in relative to 3.1 Zeta potential of solutions
non-crosslinked microspheres as a control (n The zeta potentials of G solution at
= 4). pH 7.4 was positive (3.42 mV), since pH of
2.7 Swelling ratio and gel fraction G solution (type A) was less than the
analysis isoelectric point (pI = 9) of the solution.
For the swelling ratio measurement, While, SF is in deprotonated form (negative
5 mg of G/SF microspheres were completely charge, -10.2 mV). The solution with higher
swollen by immersion in DI water at 37 C SF content showed more negative charge
for 24 h, and compared with immersion of than those with higher G content as the
G/SF microspheres in acetone which is non- positive charge of G was obstructed by the
swollen solvent. Diameter of swollen and negative charge of SF. Both G and SF
non-swollen G/SF microspheres were solutions can be interacted through
observed on a microscope (CKX41, electrostatic force of attraction.
Olympus, Tokyo, Japan) and calculated the 3.2 Morphology and size of G/SF
swelling ratio as follows; microspheres
The G/SF microspheres prepared at
Swelling ratio = (Sd/Sa) 100 different weight blending ratios (G90SF10
and G80SF20) with all solution blending at
where; Sd and Sa represent the diameter of constant total solid weight of 7.5%wt were
swollen G/SF microspheres in DI water and spherical with smooth surface as shown in
acetone, respectively (n=4). Figure 1. Some irregular shaped particles
For the gel fraction measurement, 5 were observed on microspheres at weight
mg of G/SF microspheres were completely blending ratio of G100, possibly being a
swollen by immersion in DI water at 37 C result of mechanical damage from
for 24 h. The swollen G/SF microspheres continuous stirring during washing out of
were then freeze-dried and weighted.The gel residual chemicals.
fraction was calculated as follows;
Gel fraction (%) = (Gr/Gi)100
a. b. c.
d. e.
Figure 1. Morphology of the dried GA-crosslinked G/SF microspheres (a.) 7.5%wt G100,
(b.) 7.5%wt G90SF10, (c.) 7.5%wt G80SF20, (d.) 2.5%wt G80SF20, and (e.) 5%wt
G80SF20 observed by SEM (scale bar 100 m)
All microspheres were size- crosslinked G/SF microspheres, the

fractionated by sieves, which gave yields of crosslinking degree of the microspheres
the microspheres slightly over 40% in the were around 20-25%. The higher the SF
size range of 32-75 µm. Comparing with contents, the higher the crosslink degree. On
different total solid weights (2.5, 5, and the other hand, the crosslinking degree of
7.5%wt) at constant weight blending ratio of GA-crosslinked G/SF microspheres at
G80SF20, all microspheres have round different total solid weight seemed to be
shape with smooth surface and the size of all slightly decreased with the increasing total
microspheres was in the range of 32-75 µm solid weight as shown in Table 1. In general,
which gave yields over 50%. These were G and SF composes of active free amino
sizes in the appropriate range for cell groups that can be crosslinked by GAat 30
transplant by injection through medical and 5%, respectively.9,21 For the swelling
needle.20 ratio analysis, the microspheres with higher
3.3 Crosslinking degree, swelling ratio of gelatin content and total solid weight
and gel fraction of G/SF microspheres showed a higher amount of swelling ratio
By GA crosslinking reaction, owing toits low percentage of crosslinking
covalent bonds could be formed between degree due to the hydrophilic property of
two amine groups (–NH2) of SF and G and gelatin. The swelling ratio was inversely
aldehyde groups of GA. Considering at proportional to crosslinking degree as shown
different weight blending ratios of GA- in Table 1.
Table 1. The crosslinking degree, swelling ratio, gel fraction and half-life degradation of the
obtained G/SF microspheres
Total solid weight Crosslinking degree Half-life
G/SF ratio Swelling ratio Gel fraction (%)
(%) (%) degradation (h)
7.5 100/0 20.29  0.98 1.55  0.26 71.67  2.12 3.54  1.87
7.5 90/10 22.57  1.08 1.47  0.14 77.33  6.43 5.05  0.48
7.5 80/20 24.01  0.82 1.46  0.07 78.00  4.00 18.30  2.89a,b
5.0 80/20 24.86  0.46 1.45  0.09 82.67  5.03 2.22 0.28
2.5 80/20 26.64  0.81 1.29  0.06 86.01  3.45 1.72  0.06
a
p< 0.05 significant against the value of blending ratio of G/SF microspheres (80/20), bp< 0.05 significant
against the value of total solid weight percentage of G/SF microspheres (7.5%wt)
The gel fraction, which can be used Acknowledgements
to describe stability and water solubility of This work has been supported by the
microspheres, revealed that increasing the Ratchadaphiseksomphot Endowment Fund
amount of gelatin decreased the gel fraction of Chulalongkorn University (CU-58-002-
because crosslinking degree was lowered, HR).
causing the percentage of total solid weight
to decrease thus increasing gel fraction. The References
relationship between Gel fraction was direct 1. Chang, C. Biomater. 2003, 24, 4853-
proportional to crosslinking degree 4858.
3.4 In vitro biodegradation of G/SF 2. Kawai, K.; Suzuki, S.; Tabata, Y.; Ikada,
microspheres Y.; Nishimura, Y. Biomater. 2000, 21,
The degradation rate of GA- 489-499.
crosslinked G/SF microspheres with higher 3. Jetbumpenkul, P.; Amornsudthiwat, P.;
total solid weight showed slightly higher Kanokpanont, S.; Damrongsakkul,
half-life of degradation. Meanwhile, the S. Int. J. Biol. 2012, 50, 7-13.
half-life of degradation of GA-crosslinked 4. Okhawilai, M.; Rangkupan, R.;
G/SF microspheres was increased with the Kanokpanont, S.; Damrongsakkul,
increasing SF content. Generally, protease S. Int. J. Biol. 2010, 46, 544-550.
XIV is an enzyme that specifically breaks 5. Sinlapabodin, S.; Amornsudthiwat, P.;
the peptide bonds in SF and G molecules. Damrongsakkul, S.; Kanokpanont,
Since SF provides hydrophobic domains S. Mater. Sci. Eng. C 2016, 58, 960-970
which refer to its H-bonding in -sheet 6. Lerdchai, K.; Kitsongsermthon, J.;
secondary structure, it can prolong Ratanavaraporn, J.; Kanokpanont, S.;
biodegradation rate. After 10 days of Damrongsakkul, S. J. Pharm. Sci.
incubation, all GA-crosslinked G/SF 2016, 105, 221-230.
microspheres were then completely 7. Sinthop, S.; Patumraj, S.; Kanokpanont,
degraded (data not shown). S. Adv. Mater. Res. 2014, 931-932, 200-
204.
4. Conclusion 8. Chankow, S.; Luemunkong, S.;
The G/SF microspheres were Kanokpanont, S. In Biomedical
successfully prepared using water in oil Engineering International Conference
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They provided an appropriate size in the Biomed. Mater. Res. B 2014, 102, 1639-
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microspheres with higher SF content Y. Biochem. Biophys. Res. Commun.
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Bacteria removal in wastewater by using silver nanoclusters materials
Nittaya Foansoongnoen1, Wittaya Ngeontae2, Pornsawai Praipipat1*
1
Department of Environmental Science, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
2
Department of Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
*E-mail: pornprai@kku.ac.th
Abstract:
Many parameters are determined to identify the clean water for use. From those
parameters, the total coliform bacteria (TCB) and fecal coliform bacteria (FCB) are normally
used to estimate the water quality. The contamination of bacteria and other microbes in the
water can cause many waterborne diseases, so disinfection method is necessary to apply.
Chlorination method is normally used for this purpose; however, this method has the side
effects. The alternative methods are developed to avoid these, and silver nanotechnology is
one of interesting alternative methods. Many studies indicated that silver nanomaterial
(1-100 nm) can efficiently kill bacteria in water. Due to smaller particle size, it may achieve
the higher efficiency of bacteria removal. This study synthesized the silver nanoclusters (1-5
nm) called AgNCs and studied its efficiency on bacteria removal in wastewater of Khon
Kaen University wastewater treatment plant. The batch tests (supporting material, dose,
contact time, and pH) are used to study its efficiency. The result showed that PAD 610 with
AgNCs, 0.5 g/L, 3 hours and pH 7 was the best optimum condition of silver nanoclusters
materials (AgNCs-Ms) for the highest TCB and FCB removals with measuring by most
probable number method (MPN).
1. Introduction university’s wastewater after passing of

Wastewater is discharged by many wastewater treatment processes determines
sources such as industries, agricultures, and TCB and FCB standard values in the water
households that may contain many not exceed 5,000 and 1,000 MPN /100ml,
pollutants including bacteria contamination.1 respectively, and the most probable number
It is also a major cause of deterioration of (MPN) method is standardized method that
water quality in water bodies and can cause is used to estimate TCB and FCB
many consequences, such as water pollution concentrations in the water.4,5
problems and human health harms.2 Currently, chlorine is used to remove
Formally, total coliform bacteria bacteria as a disinfectant for wastewater
(TCB) and fecal coliform bacteria (FCB) are treatment. Chemicals such as hypochlorite
used to indicate the bacterial contamination (ClO-) and Chlorine Dioxide (ClO2) were
in the water. TCB indicates the risk of used for disinfection. When chlorine is
spreading of pathogens in the added to the water, it reacts quickly. If
gastrointestinal tract whereas FCB can chlorine is added too much, it will react with
present in the human intestines and warm- the substance in water and so there will be
blooded animals that may affect to human chlorine residue. It makes water unpleasant
health.3 smell, highly alkaline and highly corrosive.6
TCB and FCB are also used as the Therefore, this study needs to investigate the
main parameters to consider the surface alternative choice to eliminate bacteria in
water quality standards creating by the wastewater to improve the wastewater
pollution control department in Thailand treatment efficiency and not to discharge the
(PCD). The surface water quality standard residue into the environment.
(type 2) that is the receiving water source The rapid growth in nanotechnology
from the discharge water such as has significant interest in the environmental
applications of nanomaterials because its 2. Materials and Methods
potential to revolutionize century-old 2.1 Chemicals
conventional water treatment processes have All chemicals were the analytical
been enunciated recently.7 Nano materials grade without the purification before use
are excellent adsorbents, catalysts, and that was nitric acid (HNO3), silver nitrate
sensors due to their large specific surface (AgNO3), ferric chloride (FeCl3·6H2O).
area and high reactivity. More recently, Sodium hydroxide (NaOH) and hydrochloric
several natural and engineered acid (HCI) were used for pH adjustment.
nanomaterials have also been shown strong Poly (methacrylic acid) or PMAA was used
antimicrobial properties of silver for the synthesis of the silver nanoclusters,
8,9
nanoparticles (nAg). Another potential and four supporting materials were used for
application of antimicrobial nanomaterials is the synthesis of silver nanocluster material
their use in decentralized or point-of-use for (AgNCs-Ms) that were polymeric non-
water treatment and reuse systems.10 The functional group resin (PAD 610), cation
concept that it has attracted much attention exchange resin (C145), anion exchange resin
in recent years due to concerns on water loss (A400), and activated carbon (AC).
and quality deterioration associated with 2.2 Water sample preparation
aging distribution networks and the Water samples were collected from
increasing energy cost to transport water, as the last pond before the pond was added
well as the increasing need for alternative chlorine for antibacterial purpose of Khon
water sources and wastewater reuse for areas Kaen University’s wastewater treatment
with water shortage problems and national plant. Ferric chloride was used to precipitate
security issues.11,12 the unwanted sediments in the water
Silver nanoclusters synthesis is samples, then filtered and kept them in the
smaller and it would be more effective than refrigerator at 4ºC before use. The initial
the nanoscale.13 Silver nanoclusters bacterial concentration of TCB and FCB
(AgNCs) are commonly used to create were normally analyzed and recorded before
sensors and they are fluorescence.14 They doing the batch experiments.
are nanomaterials composed of atoms of 2.3 Synthesis of silver nanoclusters
atoms, and their sizes are in the range of 1-5 (AgNCs)
nm. Due to this property, silver nanoclusters 1.699 g of silver nitrate (AgNO3)
are suitable for research in biological studies was weighted and diluted with 80 mL of
and sensor generation.15 deionized water (DI water), and it was noted
Since the liquid silver nanoclusters as solution A. 1 mL of PMAA was diluted
(AgNCs) cannot be applied directly for with 20 mL of DI water, it is noted as
application use in the continuous flow solution B. Brought solution A and B to
system16, so it is necessary to find the sonicated by using ultrasonicator for 30
supporting material for this purpose. The minutes. Then poured solution A into three-
resin may be an interesting choice since it neck round-bottom flask to avoid air bubble,
has the porous structure that may hold the and then heated at 90 °C set as the close
liquid silver nanoclusters (AgNCs), thus this system. When the temperature was up to 90
brings out the idea for this study. C, quickly added 9 mL of solution B by
This study aims to synthesis the syringe into a round bottom flask that
silver nanoclusters (AgNCs) and silver contained a hot solution A. Then, allowed
nanocluster materials (AgNCs-Ms) to 4.5 h for the complete reaction, and left it
investigate characterization of AgNCs-Ms cool for 1 h and stored in an air tight bottle
and examine its antibacterial efficiencies by that entirely closed by aluminum foil. The
using batch experiments on the effects of mixture gradually changed from colorless to
supporting material, dose, contact time, and light umber color.
pH.
2.4 Synthesis of silver nanoclusters material for removing both bacteria types by
materials (AgNCs-Ms) AgNCs-Ms.
Prepared 70 mL of the liquid silver 2.6.2 The effect of dose
nanocluster (AgNCs) and poured it into 110 Choose the best antibacterial
mL of polyethylene bottle, and added 37 g material from 2.6.1 to study the effect of
of material into the bottle. Then, closed the dose. Five different doses (0.2 g/L, 0.4 g/L,
bottle cap and used parafilm warp and 0.5g/L, 0.6 g/L and 0.8 g/L) were used to
aluminum foil to cover the bottle. The find the best dose for removing both bacteria
mixture was shaken by a rotary agitator for types by AgNCs-Ms with MPN analysis.
24 hours to be complete the reaction, and The control condition was the best material
then kept the bottle in a place where the (2.6.1), mixing speed at 200 rpm, mixing
light cannot touch until use. time at 5 hours, and water volume at 1 L.
2.5 Characterization of silver 2.6.3 The effect of contact time
nanoclusters materials (AgNCs-Ms) Choose the best dose of materials
The internal components, the from 2.6.2 to investigate the effect of
arrangement of atoms, morphology and contact time. Five different contact times (1,
surface characteristics of silver nanocluster 2, 3, 4, and 5 h) were used to find the best
materials were studied by scanning electron contact time for removing both bacteria
microscope (SEM), and energy dispersive types by AgNCs-Ms with MPN analysis.
X-ray spectrometry (EDX) was used for The control condition was the water sample
elemental and qualitative analysis. at 1 L, mixing speed at 200 rpm, the best
2.6 The effect of various factors on the material (2.6.1), the best dose (2.6.2).
bacteria removal efficiency by using silver 2.6.4 The effect of pH
nanoclusters materials (AgNCs-Ms) in Choose the best contact times from
batch test experiments 2.6.3 to study the effect of pH. Five different
Various factors on the bacteria pH values (2, 5, 7, 9, and 11) were used as
removal efficiency by using silver acid, neutral, and base to examine the best
nanoclusters materials (AgNCs-Ms) were pH for removing both bacteria types by
studied. The factors were supporting AgNCs-Ms with MPN analysis. The control
material, dose, contact time, and pH. The condition was the water sample at 1 L,
results were analyzed by using the most mixing speed at 200 rpm, the best material
probable number method (MPN). Jar test (2.6.1), the best dose (2.6.2), and the best
was used for all batch test studies. contact time (2.6.3).
The criteria to decide the best
condition of each effecting factor was the 3. Results and Discussion
condition that AgNCs-Ms could remove 3.1 Characterization of silver
both TCB and FCB to 0 MPN/100 mL for nanoclusters materials (AgNCs-Ms)
the challenging purpose and was also below Based on morphological studies
the PCD’s surface water standard (type 2) of using the SEM-EDX 50000x tool, the
TCB and FCB concentration in the water. material had many gaps of the porous
2.6.1 The effect of supporting materials appearance shown in Figure 1. Due to this
Four materials (PAD610, C145, feature, absorption of AgNCs-Ms could take
A400, and AC) were used to synthesize the place. According to EDX analysis, it showed
silver nanoclusters materials (AgNCs-Ms). the percentage of chemical that were 0.9%
The effect of supporting materials was of Ag. It proved that synthetic materials
studied by controlling of water sample could absorb AgNCs shown in Figure 2, so
volume in 1L, mixing speed at 200 rpm, it could effectively eliminate both types of
material dose at 1 g, and mixing time at 5 bacteria (TCB and FCB). Note that, another
hours. The samples were analyzed by MPN one high peak in Figure 2 that we were not
method to investigate the best supporting mentioned was the peak of gold (Au) that
was applied in the sample for the proceeding 3.2.2 The effect of supporting materials
of FIB-SEM analysis. In this study, the supporting
materials: PAD610, C145, A400, and AC
were compared for bacteria removal
efficiency by AgNCs-Ms. The result showed
in Figure 3, and found that PAD 610 was the
best supporting material to remove both
bacteria types at 0 MPN/mL. Therefore,
PAD 610 was used as the supporting
material to synthesize the silver nanocluster
material (AgNCs-Ms) for other batch tests.
Figure 1. FIB-SEM image of silver

nanoclusters materials (AgNCs-Ms)
Figure 3. The effect of supporting materials

on TCB and FCB removal efficiencies by
AgNCs-Ms
Figure 2. EDX analysis for the percentage

of the constituent atoms of silver
nanoclusters materials (AgNCs-Ms)
3.2 Study on the effect of various factors

on the bacteria removal efficiency by
using silver nanoclusters materials
(AgNCs-Ms) with batch test studies
3.2.1 Water sample analysis before Figure 4. The effect of dose on TCB and
treatment with AgNCs-Ms FCB removal efficiencies by AgNCs-Ms
The concentrations of total coliform
bacteria and fecal coliform bacteria in the 3.2.3 The effect of dose
water samples were analyzed before In the Figure 4, it showed the result
treatment with the silver nanocluster of the effect of dose on TCB and FCB
material (AgNCs-Ms) for all batch tests. The removal efficiencies by AgNCs-Ms. The
results found the average initial result found that 0.5 g was the best dose to
concentrations of total coliform bacteria and remove both type of bacteria at 0 MPN/mL,
fecal coliform bacteria were 2,400 and 1,100 so the condition of PAD 610 and 0.5 g was
MPN/100 mL, respectively.
used for the next batch experiments (contact 4. Conclusion
time effect). The result showed that silver
3.2.4 The effect of contact time nanoclusters (AgNCs) were synthesized as
Figure 5 showed the result of the the liquid silver nanoclusters and the silver
effect of contact time on TCB and FCB nanoclusters materials (AgNCs-Ms) were
removal efficiencies by AgNCs-Ms, and 3 successfully synthesized. The
hours was the best contact time which could characterization of silver nanoclusters
remove both bacteria types at 0 MPN/ml. materials (AgNCs-Ms) was analyzed by
Therefore, the condition of PAD 610, 0.5 g, using FIB-SEM and EDX.
and 3 h was used for the last batch test study
(pH effect). The FIB-SEM’s result found the
porous structure into the synthesized
material and EDX’s result found the
chemical compositions of C, O, and Ag
were 74.5% of C, 24.6% of O, and 0.9% of
Ag.
TCB and FCB removal efficiencies
by AgNCs-Ms were studied through the
batch tests. The various factors including the
supporting material, dose, contact time, and
pH were studied to find the best optimum
condition to remove both bacteria types by
AgNCs-Ms.
The result showed the best optimum
Figure 5. The effect of contact time on TCB condition of AgNCs-Ms was PAD 610, 0.5
and FCB removal efficiencies by AgNCs- g of material, 3 h, and pH 7 to remove TCB
Ms and FCB to 0 MPN/100 mL below the
surface water quality standard (type 2) by
3.2.5 The effect of pH PCD (TCB < 5,000 MPN/100 mLand FCB
The result of the effect of pH showed < 1,000 MPN/100 mL), and this study could
in Figure 6, and found that pH 2, 5, and 7 confirm that AgNCs-Ms can remove both
could remove both bacteria types at 0 TCB and FCB in the water.
MPN/mL. However, pH 7 was chosen for
the best pH in this study to be safe treated
water after releasing. Acknowledgements
The authors are special thanks to
Prof. Dr. Sujittra Youngme for her
assistance in this project, and grateful for the
financial supported by The Office of the
Higher Education Commission and The
Thailand Research Fund grant
(MRG6080114), Coordinating Center for
Thai Government Science and Technology
Scholarship Students (CSTS) and National
Science and Technology Development
Agency (NSTDA) Fund grant (SCH-
Figure 6. The effect of pH on TCB and FCB NR2016-122), and The Synchrotron
removal efficiencies by AgNCs-Ms Research Fund grant year 2016 by Khon
Kaen University.
References Ramirez, J. T.; Yacaman, M. J.
1. Shalinee, N.; Ademola O. O. Int. J. Nanotechnology IOP Publishing. 2005,
Environ. Res. Public Health 2014, 11, 16, 2346–2353.
249-270. 10. Qilin, L.; Shaily, M.; Delina, Y.; Lyon,
2. Kanchana, A.; Phongphan, O.; Lena B.; Michael, V.; Liga, Dong L.;
Weerayuth, T.; Nuengruthai, S.; Aniwat Pedro J. J. A. Water Res. 2008, 42,
I. Naresuan University J. 2010, 18, 51. 4591–4602.
3. Eleria, A.; Vogel, R. M. J. Am. Water 11. Haas, C. N. J. Am. Water Works Assoc.
Resour. Assoc. 2005, 41 (5), 1195-1209. 2000, 92, 72–73.
4. http://www.pcd.go.th/info_serv/reg_std 12. NRC in Drinking Water Distribution
_water05.html (accessed February 21, Systems. National Academies Press
2018). Washington, DC, 2006.
5. Blodgett, R. J. Food Microbiol. 2005, 13. Xi-Feng, Z.; Zhi-Guo, L.; Wei S.;
22, 547-552 Sangiliyandi G. Int. J. Mol. Sci. 2016,
6. Alexander, J.; Knopp, G.; Dotsch, A.; 17, 1534.
Wieland, A.; Schwartz, T. Sci. Total 14. Kanokwan, C.; Thawatchai, T.;
Environ. 2016, 559, 103-112. Wittaya, N. Sens. Actuators B Chem.
7. Shannon, M.; Bohn, P. W.; Elimelech, 2015, 207, 658–667.
M.; Georgiadis, J. G.; Marinas, B. J.; 15. Kholoud, M. M.; Abou El. N.; Ala, E.;
Mayes, A. M. Nature 2008, 301–310. Abdulrhman, Al. W.; Reda A.A.
8. Qi, L.; Xu, Z.; Jiang, X.; Hu, C.; Zou, Arabian J. Chem. 2010, 3, 135-140.
X. Carbohydr. Res. 2004, 339, 2693– 16. Supranee K. B.Sc. (Environmental
2700. Science) Degree Thesis, Khon Kaen
9. Morones, J. R.; Elechiguerra, J. L.; University, 2016.
Camacho, A.; Holt, K.; Kouri, J. B.;
Development of AgVO3/C electrocatalysts for an ethanol-tolerant in
alkaline electrolyte for direct ethanol fuel cells
Chakkrapong Chaiburi1*, Sudkate Chaiyo2
1
Department of Chemistry, Faculty of Science, Thaksin University, Phatthalung Campus,
Banpraow Sub-district, Papayom District, Phatthalung 93210, Thailand
2
Department of Chemistry, Faculty of Science, Chulalongkorn University, Patumwan,
*E-mail: chakkrapong@tsu.ac.th, kchakkrapong@yahoo.com
Abstract:
Electrochemical activity characterization was conducted by cyclic voltammetry (CV)
and oxygen reduction reaction (ORR). The AgVO3/C, V2O5/C and Ag/C electrocatalysts
displayed ethanol tolerance for oxygen reduction reaction with the presence of ethanol in
KOH working electrolyte. As a result, electrocatalysts prevented ethanol oxidation reaction
from ethanol crossover via membrane from the anode to cathode compartments in alkaline
direct ethanol fuel cells (ADEFCs). The AgVO3/C electrocatalyst was also achievable at a
lower cost than non-Pt catalysts. The results presented herein demonstrated that the AgVO3/C
electrocatalyst is significant for development and may be useful in place of the state-of-the-
art ORR platinum electrocatalyst.
1. Introduction colloidal method. The number of electrons

Alkaline direct ethanol fuel cells transferred was obtained from ORR via
(ADEFCs) have been developed and working electrode as rotating disk electrode
updated for use as alternative energy sources (RDE), which was found to be
of renewable fuel. The principle of the approximately four. From the RDE results,
ADEFC involves as changed of chemical limiting the current density (il) of the Pt/C
energy to electrical energy via redox catalyst showed higher activity than the
reaction (no combustion reaction). The Ag/C catalyst. Methanol-tolerance appeared
electrons are released from the catalyst on even at concentrations higher than 0.1 M.
the anode side through the catalyst on the On the other hand, the Pt/C was found to
cathode side. ADEFCs have been utilised in have an oxidation reaction to methanol
technologically-portable devices. Ethanol concentration. Blizanac and co-workers2
has low toxicity, can be storage without studied Ag (111) single crystals to clarify
undue pressure and is inexpensive. As such, the pH impact on the number of electron
researchers have been replacing platinum- transfers. For 0.1 M KOH, the ORR
based electrocatalysts with non-Pt encouraged the 4-electron transfer process in
electrocatalysts. ADEFCs have poisonous a fuel cell at high pH. Conversely, low pH
effects form the platinum catalyst on the (0.1 M HClO4) showed relatively less impact
cathode surface owing to ethanol cross over compared to the potential required. In 0.1 M
through the membrane. The catalyst suffers of KOH, little peroxide was found, such as
performance loss because of ethanol 0.5 - 2.5%, in electrolyte. Using the Ag
adsorption on the catalyst surface, in this (111) catalyst, there was no need for specific
manner blocking the adsorption of new chemical interaction between the catalyst
oxygen molecules. Therefore, the cathode and O2 or O2- at high pH. Contrarily, low pH
electrocatalysts are usually ethanol-tolerant. made solid chemical interaction essential. In
Demarconnay et al.1 examined Ag/C 0.1 M HClO4, the catalyst performed a 2e-
electrocatalysts with various loadings. The electron exchange with 100% H2O2
electrocatalysts were prepared using a formation. According to the results, higher
potential was discovered in the early mixed Synthesis of V2O5/C electrocatalyst
2e- and 4e- for electron exchange. The final Employing a combination of 1.60 g
process exhibited a 4e- electron transfer with of Vulcan XC-72R carbon black, 30 ml of
H2O2 formation. Vanadium oxides have ultra-pure water and 3 ml of 2-propanol, the
been indicated as important catalysts for use V2O5/C electrocatalyst was made. The
in various electronic technologies.3 Further, combination was mixed gradually for 10
V2O5 is used in semiconductors, optical min. Afterwards, 0.4 g of V2O5 was included
switching devices and write-erase media.4 and blended gradually for 3 h. Finally, the
Huang et al. studied vanadium nitride (VN) V2O5/C catalyst was dried at 90 °C using an
for ORR in alkaline electrolytes. The VN oven until the following day.
catalyst was synthesized using a Synthesis of Ag/C electrocatalyst
hydrothermal method. The results suggested Utilizing the colloidal technique, the
that ORR catalytic activity of a VN catalyst Ag/C electrocatalyst was made.7 Employing
was approximate to a commercial Pt/C a combination of 2.32 mmol of trisodium
catalyst.5 In this study, AgVO3/C, V2O5/C citrate dehydrate and 0.408 mmol of AgNO3
and Ag/C electrocatalysts in 0.1 M KOH dissolved in 50 mL ultrapure water (first
electrolyte with different ethanol-containing beaker), twenty percent Ag/C catalyst by
electrolytes are of preferential interest. weight was crafted, with the resulting
solution mixed until achieving visual clarity.
2. Materials and Methods 0.410 mmol of NaBH4 was dissolved in 50
2.1 Synthesis of catalyst materials mL of ultrapure water using the second
Synthesis of AgVO3/C electrocatalyst beaker. Using a dropwise method, the
The AgVO3/C electrocatalyst6 was second mixture was added to the Ag
achieved utilizing a ratio of 1: 1: 1 for a solution from the first beaker. A dark brown
combination of substrates equivalent to 2.5 color in the colloidal solution resulted from
mmol for ethylenediamine (C2H8N2), the previous color of yellowish-brown. In
vanadium pentoxide (V2O5) and silver order to promote diffusion, the Ag slurry
nitrate (AgNO3). Subsequently, the mixed was mixed for 15-30 min. With an ultrasonic
suspension was diffused into 40 ml of ultra- probe, 176 mg of Vulcan XC-72R carbon
pure water in a beaker, which was created black was subsequently dispersed in 30 mL
by reflux distillation at 180 °C in a glycerol- of ultrapure water for 10 min, which was
bath for 48 h until achieving a blackish then joined gradually with the Ag solution
shade. The nanoparticle catalysts were used while mixing quickly in order to create
for separation with ultra-pure water and supported active nanomaterial. The solution
washing with ethanol two times. In order to of Ag/C was mixed for 3 h, followed by 10
effect removal, the electrocatalyst solvent min of centrifuge at 11400 rpm. Using
was allowed to dry at room temperature ultrapure water, the Ag/C electrocatalyst
until the following day (first beaker). After particles were separated and cleaned.
that, 30 ml of ultra-pure water with 3 ml of Ultimately, the Ag/C electrocatalyst was
2-propanol was used to dissolve 1.34 g of allowed to dry at 90 °C until the following
Vulcan XC-72R carbon black, which was day.
stirred gradually to rapidly ca. for 10 min 2.2 Characterization of the catalyst
(second beaker). Not long after, 0.53 g of materials
AgVxOy nanoparticles from the initial The surface morphologies of
beaker was combined and blended gradually AgVO3/C, V2O5/C and Ag/C
to the second beaker over 3 h. Ultimately, electrocatalysts were characterized by
the AgVO3/C catalyst was dried overnight at transmission electron microscopy (TEM),
90 °C in an oven. with the component compositions of both
electrocatalysts used for measurement by
energy-dispersive X-ray spectroscopy
(EDX). The structures of the electrocatalysts Both dark-spherical and gray-
were analyzed by X-ray powder spherical morphologies were indicated by
diffractometry (XRD). the transmission electron microscopy
2.3 Electrode preparation (TEM). The dark spherical particles are
Electrochemical measuring likely Ag or V as metal, with the gray-
techniques were conducted with standard spherical particles being carbon black
three-electrode configurations, including 0.1 (Figure 1). The EDX examination showed
M KOH as the electrolyte. The reference the majority of Ag and V metal, which
electrode was a reversible hydrogen accounted for approximately 11.6% and
electrode (RHE). The counter electrode was 11.3%, respectively.
a platinum cathode and the working
electrode was a rotating-disk electrode
(RDE) with glassy-carbon substrate. 5000 Ag
Regarding the impacts of various 4000
Intensity, arb. units

centralizations for EtOH, a reasonable 3000
amount was added into the working 2000

electrolyte (see Table 1). In addition, 1000
Nanopure water (18 MΩ-cm, Barnstead 0
Nanopure) was used for setting up 0.1 M 10 20 30 40 50 60 70 80 90
KOH. Pure nitrogen (N2) and oxygen (O2) 2-theta (Cu K-alpha), degree
were also utilised. Moreover, AgVO3/C,

V2O5/C and Ag/C were used as
electrocatalysts. The catalyst-loading on the 1000 AgVO3
working electrode (RDE) was 56 μg cm-2. 800

Electrochemical behavior was obtained 600

using a potentiostat/galvanostat from
400
Autolab.
200
0
10 20 30 40 50 60 70 80 90
3.1 Physical characterization of AgVO3/C, 2-theta (Cu K-alpha), degree
V2O5/C, and Ag/C electrocatalysts
1400
1200 V2O5
1000
800
600
400
AgVO3/C V2O5/C 200
0
10 20 30 40 50 60 70 80 90
2-theta (Cu K-alpha), degree
Figure 2. X-ray spectroscopy (XRD) for (A)

Ag/C, (B) AgVO3/C, and (C) V2O5/C
Ag/C electrocatalysts
Figure 1. Transmission electron microscopy
The peak of Ag/C identification
(TEM) images of AgVO3/C, V2O5/C, and
included Ag, which collected information
Ag/C electrocatalysts
from JCPDS files number 01-071-3762. The
peak of AgVO3/C consisted of AgVO3,
using JCPDS files no. 00-029-1154. The
diffraction pattern of V2O5/C contained 10.0
Pt/C (A)
carbon and V2O5, using JCPDS files no. 01- 7.5 AgVO3/C
Current density (mA/cm2)

074-4605 (Figure 2). V2O5/C
3.2 Electrochemical measurements 5.0 Ag/C
3.2.1 Cyclic voltammograms for 2.5

AgVO3/C, V2O5/C, and Ag/C
electrocatalysts in 0.1 M KOH 0.0
-2.5
0.2
2 0.1
0.0
-0.1
-5.0
1
-0.2
-0.3 V2O5/C-N2
0.2 0.4 0.6 0.8 1.0 1.2
-0.4
V2O5/C-O2
-0.5
0.2 0.4 0.6 0.8 1.0 1.2 1.4
Potential (V) vs RHE
0
-1
AgVO3/C 10.0
-2
V2O5/C
Pt/C (B)
7.5 AgVO3/C

-3 Ag/C
V2O5/C
0.2 0.4 0.6 0.8 1.0 1.2 1.4 5.0 Ag/C
2.5
Figure 3. Cyclic voltammograms for
AgVO3/C, V2O5/C, and Ag/C 0.0
electrocatalysts in 0.1 M KOH electrolyte
-2.5
with N2 saturation at 30 C and a sweep rate
of 10 mV s-1, inset: cyclic voltammograms -5.0
of V2O5/C catalysts in 0.1 M KOH 0.2 0.4 0.6 0.8 1.0 1.2
electrolyte with N2 and O2 saturation at 30 Potential (V) vs RHE
C and a sweep rate of 10 mV s-1

10.0
Pt/C (C)
Figure 3 shows CV curves for 7.5 AgVO3/C
AgVO3/C, V2O5/C, and Ag/C V2O5/C

electrocatalysts with N2 saturation. The 5.0 Ag/C
oxidation peaks of AgOH and Ag2O were 2.5
formed on the surface catalyst at the
potential of around 1.29 and 1.35 V, 0.0
respectively.8 The reduction peak of Ag2O
-2.5
displays a potential about 1.0 V. As seen in
the inset of the figure, cyclic -5.0
0.2 0.4 0.6 0.8 1.0 1.2
voltammograms curves for V2O5/C catalysts
in both atmosphere shows reduction peaks at
0.6 V (Figure 3). In the presence of O2 Figure 4. Linear potential scan curves for
atmosphere, a large reduction peak can be Pt/C, Ag/C, AgVO3/C, and V2O5/C on a
observed. rotating-disk electrode in O2 saturated 0.1 M
3.2.2 Electrocatalytic activity towards KOH with varied ethanol in the electrolytes
oxygen reduction for Pt/C, Ag/C, (A) 0.1 M KOH with 0.1 M EtOH, (B) 0.1
AgVO3/C, and V2O5/C electrocatalysts in M KOH with 0.5 M EtOH, and (C) 0.1 M
0.1 M KOH with various ethanol- KOH with 1.0 M EtOH at 30 C, a sweep
containing electrolytes rate of 10 mV s-1 and a rotation rate of 1600
rpm
The ORR polarization curve of the electrocatalyst displayed ethanol oxidation
Pt/C electrocatalyst showed a diffusion- reduction (Figure 5).
limiting current (il) at potential 0.20-0.50 V 3.2.3 Tafel plots of AgVO3/C and V2O5/C
vs. RHE, which followed a region of mixed electrocatalysts in 0.1 M KOH
kinetic-diffusion control at potential 0.50- Tafel plots exhibit a linear
0.70 V vs. RHE (Figure 4). The effect of correlation of the potential (E) and the log ik.
ethanol-containing electrolytes on the The kinetic current ik can be calculated by
performance of oxygen reduction for Pt/C Equation (1) as:9
working electrode is shown in Figure 4. The
results of the linear potential scan curves on (1)
the surface of Pt/C electrocatalysts exhibited
ethanol oxidation in the presence of ethanol.
Conversely, the AgVO3/C, Ag/C and where io is the observed current and il is the
V2O5/C electrocatalysts exhibited ethanol- limiting current that can be obtained directly
tolerant result for ORR with ethanol- from the ORR curve.
containing electrolytes. According to the 0.90
results, the AgVO3/C electrocatalysts are 0.85
AgVO3/C
V2O5/C
bifunctional or alloyed vanadium, which are
Potential (V) vs. RHE
0.80
Ag/C
non-Pt catalysts and cheaper cost. A 0.75 Pt/C
0.70
comparison of the cost of silver (Ag) (19.65
0.65
USD/ozt) and vanadium (V) (0.60 USD/ozt)
0.60
showed a lower price than for platinum (Pt) 0.55
(1,090 USD/ozt) (from 0.50
www.infomine.com). 0.45
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0
2
log ik (mA/cm )
Figure 6. Mass-transfer Tafel plots of
oxygen reduction for AgVO3/C, Ag/C, and
V2O5/C electrocatalysts in 0.1 M KOH
The performance of the

electrocatalysts is evaluated from kinetic
current density (ik), as seen in Figure 6. The
Tafel slope for AgVO3/C, V2O5/C, Ag/C,
and Pt/C electrocatalysts are 75.69 mV dec-
1
, 70.79 mV dec-1, 60.30 mV dec-1, and
Figure 5. Comparison of the limiting 41.43 mV dec-1, respectively. The Ag/C and
current activity (il) (mA/cm2) at 0.3 V with a AgVO3/C electrocatalyst shows higher mass
rotation rate of 1600 rpm with and without activity than V2O5/C electrocatalyst.
0.1 M, 0.5 M, and 1.0 M EtOH in 0.1 M According to, the diffusion-limiting current
KOH (il) of AgVO3/C electrocatalyst close to Pt/C
electrocatalyst in 0.1 M KOH with various
The limiting current (il) was obtained ethanol-containing electrolytes (Figure 4).
directly from the ORR curve with and The AgVO3/C electrocatalyst shows
without EtOH at different rpm. The il in the significant for development.
presence of ethanol in KOH electrolyte
demonstrated poor activity (Figure 5). On 4. Conclusion
the other hand, the Pt/C catalyst showed The AgVO3/C, Ag/C, and V2O5/C
increasing activity in the presence of ethanol electrocatalysts exhibited ethanol-tolerance
because the surface of the Pt/C for ORR with ethanol-containing
electrolytes. Therefore, the AgVO3/C 4. Chain, E. E. Appl. Opt. 1991, 30, 2782.
electrocatalyst may be developed and 5. Huang, T.; Mao, S.; Zhou, G.; Wen, Z.;
applied instead of the platinum catalyst (as a Huang, X.; Ci, S. Nanoscale 2014, 6,
noble metal) at the cathode side of ADEFCs. 9608-9613.
6. Mao, C.; Wu, X.; Pan, H.; Zhu, J.;
References Chen, H. Nanotechnol. 2005, 16, 2892-
1. Demarconnay, L.; Coutanceau, C.; 2896.
Léger, J. M. Electrochimica Acta. 2004, 7. Meng, H.; Shen, P. K. Electrochem.
49, 4513-4521. Commun. 2006, 8, 588–594.
2. Blizanac, B. B.; Ross, P. N.; Markovic, 8. Park, S. A.; Lim, H.; Kim, Y. T. ACS
N. M. Electrochimica Acta. 2007, 52, Catal. 2015, 5, 3995-4002.
2264-2271. 9. Mayrhofer, K. J. J.; Strmcnik, D.;
3. Chakrabarti, A.; Hermann, K.; Druzinic, Blizanac, B. B.; Stamenkovic, V.;
R.; Witko, M.; Wagner, F.; Petersen, M. Arenz, M.; Markovic, N. M.
Phys. Rev. B. 1999, 59, 10583-10590. Electrochimica Acta. 2008, 53, 3181-
3188.
Influence of Ce ions in the La1-xCexCoO3+ studied by XAS and XPS
Sutasinee Kityakarn1*, Anupap Pungsomchit1, Suphannipha Thara1,
Rachadaporn Supruangnet2, Hideki Nakajima2, Yingyot Poo-arporn2
1
2
Synchrotron Light Research Institute (Public Organization), Nakhon Ratchasima 30000, Thailand
*E-mail: fscistsn@ku.ac.th
Abstract:
The La1-xCexCoO3 (x = 0.00-0.20) was prepared by the Pechini method, using metal
nitrate salt, critic acid and ethylene glycol. The crystal system of La1-xCexCoO3+ was
rhombohedral and the crystallinity decreased with increasing Ce content. The maximum
molar ratio of Ce in the La1-xCexCoO3+ was 0.15 which showed the maximum of Ce
solubility in the perovskite structure without distortion of rhombohedral unit cell. The O 1s
XPS of the chemisorbed oxygen was at 529.3-529.9 eV and that of the lattice oxygen was at
531.7 and 532.2 eV. The ratio of chemisorbed and lattice oxygen was less than 1 studied by
O 1s XPS and O K-edge XANES. It relates to the oxygen efficient non-stoichiometric
perovskite, La1-xCexCoO3+. The Co LIII-edge XANES spectrum showed the feature of Co3+
for the lower Ce concentration, and the Co2+ and the Co3+ in the perovskite for the higher Ce
concentration owing to the substituted Ce ions into the La-site and the reduction of Co3+ ions.
The Ce LIII-edge XANES spectrum presented the characteristic peak of the Ce3+/Ce4+ for the
lower Ce content and the Ce4+ for the high Ce content. The compensate oxidation state of
metal ions, Co2+ and Ce3+/Ce4+, was observed by the consistent adsorbed and lattice oxygen
ratio and the reduced Co ions.
1. Introduction
The designable material, perovskite, performance due to the redox properties of
ABO3, is well known as a semiconductor for Ce3+/Ce4+.5-7 Royer et al. prepared La1-
catalysts, sensors and batteries. In the xA’xBO3 (A’ = Sr and Ce, B = Co or Mn) for
perovskite lattice, A ions are in octahedral the decomposition of methane.8 The
holes and B ions are in the dodecahedral catalytic performance of LaCoO3 was higher
holes. The oxidation state of A and B ions in than LaMnO3 due to its surface area.
the structure can be varied, offering LaCoO3 showed the high catalytic activity at
plausibility to design the materials for low temperature due to the redox couple
various applications. The properties of Co3+/Co2+ forming suprafacial oxidation
perovskite mainly depends on the electronic based on Mars-Van-Krevelen.5,9 The Sr- and
structure of A and B ions. The non- Ce-substituted LaMnO3 showed the better
stoichiometric perovskite, AA’BB’O3, is the catalytic performance than the rare LaMnO3.
substituted perovskite that can modify Leanza et al. prepared the La1-xMxCoO3+
perovskite properties. For example, (M = Ce and Eu, and x = 0, 0.05, 0.1, 0.2)
strontium(II) ion (Sr2+) substituted in La site by flame hydrolysis10. It was found that the
of the LaCoO3, La1-xSrxCoO3, showed the additional Ce ions in perovskite structure
better performance in hydrogen peroxide presented the higher catalytic performance
sensing and solid oxide fuel cell compared than the Eu-substituted one due to the redox
to the rare LaCoO3.1-4 La1-xCexCoO3, where property of Ce. They also found the Co3+/O2-
La sites in the rare LaCoO3 were partially formed by the reduction of Co2+ under
substituted by cerium ions, showed efficient oxidizing atmosphere as studied by Electron
thermoelectric properties and high catalytic paramagnetic resonance (EPR). In this work,
we present the maximum of Ce solubility in XANES spectrum were recorded by the total
the LaCoO3 perovskite. Further, as electron yield (TEY) and the total
investigated by the synchrotron X-ray fluorescence yield (TFY) mode.
absorption and X-ray photoemission Ce L3-edge XANES spectrum were
spectroscopy, the results have suggested that measured at BL2.2, Synchrotron Light
these materials are oxygen efficient non- Research Institute (Public organization).
stoichiometric perovskites. The bent crystal Si (111) in the energy-
dispersive monochromator was used and the
2. Materials and Methods NMOSS linear image sensor was performed.
2.1 Preparation of La1-xCexCoO3+ (x = The CeO2 powder was used to calibrate the
0.0-0.20) Ce L3-edge absorption peak at 5731.5 eV.
The Ce-substituted LaCoO3 perovskite The absorption measurement was in the
materials were prepared by the Pechini range of 5661-5851 eV.
method, resulting in La1-xCexCoO3 where x
= 0-0.20. Lanthanum(III) nitrate, cobalt(II) 3. Results and Discussion
nitrate and cerium(III) nitrate were used as The metal citrate complex was
the starting materials. The citric acid and synthesized from metal nitrate salts and
ethylene glycol were added to the mixture of citric acid. The ethylene glycol was added to
metal nitrate solution.11 The molar ratio of the metal citrate to form the trimetallic in
metal nitrate, citric acid and ethylene glycol polyester observing the C=O, C-O and M-O
was 0.01:0.02:0.03. The mixture were group for all molar percentage of cerium ion
refluxed at 358 K for 3 h and dried at 383 K in the LaCoO3 by FTIR. The polyester type
for 24 h. The dried gel was calcined at 473 resin decomposed at 473 K under nitrogen
K for 4 h under nitrogen atmosphere and atmosphere and then the perovskite crystal
then at 973 K for 4 h to obtain LaCoO3 or formation was observed after calcination at
La1-xCexCoO3 powder. 973 K for 4 h under oxidized atmosphere.
2.2 Characterization La1-xCexCoO3+ (x = The lattice symmetry of perovskite is
0.0-0.20) rhombohedral and the crystallinity decreases
2.2.1 X-ray diffraction (XRD) with increasing molar percentage of cerium
The crystal structure of LaCoO3 and ions as studied by XRD (Figure 1). CeO2
La1-xCexCoO3 powder was investigated by segregation was observed in the XRD
XRD. The Cu K x-ray is generated by x-ray pattern of La0.8Ce0.2CoO3 by presenting a
tube operated at 40 kV and 30 mA. The small board peak around 29 degree,
XRD patterns were recorded in the range of suggesting that the maximum threshold of
20-70 degree. The XRD patterns had been solubility of cerium ions into the perovskite
indexed by comparison with the 00-048- structure were reached at x = 0.20. The
0123 JCPDS file. addition of Ce ions into the structure
2.2.2 Synchrotron X-ray absorption near affected the stoichiometry of the perovskite
edge spectroscopy and synchrotron X-ray since the change of oxidation state of the
photoelectron spectroscopy (XANES and cations from Co3+ to Co2+ caused the change
XPS) in the anion content used and the redox
O1s XPS and O K-edge XANES of property of Ce4+/Ce3+ to balance the
the LaCoO3 and La1-xCexCoO3 were oxidation state of all metal ions in the
measured at BL3.2a, Synchrotron Light structure.12
Research Institute (Public organization). The Since XRD technique cannot
powder was pressed on carbon tape and characterize the substituted cerium ions in
mounted on a molybdenum plate. C1s at the La1-xCexCoO3, XPS and XANES were
284.5 eV was the reference. The O 1s XPS employed to reveal the chemical information
measurement was in the range of 520-540
eV. The O K-edge XANES and Co L3-edge
highest substituted cerium ions, this ratio
were slightly decreased. The decrease in the
ratio of adsorbed and lattice oxygen with the
increasing cerium were observed as a result
of the presence of higher oxidation state of
metal ions in the perovskite, which required
more lattice oxygen to neutralize the
additional positive charge. The chemisorbed
oxygen relates to the recombination of e-/h+
suppression and creates the highly active
species such as OH.13
The O K-edge XANES spectrum
Figure 1. XRD-patterns of La1-xCexCoO3 (Figure 2b) showed three peaks
((x = 0.00 (a), 0.05 (b), 0.10 (c), 0.15 (d), corresponding to the excitation of O 1s to
and 0.20 (e)) synthesized powders (JCPDs hybridization of O 2p-Co 3d (527-530 eV),
file 00-048-0123, LaCoO3) hybridization of O 2p-La 5d (530-539 eV),
and hybridization of O 2p-Co 4sp (539-550
such as the chemical composition and the eV), respectively.14 The feature of spectrum
oxidation state of metal ions in the structure. depends on the molar percentage Ce
We have shown that for low Ce content addition. At the lower Ce substitution, x =
perovskite, the limit of Ce solubility in the 0.00-0.15, the spectrum were similar to the
perovskite structure without segregation of spectrum of the LaCoO3. At the higher Ce
CeO2 were at x = 0.00-0.20. The O 1s XPS substitution, x = 0.20, the feature of
spectra of La1-xCexCoO3 (x = 0.00-0.20) spectrum significantly changed, in which the
show the chemisorbed oxygen at 529.3- first peak at 527-530 eV corresponding to
529.9 eV and lattice oxygen at 531.7-532.2 the hybridization between O and Co slightly
eV (Figure 2), while the ratio of adsorbed shifted to the higher energy owing to the
and lattice oxygen in the range 0.84-0.92 reduced Co ions. The second peak
(Table 1). The ratio of adsorbed oxygen and significantly changed with the increasing
lattice oxygen sustained and implied the percentage of Ce ions can serve as the
oxygen efficiency of the perovskite structure electron donors, which reduces the Co ions
showing as non-stoichiometric perovskite, from Co3+ to Co2+ and change the final state
La1-xCexCoO3+. Even though the ratio of of O K-edge excitation at hybridized of La-
adsorbed oxygen and lattice oxygen for all O and Co-O.
materials were similar, for the one with
a) b)
Figure 2. a) O 1s XPS spectrum and b) O K-edge XANES of La1-xCexCoO3 (x = 0.00 (a), 0.05
(b), 0.10 (c), 0.15 (d), and 0.20 (e)) synthesized powders
Table 1. The binding energy and adsorbed and lattice oxygen ratio of La1-xCexCoO3 (x =
0.00-0.20
Sample Binding energy (eV) Ratio of /*

LaCoO3 529.6 531.9 0.91
La0.95Ce0.05CoO3 529.8 532.1 0.90
La0.90Ce0.10CoO3 529.3 531.7 0.89
La0.85Ce0.15CoO3 529.7 531.9 0.92
La0.80Ce0.20CoO3 529.9 532.2 0.84
Note *  presents the adsorbed oxygen and  presents the lattice oxygen.
The Co LIII-edge XANES spectrum result supports the XRD pattern that showed
showed mainly Co3 + when compared to the the small board characteristic peak of CeO2.
Co3 O4 at 781 eV (Figure 3a). However, the The perovskite with lower percentage of Ce
shoulder peak at 779 eV corresponding to ions showed the feature of Ce3+/Ce4+,
Co2+ increased with the cerium addition. The indicating that the Ce ions were well
Co2+ ions in perovskite increase by dissolved in the LaCoO3 perovskite lattice.
increasing the Ce addition and increase of All results have shown that the Ce
oxygen lattice. We present that the ratio of substituted LaCoO3 is oxygen efficient non-
adsorbed oxygen/lattice oxygen is less than stoichiometry perovskite with the formula of
1 and slightly decreases with increase of Ce La1-xCexCoO3+. The non-stoichiometry was
addition. These results related to the obtained as a result of the neutralization of
observation of the reduced Co in the the perovskite charge. The maximum of Ce
substituted Ce in LaCoO3. The oxidation solubility was achieved when 20% molar
state of Ce ions was studied by Ce LIII-edge ratio of Ce was reached in the perovskite
XANES, in Figure 3b. The feature of structure. This plays an important role in the
XANES spectra of the perovskite materials catalytic activity discussion.
was similar to that of the CeO2 XANES
spectrum, especially La0.80Ce0.20CoO3. This
a) b)
Figure 3. (a) Co LIII-edge XANES spectrum of La1-xCexCoO3 (x = 0.00 (a), 0.05 (b), 0.10 (c),
0.15 (d), and 0.20 (e)) and (b) Ce LIII-edge XANES spectrum of La1-xCexCoO3 (x = 0.05 (a),
0.10 (b), 0.15 (c), 0.20 (d), CeO2 and Ce(NO3)3)
4. Conclusion 3. Wang, P.; Yao, L.; Wang, M.; Wu, W.
The oxygen efficient perovskite, La1- J. Alloys Compd. 2000, 31, 53–56.
xCe xCoO 3+ was prepared by the Phecini 4. Bin, L.; Jinfan, C.; Yihan, L.;
method using metal nitrate salt. When extra Xiaozhen, Z.; Yinzhu, J.; Ling, Z.;
cerium ions could no longer substitute for Xingqin, L; Guangyao, M. J. Power
the La ions in the perovskite structure, the Sources 2010, 195.
characteristic XRD pattern of CeO2 5. Yang, W.; Yu, S.; Xianjie, W.; Wenhui,
appeared. The maximum molar ratio of Ce S.; Wenwu, C.; Xiaoyang, L. ACS Appl.
was 20% presenting the segregation of CeO2 Mater. Interfaces 2010, 8, 2213–2217.
in the XRD pattern and the feature of Ce 6. Mohammad, G.; Houshang, A.;
LIII-edge XANES. The oxidation state of Ce Sébastien, R.; Alain, A. Sens. Actuators
in the perovskite showed the Ce3+/Ce4+ for B 2011, 156, 147–155.
lower Ce concentration and Ce4+ for high Ce 7. Saira, K.; Richard, J.; Oldman, C.;,
concentration, and the Co2+ presented for the Richard A. Catlow; Samuel, A.; French,
Ce substituted LaCoO3 The compensate Sean, A. A. J. Phys. Chem. 2008, 112,
oxidation state of metal ions was observed 12310–12320.
by the sustain adsorbed and lattice oxygen 8. Royer, S.; Duprez, D.; Kaliaguine, S.
ratio and the reduced Co ions. Catal. Today 2006, 112, 99-102.
9. Leanza, R.; Rossetti, I.; Fabbrini, L.;
Acknowledgements Oliva, C.; Forni, L. Appl. Catal. B 2000,
We are pleased to thank the 28, 55-64.
Synchrotron Light Research Institute (Public 10. Imamura, M.; Matsubayashi, N.;
organization) for synchrotron facility Shimada, H. J. Phys. Chem. B 2000,
technique support. We are fully 104, 7348-7353.
acknowledged to Kasetsart University 11. Worayingyong, A.; Kangvansura, P.;
Research and Development Institute and Ausadasuk, S.; Praserthdam, P. Colloids
Department of Chemistry, Faculty of Surf. A 2008, 315, 217-225.
Science, Kasetsart University for financial 12. Forni, L.; Oliva, C.; Vatti, F. P.;
support. Kandala, M. A.; Ezerets, A. M.;
Vishniakov, A. V. Appl. Catal. B 1996,
1. Srdi, V. V.; Nikoli, L.; Pejov V. J. 13. Xin, Z.; Qiuhua, Y.; Jinjin, C. J. Rare
Optoelectron Adv. M. 2003, 5, 337 – Earths 2008, 26 (4), 511-514.
341. 14. Suntivich, J.; Hong, W. T.; Lee, Y. L.;
2. Dam, T. V. A.; Olthuis, W.; Bergveld, Rondinelli, J. M.; Yang, W.;
P. Sens. Actuators B 2004, 103, 165– Goodenough, J. B.; Dabrowski, B.;
168. Freeland, J. W.; Shao-Horn, Y. J. Phys.
Chem. 2014, 118, 1856-1863.
Effects of surfactants as additives in electronic-grade lubricant
for magnetic recording head lapping processes
Chuenkamol Khongphow1, Duangamol Tungasmita2, Benjie L. Fernandez3,
Chakkrit Supavasuthi3, Sukkaneste Tungasmita4*
1
Petrochemistry and Polymer Science Program, Faculty of Science, Chulalongkorn University,
2
3
Western Digital (Thailand) Co., Ltd., Bang Pa-in, Phra Nakhon Si Ayutthaya 13160, Thailand
4
Department of Physics, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
*E-mail: sukkaneste.t@chula.ac.th
Abstract:
Grinding and lapping are important processes in microelectronic device manufacture,
i.e. hard disk drive magnetic recording head (slider) fabrication. In lapping process, diamond
embedded Bi-Sn alloy plate is used together with functional lubricant to reduce excessive
friction side effects and protect the surface from corrosion. In this research, we studied the
effects of surfactants which play an important role as the lubricant additives to reduce the
contamination. The scanning electron microscopy results showed that the surfactants acted as
micelle formation additive to reduce and prevent the deposition of metallic particle
contaminants. The characteristics and performances of modified lubricants were measured
using quasi-static test (QST). Additional surfactant gave the change magneto resistive
resistance value (%MRR) and lapping rate lower than the standard glycol-based lubricant.
The potentiodynamic polarization indicated surfactant can also yield a better performance in
term of corrosion protection during lapping processes.
1. Introduction the metallic particles of the materials that

Hard disk drive (HDD) is an were removed from the lapping process and
essential device which is used to store data re-deposited on the surface by electrostatic
in computer, data server, cloud storage and effects. To solve this contamination issue
other electronic devices.1 In many electronic without introduce any side effects,
manufacturing, such as HDD, grinding and functional surfactants were considered to
lapping are the common processes to flatten add into the standard glycol-based lubricant
the rough surface of HDD read/write head or (SL) as an extra additive. These additional
slider after the cutting process. During the surfactants can work as micelle formation
grinding and lapping process, the lubricant agents to reduce and protect the surface
was used in order to reduce damage and from contaminant deposition. Several types
defects due to the friction and heat at the of surfactants, such as non-ionic, cationic
contact surface between specimen and and amphoteric surfactants, were selected to
lapping plate. The flow of lubricant can also modify the standard glycol-based lubricant,
help to remove debris out from the contact in order to improve properties and reduce
surface during the process. However, some the contaminant deposition at the same time.
debris and particles of removed materials There were 3 non-ionic surfactants chosen,
can re-deposit on the metallic surface of ethoxylated alcohols (EAL), polyethoxylate
specimen and causes contamination alcohol (PEAL) and polyoxyethylene
problems. sorbitan monolaurate (PO). These 3
In this work, a contamination was surfactants have been widely used in many
found at the electrical contact pads of the fields because of their good dispersion,
device. This contaminant was identified as emulsification and surface cleaning
properties2-5 for cationic surfactants, using JEOL JSM-7610F field-emission
cetyltrimethyl ammonium bromide (CTAB) scanning electron microscope (FESEM) and
and quaternary amines (QA) were chosen. energy-dispersive x-ray spectroscopy (EDS),
These 2 surfactants are used for detergent in order to identify the elemental
boosting, corrosion inhibition, anti-static composition of the contaminant material on
ingredients and emulsification.6 For surface and its morphology.
amphoteric surfactant, alkyl ethoxylated 2.3 Fluorescence spectrophotometry
amine oxide (AAO) was selected which its Fluorescence spectrophotometer
properties are detergent and oxidation (Cary Eclipse, Algilent Technologies) with
resistance.7 The effects and mechanisms of pyrene solution as a fluorescent probe, was
surfactants in modified lubricants were used to study the critical micelle
investigated in different perspectives, concentration (CMC) from different
including the anti-metal deposition, micelles concentrations of surfactant in the modified
formation above the critical micelle lubricants. In the analysis, the ratio between
concentration (CMC) and anticorrosion the first fluorescence intensity of the pyrene
function. emission spectrum at 375 nm and the third
peak at 395nm, known as I1/I3 were
2. Experimental calculated.8,9
2.1 The surfactant-modified lubricants 2.4 Potentiodynamicpolarization mea-
Surfactants: ethoxylated alcohols surement
(EAL) (Air Product Industry Co., Ltd), To investigate the corrosion
polyethoxylate alcohol (PEAL) (Sigma behaviors between the modified lubricants
Aldrich), cetyltrimethyl ammonium bromide and sliders, the polarization curve from
(CTAB) (EMD chemical), alkyl ethoxylated different surfactant-modified lubricants were
amine oxide (AAO) (Air Product industry measured, using potentiodynamic
Co., Ltd), quaternary amines (QA) (Air polarization (EG&G Model 273). Ag/AgCl,
Product industry Co., Ltd) and Pt wire and IrMn were used as the reference
polyoxyethylene sorbitan monolaurate (PO) probe, counter electrode and working
(Chemipan Corporation Co., Ltd.) were electrode, respectively. There were three
selected to modified the standard glycol- different modified lubricants that used as
based lubricant. The different concentrations electrolyte, each contained 1wt% of PEAL
of surfactants (0.2, 0.4, 0.6, 0.8 and 1.0 in based lubricant, 1wt% of QA in based
wt%) were added into standard lubricant for lubricant and 0.2wt% of PO in based
the modified lubricant preparation. lubricant, respectively. The corrosion
In order to study the effects of potential (Ecorr) and corrosion current (Icorr)
selected surfactants on the protection of the are measured and the corrosion rate were
contaminant deposition, 1wt% of each calculated to determine the corrosion effect
surfactant was added into sludge, collected of each modified lubricant.
from the lapping process. The sludge, which 2.5 Performance test of sliders
contained some debris from the lapping The performances of modified
process mixed with the standard lubricant, lubricants were tested in the production. The
were used as electrolyze in electroplating on quasi-static test (QST) was measured to
the copper foils. This experiment was to analyze the performance of the modified
simulate the deposition of the contaminant lubricants. The percent change of
on the electrical contact, in order to study magnetoresistive resistance values (%
the effects of the surfactants added in the MRR), before and after lapping, were
standard lubricant. extracted from the QST, together with the
2.2 Contaminant analysis lapping time.
The contamination, deposited on the
electrical contact of slider was investigated,
(a) (b)
Counts (cps/eV)
Counts (cps/eV)
Energy (keV) Energy (keV)
Figure 1. EDS spectrum of (a) clean copper foil and (b) copper foil with contamination layer
3. Results and Discussion from the tribocharging effect during the

3.1 Contaminant analysis lapping process. The particles were
The EDS spectrum of a clean copper deposited on the surface of electrical contact
electrical contact and a copper contact with by electrostatic effect.
deposited layer of contaminant are shown in The electron micrographs of the
Figure 1 (a) and (b), respectively. The electroplated Bi particles on copper foil,
spectrum 1b showed the present of Bi as deposited from sludge with different
majority signal, together with Al and C surfactants are shown in Figure 2. The SEM
peaks. From the result, it clearly indicates showed clean surface of copper foils can be
that the contaminant layer was mostly obtained when were added into the sludge
bismuth (Bi) particles. These Bi-particles before electoplating process. From the
came from the lapping plate due to wear result, it showed that some surfactants
during the process, since Bi is one of the (PEAL, QA and PO) can help to protect the
major components in the lapping plate. surface from Bi particle contamination.
From the lapping system and circuit These surfactants were then characterized to
analysis, it was found that the Bi-particles understand the mechanisms and functions.
were mixed with the lubricant and charged
(a) (b) (c)
(d) (e)
Figure 2. SEM of (a) clean copper surface, (b) copper surface after electrodeposition using
pure sludge, (c) copper surface after electrodeposition using sludge mixed with PEAL, (d)
copper surface after electrodeposition using sludge mixed with QA, and (e) copper surface
after electrodeposition using sludge mixed with PO
Figure 3. Pyrene intensity ratio I1/I3 versus log [concentration] with total surfactant molar
concentration for (●) PEAL modified lubricant, (■) QA modified lubricant, and (▲) PO
modified lubricant
3.2 Critical micelle concentration groups are attacked, leading to acetoxy

The optimum concentration of each group.16 For QA (cationic surfactant), the
selected surfactant was obtained from QST spherical micelles contain alkyl chain can be
technique which PEAL, QA and PO formed at using 1.0wt% concentration. It
exhibited low delta MRR at concentration of also has both cationic ion (amine ion) and
1.0, 1.0, and 0.2wt%, respectively. Then ionic site (polyoxyethylene group) at the
mixed lubricants with these optimum head of QA chain. The association of
concentrations were used in further study. charged bismuth with cationic of QA
For micelle formation analysis, graph of micelles involve electrostatic interaction.
selected modified lubricants are presented in Thus, charged bismuth is expected to be
Figure 3. PEAL, QA and PO reached their present near the stern layer and does
CMC values at 0.64 mM (0.04wt%), 0.88 not deposit on surface.17 In case of PO
mM (0.03wt%) and 4.69 mM (0.52wt%) for (nonionic surfactant), at 0.2wt% working
PEAL, QA and PO, respectively. The concentration of modified lubricant cannot
obtained CMC values of modified lubricants form spherical micelle due to higher
were higher than those in the literatures. solubility of PO in employed based
Hence, the standard lubricant contains lubricant. Moreover PO is added, the
glycol chain that has an effect to interrupt presence of the polyoxyethylene units are
the aggregation of micelle.10-15 This CMC considerable. This phenomenon can be
value can be referred to the self-assembly of attributed to intra- and interchain steric
micelle formation for the modified repulsions18 so the concentration at 0.2wt%
lubricants and indicated the efficiency of of PO is not sufficiency to form micelle.
contamination protection. Nevertheless, PO mainly consists of the
At our working concentration polyoxyethylene chains to determine the
1.0wt% of PEAL (nonionic surfactant) can formation of bismuth particles. Since PO is
form spherical micelles. Their alkyl chains able to reduce charged bismuth to bismuth
are radially arranged to form the micelle’s particles through oxidation of oxyethylene
hydrophobic core and have polyoxyethylene group into acetoxy group and prevent the
group outside the micelles.10-15 These deposited.19 Based on all CMC results, the
micelles can accommodate and protect the modified lubricants have potential to prevent
growth of deposited bismuth on surface the deposited contaminant on the electrical
because the negative charges of contact surface.
polyoxyethylene group of micelle are 3.3 Corrosion test
oxidized and reduction of charged bismuth
to neutral bismuth. Several polyoxyethylene
Figure 4. Polarization curve of surfactant-modified lubricants compare to standard lubricant
The modified lubricants of 1wt% of 4. Conclusion

PEAL, 1wt% of QA and 0.2wt% of PO were The solution for solving contaminant
used in the corrosion test, based on their deposition during lapping process can be
protective performances from QST result. obtained by adding functional surfactants
(b)
Figure 4 showed the polarization curves of into standard lubricant. The surfactants, such
surfactant-modified lubricants, compared to as PEAL and QA, functioned by the micelle
standard lubricant. It showed the Ecorr of formation in glycol-based lubricant at the
1wt% PEAL, 1wt% QA and 0.2wt% PO ratio of 1wt%. In case of PO at working
modified lubricants were at -0.053, -0.128 concentration of 0.2wt% could protect Bi
and -0.077 V, respectively. They were deposit by intra- and inter-repulsions. The
complicated to corrode than standard modified lubricants were proven to protect
lubricant that standard lubricant’s Ecorr was - the deposited contaminant on electrical
0.460 V. The corrosion rate, calculated from contacts of HDD slider. The micelles of
the plot, showed that the standard lubricant modified lubricants interrupted the
has the highest corrosion rate at about deposition of contaminant layer.
0.15Å/min but the modified lubricants with Furthermore, corrosion rates of modified
1wt% PEAL, 1wt% QA and 0.2wt% PO lubricants were also lower than the standard
have corrosion rates only at about 0.03, lubricant, which indicated an anti-corrosion
0.07, and 0.08 Å/min, respectively. This property, as an additional function. The
result indicates that all modified lubricants ∆%MRR value and the lapping time
provide lower corrosion rate than the confirmed that the PEAL, QA and PO can
standard lubricant. Thus, the anti-corrosion be used as functional additives for the
property was also obtained with the standard lubricant, without introduce
modified lubricants. additional failure to the lapping process.
3.4 Performance test of slider stability
From the QST, the obtained values Acknowledgements
of ∆%MRR and the lapping time for all the The authors would like to
modified lubricants were not significantly acknowledge Western Digital (Thailand)
different from the values of standard Co., Ltd. and graduate school,
lubricant. This indicated the modified Chulalongkorn University for the all the
lubricants does not introduce any additional supports on this research.
failures to the quality of slider in the lapping
process.
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Minerals Eng. 2013, 40, 12–15.
Effects of corrosion inhibitors in lubricant for microelectronic
lapping processes
Natkritta Maipul1, Duangamol Tungasmita2, Benjie L. Fernandez3, Chakkrit Supavasuthi3,
Sukkaneste Tungasmita4*
1
Petrochemistry and Polymer Science Program, Faculty of Science, Chulalongkorn University,
2
3
Western Digital (Thailand) Co., Ltd., Bang Pa-in, Phra Nakhon Si Ayutthaya 13160, Thailand
4
Department of Physics, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
*E-mail: sukkaneste.t@chula.ac.th
Abstract:
Lapping is one of the important processes in microelectronic device fabrication. In
hard disk drive production, the functional lubricants are used in the lapping processes to
reduce the heat, stress and material debris during the process. However, the lubricant can
react with some materials in device structure and causes the corrosion. In this work, selected
functional polymers were added as corrosion inhibitors into glycol-based standard lubricant.
The formation of protective films over the surface by their functional groups, was
investigated. The corrosion behaviors on antiferromagnetic materials were studied by
using the potentiodynamic polarization. The results showed that corrosion rate of
antiferromagnetic materials can be reduced by using modified lubricant. Scanning electron
micrographs showed the morphology of the film that was formed as the corrosion
protective layer on the device surface. Furthermore, the performance parameters from quasi-
static test (QST) indicated that the polymer modified lubricants have better corrosion
protection and lapping performance than the standard lubricant.
1. Introduction from the fabrication process by the use of

Microelectronic devices have been lubricant and other solvents. As a result,
developed continuously for hence efficiency and lifetime of the device were
capabilities to work and levels of quality decreased.
excellence that match the high standard In this work, we focused on
needed. Lapping process is one of the functions and mechanisms of selected
processes that can be used to obtain a corrosion inhibitors. These inhibitors can
specific surface roughness. It also reserved work as an extra additive for the glycol-
for products that demand very tight based standard lubricant, in order to
tolerances of flatness, parallelism, increase corrosion protection on some
1
thickness or finish. In hard disk drive sensitive materials of the slider during
(HDD) fabrication industry, the read-write lapping process. Polypyrrolidone (PP),
head or slider is one of important polyvinyl pyridineoxide (PVO) and
microelectronic components in the HDD polyoxy-ethylenestearate (PO) were
assembly for reading and writing data. For selected as functional polymer corrosion
modern higher capacity data storage, very inhibitors for this purpose. They have been
high precision in the fabrication process as widely examined for corrosion inhibition
lapping is required due to the complexity for mild steel.2-4,6-8 These polymers have
of the slider nanostructure. Some of the been reported as effective corrosion
sensitive material layers in modern device inhibitors because of their high molecular
structure can be removed by corrosion weight and large size result in they
provide high corrosion inhibition.2,5 They lubricants and the surface of IrMn thin film
also have large repeating units that may which is the most sensitive material in the
cover more area on the target surface and slider structure. The Potentiostat
form polymer complex, which lead to /galvanostat (EG&G Model 273) was used
anticorrosion property. Moreover, to measure the I-E polarization curve. For
Propylene glycol stearate (PG) also the cell electrodes, Ag/AgCl was used as
represented fatty ester for using as reference electrode, Pt wire as counter
corrosion inhibitor because it can form electrode and IrMn coupon as the working
film coating by functional group.5 The electrode. The measurements were done by
corrosion behaviors and lapping using the modified lubricants as electrolyte.
performance of all inhibitor modified 2.2.3 Film morphology
lubricants were characterized and The morphology of the corrosion
investigated to optimize the modified protective films on the surface of IrMn,
lubricant for highest corrosion protection caused by the modified lubricants were
in lapping process. investigated by using scanning electron
microscope (Jeol JSM 6400), operated at
2. Materials and Methods an accelerating voltage of 5.0 kV.
2.1 Preparation of lubricants 2.2.4 Performance test in lapping process
Several corrosion inhibitors were To study the performance of
selected to modify the properties of the modified lubricants in production line, the
standard glycol-based lubricant. PP and modified lubricants were applied to use in
PVO were obtained from Ashland Inc, the actual lapping system. In the fabrication
America. PG and PO were obtained from process, the lapping machine polished the
Chemipan Corporation, Thailand. The sliders until they reached the design
modified lubricants were prepared by criteria for thickness and surface
adding the corrosion inhibitor as an extra roughness. Lapping time was measured and
additive into the standard glycol-based the quasi static test (QST) was used to
lubricant (SL) at 4 different concentrations analyze the electrical performance of
for each corrosion inhibitor, as 0.01, 0.03, sliders. The percent change of
0.06, and 1.0wt% of the standard glycol- magnetoresistive resistance values
based lubricant. These modified lubricants (∆%MRR) were extracted, before and after
were then characterized and tested for their lapping with modified lubricants. These
performance in the process. ∆%MRR values can be referred to
2.2 Characterization of lubricants corrosion occurrence and electrical stability
2.2.1 Fundamental properties of the sliders, incorporated with the
The pH values and conductivity of surface finishing by modified lubricants
the modified lubricants were measured by
using pH/conductivity meter (Seven- 3. Results and Discussion
ExellenceTM, Mettler Toledo). Then, the 3.1 Fundamental properties
total base number (TBN) was measured, The pH values of all modified
following the ASTM D2896. TBN can be lubricants were found in range of 8.50-
used to indicate basicity of the modified 8.60, which closed to the standard glycol-
lubricants which can be expressed in terms based lubricant (8.40). The TBN of the
of the equivalent number of milligrams modified lubricants were also measured.
potassium hydroxide per gram (mg The result showed that the TBN values of
KOH/g). all modified lubricants were slightly
2.2.2 Corrosion test increased with an increasing of
Potentiodynamic polarization concentration of corrosion inhibitors. The
measurements were done to investigate the TBN value of standard glycol-based
corrosion behaviors between the modified lubricant was at 3.72 mg KOH/g while
TBN of modified lubricants were in range corrosion inhibitor used in modified
21-24 mg KOH/g. This can be due to the lubricants were selected and plotted,
corrosion inhibitors induced an increasing shown in Figure 1.
in basicity to the lubricants from their Good corrosion protection can be
chemical structures. Thus, an increasing of analyzed by the highest Ecorr and low
basicity of lubricants can help extend the corrosion rate. PG at 0.06wt% exhibited
lifetime by reducing the corrosion of the the best Ecorr value at -0.19 V and corrosion
devices and decreasing acidity from other rate at 0.084 Å/min, followed by PP at
additives.8,9 The corrosion rate can be 0.06wt%, PVO and PO at 0.1wt%,
affected by the conductivity value of the respectively. However, all modified
lubricant. A high conductivity can induce lubricants provided lower corrosion rate
high corrosion occurrence. The results than the standard glycol-based lubricant
showed that the conductivity values of PP (showed in Table 1). From the results, PG
and PG for all concentrations were in range provided the best Ecorr and Icorr values. This
of 112-117 µs/cm, which were less than PO, indicated PG-modified lubricant has the
PVO and standard glycol-based lubricant lowest corrosion effect on IrMn material,
(119-148 µs/cm). For PP, its functional followed by PP-modified lubricant.
group is difficult to ionize due to its polymer
structure. In case of PG, it has only one Table 1. Ecorr values and corrosion rate of
functional group that can ionize. In contrast, standard and modified lubricants (at their
PO and PVO increase more conductivity optimum concentrations for each corrosion
values because they contained more inhibitor)
functional groups that can be ionized. Thus, Optimum E Corrosion
modified lubricant contained PP or PG can Chemicals conc. (wt%) corr rate
in SL (V) (Å/min)
help to reduce corrosion occurrence.
SL 0.00 -0.47 0.111
3.2 Corrosion Test
SL+ PP 0.06 -0.21 0.086
SL+ GP 0.06 -0.19 0.084
SL+ PO 0.10 -0.38 0.097
SL+ PVO 0.10 -0.25 0.088
3.3 Film morphology analysis

Scanning electron micrographs
showed the thin films, formed by PP- and
PG- modified lubricants on IrMn surface,
are illustrated in Figure 2. The morphology
showed clearly the formation of the
protective film on the surface after dipped
IrMn specimens into those modified
Figure 1. Polarization curves of standard lubricants. The formation of the protective
and modified lubricants (at their optimum films on the surface can be due to the
concentrations for each corrosion inhibitors) chemical structure of added corrosion
inhibitors. For PP, it can be hydrolyzed in
Potentiodynamic polarization test neutral solution, which produced a positive
was done for all modified lubricants, at all charge on the nitrogen atom and a negative
concentration of 4 corrosion inhibitors. charge on the oxygen atom.1 On the other
The corrosion rate of the modified hand, PG has OH group that can
lubricants with IrMn material can be deprotonated H from OH group of glycol
analyzed from their polarization curves. and became alkoxyl group. These
The optimized concentration of each mechanisms are illustrated in Figure 3.
These two corrosion inhibitors also have side effect to the electrical performance and
oxygen atom with negative charge that can stability of the sliders
interact with partial positive charge of metal
surface.1,2 Therefore, PG- and PP-
a
modified lubricants can be adsorbed on the
surface of IrMn by partial charging effect hydrolyzed
and form protective film, coated on the
surface for corrosion protection.
a
b
deprotonated
1 µm
Figure 3. Functional groups mechanisms

b of (a) PP and (b) PG
4. Conclusion
The polymer type and fatty ester
1 µm type corrosion inhibitors were used as
additive in glycol-based lubricant for
c corrosion protection of materials of HDD
heads during lapping process. The
functional group of selected inhibitors (PP
and PG) can be adsorbed on the surface
1 µm of antiferromagnetic material by partial
charging effect and formed protective films
Figure 2. Scanning electron micrographs on device surface. PP- and PG-modified
of (a) IrMn surface, (b) IrMn surface after lubricants, at concentration of 0.06wt%,
dipped into PP-modified lubricant, and (c) exhibited the best corrosion protection due
IrMn surface after dipped into PG- to their high basicity and conductivity
modified lubricant, respectively values. Those modified lubricants also
gave lower corrosion rate, compared to
3.4 Performance test in lapping process standard lubricant. Both PP- and PG-
Lapping test results of the PP- and modified lubricants can form a thin layer of
PG- modified lubricants showed slightly protective film on the surface of the device,
less lapping time than the standard glycol- reducing the corrosion. The ∆%MRR
based lubricant. This may due to both values from QST measurement and the
modified lubricants has slightly lower lapping time indicated that these modified
viscosity, compared to standard glycol- lubricants can be used as alternated
based lubricant. The QST tests were lubricants in the production process and
performed before and after lapping process help to reduce the corrosion of sensitive
with modified lubricants. The QST results materials in the device during in lapping
showed the values of ∆%MRR before and process, without losing quality and electrical
after lapping test with modified lubricants performance.
were not significantly different to the
standard glycol-based lubricant. Therefore, Acknowledgements
PP- and PG-modified lubricants have no The authors would like to
acknowledge Western Digital (Thailand)
Co., Ltd. and graduate school, 5. Maltanava, H. M.; Vorobyova T. N.;
Chulalongkorn University for all the Vrublevskaya O. N. Surf. Coatings
supports on this project. Technol. 2014, 254, 388-397.
6. Karthikaiselvi, R.; Subhashini, S.
References Arabian J. Chem. 2017, 10, S627-S635.
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Hardik, B. Kalpa. Engineering 2017, 1, Senthil, K. S. Alloys compd. 2016, 675,
32–40. 139-148.
2. Layla, A.; Al. J.; Amal, A. M.; 8. Larabi, L.; Harek, Y.; Traisnel, M.;
Mekhamer, W. K. Int. J. Electrochem. Mansri, A. Appl. Electrochem. 2004,
Sci. 2012, 7, 8578 – 8596. 34, 833-839.
3. Saviour, A, U.; Ime, B. O.; Isaac, O. I. 9. Norio, T.; Atsunobu, M. IEEE
The Open Corrosion 2009, 2, 1-7. Transaction on Magnetics 2005, 41,
4. Ayyoubi El, S.; Chetouani, A.; 825-830.
Hammouti; Warthan, A.; Mansri, A.;
Deyab Al, S. S. Int. J. Electrochem. Sci.
2012, 7, 1639–1655.
Investigation of purification procedures for MIL-101(Cr) metal-organic
framework obtained from HF-free synthesis
Ratchadaporn Srikhong1, Somsak Supasitmongkol2, Ramida Rattanakam1*
1
Department of Chemistry, Faculty of Science, Kasetsart University, Chatuchak, Bangkok 10900, Thailand
2
National Metal and Materials Technology Center, Khlong Luang, Pathum Thani 12120, Thailand
*E-mail: fscirdr@ku.ac.th
Abstract:
MIL-101(Cr) is one of the most widely studied metal-organic framework materials
used greatly in many scientific fields. Its high porosity and robust framework towards
humidity and various organic solvents have promised applications in gas storage and
separation. The initial MIL-101(Cr) synthesis reported the use of chromium salt and
terephthalic acid together with some amount of hydrofluoric acid (HF) to ensure unsurpassed
crystallinity. However, the use of dangerous HF is undesirable for large-scale synthesis. In
this work, MIL-101(Cr) was hydrothermally synthesized under HF-free condition. To obtain
pure MIL-101(Cr) with high porosity, three purification procedures were investigated: (I)
treating with methanol and drying under vacuum; (II) treating with methanol and drying
under N2 flow; (III) treating with NH4F and drying under vacuum. Based on the BET surface
area and pore volume analysis, the results have suggested that, at the same drying
temperature, procedure (III) offered the best mean to remove solvent and residual reactants
from MIL-101(Cr) framework, but this also required the additional washing step to remove
NH4F. Procedure (II) was less effective compared to procedure (III) while procedure (I)
showed lowest performance.
1. Introduction and product purification. The most common

Metal organic frameworks (MOFs) synthesis approach is to use hydrothermal
are a class of crystalline organic–inorganic reaction between chromium(III) nitrate and
hybrid materials formed by coordination of terephthalic acid and fluoric acid (HF) at
metal clusters or ions with multi-dentate 220 C for 6-10 h.1 It has been noted that the
organic linkers.1 Among numerous MOFs, presence of HF led to better crystallization
chromium terephthalate MIL-101 takes a of the product. Still, the mechanism of how
special places due to a combination of high of fluoride ions involve in crystallization is
specific surface area with outstanding unclear. Moreover, the use of dangerous HF
thermal and chemical stability. These is undesirable for a large-scale synthesis and
advantages have motivated great interest in the presence of fluoride ions, in some case,
this material, in particular, to be used as decrease adsorption capacity of this
solid adsorbent and catalyst. The MIL-101 material.2,3 Thus, many recent works have
framework has a positive charge been devoted to the establishment of new
([(Cr3O(bdc)3]+) which is balanced by coordinated direct synthetic routes in the absence of
or guest anions located within the framework HF.4,5
cavities ([(Cr3O(H2O)2(bdc)3X]nH2O (X = F In spite of rather simple preparation
or OH; bdc2 = terephthalate ion p- procedure, MIL-101 with a BET (Brunauer,
C6H4(COO)22; n  25). The anion Emmett, and Teller) surface area greater
composition as well as the extent of species than 4000 m2g-1 is very difficult to obtain.
occupying the cavities depends strongly on This is primarily due to the incorporation of
the synthetic parameters including the ratio the impurities produced by the unreacted or
of starting reagents, the method of heating, recrystallized terephthalic acid both outside
and within the pores of MIL-101.6 were taken at 20 kV with a SEM Quanta 450
Consequently, many attempts have been (FEI Company). FTIR spectra (4000–400
made to increase the purity of MIL-101.7-8 It cm-1) were recorded on KBr discs in a
is crucial to have a MOF with the best Vertex 70 FTIR spectrophotometer. Thermal
textural properties possible to exercise its analysis was carried out with Perkin Elmer
full potential for applications in adsorption, TGA 7 machine. The specific surface area
gas storage and catalysis. of MIL-101(Cr) was calculated using a
Herein, MIL-101(Cr) was Micromeritics 3Flex surface characterization
synthesized by the hydrothermal method in with N2 at 77.4 K. Prior to the N2
the absence of HF, and then characterized adsorption/desorption measurement the
with various techniques including X-ray sample was degassed under vacuum for 6 h
diffraction (XRD), Scanning electron at 150 C. The specific surface area was
microscopy (SEM), Fourier transform calculated according to the BET (Brunauer–
infrared spectroscopy (FTIR), and thermo- Emmett–Teller) method.
gravimetric analysis (TGA). Three novel
approaches were applied to purify for MIL- 3. Results and Discussion
101. The effect of purification method on 3.1 Sample characterization
the purity of MIL-101 was evaluated by The resultant samples subject to
analyzing the BET surface area and pore purification method (I), (II) and (III) were
volume. labelled MIL101-vac, MIL101-N2 and
MIL101-NF, respectively. X-ray diffraction
2. Materials and Methods experiments were carried out in order to
2.1 Synthesis characterize the crystalline structure of as-
Briefly, 4 g of chromium nitrate synthesized MIL-101(Cr) and the purified
nanohydrate (Cr(NO3)39H2O, Sigma- MIL-101(Cr) samples. As seen in Figure 1,
Aldrich, ≥98%) was mixed with 50 mL of the XPD patterns of the as-synthesized MIL-
deionized water. Then, 1.67 g of terephthalic 101 shows well-resolved diffraction peaks
acid (H2bdc, Sigma-Aldrich, 98%) was which are in excellent agreement with the
added. The mixture was placed inside a simulated XRD pattern, presenting the
PTFE-lines stainless steel autoclave, and characteristic diffraction peaks at 2 =
then heated in an electric oven to 200 C for 2.81, 3.30, 3.44, 3.98, and 5.95.
10 h. The sample was recovered by filtration
and washed with small amount of hot DMF
and water to dissolve terephthalic acid MIL101-NF
crystals.
2.2 Purification MIL101-N2
Three purifying methods were

chosen: method (I), MIL-101 sample was MIL101-vac
treated with methanol and dried in vacuum
at 200 C for 6 h; method (II), MIL-101 Assyn MIL-101
sample was treated with methanol and dried

under N2 flow at 200 C for 6 h; method
MIL-101(Cr) (simulated)
(III), MIL-101 sample was reacted 2 4 6 8 10 12 14

o
with 30 mM NH4F solution and dried in 2 ( )
vacuum at 200 C for 6 h. Figure 1. XRPD patterns of as-synthesized
2.3 Characterization MIL-101(Cr) and purified MIL-101(Cr)
Powder XRD analysis was made samples
using a D8 Bruker X-ray diffractometer with
a copper anode (Cu Kα radiation, λ = 1.5418 This confirms the successful
Å). SEM images of the obtained powder synthesis of MIL-101(Cr) without a
significant amount of impurity. After The bands observed between 600-1600 cm-1
purification, the characteristic diffraction are attributed to benzene ring, including the
peaks remained unchanged, with no stretching CC at 1510 cm-1 and weak and
additional peaks and no apparent loss of narrow bands of deformation vibrations
crystallinity, which indicates the high (CH) at 1165, 1018, 880, and 750 cm-1.9,10
stability of this MOF upon purification These bands confirms the presence of
processes. terephthalate linker in MIL-101 framework.
Figure 2 shows the SEM images of No peak shift is observed in the FTIR
the as-synthesized MIL-101(Cr) and the spectra after purification, meaning that the
purified MIL-101(Cr) samples. It is clear functional groups in MIL-101 do not change
that all the samples took regular octahedral and the framework are chemically stable
shape, which is a characteristic shape of this towards the purification processes.
MOF, ranging in size between 200 and 500 The intense and broad bands around
nm. The octahedral morphology was well 3424 cm-1 can be attributed to the stretching
retained regardless of purification vibration of water molecules, suggesting that
procedures MIL-101 has been taken into, H2O molecules were present within the
indicating that the structure is preserved framework.11 As for the as-synthesized
throughout the course of purification. It MIL-101, the bands in this area are very
should be noted that the as-synthesized intense compared to those in the purified
sample also exhibits some irregular-shape MIL-101, denoting that the removal of
particles, which could be a result of the coordinating and pore-filling H2O molecules
small amounts of residual terephthalic acid were successful in the purification steps.
in the pores of MIL-101.
3100*
FTIR characterization was conducted
1405
1018
1510
1165
to detect the identity of the MIL-101(Cr)
1620
3060*
750
880
3424
functional groups and their status after being MIL101-NF

purified. As seen in Figure 3, the FTIR
spectra of all samples exhibits the MIL101-N2
characteristic bands at 1405 and 1620 cm-1
corresponding to the symmetric and
asymmetric vibrational stretching (OCO) MIL101-vac
of carboxylate groups.9
AssynMIL-101
4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )
Figure 3. FTIR spectrum of as-synthesized

MIL-101(Cr) and purified MIL-101(Cr)
samples
Moreover, for MIL-101(Cr) the band

at 1629 cm-1 can be assigned to adsorbed
water molecules.12 This band can be
Figure 2. SEM images of as-synthesized observed in the as-synthesized MIL-101 and
MIL-101(Cr) and purified MIL-101(Cr) MIL101-vac spectra but not in MIL101-N2
samples and MIL101-NF, implying that purification
methods II and III are more effective in
removing H2O molecules from the NF TGA curve. This indicates that water
framework. and residual terephthalic acid molecules
It was also reported that the within the pores could be removed more
unreacted terephthalic acid can be effectively by treating the MOF with NH4F
characterized by FTIR spectroscopy by solution and applying heat and vacuum to it.
observing the presence of 3100 and 3060 In comparison with other samples, MIL101-
cm-1 bands due to the carboxyl OH group of NF also exhibits enhanced thermal stability
unreacted terephthalic acid.11 However, as demonstrated by a rise in decomposition
here, these peaks were swamped by the high temperature from approximately 280 (in the
intensity OH bands of the adsorbed water as-synthesized MIL-101) to 330 C.
molecules. 3.2 Purity evaluation based on nitrogen
Figure 4 shows a thermal adsorption properties
gravimetric analysis, which was performed Most reports on MIL-101 derived
to investigate the content of pore-filling from HF-free synthesis revealed BET
species, including water and terephthalic surface area (SBET) of 2800-3900 m2g-1,
acid molecules. The weight losses can be even after conducting vigorous purification
divided to three stages. The weight loss from steps8,14-16 as a result of the presence of the
ambient temperature to below 200 C (5- unreacted or recrystallized terephthalic acid
8%) could be assigned to the loss of water within the pores. Therefore, the extent of
molecules from the larger cages (34 Å coordinating and pore-filling molecules
diameter).2,12 The weight loss from 200 to removed are varied greatly depending on a
280 C could be a result of the release of purification method.
water molecules from the middle-sized Nitrogen adsorption-desorption
cages (29 Å diameter)2,12 along with the isotherms of the as-synthesized MIL101,
removal of residual terephthalic acid within MIL101-vac, and MIL101-N2 and MIL101-
the pores.13 The final weight loss above 280 NF are shown in Figure 5. The isotherms of
C corresponds to the degradation of all samples indicate type-I adsorption
terephthalate linkers, accompanied by the characteristic of microporous materials with
complete collapse of the MOF structure. the secondary uptakes of N2 close to 0.1 and
0.2 relative pressures which are due to the
100 presence of two types of porous windows in
90 Assyn MIL-101
MIL101-vac
the MIL-101 framework structure.17 The
80 MIL101-N2 BET surface areas (SBET) and micropore
70 MIL101-NF
% wieght loss (%)
volumes of the samples are listed in Table 1.

60
60
50
40 50
N2 adsorbed (mmol/g)
30
40
20
10 30
0
100 200 300 400 500 600 700 20
 MIL101-NF
Temperature ( C) MIL101-N2
10
Figure 4. TGA curves of as-synthesized MIL101-vac
Assyn MIL-101
MIL-101(Cr) and purified MIL-101(Cr) 0
0.0 0.2 0.4 0.6 0.8 1.0
samples P/P0
Figure 5. N2 adsorption-desorption
It is found that the second weight isotherms of as-synthesized MIL-101(Cr)
loss, from 200 to 280 C, is observed in the and purified MIL-101(Cr) samples
as-synthesized MIL-101, MIL101-vac and
MIL101-N2 but disappears from MIL101-
Table 1. Textural properties of as-synthesized MIL-101(Cr) and the purified MIL-101(Cr)
samples
Sample SBETa (m2g-1) MAb (m2g-1) EAc (m2g-1) MVd (cm3g-1) ADe (nm)
As-syn MIL101 2,600 2,415 186 0.62 2.81
MIL101-vac 2,807 2,565 242 0.66 2.88
MIL101-N2 3,212 2,970 242 0.86 2.67
MIL101-NF 4,014 3,727 288 1.15 2.62
a
SBET = BET surface area, bMA = t-plot micropore area, cEA = t-plot external surface area, dMV =BJH
adsorption cumulative pore volume, eAD = BJH adsorption average pore diameter
Compared with the as-synthesized the MIL-101 structure has been scarcely
MIL-101, the MIL101 samples that were mentioned, at least to the best of our
purified exhibit higher SBET and total pore knowledge. However, our results show the
volume and they are in the following order, evidence for superior performance of this
MIL101-NF > MIL101-N2 > MIL101-vac > method in terms of the removal of pore-
as-synthesized MIL101. SBET of MIL101- filling molecules as demonstrated by the
vac, and MIL101-N2 and MIL101-NF are greater SBET and pore volume of MIL101-N2
found to be 2807, 3212 and 4014 m2g-1, in comparison with MIL101-vac.
respectively. This indicates that coordinating NH4F solution has been
and pore-filling molecules in as-synthesized continuously reported as the most effective
MIL-101 were removed during purification reagent to remove pore-filling molecules
step. from MIL-101 structure.2,18 Typically, the
The most commonly used method NH4F-treatment is carried out for 12-24 h
for the removal of those pore-filling prior to applying heat and vacuum.
molecules is typically performed by However, for the consistency of the study,
applying heat and vacuum after a MOF herein, MIL-101 sample was treated with
sample is pre-treated with a lower boiling NH4F solution for only 6 h prior to heating
point solvent such as ethanol and methanol. in vacuum. Still, MIL101-NF exhibited the
It was reported that by applying heat and greatest SBET and pore volume even though
vacuum without the pre-treatment step, the the treatment time was reduced confirming
SBET of 2250 m2g-1 were obtained8 whereas the efficiency of NH4F solution as a washing
the MIL-101 sample, which were washed by solution.
DMF several times and then pre-treated with According to TGA analysis and the
ethanol at 80 C for over 24 h, showed the results in Table 1, we can conclude that the
SBET of 3900 m2g-1.15 In this work, we have absence of weight loss during 200 to 280 C
shown that by treating MIL-101 with is necessary to make the pores of MIL-101
methanol for 30 min combining with the more accessible to N2 molecules. As clearly
application of heat and vacuum, the SBET of seen in Figure 4, despite the similar weight
2807 m2g-1 can be reached (in MIL101-vac) loss below 200 C of MIL101-NF and
without using vigorous pre-treatment MIL101-vac, SBET value of MIL101-NF is
condition. This confirms that the purity of 4,014 m2g-1, which is 1.4 times higher than
MIL-101 purified by applying heat and that of MIL101-vac (2,807 m2g-1). This is
vacuum rely significantly on the pre- because of the lack of second step weight
treatment step. And the replacement of pore- loss in MIL101-NF since water and residual
filling solvents with a lower boiling point terephthalic acid molecules within the pores
solvent results in better removal of pore- were removed. It should be noted that
filling molecules, and thus, more purified although the use of NH4F solution are most
MIL-101. effective for MIL-101 purification, the
The application of thermal activation NH4F solution is environmentally unfriendly
combining with flowing gas into MOFs in and additional washing step is also required
order to remove pore-filling molecules from to remove NH4F from the sample.
In cases of MIL101-vac and 2. Hong, D. Y.; Hwang, Y. K.; Serre, C.;
MIL101-N2, their TGA curves show the Férey, G.; Chang, J. S. Adv. Funct.
second weight loss, implying that water and Mater. 2009, 19, 1537-1552.
residual terephthalic acid molecules still 3. Berdonosova, E. A.; Kovalenko, K. A.;
occupied the pores. However, since the loss Polyakova, E. V.; Klyamkin, S. N.;
found in MIL101-N2 were smaller than that Fedin, V. P. J. Phys. Chem. C 2015,
found in MIL101-vac, the greater SBET of 119, 13098-13104.
3212 m2g-1 were observed for MIL101-N2, 4. Kim, J.; Lee, Y. R.; Ahn, W. S. Chem.
higher than 2807 m2g-1 of MIL101-vac. Commun. 2013, 49, 7647-7649.
This suggests that the combination of 5. Leng, K.; Sun, Y.; Li, X.; Sun, S.; Xu, W.
thermal activation and gas flowing performs Cryst. Growth Des. 2016, 16, 1168-1171.
better in removing pore-filling molecules 6. Henschel, A.; Gedrich, K.; Kraehnert,
from MIL-101 networks in comparison with R.; Kaskel, S. Chem. Commun. 2008,
the combination of thermal activation and 4192-4194.
vacuum. 7. Pan, Y.; Yuan, B.; Li, Y.; He, D. Chem.
Commun. 2010, 46, 2280-2282.
4. Conclusion 8. Yang, J; Zhao, Q.; Li, J.; Dong, J.
In summary, we have investigated Microporous Mesoporous Mater. 2010,
the potential of three purification methods in 130, 174-179.
purifying MIL-101(Cr) synthesized in the 9. Jhung, S. H.; Lee, J. H.; Yoon, J. W.;
absence of HF. The results have shown that Serre, C.; Ferey, G.; Chang, J. S. Adv.
treating the MOF with NH4F solution Mater. 2001, 19, 121-124.
followed by applying heat and vacuum was 10. Zhang, Z.; Huang, S.; Xian, S.; Xi, H.;
the most effective method for the removal of Li, Z.; Energy Fuels 2011, 25, 835-842.
water and residual terephthalic acid 11. Kayal, S.; Sun, B.; Chakraborty, A.
molecules, and resulted in MIL-101 with the Energy 2015, 91, 772-781.
highest BET surface area and pore volume. 12. Liu, Q.; Ning, L.; Zheng, S.; Tao, M.; Shi, Y.;
The application of N2 gas to flow into the He, Y. Sci. Rept. 2013, 3, 1-6.
methanol-washed sample under the elevated 13. Ren, J.; Musyoka, N. M.; Langmi, H.
temperature also allowed the removal of W.; Segakweng, T.; North, B. C.;
water and residual terephthalic acid Mathe, M.; Kang, X. Int. J. Hydrog.
molecules even though less effective than Energy 2014, 39, 12018-12023.
the NH4F washing method. The purification 14. Bromberg, L.; Diao, Y.; Wu, H.;
using methanol-treatment combining with Speakman, S. A.; Hatton, T. A. Chem.
heat and vacuum was least effective in Mater. 2012, 24, 1664-1675.
comparison with the other two methods, 15. Liang, Z.; Marshall, M.; Ng, C. H.;
however, an acceptable BET surface area Chaffee, A. L. Energy Fuels 2013, 27,
could still be achieved using this approach. 7612-7618.
16. Khan, N. A.; Kang, I. J.; Seok, H. Y.;
Acknowledgements Jhung, S. H., Chem. Eng. J. 2011, 166,
The work was supported by 1152-1157.
Kasetsart University Research and 17. Férey, G.; Mellot-Draznieks, C.; Serre, C.;
Development Institute and Faculty of Millange, F.; Dutour, J.; Surble, S.; Margiolaki,
Science, Kasetsart University. I. Science 2005, 309, 2040-2042.
18. Hwang, Y. K.; Hong, D.-Y.; Chang, J.-
References S.; Jhung, S. H.; Seo, Y.-K.; Kim, J.;
1. Zhou, H. C.; Long J. R.; Yaghi, O. M. Vimont, A.; Daturi, M.; Serre, C.;
Chem. Rev. 2012, 112, 673-1268. Férey, G. Angew. Chem. Int. Ed. 2008,
47, 4144–4148.
Colourtuning of organic light emitting diodes based on heteroleptic
iridium(III) complexes
Thanyanat Saiboh1, Laongdao Kangkeaw2, Somboon Sahasithiwat2, Rukkiat Jitchati1*
1
2
National Science and Technology Development Agency, Thailand Science Park (TSP),
*E-mail: rukkiat_j@hotmail.com
Abstract:
Four heteroleptic iridium(III) complexes bearing 4,5-diaza-9,9-spirobifluorene
and aryl pyridine ligands were synthesized and characterized for organic light emitting
diodes (OLEDs) coded [Ir(spiro)(ppy)2]PF6 (KM01), [Ir(spiro)(thio)2]PF6 (KM02),
[Ir(spiro)(difluoro)2]PF6 (KM03) and [Ir(spiro)(ppz)2]PF6 (KM04). The photophysical
and electrochemical characteristics were examined. Then, KM01-KM04 were fabricated with
OLEDs structure; ITO/PEDOT:PSS/KM01-KM04:BMIMPF6 (1:1 by mole)/TPBi/LiF/Al.
The results showed that the devices generated blue, green, yellow and orange emission
colours related to band-gap energies. KM01 showed maximum current efficiency at 1.72
cd/A and brightness at 2,027 cd/m2.

Luminescent iridium complexes 2.1 General
have been interested as multifunctional All reactants and solvents were
chromophores for advanced optoelectronic purchased from commercial sources and
applications. The photophysical properties used without further purification
of these phosphors can be tuned by varying unlessotherwise noted. H and 13C NMR
1
the ligandsor additional ancillary ligands. spectra were recorded on a Bruker ARX-300
Moreover, theiridium complexes showed spectrometer.
high stability and luminescence quantum
yields.1-3 Recently, the heteroleptic
iridium(III) complex has been attracted
attention which the structure composed
of two cyclometallating ligands and
a neutral diimine ligand of generic formulae
(C^N)2Ir (N^N)+PF6.4-9 With the additional,
the presence of counter-ions showed
beneficial for device applications.10
In this paper, we reported the tuning
emission colour of OLEDs from four
heteroleptic iridium(III) complexes using
4,5-diaza-9,9′-spirobifluorene as a bulky
N^N ligand. The molecular structures are
shown in Figure 1 which were investigated
their performances as emitter in OLEDs.
Figure 1. The molecular structures of

heteroleptic iridium(III) complexes
Molecular weight was obtained using (89%); 1H NMR (300 MHz, CDCl3) δ 8.35
a Bruker MicrOTOF-Q II. Infrared spectra (d, J = 8.6 Hz, 1H), 7.98 – 7.82 (m, 3H),
were measured on a Perkin Elmer spectrum 7.78 (dd, J = 4.2, 1.5 Hz, 1H), 7.52 – 7.29
RXI spectrometer. UV-visible absorption (m, 4H), 7.23 (dd, J = 13.9, 6.3 Hz, 1H),
and emission spectra were studied with 6.70 (d, J = 7.6 Hz, 1H), 6.65 – 6.47 (m,
Perkin Elmer spectrophotometer (model 1H), 5.82 (dd, J = 8.3, 2.0 Hz, 1H); ATR-
LS50). The synthesis of iridium complexes FTIR (neat) 3087, 1602, 1478, 1248, 1165
were used with similar report.11,12 and 1105 cm-1; MS (ESI) m/z calcd for
2.2 Synthesis and characterization C45H26F4IrN4 (M+-PF6) 891.1723, found
[(4,5-Diaza-9,9′-spirobifluorene-N- 891.1766
N′)-bis-(2-phenylpyridine-C 2 ′,N)iridium [(4,5-Diaza-9,9′-spirobifluorene-N-
(III)] hexafluorophosphate (KM01) was N′)-bis-(1(2′,4′-difluorophenyl) -1H-
obtained as the yellow solid (87%); 1H pyrazole-C6,N2) iridium(III)] hexafluoro
NMR (300 MHz, CDCl3): δ 7.95 (t, J = 6.5 phosphate (KM04) was obtained as a white
Hz, 4H), 7.86 (t, J = 7.8 Hz, 4H), 7.79 – solid (85%); 1H NMR (300 MHz, CDCl3) δ
7.64 (m, 4H), 7.46 (t, J = 7.5 Hz, 2H), 7.35 8.42 (d, J = 2.6 Hz, 1H), 7.97 – 7.80 (m,
(d, J = 10.1 Hz, 4H), 7.32 – 7.19 2H), 7.57 – 7.33 (m, 4H), 7.32 – 7.17 (m, 1
(m, 4H), 7.04 (t, J = 7.4 Hz, 2H), 6.94 H), 6.83 – 6.63 (m, 3H), 5.86 (d, J = 7.6 Hz,
(t, J = 7.3 Hz, 2H), 6.70 (d, J = 7.5 Hz, 2H), 1H); ATR-FTIR (neat) 3166, 1615, 1416,
6.45 (d, J = 7.5 Hz, 2H);13C NMR 1258, 1108 and 1037 cm-1; MS (ESI) m/z
(75 MHz, ) δ 166.8, 161.8, 158.8, 149.4, calcd for C41H24F4IrN6 (M+-PF6) 869.1628,
148.6, 144.2, 143.9, 143.7, 141.9, 141.5, found 869.1676.
138.5, 134.6, 132.0, 130.6, 129.6, 128.8,
128.2, 124.4, 123.8, 123.8, 122.9, 120.8, 3. Results and Discussion
119.5; ATR-FTIR (neat) 3037, 1607, 1478 3.1 Photophysical and electrochemical
and1163 cm-1; MS (ESI) m/z calcd for study
C45H30IrN4 (M+-PF6) 819.2100, found The photophysical and electro-
819.2100. chemical characteristics of the complexes
[(4,5-Diaza-9,9′-spirobifluorene-N- are summarized in Table 1. The results
N′)-bis-(2-thiophen-2′-yl-pyri-dineC 5 ′,N)- showed the tuning emission colors from 488
iridium(III)]hexafluorophosphate (KM02) nm to 586 nm which affected from a varied
was obtained as orange solid (73%); 1H energy gaps (Eg).
NMR (300 MHz, CDCl3) δ 7.95 (dd, J = 3.2 OLED study
13.9, 6.6 Hz, 2H), 7.85 (dd, J = 8.8, 6.7 Hz, The electroluminescences of OLEDs
2H), 7.58 (m, 2H), 7.52 – 7.35 using KM01-KM04 were investigated. The
(m, 8H), 7.22 (m, 2H), 7.10 (dd, J = 15.0, device configuration was designed by
8.3 Hz, 2H), 6.67 (dd, J = 16.7, 7.8 Hz, 2H), ITO/PEDOT:PSS/KM01-KM04:BMIMPF6
6.41 (d, J = 4.7 Hz, 2H); 13C NMR (1:1 by mole)/TPBi/LiF/Al. PEDOT:PSS
(75 MHz, ) δ 164.2, 161.9, 149.8, 149.0, acts as hole injecting layer to facilitate hole
145.8, 143.6, 141.9, 141.3, 138.9, 136.9, transport into the light-emitting layer. The
134.7, 130.6, 129.8, 129.6, 128.8, 128.3, ionic liquid (BMIMPF6) was added to
123.9, 121.0, 120.9, 120.9, 118.3; ATR- reduce the turn-on time of the device.13
FTIR (neat) 2950, 1604, 1473 and 1157 TPBi was used as an electron transporting
cm-1; MS (ESI) m/z calcd for C41H26IrN4S2 layer. LiF and Al act as a buffer layer and a
(M+-PF6) 831.1228, found 831.1243. cathode, respectively. The current density-
[(4,5-Diaza-9,9′-spirobifluorene-N- voltage-luminance (J-V-L) characteristic of
N′)-bis-(2-(2′,4′-difluorophenyl)pyridine- all OLEDs are shown in Figure 2 and
C 6 ′,N)-iridium(III)]hexafluorophosphate summarized in Table 2.
(KM03) was obtained as yellow solid
Table 1. Photophysical and electrochemical characteristics of compounds
λmax,absa λmax,ema Eoxb Egc HOMOd LUMOe
Complex
(nm) (nm) (V) (eV) (eV) (eV)
KM01 418 550 1.33 2.53 -5.77 -3.24
KM02 424 586 1.23 2.43 -5.67 -3.24
KM03 429 505 1.66 2.75 -6.10 -3.35
KM04 411 488 1.72 2.96 -6.16 -3.20
a
Measured in dichloromethane (2×10-5 M)
b
Measured in CH3CN containing 0.1 M n-Bu4NPF6 as a supporting electrolyte at a scan rate of 100 mV/s
c
Estimated from the onset of the absorption spectra (Eg = 1240/onset)
d
Calculated from HOMO = – e (Eox + 4.8eV – Eox(ferocene)); Eox (ferocene) = 0.44 V
e
Calculated from LUMO = HOMO + Eg
The maximum brightness at 2,027,

103, 879 and 27cd/m2 and the maximum
current efficiency at 1.72, 0.07, 1.68, and
0.19 cd/A were observed with KM01-
KM04, respectively. We found that KM01
gave a high brightness to 2,027 cd/m2
compared with others. The CIE coordinates
and their pictures of emission colours for
OLED devices are shown in Figure 3. We Figure 3. CIE coordinates (x,y) and the
found that the heteroleptic iridium (III) pictures of emission colours for OLED
complexes ssuccessfully generated emission devices of KM01-KM04
colours from blue, green, yellow and orange.
These emission colours are related with 4. Conclusion
energy gaps in Table 2. Four heteroleptic iridium(III)
complexes were successfully synthesized
240
KM01 and generated emission colour from 488-586
1000
200
KM02 nm. Then, KM01-KM04 were fabricated to
KM03
KM04 OLEDs based on ITO/PEDOT:PSS/KM01-

)
2
160
Brightness (cd/m
100 KM04:MIMPF6(1:1 by mole)/TPBi/LiF/Al.

120
The results showed that the devices
80
10
generated blue, green, yellow and orange
emission colours related to band-gap
40
energies. This study could be a beneficial for
0
0 2 4 6 8 10 12
1
OLED material design in the future.
Voltage (V)
Figure 2. The J-V-L characteristics of the Acknowledgements

devices of complexes The authors would like to
acknowledge the financial support from
Table 2. Performance summary of OLEDs Ubon Ratchathani University and Graduate
Vturn-ona Bmaxb CEmaxc PEmaxd Institute of Science and Technology
Complex 2
(V) (cd/m ) (cd/A) (lm/W)
(TGIST) for Thanyanat Saiboh (TG-33-24-
KM01 4.7 2027 1.72 0.77
KM02 5.7 130 0.07 0.27 60-071M).
KM03 5.3 879 1.68 0.70
KM04 4.9 27 0.19 0.10 References
a
The voltage at luminance of 1 cd/m2 1. Choy, W. C.; Chan, W. K.; Yuan, Y.
b
Maximum brightness Adv. Mater. 2014, 26, 5368-5399.
c
Maximum current efficiency
d
Maximum power efficiency 2. Slinker, J. D.; Rivnay, J.; Moskowitz, J.
S.; Parker, J. B.; Bernhard, S. A.;
Héctor D.; Malliaras, G. G. J. Mater. 8. Deng, Y.; Liu, S. J.; Fan, Q. L.; Fang,
Chem. 2007, 17, 2976-2988. C.; Zhu, R.; Pu, K. Y.; Yuwen, L. H.;
3. Jou, J. H.; Kumar, S.; Agrawal, A.; Li, Wang, L. H.; Huang, W. Synt. Met.
T. H.; Sahoo, S. J. Mater. Chem. C 2007, 157, 813-822.
2015, 3, 2974-3002. 9. Wongkaew, P.; Mahanitipong, U.;
4. Ma, D.; Tsuboi, T.; Qiu, Y.; Duan, L. Wongsang, N.; Sahasithiwat, S.;
Adv. Mater. 2017, doi: Jitchati, R. J. Surf. Sci. Nanotech. 2014,
10.1002/adma.201603253. 12, 141-144.
5. Liu, B.; Dang, F.; Feng, Z.; Tian, Z.; 10. Kwon, T.; Oh, Y.; Shin, I.; Hong, J.
Zhao, J.; Wu, Y.; Yang, X.; Zhou, G.; Adv. Funct. Mater. 2009, 19, 711-717.
Wu, Z.; Wong, W. Y. J. Mater. Chem. 11. Mothajit, K.; Wongkhan, K.; Jitchati, R.
C 2017, 5, 7871-7883. Appl. Mech. Mater. 2012, 229, 192-196.
6. Mydlak, M.; Bizzarri, C.; Hartmann, D.; 12. Wongsang, N.; Wongkhan, K.;
Sarfert, W.; Schmid, G.; Cola, L. D. Kangkeaw, L.; Sahasithiwat, S.;
Adv. Funct. Mater. 2010, 20, 1812- Jitchati, R. Key Eng. Mater. 2017, 751,
1820. 500-506.
7. Wong, W. Y.; Zhou, G. J.; Yu, X. M.; 13. Costa, R. D.; Pertegás, A.; Ortí, E.;
Kwok, H. S.; Lin, Z. Adv. Funct. Mater. Bolink, H. J. Chem. Mater. 2010, 22,
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A comparative study on glutaraldehyde crosslinking methods of three-
dimensional Thai silk fibroin/gelatin scaffolds for medical application
Wanwarang Wannaborvorn1, Anita Manassakorn2, Rath Itthipanichpong2,
Prin Rojanapongpun2, Visanee Tantisevi2, Sunee Chansangpetch2, Siriporn Damrongsakkul3,
Juthamas Ratanavaraporn1*
1
Biomedical Engineering Program, Faculty of Engineering, Chulalongkorn University, Phaya Thai Road,
2
Department of Ophthalmology, Faculty of Medicine, Chulalongkorn University, Phaya Thai Road,
3
Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Phaya Thai Road,
*E-mail: juthamas.r@chula.ac.th
Abstract:
Thai silk fibroin (SF100), type A gelatin (GA100), and the mixed SF/GA
(SF50/GA50) scaffolds were fabricated by freeze-drying and crosslinked with glutaraldehyde
(GTA) using 2 techniques, blending GTA with the SF/GA solution (blending) and immersion
of SF/GA scaffolds in a GTA solution (immersion). Properties of the scaffolds were
characterized while the concentration of GTA for the crosslinking by 2 methods were
systematically optimized. We showed that GTA could successfully crosslink all scaffolds and
the crosslinking degree depended on crosslinking method, GTA concentration, and scaffold
type. The SF100 scaffolds achieved the high crosslinking degree by using the immersion
technique. GTA blending technique could crosslink the GA100 and SF50/GA50 scaffolds in
a dose-dependent manner. The SF100 and SF50/GA50 scaffolds had significantly higher gel
fraction than the GA100 scaffolds, particularly those crosslinked by immersion technique. In
addition, all GTA-crosslinked scaffolds remained good water swelling ability with the
swelling ratios around 6-14-fold. The SF100 and SF50/GA50 scaffolds crosslinked by
immersion technique seemed to have better swelling ability than the GA100 scaffolds. In
summary, the method of crosslinking and concentration of GTA had crucial influence on
crosslinking degree and properties of the obtained scaffolds. The GTA-crosslinked SF/GA
scaffolds can be used in various medical applications.
1. Introduction Gelatin (GA) is another protein

Natural-derived biomaterials are derived from collagen which is a major
widely applied in various medical component in bone, cartilage and skin. It is
applications, for example, tissue extracted by acidic or basic process to
engineering and drug delivery system. transform the triple helix structure of
Silk is a natural protein that can be collagen into a random coil which is weaker
extracted from spiders, silkworms, mites and more water soluble.3 Furthermore,
and bees. Silk produced from Bombyx mori gelatin has similar biological characteristics
silkworm composed of 2 proteins which are to collagen.4 Therefore, the blend of silk
fibroin (70-80%) and sericin (20-30%).Silk fibroin and gelatin would optimize the
fibroin (SF) is a fibrous protein containing overall properties of materials to be suitable
strong secondary structure in a form of β- for various applications. However,
plated sheet. Therefore, it has good crosslinking is required to stabilize gelatin
mechanical properties and slow degradation and silk fibroin and to reduce the solubility
rate when compared to other natural- of gelatin.
derived polymers.1,2
Glutaraldehyde (GTA) is one of the temperature ( 27 °C) for 1 min. The mixed
most effective agents for crosslinking solution was poured into Teflon mold and
proteins.5,6 This study systematically freeze-dried. After that, the scaffolds were
investigated the chemical and physical immersed in 0.1 M glycine solution under
properties of the freeze-dried Thai SF/GA stirring for 3 h to block the residual
blended scaffolds crosslinked by aldehyde groups of GTA. Following
glutaraldehyde using 2 different techniques, washing three times with deionized water,
(1) blending GTA with the SF/GA solution the scaffolds were freeze-dried again.
(blending) and (2) immersion of SF/GA Immersion: The SF/GA scaffolds
scaffolds in a GTA solution (immersion). were fabricated by freeze-drying. Then, the
The effects of GTA crosslinking technique dried scaffolds were immersed in GTA
and concentration on properties of each solution (0, 0.025, 0.05, and 0.075%)
scaffold type were compared. prepared in the mixed solvent
(acetone/water = 3:1) at room temperature
2. Materials and Methods ( 27 °C) for 12 h. After that, the scaffolds
2.1 Materials were immersed in 0.1 M glycine solution.
Type A gelatin prepared by an acidic Following washing three times with
treatment of porcine skin collagen deionized water, the scaffolds were freeze-
( isoelectric point = 9) was kindly supplied dried again.
by Nitta Gelatin Inc, Japan. Thai silk 2.4 Determination of free amino group
cocoons from Bombyx mori silkworms content and crosslinking degree of the
“hybrid silkworm (J108 X Nanglai strain)”, SF/GA scaffolds
were kindly provided by Queen Sirikit The content of free amino groups of
Sericulture Center, Nakhon Ratchasima SF/ GA scaffolds was analyzed by 2,4,6-
province, Thailand. trinitrobenzene sulphonic acid ( TNBS)
Glutaraldehyde, glycine, sodium assay.8 The absorbance of reacting solution
carbonate (Na2CO3), lithium bromide (LiBr, was measured at 415 nm using a
and other chemicals used were analytical spectrophotometer. The content of free
grade. amino group was calculated from a standard
2. 2 Preparation of regenerated Thai SF curve prepared from β-alanine at known
and GA solution concentrations (n =3). The crosslinking
Regenerated Thai SF solution degree was calculated by the following
was prepared according to the method equation:
previously described by Lerdchai et al.7 The
final concentration of the SF solution was
approximately 6-7wt%. 2.5 Determination of gel fraction of the
SF/GA scaffolds
GA was dissolved in deionized
The gel (crosslinked) fraction of the
water at 40 oC for 2 h to prepare gelatin
GTA-crosslinked SF/ GA scaffolds was
solution at a concentration of 4wt%.
determined by immersion the dried
2.3 Fabrication of the SF/GA scaffolds
scaffolds in deionized water at 37 oC for 24
The SF100, GA100, and
h. After that, the remained scaffolds were
SF50/GA50 solution at 50/50 was prepared
collected, dried overnight at 60 oC, and
at the final concentration of 4%wt. The
weighed. The gel fraction of the scaffolds
SF/GA scaffolds were fabricated by freeze-
was calculated by the following equation:
drying and crosslinked with GTA using 2
different methods, as follow;
Blending: The GTA solution (0,
0.05, 0.1, 0.2, and 0.34%) was added to the where W1 is the weight of the remained
SF/GA solutions and stirred at room scaffolds after immersed in deionized water
for 24 h and WO is the weight of scaffolds slower rate than the solubilization of gelatin.
before immersion (n = 3).
2.6 Swelling test of the SF/GA scaffolds
The swelling ability of the GTA-
crosslinked SF/ GA scaffolds was evaluated
by immersion the dried scaffolds in
phosphate buffer saline solution PBS, pH
7.4) at 37 oC for 24 h. After that, the swollen
scaffolds were weighted after the excess
water was removed out. The swelling ratio
of the SF/GA scaffolds was calculated as
follows:
where WS is the weight of swollen

scaffolds after immersed in PBS for 24 h
and WO is the weight of dried scaffolds
before immersion (n = 3).

3.1 Free amino group content and
crosslinking degree of the SF/GA scaffolds
The amounts of free amino groups
and crosslinking degree of the SF/GA Figure 1. Concentration of free amino
scaffolds crosslinked by GTA using groups of SF/GA scaffolds before and after
blending and immersion techniques are crosslinking with GTA using (1.1) blending
presented in Figures 1 and 2. The non- and (1.2) immersion techniques; * and †
crosslinked GA100 scaffold had more free represent significant difference at p<0. 05
amino groups than SF50/ GA50 and SF100 when compared to SF100 and GA100
scaffolds. The free amino groups seemed to scaffolds, respectively, at the corresponding
decrease with the increasing glutaraldehyde GTA concentration, # represents significant
concentration.9 This effect was obvious in difference at p<0. 05 when compared to the
case of scaffolds crosslinked by blending non-crosslinked scaffold (GTA 0%) at the
technique (Figure 1.1). The crosslinking corresponding type of scaffold
degrees of the SF50/GA50 and GA100
scaffolds were up to 60-80% for those 3.2 Gel fraction of the SF/GA scaffolds
crosslinked with 0.2 and 0.34% GTA The gel or crosslinked fractions of
(Figure 2.1). On the other hand, the the SF/GA scaffolds are presented in Figure
immersion crosslinking could reduce the 3. The gel fractions of the non-crosslinked
free amino groups of SF100 scaffolds GA100, SF50/GA50, and SF100 scaffolds
significantly (Figure 1.2) and achieved the were around 10%, 30%, and 60%,
respectively. Silk fibroin has more stable β-
highest crosslinking degree (60%) while it
plated sheet structure1 than the random coil
did not much effective for the GA100 and
of gelatin,10 then SF100 scaffolds was less
SF50/GA50 scaffolds (Figure 2.2). This
water-soluble than GA100 scaffolds. For the
might be explained that, in the immersion
blending technique, all types of scaffolds
technique, gelatin component of GA100 and
had more than 80% gel fraction,
SF50/GA50 was solubilized in the
irrespectively of the GTA concentration.
crosslinking solution before it was
crosslinked. In the other words, the
crosslinking reaction may be occurred at
Figure 2. Crosslinking degree of SF/GA Figure 3. Gel fraction of SF/GA scaffolds
scaffolds after crosslinking with GTA using before and after crosslinking with GTA
(2.1) blending and (2.2) immersion using (3.1) blending and (3.2) immersion
techniques; * and † represent significant techniques; * and † represent significant
difference at p<0.05 when compared to difference at p<0. 05 when compared to
SF100 and GA100 scaffolds, respectively, at SF100 and GA100 scaffolds, respectively, at
the corresponding GTA concentration, # the corresponding GTA concentration, #
represents significant difference at p<0.05 represents significant difference at p<0. 05
when compared to the scaffold crosslinked when compared to the non-crosslinked
with 0.05% GTA at the corresponding type scaffold (GTA 0%) at the corresponding
of scaffold type of scaffold
This meant that the crosslinking of For the blending technique, the swelling
GTA by blending technique was successful ratios of all scaffolds were up to 9-15,
with all scaffolds. For the immersion irrespectively of the GTA concentration.
technique, the SF100 scaffolds showed the SF100 scaffolds showed slightly higher
highest gel fraction (>90%) while GA100 swelling ratio than the GA100 and
scaffolds showed the lowest gel fraction (60- SF50/GA50 scaffolds. The swelling ratio of
80%) due to the high water solubility of the scaffolds crosslinked with GTA by
gelatin component as discussed previously. immersion technique were in the range of 6-
The results of gel fraction seemed not to 15 and also not related to the GTA
correspond with the concentration of GTA concentration. In overall, all GTA
used for both crosslinking methods. crosslinked SF/GA scaffolds remained good
3.3 Swelling ability of the SF/GA scaffolds water swelling ability.
Figure 4 shows the swelling ratios of
the SF/GA scaffolds crosslinked with GTA
using blending and immersion techniques.
GA and SF/GA scaffolds and could
crosslink the scaffolds in dose-dependent
manner. The GTA crosslinking can
optimize properties of the SF/GA scaffolds
to suit their applications.
Acknowledgements
We acknowledge the financial
support from Graduate School Thesis Grant,
Chulalongkorn University.
References
1. Kundu, B.; Rajkhowa, R.; Kundu, S. C.;
Wang, X. Adv. Drug Deliv. Rev. 2013,
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3. Fonseca, A.; Ferreira, P.; Cordeiro, R.;
Mendonça, P.; Góis, J.; Gil, H.; Coelho,
J. New Strategies to Advance
Pre/Diabetes Care: Integrative
Figure 4. Swelling ratio of SF/GA scaffolds Approach by PPPM.; Mozaffari M.S.
after crosslinking with GTA using (4.1) Ed. Springer: Dordrecht, 2013; pp 399-
blending and (4.2) immersion techniques; * 455
and † represent significant difference at 4. Ayre, A. P.; Pawar, H.; Khutle, N. M.;
p<0.05 when compared to SF100 and Lalitha, K. G. RJPT. 2014, 5, 2809-
GA100 scaffolds, respectively, at the 2823.
corresponding GTA concentration, # 5. Migneault, I.; Dartiguenave, C.;
represents significant difference at p<0.05 Bertrand, M.J.; Waldron, K. C. Adv.
when compared to the scaffold crosslinked Drug Deliv. Rev. 2004, 37 (5), 790-796,
with 0.05% GTA at the corresponding type 798-802.
of scaffold 6. Habeeb, A. F. S. A.; Hiramoto, R. Arch.
Biochem. Biophys. 1968, 126 (1), 16-26.
4. Conclusion 7. Lerdchai, K.; Kitsongsermthon, J.;
The SF/GA scaffolds could be Ratanavaraporn, J.; Kanokpanont, S.;
successfully crosslinked by GTA using Damrongsakkul, S. J. Pharm. Sci. 2016,
either blending or immersion technique. 105 (1), 221–230.
We showed that crosslinking technique and 8. Bubnis, W. A.; Ofner, C. M. Anal.
GTA concentration influenced on the Biochem. 1992, 207, 129-133.
crosslinking degree and properties of each 9. H. H. Olde Damink, L.; J. Dijkstra, P.;
type of SF/GA scaffolds obtained. The J. A. Van Luyn, M.; B. Van Wachem,
immersion technique was effective for
P.; Nieuwenhuis, P.; Feijen, J. J. Mater.
crosslinking of SF100 scaffolds while the
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10. Karim, A. A.; Bhat, R. Food Hydrocoll.
2009, 23 (3), 563-576.
A simple and cost-effective bimetallic cobalt/iron biosensing catalyst on
activated carbon support for non-enzymatic glucose detection in
human urine
Metini Janyasupab1*, Chen-Wei Liu2, Chamras Promptmas3, Werasak Surareungchai4
1
Faculty of Engineering, King Mongkut’s Institute of Technology Latkrabang, Latkrabang,
2
Industrial Technology Research Institute, Hsinchu 31057, Taiwan
3
Department of Biomedical Engineering, Mahidol University, Nakhon Pathom 73170, Thailand
4
School of Bioresources and Technology, King Mongkut’s University Thonburi,
Bang Khun Thian, Bangkok 10150, Thailand
*E-mail: metini@kmitl.ac.th
Abstract:
Non-enzymatic glucose detection in urine can potentially provide economic viability
and great impacts toward healthcare and at-home diagnosis technologies. In our current
study, a simple, robust, and cost-effective bimetallic cobalt (Co) and iron (Fe) catalyst
prepared via a self-assembly based oleylamine/oleic acid reduction supported on activated
carbon was investigated. By modifying electronic structures of Co/Fe nanoparticles, the
bimetallic catalyst system exhibited an intrinsic electrocatalytic property toward glucose
detection without the use of enzymes in synthetic urine, standard negative control (surine)
and human urine samples on a glassy carbon rotating disk electrode. The kinetic studies show
that the Co-Fe catalyst on activated carbon support can provide a trend in concentrations from
0 to 3 milimolar glucose in synthetic urine and demonstrate a direct electron transfer property
of electrocatalytic behavior in human urine samples by differential pulse voltammetry
without any electron facilitators, thereby leading to a promising result for future smart toilet
applications.
1. Introduction reactions and many non-enzymatic

A search of non-enzymatic key reactions.7 However, only few studies
element to provide a sustainable reported the use of bimetal approach to
electrocatalytic performance is economically detect glucose in non-enzymatic detection
viable for a cutting-edge sensing technology without any electron facilitator.
platform. The essence of this component Herein, this study demonstrated a
will lead to a disruptive integrated platform simple and cost-effected preparation of self-
of smart technology to monitor a self- assembled Co/Fe on carbon power
diagnosis and individual health supported via oleylamine and oleic acid
management. Many functional reduction to enhance an electrocatalytic
nanomaterials have been investigated to activity of enzyme-free glucose detection in
non-enzymatic detection especially for synthetic urine, surine, and human urine
human samples for diabetes management.1,2 sample. The as-prepared Co/Fe catalyst was
Among these materials, cobalt (Co) and iron loaded 10%wt to detect 3 µM glucose of
(Fe) have shown profound effects of non- direct electron transfer in cyclic
precious metals toward low cost, facile voltammetry and differential voltammetry,
preparation,3,4 controllable size and showing a unique property for future sensing
morphology5,6 and excellent electrochemical diagnosis tools.
performance to enhance catalytic behaviors,
comparable to those of conventional
precious metals in oxygen reduction
2. Materials and Methods 0.05 μm alumina powder. The modified
2.1 Chemicals electrode was prepared by dissolving 4 mg
Activated carbon powder, Vulcan of Co/Fe catalyst powder into 100 µL
XC-72R was purchased from Fuel Cell isopropanol. Then, the as-prepared mixture
Earth LLC (USA). All other chemicals, Iron was sonicated for 30 min. Finally, 4 µL of
(III) Acetylacetonate (>99.9%), cobalt(II) the as-prepared Co/Fe ink was dropped onto
acetylacetonate (>99.0%), 1-ocadecene the GCE, followed by 4 µL of 1% Nafion
(>95.0% GC), olelamine (70%), (oleic acid 117 solution.
(90%), trioctyl phosphine (90%), Nafion 2.4 Material characterization
117 solution 5% mixture and glucose were Transmission electron microscopy
purchased from Sigma Aldrich and used as (TEM) images and Energy dispersive X-ray
such with no further purification. spectroscopy (EDS) were performed by a
2.2 Synthesis of Co/Fe catalyst JEM-200CX operated at 160 kV.
Bimetallic Co/Fe nanoparticle 2.5 Electrochemical characterization
catalyst was synthesized via self-assembled Rotating disk electrode (Pine
oleylamine reduction with modified metal Research Instrument, USA) was used for
ratio and surfactants as follow: 0.61 mmol of kinetic studies to analyze electrocatalytic
Co precursor was initially injected in the performance of Co/Fe/Vulcan XC72R on
surfactant mixture of 1-ocadecene (4 mL), glassy carbon electrode with a well-defined
oleylamine (8 mL), oleic acid (4 mL), and convection and controlled charge separation
trioctyl phosphine (2 mL) at room factors. The reference electrode and the
temperature. Subsequently, the mixture was counter electrode were Ag/AgCl and Pt coil,
stirred and heated at 110 C under N2 for 1 respectively.
h. Immediately, the temperature of the
mixture was raised to 230 C for 20 min and 3. Results and Discussion
cool down to room temperature with 3.1 Morphological and elemental analysis
isopropanol addition for stabilization. The Figure 1 shows the morphology and
Co nanoparticle was centrifuged at a high size of bimetallic Co/Fe nanoparticles. The
speed of 10,000 rpm and washed with cluster of bimetallic nanoparticles shows the
hexane and isopropanol at least 3-4 times. preferential aggregation of self-assembly
In the process of bimetal formation, with some contamination of surfactants. At a
0.61 mmol of Fe acetate precursor was higher magnification of TEM image as
mixed with the same ratio volume of the shown in Figure 2, the diameter of Co-Fe
surfactant mixture and undergone N2 flow at nanoparticle on carbon supported was
110 C for 1 hour. Then, 0.2 mmol of Co estimated to 50-90 nm. Both figures
nanoparticle was injected and raised the revealed that each cluster was not comprised
temperature to 230 C for 20 min. Finally, of a single crystal but instead interconnected
the bimetal nanoparticle was cool down at with small Co/Fe nanoparticles.
room temperature with addition of Furthermore, elemental analysis was also
isopropanol and followed by the same performed by EDS, sampling for various
washing procedure and centrifugation as areas in Figure 2. As shown in Figure 3, the
mentioned. Finally, 10%wt of metal solution EDS spectra of cobalt and iron confirmed
was loaded onto Vulcan XC72 carbon the co-existing of bimetal atoms distributed
powder. The mixture was sonicated, stirred within the interior of the same nanoparticles
for 3 h, and dry in an oven at 80 C (denoted “Sample 2” in Figure 2). In
overnight. addition, the EDS indicates the elemental
2.4 Preparation of modified GCE composition of carbon, oxygen, and copper,
Prior to each trial, a glassy carbon corresponding to the Vulcan XC carbon
electrode (GCE), diameter of 5 mm, was power, oxide formation of iron metal and the
clean with ethanol, and then polished with copper grid substrate, respectively.
Figure 1. TEM image of self-assembly Figure 2. TEM image of 10%wt Co/Fe
Co/Fe nanoparticle nanoparticle catalyst on activated carbon
support, labelled with various area of EDS
sampling analysis
3.2 Electrochemical and catalytic performances The flow profile of urine was convex
The bimetallic Co/Fe catalysts was in upward directed to the electrode surface.
loaded on GCE rotating disk electrode. At a Therefore, the overall current of
high speed of 900 rpm, the controlled electrochemical studies can be primarily
convection and charge separation were well- represented by catalytic behaviors of Co/Fe.
define. Hence, the overall current was In this study, cyclic voltammetry and
primarily contributed by kinetically surface differential pulse voltammetry were carried
reaction of electron transfer. out on RDE with a series of glucose
injection from 0 to 3 mM with 0.25 mM
increment.
Figure 3. Energy dispersive X-ray spectrum of Co/Fe nanoparticles on Vulcan XC power
As shown in Figure 4, each glucose
injection of 0, 0.5, 1, 1.8, 2, 2.5, and 3 mM
in synthetic urine indicated a decreasing
current of cyclic voltammogram at 50 mV/s
of scan rate, implying an ability to detect
glucose in non-enzymatic detection.
Figure 5. Differential pulse voltammogram

of 0-3 mM glucose concentration in
synthetic urine on Co/Fe activated carbon
supported on glassy carbon electrode, 900
rpm, 50 mV/s scan rate
Figure 4. Cyclic voltammogram of 0-3 mM

glucose concentration in synthetic urine on
Co/Fe activated carbon supported on glassy
carbon electrode, 900 rpm, 50 mV/s scan
rate
Similar trend of differential pulse

voltammogram in the addition of glucose
concentration of 0 to 3 mM with 0.25 mM Figure 6. Calibrated plot of two different
increment on RDE 900 rpm can be observed linear region, cut-off line at 1.0 mM glucose
in Figure 5, leading to a higher resolution of
catalytic current toward non-enzymatic
reaction on Co/Fe/C. This finding suggested
that the as prepared bimetallic catalyst can
exhibit a good separation with decreasing
current as increasing glucose concentration.
The relationship of catalytic current and
glucose concentration can be calculated in a
calibration plot shown in Figure 6. With the
estimated trend of the calibration plot, the
linear range can be justified in two region of
(i) at lower than 1.0 mM and (ii) at higher Figure 7. Differential pulse voltammo-
than 1.0 mM. The sensitivities of both gram of CoFe/C in addition of glucose
regions can be calculated to -9.19 µA/mM from 0 to 3 mM with 0.25 mM increment
(R2=0.94) and -2.27 µA/mM (R2 = 0.94), in human urine sample (repeated 3 times)
respectively. For our best knowledge, we
report for the first time of catalytic behavior These values of both sensitivities
for non-enzymatic reaction without the use may not as high as those with enzyme or
of any electron facilitator.
NaOH promotor. However, the interesting Acknowledgements
property of CoFe can be initiated to improve This research study was supported by
the performance by further synthesis The Thailand Research Funding (grant
methods and tailored structure in bimetallic number MRG 5980159) and Faculty of
catalysts. Furthermore, a sample of human Engineering, King Mongkut’s Institute of
urine sample obtained at Ladkrabang Technology Ladkrabang (grant number
Hospital, Bangkok, Thailand, was 2560-01-01006)
demonstrated an ability of Co/Fe/C catalyst
in non-enzymatic glucose detection from 0 References
to 3 mM injection on RDE in Figure 7. The 1. Zhang, Z.; Chen, Z.; Cheng, F.; Zhang,
behavior of catalytic currents are similar to Y.; Chen, L. Biosens. Bioelectronics
that of in synthetic urine with an inverse 2017, 89, 932-936.
relationship of current and glucose 2. Balamurugan, J.; Thanh, T. D.;
concentration. However, the distinct peak of Karthikeyan, G.; Kim, N. H.; Lee, J. H.
glucose at 0.87 V vs Ag/AgCl was Biosen. Bioelectronics 2017, 89, 970-
submerged and resulted to a relatively linear 977.
line, clear shown from 0.6 to 1.0 V vs 3. Bu, W.; Chen, Z.; Chen, F.; Shi, J. J.
Ag/AgCl. This different shape of Phys. Chem. C 2009, 113 (28), 12176-
voltammogram could suggest that there are 12185.
many other oxidizing species in real human 4. Mourdikoudis, S.; Liz-Marzán, L. M.
urine added into the total peak at 0.87 V. As Chem. Mater. 2013, 25 (9), 1465-1476.
a result, the distinctive peak was 5. Dong, A.; Chen, J.; Vora, P. M.;
disappeared. More samples are needed to Kikkawa, J. M.; Murray, C. B. Nature
verify in this behavior. 2010, 466, 474.
6. Sayed, F. N.; Polshettiwar, V. Sci. Rep.
4. Conclusion 2015, 5, 9733.
The as-prepared bimetallic CoFe/C 7. Xu, Y.; Ruban, A. V.; Mavrikakis, M. J.
catalyst can demonstrate an ability to detect Am. Chem. Soc. 2004, 126 (14), 4717-
glucose in synthetic urine and human urine 4725.
from 0 to 3 mM, leading to a promising
result to develop in practical self-monitoring
and diabetic management applications.
The effect of phosphoric acid concentration in crude oil pretreatment
process on the phosphorus content of crude palm oil
Boonruen Sunpetch*, Oangkana Champanon
Department of Science, Faculty of Science and Technology, Rajamangala University of Technology Srivijaya,
Nakhon Sri Thammarat Saiyai Campus, Nakhon Sri Thammarat 80110, Thailand
*E-mail: b_sunpetch@hotmail.com
Abstract:
Non-triglyceride contaminant in vegetable oil is the big problem in oil refining
process, they have to remove from the crude oil in the first step. Phospholipid, the key
contaminant in crude palm oil (CPO), can be determined in terms of phosphorus (P) content
and usually treated with phosphoric acid. Even though the phosphoric acid is get rid
phospholipid in CPO but also increased the P in palm oil. However, the optimal concentration
of phosphoric in oil degumming process is still unknown. The objective of this work is to
elucidate the effect of phosphoric acid concentration in CPO preparation. P content of 352
ppm was found in crude palm oil with UV-Vis spectrometry. The amount of content changed
in the mixture of 0-1%wt phosphoric acid in CPO for 30 minutes at the temperature range of
855 ˚C. At the proper degumming process, P content decreased by over 70%. It was found
that the higher the concentration of hot water was used, the lower P content in CPO became.
However, the P content increased when treated with the concentration of phosphoric
concentration higher than 0.25%wt. The lower level of P content was also found when the
combination the degumming process of water degumming and acid degumming together. The
optimum condition for CPO degumming is degumming with 1%wt hot water following with
phosphoric acid for 15-30 minutes at 805 ˚C. The results is also point out that, using higher
phosphoric concentration in CPO degumming process will increase the P content in palm oil
product.
1. Introduction Acid degumming or enzymatic degumming

Both animal and vegetable oil are have been applied to remove the NHPL.
contaminated with a number of impurities. There are many types of degumming process
Most of them are effect on oil further such as dry degumming, water degumming,
processing and oil product qualities. Gums acid degumming, enzymatic degumming,
are phospholipid contaminants which one of EDTA degumming, and membrane
1-4
fatty acid in triglyceride are substituted by a degumming processes. Steam and
phosphatide such as phosphotidyl choline, phosphoric acid are normally use as
phosphotidyl inositol, phosphotidyl degumming agent in vegetable oil industry.
ethanoamine and phosphatidic acid etc. Crude palm oil (CPO) is one of the
Phospholipid which contains phosphotidyl most important vegetable oil in world
ethanoamine is patial hydratable and market.5 Flying oil, consuming product and
phosphatidic acid is non-hydratable fuel application are the crucial final products
phospholipid (NHPL), while the other two of palm oil. The contamination of dirt and
are completely hydratable phospholipid minor component have to remove from CPO
(HPL).1,2 HPL can absorb water and form in the first place because there are disturb in
insoluble heavy particle, prompt to settle out many CPO refining process. So that,
from oil. Hydratable gums can eliminated by degumming process is the important refining
adding hot water into the oil at temperature step in CPO pretreatment before using as
of 70-80 ºC and stirred for 10-15 minutes. food and/or raw material for oleo chemical.
The main phospholipid composition of palm For synergetic effect was test by
oil are phosphatidyl choline, phosphotidyl eliminated the remaining phospholipid with
ethanoamine, phosphatidyl inositol, different chemical. The oil that degummed
phosphatidyl glycerol, phosphatidic acid, with hot water was further treatment with
etc.6-9 Phosphoric acid is normally used for phosphoric. While the oil that degummed
any type of vegetable oil, including palm oil, with phosphoric acid was following with hot
because of lower unit cost and easier water treatment for specific concentration in
handling. specific time. The degummed oil then heat
The separated gums are also purified at 110 ˚C to remove all water contamination
and sold as value added product for before further characterization.
vegetable oil manufacture. However, the 2.3 Palm oil characterizations
effect of degumming agent amount on Degummed crude palm oil samples
degumming process are not available. This were took the free fatty acid content
studied was determine the effect of (%FFA) by titration with potassium
degumming agent amount on phospholipid hydroxide solution. The exact concentration
content in CPO. The change of phosphorus of KOH are standardized by KHP. Gums in
content after degum with hot water (for form of phosphorous content were
HPL) and phosphoric acid (for NHPL) were determined by following Ethiopian Standard
studied in this work. Moreover, free fatty (ES ISO 10540-1:2012).10,11 The phosphorus
acid (FFA) of crude palm oil is effect on the content in oil sample is specified by
production cost and concerning with carbonized oil with magnesium
purification process. So that, the %FFA of hydroxycarbonate at 350 ˚C for 2 h then
degummed crude palm oil was also studied heated to 550 ˚C and held at this
in this work. temperature for 2 h. Dissolve the residue
from ashing procedure in HCl acid and
2. Materials and Methods neutralize the content by adding NaOH
2.1 Materials solution. Add sulfate/molybdate reagent and
Crude palm oil (CPO) was received mix before keep in a dark place 20 min. Fill
from Kanjanadit palm oil industry. sodium acetate solution until the volume of
Magnesium hydroxyl carbonat, hydrochloric the mixture reach 15 mL. Measure the
acid, sodium hydroxide, sulfuric acid, p- absorbance of the solution against the blank
methyl-aminophenol sulfate, sodium sulfite at 720 nm. Phosphorus contents were
heptahydrate, sodium metabisulfite, calculated using absorbance and calibration
ammonium molypdate tetra hydrate sodium factor which prepare from standard
acetate tri-hydrate, and potassium phosphate solution.
dihydrogen phosphate were used for
determination of phosphorous content. 3. Results and Discussion
2.2 Crude palm oil degumming CPO degumming with different
Heating 100 g of CPO to 85±5 ºC chemical and different concentration were
and hold at this temperature for heat tested. The degummed oil properties are as
equilibrium at least 15 min. Added 0.00- following.
2.00%v/w of degumming agent into the hot 3.1 Effect of phosphoric concentration on
oil and stirred with magnetic bar for 15, 30, phosphorus content of degummed oil
45, and 60 min. Insoluble gums is separated Gum content of CPO in the form of
from oil product by specific density in a phosphorus concentration were determined
separating funnel. Let gums settling down for CPO and degummed oil. Phosphorus
for 30 min and then drained it out. Washed concentration in CPO in this study are vary
the remaining oil with warm water (60 ºC) from 84 mg/kg to be 352 mg/kg. Phosphorus
for 4 times. Finally, heat the received oil concentrations were concern the
again to eliminate the contaminated water. phospholipid in oil sample which decreased
in degummed oil. The phosphorus phosphorus from acid contaminated in
concentration of palm oil degumming with degummed oil made phosphorus
phosphoric acid in different concentration concentration increase with phosphoric
for 15-45 min were shown in Table 1. The concentration. The phosphorus
phosphorus content in crude palm oil concentration of oil that degummed longer
without any degumming process was 352 than 45 min are almost constant and increase
mg/kg. It was found that, the phosphorus with the phosphoric acid concentration. The
content of palm oil heating at 85±5 ºC for results suggest that, gums in CPO are mainly
various time without acid were lower than HPL and using phosphoric acid showed
CPO and decreasing with reaction time. reverse effect on phosphorus concentration
Gum was removed in form of black solid in crude palm oil. So that, phosphoric acid
settle out from oil like Figure 1. are not recommended for degumming in
CPO.
Table 1. Phosphorus content in crude palm 3.2 Effect of hot water concentration on
oil degummed with phosphoric acid phosphorus content of degummed oil
Phosphoric Phosphorus content in The effect of water in CPO
concentration degummed oil (mg/kg) degumming were determined by using 1.00-
(%) 15 30 45 60 2.00%wt of hot water addition into oil at
min min min min 85±5 ºC. The change of phosphorus content
0.00 258 263 195 178
0.25 256 250 236 263
showed in Table 2. Addition of hot water
0.50 288 304 267 285 can decreased phosphorus content in CPO.
0.75 290 327 277 295 These results are also supported the
1.00 344 328 325 304 hypothesis that phospholipid in CPO is
HPL.
Table 2. Phosphorus content in crude palm

oil degummed with hot water
Water Phosphorus content in
concentration degummed oil (mg/kg)
(%) 30 min 45 min 60 min
0.00 219 253 201
1.00 206 227 148
2.00 178 171 144
Figure 1. Black gummed removed from CPO by 3.3 The free fatty acid content of the
using degumming agent degummed oil
Phosphoric is the most often use
Phosphoric acid addition can reduce chemical in vegetable oil industry because
the phosphorus content of CPO. However, of the efficiency and price. However,
the efficiency of phosphoric degumming are increasing phosphoric concentration in
decrease when increasing the acid degumming process is increase FFA content
concentration. Since gums are phospholipid in crude palm oil. Table 3 shows the
which hydratable and nonhydratable. The increasing of FFA content of CPO which
settle gummed after heat without phosphoric degummed with higher phosphoric acid
acid should be hydratable gummed reacted concentration at 15 min. Since, the main
with moisture contained in CPO. When gums composition in CPO are HPL which
using phosphoric acid, NHPL were also can eliminated by water. Using higher hot
remove but the total phosphorus content in water concentration can removed more gum.
degummed sample is higher than the sample However, water can also effect other
without phosphoric. May be the accesses properties of degummed palm oil. One of
the most important characteristic is FFA suggest that degum with 1.00%wt hot water
content. FFA content of water degummed following with 0.25% phosphoric acid is the
palm oil showing in Table 4. The CPO best condition for CPO degumming process.
sample which degummed with hot water Table 5. Phosphorus content in crude palm
also contains high FFA. The FFA properties oil degummed with 1.00%wt hot water and
of degummed oil have to be improve before following with phosphoric acid (PA)
use in another process.
Phosphorus content in
Phosphoric degummed oil (mg/kg)
Table 3. FFA of crude palm oil after
degummed with phosphoric acid H2O 15 min H2O 30 min
Concentration PA PA PA PA
Phosphoric FFA of degumming at 15 30 15 30
concentration different time (%) (%) Min min min min
(%) 15 min 30 min 45 min 0.00 173 90 116 111
0.00 4.82 4.52 - 0.25 149 134 101 97
0.25 4.80 5.26 - 0.50 191 110 115 197
0.50 4.80 5.04 - 0.75 218 100 123 139
0.75 4.97 5.23 - 1.00 194 141 107 146
1.00 4.94 5.30 5.31
Table 6. Phosphorus content in crude palm
Table 4. FFA of crude palm oil after oil degummed with phosphoric acid (PA)
degummed with hot water and following with 1%wt hot water
Water FFA of degumming at Phosphorus content in
concentration different time (%) degummed oil (mg/kg)
(%) 30 min 45 min 60 min Phosphoric phosphoric phosphoric
Concentration 15 min 30 min
0.00 5.03 5.08 5.01 (%) H2O H2O H2O H2O
1.00 5.41 5.41 5.16 15 30 15 30
2.00 5.12 5.62 5.09 min min min min
0.00 182 170 160 171
3.4 Effect of combination of hot water and 0.25 242 180 168 222
phosphoric acid on phosphorus content of
0.50 176 215 183 224
degummed oil
Phospholipid composition in CPO 0.75 205 220 240 184
are HPL and NHPL which can eliminated by 1.00 175 225 218 243
water and phosphoric acid. The effect of
chemical sequence on phosphorus content 4. Conclusion
also studied. Phosphorus concentration in oil Using phosphoric acid as
degummed with hot water 1%wt following degumming agent for crude palm oil can
with phosphoric acid (PA) showing in Table improved only small value of phosphorus
5. The results show that degumming with concentration. Hot water is better
hot water following with PA can eliminate degumming agent than phosphoric acid
grater phospholipid than using single because can eliminated more phospholipid
degumming agent. Since the phospholipid in than using phosphoric acid. Since
CPO compose of HPL which and react with phospholipid in CPO are HPL and NHPL,
water and NHPL which can react with PA. the combination of water and acid is
The phosphorus content in degummed oil recommend for CPO degumming process.
with phosphoric acid following with hot The synergy of hot water and phosphoric
water in Table 6 also better than in oil acid degumming show the most phosphorus
sample which degummed with phosphoric decreasing in palm oil. The oil from hot
acid in Table 1. The phosphorus results water degummed also have high FFA
content like phosphoric acid degumming. Araujo, E. C. E. Renew. Energy 2014,
The best degumming process in this studies 71, 495-501.
is degumming with 1.00%wt hot water 5. Office of global analysis, foreign
following with 0.25%wt phosphoric acid. agricultural service/USDA, January,
2017.
Acknowledgements 6. Gold, I. L.; Oseni, N. T.; Imosi, O. B.
Faculty of Science and Technology, Afr. J. Biochem. Res. 2016, 10 (4), 25-
Rajamangala University of Technology 29.
Srivijaya 7. Gomes, M. C. S.; Arroyo, P. A.;
Pereira, N. C. J. Membrane Sci. 2011,
1. Panpipat, W.; Chaijan, M. Polar Lipids: 8. Szydlowska-Czerniak, A.; Szlyk, E.
Biology, Chemistry, and Technology, Food Chem. 2003, 81, 613-619.
Elsevier, 2015. 9. Tyagi, K.; Ansari, M. A.; Tyagi, S.;
2. Paisan, S.; Chetpattananondh, P.; Tyagi, A. Adv. App. Sci. Res. 2012, 3,
Chongkhong, S. J. Env. Chem. Eng., 1435-1439.
2017, 5, 5115-5123. 10. Ethiopian standard ES ISO 10540-
3. Yang, B.; Zhou, R.; Yang, J. G.; Wang, 1:2012, 1st ed. Animal and vegetable
Y. H.; Wang, W. F. J. Am. Oil Chem. fats and oils determination of
Soc. 2008, 85, 421-425. phosphorus content. Part1: Colorimetric
4. Araujo, F. D.; Araujo, I. C.; Costa, I. C. method. Ethiopian standards agency.
G.; Moura, C. V. R.; Chaves, M. H.; 11. Zufarov, O.; Schmidt, S.; Sekretár, S.
Acta Chimica Slov. 2008, 1, 321 – 328.
Preservation of nicotine by nanoencapsulation technique using
β-cyclodextrin
Nathasak Sinlikhitkul1, Harid Pataveepaisit1, Waritsara Noodang1, Arayaporn Krissawong1,
Suriyawut Kulatee1, Anotai Suksangpanomrung2, Luckhana Lawtrakul1, Pisanu Toochinda1*
1
2
Department of Mechanical Engineering, Academic Division, Chulachomklao Royal Military Academy,
Nakhon Nayok 26001, Thailand
*E-mail: pisanu@siit.tu.ac.th
Abstract:
Nicotine, an alkaloid compound, can be extracted from waste tobacco materials found
in cigarette industry. Nicotine can be used in electronic cigarette liquid, agricultural
insecticide, and nicotine replacement therapy. However, nicotine is highly volatile, easily
oxidized, and hygroscopic. These properties limit the use of nicotine as products at normal
storage condition. Therefore, the prevention of nicotine from volatilization and degradation is
required. In this study, the nanoencapsulation technique using β-cyclodextrin (βCD) was used
to preserve nicotine. Nicotine-βCD inclusion complex was formed by the co-precipitation
method. The encapsulation efficiency, determined by GC-FID, can be achieved up to 73.97%
and the inclusion complex was also characterized by FTIR. The preservation capability of
nicotine in the form of inclusion complex was evaluated at different storage temperatures and
compared to that of nicotine in form of free compound. The durations for completed loss of
nicotine in form of free compound at 25 °C and 50 °C were within 7 days and 6 hours,
respectively. The loss of nicotine in form of inclusion complex reduced and the complex can
preserve up to 80% of nicotine in 28 days at 25 °C and 50 °C. The results showed that the
stability of nicotine was greatly improved by using the nanoencapsulation technique.
1. Introduction outer surface of CD is hydrophilic and inner

Nicotine is an alkaloid compound cavity of CD is hydrophobic.10,11 Therefore,
composed of pyrrolidine and pyridine rings CD can be prepared in aqueous solution. It
mainly found in tobacco (Nicotiana also creates strong interactions between
tabacum) leaves.1–3 Nicotine can be used in hydrophobic parts of compounds and the
electronic cigarette liquid, agricultural inner cavity of CD to form of an inclusion
insecticide application, and nicotine complex.12 CD is widely used in food,
replacement therapy for smoking pharmaceutical, and cosmetic industries to
cessation.4,5 However, nicotine is highly stabilize active compounds.13 βCD was
volatile, hygroscopic, and easily oxidized in selected due to its inexpensive price and low
the presence of air and light. These solubility to provide an ease for inclusion
properties cause the loss of nicotine from the complex recovery from solution.11
products at the normal storage conditions.6–9 In this study, the prediction of
Therefore, a preservation of nicotine to nicotine-βCD inclusion complex formation
prolong the product shelf life is required. was initially evaluated by computational
The preservation of nicotine can be simulation. The nicotine-βCD inclusion
performed by using nanoencapsulation complex was then prepared by using the co-
technique by forming an inclusion complex precipitation method at the conditions
between nicotine and cyclodextrin (CD). suggested by simulation. The preservation of
CD is a non-toxic cyclic oligomer of nicotine in the form of inclusion complex
glucoses with a truncated cone shape.10 The was evaluated and compared to that of
nicotine in free form at 25 °C and 50 °C. from the inclusion complex. IR grade
The results of this study provide the potassium bromide (Specac, USA) was used
preservation technique for nicotine related for FTIR analysis.
products. 2.3 Inclusion complex formation by
nanoencapsulation technique
2. Materials and Methods Nicotine-βCD inclusion complex
2.1 Computational simulation method was prepared by using the co-precipitation
The structure (S)-nicotine was method. βCD solution was prepared by
constructed using GaussView5 and the dissolving βCD in water at 25 °C. Then,
molecular geometry was further optimized nicotine was added to the βCD solution at
by Gaussian09 using semi-empirical PM3 the molar ratio of 1:1 (Nicotine:βCD),
basis set in gaseous phase.14,15 The crystal suggested by computational simulation, to
structure of βCD (CCDC number: form the inclusion complex. The solution
BCDEXD03) was downloaded from the was mixed using an orbital shaker
Cambridge Crystallographic Data Center (N-Biotek, South Korea). The mixture was
(CCDC).16 Hydrogen atoms were then added kept in the refrigerator (4 °C) overnight to
and water molecules were removed from enhance precipitation of the inclusion
βCD via Discovery Studio Visualizer4.0.17 complex. The inclusion complex was
Optimized nicotine was then docked into the filtered out by vacuum filtration and dried in
binding pockets of βCD via AutoDock4.2.17 the oven at 40 °C for 24 h. The obtained
Nicotine was kept flexible while βCD was inclusion complex was stored in the freezer
kept rigid. A grid size of 40×40×40 with (-20 °C) before analysis. The inclusion was
0.375 Å spacing was used. One hundred characterized by FTIR. The amount of
docking calculations were performed. The encapsulated nicotine was determined by
results obtained were classified into GC-FID. The encapsulation efficiency and
different clusters with different binding nicotine loading were calculated as in
energies. After obtaining the results from equation (2) and (3), respectively.
molecular docking calculation, they were
further optimized by Gaussian09 using Efficiency = Recovered nicotine (2)
semi-empirical PM3 basis set.15 The final Initial nicotine added
geometry obtained from each inclusion
Loading = Nicotine weight (3)
complex optimization was used for the
Inclusion complex weight
binding energy (∆E) calculation as in
equation (1).
2.4 Extraction of nicotine from inclusion
complex
∆E = Ecomplex – (Eguest + Ehost) (1)
Ethanol was added to inclusion
where, Ecomplex is the energy of the
complex powder to extract nicotine out. The
inclusion complex
mixture was placed in the ultrasonic bath
Eguest is the energy of nicotine
(Crest Ultrasonics, USA) for 1 hour. The
Ehost is the energy of βCD
mixture was then filtered out and the amount
of nicotine in filtrate was determined by
2.2 Materials
GC-FID.
(S)-nicotine, 99% purity (Alfa Aesar,
2.5 Nicotine determination by GC-FID
UK) was used as a guest molecule for
Determination of the nicotine
nanoencapsulation. βCD, 97% purity
amount was performed by using GC–FID,
(Sigma-Aldrich, USA) was used as a host
Clarus 580 GC (Perkin Elmer, USA)
molecule for nicotine. Absolute ethanol,
coupled with a Rtx-1 (Restek, USA), 30 m ×
99.9% purity (Merck, Germany) was used as
0.32 mm × 0.5 µm column.
a solvent to prepare nicotine standard
solution for GC-FID and to extract nicotine
2.6 Characterization of nicotine-βCD between nicotine and βCD is at a molar ratio
inclusion complex by FTIR of 1:1. Nicotine aligns itself perpendicular to
Nicotine in the form of inclusion the βCD horizontal plane with the pyridine
complex was characterized by using Nicolet ring facing up towards the wider rim and the
iS50 FTIR (Thermo Scientific, USA) via pyrrolidine ring facing down towards the
transmission. The sample was prepared by narrower rim of βCD.
mixing 1% (w/w) of inclusion complex in
potassium bromide powder. The solid
mixture was ground and pressed into a thin
disc. The spectrum of the inclusion complex
was also compared to the spectra of βCD
and nicotine.
2.7 Evaluation of nicotine preservation
capability in form of inclusion complex
The preservation capability of
nicotine in form of inclusion complex was
evaluated comparing to nicotine in form of
free compound at 25 °C and 50 °C for 0 – 28 Figure 1. The conformation of the inclusion
days. The same amount of nicotine in form complex calculated by PM3
of inclusion complex and free compound
were kept in beakers and stored in batch. 3.2 Inclusion complex formation by
The remaining nicotine in form of inclusion nanoencapsulation technique
complex and free compound were extracted Nanoencapsulation of nicotine was
by ethanol. The amount of nicotine was performed by the co-precipitation method.
determined by GC-FID. Nicotine can be encapsulated up to
92.51 µg nicotine per mg complex (The
3. Results and Discussion encapsulation efficiency is equivalent to
3.1 Computational simulation results 73.97%).
The possibility of using βCD to 3.3 Characterization of nicotine-βCD
encapsulate nicotine was predicted by inclusion complex by FTIR
computational simulation. The binding FTIR spectra of nicotine-βCD
energy of the inclusion complex calculated inclusion complex, βCD, and nicotine are
by PM3 is shown in Table 1. shown in Figure 2. The inclusion complex
spectrum shows the presence of N-CH3
Table 1. Frequency and binding energy of stretching, C=N stretching, C-N stretching,
nicotine-βCD inclusion complex and out-of-plane C-H bond bending at 2781,
Rank Frequency ∆E (kcal/mol) 1578, 1429, and 757 cm-1, respectively.
1 57 -16.45 These peaks indicate the presence of
2 3 -14.29
3 29 -14.25 nicotine in the inclusion complex.18–20
4 11 -13.28 There is a subtle shift between the -OH
stretching peaks of βCD in form of inclusion
The results show the negative complex (3362 cm-1) and pure compound
binding energies which indicate the (3381 cm-1). The shift indicates the
possibility of the inclusion complex interaction between nicotine and βCD,
formation. The conformation of the lowest which confirms the inclusion complex
energy inclusion complex is shown in Figure formation.20
1. The formation of the inclusion complex
Figure 2. FTIR spectra of (a) nicotine-βCD inclusion complex, (b) βCD, and (c) nicotine
3.4 Evaluation of nicotine preservation

capability in form of inclusion complex
The remaining nicotine in form of
free compound after exposure is shown in
Figure 3. The durations for completed loss
of nicotine at 2 5°C and 50 °C were within 7
days and 6 h, respectively. The high
temperature accelerates the loss of nicotine.
The result implies that nicotine is not stable.
Therefore, nanoencapsulation is used to
preserve nicotine.
The remaining nicotine in form of Figure 3. Nicotine (free compound)
inclusion complex after exposure is shown remaining after exposed to storage
in Figure 4. The inclusion complex slows temperatures of 25 °C and 50 °C for 7 days
down the loss of nicotine comparing to
nicotine in form of free compound. There is
a slightly higher loss at 50 °C (1 – 4%) than
the loss at 25 °C. The inclusion complex can
preserve up to 80% of nicotine after 28 days
at both 25 °C and 50 °C. Thus, nicotine can
be preserved in form of inclusion complex
using βCD.
Figure 4. Nicotine (inclusion complex)

remaining after exposed to storage
temperatures of 25 °C and 50 °C for 28 days
4. Conclusion 8. Wada, E.; Kisaki, T.; Saito, K. Arch.
Computational simulation shows that Biochem. Biophys. 1959, 79, 124–130.
nicotine can be encapsulated by βCD in 9. Hǎdǎrugǎ, D. I.; Hǎdǎrugǎ, N. G.;
form of inclusion complex at a molar ratio Butnaru, G.; Tatu, C.; Gruia, A. J. Incl.
of 1:1. The inclusion complex prepared by Phenom. Macrocycl. Chem. 2010, 68
the co-precipitation method resulted in the (1), 155–164.
encapsulation efficiency of 73.97%. The 10. Pinho, E.; Grootveld, M.; Soares, G.;
preservation of nicotine in form of inclusion Henriques, M. Carbohydr. Polym. 2014,
complex was further evaluated comparing to 101 (1), 121–135.
that of nicotine in form of free compound. 11. Del Valle, E. M. M. Process Biochem.
The results showed that the inclusion 2004, 39 (9), 1033–1046.
complex can preserve nicotine at both 25 °C 12. Astray, G.; Gonzalez-Barreiro, C.;
and 50 °C in 28 days. This preservation Mejuto, J. C.; Rial-Otero, R.; Simal-
technique could be further used to protect Gándara, J. Food Hydrocoll. 2009, 23
other active compounds from environmental (7), 1631–1640.
factors to prolong the shelf life of their 13. Szejtli, J. Chem. Rev. 1998, 98 (5),
products. 1743–1754.
14. Dennington, R.; Keith, T. A.; Millam, J.
Acknowledgements GaussView, Version 5 . USA, 2016.
The authors gratefully acknowledge 15. Frisch, M. J.; Trucks, G. W.; Schlegel,
the financial support from Thailand Tobacco H. B.; Scuseria, G. E.; Robb, M. A.;
Monopoly. Cheeseman, J. R.; Scalmani, G.;
Barone, V.; Petersson, G. A.; Nakatsuji,
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Toxicol. 2009, 49 (1), 57–71. W.; Sanner, M. F.; Belew, R. K.;
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4. White, H. K.; Levin, E. D. Chem. 2009, 30 (16), 2785–2791.
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5. Hughes, J. R.; Pembroke, A. C. Clin. 512.
Exp. Dermatol. 1994, 19 (3), 280. 19. Coates, J.; Coates; John. Encyclopedia
6. de Ong, E. R. Ind. Eng. Chem. 1924, 16 of Analytical Chemistry. John Wiley &
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7. Gorrod, J. W.; John W.; Jacob, P. 20. Veer, V.; Gopalakrishnan, R. Herbal
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Improvement of mechanical properties of jute fabric by bio-scouring and
bio-polishing process with enzymes
Chatdanai Thongsamut1, Chutimon Satirapipathkul2*, Rattanapol Mongkholrattanasit3*
1
Chemical Engineering Research Unit for Value Adding of Bioresources, Department of Chemical Engineering,
2
Faculty of Industrial Textiles and Fashion Design Rajamangala University of Technology Phra Nakhon,
399 Samsen Rd. Vachira Phayaban Dusit, Bangkok 10300, Thailand
*E-mail: chutimon.s@chula.ac.th, rattanaphol.m@rmutp.ac.th
Abstract:
The effect of pH on bio-scouring using commercial pectinase and xylanase on the
physical characteristics of jute fabric was investigated. The ratio of pectinase to xylanase (at
4% owf.) at 1:4, 40 ºC and pH 4 were optimized in bio-scouring. The physical characteristics
of jute fabric from bio-scouring and conventional scouring were compared in this study. The
conventional scouring produce brown surface while bio-scouring produce bright surface.
After bio-scoured jute fabric was treated with commercial cellulase at pH 4, 65 ºC, 2 h, to
improve mechanical properties and eliminate fuzz. The physical characteristic, such as water
absorbency, weight loss, L* a* b* values, tensile properties, and stiffness of jute fabrics have
been analyzed. Surface morphology of jute fabric have also been analyzed by stereo
microscopic.
1. Introduction in textile industry in order to protect

Jute (Corchorus olitorius) is environment.2 This process can be applied at
lignocellulose fiber which is used in the mid condition which avoid high energy
manufacturing of sack, hessian, carpet, consumption and pollution problem. Several
cordage and mat due to its unique properties, enzymes have been used in bio-scouring,
such as high stiffness, high tensile strength, such as cellulase, laccase, lipase, pectinase,
low extensibility and unattractive surface. protase and xylanase.3 Gaurav Grag et. al.4
Nowadays home textile and decorative studied bio-scouring of jute fabric by
article are produced from jute fiber. xylanase from Bacillus pumilus ASH. They
However, jute has some disadvantages due reported that the enzyme made jute finer,
to its natural properties, such as high softer, cleaner and brighter than the
stiffness, poor drape ability, crease chemical scouring. Bio-polishing is the
resistance, brittleness and fuzz. Chemical or process that eliminates fuzz on surface
bio-chemical process such as scouring or fabric and makes jute fabric soft and smooth
polishing was applied to improve these with cellulase. Recently, many studies using
properties.1 Scouring is the process for cellulase and mixed enzyme (cellulase,
removing non-cellulosic impurities (pectin, pectinase and xylanase) treat jute fiber in
fats, waxes, partially lignin and bio-polishing. Vigneswaran, C. and J.
hemicellulose) which limit the dyeing and Jayapriya,2 studied the effect of cellulase
finishing processes due to poor wet ability. and mixed enzymatic systems (cellulase,
Conventional scouring uses alkaline solution pectinase and xylanase) on physical
at high temperature. Alkaline solution can characteristics of jute fiber. They reported
dissolve cellulose of the fiber leading that the enzymatic treatment improved the
strength and weight loss. 2 This process also physical characteristic, such as flexural
causes high energy consumption and a large rigidity, elongation, whiteness index,
amount of waste water. Therefore, brightness index and reflectance. This
enzymatic technology is selected to be used research studied effect of bio-scouring and
bio-polishing to improve the physical 2.2.3 Bio-polishing treatment
properties of jute fabric for home textile. Bio-polishing Jute fabrics were
The commercial enzymes, such as cellulase optimally treated by commercial cellulase,
pectinase and xylanase were used in bio- using color fastness tester. The
scouring and bio-polishing. In this work, concentration of enzymes was 4% owf. The
the effect of pH on bio-scouring jute fabrics bio-polishing was done at pH 4, 65 ºC, for 2
by pectinase and xylanase was investigated. h with sodium acetate buffer at 100 mM.
The optimal pH of bio-scouring was used in After bio-polishing, the temperature was
pretreatment before bio-polishing. In bio- raised to 90 °C and maintained for 15 min to
polishing, jute fabric was treated with deactivate the action of the enzyme. Then,
commercial cellulase at pH 4, 65ºC and 2 h. fabric was washed in water, dried in oven at
Weight loss, stiffness, breaking strength, 50 °C for 24 h, and kept in a desiccator.
breaking extension, L* a* b* values and 2.3 Determination of weight loss
surface morphology were analyzed after bio- Weight of the fabrics before and
scouring and bio-polishing. after treatment was measured. To calculate
the weight loss used the following formula
2.1 Materials Weight loss (%) = 100× (w1-w2)/w1
Jute fabric was purchased from
Janata Jute Mills Ltd (Thickness 1 mm, where, w1 and w2 are the weights of fabric
weight 290 g/m2, fabric count per inch before and after the treatment respectively.
13×13). All commercial enzymes such as 2.4 Water absorbency test
cellulase (40,000 u/g), pectinase (50,000 The water absorbency of the fabrics
u/g) and xylanase (200,000 u/g) were was evaluated according to the AATTC
purchased from EN Bio-tech. Co. Ltd. Testing Method 79-2014. A drop of water
Acetic acid, EDTA, sodium acetate and was allowed to fall onto the surface of the
wetting agent were purchased form Sigma fabrics. The time (Seconds) between the
Aldrich. contract of water drops with the fabric and
2.2 Methods its disappearance in to the fabric was
2.2.1 Chemical scouring measured and recorded as a wetting time.
In conventional scouring, alkaline 2.5 Measurement of L* a* and b* values
solution (NaOH 10 g/L) and wetting agent The L*, a*, b* values of fabrics were
(2 g/l) were used to treat fabric (fabric to analyzed by Chroma meter (cr-410).
liquor ratio of 1:40) at 90 °C for 1 h. After The maximum and minimum value for L*
alkaline scouring process, fabrics were was 100 and 0 which represents a reflecting
washed in water, dried in oven at 50 °C for diffuser and black, respectively. Positive and
24 h, and kept in desiccator. negative of a* was red and green,
2.2.2 Bio-sourcing treatment respectively. Positive and negative of b*
Fabrics were scoured with was yellow and blue, respectively.
commercial pectinase and xylanase, using 2.6 Flexural rigidity
color fastness tester. The concentration of The bending length of fabrics was
enzymes was 4% owf. The bio-scouring was measured according ASTM D 1388-96
done at the ratio of pectinase to xylanase method with shirey’ stiffness tester using the
1:4, 40 ºC, 2 h and pH from 4-5.5, using specimen size 200 mm 25 mm. The test
sodium acetate buffer at 100 mM. After bio- result are an average of 5 reading in each
scouring process, the temperature was raised case. Flexural rigidity was calculated using
to 90°C and maintained for 15 min to the following formula:
deactivate the action of the enzymes. Then,
fabric was washed in water, dried in oven at
50°C for 24 h, and kept in desiccator.
G = w×c3 hydrolysis of cellulose portion on fabric
surface.
where, G is flexural rigidity, w is mass 3.2 Water absorbency
fraction per unit area (mg/cm2), and c is From the results in Table 1, wetting
bending length (cm). time of raw jute was more than 10 min. The
2.7 Tensile properties raw jute fibers were covered with some of
Breaking strength and breaking fat or wax and protein, which are insoluble
extension values of fabrics were measured in water. The wetting time of conventional
by the strip method (ASTM-D5035-95) by scouring was higher (264 s), as alkaline
using Tinius Olsen Universal tensile tester solution reacted with fat and wax on jute
with a traverse speed of 300 mm/min and fiber and dissolved partial lignin (water
pre tension of 5 N. Reported results are an insoluble component). All values of wetting
average of 5 tests for each sample. time in bio-scouring treated fabrics were
2.8 Surface morphology found to be lower than those of conventional
The surface morphology of fabric scouring treated fabrics and raw jute. The
was examined using stereomicroscopes values ranged from 125.67 s to 147.67 s.
(Nikon smz 1500). The reduction of wetting time was from the
degradation of some insoluble matter (wax,
3. Results and Discussion fat, pectin and lignin) on surface by
3.1 Weight loss (%) pectinase and xylanase in bio-scouring
From table 1, the weight loss of treatment.7 The shortest wetting time of bio-
control was 3.98% due to dissolving of scouring treated fabric was 125.67 s at pH 4
partial hemicellulose and some component in (optimal pH of pectinase), Pectinase affects
acid buffer solution. The weight loss of pectin, fat and wax covered on primary cell
conventional scouring was 10.93% because wall of treated fabric but xylanase affect
alkaline solution partially dissolved xylan in hemicellulose inside fiber.
hemicellulose, lignin, cellulose and other 3.3 L* a* and b* values
impurities. Alkaline solution enabled to From Table 1, L* a* and b* values
react with fat component on primary cell of all treated fabric from each process were
wall and provides soap and glycerol. compared with the values of raw jute. L*
Oxidization of OH group of lignin by values of conventional scouring treated
alkaline solution also caused change of fabric (18%) was lower than values of raw
chemical structure. jute, The a* (16.36%) and b* (9.32%) values
By using bio-scouring at different pH were higher than values of raw jute.
from 4 to 5.5, weight loss of fabric ranged Reduction of L* values caused brown
from 5.42% to 6.27%. Three enzymes in surface, as alkaline solution oxidized OH
bio-scouring provided hydrolysis of surface group of lignin. The L* values of bio-
of fabric. Pectinase could hydrolyze α-(1,4)- scouring treated fabric at pH 4.0-5.5 were
polygalacturonic acid in pectin structure. ranged from 1.66% to 2.74%. Each value
Xylanase contributed to breakdown xylan in was higher than values of raw jute, because
hemicelluloses.6 Weight loss from bio- the treated fabrics were brighter. All
scouring was inversely proportional with pH enzymes in bio-scouring process affected
condition. The scouring at pH 4 showed the specific substrate and did not change in
highest weight loss because pectinase chemical structure of fiber. The a* values of
optimally reacted at pH 4 and denature when bio-scouring treated fabric at pH 4-5.5 were
pH increased. The synergism of pectinase ranged from 21.23% to 33.06%. The b*
and xylanase provided increase of the values at pH 4-5.5 were ranged from 3.39%
weight loss.7 Weight loss of fabric treated by to 9.46%. All values of a* and b* were
bio-scouring/bio-polishing was 5.40%. higher than values of raw jute. All values of
Cellulase enzyme in bio-polishing provided bio-scouring/bio-polishing treated fabric
were similar to those of bio-scouring treated found to be most efficient at pH 4. After bio-
fabric. polishing, it was found that the stiffness of
3.4 Flexural rigidity jute fabric was reduced to 22.51% in weft
From Table 2, the stiffness value of and 35.16% in warp direction. Cellulose
raw jute was 351.96 and 505.16 (mg.cm) in affected on strength of fiber by degradation
weft and warp direction. Lignin and of some cellulose on fabric surface.
hemicellulose affect stiffness and strength of 3.5 Breaking strength
fiber. The stiffness of treated fabric was From Table 2, the breaking strength
reduced to 3.85% and 1.64% in weft and values of raw jute was 214.34 N in weft and
warp direction, due to solubility of lignin 290.57 N in warp direction. Breaking
and hemicellulose in acid buffer solution. strength of raw jute as reduced to 6.04% in
After conventional scouring process, weft and 10.44% in warp direction, because
stiffness of treated fabric was reduced to partial hemicellulose and lignin were
34.22% in weft direction and 36.72% in dissolved in acid buffer solution. As some
warp direction. The values of stiffness was portion of hemicellulose, cellulose and
reduced, as alkaline solution dissolved lignin were dissolved in alkaline solution, so
partial hemicellulose and lignin and also the breaking strength of conventional
attacked cellulose chains. The stiffness of all scouring treated fabric was reduced to
bio-scoured jute fabrics at each pH were 41.45% in weft and 31.87% in warp
reduce. The values in weft direction were direction. The breaking strength values of
ranged from 9.80% to 23.97%. Those in fabrics treated by bio-scouring at pH 4-5.5
warp direction were ranged from 11.60% to was reduced. They were ranged from
30.19%. The stiffness values of bio-scouring 17.78% to 29.61% in weft direction. Those
treated fabrics were higher than those of in warp direction were ranged from 15.13%
conventional scouring treated fabrics, as to 23.79%. The breaking strength of
pectinase and xylanase reacted with conventional scouring treated fabrics was
individual substrate and did not attack lower than those of bio-scouring treated
cellulose chains. Xylanase contributed to fabrics. The breaking strength of bio-
breakdown xylan which made jute softer and scouring treated fabric at pH 4 was the
improved various properties. Pectinase lowest. The increase of breaking strength
contributed to breakdown pectin in primary was the result from synergism action of
cell wall. The bio-scouring process at pH 4 pectinase and xylanase on surface fabric.
caused the softest of fabric as enzymes were
Table 1. Weight loss, water absorbency, and L*, a*, b* values of untreated and treated jute
Treatment Weight loss Wetting time (s) L* a* b*
Raw - >10min 60.19 6.17 21.56

Control 3.98 156.33 62.69 7.4 23.54
Conventional Scouring 10.93 264.00 49.35 7.18 19.55
Bio-scouring
4 6.27 125.67 61.37 8.21 23.56
4.5 6.16 136.67 61.64 8.03 23.6
5 5.91 139.67 61.84 7.19 22.29
5.5 5.42 141.67 61.19 7.48 22.93
Bio-Scouring/Bio-polishing 5.40 128.00 61.52 7.99 23.49
Table 2. Flexural rigidity stiffness, shrinkage, breaking strength (N), and breaking extension
(%) of untreated and treated jute
Stiffness
Breaking strength (N) Breaking Extension (%)
Treatment (mg×cm2)
Weft Warp Weft Warp Weft Warp
Raw 351.96 505.16 214.34 290.57 3.60 3.94
Control 338.41 496.90 201.40 260.22 3.45 3.84
Conventional
231.53 319.69 125.50 180.53 3.19 3.42
Scouring
Bio-Scouring
4 267.59 352.65 150.87 221.43 2.78 3.62

4.5 310.40 384.27 151.52 226.93 2.60 3.47
5 310.74 396.77 176.22 232.12 2.66 3.28
5.5 317.46 446.54 174.45 246.6 2.48 2.87
Bio-Scouring/Bio-
272.74 327.57 150.68 202.62 2.73 3.22
Polishing
a b
c d
Figure 1. Surface morphology of jute fabrics. (a) raw jute fabric, (b) conventional scoured
jute fabric, (c) bio-scoured jute fabrics, and (d) bio-scoured/bio-polished jute fabric
3.6 Breaking extension scouring treatment at pH 4 provided the

From the results in Table 2, breaking highest values of breaking extension,
extension values of raw jute were 3.6 in weft because of the optimal pH for enzymes.
and 3.94 in warp direction. Breaking 3.7 Surface morphology study
extension values of conventional scouring Figure 1 showed all surface
treated fabric were reduced to 11.39% in morphology of each samples. The raw
weft and 13.20% in warp direct. As lignin fabrics were fuzz (Figure 1a). At
and hemicellulose were dissolved in alkaline conventional scouring, treatment with
solution, these affected brittle and strength alkaline solution changed chemical structure
of fiber. The breaking extension of bio- of lignin and produced brown surface for
scouring treated fabric at pH 4-5.5 was treated fabric (Figure 1b). The treated
reduced by the synergistic action of fabrics were less fuzz than raw jute fabric.
pectinase and xylanase. The values in weft The samples from bio-scouring treatment
direction were ranged from 22.78% to were clearer and brighter than those (Figure
31.11%. Those in warp direction were 1c). Bio-scoured fabric surface was less
ranged from 8.12% to 27.16%. The bio- fuzz than raw and conventional scoured jute
fabrics. The bio-scouring/bio-polishing of Textiles. Vigneswaran, C.;
treatment removed fuzz on the fabric surface Ananthasubramanian, M.;
(Figure 1d). Cellulase degraded shot fuzz Kandhavadivu, P. Eds. WPI: India,
from the surface, so the surface fabric was 2014; pp 87-112.
the clearest and brightest. 2. Vigneswaran, C.; Jayapriya, J. J. Eng.
Fiber. Fabr. 2010, 101, 506-513.
4. Conclusion 3. Dhiman, S. S.; Sharma, J.; Battan, B. J.
Results of this study indicated that Enz. Mic. Tec. 2008, 43 (3), 262-269.
the use of all enzymes in bio-scouring and 4. Garg, G.; Dhiman, S. S.; Gautam, R.;
bio-polishing could improve properties of Mahajan, R.; Patra, A. K.; Sharma, J. J.
jute fabric, such as wetting time, stiffness, Mol. Catal. B Enzym. 2013, 85, 43-48.
tensile properties and color values. The 5. P’erez, J.; Munoz-Dorado, J,; De la
optimal condition of bio-scouring process Rubia, T,; Martinez, J. Biodegradation
for pectinase: xylanase was at pH 4, 40 ºC and biological treatment: Handbook of
for 2 h. The bio-polishing could eliminate Natural Fibres; Kozłowski, R. Eds.
fuzz on fabric surface. The application of Woodhead: Cambridge, 2012; pp 53-63.
bio-scouring and bio-polishing processes for 6. Samata, A.K,; Singhee, D,; Basu, G,;
jute fibers was more environmental friendly Mahabalanabis, K, K, J. Indian J. Fib.
than chemical scouring. Tex. Res. 2005, 30, 451-467.
7. Singh, A,; Kuar, A,; Petra, A, K,;
References Mahajan, R. J. Clean Prod. 2016, 124,
1. Vigneswaran, C. Bioprocessing of jute 120-125.
and their characteristics; Bioprocessing
Characterization of epoxidized castor oil blended with epoxy phenol
novolac resin for adhesive application
Thanapat Supannasud, Chavakorn Samthong, Anongnat Somwangthanaroj*
*E-mail: anongnat.s@chula.ac.th
Abstract:
Epoxy adhesive is one of the most important thermosetting polymers that presents
good thermal and mechanical properties. However, the cured epoxy exhibits intrinsically
brittle behavior in nature. To solve this problem, the modification of epoxy is required to
enhance the flexibility. In this work, epoxidized castor oil (ECO) blended with epoxy phenol
novolac (EPN) resin was used as adhesive. Epoxy systems were prepared by an addition of
ECO at 20-50wt% to the EPN epoxy resin using m-phenylenediamine (PDA) as curing agent.
Effect of ECO amount on the curing reaction, thermal and mechanical properties of EPN
resin was investigated by FTIR, DSC and universal testing machine. FTIR results showed
that a decrease in intensity of epoxide group at 837 cm-1 confirmed the formation of three-
dimensional network structures in the epoxy systems. The heat of reaction of EPN/ECO
blends increased when adding more multifunctional EPN as confirmed by DSC. On the other
hand, the glass transition temperature of EPN/ECO adhesives decreased with increasing ECO
loading. This is due to the increase of soft part by ECO molecules. From the 90 peel strength
test, the maximum value was obtained at 80/20 w/w of EPN/ECO blends, implying the
suitable content of ECO for increasing chain mobility and flexibility in the epoxy matrix.
1. Introduction epoxy system and poor flexibility.2 From

Epoxy adhesives are the this problem, the modification of epoxy is
thermosetting resins that used to joint required to enhance the flexibility. In this
components by making a bond between two work, a long chain aliphatic epoxy resin,
surfaces. They have been used for wide epoxidized castor oil (ECO), was added into
applications including automotive, EPN epoxy resin at various concentra-
construction industries, and electronics. tions.3,5,6
Among many types of adhesives, epoxies The main aim of this work is to study the
are probably the best adhesives because they effect of ECO addition on the properties of
have strong bonding strength to many EPN resins for adhesive application. The
substrates and possess broad outstanding curing studies, thermal properties, curing
properties, depending on their chemical reaction were analyzed by using differential
structures, such as good mechanical scanning calorimetry (DSC) and Fourier
properties, high thermal stabilities, and high transform infrared spectroscopy (FTIR). The
chemical resistances.1 peel strength of adhesive was studied by
Epoxy phenol novolac resins (EPNs) using a universal testing machine.
are polyglycidyl ethers of novolac resin,
which have high thermal resistance, high 2. Materials and Methods
crosslink density, and multifunctionality. 2.1 Materials
EPNs also bond well to aluminum substrate Epoxy phenol novolac resin (EPN,
because they have many hydroxyl groups on eq. epoxy = 172.7 g/eq.) as multifunctional
the surface that can be reacted with the epoxy resin and epoxidized castor oil (ECO,
aluminum surface. However, EPNs are eq. epoxy = 575 g/eq.) as trifunctional epoxy
brittle because of their Multifunctionality, resin were kindly supplied by Aditya Birla
leading to high crosslink density of cured Chemical (Thailand) Ltd. 1,3-
phenylenediamine (mPDA) as amine curing cured epoxy systems including EPN, ECO,
agent was purchased from Sigma-Aldrich. and EPN/ECO blends (50/50 and 60/40
The chemical structures of epoxidized castor w/w) were measured in a spectral range of
oil (ECO) and epoxy phenol novolac resin 4000-400 cm-1 with 128 scans at a resolution
(EPN) are shown in Figure 1 and 2. of 4 cm-1.
2.3.3 Peel strength test
The 90 peel strength was measured
by a universal testing machine (Instron,
model 5567) based on IPC-TM-650-249
Figure 1. Structure of epoxidized castor oil from Instron (Thailand) Co.,Ltd. The
(ECO) specimens with 5 mm width prepared from
the adhesive joints between an aluminum
and polyimide film were evaluated with a
peel rate of 50 mm/min. The peel strength
was the average peel force divided by
specimen width.
Figure 2. Structure of epoxy phenol novolac 3. Results and Discussion

resin (EPN) 3.1 Curing behavior analysis
The DSC thermograms as well as
2.2 Preparation of EPN/ECO blends. maximum curing temperature and heat of
EPN was stirred with ECO for 30 curing of neat EPN, neat ECO, and epoxy
min at room temperature following by mixtures at 80/20, 70/30, 60/40, and 50/50
stirring with mPDA at a stoichiometric w/w were shown in Figure 3 and Table 1.
molar balance of hydrogen of amine to The maximum curing temperature
epoxide ring of 1:1 for 1 hr at room decreased, and heat of curing increased as
temperature to obtain the homogeneous increasing amount of EPN epoxy resin,
mixture.4 Subsequently, the mixture was meaning that EPN can provide faster and
laminated between aluminum and polyimide high curing reaction than ECO. This is
film. The substrate of aluminum and because EPN has larger numbers of epoxy
polyimide film was placed in an oven at 170 groups than ECO, which effectively
C for 10 min to cure the adhesive film. facilitate the curing reaction. Figure 4 shows
2.3 Characterization the isothermal curing behavior of EPN/ECO
2.3.1 Differential scanning calorimetry blends at 170 C (average of Tcure from
The curing characteristics of the Table 1) for 30 min. The result shows that
epoxy adhesives were evaluated by a the heat of reaction increased with the
differential scanning calorimeter (DSC) 5- addition of EPN content and EPN/ECO
10 mg of samples were sealed in the blends can be fully cured with in 10 min.
aluminum pan. The samples were heated Table 2 shows the Tg of neat EPN,
from 0 C to 250 C with a heating rate of neat ECO and EPN/ECO blends after curing
10 C/min under nitrogen flow rate of 50 at 170 C for 10 min. The Tg decreased with
ml/min. Glass transition temperature (Tg), the increase of ECO content because that
maximum curing temperature, and heat of EPN has many aromatic rings attached with
curing were obtained from the DSC epoxy. This aromatic is bulky. Therefore,
thermograms. EPN has higher Tg than ECO which mainly
2.3.2 Fourier transform infrared contains tri-aliphatic chain of epoxy.
spectroscopy EPN/ECO blends have Tg between pure
The curing reaction was further epoxy and only single Tg found means that
confirmed by Fourier transform infrared there is no phase separation because this is
spectroscopy (FTIR). FTIR spectra of the the characteristic of miscible blend.
Neat ECO
Neat ECO EPN/ECO (50/50)
EPN/ECO (50:50) EPN/ECO (60/40)
EPN/ECO (60:40) EPN/ECO (70/30)
EPN/ECO (70:30) EPN/ECO (80/20)
Heatflow (J/g) exo up
Neat EPN
EPN/ECO (80:20)

Neat EPN
50 100 150 200 250 300

o
Temperature ( C) 50 100 150 200 250
o
Figure 3. DSC heating thermograms of Temperature ( C)
uncured EPN, ECO and their blends at Figure 5. Differential scanning calorimetry
heating rate of 10 C/min thermograms (Temperature range 0 oC to
300 oC, heating rate 10 C/min.) for Epoxy
Table 1. Thermal properties from DSC phenol novolac/epoxidized castor oil
heating thermograms of uncured epoxy systems
systems
EPN/ECO Heat of Curing Table 2. Glass transition temperature (Tg)
Mass ratio reactions (J/g) temperature (C) obtained from DSC heating curves of cured
Neat EPN 422 149 epoxy systems
80/20 325 153 EPN/ECO Glass transition
70/30 256 176 Mass ratio temperature (C)
60/40 252 178 Neat EPN 161
50/50 216 179 80/20 146
Neat ECO 40 237 70/30 137
60/40 126
EPN/ECO (50:50)
50/50 122
EPN/ECO (60:40) Neat ECO 115
EPN/ECO (70:30)
EPN/ECO (80:20)
3.2 Confirmation of curing reaction

The FTIR spectra of EPN, ECO and
EPN/ECO blends (100/0, 0/100, 60/40, and
50/50 w/w) are shown in Figure 6. The
uncured ECO spectrum indicated -OH
stretching at 3490 cm-1, -CH2 symmetric
stretching at 2919 cm-1, and epoxide group
vibration at 834 cm-1. The FTIR spectrum of
0 80 160 240 320 400 480 560
cured EPN/ECO (50/50) showed the
Time (s) presence of intensity decreased from
uncured EPN/ECO (50/50). This is mean
Figure 4. DSC isotherms of cured EPN, that epoxy groups confirmed the reaction of
ECO, and their blends at 170 C and a epoxide group.
heating rate of 10 C/min for 30 min
4. Conclusion
Epoxy based adhesive in binary
system of multifunctional aromatic epoxy
D
(EPN) and trifunctional aliphatic epoxy
(ECO) was successfully used as the
Absorbance
C adhesive between aluminum and polyimide

-1 -1
film substrates. From the results, DSC
2919 cm 834 cm
3490 cm
-1 thermograms showed maximum curing
B temperature of epoxy systems decreased and
820 cm
-1
heat of curing increased with increasing
A
EPN content, resulting from the
multifunctionality of EPN which supports
the curing behavior of epoxy resin with
4000 3500 3000 2500 2000 1500 1000 500
-1 diamine. FTIR results indicated that epoxy
Wavenumbers (cm )
groups confirmed the reaction of epoxide
Figure 6. FTIR spectra of (A) uncured group in the blend systems. Finally, peel
ECO, (B) uncured EPN, (C) uncured strength of EPN/ECO blends was higher
EPN/ECO (50/50), and (D) cured EPN/ECO than that of pure EPN and pure ECO due to
(50/50) the increase of flexibility in the epoxy
matrix.
3.3 Mechanical properties
Figure 7 shows the 90 peel strength Acknowledgements
of EPN, ECO, and EPN/ECO blends at This work was financially supported
various ratios. The maximum value of 90 by the Mektec Manufacturing Corporation
peel strength was found at 80/20 w/w of (Thailand) Ltd. Authors thanks Aditya Birla
EPN/ECO blends. The 90 peel strength Chemicals (Thailand) Ltd. for material
showed an increased adhesive ability with support.
binary resin mixtures of EPN/ECO due to
the enhanced toughness and flexibility of References
aliphatic structure of ECO. When further 1. Petrie, E. M. Epoxy adhesive
increasing ECO content, the peel strength formulations. McGraw-Hill: Boston,
was dropped because the long chain 2006.
branched aliphatic structure of ECO reduced 2. Gouri, C. R. R.; Ninan, K. N. I. J.
the crosslink density and excess ECO in the Adhesion 2000, 20, 305-314.
binary resin mixtures could be resulted in 3. Sudha, G. S.; Kalita, H.; Mohanty, S.;
decrease of adhesive ability. Nayak, S. K. Macromol. Res. 2017, 25,
420-430.
4. Razack-Varghese L. A. IJERT. 2014, 3,
2662-2665.
5. Ratna D.; Banthia A. K. I. J. Adhesion
2012, 14, 15-25.
6. Ratna, D. Polym. Int. 2012, 50, 179-
184.
Figure 7. 90 peel strength of EPN, ECO,

and EPN/ECO blends
Electrochemical oxidation of ethanol on palladium
electrodeposited on nickel catalyst
Wilaiwan Supap1, Thaneeya Hawiset2, Prachak Inkaew1,3*
1
School of Science, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand
2
School of Medicine, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand
3
Center of Chemical Innovation for Sustainability (CIS), Mae Fah Luang University, Muang,
*E-mail: prachak.ink@mfu.ac.th
Abstract:
Direct ethanol fuel cells have a potential to be used as power sources in the future.
Platinum is the main catalyst used in the system. Because Pt is very expensive, other catalysts
have been investigated to be used instead of Pt. In this work, the reaction at the anode side of
the ethanol fuel cells was investigated. Palladium was electrodeposited onto nickel substrate
(0.5 x 8 x 0.01 cm) and used as the catalyst for ethanol oxidation reaction. The
electrodeposition was performed by applying -0.2 V (versus Ag/AgCl) to nickel substrate.
Cyclic voltammograms confirmed the deposition of palladium onto the nickel surface. The
coverages of palladium on the nickel surface were controlled by the deposition time which
resulted in palladium coverage between 21 and 93%. The addition of palladium to nickel led
to some improvement in electrocatalytic activity and stability of nickel catalyst. The results
showed this modified catalyst was effective and had a potential to be used as the catalyst for
ethanol oxidation in direct ethanol fuel cells.
1. Introduction prone to the adsorption of by-products to use

Fuel cells (FCs) convert chemical instead of Pt for DEFCs. This work
energy from a fuel such as hydrogen gas or investigated the application of Ni-based
small organic molecules (SOMs) into catalysts to replace Pt-based catalysts in
electricity. Ethanol fuels have been order to reduce the cost of the catalyst and
recognized as a substantial energy source in provide alternative catalysts. Nickel (Ni) is
the future of ‘green’ technology. Direct active toward oxidation of small organic
ethanol fuel cells (DEFCs) will be the molecules and is less expensive than Pt.
important renewable and clean energy Because Ni-based catalysts are
sources in the near future. Ethanol has the electrocatalytically active for the ethanol
advantages over methanol including lower oxidation reaction (EOR) in alkaline
toxicity and higher energy density (12 e- condition, the use of Ni-based catalysts will
transfer in complete oxidation reaction).1 be widely used in the future.2-4 Various
The important part in the fuel cells system is research groups reported the enhancement of
catalyst which required some improvement. the electro-catalytic performance of Ni
Platinum (Pt) is a major catalyst used in catalyst by adding second element to form
ethanol fuel cells because of its high binary, Ni/M (M: Ru, Co, Sn, Pd, Mo, Bi,
catalytic activity. However, the use of Pt Mn, etc.), ternary or multicomponent
catalyst is limited because of its high cost electrocatalysts.
and low availability. Pt catalyst surface is In this work, modified binary
hindered when by-products of oxidation catalysts were prepared by electrodepositing
reaction adsorb onto its surface and reduce Pd onto Ni plate. The catalytic activity and
its catalytic activity. It is hence, important to stability of Pd electrodeposited on Ni
find other catalysts which have higher or substrate (Ni/Pd) were evaluated for ethanol
comparable catalytic activity and are less oxidation reaction in an alkaline electrolyte.
The catalytic activity and stability of Ni/Pd densities were evaluated by the current over
toward ethanol oxidation were evaluated the active surface areas of the electrodes.
using cyclic voltammetry (CV) and Ethanol oxidation was evaluated in 1 M
chronoamperometry (CA). The results were CH3CH2OH + 1 M NaOH solution by
compared to those of Ni catalyst in similar scanning at 100 mVs-1 between -1.1 and 0.6
condition. V for Ni/Pd and -1.1 and 0.4 V for Ni
catalyst.
2. Materials and Methods 2.3 Images of the surface
2.1 Chemicals Scanning electron microscope
The chemicals used in this work (SEM) was used to determine surface micro-
were obtained from Sigma-Aldrich (St. structure. The voltage applied was 20 kV.
Louis, USA). All materials were reagent
grade and used as received without any 3. Results and Discussion
further purification. Ethanol was obtained 3.1 Voltammetric characterization of Ni
from QRec, Australia. Electrolyte was and Ni/Pd
prepared fresh each day. Double distillation
water (Milli-Q water system, Quantum) was
used to prepare all the solutions.
2.2 Electrochemical analysis
A Ni plate (0.5 × 8 × 0.01 cm) was
used as the working electrode. Pt wire was
used as the counter electrode, and a
Ag/AgCl (3 M KCl) electrode was used as
the reference electrode. Electrochemical
measurement was carried out in three-
electrode system. Potentiostat/Galvanostat
(eDAQ) was used to control the potential Figure 1. Cyclic voltammograms for Ni
and record the current. Ni substrate was plate and Ni/Pd, Ni and Ni/Pd electrode in
rinsed with acetone and double distillation 1 M NaOH, scan rate 100 mVs-1
water before use. To electrodeposit Pd onto
Ni substrate, the substrate was immersed in The voltammograms of the Ni and
electrolyte solution about 0.8 cm. 1 mM Ni/Pd are shown in Figure 1. The peaks are
Na2PdCl4 was used as the precursor for different because of different electro-
electrodeposition. A cyclic voltammetry was chemical processes over each surface. Ni/Pd
used as a method to check Pd deposition. Pd catalysts show peaks at -0.8 V and -0.5 V
was deposited at -0.2 V (vs. Ag/AgCl) in 0.5 which are the oxidation/reduction peaks of
M H2SO4 for different controlled time. The Pd. The voltammogram of Ni/Pd in Figure 1
electrodeposited electrode was rinsed with shows the adsorption/desorption peak of Ni
double distillation water before use for at about 0.4 and 0.3 V confirming the
ethanol oxidation. The active surface area of presence of Ni surface. In the backward
Pd modified electrode was determined from scan, the reduction peak of Pd-oxides can be
cyclic voltammetry by integrating the peak observed at -0.4 V. These peaks confirm that
area between -0.9 to -0.3 V, and subtracting Pd was successfully deposited on the surface
the capacitive charges. For Pd(II)-oxide of Ni substrate. Cyclic voltammograms
surface, 424 µC/cm2 was used to evaluate (CV) of Ni/Pd are similar to CV of Pd
the electroactive surface area (EASA).5 The catalysts and Pd-Ni/C catalysts in alkaline
active surface area of Ni was evaluated by solution reported in literature.7,8 The
integrating the oxidation peak area between oxidation of hydroxide adsorbed and
0.1 to 0.6 V. 514 µC/cm2 was used to desorbed on the surface of Pd-based
evaluate the EASA of Ni.6 The current catalysts in the alkaline electrolyte are
showed in equations (1)-(3).9 In Figure 1, 3.2 Ethanol oxidation of Ni and Ni/Pd
adsorption/desorption of Ni(OH)2/ (NiO)OH Figure 3 shows the voltammograms
are observed at 0.4 and 0.28 V respectively. of ethanol oxidation in 1 M NaOH of Ni/Pd
These peaks are the characteristic peaks of and Ni. For ethanol oxidation reaction on the
Ni in basic solution.8 In acid solution, surface of Ni/Pd, the onset potential is at
adsorption/desorption peaks are not -0.6 V and the peak is at -0.1 V. The
observed. The electrochemical signals of backward scan shows the oxidation of by-
Ni(II)/Ni(III) peak of Ni-based catalysts products such as CH3COOH, CH3CHO, CH4
surface can be described by equations (4)- and species generated from ethanol
(5). The anodic peak at 0.4 V is observed for oxidation.10 However, on the surface of Ni
the transition of Ni(OH)2 to (NiO)OH. The the ethanol oxidation was not observed. Ni
integration of the area under the plate is not active toward ethanol oxidation.
voltammogram of Ni(OH)2/(NiO)OH peak The direct ethanol oxidation pathway is
was used to estimate the current density and shown in equation (6). High forward peak
EASA of the catalyst. The increased anodic represents the amount of ethanol oxidation
were toward the positive potentials is related in direct pathway and yields more electrons
to oxygen evolution or oxidation. However, in the process than the indirect pathway.
the ratio of Ni and Pd were estimated from High backward peak indicates that the
charges of peak at 0.3 V for Ni and charge oxidation proceeds via indirect pathway and
under the cathodic peak at -0.4 V of Ni/Pd
as shown in Figure 2.
Figure 3. Cyclic voltamograms for ethanol

oxidation of Ni and Ni/Pd in 1 M NaOH + 1
Figure 2. Cyclic voltammograms for Ni M ethanol, scan rate 100 mV/s; The current
plate (Ni) and Ni/Pd in different deposition density was obtained from the current over
ratio, Ni and Ni/Pd electrodes in 1 M NaOH, the electroactive surface area.
scan rate 100 mVs-1
Pd + OH¯ ⇋ Pd-OHads + e¯ (adsorption of OH¯) (1)

Pd-OHads + OH¯ ⇋ Pd-O + H2O + e¯ (formation of Pd oxide layer) (2)
Pd-O + H2O + e¯ ⇋ Pd + 2OH¯ (reduction of oxide to Pd) (3)
NiO + OH¯ _ e¯ ⇋ (NiO)OH (4)
Ni(OH)2 + OH¯ _ e¯ ⇋ (NiO)OH + H2O (5)
C2H5OH + 3H2O → 2CO2 + 12H++ 12 e¯ (6)
Pd + C2H5OH ⇋ Pd-(CH3CH2OH)ads (7)
Pd-(CH3CH2OH)ads+ 3OH¯  Pd-(CH3CO)ads + H2O + 3e¯ (8)
Pd + OH¯ ⇋ Pd-OHads + e¯ (9)
Pd-( CH3CO)ads + Pd-OHads  Pd-(CH3COOH) + Pd (10)
Pd-(CH3COOH) + OH¯  Pd + CH3COO¯ + H2O (11)
yields many by-products of small organic and Ni/Pd in different ratios. At the
molecules. The mechanism of ethanol beginning, Ni/Pd has higher current density
oxidation on the Pd surface in alkaline than Ni when the potential of 0.3 V was
media is shown in equations (7) - (11).11,12 applied to the electrode. However, for longer
Cyclic voltammograms in Figure 3 were period of time, the current from Ni/Pd decay
used for calculating charges of the ethanol to lower values and be come stable. Ni/Pd
oxidation and the calculated value are shown (39:61) shows the highest current density
in Figure 4. Charges of ethanol oxidation compared to other ratios.
significantly increase with Pd amount on Ni
surface (Figure 4). Ni/Pd (39:61) exhibits
the highest activity with charge density of
125.06 mC/cm2 at -0.1 V in the forward scan
(Figure 3). The charge densities decrease
with high ratio of Pd. Lović and co-workers
reported that Pd0.74Ni0.26 alloy was the most
active surface for electrochemical oxidation
of ethanol.13 In Ni/Pd, Ni helps to enhance
ethanol oxidation because Ni increases OH Figure 5. Current-time transient of ethanol
species at the surface of Pd-based catalyst. oxidation on Ni and Ni/Pd in different
As a result, the surface of Ni/Pd is more deposition ratios, step potential was 0.3 V
active toward ethanol oxidation. Hence, Pd (vs. Ag/AgCl) for 10 min in 1 M NaOH+1
surface coverage needs to be optimized. M ethanol; The current density was calculate
based on the EASA of each surface.
3.3 Characterization of Ni/Pd
Figure 4. The corresponding columbic

charges versus ratio of Ni:Pd for ethanol
oxidation of Ni and Ni/Pd of different
deposition ratio, Ni and Ni/Pd in 1 M NaOH
+ 1 M ethanol, scan rate 100 mV/s
Stability was also very important

property for a desirable catalyst. High-
stability catalyst could be used for long
period of time with very small decrease in
the performance or with no decrease in the
performance at all. The desired catalyst
should high produce and stable current. For
catalyst in ethanol oxidation reaction, plot of
current-time was used to evaluate the
stability of the catalyst.14 Figure 5 shows the
current-time plot of ethanol oxidation on Ni Figure 6. Top view of SEM micrographs of
(a) Ni, 1000X, (b) Ni/Pd (35:65), 3000X
The morphology of Ni substrate and C. W.; Lee, C. H.; Lee, J. F.; Wang, K.
Ni/Pd catalyst surface were determined by W. Inter. J. Hydrogen Energy 2013, 38,
scanning electron microscope (SEM). Figure 4474-4482.
6 shows the top view micrographs of Ni 4. Ferdowsi, G. S.; Seyedsadjadi, S. A.;
(Figure 6a) and electrodeposited Ni/Pd Ghaffarinejad, A. J. Nanostruct. Chem.
catalyst (Figure 6b). In Ni:Pd (35:65), large 2015, 5, 17-23.
aggregate of Pd was observed on Ni 5. Pandey, R. K.; Lakshminarayanan, V. J.
substrate. Plates of about 5-10 m were Phys. Chem. C 2009, 113, 21596-
observed. The surface was different from Pd 21603.
electrode deposited onto Pt surface reported 6. Jiang, Q.; Jiang, L.; Hou, H.; Qi, J.;
by Hoyer and coworkers.15 Wang, S.; Sun, G. J. Phys. Chem. C
2010, 114, 19714-19722.
4. Conclusion 7. del Rosario, J. A. D.; Ocon, J. D.; Jeon,
In this work, Pd was electrodeposited H.; Yi, Y.; Lee, J. K.; Lee, J. J. Phys.
onto Ni plate to prepare binary catalyst for Chem. C 2014, 118, 22473-22478.
ethanol oxidation. Ni/Pd surface is active 8. Obradović, M. D.; Stančić, Z. M.;
toward ethanol oxidation reaction whereas Lačnjevac, U. Č.; Radmilović, V. V.;
Ni was not active. The ratio of Pd Gavrilović, W. A.; Radmilović, V. R.;
significantly affects ethanol oxidation Gojković, S. L. Appl. Catal. B: Environ.
reaction of the modified surface. Ni/Pd 2016, 189, 110-118.
(39:61) provides the best activity and 9. Wang, X.; Ma, G.; Zhu, F.; Lin, N.;
stability. Tang, B.; Zhang, Z. Electrochim. Acta
2013, 114, 500-508.
Acknowledgements 10. Antoniassi, R. M.; Otubo, L.; Vaz, J.
Financial support from the office of M.; Oliveira, N. A.; Spinacé, E. V. J.
the Postgraduate Studies, Mae Fah Luang Catal. 2016, 342, 67-74.
University is greatly appreciated. We also 11. Yin, J.; Shan, S.; Ng, M. S.; Yang, L.;
would like to thank the Scientific and Mott, D.; Fang, W.; Kang, N.; Luo, J.;
Technological Instrument Center (STIC) at Zhong, C. J. Langmuir 2013, 29, 9249-
Mae Fah Luang University for providing the 9258.
electrodes and potentiostat used in this 12. Cerritos, R. C.; Guerra, B. M.; Ramírez,
work. R. F.; Ledesma, G. J.; Arriaga, L. G.
Materials 2012, 5, 1686-1697.
References 13. Lović, J. D.; Vladimir D. J. J. Solid
1. Hoogers, G. Fuel Cell Technology- State Electrochem. 2017, 21, 2433-
Handbook. Society of Automotive 2441.
Engineers. New York, 2002. 14. Behling, N. H. Fuel cells: current
2. Tan, J. L.; De Jesus, A. M.; Chua, S. L.; technology challenges and future
Sanetuntikul, J.; Shanmugam, S.; research needs. Elsevier, 2012.
Tongol, B. J. V.; Kim, H. Appl. Catal. 15. Hoyer, R.; Kibler, L. A.; Kolb, D. M.
A: Gen. 2017, 531, 29-35. Electrochim. Acta 2003, 49, 63-72.
3. Su, P. C.; Chen, H. S.; Chen, T. Y.; Liu,
The antibacterial activities of silver nanomaterials against gram positive
and gram negative bacteria
Romrawin Rooblert1, Sujittra Youngme2, Pornsawai Praipipat1*
1
Department of Environmental Science, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
2
Department of Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
*E-mail: pornprai@kku.ac.th
Abstract :
The disinfection is the final step of domestic wastewater treatment to ensure treated
water without bacteria or other microbes that cause waterborne diseases. Different techniques
have been developed to eliminate gram positive and gram negative bacteria to achieve clean
water for use. This study presents a new alternative choice by using silver nanomaterials
which synthesizes by impregnating silver nitrate (AgNO3-) to cation exchange resin (C145)
called AgNPs-C145 to be against both gram positive bacteria (Staphyloloccus aureus:
S. aureus) and gram negative bacteria (Escherischia coli: E. coli) in the water. The results
showed the formation of a prominent zone of inhibition (ZOI) for both bacteria, and the
average of ZOI size of E. coli (0.441 mm) was bigger than S. aureus (0.354 mm). The batch
tests (dose, contact time, pH, and concentration) were used to investigate the efficiency of
silver nanomaterials to be against two bacteria types. The result showed that 1.5 g/200 mL, 1
h, pH 7, 106 CFU/mL and 3 g/200 mL, 5 h, pH 7, 106 CFU/mL were the best optimum
condition to be against E. coli and S. aureus, respectively with measuring by the plate count
technique. This confirmed that AgNPs-C145 can be against both bacteria types.
1. Introduction nanoparticle mechanism can inhibit the

Gram-positive and gram-negative function of enzymes that acts by consuming
bacteria can be found in nature such as soil, oxygen and energy of bacteria can destroy
water, air, and also generally found in the the cells of bacteria by using only a small
environment. Some types of bacteria cause quantity.6
diseases in humans; however, some species In this study, the researchers aim to
are benefits to humans. Pathogenic bacteria synthesize the silver nanomaterials called
such as Staphylococcus aureus can cause AgNPs-C145 for antibacterial activities
foodborne disease and Escherichia coli can against gram-positive (Staphylococcus
cause Diarrhea.1 For the beneficial bacteria, aureus: S. aureus) and gram-negative
Lactobacillus Oenococcus oeni and (Escherichia coli: E. coli) bacteria to
Pediococcus bacteria are used in the wine investigate their characterizations, examine
industry.2 the inhibition zone study, and find the best
Therefore, the development of conditions for antibacterial activities against
antimicrobial agents of harmful bacteria and both bacteria types by using the batch test
causing diseases in humans are necessary to experiments on the effects of does, contact
protect human health.3 time, pH, and concentration.
For the previous studies showed that
silver nanoparticles can effectively use for 2. Materials and Methods
resistant and antibacterial activities of 2.1 Synthesis of the synthesized silver
bacteria.4 Silver compounds have also been nanomaterials (AgNPs-C145)
applied in the pharmaceutical industry, food Prepared 100 mL of 2% solution of
and water with the property against and silver nitrate (AgNO3) in the 200 mL
inhibit the growth of bacteria.5 The silver polyethylene bottle, then added 37.5 g of the
cation exchange resin (C145) into the tests by controlling the mixing speed at 200
solution. The mixture was shaken in a rpm.
gyratory shaker for 24 h at a room For three batch test experiments of
temperature 24±1.0 ºC, and then filtered and dose (0, 1.5, 3 g), contact time (1, 3, 5 h),
was air dried in 12 h. After that, transferred and pH (5, 7, 9) were used the initial
to 80 mL of 2% of sodium borohydride concentration of S. aureus and E. coli
(NaBH4) in the polyethylene bottle and was bacteria were at 106 CFU/mL with 200 mL
shaken in a gyratory shaker for 1 h, and then water sample volume per experiments.
filtered and preserved it into 70 mL of The last experiment would be
ethanol in 100 mL of glass bottle until use. varying the concentration at 104, 105, 106,
2.2 Characterization of silver 107, and 108 CFU/mL with 200 mL water
nanomaterials (AgNPs-C145) sample volume per experiments and used the
The internal components, the best conditions of does, contact time, and
arrangement of atoms, morphology and pH experiments. The results were analyzed
surface characteristics of silver by using the plate count technique.
nanomaterials (AgNPs-C145) were studied
by Focused Ion Beam Scanning Electron 3. Results and Discussion
Microscope (FIB-SEM), and Energy 3.1 Characterization of silver
Dispersive X-ray Spectrometry (EDX) was nanomaterials (AgNPs-C145)
used for elemental and qualitative analysis. Based on morphological studies
2.3 Comparison of AgNPs-C145 inhibited using the FIB-SEM and EDX 50000x tool,
abilities on of S. aureus and E. coli the material had many gaps of the porous
bacteria by using the inhibition zone appearance shown in Figure 1. According to
study EDX analysis, it showed the percentage of
Two bacteria types (S. aureus and E. chemical that were carbon (C), oxygen (O),
coli) were prepared at 106 CFU/ml sodium (Na), sulfur (S), and silver (Ag) at
concentrations by using a standard 39.5%, 29.8%, 14%, 11.6%, and 5.1%,
McFarland 0.5 method, and used the cotton respectively. It proved that synthetic
swab technique for applying bacteria into materials could absorb silver (Ag) into the
the nutrient agar for the inhibition zone cation exchange resin shown in Figure 2.
study. Triplicated experiments of each
bacteria test were used to confirm the results
and the average was reported. For each plate
test, applied the bacteria into the nutrient
agar and put 4 pieces of AgNPs-C145 in 4
different points. Then, took the plate into the
incubation oven at 37 ºC for 24 h. The result
was analyzed by measuring the inhibition
zone diameter.
2.4 Batch test experiments were used to
investigate the efficiency of AgNPs-C145
against two bacteria types
Finding the synthesized silver
nanomaterials (AgNPs-C145) ability to Figure 1. FIB-SEM image of silver
against both bacteria types were studied with nanomaterials (AgNPs-C145)
the effects of dose, contact time, pH, and
concentration in order to find the best
condition of each bacteria inhibition by
AgNPs-C145. Jar Test was used for all batch
study, and the measuring the inhibition zone
diameter was reported. The results showed
the formation of prominent zones of
inhibition (ZOI) for both bacteria shown in
Figure 3, and found that the average of ZOI
size of E. coli (0.441 mm) was bigger than
S. aureus (0.354 mm). It meant that AgNPs-
C145 had an antibacterial ability on E. coli
higher than S. aureus.
3.3 Batch tests were used to investigate
Figure 2. EDX analysis for the percentage efficiency of AgNPs-C145 against two
of the constituent atoms of nanomaterials bacteria types
(AgNPs-C145) 3.3.1 The effect of dose
The results showed in Figure 4
which found the inhibition of E. coli to zero
detectable CFU/ml by using 1.5 grams of
AgNPs-C145 whereas S. aureus used 2
times amount of AgNPs-C145 at 3 g. That
meant AgNPs-C145 had the higher inhibited
ability on E. coli than S. aureus.
S. aureus
Figure 4. The effect of dose of inhibited

E. coli abilities of AgNPs-C145 on E. Coli and S.
aureus
Figure 3. The formation of prominent zones
of inhibition (ZOI) for of E. coli and S. 3.3.2 The effect of contact time
aureus Figure 5 showed the effect of contact
time of AgNPs-C145 inhibited abilities on
3.2 Comparison of AgNPs-C145 inhibited both bacteria types. The results showed that
abilities of S. aureus and E. coli bacteria E. coli spent only 1 h to zero detectable
by using the inhibition zone study CFU/mL whereas S. aureus spent 5 h. It
The use of the synthesized silver could conclude that AgNPs-C145 had the
nanomaterials (AgNPs-C145) for inhibiting higher inhibited ability on E. coli than S.
of gram-positive and gram-negative bacteria aureus.
with the bacteria concentration at 106
CFU/mL was used for the inhibition zone
E. Coli and S. aureus, respectively. These
conditions were used for each bacteria type
in the last experiment with varying bacteria
concentration at 104, 105, 106, 107, and 108
CFU/mL to find which concentration was
the best optimum concentration of each
bacteria type.
3.3.4 The effect of concentration
Varying of bacteria concentrations at
104, 105, 106, 107, and 108 CFU/mL and the
best conditions of does, contact time, and
pH experiments were used to study the
effect of concentration.
Figure 5. The effect of contact time of In Figure 7, the result showed that
AgNPs-C145 inhibited abilities on both both bacteria types could inhibit to zero
bacteria types detectable CFU/mL from 104 to 106
CFU/mL. Therefore, the best bacteria
3.3.3 The effect of pH concentration was 106 CFU/mL for
Three different pH ranges (5, 7, and inhibiting of E. coli and S. aureus by using
9) to represent as acid, neutral, and base AgNPs-C145.
were used for the pH effect study of AgNPs-
C145 on both bacteria types. The results
showed in Figure 6. All three pH could
inhibit E. coli to zero detectable CFU/ml
whereas S. aureus was value to zero
detectable CFU/mL at pH 7. From this
result, it could conclude that pH 7 was the
best pH for both bacteria type inhibitions by
using AgNPs-C145.
Figure 7. The concentration effect on the

inhibited abilities of AgNPs-C145 on both
bacteria types
4. Conclusion
This study was successful to
synthesis silver nanomaterials namely
AgNPs-C145 for antibacterial activities
against gram-positive (E. coli) and gram-
Figure 6. The effect of pH on AgNPs-C145 negative (S. aureus) bacteria. The
inhibited abilities on both bacteria types characterization of silver nanomaterials
(AgNPs-C145) was analyzed by using FIB-
From three experiments (does, SEM and EDX. The FIB-SEM’s result
contact time, and pH), the best optimum found the porous structure into the
condition of AgNPs-C145 inhibited abilities synthesized material and EDX’s result found
were 1.5 g, 1 h, pH 7 and 3 g, 5 h, pH 7 for the chemical compositions of C, O, Na, S,
and Ag were 39.5%, 29.8%, 14%, 11.6%, assistance for both bacteria types for this
and 5.1%, respectively. The inhibition zone experiment.
study could confirm that AgNPs-C145 could
inhibit both E. coli and S. aureus with the References
higher inhibition found on E. coli than S. 1. Jiraporn, B.; Reuankeaw, P. J. Thai
aureus. The batch tests showed the best Tradit. Alternative Med. 2012, 10 (1),
optimum condition on does, contact time, 11-22.
pH, and concentration of each bacteria type 2. Michlmayr, H.; Schumann, C.; Wurbs,
were 1.5 g, 1 h, pH 7, 106 CFU/mL and 3 g, P.; Nuno, M.; da Silva, B. B.; Rogl, V.;
5 h, pH 7, 106 CFU/mL CFU/mL for E. coli Kulbe, K. D.; del Hierro, A. M. World
and S. aureus, respectively. This study J. Microbiol. Biotechnol. 2010, 26 (1),
confirmed that AgNPs-C145 can be against 1281–1289.
both gram-positive and gram-negative 3. Uamporn, L.; Pornpen, A.; Wisatre, K.
bacteria. Burapha Sci. J. 2015, 211-217.
4. Kyung-Hwan, C.; Jong-Eun, P.;
Acknowledgements Tetsuya, O.; Soo-Gil, P. Electrochimi.
The authors are grateful for financial Acta 2005, 51 (5), 956–960.
supported by The Office of the Higher 5. Chaloupka, K.; Malam, Y.; Seifalian, A.
Education Commission and The Thailand M. Trends Biotecnol. 2010, 28 (11),
Research Fund grant (MRG6080114), 580-588.
Coordinating Center for Thai Government 6. Chatkaew, P. Ph.D. Silvernano:
Science and Technology Scholarship synthesis production and effective
Students (CSTS) and National Science and against bacteria of plastic compound.
Technology Development Agency Degree Thesis, King Mongkut’s
(NSTDA) Fund grant (SCH-NR2016-122), University of Technology North
and special thanks to Assist. Prof. Dr. Bangkok, 2010.
Jutaporn Swangkeaw for her kindly
Effect of the sample preparation on the composition of hydroxyapatite
derived from waste anchovy fish bone
Hasan Daupor1*, Pateeroh Kuwae1, Anugrah Ricky Wijaya2, Isma-ae Chelong3,
Abd Naser Haji Samoh1
1
Chemistry Major, Faculty of Science Technology and Agriculture, Yala Rajabhat University,
Yala 95000, Thailand
2
Chemistry Department, Faculty of Mathematics and Natural Science, Universitas Negeri Malang,
State University of Malang, Jln. Semarang No. 5 Malang, Indonesia
3
Biology Major, Faculty of Science Technology and Agriculture, Yala Rajabhat University,
Yala 95000, Thailand
*E-mail: hasan.d@yru.ac.th
Abstract:
Calcium hydroxyapatite (HAp) powder was synthesized using waste anchovy fish
bone through maceration process and subsequently heat treatment. The percent yield of
obtaining product was 60%. On XRD analysis, the high purity of HAp phase was formed
after immersing of each HCl and NaOH for 12 h and followed by heat
treatmentat900°Cfor12h, resulting in thetriphasicmaterialofhydroxyapatite [Ca5(PO4)3OH],
magnesium phosphate [Mg2(P2O7)] and whitlockite [Ca5Mg2H2(PO4)14], which contained
weight percentage of 79:11:10 determined by the reference intensity ratio. The HAp powders
had the degree of crystallinity equal to 98% and crystal size equal to 56 mm. The lattice
parameter values of the HAp hexagonal crystal, a and c, were 9.4260 and 6.8845 Å,
respectively. Moreover, the results of the FTIR analysis showed vibrational modes of at
569, 962, 1036, and 1189 cm−1, and the weak peak located at 3571 and 633 cm−1 corresponds
to the vibration of ion in the HAp lattice. Meanwhile, there was also band at 371 cm-1
assigned to the bonding in calcium hydroxyl phosphates. This paper showed the great
potential for the conversion of this by-product into highly valuable compounds.
1. Introduction black color or rather blackcalled budu.

Budu or fish sauce southern Thailand Liquid budu will leave the residue
style is a favorite side dish of the Southern containing fish bones, fins, and also
Border Provinces with various cooked forms contained fish meat. Therefore, it is the
such as kneading rice, mixed with source of calcium, phosphorus, and other
vegetables, peper source and so on, proteins and nutrients which can be used to
especially in Saiburi district, Pattani prepare hydroxyapatite [Ca5(PO4)3(OH)-
Province Industrial production. The budu Hap].1 The protein and other nutrients were
bottle is sold in the form of One Tumbon decomposed when calcined at high
One Product (OTOP) there are two brands: temperature up to 900 °C resulting in HAp
Budu Heng and Budu Yiseng. Budu is based growth.2
on digestion of enzymes and HAp has been synthesized from
microorganisms from naturally occurring various sources such as animal bone. Pulp
fish, which uses small fish such as and paper tuna bones (Thunnus obesus) were
Stolephorus indicus, Clupeoides sp., Sardine used to synthesis hydroxyapatite. It was
sp., Pinialo pingalo or Decapterus russelli. washed with hot tap water to remove the
These fishes are taken without removingit’s remaining fish meat, followed with NaOH,
tripe and by fermentation with sea salt in the acetone to remove dirt, proteins, fat and
ratio of 3:1 for 8-12 months. Fish and bone other organicsubstances. The collagen was
are fermentated and produced a liquid of hydrolyzed with 2 M NaOH for 5 h at 250
°C and finally burned at 900 °C for 5 h for To hydrolyze collagen and other organic
completely removing the organic matter moieties, the alkaline hydrolysis method was
from the bone and furnish Hap.1 Calcium followed. Briefly, 20 g of grounded anchovy
phosphates are a class of compounds with bone was treated with 1 M HCl with
very high value due to their properties and continuously stirring for 12 h (solid liquid
technological applications. Several calcium ratio 1:2). After that, the treated sample was
phosphates are well known for their use as strirred in 1 M NaOH for 12 h. The mixture
biomaterials.3 Hydroxyapatite, the major was filtered in a suction pump with
component of human bone, is probably the continuous washing with water and dried in
most important one; it has very high an oven at 100 ºC. 1 g of the dried fish bone
biocompatibility, and for this reason it is was placed in a silica crucible and subjected
widely used in many applications, e.g. fillers to a temperature of then calcined in an
for improving the properties of dental electrical muffle furnace at 900 °C for 12 h.
adhesives, drug delivery agents, and The white powder of HAp was obtained.
biosensor applications.4 2.3 Sample characterization
Recently, HAp production from X-ray Diffraction (XRD) was
different sources of bones such as Japanese applied to monitor the phase composition
sea bream,5 Brazilian river fish6 and Atlantic features of the samples after calcination at
swordfish7using a subcritical water process 900 ºC. The sample spectra were collected
and alkaline hydrothermal hydrolysis was using a Philips PW 3710 powder
reported.8 All possible sources were diffractometer (PHILIPS X'Pert MPD, The
successfully employed to obtain HAp using Netherlands), Cu Kα (Ni filtered) radiation
thermal treatment methods. In this paper, we λ=1.5406 Å. Intensity data were collected by
report the use of anchovy fish bone collected the step counting method in the 2θ range of
from the budu residue to convert into HAp, = 10-90º. Fourier transform infrared
an alkaline-hydrolysis process was spectroscopy (FT-IR) was used to
employed. The bones were also treated in investigate the chemical composition of the
solution prior to calcination to change their control and treated samples. The FT-IR
composition. This sample, never reported spectra were performed using a Spectrum
before for this method of preparation, was JASCO 6800 spectrometer in the range of
employed to achieve materials. 400-4000 cm-1. The morphological and
microstructure analysis of all samples was
2. Materials and Methods carried out using the instrument Quanta 400
2.1 Materials scanning electron microscope (SEM, Quanta
Anchovy fish bone as a waste was 400, FEI). The elemental composition was
obtained from the end of the budu analyzed by an energy dispersive
production process from Budu Yiseng spectrometer (EDS) equipped with the SEM
Factory, Amphor Saiburi, Pattani Province, microscope system.
Thailand. Sodium hydroxide 1 M (BDH),
hydrochloric acid 1 M (BDH). All chemicals 3. Results and Discussion
were reagent grades and used without 3.1 XRD result
further purification. The relative content of HAp,
2.2 Synthesis of hydroxyapatite NaCaPO4 and whitlockite phases were the
The anchovy fish bone was collected ratio closest to the 79:11:10 ratios. Figure 1
from budu Yiseng residue and washed with showed powder XRD pattern of calcined
water to remove the traces of meat and skin. anchovy fish bones at 900 ºC. The
After thorough washing the bones were diffraction peaks can be assigned to the
dried at 60 ºC and ground in a mortar pestle. hexagonal HAp (JCPDS No. 01-084-1998).
Figure 1. The XRD pattern of sample
Table 1. Calcination data and HAp crystal size D (nm) and lattice parameters calculated from
XRD using the Scherrer equation
Initial After Average Unit cell
Yield
weigth calcination a-exes (Å) c-exes (Å) Crystal size volume (Å3) Xc (%)
(%)
(g) (g) (nm)
1.0 0.6 60 9.4260 6.8845 56 529.73 98
More specifically, the diffraction peaks at where Dhkl is the average crystallite size, K
= 25.8º, 31.8º, and 32.9º are consistent is the broadening constant, λ is the
with (002), (121), and (300) reflections of wavelength of Cu Kα radiation (1.5406 Å),
hexagonal Hap9 respectively. In addition, β1/2 is the full-width at half-maximum of
sodium calcium phosphate (NaCaPO4; (002) peak, and θ is the diffraction angle.
JCPDS No. 01-084-1998) and calcium The lattice parameters (a and c) of
magnesium phosphate (Ca18Mg2H2(PO4)14; HAp were calculated by the method of least
JCPDS No. 01-084-1998), as well as HAp, squares using the following equation4:
were detected. (
The fraction of crystalline phase (3)
(Xc%) in bioceramic powders can be
evaluated by the following equation:10 where d is the spacing between the planes in
( the atomic lattice. The volume (V) of the
(1) hexagonal unit cell of each HAp formulation
where Xc% is the crystallinity degree, was calculated using the below relation:9
V112/300 is the intensity of the hollow between (4)
(112) and (300) diffraction peaks, I300 is the
intensity of (300) diffraction peak. The lattice parameters on a-axis
The average crystallite size was increased but on c-axis decreased upon
calculated from the broadening in the XRD impurities addition, and also the volume of
pattern according to the Scherrer’s the lattice undergoes a large contraction as
equation:11 shown in Table 1. This behavior is due to
( the many compositions contained in unit
(2) cell, which is in agreement with the clearly
demonstrated NaCaPO4 and whitlockite
were structurally incorporated into HAp (a)
crystals.
3.2 FTIR
In FTIR pattern (Figure 2c), the low
intensity peak around 3567-3574 cm-1 was
due to stretching vibration of O-H groups.
The bands corresponding to 1025 cm-1
signify the stretching vibration of P-O
bonds. The bands at 601 and 548 cm-1
representedthe bending mode of P-O groups.
The band of the carbonyl group appeared at
1744 cm-1. The band at 371 cm-1 was due to
the presence of Ca-O bonding. In the case of
uncalcined sample (Figure 2a), the peaks at (b)
2926 and 2854 cm-1 were assigned to
vibrations of C-H groups, and the peaks at
1655, 1542, and 1165 cm-1 were assigned to
amide I and II bands that are not found in
the treated bone by the proposed methods.
These peaks were derived from the organic
compounds in bone.12
(c)
Figure 2. Infrared spectra of (a) raw bone,

(b) the reference spectrum of calcium
phospate from Sigma, (c) synthesized HAp
sample
3.3 SEM-EDX
Formation of HAp particles can be (d)
observed in the SEM image presented in
Figure 3. It can be seen that HAp has formed
as much large irregular in the order of
microns. The average particle size of HAp
powders was determined from these
micrographs with approximately 0.5 µm. In
high magnification of Figure 3c, clearly,
some hexagonal morphologies of HAp were
obtained with a crystallite size of 0.5 µm
high, 1 µm wide corresponding to the lattice Figure 3. (a)-(c) SEM images of sample
paremeters of a-axes longer than c-axes. with different magnification, (d) EDX
spectrum of its sample
Figure 3d showed the typical EDX 33, 3111-3120.
spectrum of HAp powder, which confirmed 2. Cui, H.; Yang, X.; Qin, J.; Tang, H.;
the impurity of the material by showing the Liu, H.; Li, Y. Micro & Nano Lett.
Na and Mg elements in the spectrum. 2012, 7, 1292-1295.
Moreover, the Ca/P atomic ratios in the 3. Dorozhkin, S. V. Biomater. 2010, 31,
synthesized HAp powders determined 1465-1485.
through EDS analysis closed to 1.67 of the 4. Pal, A.; Paul, S.; Roy, A.; Krishna, V.;
natural enamel. Das, M.; Sinha, A. Mater. Lett. 2017,
203, 89-92.
4. Conclusion 5. Ozawa, M.; Suzuki, S. Commun. Am.
This work showed the studied Soc. 2002, 85, 1315–1317.
anchovy fish bone from budu residue 6. Coelho, T. M.; Nogueira, E. S.;
samples calcined at 900 ºC exhibit a most Weinand, W. R.; Lima, W. M.;
promising chemical composition and Steimacher, A.; Medina, A. N.; Baesso,
structure that could be exploited to provide M. L. J. Appl. Phys. 2007, 101, 084701
good alternatives to synthetic 7. Boutinguiza, M.; Pou, J.; Lusquiños, F.;
hydroxyapatite. The lattice parameter and Comesaña, R.; Riveiro, A. Phys.
the volume began to increase, and this is Procedia 2016, 12, 54–59.
may be due to the crowdeness of the other 8. Nasser, A. M.; Barakat, K. A.; Khalil
compound in the HAp crystal. Faheem, A.; Sheikh, A. M.; Omran B.
G. Mater. Sci. Eng. C 2008, 28, 1381-
We acknowledge the Budu Yiseng 9. Kumar, G. S.; Rajendran, S.; Sekar,
Factory, Amphor Saiburi, Pattani Province, Karthi, S.; Govindan, R.; Girija, E. K.;
Thailand, for supporting the budu residue Karunakaran. G.; Kuznetsov. D. MRS
samples. Our appreciation is also extended Commun. 2017, 1-6.
to Mr. Tata Kwawi Mbinglo for assistance 10. Figueiredo, M.; Fernando, A.; Martins,
with English corrections. G.; Freitas, J.; Judas, F.; Figueiredo, H.
Ceram. Inter. 2010, 36, 2383-2393.
References 11. Goto, T.; Sasaki, K. Ceram. Inter. 2014,
1. Ferraro, V.; Carvalho, A. P.; Piccirillo, 40, 10777–10785.
C.; Santos, M. M.; Castro, P. M. L.; 12. Venkatesan, J.; Qian, Z. J.; Ryu, B.
Pintado M. E. Mater. Sci. Eng. C 2013, Biomed. Mater. 2011, 6, 035003.
Synthesis of mesoporous silica with site-isolated acid and base – based
catalysts and their bifunctional application in a one-pot cascade reaction
Apichat Klayanon, Wipark Anutrasakda*
Department of Chemistry, Faculty of Science, Chulalongkorn University, Pathumwan,
*E-mail: wipark.a@chula.ac.th
Abstract:
In this work, mesoporous silica supported acid (-SO3H)-base (-NH2) catalysts were
synthesized using a combination of co-condensation and post-synthetic grafting methods. The
prepared acid-base catalysts were characterized by XRD, FT-IR, and N2 adsorption-
desorption techniques. XRD and N2 adsorption-desorption indicated that the synthesized
mesoporous silica supported acid-base catalysts preserved the hexagonal structure with the
surface area in the range of 609-691 m2g-1. The S=O and N-H stretching frequencies, as
indicated by FT-IR, occurred at 1,032 and 1,536 cm-1, respectively. The acid and base
concentrations of the catalysts, examined by back titration method, were in the range of 0.89-
1.10 mmol g-1 and 0.38-0.57 mmol g-1, respectively. The synthesized catalysts were tested for
a one-pot deacetylation-aldol reaction of benzaldehydedimethylacetal (A) with malononitrile
to produce benzylidenemalononitrile (C). Under the 5 h reaction time at 90 C, the reaction
gave a 71% yield of benzylidenemalononitrile as the sole product, suggesting that the
synthesized materials are ones of efficient catalysts for one-pot cascade reactions requiring
both acid and base active species.
1. Introduction containing sulfonic acid and amine groups.5

A cascade reaction is an interesting These bifunctional catalysts showed various
subject in catalysis due to many advantages degrees of success in cascade reactions.
such as a decrease in the amount of waste, However, target products were obtained in
low operating cost and environment- low yield. Therefore, the development of
friendliness.1 This process can be achieved bifunctional catalysts is still needed.
by using multifunctional catalysts such as In the present study, we synthesized
acid-base catalysts. Acid-base bifunctional bifunctional mesoporous silica nanoparticle
catalysts have gained a great attention; with sulfonic acid as the acid source on its
however, a major problem is that acids and internal surface and using aminosilanes as
bases can neutralize each other when the amino source on its external surface. The
brought together. In order to avoid this obtained catalysts were used in a one-pot
problem, incorporating acid and base deacetylation-aldol reaction of benzaldehyde
functional groups onto solid supports has dimethylacetal (A) with malononitrile to
been developed. One of the commonly used produce benzylidenemalononitrile (C)
supports is mesoporous silica, which has a (Scheme 1).
high surface area and tunability of its pore
size.
Bifunctional acid-base catalysts on
solid supports have been reported such as
phosphotungstic acid immobilized on
amine-grafted graphene oxide,2 SBA-15 Scheme 1. One-pot deacetylation-aldol
containing benzenesulsulfonic acid and a reaction
secondary amine4 and MCM-41 silica
2. Materials and Methods APTES was added into the solution under
2.1 General stirring for 6 h. Finally, the product was
The chemicals consisting of cetyltri- filtered, washed with toluene, and dried at
methyl ammonium bromide (CTAB), tetra- 120 C overnight and designated as SHxN2-
ethyl orthosilicate (TEOS), (3-amino- CTAB, where 2 represents the amount (in
propyl) triethoxysilane (APTES, N), 3- mmol) of APTES.
(trimethoxysilylpropyl) diethylenetriamine In order to convert the thiol
(DETTMS, NNN), and 3-mercapto- functional groups to sulfonic acid groups,
propyltrimethoxysilane (MPTMS) were each SHx-N2-CTAB material was refluxed
purchased from Sigma-Aldrich. 3-(2- in a mixed solution of glacial acetic acid and
Aminoethylamino) propyltrimethoxysilane H2O2 at 100 C for 6 h. Then, the solid was
(AAPTMS, NN) was purchased from Alfa filtered, washed with deionized water, and
Aesar. Acetic acid, toluene, hydrogen dried at 120 C overnight. The template
peroxide solution, hydrochloric acid and remained in the materials was then removed
methanol were acquired from Merck. by extraction with acidic methanol at 60 C
Standard solution of benzaldehyde- for 6 h. Finally, the solid products were
dimethylacetal, benzaldehyde, benzylidene filtered, washed with deionized water and
malononitrile, p-xylene, and malononitrile dried at 150 C overnight. The final
were purchased from Sigma-Aldrich. All materials were designated as SAxN2.
commercial chemicals were used without 2.2.2 Synthesis of SA0.5N4
further purification. SA0.5N4 was prepared using the same
2.2 Synthesis of catalysts manner as Section 2.2.1 but 4 mmol of
2.2.1 Synthesis of SAxN2 APTES was used instead.
Bifunctional acid-base catalysts was 2.2.3 Synthesis of SA0.5NN4 and
prepared by a co-condensation method to SA0.5NNN4
functionalize the internal surface of SA0.5NN4 and SA0.5NNN4 were
mesoporous silica with acid groups and prepared using the same manner as Section
subsequent functionalize the external 2.2.2 but using AAPTMS and DETTMS
surface with the base groups. Briefly, 4 g of aminosilanes instead of APTES,
CTAB was dissolved in a solution of 960 respectively.
mL of H2O and 14 mL of 2 M NaOH with 2.2.4 Catalytic testing
stirring until the solution became clear. Benzaldehydedimethylacetal (0.15
Then, 20 mL of TEOS was added dropwise mmol), malononitrile (1.2 mmol), p-xylene
to the above solution under continuous (0.5 mmol), and each catalyst (40 mg) were
stirring for 1 h at 80 C. After that, a desired added in a two-necked round bottom flask
amount of MPTMS (0.5, 1, or 2 mmol) was equipped with an oil bath, schlenk line, and
added to the reaction mixture and the a magnetic stirring bar under nitrogen
mixture was stirred for an additional 2 h. atmosphere. The reaction mixture was
Finally, the product was filtered, washed magnetically stirred at 90 C for 0.5 h. The
with deionized water, and dried at 120 C catalyst was separated from the reaction
overnight. The obtained materials were mixture by centrifugation, and the filtrate
designated as SHx-CTAB, where x = 0.5, 1, was analyzed by a Varian CP-3800 gas
and 2 represents the initial amount (in chromatograph equipped with a flame
mmol) of MPTMS. ionization detector (FID).
In order to selectively functionalize 2.3 Catalyst characterization
the external surface of mesoporous silica IR spectra were recorded on the
with APTES by post-synthesis grafting, the Impact 410 (Nicolet). XRD patterns of the
as-made solids with template remained samples were measured using the DMAX
inside mesoporous channels were refluxed 2200/Ultima (Rigaku) with Cu Kα radiation.
in toluene at 120 C. Then, 2 mmol of Surface area analyzer was measured using
the BELSORP-mini (BEL Japan). A back 3.1.2 N2 sorption isotherms analysis and
titration method was used to measure the acidity-basicity
amount of acid and base of the synthesized The textural properties of the
materials.7 synthesized materials were examined by N2
sorption technique. Surface areas of the
3. Results and Discussion synthesized materials were calculated from
3.1 Characterization of materials the BET method while their pore diameters
3.1.1 Powder X-ray diffraction were calculated from the BJH equation. The
The X-ray diffraction patterns of results were listed in Table 1. Among the
SH0.5-CTAB, SH0.5N4-CTAB and SA0.5-N4, materials with the same base loading (2
representing all the synthesized materials are mmol) but different acid loadings (0.5, 1,
shown in Figure 1. The patterns showed and 2 mmol), SA0.5N2 had the highest
three peaks at 2θ = 2.0-2.2, 3.7-3.8 and 4.3- surface area of 691 m2 g-1 and total pore
4.4, corresponding to (100), (110), and (200) volume of 0.35 cm3 g-1. In the case of SA1N2
reflection planes of hexagonal MCM-41 and SA2N2, their surface areas and total pore
silica,8 respectively. This information volumes were decreased because their
suggests that the crystallographic ordering mesopores were occupied with higher
of the mesopore was retained after all steps amounts of acid-organic groups. Moreover,
of the modification.9 the effect of the number of amino groups
was investigated for SA0.5N4, SA0.5NN4, and
SA0.5NNN4 catalysts. The results show that
surface area, pore diameter and total pore
volume were decreased with increasing the
number of amino groups mainly because
SA0.5NNN4 contained the bulkier
aminosilane molecules than SA0.5NN4,
SA0.5N4, respectively.
The amounts of acid and base loaded
in catalysts were determined using the back
titration method as shown in Table 1. The
results confirmed the coexistence of acid
Figure 1. XRD patterns of representative and base in all samples. The acidity of
synthesized materials SA0.5N2 was found to be 0.89 mmol g-1
which was lower than that of SA1N2 (0.96
mmol g-1), and SA2N2 (1.10 mmol g-1)
Table 1. Textural properties, acidity, and basicity of the synthesized materials

BET surface Pore size diametersa Total pore volume Acidityb Basicityb
Materials (mmol g-1) (mmol g-1)
area (m2g-1) (nm) (cm3g-1)
SA0.5N2 691 2.05 0.35 0.89 0.38
SA1N2 660 1.97 0.32 0.96 0.40
SA2N2 615 2.09 0.31 1.10 0.42
SA0.5N4 660 2.09 0.34 0.89 0.47
SA0.5NN4 646 2.01 0.32 0.90 0.50
SA0.5NNN4 609 1.99 0.30 0.89 0.57
a
Calculated from the N2 sorption isotherms by BJH method
b
Calculated from back titration
while the amount of base remained roughly
constant (~0.4 mmol g-1). Additionally,
when the number of amino groups was
increased in SA0.5N4, SA0.5NN4, and
SA0.5NNN4 catalysts, the amount of base
was also increased: 0.47, 0.50, and 0.57
mmol g-1, respectively. This occurrence
might be explained that the increasing of
basicity in catalysts resulted from increasing
the number of amino groups from
aminosilanes used.
3.1.3 Fourier transform infrared Figure 2. FT-IR spectra of the synthesized
spectroscopy (FT-IR) materials
The functional groups present in
mesoporous silica supported acid-base followed by the base-catalyzed aldol
catalysts were characterized using FT-IR. condensation of B with malononitrile to
The peaks of the template in the range of produce benzylidene malononitrile (C) in
2850-3000 and 1460 cm-1 corresponded to the second step.
the C–H stretching and bending vibrations, 3.2.1 Effect of acid loading
respectively. These peaks disappeared or Three catalysts containing different
almost disappeared in the spectra of all acid loadings (0.5, 1, and 2 mmol) with
catalysts, suggesting that the template was fixed base loading (2 mmol) were tested to
efficiently removed after extraction.9 The compare their activity for the one-pot
peak at 1032 cm-1 was assignable to the reaction of A with malononitrile using the
asymmetric stretching vibration of S=O reaction conditions as follows: 90 °C and 0-
bands and 1125 cm-1 was correlated to the 5 h. The results show that, in the first step,
symmetric vibration of S=O bands. These all catalysts could convert A to B in 100%
results confirmed the presence of sulfonic conversion within 5 h (Figure 3a). However,
groups into the mesoporous silica. In it is found that SA2N2 could completely
addition, an absorption of 610 cm-1 was convert A within 0.5 h while the conversion
observed, which was attribute to the bending of A using SA0.5N2 and SA1N2 gradually
vibration of -OH groups of SO3H groups.10 increased and took about 4 h to reach 100%
The presence of two peaks at 1536 and 801 conversion. This finding can be explained
cm−1 could be attributed to the symmetric by the acidity of the catalysts. Specifically,
N−H stretching band and the N-H out-of- the acidity of SA2N2 (1.10 mmol g-1) was
plane bending vibration from the NH2 higher than that of SA1N2 (0.96 mmol g-1)
group, respectively.11 These results and SA0.5N2 (0.89 mmol g-1), leading to
confirmed that the amino groups were higher conversion of A.
successfully grafted on the surface of At the same time, yield of B was also
materials in all samples. investigated (Figure 3b). The result suggests
3.2 Catalytic activity that in the case of SA1N2 and SA0.5N2, the
The catalytic activity of the yield of B gradually increased in the first 4 h
synthesized acid-base catalysts in the one- before slightly decrease in the last 1 h of
pot deacetalization-aldol reaction was SA0.5N2 and SA1N2 while SA2N2 could
investigated. Mechanistically, the acid- produce B in nearly 100% yield within 0.5 h
catalyzed deacetalization of benzaldehyde- before slowly decreasing.
dimethylacetal (A) to produce
benzaldehyde (B) occurred in the first step,
a b c
Figure 3. Effect of acid loading in catalysts for the one-pot reaction of A with malononitrile
(a) conversion of A, (b) yield of B, (c) yield of C, conditions: 90 °C and 0-5 h
In the second step, yield of C were Figure 4a. This could be explained by the
observed as shown in Figure 3c. For all similar amount of acidity (~0.9 mmol g-1) of
catalysts, the yield of C gradually increased each catalyst. The intermediate B was also
during the 5 h reaction time. However, it is monitored as shown in Fig. 4b. For all
seen that the reactivity of SA2N2 to produce catalysts, yield of B were gradually
C is poorer than the other two catalysts increased when increasing the reaction time.
probably because high density of B (~80%) However, the yield of B from reactivity of
inside pore could not rapidly diffuse to react SA0.5N4 was slowly decreased after the first
with amine groups outside the pore to 3h of reaction time probably because it
produce C. This finding was supported by could easily diffuse to react with amine
the lowest surface area of SA2N2 (615 m2 g- functional groups outside the pores. This
1
), which was lower than that of SA1N2 (660 result was confirmed by the largest surface
m2g-1), and SA0.5N2 (691 m2g-1), area of SA0.5N4 (660 m2 g-1), which was
respectively. Therefore, 0.5 mmol of acid higher than that of SA0.5NN4 (646 m2g-1),
loading was used for further study. and SA0.5NNN4 (609 m2g-1).
3.2.2 Effect of aminosilane types In the second step, yield of C was
The effect of different aminosilanes observed as shown in Figure 4c. All
was investigated by varying the number of catalysts showed a good catalytic activity
amino groups. The conversion of A and the that C yield was gradually increased when
yield of products are shown in Fig. 4. In the the reaction time increased. Interestingly,
first step, when the number of amino groups SA0.5N4 exhibited the higher reactivity than
was increased from one (N) to three (NNN), the other two catalysts because it contained
all catalysts could convert A to B in a the smallest aminosilane molecules, which
similar trend. The conversion of A was were less steric hindrance in the reaction to
virtually completed within 5 h as shown in form C.12
a b c
Figure 4. Effect of amino-organosilane types for the one-pot reaction of A with malononitrile
(a) conversion of A, (b) yield of B, (c) yield of C, conditions: 90 °C and 0-5 h
Therefore, the efficiency of catalytic Acknowledgements
activity of SA0.5N4 containing one amino This work received financial support
group gave the highest yield of the target from the Research Grant for New scholar
product C. CU Researcher’s Project from the
3.2.3 Catalyst reusability Ratchadaphiseksomphot Endowment Fund
For practical applications of of Chulalongkorn University.
heterogeneous catalysts, the reusability of
the catalysts is an important factor to References
investigate. Based on the above results, the 1. Felpin, F. X.; Fouquet, E. Chem. Sus.
SA0.5N4 catalyst showed the highest catalytic Chem. 2008, 1 (8-9), 718-724.
activity in this study. Therefore, SA0.5N4 was 2. Zhang, W.; Zhao, Q.; Liu, T.; Gao, Y.;
chosen for catalyst reusability test. Staring Li, Y.; Zhang, G.; Zhang, F.; Fan, X.
with the recovery of catalyst by using Indus. Eng. Chem. Res. 2014, 53 (4),
centrifugation, then washed with ethanol, 1437-1441.
and reused in the next run. As shown in 3. Zeidan, R.; Davis, M. J. Catal. 2007,
Figure 5, the results demonstrate that the 247 (2), 379-382.
catalyst can be reused for four times without 4. Shao, Y.; Guan, J.; Wu, S.; Liu, H.; Liu,
a significant decrease in activity, suggesting B.; Kan, Q. Micropor. Mesopor. Mater.
that covalently attached bifunctional groups 2010, 128 (1-3), 120-125.
were stable and robust. 5. Shang, F.; Sun, J.; Wu, S.; Yang, Y.;
Kan, Q.; Guan, J. Micropor. Mesopor.
Mater. 2010, 134 (1-3), 44-50.
6. Huang, Y.; Xu, S.; Lin, V. S. Y. Angew.
Chem. Inter. Ed. 2011, 50 (3), 661-664.
100 100 98 97 7. Varadwaj, G. B. B.; Rana, S.; Parida,
K.; Nayak, B. B. J. Mater. Chem. A
2014, 2 (20), 7526.
8. Huh, S.; Wiench, J. W.; Yoo, J.-C.;
Pruski, M.; Lin, V. S. Y. Chem. Mater.
2003, 15 (22), 4247-4256.
9. Shang, F.; Sun, J.; Wu, S.; Yang, Y.;
Figure 5. Reusability test using SA0.5N4 Kan, Q.; Guan, J. Micropor. Mesopor.
catalyst Mater. 2010, 134 (1), 44-50.
10. Yadav, G. D.; Murkute, A. D. Adv. Syn.
4. Conclusion Catal. 2004, 346 (4), 389-394.
Heterogeneous bifunctional acid- 11. Zhang, F.; Jiang, H.; Li, X.; Wu, X.; Li,
base catalysts were successfully synthesized. H. ACS Catal. 2013, 4 (2), 394-401.
SA0.5N4 showed good catalytic activity and 12. Suzuki, T. M.; Nakamura, T.;
reusability of the catalyst in one-pot Fukumoto, K.; Yamamoto, M.;
deacetylation-aldol reaction A with Akimoto, Y.; Yano, K. J. Mol. Catal. A:
malononitrile. Specifically, high conversion Chem. 2008, 280 (1), 224-232.
of A (98%) and high yield of C (71%) were
achieved within 5 h which was higher than
that of SA0.5NN4 and SA0.5NNN4. Moreover,
SA0.5N4 could be reused for at least 4 times
without significant loss in activity.
Formulation of facial cream and body mist from avocado oil
Thitiphan Chimsook1*, Dorngporn Amornlerdpisan2
1
Department of Chemistry, Faculty of Science, Maejo University, Chiangmai 50290, Thailand
2
Faculty of Fisheries Technology and Aquatic Resources, Maejo University, Chiangmai 50290, Thailand
*E-mail: thitiphan.cs@gmail.com
Abstract:
The formulation of facial cream and body mist from avocado oil can help to value-add
of fresh avocado (Persea americana) and increase marketing channel. Pure cold pressed
avocado oil containing high nourishing oil can be extracted from fresh avocado in high
quantity and with low cost. The chemical properties of the cold pressed avocado oil were
examined. The results showed that acid value, free fatty acids and peroxide values were 1.20
mg KOH/g, 0.82 % as oleic acid and 5.56 meq O2/kg, respectively. The physical properties,
stability test and testing by volunteers of cosmetic products from cold pressed avocado oil
including facial cream and body mist were investigated. The texture of white facial cream
(pH = 6.29 at room temperature) was smooth and creamy. For the body mist, the texture of
white body mist (pH = 6.22 at room temperature) was light. Both white facial cream and
body mist were tested by 50 volunteers for one month. The results of testing by volunteers
revealed that facial cream and body mist showed an increased hydration, elasticity and no
skin irritation when compared to other skin care cosmetics.
1. Introduction These days we have so many types of skin

Avocado oil extracted from avocado moisturizers for skin treatments because it is
pulp has been known to utilize as a cosmetic always safe to use natural products likewise
material. The minimal refinement of cold natural moisturizers seem to show much
pressed avocado oil preserves more of the better results with very little or no side
important plant sterols, chlorophyll, vitamin effects. Natural oil such as cold pressed
E and other antioxidants. The bioactivity of avocado oil and argan oil can be used in
avocado oil has been reported1 for example formulating the cosmetic for face and body
antioxidant activity, total phenolic contents including facial cream, facial moisturizer
and vitamin E. For vitamin E, it is found to cream, lotion and also body mist,
be effective in smoothening up the skin respectively.
stature thereby maintaining a silky skin tone. Basically cream is emulsion which is
High level of vitamin E can prevent the skin relatively stable mixtures of oils and fats and
from inflammations and itchiness thereby water. These are generally made by mixing
helping skin to maintain its health and oil soluble and water soluble substances
softness. Avocado oil can prepare from both together in the presence of an emulsifying
natural and chemical methods. Organic oil agent. Cosmetics facial creams are marketed
extracted from the natural method is and sold on the basis of their function which
considered as extremely healthy and can be is generally advertised on the packaging
stored for longer periods. Nowadays, cold which contains them.2 Body mist or body
pressed avocado oil is the famous active spray is lightweight and easily absorbed, this
ingredient in many cosmetic skin care or mist formula helps to soften and moisturize
hair care products because it has one of the the skin.2,3
best moisturizing ability among all oils and In this work, cold pressed avocado
that is exactly why it is used in many of oil was used as an ingredient for formulating
those famous cosmetic products. the facial cream and body mist. The physical
and chemical properties of facial cream and
© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) NP1
body mist were investigated for example stirred after each addition. It was
pH, color and viscosity. These cosmetic recommended to package into light
products were tested by volunteers. protected bottles as shown in Figure 1. The
physical and chemical properties of cream
2. Materials and Methods were investigated.5 The stability test was
2.1 Extraction of avocado oil performed at least 3 months at 4 C, 45 C
The cold pressed avocado oil was and room temperature. Moreover, the
extracted from the 20 kg of fresh pulp heating-cooling cycle was determined for
avocado (Persea americana) using hot air the avocado body mist formulation.6-8
drying process4 and natural oil cold pressed
machine. The physical and chemical
properties of cold pressed avocado oil were
examined before used as the ingredient to
formulate the facial cream and body mist.
2.2 Formulation of avocado facial cream
and avocado body mist
The formulation of avocado facial
cream was presented in Table 1. All
chemical ingredients have 4 phases (A, B, C
and D). All phases were prepared separately.
This formulation shown in Table 1 was the
best ingredient compared to the other
formulations which contained no avocado
oil and 2% avocado oil.
Table 1. Formulation of avocado facial Figure 1. The package of avocado facial

cream cream
Phase INCI name %(w/w)
A Aqua 83.0 The formulation of avocado body
Hyaluronic acid 2.0
mist was presented in Table 2. All chemical
Panthenol 1.3
Transexamic acid 2.0
ingredients have performed in 5 phases (A,
Dipotassium glycyrrhizate 0.1 B, C, D and E). All phases were prepared
B Sodium separately. This formulation shown in Table
acrylates/Beheneth-25 2 was the best ingredient compare to the
methacrylate crosspolymer other formulations which contained no
(and)
avocado oil and 2% avocado oil.
Hydrogenated polydecene
(and) Lauryl glucoside 3.0
C Avocado oil 5.5 Table 2. Formulation of avocado body mist
VCIP 0.6 Phase INCI name %(w/w)
Squalen 1.0 A Aqua 80.0
D Caprylhydroxamic Acid Trisodium
(and) 1,2-Hexanediol (and) ethylenediamine
Propanediol 1.5 dissuccinate 0.1
Acrylate
crosspolymer/aqua 8.0
Firstly, all of the ingredients in each
B Sodium hydroxide 0.2
phase were blended well until dissolved. C Hydrogenate castor oil 5.1
Phase B was mixed in phase A and stirred D Caprylhydroxamic acid 1.4
using a homogenizer at room temperature to Ethylhexylglycerin 0.1
ensure that they were all fully incorporated. Methylpropanediol 0.1
Secondary, the residues of phase C and D E Vitamin E 1.0
were added in mixed phase A and B and Avocado oil 4.0
Firstly, all of the ingredients in each appearance of your skin. Not only is
phase were combined. Phase A was mixed avocado oil extremely good at hydrating and
and stirred using a homogenizer at room softening your skin, is it also often
temperature to ensure that they were all fully recommended as a beneficial for
incorporated. Secondly, the residues diminishing age spots and smoothing out
including phase B, C, D and E were added fine lines and wrinkles because avocado oil
in phase A one by one, stirred after each is capable of penetrating deep into the skin
addition. The avocado body mist was unlike other oils and this helps in achieving
packed into bottle with a spray dispenser as quicker results. This helps in making skin
shown in Figure 2. The physical and soft and hydrated. One of the amazing
chemical properties of body mist were properties of avocado oil is humectant,
investigated.5 The stability test was which prevents the skin from drying out and
performed at least 3 months at 4 C, 45 C allows the skin to be on hydrated mode for
and room temperature. Moreover, the longer period.
heating-cooling cycle was determined for 3.2 Formulation of avocado facial cream
the avocado body mist formulation.6,7,8 and avocado body mist
Avocado facial cream with a
combination of avocado oil and other
ingredients was formulated and examined
the stability of product. The texture and
color of avocado facial cream and avocado
body mist were shown in Figure 3a and
Figure 3b. The texture of white facial cream
(pH = 6.29 at room temperature) was
smooth and creamy. For the body mist, the
texture of white body mist (pH = 6.22 at
room temperature) was light. The physical
properties and stability test of facial cream
and body mist were presented in Table 1-4.
Figure 2. The package of avocado body

mist

3.1 Extraction of avocado oil (a)
Avocado oil was produced using
cold pressed machine after dried using hot
air drying. The color of avocado oil was
light green. The chemical properties of
avocado oil was investigated and found that
acid value, free fatty acids and peroxide
values were 1.20 mg KOH/g, 0.82 % as (b)
oleic acid and 5.56 meq O2/kg, respectively. Figure 3. The texture of (a) avocado facial
The percentage yield of avocado oil was cream and (b) avocado body mist
20% and the cost was 120 baht/20 kg. In
general, the cold pressed avocado oil has an
immediate effect on the texture and
Table 1. Stability study of facial cream by heating and cooling of 6 cycles
Viscosity*
Storage cycle pH Color Texture Feel on skin
(mPas)
1 6.33 white smooth, creamy soft 4988.8
2 6.38 white smooth, creamy soft ND
Viscometer (Brookfield, DV-1, R5 probe, 60 rpm, %wresting square = 74.8%)
ND = non detected
Table 2. Stability study of body mist by heating and cooling of 6 cycles

Viscosity*
Storage cycle pH Color Texture Feel on skin
(mPas)
1 6.48 white light soft 1142.7
2 6.47 white light soft ND
Viscometer (Brookfield, DV-1, R5 probe, 60 rpm, %wresting square = 74.8%)
ND = non detected
Table 3. Study of stability of avocado facial cream at various temperatures for 45 days
Condition pH Color Texture Feel on skin
Room 6.29 white smooth, creamy soft
4 C 6.27 white smooth, creamy soft
45 C 6.28 white smooth, creamy soft
Table 4. Study of stability of avocado body mist at various temperatures for 45 days
Condition pH Color Texture Feel on skin
Room 6.22 white light soft
4 C 6.21 white light soft
45 C 6.23 white light soft
Most of cosmetic products are mist delivered instant moisture for achieving
emulsions that function as delivery vehicle soft and silky results. Body mist infused the
to the skin and are classified according to skin with the nourishing properties of active
different body parts to be applied i.e., skin, ingredient and avocado oil. The weightless
teeth, hair and mouth. Emulsions are formula absorbs quickly to lock in moisture,
heterogeneous systems and consist of whilst balanced emollients soothe dry areas
mixture of two immiscible liquids i.e., and improve skin comfort. The mist can be
aqueous phase and oil phase, and system is sprayed on wet or dry skin after showering
stabilized by the presence of third substance as an alternative to traditional body lotion.
known as emulsifying agent or emulsifier.9 Stability study of facial cream and body mist
In this work, avocado facial cream and body was done by heating and cooling method.
mist were performed in order to formulate Viscosity of the facial cream and body mist
the moisturize cosmetic formulations which were measured using a Brookfield
contained the avocado oil to improve the viscometer at 60 rpm at 25 °C. The viscosity
nourishing dry skin. The facial cream of products showed good spreadability with
delivers as a light moisturizer cream and 4988.80 and 1142.70 mPas in facial cream
toner which may sooth damage skin and and body mist, respectively. The stability
give the moisture to face. Moreover, a body test of formulated cream by heating and
cooling of 6 cycles indicated that all tested Acknowledgements
facial creams and body mist were still stable The authors thank the ARDA,
with more viscous and white color (Table 1 Thailand and Faculty of Science, Chemistry
and Table 2). Stability assessment of facial and Applied Chemistry, Maejo University
cream and body mist were performed at for scholarship and all supports.
various storage conditions 4 C, 45 C and
at room temperature for 45 days. The References
physical properties of both products were 1. http://www.goodhealthacademy.com
constant in texture, color pH and feel on 2. http://superfoodprofiles.com/how-to-
skin, respectively. Results indicated that apply-avocado-oil-face-moisturizer
both facial cream and body mist remained 3. Barel, A. O.; Paye, M.; Maibach, H. I.
stable at all storage conditions (Table 3 and Handbook of cosmetic science and
Table 4). Both white facial cream and body technology. Marcel Dekker: New York,
mist were tested by 50 volunteers for one 2001; pp 19–27.
month. The results of testing by volunteers 4. Chimsook, T.; Assawarachan, R. Key.
revealed that facial cream and body mist Eng. Mater. 2017, 735, 127–131.
increased hydration and elasticity and 5. Genovese, M. I.; Lajolo, F. M.; Barbosa,
showed no skin irritation when compared to A. C. L. Food Chem. 2006, 98, 757–766.
other skin care cosmetics. 6. Kommanaboyina B.; Rhodes, C. T. Drug
Dev. Ind. Pharm. 1999, 25, 857–867.
4. Conclusion 7. Anderson, G.; Scott, M. Clin. Chem.
This study showed that avocado 1991, 37, 398–402.
facial cream and avocado body mist 8. Bajaj, S.; Singla, D.; Sakhuja, N. J.
contained beneficial cosmeceutical Appl. Pharm. Sci. 2012, 02, 129–138.
properties including moisture retention and 9. Arshad, A. I.; Khan, S. H. M.; Akhtar,
skin regeneration, had a promising potential N. Acta. Pol. Pharm. 2016, 73, 485–494.
for use as an effective and economical
cosmetic that could be applied in skincare
especially for sensitive skin.
Depsidones and a γ-butenolide from Simplicillium sp. PSU-H168,
an endophytic fungus from Hevea brasiliensis leaves
Supaporn Chinpha1, Souwalak Phongpaichit2, Jariya Sakayaroj3, Vatcharin Rukachaisirikul1*
1
Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
2
Department of Microbiology, Faculty of Science, Prince of Songkla University,
3
National Center for Genetic Engineering and Biotechnology (BIOTEC),
Thailand Science Park, Klong Luang, Pathumthani 12120, Thailand
*E-mail: vatcharin.r@psu.ac.th
Abstract:
Four known compounds, three depsidone derivatives, named botryorhodine C (1),
simplicildones A (2) and B (3), and one known γ-butenolide derivative, named penicillic acid
(4), were isolated from an endophytic fungus Simplicillium sp. PSU-H168. Their structures
were elucidated by the spectroscopic evidence. The absolute configuration of 4 was assigned
on the basis of similar circular dichroism data to those of dihydropenicillic acid. Among
them, compound 1 displayed antibacterial activity against Staphylococcus aureus and
methicillin-resistant S. aureus with equal MIC values of 32 µg/mL and was not cytotoxic to
noncancerous cell lines.
1. Introduction The circular dichroism (CD) spectrum was

Endophytic fungi in the genus recorded on a JASCO J-810 polarimeter.
Simplicillium have produced natural products The ultraviolet (UV) spectra were measured
with a variety of chemical structures and in MeOH on a PerkinElmer Lambda 45
interesting antimicrobial activities, for example, spectrophotometer. The infrared (IR) spectra
antibacterial aogacillins A and B from were recorded neat using a Perkin-Elmer
Simplicillium sp. FKI-5985,1 antibacterial 783 FTS 165 FT-IR spectrometer. The 1H
halymecins F and G from S. lamellicola and 13C NMR spectra were recorded on a
BCP,2 and antifungal preussin B from S. 300 MHz Bruker FTNMR Ultra ShieldTM
lanosoniveum TAMA 173.3 We investigated spectrometer using tetramethylsilane (TMS)
an endophytic fungus Simplicillium sp. PSU- as an internal standard. Thin-layer
H168 which was isolated from a leaf of chromatography (TLC) and preparative TLC
Hevea brasiliensis, collected in Songkhla were performed on silica gel 60 GF254
Province, Thailand. Chemical investigation (Merck). Column chromatography (CC) was
of the broth extract afforded four known carried out on Sephadex LH-20, silica gel
compounds, botryorhodine C (1),4 (Merck) type 60 (230-400 mesh ASTM) or
5
simplicildones A (2) and B (3), and type 100 (70-230 mesh ASTM), or on
penicillic acid (4).6 The antibacterial and reversed phase C18 silica gel.
cytotoxic activities of compounds 1, 2 and 4 2.2 Fungal material
were evaluated. The fungal endophyte PSU-H168
(accession no. KX020563) was isolated
2. Materials and Methods from a Para rubber leaf (Hevea brasiliensis).
2.1 General Based on ITS sequence analysis, the fungus
The melting points were determined PSU-H168 was closely related to
on an Electrothermal 9100 melting point Simplicillium sp. KT318874, KP184311 and
apparatus and reported without correction. KX020567 with high sequence identity of
100%. Therefore, the fungus PSU-H168 113.6 (CH, C-1'), 56.1 (CH2, C-8), 21.1
could be identified as Simplicillium sp. (CH3, C-9), 16.7 (CH3, C-7'), 9.2 (CH3, C-
2.3 Fermentation, extraction and purification 8').
The crude EtOAc extract (2.7 g, a 2.3.2 Simplicildone A (2)
dark brown gum) from the culture broth of Colorless solid; mp 215-218 C; UV
the fungus PSU-H168 (15 L) was prepared (MeOH) max nm (log ): 204 (4.16), 269
using the same procedure as described (3.48); IR (neat) νmax cm-1: 3429, 1703,
previously.5 The broth extract was subjected 1612; 1H (300 MHz, CDCl3) H 8.40 (s, 1H,
to CC over Sephadex LH-20 using 100% 4-OH), 6.61 (s, 1H, H-5), 6.42 (s, 1H, H-1'),
MeOH as an eluent to afford five fractions 5.02 (s, 2H, H2-8), 4.89 (brs, 1H, 2'-OH),
(A-E). Fraction C (482.2 mg) was purified 3.53 (s, 3H, H3-10), 2.45 (s, 3H, H3-9), 2.32
using the same procedure as the crude (s, 3H, H3-7'), 2.22 (s, 3H, H3-8'); 13C NMR
extract to give four subfractions (C1-C4). (75 MHz, CDCl3) C 163.5 (C, C-7), 160.8
Subfraction C2 (241.4 mg) was subjected to (C, C-4), 160.2 (C, C-2), 151.0 (C, C-2'),
CC over silica gel using 0.5% MeOH/CH2Cl2 145.4 (C, C-6), 143.7 (C, C-5'), 143.6 (C, C-
to obtain five subfractions (C2A-C2E). 4'), 126.9 (C, C-6'), 116.5 (CH, C-5), 114.4
Subfraction C2E (76.8 mg) was subjected to (C, C-3'), 113.2 (2xC, C-1 and C-1'), 110.6
CC over reverse phase C18 silica gel using a (C, C-3), 68.1 (CH2, C-8), 58.8 (CH3, C-10),
gradient of MeOH and H2O, starting from 21.3 (CH3, C-9), 16.9 (CH3, C-7'), 9.2 (CH3,
40% MeOH/H2O until pure MeOH, to C-8').
obtain three subfractions (C2E1-C2E3). 2.3.3 Simplicildone B (3)
Subfraction C2E1 (7.0 mg) was purified by Pale yellow solid; mp 206-207 C;
preparative TLC using 2% MeOH/CH2Cl2 UV (MeOH) max nm (log ): 203 (2.76),
as a mobile phase (3 runs) to afford 4 (2.1 267 (1.07); IR (neat) νmax cm-1: 3419, 1715,
mg). Purification of subfraction C3 (135.8
1639; 1H (300 MHz, CDCl3) H 8.64 (s, 1H,
mg) using the same procedure as subfraction
4-OH), 6.60 (s, 1H, H-5), 6.41 (s, 1H, H-1'),
C2 gave seven subfractions (C3A-C3G).
5.06 (s, 2H, H2-8), 4.74 (brs, 1H, 2'-OH),
Subfraction C3D (10.9 mg) was purified by
3.70 (q, J = 7.0, 2H, H2-10), 2.45 (s, 3H, H3-
preparative TLC using 1% MeOH/CH2Cl2
9), 2.32 (s, 3H, H3-7'), 2.23 (s, 3H, H3-8'),
as a mobile phase (3 runs) to yield 3 (1.6
1.32 (t, J = 7.0, 3H, H3-11); 13C NMR (75
mg). Subfraction C3F (17.3 mg) was
MHz, CDCl3) C 163.6 (C, C-7), 161.0 (C,
purified by CC over silica gel using 1%
C-4), 160.2 (C, C-2), 151.0 (C, C-2'), 145.4
MeOH/CH2Cl2 to afford five subfractions
(C, C-6), 143.8 (C, C-5'), 143.8 (C, C-4'),
(C3F1-C3F5). Subfractions C3F4 (4.0 mg)
127.1 (C, C-6'), 116.7 (CH, C-5), 114.5 (C,
and C3G (3.8 mg) were purified using the
C-3'), 113.3 (2xC, C-1 and C-1'), 111.0 (C,
same procedure as subfraction C3D to yield
C-3), 67.1 (CH2, C-10), 66.3 (CH2, C-8),
1 (1.9 mg) and 2 (2.9 mg), respectively.
21.5 (CH3, C-9), 17.0 (CH3, C-7'), 15.2
2.3.1 Botryorhodine C (1)
(CH3, C-11), 9.3 (CH3, C-8').
Colorless solid; mp 237-239 C; UV
2.3.4 Penicillic acid (4)
(MeOH) max nm (log ): 203 (4.63), 270
Colorless gum; UV (MeOH) max nm
(3.95); IR (neat) νmax cm-1: 3337, 1701,
(log ): 203 (2.53), 222 (1.45); CD (MeOH,
1607; 1H (300 MHz, acetone-d6) H 8.51 (s,
c 0.0001) λmax (Δε) 213 (+3.15) nm; [lit.
1H, 4-OH), 6.65 (s, 1H, H-5), 6.54 (s, 1H,
(MeOH, c 1x10-4) λmax (Δε) 219 (+5.04)
H-1'), 5.09 (s, 2H, H2-8), 2.39 (s, 6H, H3-9
nm];7 IR (neat) νmax cm-1: 3444, 1745; 1643;
and H3-7'), 2.15 (s, 3H, H3-8'); 13C NMR (75 1
H (300 MHz, CDCl3) H 5.48 (s, 1H, H-6a),
MHz, acetone-d6) C 163.7 (C, C-7), 161.6
5.23 (s, 1H, H-6b), 5.13 (s, 1H, H-2), 3.92
(C, C-4), 161.2 (C, C-2), 153.3 (C, C-2'),
(s, 3H, H3-8), 1.79 (s, 3H, H3-7); 13C NMR
144.7 (C, C-6), 144.4 (C, C-4'), 143.7 (C, C-
(75 MHz, CDCl3) C 178.8 (C, C-3), 169.7
5'), 128.0 (C, C-6'), 116.3 (CH, C-5), 116.1
(C, C-1), 140.2 (C, C-5), 116.5 (CH2, C-6),
(C, C-3), 114.8 (C, C-3'), 113.8 (C, C-1),
102.4 (C, C-4), 89.6 (CH, C-2), 59.9 (CH3,
C-8), 17.6 (CH3, C-7).
2.4 Bioassays
Antimicrobial activity was determined
as described by the Clinical and Laboratory
Standards Institute.8 Antibacterial activity
was evaluated against S. aureus, methicillin-
resistant S. aureus and Escherichia coli
while antifungal activity was determined Figure 1. Structures of compounds 1-4
against a clinical isolate of Microsporum isolated from Simplicillium sp. PSU-H168
gypseum SH-MU-4. Vancomycin (MIC 0.50
and 1.00 μg/mL) and gentamicin (MIC 0.50 two pentasubstituted benzenes (H 6.65 and
μg/mL) were used as positive controls for 6.54, each s, 1H), two oxymethylene protons
bacteria whereas micronazole (MIC 2.00 (H 5.09, s) and three methyl groups [(H
μg/mL) was used for M. gypseum. 2.39 (6H) and 2.15 (3H), each s)]. The 13C
Cytotoxic activity against Vero cells NMR spectrum displayed signals for one
was assessed employing the colorimetric conjugated ester carbonyl (C 163.7), ten
method,9 and the positive control ellipticine quaternary (C 161.6, 161.2, 153.3, 144.7,
displayed IC50 values of 5.97 μM. The 144.4, 143.7, 128.0, 116.1, 114.8 and
activities against KB and MCF-7 cell lines 113.8), two methine (C 116.3 and 113.6),
were conducted using the method described
one oxymetylene (C 56.1) and three methyl
by O’Brien and coworkers.10 Ellipticine
(C 21.1, 16.7 and 9.2) carbons. The
(IC50 15.83 μM) and doxorubicin (IC50 1.21
μM) were used as standard drugs for KB cell aromatic proton at H 6.65 was assigned as
lines while the standard compounds for H-5 and displayed the HMBC correlations
MCF-7 cell lines were doxorubicin (IC50 with C-1 (C 113.8), C-3 (C 116.1) and C-4
15.80 μM) and tamoxifen (IC50 18.47 μM). (C 161.6). The methyl group at H 2.39 (H3-
9) was attached at C-6 (C 144.7) on the
3. Results and Discussion basis of the HMBC correlations of H3-9 to
The broth ethyl acetate extract of the C-1, C-5 (C 116.3) and C-6. In addition, the
fungus Simplicillium sp. PSU-H168 was HMBC correlations of H-5 and H3-9 with
purified using various chromatographic the ester carbonyl carbon (C-7, δC 163.7)
techniques to provide four compounds (1-4). indicated the presence of an ester
Their structures (Figure 1) were elucidated functionality at C-1. The attachment of the
by spectroscopic analysis and by comparison hydroxymethyl group at C-3 was established
of the 1H and 13C NMR data with those by the HMBC correlations from H2-8 (H
previously reported in the literature. The 5.09) with C-2 (C 161.2), C-3 and C-4
absolute configuration of compound 4 was together with the chemical shift of C-8 (C
assigned by circular dichroism spectroscopy. 56.1). Furthermore, the chemical shifts of C-
Botryorhodine C (1) was obtained as 2 and C-4 indicated that the substituents at
a colorless solid, melting at 237-239 C. The these carbons were oxy groups. The
UV spectrum displayed absorption bands at aromatic proton resonating at H 6.54 was
203 and 270 nm, indicating the presence of a assigned as H-1' and showed the HMBC
conjugated carbonyl chromophore.4 The IR correlations with C-2' (C 153.3), C-3' (C
spectrum showed absorption bands at 3337, 114.8) and C-5' (C 143.7). The HMBC
1701 and 1607 cm-1 for hydroxy, ester correlations from H3-8' (H 2.15) to C-2', C-
carbonyl and double bond functional groups, 3' and C-4' (C 144.4) and from H3-7' (H
respectively. The 1H NMR spectroscopic 2.39) to C-1' (C 113.6), C-5' and C-6' (C
data contained signals for one hydroxy 128.0) indicated the attachment of the
proton (H 8.51, s), two aromatic protons of methyl groups at C-3' and C-6'. Due to the
chemical shifts of C-2' and C-5', the carbon of the ethoxy group at C 67.1 and
substituents at these carbons were oxy 15.2, respectively, instead of the signal of
groups. According to the chemical shifts of the methoxy carbon. The 1H-1H COSY
C-2, C-7, C-4' and C-5' and signal correlation from H2-10 (H 3.70) to H3-11
enhancement of 4-OH (H 8.51) upon (H 1.32) and the HMBC correlation from
irradiation of H-5, a depsidone skeleton H2-10 to C-8 (C 66.3) supported that the
having hydroxy groups at C-4 and C-2' was methoxy group in 2 was replaced with the
established. In the NOEDIFF experiment ethoxy group in 3. Signal enhancement of
(Figure 2), irradiation of H3-7' enhanced H2-8 after irradiation of H3-7' in the
signal intensity of H2-8, supporting the NOEDIFF experiment (Figure 3) supported
assigned structure. Botryorhodine C was the above assignment. Simplicildone B was
previously isolated from Botryosphaeria previously isolated from Simplicillium sp.
rhodina.4 PSU-H41.5
Figure 2. Key NOEDIFF data of 1 and 2 Figure 3. Key HMBC and NOEDIFF data
of 3
Simplicildone A (2) was obtained as
a colorless solid, melting at 215-218 C. The Penicillic acid (4) was isolated as a
UV and IR spectra were similar to those of colorless gum. The UV spectrum showed
1. Their 1H NMR spectroscopic data were absorption bands at 203 and 222 nm,
also similar except for an additional signal indicating the presence of α ,β-unsaturated
for a methoxy group at H 3.53. In addition, lactone chromophore.6 The IR spectrum
a signal for a methoxy carbon at C 58.8 was displayed absorption bands at 3444, 1745
observed in the 13C NMR spectrum. A HMBC and 1643 cm-1 for hydroxy, γ-lactone
correlation of the methoxy protons with C-8 carbonyl and double bond functional groups,
(C 68.1) established the attachment of the respectively. The 1H NMR spectrum
methoxy group at C-8. Signal enhancement demonstrated signals for two geminal olefinic
of H2-8 and H3-10 after irradiation of H3-7' protons (H 5.48 and 5.23, each s, 1H), one
in the NOEDIFF experiment (Figure 2) olefinic proton of a trisubstituted alkene (H
supported the above assignment. 5.13, s, 1H), one methoxy group (H 3.92, s,
Simplicildone A was previously isolated 3H) and one methyl group (H 1.79, s, 3H).
from Simplicillium sp. PSU-H41.5 The 13C NMR spectrum showed one
Simplicildone B (3) was isolated as a carbonyl carbon (δ C 169.7), three quaternary
pale yellow solid, melting at 206-207 C. carbons (δ C 178.8, 140.2 and 102.4), one
The UV, IR and 1H NMR spectroscopic data methylene carbon (δ C 116.5), one methine
were similar to those of 2. However, the 1H carbon (δ C 89.6), one methoxy carbon (δ C
NMR spectrum displayed the replacement of 59.9) and one methyl carbon (δ C 17.6). The
the methoxy signal in 2 with signals for an HMBC correlations of H-2 (H 5.13) with C-
ethoxy group at H 3.70 (q, J = 7.0 Hz, 2H) 1 (δ c 169.7), C-3 (δ C 178.8) and C-4 (δ C
and 1.32 (t, J = 7.0 Hz, 3H). The 13C NMR 102.4) together with the chemical shifts of
spectrum contained signals for one C-1 and C-4 established an α ,β-unsaturated
oxymethylene carbon and one methyl γ-lactone unit. The HMBC correlation from
H3-8 (H 3.92) to C-3 attached the methoxy 200 µg/mL. For cytotoxic activity toward
group at C-3. This assignment was KB, MCF-7 and Vero cells, 1 displayed
confirmed by signal enhancement of H3-8 weak activity with the MIC values of 90.48,
upon irradiation of H-2 in the NOEDIFF 60.13 and 40.28 μM, respectively.
experiment (Figure 4). A 2'-substituted
propenyl unit was established by the HMBC 4. Conclusion
correlations from H3-7 (H 1.79) to C-5 (C The fungus PSU-H168 isolated from
140.2) and C-6 (C 116.5) as well as the 1H- a leaf of Hevea brasiliensis, produced four
1
H COSY correlations of H3-7 with Hab-6 known compounds including botryorhodine
(H 5.48 and 5.23). This unit was attached at C (1), simplicildones A (2) and B (3), and
C-4 of the γ-lactone moiety on the basis of a penicillic acid (4). Among the isolated
HMBC correlation of H3-7 with C-4. A compounds, compound 1 displayed weak
hydroxy group was attached at C-4 based on antibacterial activity against S. aureus and
the chemical shift of this carbon. The methicillin-resistant S. aureus with equal
absolute configuration of C-4 was assigned MIC values of 32 µg/mL as well as weak
as S according to the CD spectrum of 4 activity against KB, MCF-7, and Vero cell
which showed a positive Cotton effect at lines with the IC50 values in the range of
213 nm (Δ +3.15, c = 1 x 10-4 M, MeOH), 40.28-90.48 μM.
the same sign as that of dihydropenicillic
acid, CD (MeOH) max: 219 nm (Δ +5.04).7 Acknowledgements
Penicillic acid was previously isolated from V.R. thanks the NSTDA Chair
Aspergillus melleus.6 Professor grants of the Crown Property
Bureau Foundation and the National Science
and Technology Development Agency. P.S.
acknowledges the Faculty of Science, Prince
of Songkla University for the research
assistantship, and the Center of Excellence
for Innovation in Chemistry (PERCH-CIC),
the Graduate School and the Department of
Chemistry, Faculty of Science, Prince of
Songkla University for partial support.
Figure 4. Key HMBC and NOEDIFF data References

of 4 1. Takata, K.; Iwatsuki, M.; Yamamoto, T.;
Shirahata, T.; Nonaka, K.; Masuma, R.;
Compounds 1, 2 and 4 which were Hayakawa, Y.; Hanaki, H.; Kobayashi,
obtained in sufficient amount were tested for Y.; Petersson, G. A.; Ōmura, S.; Shiomi,
antimicrobial (S. aureus ATCC25923, K. Org. Lett. 2013, 15, 4678-4681.
methicillin-resistant S. aureus, E. coli and M. 2. Dang, Q. L.; Shin, T. S.; Park, M. S.;
gypseum) and cytotoxic (KB and MCF-7 cell Choi, Y. H.; Choi, G. J.; Jang, K. S.;
lines as well as Vero cells) activities. Kim, I. S.; Kim, J.-C. J. Agric. Food
Compounds 1, 2 and 4 showed weak Chem. 2014, 62, 3363-3370.
antibacterial activity against S. aureus with 3. Fukuda, T.; Sudoh, Y.; Tsuchiya, Y.;
MIC values of 32, 32 and 128 µg/mL, Okuda, T.; Igarashi, Y. J. Nat. Prod.
respectively, whereas compounds 1 and 4 2014, 77, 813-817.
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aureus with the respective MIC values of 32 Sattler, I.; Hertweck, C. Phytochemistry.
and 200 µg/mL. Only compound 4 showed 2010, 71, 110-116.
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Sakayaroj, J.; Borwornpinyo, S.; 8. Phongpaichit, S.; Rungjindamai, N.;
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165-173.
Phytochemical screening and antioxidant activity of 10 Araceae rhizomes
in the Dong Phayayen, Khao Yai forest
Sawittree Rujitanapanich1*, Duangchai Sookchaloem2,
Pannee Denrungruang3, Chaleaw Petchthong4
1
Phranakhon Rajabhat University, Bang Khen, Bangkok 10220, Thailand
2
Faculty of Forestry, Kasetsart University, Chatuchak, Bangkok 10900, Thailand
3
Forest Products Development Division, Royal Forest Department, Chatuchak, Bangkok 10900, Thailand
4
Kanchanaburi Rajabhat University, Kanchanaburi 71190, Thailand
*E-mail: swt.ruji@gmail.com
Abstract:
The aims of this work were to investigate phytochemicals and anti-oxidant activity of
rhizomes of 10 plants in the Araceae family, collected from the Dong Phayayen, Khao Yai.
Rhizomes were dried at room temperature and extracted with ethanol. The phytochemical
methods developed by Trease & Evans and Harbone were used in the analysis. The alkoloids
were found in Colocasia esculenta (L.) Schott, Aglaonema simplex (Blume) Blume, Lasia
spinosa (L.) Thwaites and Amorphophallus paeoniifolius (Dennst.) Nicolson. The phenolics,
tannins and flavonoids were detected in most of the rhizomes except for Alocasia acuminata
Schott, Amorphophallus asterostigmatus Bogner & Hett. and Amorphophallus brevispathus
Gagnep. Coumarins were detected in Alocasia longiloba Miq. and A. paeoniifolius. However,
anthocyanins were not detected in any species. Triterpenes were found in A. simplex, L.
spinosa, and A. asterostigmatus, while diterpenes were detected in A. longiloba,
Amorphophallus putii Gagnep., A. acuminate, A. asterostigmatus and A. brevispathus.
Steroids were detected in C. esculenta, A. simplex, Homalomena aromatica (Spreng.)
Schott, L. spinosa, and A. acuminata. The antioxidant activity was determined by DPPH
radical scavenging assay. The highest antioxidant activity was found in the crude ethanol
extract from A. simplex (IC50 11.760.66 g/mL) followed by L. spinosa (17.860.10 g/mL)
and A.paeoniifolius (38.291.81g/mL).
1. Introduction antioxidant and antibacterial compounds

The Araceae is a large family especially from plants. Antioxidants are
comprising about 105 genera and important substances which possess the
approximately 3000 species of herbaceous potential of protecting organisms from
monocotyledons.1 In tropical jungle of damage caused by free radical. Phenolic and
Thailand, the Araceae family consists of polyphenolic compounds constitute the main
approximately 30 genera and 210 species.2 class of natural antioxidants present in
The family contains several well-known cultivated plants, food and beverages. 3 These
flowering and foliage plants, such as Alocasia compounds include alkaloids, catechins,
longifolia, Anthurium andraeanum, Monstera flavonols, tannins, terpenoids, etc. Most of
deliciosa, Spathiphllum cochlearispathum, etc. the numbers of Araceae are used in
A number of food crops also belong to Araceae, traditional medicine. The phytochemical
remarkably, Amorphophallus paeoniifolius investigation of the petroleum ether and
(elephant yam), A. konjac (konnyaku), ethanol extracts of Raphidophora pertusa
Colocasia esculenta, etc. One of the important revealed the presence of alkaloids, glycosides,
characters of this family is containing crystals of phenols, flavonoids, tannins and saponins.4
calcium oxalate which are often cited as causing The ethanolic extract of Arisaema leschenaultia
intense irritation when handling. In recent years, Blume possesses potent antibacterial
there has been increasing scientific interests in activity against Staphylococcus aureus,
Bacillus subtilis, Sarcina lutea.5 The ethanol brownish-red and/or yellow-precipitate
extract of tubers of A. paeoniifolius showed a indicates the presence of alkaloids.
maximum of 68.6% of DPPH scavenging Detection of phenols
activity.6 Therefore, the present investigation is Two mL of the extracts were mixed
aimed at the evaluation of phytochemical and with few drops of 5%FeCl3. The formation
antioxidant activity of rhizomes of 10 plants of blue-black colored solution indicates the
in the Araceae family collected from the presence of phenols.
Dong Phayayen, Khao Yai forest. Detection of tannins
Two mL of the extracts were mixed
2. Materials and Methods with 10 mL of distilled water, filtered
2.1 Plant material and extract preparation followed by few drops of 1%FeCl3.
The 10 Araceae rhizomes were Occurrence of blue-black, green or blue-green
collected from the Dong Phayayen, Khao precipitate indicates the presence of tannins.
Yai forest. There were Colocasia esculenta Detection of flavonoids
(L.) Schott, Aglaonema simplex (Blume) Two mL of the extracts were added
Blume, Alocasia longiloba Mig, with few pieces of magnesium chips
Amorphophallus putii Gagnep, Lasia followed by addition of few drops of
spinosa (L.) Thwaitei, Alocasia acuminate concentrated HCl. The appearance of pink,
Schott, Amorphophallus brevispathus Gagnep orange, or red to purple colored solution
and Amorphophallus asterostigmatus Bogner indicates the presence of flavonoids.
& Hett, Amorphophallus paeoniifolius Detection of coumarins
(Dennst.) Nicolson and Homalomena Two mL of the extracts were taken
aromatica (Spreng.) Schott (Figure 1). The in test tubes, covered with filter papers and
entire rhizomes were washed and then dried treated with NaOH solution. Test tubes were
in the shade at room temperature. After placed for few minutes in boiling water, the
drying, the rhizomes were then grinded well filter papers were removed and examined
by a blender. The dry powders were under UV light. Intense fluorescence indicates
extracted three times with ethanol. The the presence of coumarins.
extracts were filtered and dried in a rotary Detection of anthocyanins
evaporator then stored at 4 C until used. Two mL of the extracts were mixed
2.2 Chemical reagents with 2 M HCl. The appearance of pink-red
Chemical reagents used in this study color that turns purplish blue after adding of
were analytical grade ascorbic acid and 1,1- NH4OH indicates the presence of
diphenyl-2-picrylhydrazyl (DPPH) which anthocyanins.
were purchased from Sigma-Aldrich. Detection of diterpenes (Copper acetate test)
2.3 Phytochemical screening Two mL of the extracts were treated
Qualitative phytochemical tests (for with few drops of Cu(CH3COO)2 solution.
alkaloids, phenols, tannins, flavonoids, Formation of emerald green colored
coumarins, anthocyanins, diterpenes, solution indicates the presence of diterpenes.
triterpenes and steroids) were carried out Detection of triterpenes (Salkowski’s test)
by the method described by Trease & Evans Two mL of the extracts were treated
and Harbone7,8 as follows. with few drops of CHCl3 and then filtered.
Detection of alkaloids A few drops of concentrated H2SO4 were
Two mL of the extracts were carefully added in to the filtrates. Formation
warmed with 15 mL of 2 M HCl in a water of golden yellow colored solution indicates the
bath for 10 min and filtered. After that, 2 presence of triterpenes.
drops of Dragendorff’s reagent / Wagner’s Detection of steroids (Liebermann Burchard’s
reagent/ Mayer’s reagent were added and test)
mixed separately. The formation of orange, Two mL of the extracts were treated
Figure 1. 10 Plants in the Araceae family
with CHCl3 and filtered. To the filtrates on concentration of the extract (g/mL) a trend
ice, 1 mL of CH3COOH was added and then line equation: y =aln(x) ± b was determined.
few drops of concentrated H2SO4 were run
down the side of the test tube. The 3. Results and Discussion
appearance of blue, bluish-green solution 3.1 Phytochemical screening
indicates the presence of steroids. Phytochemical screening for ethanolic
2.4 DPPH radical scavenging assay extracts of 10 Araceae rhizomes showed the
The free radical scavenging activities antioxidant phytochemical classes of
were modified from the microplate- alkaloids, phenols, tannins, flavonoids,
based method as described by Ronald.9 coumarins, diterpenes, triterpenes and steroids as
Test samples were dissolved in ethanol and presented in Table 1. The alkaloids were found
serially diluted into different concentrations. in C. esculenta, A. simplex, L. spinosa and
Each solution 100 L of each samples was A. paeoniifolius. In the previous study, the
added to 100 L freshly prepared in DPPH methanolic and aqueous extracts of L.
ethanol (60 g/mL) on a 96-well microplate at spinosa leaves revealed positive test of
room temperature. After incubation in the alkaloids. 10 The phenolics, tannins and
dark for 30 min, DPPH level of each well flavonoids were detected in the rhizomes
was evaluated by detecting absorbance at 515 except for A. acuminate, A.
nm with Microplate Reader (Biotek Instruments asterostigmatus and A. brevispathus.
Inc., USA). The activity is given as the Day et al. (2016) has reported that the
percentage of scavenged DPPH, which is methanolic extract of A. paeoniifolius tuber
calculated using the following equation: showed alkaloids, flavonoids, sterols,
DPPH radical scavenging activity (%) phenolic compounds and tannins. 11
A c - As Coumarins were detected in A.longiloba and
= x 100 A. paeoniifolius. However, anthocyanins were
Ac not detected in any species. Triterpenes were
where Ac was the absorbance of the control found in A. simplex, L. spinosa, and A.
and As was the absorbance in the presence asterostigmatus, while diterpenes were
of samples. All determinations were performed detected in A. longiloba, A. putii, A. acuminate,
in triplicate. Results were expressed as IC50 A. asterostigmatus and A. brevispathus.
concentration. IC50 values were calculated Steroids were detected in C. esculenta,
using a calibration curve. On a graph section A. simplex, H. aromatica, L. spinosa and
of the percentage of inhibition according to the
Table 1. Preliminary phytochemical screenings of 10 Araceae rhizomes
Phytochemical Name of plants

compounds C. A. A. A. L.spinosa A. A. A. A. H.
esculenta simplex longiloba putii acuminate brevispathus asterostigmatus paeoniifolius aromatic
Alkaloids
Dragendorff’s + + - - + - - - + -
reagent
Wagner’s + + - - - - - - - -
reagent
Mayer’s + - - - - - - - - -
reagent
Phenols + + + - + - - - + +
Tannins - - + - + - - - + +
Flavonoids + + + + + + + + + +
Coumarins - - + - - - - - + -
Anthocyanins - - - - - - - - - -
Diterpenes - - + + - + + + - -
Triterpenes + + - - + - + + - +
Steroids + + - - + + - - - +
(+), presence (-), absence
A. acuminata. In the previous report, H. tuber displayed higher inhibitory activity

aromatic showed the presence of steroids in with IC50 value of 30 g/mL.15 However, the
methanolic extract of rhizomes.12 antioxidant ascorbic acid was found to be
3.2 DPPH radical scavenging activity more potent for providing the hydrogen
The DPPH scavenging assay is a donating/electron transfer ability than all
widely used method to assess antioxidant other rhizome extracts.
activities in a relatively short time. DPPH is
a stable free radical at room temperature and Table 2. DPPH radical scavenging activities
accepts an electron or hydrogen radical to of 10 Araceae rhizome extracts
develop into a stable diamagnetic molecule.13 Sample DPPH radical scavenging
The results show that the highest antioxidant activities
activity was found in the crude ethanol conc.range IC50(g/ml)
(g/ml) SD
extract from A. simplex (IC50 11.760.66
A. simplex 2-45 11.76±0.66
g/mL) followed by L. spinosa (17.860.10 L. spinosa 10-60 17.86±0.10
g/mL) and A. paeoniifolius (38.291.81g/mL). A. paeoniifolius 20-200 38.29±1.81
The results reveal that extracts contain H. aromatic 20-200 49.32±0.21
powerful inhibitor compounds, which may C. esculenta 100-1,000 290.71±9.01
act as primary antioxidants that react with A. longiloba 200-2,000 375.18±1.62
free radicals. Meanwhile, the IC 50 value A. putii 400-3,000 645.79±14.52
of A. acuminate (2226.39±20.98 g/mL) A. acuminate 1,000-4,000 2226.39±20.98
A. asterostigmatus 1,000-4,000 2,272.59±44.92
followed by A. asterostigmatus (2,272.59±44.92
A. brevispathus 1,000-5,000 4,603±177.17
g/mL) and A. brevispathus Ascorbic acid 2-15 3.92±0.12
(4,603±177.17g/mL) were extremely low
due to very poor free radical scavenging 4. Conclusion
activity as shown in Table 2. This finding Qualitative screening of 10 Araceae
coincides with a previous reported that ethyl rhizomes show the presence of some
acetate fraction of L. spinosa leaves showed phytochemicals such as alkaloids, phenols,
the highest free radical scavenging activity tannins, flavonoids and steroids. Flavonoids
with IC50 value 16.42 µg/ml.14 In addition, were present in more number of plants. In
the ethanolic extract of A. paeoniifolius the DPPH assay, the highest antioxidant
activity was found in the crude ethanol 6. Angayakanni, J.; Ramkumar, K. M.;
extract from A. simplex followed by L. Priyadarshini, U.; Ravendran, P. J.
spinosa and A. paeoniifolius. The results Pharm. Biol. Szeged. 2009, 48, 659–665.
indicate that A. simplex, L. spinosa and A. 7. Trease, G. E.; Evans, W. C.
paeoniifolius are rich in anti-oxidative Pharmacognosy. 13th ed. Balliere Tindall:
compounds which are worth for further London, 1987; pp 61–62.
investigations. 8. Harborne, J. B. (eds.). Phytochemical
Methods: A guide to modern techniques of
Acknowledgements plant analysis. 3rd ed. Chapman and
The authors are grateful to Hall: London, 1998.
Biodivesity-Based Economy Development 9. Ronald, L. Prior, X. W.; Karen Schaich. J.
Office (BEDO) for financial support. We Agric. Food Chem. 2005, 53, 4290–
also thank Phranakhon Rajabhat 4302.
University for providing the facilities to 10. Kumar, M.; Mondal, P.; Borah, S.;
complete this research. Mahato, K. J. Pharm. Pharm. Sci. 2013, 5,
306–310.
References 11. Dey, Y. N.; Wanjari, M. M.; Kumar, D.;
1. Roy, S.; Dutta, C. M.; Paul, S. B. J. Res. Lomash, V.; Jadhav, A. D. J.
Ayur. Pharm. 2013, 4, 15–17. Ethnopharmacol. 2016, 192, 183–191.
2. Boyce, P. C.; Sookchaloem, D.; Hetterscheid 12. Das, P. S.; Talukdar, A. D. J. Biol.
W. L. A.; Gusman, G.; Jacobsen, N.; Idei, Environ. Sci. 2010, 6, 71–74.
T.; Du, N. V. Araceae. Flora of Thailand: 13. Yesilyurt, V.; Halfon, B.; Ozturk, M.;
Bangkok, 2012; pp 101–321. Topcu, G. J. Food Chem. 2008, 108, 31–
3. Kalpna, R.; Mital, K.; Sumitra, C. J. 39.
Med. Plants. Res. 2011, 5, 63–71. 14. Goshwami, D.; Rahman, M. M.; Muhit,
4. Kalairasan, A.; John, S. A. J. Pharm. Res. MA.; Islam, MS. J. Arch. Appl. Sci. Res.
2011, 4, 1–2. 2012, 4, 2431–2434.
5. Jain, V.; Jat, R. C.; Bhardwaj, S.; Jain, 15. Angayarkanni, J.; Ramkumar, K. M.;
S.; Pathak, S. Pharmacognosy Research Priyadharshini, U.; Ravendran, P. J.
Laboratory, Department of Pharm. Biol. 2010, 48, 659–665.
Pharmaceutical Sciences. Barkatullah
University, Bhopal. 2010.
Volatile compositions of Clausena harmandiana essential oil
Chalermporn Thongpoon1*, Chalida Phungpanya2,3, Theeraphan Machan2,3
1
Program of Chemistry, Faculty of Science and Technology, Pibulsongkram Rajabhat University,
2
Program of Applied Chemistry, School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
3
Center of Chemical Innovation for Sustainability, Mae Fah Luang University, Chiang Rai 57100, Thailand
*E-mail: cthongpoon@gmail.com
Abstract:
The essential oil extracted from dried Clausena harmandiana leaves was analyzed for
volatile compositions by using GC–MS technique. The antibacterial activity of the essential
oil was evaluated by using resazurin microtiter assay (REMA). The total 24 compounds
(97.41%) was identified which can be categorized into 5 component groups including
monoterpene (93.67%), sesquiterpene (2.59%), alcohol (0.73%), aldehyde (0.28%) and
ketone (0.14%). E-anethole (92.43%) was the predominant and classified in monoterpene
group. In this study, C. harmandiana essential oil failed to show any antibacterial activity.
1. Introduction dried under shade, then, stored in a plastic

Clausena harmandiana or Song Fah bag with tightly tied before used.
in Thai name is a plant in Rutaceae family 2.2 Hydrodistillation
which is pervasively found in the 60 g of dried C. harmandiana leaves
northeastern of Thailand.1 The leaves are was crushed into small pieces, then,
pinnate and alternate.2 This plant has been subjected to a 2 L round bottom flask. The
recorded for treatment of ailments e.g. distilled water was poured into the flask and
headache, stomachache and fever as the connected to a Clevenger apparatus. The
traditional medicine.3 From the previous hydrodistillation was carried out for 4 h
publications, the non–volatile constituents of followed by collecting the EO from the
C. harmandiana extract were isolated and receiving part of the apparatus. The obtained
purified from several parts of the plant such EO was dried with anhydrous sodium
as roots, twigs, barks and fruits. The sulfate. After filtration, the oil was kept in a
phytochemicals found in C. harmandiana small amber glass bottle with tightly capped
were carbazole alkaloids, coumarins and and stored at 4 ºC prior to analysis.
ferulate.4-8 In Thailand, however, the volatile 2.3 Analysis of chemical compositions
composition of essential oil from this plant The GC instrument HP6980 model
has never been reported. The purposes of (Agilent Technologies, USA) equipped with
this study are to extract the essential oil a HP5793 mass–selective detector was used
(EO) from dried leaves of C. harmandiana for analysis of chemical compositions of the
and to analyze its volatile compounds, in C. harmandiana oil. The EI mass spectra
addition, to evaluate the antibacterial were collected at 70 eV ionization voltages
activity of the extracted essential oil by with the range of m/z 29–300. The ion
using resazurin microtiter assay (REMA). source and quardrupole temperatures were
set at 230 and 150 ºC, respectively. The
2. Materials and Methods injector and detector temperatures were 250
2.1 Plant preparation and 280 ºC, respectively. The carrier gas,
The C. harmandiana fresh leaves purified helium (He), was flowed at 1
were collected from Phitsanulok province, mL/min flow rate. The column used was
Thailand in July 2014. The leaves were HP–5MS capillary column (30m×0.25mm
i.d., film thickness 0.25μm). The oven
temperature was initially started at 60 ºC and inhibition of bacterium. The active bacterial
then gradually increased to 250 ºC by 3 cells changed the resazurin (blue) to the
ºC/min. The components were examined resorufin (pink), however, the color of
with NIST 98 and Wiley 275 databases with resazurin (blue) still claimed for inactive
unanimous acceptance at over 80% quality bacterial cells.9 The investigation was done
matches. In addition, the identification was in triplicate.
not only collaborated with Kovát retention
indices, related to n–alkanes (C8–C22), but 3. Results and Discussion
also compared to the previous publications 3.1 Essential oil from hydrodistillation
with similar analytical condition. The C. harmandiana EO obtained
2.4 Investigation of antibacterial activity from hydrodistillation process by using a
Seven selected pathogenic bacterial Clevenger apparatus was a pale yellow oil.
organisms for investigation of antibacterial The percentage yield of the obtained EO
activity were negative gram bacteria was 2.35.
(Pseudomonas aeruginosa TISTR 781, 3.2 Analysis of chemical compositions
Salmonella typhimurium TISTR 292 and According to the analysis of the
Escherichia coli TISTR 780) and positive chemical compositions by using GC–MS
gram bacteria (Staphylococcus aureus technique, a gas chromatogram of EO was
TISTR 1466, Bacillus subtilis TISTR 008, presented in Figure 1. The total twenty four
Bacillus cereus TISTR 687 and compounds were identified which could be
Micrococcus luteus TISTR 884). All calculated of the relative peak area
bacterial organisms were streaked in nutrient percentage to be 97.41. The identified
agar (NA) and selected the single colony of compounds could be classified into 5 groups
each bacterium, then, transferred to nutrient including monoterpenes (93.67%),
broth (NB). All NBs were incubated at 37 sesquiterpenes (2.59%), alcohols (0.73%),
ºC with vigorously shaken at 150 rpm aldehydes (0.28%) and ketones (0.14%).
except M. luteus was shaken at 30 ºC. The The major component was E–anethole
optical density (OD) was measured at (92.43%) which is in the monoterpene
wavelength of 600 nm to check the suitable category. The identified compounds of the
turbidity of bacterium in range of 0.4–0.6 C. harmandiana essential oil were shown in
absorbance. Table 1.
The microdilution assay by using the
96–well microplate was performed to test
the minimal inhibitory concentration (MIC).
The EO solution was prepared at 10000 ppm
as the initial concentration in current test by
dissolving the 10 mg of EO with 1 mL of the
5% (v/v) dimethyl sulfoxide (DMSO). Then,
the microplates were incubated at 37 ºC for
8–12 h for B. subtilis and E. coli whereas
16–18 h was carried out for other bacteria.
M. luteus was incubated at 30 ºC for 8–12 h.
The 30 μL of resazurin was added to each
well and incubated once again for 1 and 2 h
for the positive and negative gram bacteria,
respectively. The color changing of Figure 1. Gas chromatogram of EO from C.
resazurin is able to demonstrate the harmandiana dried leaves
Table 1. Chemical compositions of essential oil from C. harmandiana dried leaves
No. RT Compound %RA MM RIExp RILit
1 9.02 (Z)-β-Ocimene 0.05 136 1044 1037
2 9.41 (E)-β-Ocimene 0.05 136 1055 1050
3 12.53 allo-Ocimene 0.05 136 1136 1132
4 15.42 Estragole 1.07 148 1206 1196
5 18.83 4-Methoxybenzaldehyde 0.28 136 1285 127010
6 19.11 E-Anethole 92.43 148 1292 1284
7 20.83 ρ-vinyl-Guaiacol 0.62 150 1333 132211
8 21.54 δ-Elemene 0.22 204 1350 1388
9 22.34 Eugenol 0.02 164 1369 1359
10 23.39 Anisyl methyl ketone 0.10 164 1394 1382
11 23.68 β-Elemene 0.10 204 1401 1390
12 23.98 Z-Jasmone t 164 1409 1392
13 24.75 β-Ylangene 0.07 204 1430 1420
14 25.14 β-Copaene t 204 1441 1432
15 26.11 α-Humulene t 204 1468 1454
16 27.23 γ-Muurolene 0.09 204 1500 1479
17 27.33 ar-Curcumene 0.07 202 1502 1480
18 27.44 β-Selinene 0.06 204 1505 1490
19 27.81 α-Selinene 0.81 204 1513 1498
20 28.90 δ-Cadinene 0.07 204 1538 1523
21 31.09 Spathulenol 1.02 220 1588 1578
22 32.60 10-epi-γ-Eudesmol t 222 1626 1623
23 33.94 α-Cadinol 0.11 222 1662 1654
24 36.37 (2Z,6E)-Farnesol t 222 1729 1723
25 40.61 6,10,14-Trimethyl-2-pentadecanone t 268 1848 184612
RT = Retention time, %RA = %Relative peak area, MM = Molecular mass, RIExp = Retention indices obtained
from the analysis with HP–5MS column and compared with n–alkanes (C8–C22), RILit = Retention indices of
reference obtained from the analysis with DB–5 column10, t = Trace amount (<0.05%),11-13 = Retention index of
previous research
3.3 Antibacterial activity anethole (92.43%) as a major compound

The inhibition of all seven bacteria along with another twenty four identified
by the C. harmandiana EO was evaluated. components. In addition, the obtained
The result indicated that all bacteria in this essential oil lacked of ability to inhibit all
study were not inhibited by the obtained EO seven pathogenic bacteria.
from dried leaves of C. harmandiana,
although the previous reports published the Acknowledgements
ability of C. harmandiana extracts from We would like to thank the National
several parts of this plant have antibacterial Research Council of Thailand (NRCT) for
activity. From previous works, almost non– financial support of this work. We also
volatile compounds from those reports were sincerely appreciate for Pibulsongkram
phenylpropanoid and carbazole alkaloid Rajabhat University, Phitsanulok, Thailand
groups which played a crucial role in and the Scientific and Technological
antibacterial activity.4,7 Accordingly, the Instruments Center (STIC) of Mae Fah
essential oil which contained a monoterpene Luang University, Chiang Rai, Thailand for
as a major component did not inhibit all instrumental support.
tested bacteria in this study.
References
4. Conclusion 1. Noipha, K.; Thongthoom, T.; Songsiang,
In the current study, the essential oil U.; Boonyarat. C.; Yenjai, C. Diabetes
of dried C. harmandiana leaves was Res. Clin. Pract. 2010, 90, 67–71.
hydrodistillated and then analyzed by GC– 2. Wiart, C. Lead Compounds from
MS. The EO contained the monoterpene E– Medicinal Plants for the Treatment of
Cancer, 1st ed.; Academic Press: Boston, Deachathai, S.; Laphookhieo, S.
2013; pp 19–21. Fitoterapia. 2012, 83 (6), 1110–1114.
3. Chaichantipyuth, C.; Pummangura, S.; 8. Thongthoom, T.; Songsiang, U.;
Naowsaran, K.; Thanyavuthi, D.; Phaosiri, C.; Yenjai, C. Arch. Pharm.
Anderson, J. E.; McLaunghlin. J. L. J. Res. 2010, 33 (5), 675–680.
Nat. Prod. 1988, 51 (6), 1285–1288. 9. Elshikh, M.; Ahmed, S.; Funston, S.;
4. Maneerat, W.; Phakhodee, W.; Dunlop, P.; McGaw, M.; Marchant, R.;
Ritthiwigrom, T.; Cheenpracha, S.; Banat, I. M. Biotechnol. Lett. 2016, 38,
Deachathai, S.; Laphookhieo, S. 1015–1019.
Phytochem. Lett. 2013, 6 (1), 18–20. 10. Adams, R. P. Identification of Essential
5. Sriphana, U.; Thongsri, Y.; Oil Components by Gas
Prariyachatigul, C.; Pakawatchai, C.; Chromatography/Mass Spectrometry, 4th
Yenjai, C. Arch. Pharm. Res. 2013, 36 ed.; Allured Publishing Corp.: Carol
(9), 1078–1083. Stream, 2017.
6. Yenjai, C.; Sripontan, S.; Sriprajun, P.; 11. Mimica-Dukić, N.; Kujundžić, S.,
Kittakoop, P.; Jintasirikul, A.; Soković, M.; Couladis, M. Phytother.
Tanticharoen, M.; Thebtaranonth, Y. Res. 2003, 17 (4), 368–371.
Planta. Med. 2000, 66 (3), 277–279. 12. Pripdeevech, P.; Saansoomchai, J.
7. Maneerat, W.; Phakhodee, W.; Chiang Mai J. Sci. 2013, 40, 214–223.
Ritthiwigrom, T.; Cheenpracha, S.; 13. Kahriman, H.; Tosun, G.; Genç, H.;
Promgool, T.; Yossathera, K.; Yayli, N. Turk. J. Chem. 2010, 34, 969–
976.
Evaluation of chemical constituents from the leaves of Oroxylum indicum as
histone deacetylase inhibitors
Laor Somsakeesit1*, Pakit Kumboonma1, Somprasong Saenglee2,
Thanaset Senawong2, Chanokbhorn Phaosiri1
1
Natural Products Research Unit, Department of Chemistry, Faculty of Science, Khon Kaen University,
Khon Kaen 40002, Thailand
2
Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
*E-mail: laor.so@kkumail.com
Abstract:
This research focused on the chemical constituents from the leaves of Oroxylum
indicum as histone deacetylase inhibitors. The ethyl acetate extract was purified by column
chromatography on silica gel to afford four-major compounds, chrysin (3), 5,7-dihydroxy-3-
methoxyflavone (5), oleanolic acid (6) and 5,7,4-trihydroxy-3-methoxyflavone (7). Their
structures were confirmed by comparison of the spectroscopic data (IR, 1H NMR and 13C
NMR) with the previous reports. The isolated compounds were evaluated for histone
deacetylase inhibitory activity. The preliminary results indicated that these compounds could
act as non-hydroxamic acid based histone deacetylase inhibitors.
1. Introduction structural classes, including hydroxamic

Histone acetylation and deacetylation acid, cyclic peptides, benzamides,
play an essential role in the epigenetic electrophilic ketones and carboxylic acids.
regulation of gene expression. Histone These molecules are active only on HDACs
deacetylases (HDACs) are a group of zinc- class I and II by binding to the zinc-
binding metalloenzymes that catalyze the containing catalytic domains of the
removal of acetyl groups from histone tails. enzymes.5 In general, HDAC inhibitor
This mechanism results in chromatin consists of three domain: (1) a metal-binding
condensation and causes down-regulation of functional group to coordinate with Zn2+ ion;
tumor suppressor genes.1 Commotion of the (2) a hydrocarbon linker to fill out the
balance between HATs and HDACs relates narrow tunnel and (3) a capping group to
with various disorders and metabolic interact with the amino acids near the
diseases.2 entrance of the active site (Figure 1).5
Recently, 18 HDAC enzymes have The FDA approved histone
been identified and grouped into four classes deacetylase inhibitor, vorinostat (SAHA, 1)6
based on homology to yeast HDACs. Class I and the natural product trichosatin A (TSA,
(HDAC1, 2, 3, and 8), class IIa (HDAC4, 5, 2) are the most potent class which is
7, and 9), class IIb (HDAC6 and 10) and hydroxamic acid. However, these
class IV (HDAC11) HDACs are zinc- compounds are non-selective and toxic.
dependent metallo proteins. Class III (Sir 1- They have poor pharmacokinetic properties.
7) HDACs do not contain zinc and their Thus, there have been a developing of
activity requires nicotinamide adenine selective non-hydroxamic acid class
dinucleotide (NAD+).3 The previous reports inhibitors with less toxicity and improved
indicated that both class I and class II pharmacokinetic properties.7 The phenolic
HDACs have been associated with the compounds are non-hydroxamic acid and
process of tumor cell proliferation, display HDAC inhibitory activity, for
angiogenesis, invasion and metastasis.4 The examples, chrysin (3) and keampferol (4)8 as
HDAC inhibitors can be divided into five shown in Figure 1.
From the previous study, the Herbarium of Khon Kaen University
investigation on the chemical constituents (KKU), Khon Kaen, Thailand.
from the bark of O. indicum gave 2.3 Extraction and isolation
flavonoids Open air-dried and crushed leaves of
O. indicum (1.36 kg) were successively
extracted 3 times with hexane, EtOAc and
MeOH at room temperature (4 days). After
filtration, the combined extracts were
evaporated to obtain a yellow-brown oil of
hexane extract (67.30 g), a dark-green solid
of EtOAc extract (46.50 g) and a dark green
solid of MeOH extract (210.50 g). The
EtOAc extract was chromatograped on a
quick column chromatography using silica
gel 60 and mixtures of hexane-EtOAc,
Figure 1. Structures of SAHA (1), TSA (2), EtOAc, and EtOAc-MeOH as eluents.
chrysin (3) and kaempferol (4) Fractions with the similar characteristic on
TLC were combined to give 9 fractions
(mainly chrysin and baicalein) and (FC1-FC9). Fraction FC8 (8.69 g) was
glucosides of the flavonoids (baicalin).9 purified by column chromatography on
Therefore, in this research, we focused on silica gel and eluted with a gradient of
the isolation of these non-hydroxamic EtOAc-hexane (1:9 to 9:1) to give 9
chemical constituents from leaves of O. fractions (FC8.1-FC8.9). 5,7-Dihydroxy-3-
indicum to further explore their HDAC methoxy-flavone (5) (27.24 mg) was
inhibitory activity. obtained from FC8.5 as a pale yellow solid.
Fraction FC8.6 (1.02 g) was purified by
2. Materials and Methods column chromatography on silica gel and
2.1 General experimental procedure: eluted with a mixture of EtOAc–hexane
Quick column chromatography and (1:4) to obtain subfractions FC8.6.1-
column chromatography were carried out on FC8.6.8. Oleanolic acid (6) was obtained
silica gel 60 (230-400 Mesh ASTM, Merck) from FC8.6.3 as a white powder. Fraction
and silica gel 100 (70-230 Mesh ASTM, FC8.6.5 (256.2 mg) was purified by column
Merck), respectively. Aluminum sheets of chromatography on silica gel and eluted
silica gel 60 F254 (Merck) were used for thin with a mixture of EtOAc-hexane (1:4) to
layer chromatography (TLC). Solvents for give chrysin (3) (35.65 mg) as a pale yellow
chromatography were distilled at their solid. Fraction FC9 (2.62 g) was purified by
boiling ranges prior to use.1H and 13C NMR column chromatography on silica gel and
spectra were recorded on a Bruker AM400. eluted with a gradient of EtOAc-hexane (1:9
When CDCl3 was used as a solvent and an to 9:1) to give 5 fractions (FC9.1-FC9.5).
internal standard, signals  1.26 and 77.00 Subfraction FC9.4 (293.7 mg) was purified
were the reference for 1H and 13C NMR using the same procedure as FC8.6.5 to give
spectra.3 5,7,4-trihydroxy-3-methoxyflavone (7)
2.2 Plant materials (7.35 mg) as a pale yellow solid.
The leaves of O. indicum were 2.4 Histone deacetylase activity assay
collected from Amphoe Phon, Khon Kaen Briefly, compounds at 100 µM
province in the northeastern part of Thailand concentration were mixed with HeLa
in 2016. The plant was identified by Mr. nuclear extract, which contains all HDAC
Boonchuang Boonsuk. A voucher specimen enzyme isoforms. TSA was used as a
(L. Somsakeesit) was deposited at the positive control. HDAC fluorometric
substrate [Boc-Lys(Ac)-AMC] which
contained an acetylated lysine side chains 7.46 (H-3, H-4, H-5), and the resonances
was added to the mixture. The substrate was of a flavone unit at  6.61 (s, H-3), 6.43 (s,
incubated in the presence of the inhibitors. H-8) and 6.25 (s, H-6) which were
Deacetylation of the substrate followed by consistent with the previous report.11
mixing with the obtained developer
generated a fluorophore and comparison of
inhibitor vs control relative fluorescence
using a standard plate reader was employed
to determine percent of HDAC inhibitory
activity. The experiments were done in
duplicate and the average values were
reported.10 Figure 2. Structures of compounds 3 and 5-
2.5 Molecular docking studies 7.
The ligand setup was carried out using
Hyperchem 8.0. Auto Dock Tools 1.5.4 Compound 5 (5,7-dihydroxy-3-
(ADT) and Auto Dock 4.2 programs were methoxyflavone) was obtained as a pale
used for molecular docking for 50 runs. The yellow solid, melting at 276-278 oC. The 1H
size of grid box was indicated as 66 x 66 x NMR spectroscopic data (Table 1) showed
66 points and Lamarckian genetic algorithm singlet signal of a chelated hydroxy group at
search. The crystal structures of human  13.00 (5-OH), a doublet of doublet signal
histone deacetylase HDAC8 [PDB entry of equivalent protons at  7.88 (H-2, H-6),
code: 1T64, complexed with the inhibitor, multiplet signals of three aromatic protons at
TSA, resolution: 1.90 Å] was obtained from  7.52-7.56 (H-3, H-4, H-5), two singlets
the Protein Data Bank. All water and non- of meta-aromatic protons at  6.67 (H-6) and
interacting ions as well as TSA were
 6.62 (H-8) and a singlet for a methoxy
removed. Then all missing hydrogen and
group at  4.06 (3-OCH3). The data were in
side chain atoms were added using the ADT
agreement with the earlier report.12
program. Gasteiger charges were calculated
Compound 6 (oleanolic acid) was
for the system.3
obtained as a white powder, melting at 310-
312 oC. The 1H NMR spectrum showed the
3.1 Chemical constituents presence of a triplet signal at  5.28 (1H, t, J
The leaves of O. indicum were = 3.6 Hz, H-12), doublet of doublet signals
extracted with hexane, EtOAc and MeOH at at  3.21 (1H, dd, J = 10.2/4.4 Hz, H-3) and
room temperature. The crude EtOAc extract 2.82 (1H, dd, J = 12.7/ 4.3 Hz, H-18) and
was purified by column chromatography to singlet signals of seven methyl groups at 
give four compounds (3 and 5-7). These 1.25-0.76. The results were consistent with
compounds (Figure 2) were identified as the previous report.13
chrysin (3), 5,7-dihydro-3-methoxyflavone
(5), oleanolic acid (6) and 5,7,4-trihydroxy- Table 1. 1H NMR (400 MHz) data of 3-7 in
3-methoxyflavone (7) by comparing their 1H CDCl3/CD3OD
Position  (multiplicity, J)
(Table 1) and 13C NMR (Table 2)
3 5 7
spectroscopic data with those previously 3 6.61 (s) - -
reported. 6 6.25 (s) 6.67 (s) 6.55 (s)
Compound 3 (chrysin) was obtained as 8 6.43 (s) 6.62 (s) 6.50 (s)
a pale yellow solid, melting at 275-277 oC. 2,6 7.85 (d, 7.6) 7.88 (d, 7.6) 7.55 (d, 8.4)
The 1H NMR spectrum (Table 1) showed a 3,4,5 7.51- 7.52-7.56 -
7.46 (m) (m)
doublet signal of equivalent protons H-2, H- - - 6.96 (d, 8.4)
3,5
6 at  7.85 (2H, d, J = 7.6 Hz), multiplet 5-OH - 13.0 (s) -
signals of three aromatic protons at  7.51- 3-OMe - 4.06 (s) 3.92 (s)
Compound 7 (5,7,4-trihydroxy-3- Oleanolic acid (6) showed the best
methoxyflavone) was obtained as a pale inhibitory activity. It may be due to two
yellow solid, melting at 345-348 oC. The 1H functional groups including the carboxylic
NMR spectrum (Table 1) showed signals for group at C-17 and the hydroxy group at C-3.
para-disubstituted aromatic protons at  The molecular docking study indicated that
7.55 (d, J = 8.4 Hz, 2H, H-2, H-6) and  oleanolic acid used hydroxy group
6.96 (d, J = 8.4 Hz, 2H, H-3, H-5). The coordinating with the Zn2+ ion, whereas the
spectrum also showed signals of meta- carboxylic acid group interacted with
coupled aromatic protons at  6.55 (H-6) PHE227 of HDAC4 (Figure 3). The
and  6.50 (H-8) and a signal of a methoxy prediction of the binding free energy of
group at  3.92 (3-OCH3) which were oleanolic acid with HDAC4 showed G
consistent with the previous report.14 value as -9.84 kcal/ mol and Ki value as
60.98 nM (Table 4). Oleanolic acid also
Table 2. 13C-NMR (400 MHz) data of 3-7 in used the carboxylic group to chelate with
CDCl3/CD3OD Zn2+ ion and the hydroxy group to interact
Position 
with PRO809 HDAC7 (Figure 3). The
3 5 7 prediction of the binding free energy of
2 164.5 164.1 152.1 oleanolic acid with HDAC7 showed G
3 105.2 130.5 131.4 value as -11.68 kcal/ mol and Ki value as
4 182.6 183.0 182.9 2.75 nM which was the best inhibition
4a 104.7 131.3 104.9
5 among the tested compounds.
161.6 152.1 160.9
6 99.4 105.3 102.6 For HDAC4 binding, oleanolic acid
7 164.4 153.2 164.9 has keys of inhibitor-enzyme interactions
8 94.4 93.4 94.3 consisting as capping group, linker and
8a 158.1 155.2 156.8 metal-binding moiety. The carboxylic acid
1 131.1 105.9 122.1 can be the capping group, the hydrophobic
2,6 126.2 126.3 128.2
3,5 128.9 129.1 115.9 skeletal terpene as the linker and the
4 131.8 131.8 - hydroxy group can act as the Zn2+ ion. In
4-OH - - 153.3 addition, the compound has the best HDAC
3-OMe - 69.0 60.4 inhibitor activity.
3.2 HDACs inhibitory activity and Table 3. HDAC inhibitory activity of

molecular docking studies compounds 3 and 5-7
We evaluated the HDAC inhibitory Compounds HDAC inhibitory
activity of compounds 3 and 5-7. They were activity (%)
screened in vitro using HeLa nuclear extract 3 60
5 61
in a fluorimetric assay at 100 µM 6 68
concentration. TSA was used as the 7 62
reference compound because of its well Control (2) 93
established HDAC inhibitory activity to
compare the effect of the compounds on
HDAC inhibitory activity (Table 3).
Table 4. AutoDock 4.01 estimated free energies of binding (G) and inhibition constant (Ki)
values
Compounds HDAC2 HDAC4 HDAC7 HDAC8
G Ki G Ki G Ki G Ki
(kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol)
3 -6.69 12.51µM -7.92 1.57 µM -6.76 11.08µM -7.07 6.61 µM
5 -6.52 16.67µM -6.81 10.23µM -6.52 16.69µM -6.96 7.88 µM
6 -6.81 10.2µM -9.84 60.98nM -11.68 2.75 nM -7.37 3.98 µM
7 -6.25 26.32µM -7.71 2.24 µM -6.75 11.2 µM -6.61 12.85µM
Figure 3. The binding modes of oleanolic acid (6) in the active site of HDAC4 and HDAC7
Conclusion 5. Moradei, O.; Maron, C. R.; Paquin, I.;

Investigation on the chemical Vaisburg, A. Curr. Med. Chem.
constituents from the leaves of O. indicum Anticancer. Agents 2005, 5, 529–560.
resulted in the isolation of four-major 6. Mann, B. S.; Johnson, J. R.; Cohen, M.
compounds. These compounds were tested H.; Justice, R.; Pazdur, R. FDA approval
against HDACs inhibitory activity. summary: Vorinostat for treatment of
Oleanolic acid showed the best inhibitory advanced primary cutaneous T-cell
activity against HDACs both in vitro and in lymphoma, Oncologist, 2007; pp 1247–
silico. The compound also binded HDAC4 1252.
and HDAC7 with the Ki values in 7. Suzuki, T.; Miyata, N. Curr. Med.
nanomolar ranges. Therefore, it can be a Chem. 2005, 12, 2867.
good candidate for further development of 8. Li, N.; Liu, J. H.; Zhang, J.; Yu, B. J.
class II HDAC inhibitor and an anticancer Agric. Food. Chem. 2008, 56, 3876–
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renal carcinoma cells.15-16 9. Fan, Q. F., Hu, Z. Y., Na, Z., Tang, H.
S., Zuo, G. Y.; Song, Q. S. Nat. Prod.
Acknowledgements Res. 2015, 29, 1828–1832.
This work was supported by the 10. Ayhan, E. P.; Seven, Ӧ.; Eymur, G.;
Natural Product Research Unit, Khon Kaen Bora, T. G.; Dayngac, E. D.; Yelekci,
University and Rajamangala University of K.; Yurter, H.; Demir, A. A. Turk. J.
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Cytotoxic and antimalarial constituents from the roots of
Atalantia monophylla
Awat Wisetsai*, Ratsami Lekphrom, Florian T. Schevenels
Natural Products Research Unit, Department of Chemistry, and Center for Innovation in Chemistry, Faculty of
Science, Khon Kaen University, Khon Kaen 40002, Thailand
*E-mail: w.awat@kkumail.com
Abstract:
Three known acridone alkaloids, atalaphylline (1), N-methylatalaphylline (2), and N-
methylatalaphyllinine (3), as well as three known coumarins, 2,2-dimethylchromenocoumarin
(4), auraptene (5) and 7-O-geranylscopoletin (6) were isolated from the n-hexane extract of
the roots of Atalantia monophylla. Their structures were elucidated on the basis of
spectroscopic analyses (1D and 2D NMR). Compounds 1, 2 and 3 possess appreciable anti-
malarial activity against Plasmodium falciparum with IC50 values of 2.9, 2.6 and 1.3 μg/mL,
respectively. Additionally, they displayed cytotoxicity against KB, NCI-H187 and MCF-7
cell lines with IC50 values ranging from 5.8 to 21.1 μg/mL.

Atalantia monophylla DC 2.1 General
(Rutaceae), is a large, thorny shrub or small IR spectra were obtained using a
tree. It usually reaches up to 6 m in height Bruker Tenser 27 spectrophotometer
and can be found widely in peninsular India (Bruker, Germany). NMR spectra were
and the northeastern and southern parts of recorded on a Varian Mercury Plus 400
Thailand. In Thailand, it is known as “Ma spectrometer (Varian Inc., USA) using
Nao Phee”.1 This medicinal plant has been CDCl3 and CD3OD as solvents. The internal
used to treat chronic rheumatism, paralysis, standards were referenced from the residue
antispasmodic, stimulant and hemiplegia.2 In of those solvents. Column chromatography
previous reports, limonoids, coumarins and was carried out on MERCK silica gel 60
acridone alkaloids have been isolated from (230–400 mesh) (Merck, Darmstadt,
the roots of this plant.3,4 The essential oil Germany). Thin-layer chromatography was
from the leaves showed antimicrobial and carried out with pre-coated MERCK silica
strong inhibitory activities against some gel 60 PF254 (Merck, Darmstadt,
pathogenic fungi. Acridone alkaloids have Germany); the spots were visualized under
several biological activities such as UV light (254 and 365 nm) and further
inhibition of Epstein-Barr virus (EBV)-EA stained by spraying anisaldehyde and then
induction, induction of human heated until charred.
promyelocytic leukemia cell (HL-60) 2.2 Plant material
differentiation and antiproliferative activity.2 The roots of A. monophylla were
In continuation of our studies of Thai collected from Kaset Somboon District,
medicinal plants, we report herein the Chaiyaphum Province, Thailand, in 2016.
isolation of three known acridone alkaloids The plant material was identified by Prof.
and three known coumarins from the n- Pranom Chantaranothai, Department of
hexane extract of the roots of A. Biology, Khon Kaen University, Thailand,
monophylla. In addition, their anti-malarial where a voucher specimen (R.
activity and cytotoxicity against KB, NCI- LekphromKKU005) was deposited.
H187 and MCF-7 cell lines are also
discussed.
2.3 Extraction and isolation AH5.4 was purified by silica gel FCC, eluting
Air-dried roots of A. monophylla (4.0 with n-hexane/CH2Cl2/MeOH (67:30:3,
kg) were ground to powder and then v/v/v) to yield 4 (30.0 mg) and 5 (17.0 mg)
extracted at room temperature with n-hexane both as colorless needles. Subfraction AH5.5
(3 x 10 L). Removal of solvent under was separated by silica gel FCC, eluting
reduced pressure gave crude hexane extract with n-hexane/acetone (90:10, v/v) to yield
(40.0 g). Forty grams of the crude hexane 2 (15.5 mg) and 3 (18.4 mg) both as orange
extract were subjected to silica gel flash needles. Fraction AH6 was separated by
column chromatography (FCC), eluting silica gel FCC, gradually eluting with n-
firstly with 95/5 (v/v) n-hexane/EtOAc. The hexane/EtOAc (90:5, v/v) to give 5
polarity was gradually increased with fractions, AH6.1-AH6.5. Subfraction AH6.4
EtOAc. Each fraction (200 mL) was was purified by silica gel FCC, eluting with
analyzed by TLC. Fractions with similar n-hexane/CH2Cl2 (50:50, v/v) to yield 6 as
TLC patterns were combined to give seven colorless needles (9.5 mg). Fraction AH6
fractions (AH1-AH7). Fraction AH5 was was purified by silica gel FCC, eluting with
subjected to silica gel FCC, gradually n-hexane/EtOAc (80:20, v/v) to yield 1 as
eluting with n-hexane/EtOAc (90:5, v/v) to yellow needles (9.5 mg).
give 5 fractions, AH5.1-AH5.5. Subfraction
Figure 1. Constituents of Atalantia monophylla (roots).
2.4 Cytotoxicity assays 3. Results and Discussion

Cytotoxicity assays against human Chromatographic separation of n-
epidermoid carcinoma (KB), human breast hexane extract yielded six known
adenocarcinoma (MCF-7), and human small compounds (1-6). The structures of all
cell lung cancer (NCI-H187) were isolated compounds were identified by
performed employing the Resazurin spectroscopic techniques (1D and 2D
microplate assay (REMA) described by NMR). The data obtained were compared
Brien et al.5 The reference substances were with published values. They were
ellipticine, tamoxifen and doxorubicin. atalaphylline (1), N-methylatalaphylline (2),
2.5 Antimalarial assay N-methylatalaphyllinine (3), 2,2-
Antimalarial activity was tested dimethylchromenocoumarin (4), auraptene
against Plasmodium falciparum (K1, (5) and 7-O-geranylscopoletin (6) (Figure
multidrug resistant strain), which was 1).
cultured continuously, according to the Compound 1 was isolated as yellow
method of Trager and Jensen.6 needles, melting at 244.0-246.0 oC. The 13C
NMR spectroscopic data (Table 1) displayed
23 carbons, attributable to four methyl, two 7.2 Hz, H-1´´), 1.88 and 1.69 (each 3H, s,
methylene, five methine and twelve Me-5´´, Me-4´´ respectively), and at δ 5.09
quaternary carbons. The 1H NMR spectrum (1H, t, J = 7.2 Hz, H-2´), 3.34 (2H, d, J =
(Table 1) showed signals at δ 8.97 (1H, s, N- 7.2 Hz, H-1´), 1.80 and 1.71 (each 3H, s,
H) and δ 7.67 (1H, m) and δ 6.97 and 6.93 Me-4´, Me-5´, respectively). Compound 1
(2H, m) which were attributed to H-8, H-6 was therefore identified as atalaphylline
and H-7, respectively. Two prenyl groups which was previously isolated from this
were inferred by the resonances at δ 5.23 plant by Govindachari et al.
(1H, t, J = 7.2 Hz, H-2´´), 3.47 (2H, d, J =
Table 1. 1H and 13C NMR spectroscopic data of compounds 1−3 (400 and 100 MHz)
Position 1a 2b 3c
δH (J in Hz) δc δH (J in Hz) δc δH (J in Hz) δc
1-OH 14.4 (1H, s) 14.73 (1H, s)
1 158.6 159.3 160.9
2 106.1 108.7 109.3
3 158.8 161.4 158.8
4 101.1 106.5 102.2
5 144.5 147.8 149.0
6 6.97 (1H, m) 115.3 7.08 (1H, m) 119.3 7.23 (1H, m) 120.3
7 6.93 (1H, m) 120.9 7.07 (1H, m) 122.6 7.17 (1H, m) 123.8
8 7.67 (1H, m) 115.7 7.78 (1H, dd, 6.0, 3.6) 116.9 7.66 (1H, dd, 7.6, 1.6) 115.8
9 181.5 182.9 181.8
4a 138.7 149.1 130.8
5a 131.0 138.1 137.2
8a 119.9 125.2 145.8
9a 104.5 107.5 106.4
1´ 3.34 (2H, d, 7.2) 21.7 3.42 (2H, d, 7.0) 21.6 3.22 (2H, d, 7.2) 21.4
2´ 5.09 (1H, t, 7.2) 121.6 5.27 (1H, t, 7.0) 121.9 5.17 (1H, t, 7.2) 122.7
3´ 135.3 135.2 130.8
4´ 1.80 (3H, s) 17.8 1.82 (3H, s) 17.8 1.74 (3H, s) 18.2
5´ 1.71 (3H, s) 25.7 1.71 (3H, s) 25.7 1.61 (3H, s) 26.0
1´´ 3.47 (2H, d, 7.2) 22.3 3.51 (2H, d, 5.8) 26.7 6.66 (1H, d, 9.6) 121.2
2´´ 5.23 (1H, t, 7.2) 121.9 5.34 (1H, t, 5.8) 122.8 5.65 (1H, d, 9.6) 124.4
3´´ 134.4 134.1 76.9
4´´ 1.88 (3H, s) 17.8 1.81 (3H, s) 18.1 1.45 (3H, s) 27.2
5´´ 1.69 (3H, s) 25.6 1.77 (3H, s) 25.8 1.45 (3H, s) 27.2
10-NH 8.97 (1H, s)
10-NMe 3.59 (3H, s) 48.2 3.70 (3H, s) 48.9
a
Measured in CDCl3 + CD3OD, bMeasured in CDCl3, cMeasured in DMSO-d6
Compound 2 was isolated as yellow were closely related to those of 2, except

needles, melting at 190.0-192.0 oC. The 1H that the OH at the 3-position cyclized with
(Table 1) and 13C (Table 1) NMR spectra the prenyl group at the 4-position to form a
were closely related to those of 1, except chromene unit, showing specific signals at δ
that the N–H proton signal at δ 8.97 in 1 was 6.66 (1H, d, J = 9.6 Hz, H-1´´), 5.65 (1H, d,
replaced by a N-methyl resonance in 2 at δ J = 9.6 Hz, H-2´´) and 1.45 (6H, s, H-4´´
H 3.59: δ C 48.2. Thence, 2 was identified and H-5´´). Compound 3 was therefore
as an N-methyl derivative of 1, named N- identified as N-methylatalaphyllinine which
methylatalaphylline, which was previously was previously isolated from this plant by
isolated from this plant by Govindachari et Fraser et al.8 and from Bosistoa transversa
al.7 by Auzi et al.9
Compound 3 was isolated as orange Compound 4 was isolated as
needles, melting at 194.0-195.0 oC. The 1H colorless needles, melting at 130.0-133.0 oC.
(Table 1) and 13C (Table 1) NMR spectra The 13C NMR spectroscopic data (Table 2)
displayed 14 carbons, attributable to two and δ 6.21 (1H, d, J = 9.6 Hz, H-3) and 7.57
methyl, six methine and six quaternary (1H, d, J = 9.6 Hz, H-4). Two methyl groups
carbons. Its 1H NMR spectral data (Table 2) showed signals at δ 1.46 (6H, s, H-11 and
showed the presence of two aromatic H-12). Compound 4 was therefore identified
protons at δ 7.04 (1H, s, H-5) and δ 6.71 as 2,2-dimethylchromenocoumarin which
(1H, s, H-10). Two sets of cis-olefinic was previously isolated from this plant by
protons resonated at δ 5.68 (1H, d, J = 10.0 Talapatra et al.10
Hz, H-7) and 6.33 (1H, d, J = 10.0 Hz, H-6),
Table 2. 1H and 13C NMR spectroscopic data of compounds 4−6 in CDCl3 (400 and 100 Hz)
Position 4 5 6
δH (J in Hz) δc δH (J in Hz) δc δH (J in Hz) δc
1
2 161.2 161.2 161.5
3 6.21 (1H, d, 9.6) 104.4 6.23 (1H, d, 9.4) 112.9 6.27 (1H, d, 9.6) 111.3
4 7.57 (1H, d, 9.6) 143.3 7.62 (1H, d, 9.4) 143.4 7.61 (1H, d, 9.6) 143.3
5 7.04 (1H, s) 131.2 7.35 (1H, d, 8.6) 128.6 6.83 (1H, s) 108.0
6 6.33 (1H, d, 10.0) 120.7 6.83 (1H, dd, 8.6, 2.4) 113.2 146.6
7 5.68 (1H, d, 10.0) 124.7 162.1 152.1
8 77.7 6.80 (1H, d, 2.4) 101.6 6.84 (1H, s) 101.2
9
10 6.71 (1H, s) 113.0
11 1.46 (3H, s) 28.3
12 1.46 (3H, s) 28.3
4a 112.7 112.4 113.3
5a 118.5
8a 155.8 149.9
9a 156.8
10a 155.4
1´ 4.58 (2H, d, 6.4) 65.5 4.68 (2H, d, 6.6) 66.3
2´ 5.45 (1H, t, 6.4) 118.4 5.47 (1H, t, 6.6) 118.4
3´ 142.3 149.9
4´ 2.14-2.05 (2H, m)b 39.5 2.13-2.06 (2H, m) a 39.5
5´ 2.14-2.05 (2H, m)b 26.2 2.13-2.06 (2H, m) a 26.2
6´ 5.06 (1H, d, 6.4) 123.6 5.06 (1H, t, 7.6) 123.6
7´ 131.9 131.9
3´-Me 1.75 (3H, s) 16.7 1.76 (3H, s) 16.8
8´-Me 1.65 (3H, s) 25.6 1.65 (3H, s) 25.6
9´-Me 1.59 (3H, s) 17.7 1.59 (3H, s) 17.7
6-OMe 3.91 (3H, s) 56.4
a
overlap
Compound 5 was isolated as geranyl group was inferred by the

colorless needles, melting at 66.0-67.0 oC. resonances at δ 5.45 (1H, t, J = 6.4 Hz, H-
The 13C NMR spectroscopic data (Table 2) 2´), 5.06 (1H, d, J = 6.4 Hz, H-6´), 4.58 (2H,
displayed 19 carbons, ascribable to three d, J = 6.4 Hz, H-1´), 2.14-2.05 (4H, m, H-4´
methyl, three methylene, seven methine and and H-5´). Three methyl groups showed
six quaternary carbons. Its 1H NMR spectral signals at δ 1.75 (3H, s, vinyl CH3), 1.65
data (Table 2) demonstrated the presence of (3H, s, 8´-CH3) and δ 1.59 (3H, s, 9´-CH3).
aromatic protons at δ 7.35 (1H, d, J = 8.6 Thus, compound 5 was identified as
Hz, H-5), δ 6.83 (1H, dd, J = 8.6, 2.4 Hz, H- auraptene which was previously isolated
6) and δ 6.80 (1H, d, J = 2.4 Hz, H-8). Two from the root bark of this plant by
cis- olefinic protons were extrapolated by Shringarpure and Sabata.11
the resonances at δ 7.62 (1H, d, J = 9.4 Hz, Compound 6 was isolated as
H-4), 6.23 (1H, d, J = 9.4 Hz, H-3). A colorless needles, melting at 82.0-84.0 oC.
The 1H (Table 2) and 13C NMR spectra falciparum with IC50 values of 2.94, 2.58
(Table 2) were closely related to those of 5, and 1.34 μg/mL, respectively. Compound 2
except that the H-6 proton signal at δ 6.83 showed cytotoxicity against MCF-7 and KB
was replaced by an O-methyl resonance in 6 cells with IC50 values of 21.10 and 17.20
at δ 3.91: δ 56.4. Therefore, 6 was identified μg/mL, respectively, while 3 showed
as an O-methyl derivative of 5, named 7-O- cytotoxicity against MCF-7 and KB cancer
geranylscopoletin, which was previously cell lines with IC50 values of 13.33 and 5.82
isolated from the roots of this plant by μg/mL, respectively. In addition, compound
Chukaew et al.12 3 also showed cytotoxicity against NCI-
Compounds 1, 2 and 3 exhibited H187 cancer cell lines with an IC50 value of
antimalarial activity against Plasmodium 9.62 μg/mL (Table 3).
Table 3. Biological activities of compounds 1-3 from the roots of A. monophylla

compound Antimalarial Cytotoxicity, IC50 (μg/mL)
IC50 (μg/mL) NCI-H187a MCF-7b KBc
1 2.94 inactive inactive Inactive
2 2.58 inactive 21.10 17.20
3 1.34 9.62 13.33 5.82
a
Human lung cancer cells
b
Human breast cancer cells
c
Human epidermoid carcinoma in the mouth
The roots of A. monophylla were 1. Sribuhom, T.; Boueroy, P.;
investigated, leading to the isolation of three Hahnvajanawong, C.; Phatchana, R.;
known acridone alkaloids, atalaphylline (1), Yenjai, C. J. Nat. Prod. 2017, 80, 403–
N-methylatalaphylline (2), and N- 408.
methylatalaphyllinine (3) as well as three 2. Patil, V. R.; Thakare, V. M.; Joshi, V. S.
known coumarins, 2,2- Phcog. J. 2015, 7, 37–43.
dimethylchromenocoumarin (4), auraptene 3. Chukaew, A.; Ponglimanont, C.;
(5) and 7-O-geranylscopoletin (6). Karalai, C.; Tewtrakul, S.
Additionally, compounds 1, 2 and 3 Phytochemistry 2008, 69, 2616–2620.
possessed antimalarial activity against P. 4. Dreyer, D. L.; Bennett, R. D.; Basa, S.
falciparum. They also showed cytotoxicity C. Tetrahedron 1976, 32, 2367–2373
against KB, NCI-H187 and MCF-7 cell 5. O’Brien, J.; Wilson, I.; Orton, T.;
lines. Pognan, F. Eur. J. Biochem. 2000, 267,
5421–5426.
Acknowledgements 6. Trager, W.; Jensen, J. B. Science 1967,
Funding for this work was provided 193, 673–675.
by The Thailand Research Fund: Grant No. 7. Govindachari, T, R.; Viswanathan, N.;
MRG6080288 (for R.L.), and by the Center Pai, B. R.; Ramachandran, V, N.;
of Excellence for Innovation in Chemistry Subramaniam, P. S. Tetrahedron 1970,
(PERCH-CIC). A.W. thanks the Science 26, 2905–2910.
Achievement Scholarship of Thailand 8. Fraser, A, W.; John R. L. J. Chem. Soc.,
(SAST) for a scholarship and Khon Kaen Chem. Commun. 1973, 615–616.
University via the Natural Products 9. Auzi, A. A.; Hartley, T. G.; Waigh, R.
Research Unit. We are indebted to the D.; Waterman, P. G. Phytochemistry
Bioassay Research Facility of the National 1996, 42, 235–238.
Center for Genetic Engineering for 10. Talapatra, S. K.; Bhattacharya, S.;
bioactivity tests. Talapatra, B. J. Indian Chem. Soc. 1970,
47, 600–604.
11. Shringarpure, J. D.; Sabata, B. K. From 12. Chukaew, A.; Ponglimanont, C.;
Indian J. Chem. 1975, 13, 24–28. Karalai, C.; Tewtrakul, S.
Phytochemistry 2008, 69, 2616–2620.
so
Semi-synthetic studies of 2-substituted huperzine A analogues

as acetylcholinesterase inhibitors
Wareepat Tiyabhorn1, Pornkanok Pongpamorn1,2, Richard Sessions2, Somsak Ruchirawat1,3,
Nopporn Thasana1,3*
1
Chemical Biology Program, Chulabhorn Graduate Institute,Chulabhorn Royal Academy of Science,
Laksi, Bangkok 10210, Thailand
2
School of Biochemistry, University of Bristol, Woodland Road, Bristol, BS8 1TD, United Kingdom
3
Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Laksi, Bangkok 10210, Thailand
*Email: nopporn@cri.or.th
Abstract:
Huperzine A (HupA), a Lycopodium alkaloid, is known as a potent and reversible
inhibitor of acetylcholinesterase (AChE) which shows a therapeutic benefit for Alzheimer’s
disease (AD). In this study, HupA, isolated from a club moss Huperzia carinata, was
derivatized at C2 position to improve its binding affinity toward AChE. The crystallographic
structure of human AChE complexed Hup A revealed that in the vicinity of the pyridone ring
at C2 position, there is still space available for introducing designed novel substitutions to
increase the binding interactions with amino acids inside the AChE pocket. Over 20 HupA
analogues have been designed and studied using BUDE (Bristol University Docking Engine),
Amber, and Waterswap Sire. The in silico results led to a number of analogues, modified at
C2 position on HupA, that have binding poses nearly similar to the parent molecule, with
lower ∆G ligand binding free energies in the range of -37.8 ± 4.7 to -50.8 ± 4.9 kcal mol-1.
Nevertheless, to our knowledge, the modification of C2 position of Hup A has not yet been
investigated. This work would be useful to attain knowledge and better understanding for
quantitative study-activity relationship (QSAR) of 2-substituted HupA analogues.
1. Introduction was claimed to be safer than other licensed

Alzheimer disease (AD) is one of the drugs.4 However, since HupA is a reversible
major diseases mostly found in aging inhibitor, it only exhibits temporary memory
population. Unfortunately, nowadays, there enhancing activities in Alzheimer’s patients.
are no curative or preventive measures for Herein, our group is interested in modifying
AD. Acetylcholinesterase (AChE) is HupA to enhance its binding affinity
involved in AD progression. The major towards AChE with the hope that modified
mechanism of AChE is to degrade analogues will become a promising
acetylcholine, a critical neurotransmitter for therapeutic drug for AD patients. However,
regulating neurotransmission at the synapses from several QSAR studies, where the
in the nervous system, including the CNS equatorial methyl (C16) or the pyridine
(central nervous system).1,2 Approved moieties have been modified,5 most
medicines for treating AD at all stages act functional groups on HupA need to be
by inhibiting the activity of AChE conserved in order to maintain the
(including Donepezil, Galantamine, interaction with AChE. Many efforts to
Rivastigmine), except for Memantine, which synthesize HupA analogues with improved
targets on regulating the activity of binding affinity towards AChE have been
glutamate. Other than those approved drugs, attempted. Among those numerous HupA
Huperzine A (HupA), a Lycopodium analogues, no HupA derivatives have been
alkaloid, is another AChE inhibitor that is a reported to have higher binding affinity to
natural product. It has been approved for AChE than HupA.6 In addition, structural
treating AD in China since 1994.3 HupA modifications at C2 position has never been
investigated. Computational chemistry study MeOH/CH2Cl2 (0.07:1) as eluent to afford
by Miss Pornkanok Pongpamorn suggested HupA (3.0 g), which was 0.103% of the
that modifying the 2,3-unsaturated ketone MeOH crude extract.
moiety of HupA should increase its binding 2.3 Identification of huperzine A
affinity towards AChE. The identity of HupA was confirmed
1 13
by H and C NMR data
+
2. Materials and Methods (Table 1) and HRMS data of M+H ion at
2.1 General m/z 243.1493 (calcd for C15H18N2O). The
All reagents were commercially spectroscopic data were consistent with
available and were used without further literature values.7
purification. The 1D and 2D NMR spectra
were measured in CDCl3 on a FT-400
spectrometer with TMS as the internal
standard. High resolution mass spectra were
obtained on a Bruker Daltonics MicroTOF
instrument using atmospheric pressure Table 1. 1H (400 MHz) and 13C (75 MHz)
chemical ionization (APCI). Sephadex LH- NMR data of huperzine Aa
1 13
20 was purchased from GE Healthcare. Thin Carbon H (ppm) C (ppm)
1 - 165.3
layer chromatography (TLC) was carried out 2 6.41 (d, 9) 117.0
on Merck silica gel (70-230 mesh) and silica 3 7.89 (d, 6) 142.4
gel column chromatography (CC) was 4 - 122.8
carried out on SiliCycle silica gel (70-230 5 - 140.2
mesh). TLC spots were visualized by UV 6 2.86 (dd, 6, 9),6a 35.2
2.71 (d, 12),6b
light (254 nm) or staining with p- 7 3.61 (t, 4.5) 32.8
anisaldeyde. 8 5.41 (d, 3) 124.2
2.2 Extraction and isolation of huperzine 10 1.67 (d, 6) 12.3
A from Huperzia carinata 11 5.48 (q, 6) 111.2
12 - 143.1
13 - 54.3
14 2.18 (s) 49.1
15 - 134.1
16 1.51 (s) 22.6
a
The coupling constants (J) are in Hertz in
parentheses, chemical shift are given in ppm.
Figure 1. Huperzia carinata
Huperzia carinata was purchased in 2.4 Computational chemistry
2016 from Chatuchak Weekend Market, Waterswap Sire8 was used to
Bangkok, Thailand and identified by calculate the binding energies of HupA
Dr. Nopporn Thasana. The fresh aerial part analogues, modified at C2 position on the
of H. carinata (54.7 kg) was chopped and pyridone ring, for human AChE.1
soaked in MeOH (3x250 L) overnight at Derivatives having the absolute binding free
room temperature (rt.). The alcoholic extract energy lower than -35 kcal·mol-1 were
was concentrated under vacuum to afford a considered for actual synthesis because the
crude extract (2.9 kg) which was suspended parent HupA’s binding free energy was -
in 5% tartaric acid (50 L) and partitioned 34.9 ± 3.4 kcal mol-1. Low binding free
with CH2Cl2. The aqueous filtrate was energy correlates with good binding affinity.
adjusted to pH 10 with Na2CO3 and 2.5 Semi-synthesis of 2-substituted HupA
partitioned again with CH2Cl2. The CH2Cl2 analogues and their spectroscopic data
extract was evaporated to dryness to give the 2.5.1 N-acetyl HupA (2) and acetyl
alkaloid fraction (32.44 g) that was further derivative of 2 (3)
purified by sequent silica columns using HupA (1, 79.4 mg) was reacted with
acetic anhydride (17.2 equiv., 532 µL) and to afford 5 (19.5 mg, 72%); 1H NMR δ
DMF (0.5 mL) in CH2Cl2 (2 mL). The (ppm): 7.89 (s), 5.38 (d, 4.8), 5.22 (q, 6.8),
reaction was stirred at rt for 1 h, then 3.63 (t, 4.8), 2.85 (dd, 4.4, 12.8), 2.55 (d,
quenched with H2O and extracted three 16.8), 2.19 (d, 12), 2.034 (s), 1.60 (d, 6.4),
times with CH2Cl2. The combined organic 1.25 (s). HRMS m/z calcd for C17H20IN2O2
layers were washed with sat. NaCl, then [M + H]+: 411.0491; found: 411.0554.
dried over anhydrous Na2SO4 and 2.5.4 Compounds 6 and 7
evaporated under reduced pressure. The The starting material, 2 (29.1 mg),
crude product was purified using CC (SiO2) was reacted with NCS (1 equiv., 13.7 mg) in
to afford N-acetyl HupA 2 (48.6 mg, 52%) CH2Cl2 (1 mL) at rt. for 18 h, then quenched
and diacetylation by-product 3 (14.3 mg, with H2O and extracted with CH2Cl2 x 3.
13%) The combined organic layers were washed
2; 1H NMR δ (ppm): 7.43 (d, 9.2), 6.40 with sat. NaCl, then dried over anhydrous
(d, 9.2), 5.43 (d, 4.8), 5.30 (q, 7.2), 3.60 Na2SO4 and evaporated under reduced
(t, 4.8), 2.96 (dd, 4.8, 12), 2.70 (d, 16), 2.27 pressure. The crude product was purified
(d, 6), 2.08 (s), 1.67 (d, 6.8), 1.55 (s). using CC (SiO2) to afford 6 (31%, NMR
HRMS m/z calcd for C17H21N2O2 [M + H]+: yield) and 7 (69%, NMR yield)
285.1525; found: 285.1573. 6; 1H NMR δ (ppm): 8.51 (br. s), 8.05 (s),
3; 1H NMR δ (ppm): 7.72 (d, 8), 6.92 (d, 5.50 (d, 5.2), 4.64 (q, 6.8), 3.72 (t, 4.8), 3.14
8.4), 5.46 (d, 4.6), 5.33 (q, 6.7), 3.70 (t, 4.6), (dd, 4.8, 12.8), 2.65 (d, 16.8), 2.19 (d, 12),
3.14 (dd, 4.8, 12.4), 2.95 (d, 8.8), 2.32 (d, 2.03 (s), 1.53 (d, 6.8), 1.26 (s). HRMS m/z
10.4), 2.31 (s), 2.07 (s), 1.72 (d, 6.4), 1.52 calcd for C17H20ClN2O2 [M + H]+: 319.1100;
(s).HRMS m/z calcd for C17H21N2O2 [M + found: 319.1196.
H]+: 327.1630; found: 327.1705. 7; 1H NMR δ (ppm): 8.51 (br. s), 8.05 (s),
2.5.2 Compound 4 5.43 (d, 5.2), 4.45 (q, 6.8), 3.67 (t, 4.8), 3.14
The starting material, 2 (19.4 mg), (dd, 4.8, 12.8), 2.65 (d, 16.8), 2.19 (d, 12),
was reacted with CuBr2 (1.2 equiv.) in 2.03 (s), 1.53 (d, 6.8), 1.26 (s). HRMS m/z
acetonitrile (2 mL) under argon at 50 °C for calcd for C17H20Cl2N2O2 [M + H]+:
4 days, then quenched with H2O and 353.0800; found: 353.0818.
extracted three times with CH2Cl2. The 2.5.5 Compound 8
combined organic layers were washed with The starting material, 5 (7 mg), was
sat. NaCl, then dried over anhydrous reacted with KOH (3 equiv., 2.4 mg) in 1,4-
Na2SO4 and evaporated under reduced dioxane (0.2 mL) with Pd2(dba)3 (5 mol%,
pressure. The crude product was purified 0.65 mg) and tBuXPhos (20 mol%, 1.2 mg)
using CC (SiO2) to afford 4 (20.5 mg, 83%); in reflux condition for 1 day, then quenched
1
H NMR δ (ppm): 7.52 (s), 5.45 (d, 4.8), with H2O and extracted three times with
5.29 (q, 6.8), 3.63 (t, 4.8), 2.93 (dd, 4.4, 12), CH2Cl2. The combined organic layers were
2.24 (d, 12), 2.107 (s), 1.60 (d, 4.4), 1.25 (s). washed with sat. NaCl, then dried over
HRMS m/z calcd for C17H20BrN2O2 [M + anhydrous Na2SO4 and evaporated under
H]+: 363.0630; found: 363.0703. reduced pressure. The crude product was
2.5.3 Compound 5 purified using CC (SiO2) to afford 8 (35%,
The starting material, 2 (28.4 mg), NMR yield), 2 (38%, NMR yield) and
was reacted with NIS (1 equiv., 15 mg) in recover of starting material (27%, NMR
CH2Cl2 (1 mL) at rt. for 5.5 h, then yield); 1H NMR δ (ppm): 6.96 (s), 5.44 (d,
quenched with H2O and extracted three 4.8), 5.30 (q, 6.4), 3.61 (t, 4.8), 2.95 (dd,
times with CH2Cl2. The combined organic 4.4, 12.8), 2.65 (d, 17.2), 2.09 (s), 1.57 (d,
layers were washed with sat. NaCl, then 6), 1.25 (s). HRMS m/z calcd for
dried over anhydrous Na2SO4 and C17H21N2O3 [M + H]+: 300.1474; found:
evaporated under reduced pressure. The 301.1422.
crude product was purified using CC (SiO2)
2.5.6 Compound (9) Table 2. Binding free energies of HupA
The starting material 5 (16.2 mg) derivatives from Sire Waterswapb
dissolved in diglyme (0.5 mL) was
subsequently added NBu3 (1.1 equiv., 9.53 NH
O
µL), 28% NH3 in water (1 equiv., 3.33 mL), NH2
Mo(CO)6 (5 equiv., 10.56 mg) and Et4NCl R1
(5 equiv., 5.2 mg). The reaction was done 4ey5

Entry R1 Compound
under argon at 150 °C for 1.5 h, then (kcal·mol-1)
1 Cl 11 -41.8±2.2
evaporated under reduced pressure. The 2 I 12 -37.1±2.7
crude product was purified using CC (SiO2) 3 OH 13 -43.5±2.7
to afford unexpected product 10 in 100% 4 CONH2 14 -38.3±4.7
conversion instead of compound 9. 1H NMR 5 CONMe2 15 -45.2±3.2
δ (ppm): 8.36 (s), 5.44 (d, 4.8), 5.30 (q, 6.4), 6 CH2COCH3 16 -43.0±4.6
3.61 (t, 4.8), 2.95 (dd, 4.4, 12.8), 2.65 (d, 7 NH(CH2)2OH 17 -37.8±4.7
b
More than 20 derivatives have been calculated using
17.2), 2.09 (s), 1.56 (d, 7.6), 1.26 (s).
Sire Waterswap, but their binding energies values
HRMS m/z calcd for C18H21N2O4 [M + H]+: were higher than -35kcal·mol-1.
329.1423; found: 329.1496.
acetyl Hup A, 2 and 3) were performed
3. Results and Discussion under different condition to give
3.1 Computational chemistry result corresponding bromo, iodo and chloro
The X-ray crystal structure of the products (Scheme 1).9,10 Coupling reaction
human AChE was downloaded as PDB data then was performed using an 2-iodo-N-
file which 4ey5 is a PDB code represents acetyl Hup A 5 as a starting material.
rhAChE protein binding with (−)-HupA.2 Hydroxylation of 5 using KOH and 5 mol%
The protein was prepared by removing all Pd2(dba)3 as catalyst and tBuXPhos as a
the unnecessary components and its dimer. phosphine ligand gave 8 in moderate yield
HupA analogues were built in silico. Then (Scheme 2).11
they were docked into the protein using To prepare carboxamide HupA 14,12
BUDE and Sire Waterswap was used to amidation of 5 was studied in presence of
calculatethe binding free energy of the Mo(CO)6 and aqueous NH3 and yielded
analogues. Analogues with binding free HupA-2-carboxylic acid 10 which
energy lower than-35 kcal·mol-1 are shown performed by C-C bond formation of the
in Table 2. carbon monoxide species with C2. It was
3.2 Semi-synthesis of 2-substituted HupA suspected that water in the reaction could be
analogues. a key player to interfere the carboxamide
Following the isolation of HupA formation. Therefore, derivative 9 is
from the club moss H. carinata, chemical proposed to be prepared by amidation from
modifications were performed on C2 10 instead, whereas derivative 14 can be
position of the α,β-unsaturated ketone on the accessed from deacetylation of 9.
pyridone ring of HupA.
Preparation procedures for acylated 4. Conclusion
derivatives of HupA and functionalization of In conclusion, we believe that
C2 position are illustrated as shown in deacetylation of 5, 6 and 8 will lead to the
Schemes 1, 2 and 3. Acetylation of the free production of 11, 12 and 13. The other two
amine group at C13 and/or O position on the 2-substituted HupA analogues 14-15 could
pyridine ring was performed in order to be prepared by amidation of 10. In future
confine halogenation reaction to only the C2 studies, after the 2-substituted HupA
position (Scheme 1). The halogenation of analogues have been synthesized, their
acetyl-HupA (a mixture of mono- and di- AChE inhibitory properties will also be
Scheme 1. Preparation of 2-halo-N-acetyl huperzine A
Scheme 2. Synthesis of N-((5R,9R,E)-11-ethylidene-3-hydroxy-7-methyl-2-oxo-1,2,5,6,9,10-

hexahydro-5,9-methanocycloocta[b]pyridin-5-yl)acetamide (8)
Scheme 3. (5R,9R,E)-5-acetamido-11-ethylidene-7-methyl-2-oxo-1,2,5,6,9,10-hexahydro-
5,9-methanocycloocta[b]pyridine-3-carboxamide (9)
evaluated. J.; Franklin, M. C.; Height, J. J. J. Med.

Chem. 2012, 55 (22), 10282–10286.
Acknowledgements 2. Wright, C. I.; Geula, C.; Mesulam, M.
This work was financially supported M. Ann. Neurol. 1993, 34 (3), 373–384.
by the Royal Project Foundation (3040- 3. YAN Han, T. X.-C. Chin. J. New.
A013), the Thailand Research Fund Drugs. Clin. Rem. 2006, 25, 682–687.
(BRG6080013) (NT), the Royal Golden 4. Chomchalow N. AU. J. Technol. 2013,
Jubilee PhD Program (PHD/0127/2557) 16 (4), 215–220.
(PP) and The King Honour Celebration 5. Tun, M. K. M.; Herzon, S. B. J. Exp.
Scholarships Project for the Scientists Pharmacol. 2012, 4, 113–123.
Development (WT). 6. Yan, J.; Sun, L.; Wu, G.; Yi, P.; Yang,
F.; Zhou, L.; Zhang, X.; Li, Z.; Yang,
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Chemical constituents from the leaves of Artocapus chama Buch.-Ham.
Suthida Rattanaburi1*, Wilawan Mahabusarakam2,3
1
Laboratory of Natural Products Chemistry, Faculty of Science and Technology,
Phuket Rajabhat University, Muang, Phuket 83000, Thailand
2
3
Natural Product Research Center of Excellence, Prince of Songkla University, Hat Yai,
Songkhla 90110, Thailand
*E-mail: suthida.r@pkru.ac.th
Abstract:
Phytochemical investigation of the leaf extract of Artocarpus chama Buch.-Ham. led
to the isolation of four known phenolic compounds including three chalcones: xanthoangelol
(1), 3-geranyl-2,3,4,4-tetrahydroxychalcone (2) and xanthoangelol B (4), and a flavonoid,
apigenin (3). Their structures were characterized by spectroscopic methods and comparison
with those of published compounds. All compounds were isolated from this plant for the first
time.
1. Introduction 2.2 Plant material

Artocarpus chama belonging to the The leaves of A. chama were
family Moraceae is distributed in the collected from Amphur Yan Ta Khao, Trang
Southern part of Thailand. Previous Province, Thailand in June, 2016. The
phytochemical investigations of plants in herbarium specimen (S. Rattanaburi 04) was
this genus have revealed the presence of deposited at the herbarium of the
prenylated flavonoids,1 prenylated Department of Biology, Faculty of Science,
stilbenes and chalcones.4 Some of these
2,3
Prince of Songkla University, Thailand.
compounds showed antioxidant4,5 and 2.3 Extraction and isolation
cytotoxic2,6 activities. In the present study, The ground-dried leaves of A. chama
we report the isolation and structural (5 kg) were extracted with EtOH at room
elucidation of compounds 1-4 from the leaf temperature for 3 days. After removal of
extract of A. chama. EtOH, a green gum extract (380.2 g) was
obtained which was partitioned with EtOAc
2. Materials and Methods and water. The EtOAc layer was evaporated
2.1 General to provide a dark green extract (118.4 g)
The IR spectra were measured with a which was separated by QCC using a
FTS 165 FT-IR Perkin-Elmer gradient of hexane/acetone and acetone as
spectrophotometer. UV spectra were eluent to obtain 12 fractions (A-L).
recorded by a SPECORD S100 Compounds 1 (1.4371 g), and 4 (3.8 mg)
1 13
spectrophotometer. H and C-NMR spectra were obtained from fraction H after repeated
were recorded in acetone-d6 using a FT- purification by QCC (1:19 acetone/hexane).
NMR Bruker Avance spectrometer (300 Fraction I was re-chromatographed by QCC
MHz for 1, 2 and 4; 500 MHz for 3). Quick (7:13 acetone/hexane) to give 3 (2.2 mg) and
column chromatography (QCC) and column 2 (18.0 mg).
chromatography (CC) were carried out on
silica gel 60H (Merck) and silica gel 60 3. Results and Discussion
(Merck), respectively. Precoated plates of Chromatographic separation of
silica gel 60 GF254 were used for TLC EtOAc fraction from the leaves of A. chama
analysis. led to the isolation of compounds 1-4
(Figure 1).
of 3350 and 1625 cm-1 for O-H stretching
and C=O stretching, respectively. The 13C
NMR spectrum showed the resonances of a
carbonyl carbon (C 192.2), three methyl
carbons (C 24.9, 15.8, 15.4), three
methylene carbons (C 39.6, 26.5, 21.3), ten
methine carbons (C 144.0, 130.1  2, 129.3,
124.3, 122.4, 118.2, 115.9  2, 107.2), three
methylene carbons (C 39.6, 26.5, 21.3) and
seven quaternary carbons (C 162.1, 160.3,
134.4, 130.8, 126.6, 115.3, 113.4). The 1H
NMR spectroscopic data of 1 (Table 1)
indicated the presence of a chalcone
derivative which was deduced from the
following NMR spectroscopic data: a set of
Figure 1. Structures of compounds 1-4 trans enone system at H 7.82 (H-) and
7.73 (H-), one H-bonded hydroxy proton at
Compound 1 was a yellow H14.00, two ortho-coupled aromatic
amorphous powder. Its UV spectrum protons at H 7.95 (d, J = 8.7 Hz, H-6) and
showed maximum absorption bands at max 6.53 (d, J = 8.7 Hz, H-5) of ring A, two
(MeOH) 225 and 368 nm whereas the IR doublet signals of a 1,4-disubstuted benzene
spectrum showed the characteristic ring at
absorption bands
Table 1. 13C, 1H NMR and HMBC spectroscopic data (300 MHz, acetone-d6) for 1 and 2
1 2
Position δC δH (mult, JHz) HMBC δC δH (mult, JHz) HMBC
1 126.6 127.2
2 130.1 7.70 (d, 8.7) 4,  114.9 7.32 (d, 3.0) 4, 6, 
3 115.9 6.91 (d, 8.7) 1, 4 145.6
4 160.3 148.5
5 115.9 6.91 (d, 8.7) 1, 4 115.5 6.88 (d, 9.0) 1, 3
6 130.1 7.70 (d, 8.7) 4,  122.4 7.18 (dd, 9.0, 3.0) 2, 4, 
 118.2 7.73 (d, 15.6) 1, 1, C=O 117.5 7.65 (d, 15.0) 1, 1, C=O
 144.0 7.82 (d, 15.6) 1, 2, 6, C=O 144.5 7.74 (d, 15.0) 1, 2, 6, C=O
1 113.4 113.4
2 164.3 164.3
3 115.3 115.2
4 162.1 162.1
5 107.2 6.53 (d, 8.7) 1, 3, 4 107.2 6.54 (d, 9.0) 1, 3, 4
6 129.3 7.95 (d, 8.7) 2, 4, C=O 129.3 7.94 (d, 9.0) 2, 4
1 21.3 3.36 (d, 7.2) 3, 4, 2, 4 21.3 3.36 (d, 6.0) 3, 4, 2, 4
2 122.4 5.28 (t, 7.2) 122.4 5.28 (t, 6.0)
3 134.4 134.3
4 39.6 1.95 (m) 39.6 1.95 (m)
5 26.5 2.04 (m) 26.5 2.03 (m)
6 124.3 5.06 (t, 6.6) 124.3 5.06 (t, 6.6)
7 130.8 130.7
8 24.9 1.55 (s) 6, 7, 9 16.8 1.53 (s) 6, 7, 9
9 15.8 1.53 (s) 6, 7, 8 24.9 1.58 (s) 6, 7, 8
10 15.4 1.78 (s) 2, 3, 4 15.4 1.78 (s) 2, 3, 4
2-OH 14.0 (s) 1, 2, 3 14.0 (s) 1, 2, 3
C=O 192.2 192.1
H 7.70 (J = 8.7 Hz, H-2, H-6) and 6.91 Hz, H-8), 6.90 (d, J = 9.0 Hz, H-3), 7.80 (d,
(J = 8.7 Hz, H-3, H-5) of ring B. The J = 9.0 Hz, H-2). Three hydroxyl groups
remaining signals were identified as a were located at C-5, C-7, and C-4 positions
geranyl unit which showed resonances at H on the basis of HMBC correlations.
5.25 (t, J = 7.2 Hz, H-2), 5.06 (t, J = 6.6 Table 2. 13C, 1H NMR and HMBC
Hz, H-6), 3.36 (d, J = 7.2 Hz, H-1), 2.04 spectroscopic data (300 MHz, acetone-d6)
(m, H-5), 1.95 (m, H-4), 1.78 (s, H-10), for 4
1.55 (s, H-8), 1.53 (s, H-9). This group Position δC δH (mult, JHz) HMBC
1 128.1
was placed at C-3 due to HMBC 2 132.1 7.73 (d, 9.0) 4, 6, 
correlations of H-1 to C-3 , C-4. 3 177.2 6.92 (d, 9.0) 1, 4, 5
Therefore, compound 1 was identified to be 4 161.5
xanthoangelol.7 5 117.2 6.92 (d, 9.0) 1, 3, 4
Compound 2 was a yellow 6 132.1 7.73 (d, 9.0) 2, 4, 
 118.9 7.75 (d, 15.3) 1, 1, C=O, 
amorphous powder. The 1H and 13C NMR
 145.4 7.84 (d, 15.3) 2, 6, C=O, 
spectroscopic data of 2 were similar to those 1 114.9
of 1 (Table 1). The main differences were 2 165.6
that signals for the 1,4-disubstuted benzene 3 116.6
ring of 1 were replaced by those of a 1,2,4- 4 163.3
trisubstuted benzene ring (H 7.32, d, J = 3.0 5 108.5 6.53 (d, 9.0) 1, 3, 4
Hz, H-2), 6.88 (d, J = 9.0 Hz, H-5), 7.18 6 130.7 7.98 (d, 9.0) 2, 4, C=O
1 22.6 3.38 (d, 7.2) 2, 3, 4, 2, 3
(dd, J = 9.0 and 3.0 Hz, H-6). Therefore,
2 123.6 5.31 (t, 7.2)
compound 2 was assigned to 3-geranyl- 3 135.9
2,3,4,4-tetrahydroxychalcone, previously 4 37.0 1.97 (m)
isolated from the leaves of Artocarpus 5 35.1 1.56 (m)
incises.8 6 75.8 3.96 (t, 6.0) 4, 7, 8, 9
Compound 4 was obtained as a 7 149.8
yellow amorphous powder. The 1H and 13C 8 110.7 4.85 (brs) 6, 7, 9
4.70 (brs)
NMR spectroscopic data of 4 were similar to 18.3 1.66 (s)
9 6, 7, 8
those of 1 (Table 2). The major differences 10 16.8 1.80 (s) 2, 3, 4
were that compound 4 showed a terminal 2-OH 14.00 (s) 1, 2, 3
alkene (H 4.85 and 4.70, H-8) and an oxy- C=O 193.5
methine (H 3.96, J = 6.0 Hz, H-6) signals
instead of the geranyl proton signals as Table 3. 13C, 1H NMR and HMBC
observed in 1. Compound 4 was thus spectroscopic data (500 MHz, acetone-d6)
identified as xanthoangelol B.9 for compound 3
Compound 3 was a yellow solid, Position δC δH (mult, JHz) HMBC
m.p. 340-342 oC. The UV spectrum 2 164.9
3 103.8 6.51 (s) 1, 2, 4, 4a
suggested a flavonoid structure (max 4 182.9
(MeOH) 265 and 336 nm). The IR spectrum 4a 105.2
showed the characteristic absorption bands 5 163.1
of O-H stretching (3350 cm-1) and C=O 6 99.5 6.13 (d, 2.0) 4a, 5, 7, 8
stretching (1625 cm-1). The 13C NMR 7 164.9
8 94.5 6.41 (d, 2.0) 4a, 6, 7
spectrum displayed 13 signals for 15 8a 158.6
carbons of flavonoids core structure (Table 1 123.1
3). The 1H NMR spectrum proved it to be a 2 128.9 7.80 (d, 9.0) 2, 4, 6
flavone with the olefinic signal at H 6.51 (s, 3 116.6 6.90 (d, 9.0) 1, 4, 5
H-3), a chelated hydroxyl proton at H 12.87 4 161.8
116.6 6.90 (d, 9.0)
(5-OH) and 2 set of aromatic signals at H 5 1, 3, 4,
6 128.9 7.80 (d, 9.0) 2, 2, 6
6.13 (d, J = 2.0 Hz, H-6), 6.41 (d, J = 2.0 5-OH 12.87 (s) 4a, 5, 6
The structure 3 was consistent to that of 2. Wang, Y. H.; Hou, A. J.; Chen, D. F.;
apigenin, previously isolated from the roots Weiller, M.; Wendel, A.; Staples, R. J.
of Astragalus propinquus.10 Eur. J. Org. Chem. 2006, 15, 3457–
3463.
4. Conclusion 3. Wang, Y. H.; Hou, A. J.; Chen, D. F. J
Four known compounds (1-4) have Integr Plant Biol. 2007, 49, 605–608.
been isolated from the EtOAc fraction of the 4. Wang Y, Xu K, Lin L, Pan Y, Zheng X.
leaves of A. chama. Their structures were Phytochemistry 2007, 68, 1300–1306.
established by spectroscopic data as well as 5. Lan, W. C.; Tzeng, C. W.; Lin, C. C.,
comparison of these data with the literature. Yen, F. L.; Ko, H. H. Phytochemistry
2013, 89, 78–88.
Acknowledgements 6. Namdaung, U.; Aroonrerk, N.;
This work was supported by the Suksamrarn S.; Danwisetkanjana, K.;
National Research Council of Thailand Saenboonrueng, J.; Arjchomphu, W.;
(NRCT) and Laboratory of Natural Products Suksamrarn, A. Chem. Pharm. Bull.
Chemistry, Faculty of Science and 2006, 54, 1433–1436.
Technology, Phuket Rajabhat University. 7. Kil, Y. S.; Nam, J. W.; Lee, J.; Seo, E.
We also thank Assoc. Prof. Dr. Wilawan K. Arch. Pharm., Res. 2015, 38, 1506–
Mahabusarakam, Department of Chemistry, 1511.
Faculty of Science, Prince of Songkla 8. Shimizu, K.; Kondo, R.; Sakai, K.;
University for supporting some scientific Buabarn, S.; Dilokkunanant, U.
materials and giving valuable suggestions. Phytochemistry 2000, 54, 737–739.
9. Baba, K.; Nakata, K.; Taniguchi, M.;
References Kido, T.; Kozawa, M. Phytochemistry
1. Wang, Y. H.; Hou, A. J.; Chen, L.; 1990, 29, 3907–3910.
Chen, D. F.; Sun, H. D.; Zhao, Q. S.; 10. Chaturvedula, V. S. P.; Prakash, I. J.
Bastow, K. F.; Nakanish, Y.; Wang, X. Chem. Pharm. Res. 2013, 5, 261–265.
H.; Lee, K.-H. J. Nat. Prod. 2004, 67,
757–761.
Extraction of phenolic compounds from fenugreek seeds using liquefied
dimethyl ether (DME)
Apinya Thungoen1, Artiwan Shotipruk2, Panatpong Boonnoun1*
1
Department of Industrial Engineering, Chemical Engineering Program, Faculty of Engineering,
Naresuan University, Phitsanulok 65000, Thailand.
2
Chemical Engineering Research Unit for Value Adding of Bioresources, Department of Chemical Engineering,
Faculty of Engineering, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand
*E-mail: panatpongb@nu.ac.th
Abstract:
Fenugreek (Trigonella foenum-graecum) is a plant originated in Eastern Europe,
Northern Africa and Central Asia. Fenugreek seeds were found to be a rich source of phenolic
compounds which show beneficial health effects to human body. The phenolic compounds
and other active substances found in the seeds were apigenin, kaempferol and quercitin
glycosides as well as the flavonoids; vitexin, tricin, naringenin, quercetin and tricin 7-O--D-
glucopyranoside. The extraction of the total phenolic compounds from fenugreek seeds was
therefore investigated in this work. Liquefied dimethyl ether (DME), the safe solvent, which
is easily removed from an extract product was employed in this work. The response surface
methodology (RSM) using spherical central composite design (CCD) was employed for the
experimental design and optimization of extraction condition. The three parameters including
water adding as co-solvent, the solvent to seeds weight ratio and extraction temperature were
optimized. From the results, the RSM gave the well-predicted quadratic regression model
which showed 0.94 of R2. The optimal extraction conditions suggested by the model were at
22.65%wt of co-solvent, 5.60 of the solvent to seeds weight ratio (g/g) and 30.89 C which
gave 233.83 mg of total phenolic compounds/g of seeds.
1. Introduction by conventional solvent extraction, which is

Fenugreek (Trigonella foenum- the combination of non-polar solvents (e.g.
graecum) seeds are a rich source of bioactive hexane) and polar solvents (e.g. ethyl
phytochemicals that have health benefits, acetate, dichloromethane, water, butanol and
such as reduction of total cholesterol and methanol).5,6 However when toxic solvents
triglyceride levels.1 The seed extract has such as hexane are removed, normally
been tested in rats, and the effects found through evaporation, some toxic residue can
include alleviation of physiological remain in the extract, which is a concern.
oxidative stress and inflammation2 as well as Evaporation also has the disadvantage of
protection against hepatotoxicity caused by being a relatively complex procedure
the anticancer drug adriamycin (ADR).3 requiring high energy consumption.
Fenugreek has therefore been widely used as Alternatively, supercritical carbon dioxide
a traditional medicine in Asia and Europe.4 (SC-CO2) can be applied for extraction of
Fenugreek seed extracts (FSE) also phenolic compounds from natural materials
have anti-oxidative properties related to their such as grape seeds7. Unfortunately, that
phenolic compounds. The extraction of process requires high operating pressure
fenugreek seeds can generally be achieved (20-60 MPa), leading to high operating
costs.
Alternatively, liquefied dimethyl extraction, approximately 10 g of the dried
ether (DME), a safe solvent, is widely used powder was loaded into a thimble (cellulose,
in various extraction applications such as 30x100 mm) along with an 8-mm-diameter
food, cosmetics and pharmaceuticals. magnetic bar. The thimble was placed into
Because of its low boiling point (-24 C), an extractor (stainless-steel, 100-mL) and
dietary exposure to dimethyl ether could be the exact amount of distilled water at the
negligible due to rapid evaporation of any desired co-solvent percentage (v/w of seeds)
residual dimethyl ether present in food was then added. To achieve the required
following processing. Compared with SC- solvent to solid weight ratio, liquefied DME
CO2, the operating pressure required for was filled into the pre-weighed extractor.
DME extraction is rather low (0.6-1 MPa), The extraction was carried out at a
resulting in lower equipment cost and controlled temperature with a stir rate of 500
operating cost. Moreover, DME is partially rpm for 30 min. After extraction, the valve
water soluble, which allows wet extraction was opened, releasing the DME and the
without a water removal step prior to extracts, which passed through a stainless-
extraction, and water can be considered a steel filter (7 µm pore diameter, Swagelok)
safe co-solvent in the DME extraction into a test tube. The resulting 17 versions of
process. the FSE were stored in the refrigerator at 4
The extraction of phenolic C while waiting to be analyzed for phenolic
compounds using liquefied DME is content.

therefore investigated in this work. Three 2.3 Design of experiment by response
extraction variables were studied for their surface methodology (RSM)
effects on total phenolic content (TPC): Design of experiment and
water amount as co-solvent, solvent to optimization of extraction conditions were
sample ratio and temperature. The optimum carried out using a spherical Central
extraction conditions were determined by Composite Design (Spherical CCD). The
Response Surface Methodology (RSM) three independent variables were defined as
using the spherical Central Composite X1 = co-solvent percentage, X2 = solvent to
Design (CCD) model. solid weight ratio and X3 = extraction
temperature. The factor levels were designed
2. Materials and Methods with =K1/2 where K is the number of
2.1 Materials and chemicals variables (K = 3). The low, middle and high
Fenugreek seeds were provided by levels of each variable were designated -1, 0,
POON PHALIT CO., LTD. (Petchabun and +1, respectively. The corresponding
Province, Thailand) Liquefied DME (Spray- actual values for each variable are listed in
work air can 420D) used for extraction was Table 1. The correlation between the
purchased from Siam Tamiya Co., Ltd.,
independent variables and the response was
Thailand. described by the following second-order
2.2 Total phenolic compound extraction polynomial model.
by liquefied DME
The fenugreek seeds were first
crushed in order to obtain approximately 60-
120 mesh of fenugreek powder. The powder
was then dried in a hot air oven at 60 C for Where Xi = actual values of input variables,
72 h. The dried sample was kept in a ß0 = interception, ßii = regression coefficient
desiccator to maintain the moisture content and ßij = cross product coefficient.
before extraction. To conduct the DME
The analysis of the spherical CCD allowed to take place for 2 min. 40 l of
experimental design, analysis of variance Na2CO3 solution (4% w/v) was then added
(ANOVA) and optimization of the to all 17 utilized wells of the microplate.
conditions were carried out using Minitab The solutions were incubated in the dark, at
Statistical Software (version 17). room temperature, for 45 min. The
2.4 Analysis of total phenolic content absorbance of each extract was measured at
(TPC) 740 nm using a spectrophotometer (EnSpire
100 L of Folin–Ciocalteu (2N) Multimode Plate Reader, Perkin Elmer, US).
reagent was diluted with 900 L of DI Pyrogallol was used as a reference standard
water. 50 L of the diluted reagent was and a calibration curve (0.375-20 mg/mL).
placed in each of 17 wells of a microplate This assay was repeated in triplicate for each
and into each well was then added 2 L of extract. The results were expressed in terms
one of the 17 FSE versions, one version for of pyrogallol equivalents (mg PGE/g
each well. The resulting reaction was fenugreek sample).
Table 1. Levels of actual and coded factors
Xi -1.73 -1 0 1 1.73
Co-solvent %wt (X1) 5.4 9 14 19 22.7
Solvent to sample weight ratio (X2) 2.8 3.5 4.5 5.5 6.2
Temperature(C) (X3) 31 36 43 50 55
Table 2. Total phenolic content (TPC) results from experimental data and along with
predicted data
Run Co-solvent Solvent to Temperature gPGE/g of sample
(%) (X1) sample ratio (๐C) (X3)
(X2) Experimental Predicted %Error
data data
1 9 3.5 36 45.90 30.40 -33.77%
2 19 3.5 36 132.42 130.84 -1.19%
3 9 5.5 36 39.77 48.99 +23.18%
4 19 5.5 36 180.00 168.79 -6.23%
5 9 3.5 50 87.75 92.95 +5.93%
6 19 3.5 50 70.49 55.21 -21.68%
7 9 5.5 50 135.15 130.72 -3.28%
8 19 5.5 50 102.90 112.34 +9.17%
9 5.4 4.5 43 48.51 46.81 -3.50%
10 22.7 4.5 43 112.64 117.79 +4.57%
11 14 2.8 43 45.24 55.93 +23.63%
12 14 6.2 43 128.66 121.42 -5.63%
13 14 4.5 31 109.20 115.35 +5.63%
14 14 4.5 55 123.33 120.63 -2.19%
15 14 4.5 43 121.75 114.78 -5.72%
16 14 4.5 43 100.76 114.78 13.91%
17 14 4.5 43 124.80 114.78 -8.03%
Table 3. Analysis of variance (ANOVA) for the quadratic polynomial mode
Source DF Adj SS Adj MS F-Value P-Value
Model 9 23221.7 2580.19 12.97 0.001
Linear 3 11018.1 3672.71 18.46 0.001
x1 1 5938.1 5938.13 29.85 0.001
x2 1 5043.3 5043.32 25.35 0.002
x3 1 36.7 36.68 0.18 0.681
Square 3 2292.5 764.15 3.84 0.065
x1*x1 1 1430.8 1430.76 7.19 0.031
x2*x2 1 923.9 923.93 4.64 0.068
x3*x3 1 14.1 14.06 0.07 0.798
2-Way Interaction 3 9911.1 3303.71 16.61 0.001
x1*x2 1 187.5 187.46 0.94 0.364
x1*x3 1 9539.7 9539.73 47.95 0.000
x2*x3 1 183.9 183.95 0.92 0.368
Error 7 1392.6 198.95
Lack-of-Fit 5 1050.1 210.03 1.23 0.506
Pure Error 2 342.5 171.24
Total 16 24614.4
R2 0.94
Adj R2 0.87
3. Results and Discussion 0.002, respectively). The regression model

3.1 Quadratic regression model and generated with actual values is as follows:
variance analysis TPC = - 659 + 54.34X1 + 54.4X2
The experimental results of total + 9.07X3 - 0.434X12
phenolic content (TPC) extracted from the - 8.72 X22 + 0.0219X32
fenugreek seeds using liquefied DME in
+ 0.968X1X2 - 0.987X1X3
each extraction condition are shown in Table
2 along with the predicted results obtained + 0.685X2X3
from the regression model. The analysis of The regression model was used to
variance using Minitab version 17 is shown create the surface plots (three-dimensional
in Table 3. Looking at the results, the plots) shown in Figure 1-3. Each plot shows
predicted TPC and the actual TPC are 2.19- the combination of two test variables with
33.77% different, with an R2 of 0.94 and an the third variable maintained at its respective
zero level. The optimal conditions that
adjusted R2 of 0.87. The statistical analysis
provide the maximum TPC were co-solvent
revealed that the amount of water added as a
co-solvent and the solvent to sample ratio content of 22.65%, solvent to sample ratio of
were significant parameters, because their p- 5.60 and temperature of 30.89 C. These
values were lower than 0.05 (0.001 and optimal conditions resulted in 233.13 g of
TPC/g of seeds, with the extraction time of explained by the fact that as the solvent
30 minutes and agitation rate of 500 rpm. increases, interactions between the solute
3.2 Effect of extraction parameters on and solvent are also enhanced, and this in
TPC turn enhances the solubility into the solvent.
The effects of the three extraction Finally, looking at the effect of temperature,
conditions (%co-solvent, solvent to sample an increase in extraction temperature
ratio and temperature) on TPC were studied generally increased solubility of the solute
using the surface plots shown in Figure 1-3. (i.e. the phenolic compounds) into the solvent.
The results in Figures 1 and 2 show that an However at too high an extraction
increase in co-solvent percentage from 5.4 to temperature, the density of DME was
significantly decreased, resulting in lower
22.7% led to a significant increase in TPC
solvent power and subsequently lower TPC.
for all solvent to sample ratios. However,
increasing the co-solvent percentage at lower 4. Conclusion
temperatures gave higher TPC. The results in Liquefied DME functioned as an
Figures 1 and 3 show that an increase in effective solvent for the extraction of
solvent to sample ratio from 2.8 to 6.2 phenolic compounds from fenugreek seeds.
yielded higher TPC in all co-solvent At optimum extraction conditions suggested
percentages and at all experimental by the regression model, 233.13 g of
temperatures of this study. However, the TPC/g of seeds can be obtained. Those
effect of temperature observed from Figures optimal conditions were a co-solvent
2 and 3 might imply that too high an percentage of 22.65%, a solvent to sample
extraction temperature would yield a lower ratio of 5.60 and a temperature of 30.89C.
TPC. Regarding the effect of co-solvent The regression model provided a good
estimate of the actual experimental results,
percentage, that might be explained by the
which showed R2 = 0.94 and adjusted R2 =
overall polarity of the solvent mixture. An
0.87. In addition, the variance analysis
increase in co-solvent percentage (i.e. the revealed that co-solvent percentage and
amount of water) might increase the polarity solvent to sample ratio were significant
of the solvent and cause the higher TPC, parameters, both with p-values lower than
since phenolic compounds dissolve better in 0.05.
high polar solvents. As for the effect of
solvent to sample ratio, the results might be
A B
Figure 1. Surface plot (A) and contour plot (B) of TPC and co-solvent percentage (X1),
solvent to sample ratio (X2), fixed temperature at 43 C
A B
Figure 2. Surface plot (A) and contour plot (B) of TPC and %co-solvent (X1), temperature
(X3), fixed solvent to sample ratio at 4.5
A B
Figure 3. Surface plot (A) and contour plot (B) of TPC and solvent to sample ratio (X2),
temperature (X3), fixed %co-solvent at 14%
Acknowledgements 4. Eidi, A.; Eidi, M.; Sokhteh, M. Nutr. Res.

This work was supported by the 2007, 27, 728–733.
Thailand Research Fund (MRG5980085). 5. Swati, K.; Suchandra, C.; Prasad, V.;
Arun, S.; Devasagayam, T. P. A.; Saroj, G.
References
1. Bordia, A.; Verma, S. K.; Srivastava, K. C. J. Funct. Foods. 2014, 6, 270–279.
Prostaglandins Leukot. Essent. Fatty 6. Kenny, O.; Smyth, T. J.; Hewage, C. M.;
Acids. 1997, 56, 379–384. Brunton, N. P. Food Chem. 2013, 141,
2. Sindhu, G.; Ratheesh, M.; Shyni, G .L.; 4295–4302.
Nambisan, B.; Helen, A .Int . 7. Ghafoor, K.; Al-Juhaimi, F. Y.; Choi, Y.
Immunopharmacol .2012, 12, 205–211. H. Plant Foods Hum. Nutr. 2012, 67, 407–
3. Sakr, S. B.; Abo-El-Yazid, S. M. Toxicol. 414.
Ind. Health. 2012, 28, 876–885.
Synthesis of cationic lipids with spermine as a polar head
and its analogues for gene delivery
Chopaka Thongbamrer1, Boon-ek Yingyongnarongkul2, Uthai Sakee1,
Widchaya Radchatawedchakoon1*
1
Creative Chemistry and Innovation Research Unit, Department of Chemistry and Center of Excellence for
Innovation in Chemistry (PERCH-CIC), Faculty of Science, Mahasarakham University, Kantharawichai,
Maha Sarakham 44150, Thailand
2
Department of Chemistry and Center of Excellence for Innovation in Chemistry (PERCH-CIC),
Faculty of Science, Ramkhamhaeng University, Bangkapi, Bangkok 10240, Thailand
*E-mail: widchaya.r@msu.ac.th
Abstract:
Cholesterol-based cationic lipids are promising non-viral vectors that potentially and
safely deliver gene into cells. Cationic lipids 14 having spermine and 1,4-bis(3-
aminopropoxy)butane as polar heads and cholesterol as a hydrophobic tail were designed and
synthesized. Their structures were characterized by spectroscopic means (IR, NMR and MS).
Gel electrophoresis was used to evaluate the interaction between cationic liposome and DNA.
Cationic liposome formulations generated from lipids 1–4 could strongly bind and retard the
migration of DNA, suggesting that full DNA condensation was achieved.
1. Introduction cholesterol can form a very stable bilayer

Gene therapy using cationic lipids as structure.5 Meanwhile, the carbamate linker
non-viral for delivery a gene into the cells was cationic lipids exhibited relatively high
first reported by Felgner et al. in 1987.1 The transfection efficiency and low toxicity in
advantages of non-viral vectors provide a vitro gene delivery.9
great potential of gene delivery with easy Helper lipids can promote
preparation, less toxicity, low immunogenicity, destabilized lipid bilayers of the transition
inexpensive and high carrying capacity.2,3 from a lamellar to a hexagonal phase. It is
Commonly, the chemical structures of cationic believed to be involved in endosomal
lipids consist of three components, namely disruption. The cationic liposomes were co-
polar headgroup, hydrophobic tail and formulated with helper lipid, dioleoyl-
linker. The polar headgroup is typically phosphatidylethanolamine (DOPE), to
established from various amines or quaternary improve the transfection efficiency. In
ammonium salts such as imidazolium, addition, cholesterol was used to stabilize
guanidinium and pyridinium. Non-polar liposomal membrane, which is important for
molecules such as cholesterol and long chain the in vivo applications.10
hydrocarbons are used as the hydrophobic In this work, we designed and
tail. The linker is made from the various synthe-sized a novel series of cationic lipids
functional groups such as ester, carbamate, containing spermine and 1,4-bis(3-
amide, or ether to form a chemical bridge aminopropoxy)butane as a headgroup,
between the polar headgroup and the carbamate as a linker and cholesterol as a
hydrophobic tail.4,5 hydrophobic tail (Figure 1). The DNA binding
In previous studies, the transfection affinities of synthesized lipids were evaluated
efficiency and cytotoxicity of each coun- using gel electrophoresis.
terpart of cationic lipids were evaluated.6–8
The cholesterol was used as a hydrophobic 2. Materials and Methods
tail to enhance the efficiency of transfection 2.1 General
with low toxicity to the target cells because
Organic solvents were purchased reaction mixture was stirred at room
from commercial suppliers and were temperature for 24 h and then washed with
distilled before used. All commercial water (3×100 mL). The organic layer was
reagents from Sigma–Aldrich, Fisher combined, dried over Na2SO4, filtered, and
Scientific and Fluka were used without concentrated. The crude product was purified
further purification. IR spectra were recorded by silica gel column chromatography
(CH2Cl2) to afford compound 6 as a white
amorphous solid which was then treated
with 20% TFA in CH2Cl2 for 2 h to provide
cationic lipid 1 (119.2 mg, 92%) as a white
solid. IR: νmax 3402, 3027, 2944, 2868, 1666,
1604, 1527, 1485, 1430, 1258, 1197, 1176,
1128, 1043 cm-1; 1H NMR (400 MHz, CDCl3 +
5 drops of CD3OD): δ 0.51 (s, 3H, CH3-18),
0.69 (d, J = 6.4 Hz, 6H, CH3-26 and CH3-27),
0.75 (d, J = 6.2 Hz, 3H, CH3-21), 0.84 (s, 3H,
CH3-19), 0.91−1.40 and 1.68−2.13 (m, protons in
cholesteryl skeleton), 1.57 (m, 4H, NHCH2CH2
CH2CH2NH), 1.67 (m, 4H, NH2CH2CH2CH2
NH, NHCH2CH2CH2NHCO2-Chol), 2.81−
2.92 (m, 8H, NH2CH2CH2CH2NH, NHCH2
CH2CH2CH2NH), 3.01 (m, 2H,NHCH2CH2
Figure 1. The structures of cationic lipids 1– CH2NHCO2-Chol), 3.17 (m, 2H, NHCH2CH2
4 containing spermine and 1,4-bis(3- CH2NHCO2-Chol), 4.24 (m, 1H, H-3-Chol),
aminopropoxy)butane as polar headgroups. 5.19 (br s, 1H, H-6-Chol); 13C NMR (100 MHz,
CDCl3 + 5 drops of CD3OD): 11.5, 18.3, 18.9,
on a Perkin-Elmer FT-IR 400 spectrometer 20.7, 22.1, 22.4, 23.5, 23.9, 27.7, 27.9, 29.8, 31.6,
(ATR). NMR spectra were recorded on a 32.0, 35.5, 35.9, 36.2, 36.7, 38.1, 39.2, 39.4, 42.0,
Bruker AVANCE400 and AscendTM 400 49.8, 55.9, 56.4, 74.6, 122.3, 139.5 (carbons in
spectrometers operating at 400 MHz for 1H cholesteryl skeleton), 26.1, 26.2, 27.7, 29.4,
and 100 MHz for 13C. All coupling constants 37.1, 39.2, 44.2, 45.1, 46.7, 46.7 (carbons in
(J values) were measured in Hertz (Hz). ESI spermine), 157.2 (CO carbamoyl); MS (ESI
mass spectra were recorded with a Finnigan positive): m/z 615.0 ([M + H]+, 100%); HRMS:
LCQ mass spectrometer. HR-ESI-MS spectra m/z calcd for C38H70N4NaO2: 637.5391; found
of separated compounds were obtained using 637.5435.
a Bruker micrOTOF-II mass spectrometer. 2.2.2 Synthesis of cationic lipid 2
Thin-layer chromatography (TLC) was run To a solution of lipid 7 (502.4 mg, 1.1
by using Silica gel 60 F254 precoated on mmol) in CH2Cl2 (10 mL) was added methyl
aluminum plate (E-Merck). TLC plates were acrylate (10.0 mL). The reaction mixture was
visualized under UV light followed by stirred at room temperature for 5 days. The
straining in ninhydrin or p-anisaldehyde solvent was removed under reduced pressure
solution. The product was isolated as a pure and the residue was purified by silica gel
material using column chromatography on column chromatography (CH2Cl2) to afford
silica gel or Sephadex LH-20 column compound 8 as a pale yellow oil.
chromatography. To a solution of compound 8 (200.6
2.2 Synthesis mg, 0.31 mmol) in MeOH was added
2.2.1 Synthesis of cationic lipid 1 spermine (125.5 mg, 0.6 mmol). The reaction
To a stirred solution of 5 (500.2 mg, mixture was stirred at room temperature for
1.1 mmol) in CH2Cl2 (10 mL) was added 5 days. The solvent was removed under
compound 9 (442.2 mg, 1.1 mmol). The reduced pressure to afford a yellow oil, which
was purified by Sephadex LH-20 column 3H, CH3-21), 0.90 (s, 3H, CH3-19), 0.91−1.40
chromatography (MeOH) to provide cationic and 0.94−2.23 (m, protons in cholesteryl
lipid 2 (272.2 mg, 89%) as a pale yellow oil. skeleton), 1.53 (m, 6H, OCH2CH2CH2CH2O,
IR: νmax 3248, 3115, 2927, 2867, 1704, 1621, NH2CH2CH2CH2O),1.93 (m 2H, OCH2CH2
1582, 1476, 1396, 1370, 1274, 1262, 1127 cm- CH2NHCO2-Chol), 2.99 (br s, 2H, OCH2
1 1
; H NMR (400 MHz, CDCl3 + 3 drops of CH2CH2CH2O), 3.12 (m, 2H, OCH2CH2
CD3OD): δ 0.58 (s, 3H, CH3-18), 0.77 (d, J = CH2NHCO2-Chol), 3.34 (m, 6H, NH2CH2CH2
6.6 Hz, 6H, CH3-26 and CH3-27), 0.82 (d, J = CH2O, OCH2CH2CH2CH2O), 3.48 (br s,
6.5 Hz, 3H, CH3-21), 0.91 (s, 3H, CH3-19), 2H, OCH2CH2CH2NHCO2-Chol), 4.35 (m,
0.98−1.44 and 1.75−2.35 (m, protons in 1H, H-3-Chol), 5.27 (br s, 1H, H-6-Chol); 13C
cholesteryl skeleton), 1.47 (m, 8H, 2×NHCH2 NMR (100 MHz, CDCl3 + 5 drops of CD3OD):
CH2CH2CH2NH), 1.61 (m, 8H, 2×NH2CH2 11.7,18.5,19.1,20.9,22.3,22.6,23.6,24.1,27.8,
CH2CH2NH, 2×NHCH2CH2CH2NHCO), 2.38 28.0, 28.1, 31.6, 31.7, 35.6, 36.0, 36.4, 36.8,
(m, 2H, NCH2CH2NHCO2-Chol), 2.45, 3.28 38.4, 39.3, 39.5, 42.1, 49.8, 55.9, 56.5, 74.3,
(m, 4H, 2×COCH2CH2N), 2.35, 3.59 (m, 4H, 122.4, 139.6 (carbons in cholesteryl skeleton),
2×COCH2CH2N), 2.55−2.69 (m, 20H, 2×NH2 26.0, 27.8, 29.5, 31.7, 38.5, 38.6, 68.6, 68.8,
CH2CH2CH2NH, 2×NHCH2CH2CH2CH2NH, 70.5, 71.1 (carbons in 1,4-bis(3-aminopropoxy)
2×NHCH2CH2CH2NHCO), 3.09−3.17 (m, 6H, butane), 156.5 (CO carbamoyl); HRMS: m/z
2×NHCH2CH2CH2NHCO, NCH2CH2NHCO2- calcd for C38H69N2O4: 617.5252; found
Chol), 4.36 (m, 1H, H-3-Chol), 5.27 (br s, 1H, 617.5221.
H-6-Chol); 13C NMR (100 MHz, CDCl3 + 3 2.2.4 Synthesis of cationic lipid 4
drops of CD3OD): 11.7, 18.5, 19.1, 20.9, 22.4, According to the procedure for the
22.6, 23.6, 24.1, 26.8, 28.0, 28.1, 31.7, 31.7, synthesis of lipid 2, compound 8 (201.3 mg,
35.6, 36.0, 36.4, 36.8, 38.4, 39.3, 39.6, 42.1, 0.31 mmol) was reacted with 1,4-bis(3-
51.6, 55.9, 56.5, 74.3, 122.4, 139.6 (carbons in aminopropoxy)butane (126 mg, 0.62 mol) to
cholesteryl skeleton), 23.6, 24.1, 27.8, 31.3, provide lipid 4 (285.2 mg, 93%) as a pale
36.4, 39.3, 39.6, 42.1, 47.1, 47.1 (carbons in yellow oil. IR: νmax 3359, 3305, 2936, 2863,
spermine), 33.6, 44.4, 52.6, 53.3 (2×COCH2 1642, 1553, 1467, 1372, 1275, 1260, 1103,
CH2N, NCH2CH2CO2-Chol), 156.5 (CO 1032 cm-1; 1H NMR (400 MHz, CDCl3 + 5
carbamoyl), 173.0, 173.6 (CO amide); HRMS: drops of CD3OD): δ 0.51 (s, 3H, CH3-18),
m/z calcd for C60H113N4O6: 985.8654; found 0.69 (d, J = 6.6 Hz, 6H, CH3-26 and CH3-27),
985.8644. 0.74 (d, J = 6.5 Hz, 3H, CH3-21), 0.84 (s, 3H,
2.2.3 Synthesis of cationic lipid 3 CH3-19), 0.86−1.86 (m, protons in
To a solution of cholesteryl cholesteryl skeleton), 1.44 (m, 12H, 2×
chloroformate (500.3 mg, 1.1 mmol) in CH2Cl2
OCH2CH2CH2CH2O, 2×NH2CH2CH2CH2
(10 mL) was added 1,4-bis(3-aminopropoxy)-
O), 1.60 (m, 4H, 2× OCH2CH2CH2NHCO),
butane (224.4 mg, 1.1 mmol). The reaction
mixture was stirred at room temperature for 2.14 (t, J = 6.2 Hz, 6H, NCH2CH2NHCO2-
24 h and then washed with water (3×100 Chol, 2×COCH2CH2N), 2.54 (t, J = 6.6 Hz,
mL). The organic layer was combined, dried 4H, 2×NH2CH2CH2CH2O), 3.09 (t, J = 7.0
over Na2SO4, filtered, and concentrated. The Hz, 4H, 2×NH2CH2CH2CH2O), 3.19−3.36
residue was purified by silica gel column (m, 16H, 2×OCH2CH2CH2NHCO,
chromatography (1:1 hexane/CH2Cl2 to 80:20 2×OCH2CH2CH2CH2O), 4.25 (m, 1H, H-3-
CH2Cl2/MeOH) to afford cationic lipid 3 Chol), 5.20 (br s, 1H, H-6-Chol); 13C NMR
(536.4 mg, 79%) as a pale yellow oil. IR: νmax (100 MHz, CDCl3 + 5 drops of CD3OD):
3356, 3346, 2936, 2866, 1695, 1523, 1466, 11.7, 18.5, 19.2, 20.9, 22.4, 22.6, 23.7, 24.1,
1376, 1366, 1249, 1105, 1031, 1011 cm-1; 1H 27.9, 28.0, 29.1, 31.8, 31.8, 36.1, 36.4, 36.8,
NMR (400 MHz, CDCl3 + 5 drops of CD3OD): 36.9, 38.3, 38.5, 39.6, 42.2, 49.7, 56.0, 56.6,
δ 0.58 (s, 3H, CH3-18), 0.76 (d, J = 6.4 Hz, 74.4, 122.5, 139.7 (carbons in cholesteryl
6H, CH3-26 and CH3-27), 0.83 (d, J = 6.2 Hz, skeleton), 26.2, 26.2, 28.1, 33.7,
Scheme 1. Synthesis of cationic lipids 1–4. Reagents and conditions: (a) BocNH(CH2)3NBoc
(CH2)4NBoc(CH2)3NH2 (9) or 1,4-bis(3-aminopropoxy)butane (1.0 equiv), CH2Cl2, 24 h; (b)
20% TFA in CH2Cl2, 2 h; (c) methyl acrylate, MeOH, 5 days; (d) spermine or 1,4-bis(3-
aminopropoxy)butane (2 equiv), MeOH, 5 days
39.0, 39.4, 68.4, 68.9, 70.6, 70.8 (2× carbons in 3. Results and Discussion
1,4-bis(3-aminopropoxy)butane), 35.7, 44.9, 3.1 Synthesis of lipids 14
49.9, 52.5 (2×COCH2CH2N, - Starting from cholesteryl
NCH2CH2CO2-Chol), 156.9 (CO chloroformate, the synthesis of lipid 3 with
carbamoyl), 173.2 (CO amide); HRMS: m/z carbamate linker and amine headgroup can
calcd for C56H105N6O8: 989.7988; found be achieved in only one step by reacting
989.8028. with 1,4-bis(3-aminopropoxy)butane.
2.2 Gel electrophoresis Compound 6 was also reacted with Boc-
Gel electrophoresis was used to protected spermine (9) followed by Boc
evaluate the formulated between DNA and deprotection to furnish cationic lipid 1 in
cationic lipids. The complexes were prepared excellent yield (Scheme 1).
by adding liposome into DNA (0.1 µg). The The two-headgroup cationic lipids 2
DNA/lipid ratios of 1:5, 1:10 and 1:20 were and 4 were synthesized from compound 7
examined for the ability to retard DNA prepared from the reaction between ethylene-
migration through 1.0% agarose gel diamine and cholesteryl chloroformate. In
electrophoresis. Ethidium bromide was also this case, we elongated the structure by
added in agarose plate. Loading dye reagent installing two methyl ester groups at the
was then added to the lipoplexes. The primary amino group of compound 7 by
solutions (10 µL) were loaded into the gel reacting with methyl acrylate to provide
and operated at 100 V for 30 min. compound 8. Treating 8 with 2 equiv. of
spermine and 1,4-bis(3-aminopropoxy)butane Center of Excellence for Innovation in
afforded lipids 2 and 4, respectively in good Chemistry (PERCH-CIC). This project was
yields. financially supported by the Faculty of
3.2 Gel electrophoresis assay Science, Mahasarakham University.
Gel electrophoresis was used to
evaluate the ability of cationic lipids to form References
complexes with DNA. As shown in Figure 1. Felgner, P. L.; Gadek, T. R.; Holm, M.;
2, cationic lipids 14 with and without Roman, R.; Chan, H. W.; Wenz, M.;
DOPE could retard a strong DNA movement Northrop, J. P.; Ringold, G. M.;
at all ratios of DNA/cationic lipids. The Danielsen, M. Proc. Natl. Acad. Sci.
result suggested that the synthesized lipids U.S.A. 1987, 84, 7413–7417
could fully condense the DNA. 2. Ju, J.; Haun, M. L.; Wan, N.; Hou, Y. L.;
a. Ma, X. X.; Jia, Y. Y.; Li, C.; Zhou, S.
1 2 3 4
Y.; Zhang, B L. Bioorg. Med. Chem.
control
1:20
1:20
1:10
1:10
1:20
1:10
1:10
1:20
Lett. 2016, 26, 2401–2407.
1:5
1:5
1:5
1:5
3. Schmidt-Wolf, G. D.; Schmidt-Wolf, I.

G. Trends Mol Med. 2003, 9, 67–72.
4. Caroline, C.; Vandenbranden, M.;
b. Ruysschaert, J. M. Prog. Lipid Res. 2008,
1 2 3 4
47, 340–347.
control
1:10
1:10
1:20
1:10
1:20
1:20
1:10
1:20
5. Zhi, D.; Zhang, S.; Wang, B.; Zhao, Y.;

1:5
1:5
1:5
1:5
Yang, B.; Yu, S. Bioconjug. Chem.

2010, 21, 563–577.
6. Radchatawedchakoon, W.; Watanapokasin,
Figure 2. Electrophoretic gel retardation R.; Krajarng, A.; Yingyongnarongkul, B.
assays (a) lipoplexes 14 with DOPE (b) Bioorg. Med. Chem. 2010, 18, 330–342.
lipoplexes 14 without DOPE 7. Sheng, R.; Luo, T.; Li, H.; Sun, J.; Wang,
Z.; Cao A. Colloids Surf B Biointerfaces.
2014, 116, 32–40.
4. Conclusion
The synthesized lipids containing 8. Bajaj, a.; Mishra, S. K.; Kondaiah, P.;
spermine and 1,4-bis(3-aminopropoxy)butane Bhattacharya, S. Biochim. Biophys. Acta.
as headgroups, carbamate as linker and 2008, 1778, 1222–1236.
choles-terol as hydrophobic tail exhibited 9. Zhi, D.; Zhang, S.; Qureshi, F.; Zhao,
high DNA binding affinities. The Y.; Cui, S.; Wang, B.; Chen, H.; Wang,
condensation between DNA and cationic Y.; Zhao, D. Bioorg. Med. Chem. Lett.
liposome is the crucial step to evaluate the 2012, 22, 3837–3841.
transfection efficiency both in vitro and in 10. Kurosaki, T.; Kitahara, T.; Teshima, M.;
vivo applications. Nishida, K.; Nakamura, J.; Nakashima,
M.; To, H.; Fukuchi, H.; Hamamoto, T.;
Sasaki, H. J. Pharm. Pharm. Sci. 2008,
Acknowledgements
CT acknowledges the Science 11, 56–67.
Achievement Scholarship of Thailand (SAST)
for scholarship. Partial support was from the
Fungal metabolites from the endophytic fungus Xylaria sp. PSU-STD70
Wipapan Kongyen1*, Vatcharin Rukachaisirikul2, Souwaluk Phongpaichit3, Jariya Sakayaroj4
1
Program of Chemistry and Applied Chemistry, Faculty of Science and Technology,
Songkhla Rajabhat University, Songkhla 90000, Thailand
2
Prince of Songkla University, Songkhla 90112, Thailand
3
Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand
4
National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Klong Luang,
*E-mail: pwipapan@yahoo.com
Abstract:
Fungi belonging to the genus Xylaria are a source of bioactive metabolites. The ethyl
acetate extracts from the culture broth and mycelia of Xylaria sp. PSU-STD70, which was
isolated from midrib of a mangrove plant, Avicennia alba Bl., exhibited mild antimicrobial
activities against Staphylococcus aureus, methicillin-resistant S. aureus and Microsporum
gypseum. We describe herein the isolation and structural elucidation of cytochalasin D (1),
5-methoxycarbonylmellein (2), (R)-(-)-mellein methyl ether (3), 5-carboxymellein (4), tyrosol
(5), stachyline C (6) and griseoxanthone C (7). The structures were assigned by spectroscopic
methods. All compounds were weakly active against both bacterial strains with equal
minimum inhibitory concentration (MIC) values of 200 g/mL. Compound 2 displayed better
activity against M. gypseum with the MIC value of 128 g/mL while other compounds
showed mild activity.

The genus Xylaria is a rich source of 2.1 General
biologically active secondary metabolites. Infrared spectra (IR) were recorded
These include cytotoxic cytochalasins,1 as neat on a Perkin Elmer 783 FTS165 FT-
antibacterial fatty acids2 and antimalarial IR spectrometer. Ultraviolet (UV)
chromanones.3 The ethyl acetate extracts absorption spectra were measured in
from the culture broth and mycelia of methanol solution on a SHIMADZU UV-
Xylaria sp. PSU-STD70, which was isolated 160A spectrophotometer. 1H and 13C NMR
from midrib of a mangrove plant, Avicennia spectra were recorded on a 500 MHz Bruker
alba Bl., exhibited mild antimicrobial FTNMR Ultra ShieldTM spectrometer with
activities against Staphylococcus aureus, tetramethylsilane (TMS) as an internal
methicillin-resistant S. aureus and standard. Mass spectra were obtained on a
Microsporum gypseum. Investigation of the MAT 95 XL mass spectrometer (Thermo
extracts led to the isolation and structural Finnigan). Optical rotations were measured
elucidation of cytochalasin D (1),4 5- in methanol solution on a JASCO P-1020
methoxycarbonylmellein (2),5 (R)-(-)- polarimeter. Thin-layer chromatography
6
mellein methyl ether (3), 5-carboxymellein (TLC) and precoated TLC were performed
(4),5 tyrosol (5),7 stachyline C (6)8,9 and on silica gel 60 GF254 (Merck). Column
griseoxanthone C (7).10 All isolated chromatography (CC) was carried out on
compounds were tested for antimicrobial silica gel (Merck) type 100 (70-230 Mesh
activity against S. aureus, methicillin- ASTM) or on Sephadex LH-20 with MeOH,
resistant S. aureus and M. gypseum. or as otherwise stated.
2.2 Fungal material
The endophytic fungus Xylaria sp.
PSU-STD70 was isolated from midrib of A.
alba Bl., which was collected in Suratthani fractions (D1-D3). Fraction D2 (38.1 mg)
Province, Thailand, in 2006. This fungus was separated by CC over silica gel with the
was deposited as PSU-STD70 at the same gradient system as fraction B2 to yield
Department of Microbiology, Faculty of 6 (6.6 mg). Fraction F (35.4 mg) was further
Science, Prince of Songkla University. subjected to CC over silica gel with gradient
2.3 Fermentation and isolation systems of EtOAc-light petroleum and
The endophytic fungus Xylaria sp. MeOH-EtOAc to afford 7 (1.6 mg). The wet
PSU-STD70 was grown on potato dextrose mycelia were extracted twice with 500 mL
agar (PDA) and incubated at 25 °C for 5 of MeOH for 2 days. The aqueous MeOH
days. Six agar plugs (1x1 cm2) from the layer was concentrated under reduced
edge of the mycelium from an actively pressure. The extract was added H2O (50
growing culture were inoculated into 500 mL), and the mixture was washed with
mL Erlenmeyer flasks containing 300 mL hexane (700 mL). The aqueous layer was
potato dextrose broth (PDB) and incubated extracted three times with an equal volume
for 3 weeks at room temperature.11 The of EtOAc (300 mL). The combined EtOAc
culture broth (15.6 L) was filtered through extracts were dried over anhydrous Na2SO4
Whatman #1 to separate the filtrate and and then evaporated to dryness under
mycelia. The filtrate (300x5 mL) was reduced pressure to give a dark brown gum
extracted three times with an equal volume (485.0 mg). The mycelial extract was
of EtOAc (300 mL). The combined EtOAc fractionated using the same procedure as the
layer was dried over anhydrous Na2SO4, and broth extract to afford three fractions. The
evaporated to dryness under reduced second fraction (70.8 mg) was separated by
pressure to obtain a brown gum (3.02 g). CC over Sephadex LH-20 using the same
The broth extract was fractionated by CC eluent as the mycelial extract to afford five
over Sephadex LH-20 using 100% MeOH as subfractions. The third subfraction (22.7
eluent to afford seven fractions (A-G). mg) was purified using precoated TLC with
Fraction B (259.8 mg) was subjected to CC 40% EtOAc-light petroleum as a mobile
over Sephadex LH-20 using 100% MeOH as phase (2 runs) to give 2 (4.6 mg) and 3 (4.0
eluent to give three fractions (B1-B3). mg). The fourth subfraction (18.0 mg) was
Fraction B2 (195.1 mg) was separated by further separated using the same proceduce
CC over silica gel using a gradient system of as the third subfraction with 50% EtOAc-
MeOH-CH2Cl2 to give three subfractions light petroleum as a mobile phase (2 runs) to
(B21-B23). Subfraction B22 (139.4 mg) was give 2 (3.5 mg) and 4 (14.0 mg).
further separated using the same procedure 2.4 Antimicrobial activity testing
as fraction B2 to give 1 (60.6 mg). Fraction Antibacterial activity was evaluated
C (475.7 mg) upon CC over silica gel using against S. aureus and methicillin-resistant S.
the same gradient system as that of fraction aureus, using the agar microdilution
B2 yielded five fractions (C1-C5). Fraction method.12 MICs were recorded by reading
C2 (6.0 mg) was subjected to precoated TLC the lowest substance concentration that
using 40% EtOAc-light petroleum as a inhibited visible growth. Vancomycin, a
mobile phase (2 runs) to give 2 (3.0 mg). positive control drug, exhibited the MIC
Fraction C3 (118.1 mg) was subjected to CC value of 1g/mL. Growth controls were
over Sephadex LH-20 using 100% MeOH as performed on the agar containing DMSO.
eluent to give four subfractions (C31-C34). Antifungal activity was evaluated against
Subfraction C32 (93.2 mg) upon CC over M. gypseum SH-MU-4 using the hyphal
silica gel with gradient systems of EtOAc- extension-inhibition assay.13 A modification
light petroleum and MeOH-EtOAc afforded of the NCCLS M38-A broth microdilution
3 (8.5 mg) and 5 (6.2 mg). Fraction D (267.6 test14 was performed. Equal volumes of a
mg) upon purification using the same suspension of conidia (approximately 4x103
procedure as fraction B2 yielded three
CH2
5
H3C OH HO
5 7
10 H H 15 7 1
OR
24 9 22
3
3 CH3
H 1 H 13
HN 5: R = H 4'
27 O 18
20 O
H3C 30 O
1' 3'
H3C OH 6: R = 5'
23
O 1 OH
R1 O
CH3 O OH
8a
8 1
O 8a 9a
H 8 1
5
4
4a 5 4
CH3 HO 10a O 4a
OCH3
R2 9
10
2: R1 = OH, R2 = COOCH3 7
3: R1 = OCH3, R2 = H
4: R1 = OH, R2 = COOH
Figure 1. Chemical structures of compounds 1-7
conidia/mL) were added to each test dilution and 5.09) (Table 1). These indicated that the
to make final concentrations of 1-128 gem-disubstituted alkene moiety was
g/mL in triplicate. Plates were incubated at connected to C-6 (C 147.6). HMBC
25 °C for 72 h. Miconazole, a standard correlations of Hab-12 with C-5 (C 32.7), C-
antifungal agent, gave an MIC value of 9.6 6 and C-7 (C 69.8) supported the
M. The MICs were recorded for the lowest assignment. Thus, 1 was identified as
concentration that resulted in a reduction of cytochalasin D which was previously
approximately 50% of the fungal growth. isolated from Xylaria sp. BCC 9653.15 In
addition, the observed optical rotation of 1
3. Results and Discussion ([]D27 -31.05, c = 0.33, CHCl3) was almost
The crude extracts from the culture identical to that of cytochalasin D ([]D20 -
broth and mycelia of the fungus PSU- 28.00, c = 0.33, CHCl3),4 indicating their
STD70 were purified using various identical absolute configuration.
chromatographic techniques. All compounds Compound 2 was obtained as a
except for 4 were obtained from the broth yellow gum. Its NMR spectroscopic data
extract while the mycelial extract provided (Table 2) were similar to those of 5-
compounds 2, 3 and 4. Their structures were methoxycarbonyl-mellein. Compound 2
elucidated by spectroscopic methods. 1H gave almost identical optical rotation to that
NMR data were compared with those of 5-methoxy-carbonylmellein, []D27 of 2 =
previously reported. -158.00 (c = 1.00, CHCl3) and []D22 of 5-
Compound 1 was obtained as a white methoxy-carbonylmellein = -163.00 (c =
solid and melted at 267.0-267.3 C. The 1H 1.00, CHCl3).5 Therefore, 2 was assigned as
NMR spectral data were similar to those of 5-methoxycarbonylmellein which was
cytochalasin O4 except that the singlet previously isolated from xylariaceae family.5
methyl signal (H 1.22) in cytochalasin O Compound 3 was obtained as a
was replaced, in 1, by signals of two yellow gum. The NMR spectral data (Table
geminal olefinic protons, Hab-12 (H 5.30,
2) were similar to those of 2 except for the methoxy signal (H 3.88). These suggested
absence of the ester carbonyl group. In that the methoxy carbonyl group in 2 was
addition, signal of the chelated hydroxy replaced, in 4, by a carboxyl group at C-5
proton (H 11.83) in 2 was replaced, in 3, by (C 128.0). The 13C NMR spectrum
a methoxy signal (H 3.95). The methoxyl displayed two carbonyl carbons at C 170.6
group was then located at C-8 (C 161.3). and 166.9. The carbon resonance at C 170.6
Compound 3 gave almost identical optical was assigned as C-1 on the basis of HMBC
rotation to that of (R)-(-)-mellein methyl correlations of H-7 (H 6.84)/C-1, C-5 (C
ether, []D27 of 3 = -232.73 (c = 0.50, 128.0) and C-8a (C 107.8). According to
CHCl3) and []D of (R)-(-)-mellein methyl the chemical shift of C-5, the substituent at
ether = -259.00 (c = 0.50, CHCl3).6 C-5 must be the carboxyl group. Therefore,
Therefore, 3 was assigned as (R)-(-)-mellein 4 was assigned as 5-carboxymellein which
methyl ether which was previously isolated was previously isolated from Xylaria sp.
from Xylaria sp. BCC 9653.15 BCC 9653.15
Compound 5 was obtained as a
Table 1. The NMR data of 1 in CDCl3 colorless gum. Comparison of the 1H NMR
Position H C (C-Type) data of 5 (Table 3) with those of tyrosol
1 - 173.5 (C=O)
2-NH 5.60 (brs) -
indicated that 5 was tyrosol7 which was
3 3.24 (m) 53.6 (CH) previously isolated from X. longipes.16
4 2.15 (dd, 6.6, 3.6) 50.0 (CH) Compound 6 was obtained as a
5 2.75 (m) 32.7 (CH) yellow gum with []28D -0.72 (c 0.90,
6 - 147.6 (C) MeOH). The HREI-MS showed the
7 3.81 (d, 10.8) 69.8 (CH)
8 2.85 (m) 47.0 (CH)
molecular formula C13H18O3. The NMR
9 - 50.9 (C) spectral data (Table 4) displayed signals
10 a: 2.83 (m) 45.3 (CH2) similar to those of 5 except for an additional
b: 2.68 (m) signal of a 1,2-dioxy-3-methyl-3-butenyl
11 0.94 (d, 6.6) 13.6 (CH3) side chain. The 1H-1H COSY spectrum
12 a: 5.30 (brs) 114.4 (CH2)
b: 5.09 (brs) supported that the Hab-1’ (H 4.04 and 3.91)
13 5.69 (dd, 15.6, 9.9) 130.6 (CH) were coupled with the H-2’ (H 4.47). Hab-4’
14 5.34 (ddd, 15.6, 10.5, 134.1 (CH) (H 5.15 and 5.01) and H3-5’ (H 1.82)
4.8) showed cross peaks in the HMBC spectrum
15 a: 2.51 (dd, 12.6, 10.5) 37.7 (CH2)
b: 2.02 (dd, 12.6, 4.8) with C-2’ (C 73.6) and C-3’ (C 143.4). The
16 2.74 (m) 42.3 (CH) attachment of this side chain at C-4 (C
17 - 210.3 (C=O) 157.3) through an ether linkage was
18-OH - 77.1 (C) established according to the 3J HMBC
19 5.14 (dd, 15.6, 2.4) 127.6 (CH)
20 6.10 (dd, 15.6, 2.4) 132.3 (CH) correlation from Hab-1’ to C-4. Therefore, 6
21 5.63 (t, 2.4) 77.7 (CH) was stachyline C which was previously
22 1.20 (d, 6.9) 19.4 (CH3) isolated from Stachylidium sp.8 and
23 1.51 (s) 24.2 (CH3) Marasmiellus ramealis (Bull.).9 However,
24 - 137.2 (C) this is the first isolated report from the
25, 29 7.13 (dd, 8.1, 1.5) 129.1 (CH)
26, 28 7.27 (m) 127.1 (CH) Xylaria genus.
27 7.32 (td, 8.1, 1.5) 128.9 (CH) Compound 7 was obtained as a
30 - 169.7 (C=O) yellow gum. The chelated hydroxy proton
31 2.26 (s) 20.8 (CH3) (H 13.37) (Table 5) was placed at C-1 (C
164.0), peri position to the ketone carbonyl
Compound 4 with []D27 -52.55 (c = group (C 163.5, C-9), due to its HMBC
0.02, MeOH) was obtained as a white solid
correlations with C-2 (C 96.9) and C-9a (C
and melted at 245.5-246.3 C. The NMR
104.0). Two meta-aromatic protons at H
spectroscopic data (Table 2) were similar to
6.30 and 6.33 were then attributed to H-2
those of 2 except for the absence of a
and H-4, respectively. HMBC correlations
Table 2. The NMR data of 2, 3 in CDCl3 and 4 in CD3OD
Position 2 3 4
H C (C-Type) H C (C-Type) H C (C-Type)
1 - 170.0 (C=O) - 162.6 (C=O) - 170.6 (C=O)
3 4.67 (m) 75.6 (CH) 4.56 (qt, 6.3, 4.2) 74.1 (CH) 4.68 (m) 76.1 (CH)
4 a: 3.87 (dd, 17.7, 32.6 (CH2) 2.87 (d, 4.2) 36.1 (CH2) a: 3.74 (dd, 17.4, 32.6 (CH2)
3.3) 3.3)
b: 3.05 (dd, 17.7, b: 2.97 (dd, 17.4,
11.7) 11.7)
4a - 143.5 (C) - 141.9 (C) - 139.7 (C)
5 - 118.6 (C) 6.79 (d, 7.2) 119.2 (CH) - 128.0 (C)
6 8.13 (d, 9.0) 138.5 (CH) 7.45 (dd, 8.7, 7.2) 134.4 (CH) 7.90 (d, 8.7) 137.1 (CH)
7 6.94 (d, 9.0) 116.2 (CH) 6.92 (d, 8.7) 110.9 (CH) 6.84 (d, 8.7) 114.7 (CH)
8-OH 11.83 (s) - - - - -
8-OCH3 - - 3.95 (s) 56.2 (CH3) - -
8 - 165.5 (C) - 161.3 (C) - 162.8 (C)
8a - 108.9 (C) - 113.9 (C) - 107.8 (C)
9 1.56 (d, 6.3) 20.8 (CH3) 1.48 (d, 6.3) 20.7 (CH3) 1.49 (d, 6.3) 19.6 (CH3)
10 - 166.2 (C=O) - - - 166.9 (C=O)
10-OCH3 3.88 (s) 52.0 (CH3) - - - -
Table 3. The NMR data of 5 in CDCl3 (H 2.85, Me-11) with C-7 (C 115.7) and C-
Position H C (C-Type) 8a (C 113.0) suggested the attachment of
1 - 154.2 (C)
the methyl group at C-8 (C 145.0). Since
2, 6 6.79 (d, 9.0) 115.4 (CH)
3, 5 7.11 (d, 9.0) 130.1 (CH) there was no other signal, the substituent at
4 - 130.5 (C) C-6 (C 159.0) must be a hydroxyl group.
7 2.81 (t, 6.0) 38.3 (CH2) Thus, 7 was assigned as griseoxanthone C
8 3.83 (t, 6.0) 63.8 (CH2) which was previously unreported from
Xylaria genus. It was isolated from many
Table 4. The NMR data of 6 in CDCl3 genera for example, Ledebouria
Position H C (C-Type)
1 - 157.3 (C)
graminifolia,10 Penicillium sacculum17 and
2, 6 6.88 (d, 8.7) 114.9 (CH) Phomopsis sp.18
3, 5 7.15 (d, 8.7) 130.1 (CH)
4 - 157.3 (C) Table 5. The NMR data of 7 in CDCl3
7 2.82 (t, 6.6) 38.3 (CH2) Position H C (C-Type)
8 3.83 (t, 6.6) 63.8 (CH2) 1-OH 13.37 (s) 164.0 (C)
1’ a: 4.04 (dd, 9.6, 3.3) 71.3 (CH2) 2 6.30 (d, 2.5) 96.9 (CH)
b: 3.91 (dd, 9.6, 3.3) 3 - 166.0 (C)
2’ 4.47 (dd, 8.1, 3.3) 73.6 (CH2) 3-OCH3 3.87 (s) 55.7 (CH3)
3’ - 143.4 (C) 4 6.33 (d, 2.5) 92.1 (CH)
4’ a: 5.15 (brs) 112.7 (CH2) 4a - 157.0 (C)
b: 5.01 (brs) 5 6.67 (d, 1.0) 101.1 (CH)
5’ 1.82 (s) 18.9 (CH3) 6-OH - 159.0 (C)
7 6.60 (brs) 115.7 (CH)
of these protons supported the assignment. 8 - 145.0 (C)
Furthermore, the HMBC correlations of the 8a - 113.0 (C)
9 - 163.5 (C=O)
methoxy protons (H 3.87) with C-3 (C 9a - 104.0 (C)
166.0) established the location of the 10a - 160.0 (C)
methoxyl group at C-3. Other two meta- 11 2.85 (s) 23.3 (CH3)
aromatic protons at H 6.67 and 6.60 were
assigned as H-5 and H-7, respectively, on All compounds were weakly active
the basis of their chemical shifts and HMBC against both S. aureus and methicillin-
correlations. These results together with resistant S. aureus with equal minimum
HMBC cross peaks of the methyl protons inhibitory concentration (MIC) values of
200 g/mL. However, compound 2 7. Guzman-Lopez, O.; Trigos, A.;
exhibited better activity against M. gypseum Fernandez, F. J.; Yanez-Morales, M.;
with the MIC value of 128 g/mL, while Saucedo-Castaneda, G. World J
other compounds showed mild activity. Microbiol Biotechnol. 2007, 23, 1473-
1477.
4. Conclusion 8. Almeida, C.; Part, N.; Bouhired, S.;
Compounds 1, 3 and 4 were Kehraus, S.; Kӧnig, G. M. J. Nat. Prod.
previously isolated from Xylaria sp. BCC 2011, 74, 21-25.
965315 while 2 was produced by the fungus 9. Yang, N.; Ma, Q.; Huang, S.; Dai, H.;
in the xylariaceae family.5 In addition, the Guo, Z.; Lu, X.; Wang, Y.; Yu, Z.;
fungus X. longipes16 yield 5. Compounds 6 Zhao, Y. J. Braz. Chem. Soc. 2015,
and 7 have never been isolated from the 26 (1), 9-13.
Xylaria genus. 10. Mutanyatta, J.; Matapa, B. G.; Shushu,
D. D.; Abegaz, B. M. Phytochemistry
Acknowledgements 2003, 62, 797-804.
W. Kongyen thanks the Commission 11. Phongpaichit, S.; Rungjindamai, N.;
on Higher Education and the Thailand Rukachaisirikul, V.; Sakayaroj, J.
Research Fund for the Research Grant No. Microbiol. 2006, 48, 367-372.
RTA5180007 to Prof. Dr. Vatcharin 12. Lorian, V. Antibiotics in Laboratory
Rukachaisirikul. Medicine, 4th ed.; William and Wilkins:
Baltimore, 1996, pp. 28-32.
References 13. Huang, X.; Xie, W.; Gong, Z. FEBS
1. Chen, Z.; Chen, Y.; Huang, H.; Yang, Lett. 2000, 478, 123-126.
H.; Zhang, W.; Sun, Y.; Wen, J. 14. National Committee for Clinical
Z. Naturforsch., C: Biosci. 2017, 72 (3- Laboratory Standards. Reference
4), 129-132. Method for Broth Dilution Antifungal
2. Sun, D. W.; Cao, F.; Liu, M.; Guan, F. Susceptibility Testing of Filamentous
F.; Wang, C. Y. Chem. Nat. Compd. Fungi; Approved Standard. NCCLS
2017, 53 (2), 227-230. documents M38-A; National Committee
3. Maha, A.; Rukachaisirikul, V.; for Clinical Laboratory Standards:
Phongpaichit, S.; Poonsuwan, W.; Wayne, PA. 2002.
Sakayaroj, J. Tetrahedron 2016, 72 15. Pongcharoen, W.; Rukachaisikul, V.;
(22), 2874-2879. Isaka, M.; Sriklung, K. Chem. Pharm.
4. Merifield, E.; Thomas, E. J., J. Chem. Bull. 2007, 55 (11), 1647-1648.
Soc., Perkin Trans. 1. 1999, 3269-3283. 16. Schneider, G.; Anke, H.; Sterner, O. Z.
5. Anderson, J. R.; Edwards, R. L.; Naturforsch. C: Biosci. 1996, 51
Whalley, A. J. S. J. Chem. Soc., Perkin (11/12), 802-806.
Trans. I. 1983, 2185-2192. 17. Liu, T.; Li, Z.; Wang, Y.; Zhang, L.;
6. Dimitriadis, C.; Gill, M.; Harte, M. F. Song, J.; Tian, L.; Pei, Y.; Hua, H.
Tetrahedron: Asymmetry 1997, 8 (13), Zhongguo Yaoxue Zazhi (Beijing,
2153-2158. China) 2012, 47 (8), 577-580.
18. Yang, J. X.; Qiu, S. X.; She, Z.; Lin, Y.
Chem. Nat. Compd. 2013, 49 (1), 31-33.
Synthesis of novel neutral helper lipids with amino acids as polar heads
Prutchaya Kumpairee1, Nattisa Niyomtham1, Nuttapon Apiratikul2,
Boon-ek Yingyongnarongkul1*
1
Ramkhamhaeng University, Bangkok 10240, Thailand
2
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand
*E-mail: boonek@ru.ac.th
Abstract:
Cationic liposomes have become the most common non-viral gene delivery agents for
the future clinical applications of gene therapy. Many cationic lipids require helper lipids for
highly efficient gene delivery. Thus, the novel helper lipids 6a, 6b, 7a and 7b having
glutamic acid and aspartic acid as polar heads and oleic acid as hydrophobic tail were
designed and synthesized. The synthesized lipids were obtained in high yields and their
structures were characterized by spectroscopic methods (IR, NMR and MS).

Cationic liposomes have been 2.1 General
investigated for various applications in gene IR spectra were recorded on a
therapy.1-3 Cationic liposomes are Perkin-Elmer FT-IR 400 spectrophotometer.
1
commonly used for mediating gene transfer H NMR spectra were recorded on a Bruker
to mammalian cells. Their popularity is AVANCE400 and AscendTM 400
justified by their high transfection spectrometers using a residual solvent signal
efficiency, low toxicity and commercial as internal standard. Mass spectra were
availability among others.4-5 For maximal obtained on a Finnigan LC-Q mass
transfection efficiency, most cationic spectrometer. High resolution mass spectra
liposome formulations require a significant were obtained using a Bruker micrOTOF-
amount of helper lipid. The role of helper QII ESI mass spectrometer. Starting materials
lipids in enhancing transfection is generally and reagents were purchased from
attributed to its ability to promote the commercial suppliers and used without
lamellar to inverted hexagonal phase further purification.
transition. This leads to the fusion of 2.2 Synthesis of helper lipids
lipoplexes through cell membranes resulting 2.2.1 Synthesis of compound 2
in the entry of DNA into the cells.6 To a solution of (+/-)-3-amino-1,2-
Dioleoylphosphati- dylethanolamine propanediol (1.0 g, 10.97 mmol) in CH2Cl2
(DOPE)7 is the neutral component in all (20 ml) was added a solution of di-tert-butyl
commercially available cationic liposome carbonate (2.4 g, 10.97 mmol) in CH2Cl2 (30
preparations. However, DOPE has limited ml) and the mixture was stirred for 6 h. The
utility in vivo because it has high fusogenic mixture was quenched by addition of water
nature, which helps to promote transfection (100 ml) and extracted with EtOAc (3×50
in vitro, renders lipoplexes too unstable with ml). The combined organic phase was
respect to aggregation in the more complex washed with water, dried over anhydrous
in vivo environment.8 In the present study, Na2SO4 and concentrated in vacuo. The
we described the synthesis of neutral lipids crude product was purified by column
using aspartic acid and glutamic acid as chromatography with CH2Cl2-MeOH to
polar heads and oleic acid as nonpolar tails. yield compound 2 (1.97 g, 94 %) as a white
solid, melting point 66 C. IR: νmax 3414,
3387, 3239, 2986, 2940, 2882, 1680, 1516,
1367, 1268, 1250, 1157, 998 cm-1; 1H NMR 2), 5.31 (4H, m, (H-9′ and H-10′); ESIMS (+
(400 MHz, CDCl3): δ 1.40 (9H, s, H-Boc), ve) m/z (% rel. intensity): 621[M+H]+ (100).
3.21 (2H, m, H-1), 3.40 (2H, br s, OH), 3.50 2.2.3 Synthesis of compounds 4a and 4b
(1H, dd, J = 11.6 Hz, 5.4 Hz, H-3a), 3.53 N-α-Fmoc-L-aspartic acid β-tert
(1H, dd, J = 11.6 Hz, 3.8 Hz, H-3b), 3.70 butyl ester (510 mg, 1.2 mmol) and HBTU
(1H, quin, J = 4.9 Hz, H-2), 5.24 (1H, br s, (455 mg, 1.2 mmol) were dissolved in dry
NH); ESIMS (+ ve) m/z (% rel. intensity): CH3CN (20 ml) and stirred at 0 °C for 30
192 [M+H]+ (100). min. Compound 3 (500 mg, 0.81 mmol) and
2.2.2 Synthesis of compound 3 Et3N (282 μl, 1.62 mmol) were then added.
Oleic acid (3.3 g, 11.5 mmol) and The mixture was allowed to warm up to
dicyclohexylcarbodiimide (2.4 g, 11.5 ambient temperature and stirring was
mmol) were dissolved in dry CH2Cl2 (20 continued for 2 h. The mixture was
ml). The mixture was stirred at room quenched by addition of water (50 ml) and
temperature for 30 min, a solution of extracted with EtOAc (3×50 ml). The
compound 2 (1.0 g, 5.23 mmol) in dry combined organic phase was washed with
CH2Cl2 (10 ml) was then added and the water, dried over anhydrous Na2SO4 and
resulting mixture was stirred overnight at the concentrated in vacuo. The crude product
same temperature. The precipitated DCU was purified by column chromatography
was removed by filtration and the filtrate with CH2Cl2-MeOH to yield compound 4a
was evaporated under reduced pressure to as neat oil (700 mg, 86 %). Compound 4b
give crude product. The crude mixture was was synthesized by the procedure described
separated by column chromatography to above using N-α-Fmoc-L-glutamic acid γ-
yield the Boc-protected product (2.88 g, 76 tert butyl ester instead of N-α-Fmoc-L-
%) as neat oil. IR: νmax 3325, 3005, 2924, aspartic acid β-tert butyl ester to give
2854, 1778, 1731, 1665, 1533, 1451, 1367, compound 4b as neat oil (1.10 g, 86 %).
1275, 1259, 1157, 1049, 966, 844, 758, 750 2.2.3.1 Compound 4a
cm-1; 1H NMR (400 MHz, CDCl3): δ 0.83 IR: νmax 3319, 3062, 3004, 2924,
(6H, t, J = 5.9 Hz, H-18′), 1.27-1.31 (40H, 2854, 1730, 1668, 1533, 1451, 1367, 1242,
br d, J = 13.8 Hz, H-4′H-7′ and H-12′H- 1156, 1089, 1048, 966, 844, 758, 738 cm-1;
17′), 1.44 (9H, s, H-Boc), 1.63, (4H, br s, H- 1
H NMR (400 MHz, CDCl3): δ 0.85 (6H, t, J
3′), 2.02 (8H, m, H-8′ and H-11′), 2.32 (2H, = 6.5 Hz, H-18′), 1.24 (40H, s, H-4′H-7′
t, J = 7.4 Hz, H-2′), 3.35 (2H, m, H-1), 4.13 and H-12′H-17′), 1.42 (9H, s, H-Boc),
(1H, dd, J = 11.6 Hz, 5.5 Hz, H-3a), 4.28 1.56, (4H, br t, J = 6.2 Hz, H-3′), 1.97 (8H,
(1H, dd, J = 12.0 Hz, 3.4 Hz, H-3b), 5.10 br d, J = 5.5 Hz, H-8′ and H-11′), 2.27 (4H,
(1H, q, J = 5.1 Hz, H-2), 5.35 (4H, m, H-9′ t, J = 7.5 Hz, H-2′), 2.59 (1H, m, H-3a′′),
and H-10′); ESIMS (+ ve) m/z (% rel. 2.89 (1H, m, H-3b′′), 3.46 (1H, m, H-1),
intensity): 743 [M+Na]+ (100). 4.08 (1H, dd, J = 12.6 Hz, 5.6 Hz, H-3a),
The tert-butyl protecting group was 4.21 (2H, m, H-3b and H-3′′′), 4.40 (3H, m,
removed by treating with 20% TFA in H-2′′ and H-2′′′), 5.07 (1H, m, H-2), 5.31
CH2Cl2 to afford compound 3 in quantitative (4H, m, H-9′ and H-10′), 5.95 (1H, br m, -
yield. IR: νmax 2924, 2854, 1711, 1677, 1460, CHNHCO-), 6.85 (1H, br s, -CONHCH2-),
1275, 1260, 1200, 1176, 1142, 750, 722 cm- 7.29 (2H, t, J = 7.4 Hz, H-6′′′), 7.38 (2H, t, J
; H NMR (400 MHz, CDCl3): δ 0.85 (6H,
1 1
= 7.4 Hz, H-7′′′), 7.58 (2H, br d, J = 6.7 Hz,
t, J = 6.4 Hz, H-18′), 1.23-1.27 (40H, br t, J H-5′′′), 7.74 (2H, d, J = 7.5 Hz, H-8′′′);
= 8.38 Hz, H-4′H-7′ and H-12′H-17′), HRESIMS (+ve): m/z calcd for
1.57 (4H, br d, J = 4.41 Hz, H-3′), 1.98 (8H, C62H96N2O9Na [M+Na]+ 1035.7008, found
m, H-8′ and H-11′), 2.30 (4H, m, H-2′), 3.17 1035.7005.
(2H, m, H-1), 4.11 (1H, dd, J = 11.4 Hz, 4.6 2.2.3.2 Compound 4b
Hz, H-3a), 4.36 (1H, dd, J = 12.2 Hz, 3.8 IR: νmax 3327, 3005, 2925, 2854,
Hz, H-3b), 5.10 (1H, br d, J = 10.4 Hz, H- 1726, 1665, 1532, 1451, 1367, 1275, 1260,
1152, 1092, 967, 846, 763, 750 cm-1; 1H tt, J = 11.9 Hz, 3.3 Hz, H-3b), 5.09 (1H,
NMR (400 MHz, CDCl3): δ 0.85 (6H, t, J = quin, J = 5.1 Hz, H-2), 5.30 (4H, m, H-9′
6.1 Hz, H-18′), 1.23-1.24 (40H, s, H-4′H-7′ and H-10′), 7.70 (1H, br m, -CONHCH2-);
and H-12′H-17′), 1.43 (9H, s, H-Boc), HRESIMS (+ve): m/z calcd for C47H87N2O7
1.56, (4H, br d, J = 6.0 Hz, H-3′), 1.97 (8H, [M+H]+ 791.6507, found 791.6505.
m, H-8′ and H-11′), 1.97 (2H, m, H-3′′), 2.27 2.2.4.2 Compound 5b
(2H, t, J = 6.9 Hz, H-2′), 2.27 (1H, t, J = 6.9 IR: νmax 3301, 3005, 2923, 2853,
Hz, H-4a′′), 2.43 (1H, m, H-4b′′), 3.46 (2H, 1734, 1677, 1542, 1459, 1367, 1275, 1260,
m, H-1), 4.11 (2H, m, H-2′′ and H-3a), 4.19 1155, 1092, 963, 848, 764,750 cm-1; 1H
(2H, m, H-3b and H-3′′′), 4.35 (2H, m, H- NMR (400 MHz, CDCl3): δ 0.89 (6H, t, J =
2′′′), 5.08 (1H, br s, H-2), 5.31 (4H, m, H-9′ 6.4 Hz, H-18′), 1.28-1.31 (40H, br d, J =
and H-10′), 5.75 (1H, br m, -CHNHCO-), 13.2 Hz, H-4′H-7′ and H-12′H-17′), 1.46
6.72 (1H, br s, -CONHCH2-), 7.28 (2H, t, J (9H, s, H-Boc), 1.62 (4H, br s, H-3′), 1.95
= 7.4 Hz, H-6′′′), 7.37 (2H, t, J = 7.4 Hz, H- (2H, br s, H-3′′), 2.02 (8H, m, H-8′ and H-
7′′′), 7.57 (2H, br d, J = 6.9 Hz, H-5′′′), 7.74 11′), 2.34 (2H, m, H-2′), 2.34 (2H, m, H-4′′),
(2H, d, J = 7.5 Hz, H-8′′′); HRESIMS (+ve): 3.40 (1H, m, H-1a), 3.56 (2H, m, H-1b and
m/z calcd for C63H98N2O9Na [M+Na]+ H-2′′), 4.13 (1H, dd, J = 11.9 Hz, 5.8 Hz, H-
1049.7164, found 1049.7162. 3a), 4.27 (1H, dd, J = 11.9 Hz, 3.7 Hz, H-
2.2.4 Synthesis of compounds 5a and 5b 3b), 5.15 (1H, br s, H-2), 5.36 (4H, m, H-9′
To a solution of compound 4a (570 and H-10′), 7.53 (1H, br s, -CONHCH2-);
mg, 0.56 mmol) in CH2Cl2 (20 ml) was HRESIMS (+ve): m/z calcd for C48H89N2O7
added 20% piperidine in CH2Cl2 (10 ml) and [M+H]+ 805.6694, found 805.6696.
the mixture was stirred at ambient for an 2.2.5 Synthesis of compounds 6a and 6b
hour. The mixture was quenched by addition To a solution of compound 5a (100
of water (50 ml) and extracted with EtOAc mg, 0.13 mmol) in CH2Cl2 (10 ml) was
(3×50 ml). The combined organic phase was added a solution of 20% trifluoroacetic acid
washed with water, dried over anhydrous (TFA) in CH2Cl2 (10 ml). The reaction
Na2SO4 and concentrated in vacuo. The mixture was stirred at room temperature for
crude product was purified by column 2 h. The solution was removed under steam
chromatography using CH2Cl2 and CH2Cl2- of nitrogen to dryness. The products were
MeOH as eluting solvent, with gradually further dried under reduced pressure for 2 h
increase in the concentration of more polar to give compound 6a (93 mg) in quantitative
component to yield compound 5a as neat oil yield. Compound 6b were carried out using
(260 mg, 59 %). Compound 4b was carried the same condition to that of compound 6a
out under the same condition to that of to give compound 6b (75 mg) in quantitative
compound 5a to give compound 5b as neat yield.
oil (485 mg, 65 %). 2.2.5.1 Compound 6a
2.2.4.1 Compound 5a IR: νmax 3282, 2923, 2853, 1738,
IR: νmax 3353, 2924, 2854, 1732, 1674, 1569, 1461, 1380, 1273, 1260, 1199,
1666, 1525, 1460, 1367, 1276, 1258, 1154, 1175, 1139, 764, 750, 721 cm-1; 1H NMR
764, 750 cm-1; 1H NMR (400 MHz, CDCl3): (400 MHz, CDCl3): δ 0.85 (6H, t, J = 6.8
δ 0.84 (6H, t, J = 6.4 Hz, H-18′), 1.23-1.26 Hz, H-18′), 1.23-1.26 (40H, br d, J = 8.4 Hz,
(40H, br d, J = 13.32 Hz, H-4′H-7′ and H- H-4′H-7′ and H-12′H-17′), 1.56, (2H, br
12′H-17′), 1.41 (9H, s, H-Boc), 1.57, (4H, s, H-3′), 1.97 (8H, br m, H-8′ and H-11′),
br d, J = 4.8 Hz, H-3′), 1.97 (8H, br d, J = 2.26 (4H, t, J = 6.9 Hz, H-2′), 2.79 (2H, br s,
5.8 Hz, H-8′ and H-11′), 2.28 (2H, q, J = 7.0 H-3′′), 3.38 (2H, br s, H-1), 4.02 (1H, br m,
Hz, H-2′), 2.5 (1H, m, H-3a′′), 2.77 (1H, m, H-3a), 4.21 (1H, br t, J = 7.7 Hz, H-3b),
H-3b′′), 3.40 (2H, m, H-1a), 3.49 (1H, m, H- 4.34 (1H, br s, H-2′′), 5.07 (1H, br s, H-2),
1b), 3.61 (1H, br d, J = 3.1 Hz, H-2′′), 4.08 5.30 (4H, m, H-9′ and H-10′), 8.33 (1H, br s,
(1H, dd, J = 11.6 Hz, 5.8, H-3a), 4.23 (2H, -CONHCH2-); HRESIMS (+ve): m/z calcd
for C43H78N2O7 [M+Na]+ 757.5701, found Hz, H-3b), 5.16 (1H, quin, J = 4.8 Hz, H-2),
757.5696. 5.31 (4H, m, H-9′ and H-10′); HRESIMS
2.2.5.2 6b (+ve): m/z calcd for C46H85N2O7+ [M]+
IR: νmax 3291, 2923, 2853, 1738, 777.6351, found 777.6349.
1680, 1551, 1460, 1376, 1275, 1260, 1167, 2.2.5.2 Compound 7b
1091, 764, 749, 721 cm-1; 1H NMR (400 IR: νmax 3246, 2924, 2854, 1735,
MHz, CDCl3): δ 0.84 (6H, t, J = 6.7 Hz, H- 1681, 1462, 1275, 1260, 1198, 1177, 1138,
18′), 1.23-1.26 (40H, br t, J = 6.36 Hz, H- 764, 750, 720 cm-1; 1H NMR (400 MHz,
4′H-7′ and H-12′H-17′), 1.56, (2H, d, J = CDCl3): δ 0.85 (6H, t, J = 4.4 Hz, H-18′),
1.0 Hz, H-3′), 1.97 (4H, m, H-8′ and H-11′), 1.23-1.26 (40H, br d, J = 12.48 Hz, H-4′H-
2.15 (2H, m, H-3′′), 2.28 (2H, m, H-2′), 2.28 7′ and H-12′H-17′), 1.55, (4H, br s, H-3′),
(1H, m, H-4a′′), 2.38 (1H, m, H-4b′′), 3.47 1.97 (8H, m, H-8′ and H-11′), 2.15 (1H, br s,
(2H, m, H-1), 4.10 (2H, m, H-3a and H-2′′), H-3a′′), 2.27 (6H, m, H-3b′′, H-2′ and H-
4.22 (1H, m, H-3b), 5.10 (1H, br m, H-2), 4a′′), 2.46 (1H, br s, H-4b′′), 3.30 (9H, s, -
5.32 (4H, m, H-9′ and H-10′), 8.85 (1H, br s, NCH3), 3.39 (1H, m, H-1a), 3.64 (1H, m, H-
-CONHCH2-); HRESIMS (+ve): m/z calcd 1b), 4.04 (1H, m, H-3a), 4.28 (1H, m, H-3b),
for C44H80N2O7Na [M+Na]+ 771.5857, 4.69 (1H, br s, H-2′′) 5.15 (1H, br m, H-2),
found 771.5855. 5.31 (4H, m, H-9′ and H-10′), 9.44 (1H, br s,
2.2.5 Synthesis of compounds 7a and 7b -CONHCH2-); HRESIMS (+ve): m/z calcd
Compound 5a (160 mg, 0.2 mmol) for C47H87N2O7+ [M]+ 791.6507, found
was dissolved with CH2Cl2 (10 ml). Methyl 701.6506.
iodide (50 μl, 0.8 mmol) and DIEA (70 μl,
0.4 mmol) were added into this solution. 3. Results and Discussion
The mixture was stirred at room temperature Helper lipids are considered to affect
for 24 h. All solvents were removed under various properties of DNA–cationic
reduced pressure and the crude product was liposome complexes and play a very
purified by column chromatography with important role in efficient gene transfer.
CH2Cl2-MeOH to give desired product as DOPE is helper lipid mostly used to employ
neat oil (77 mg, 56 %). This was further with cationic lipids in order to gain high
reacted with 20% TFA in CH2Cl2. The transfection efficiency. However, DOPE has
solution was removed under steam of limited utility in vivo application. Thus, it is
nitrogen to dryness. The products were necessary to design novel helper lipids to
further dried under reduced pressure for 2 h expand the choices of cationic liposome
to give compound 7a (72.0 mg, quantitative formulations. We have designed and
yield). Compound 7b was synthesized using synthesized novel helper lipids with amino
the procedure described above to yield 7b acids polar heads as shown in Scheme 1.
(170 mg, quantitative yield). Glycerol is usually used as central
2.2.5.1 Compound 7a core of helper lipids including DOPE. Thus,
IR: νmax 3276, 2923, 2854, 1733, the glycerol analogue, (+/-)-3-amino-1,2-
1671, 1554, 1462, 1276, 1260, 1173, 983, propanediol was used to prepare new neutral
764, 750, 721 cm-1; 1H NMR (400 MHz, lipids. N-Boc-protected (+/-)-3-amino-1,2-
CDCl3): δ 0.84 (6H, t, J = 6.7 Hz, H-18′), propanediol (2) was reacted with Boc2O,
1.23 - 1.26 (40H, br d, J = 8.4 Hz, H-4′H- followed by coupling with oleic acid using
7′ and H-12′H-17′), 1.56, (4H, br s, H-3′), DCC as coupling agent, and Boc
1.75 (1H, m, H-3a′′, 1.83 (1H, m, H-3b′′), deprotection to give compound 3. Aspartic
1.97 (8H, br m, H-8′ and H-11′), 2.29 (4H, t, acid and glutamic acid were chosen as polar
J = 3.9 Hz, H-2′), 2.86 (9H, s, -NCH3), 3.56 head due to bearing negative and positive
(2H, m, H-1), 4.00 (1H, br dd, J = 8.8 Hz, charges from carboxyl and amino group,
2.1 Hz, H-2′′), 4.14 (1H, dd, J = 11.8 Hz, 5.7 respectively. Thus, amino group was then
Hz, H-3a), 4.26 (1H, dd, J = 11.8 Hz, 3.4 coupled with Fmoc-protected amino acid
using HBTU as coupling agent to give lipids helper lipids with amino acid polar head.
4a and 4b. The Fmoc and tert-butyl The application of helper lipids to enhance
protecting groups were then removed with gene delivery into targeted cells of cationic
piperidine and TFA, respectively, to give lipid is under investigation and will be
target compounds 6a and 6b in good yields. reported elsewhere.
To synthesize the quaternary analogues,
compounds 5a and 5b were subjected to 4. Conclusion
react with methyl iodide followed by New helper lipids containing amino
removal of tert-butyl protecting group to acids as polar heads and oleic acid as
furnish compounds 7a and 7b in moderate nonpolar tails were synthesized in good
yields. All the structures of synthesized yields. These helper lipids will be useful for
lipids were characterized by IR, NMR and gene and drug carrier applications.
MS techniques. The synthetic method is
simple and efficient for the synthesis of new
Scheme 1. Reagents and conditions: a) Boc2O, CH2Cl2, rt., b) oleic acid, DCC, CH2Cl2, c)
20% TFA/CH2Cl2, d) Fmoc-Asp(tBu)-OH (for 4a) or Fmoc-Glu(tBu)-OH (for 4b), HBTU,
CH2Cl2, e) piperidine, CH2Cl2, f) MeI, DIEA, CH2Cl2
Acknowledgements 3. Piron, J.; Quang1, K. L.; Briec, F.;

This work was supported by the Amirault, J. C.; Leoni, A. L.; Desigaux,
Thailand Research Fund (TRF). Support L.; Escande, D.; Pitard, B.; Charpentier,
from the Center of Excellence for F. Mol. Ther. 2008, 16, 1937–1943.
Innovation in Chemistry (PERCH-CIC), 4. Gao, X.; Huang, L. J. Liposome Res.
Office of Higher Education Commission, 1993, 3, 17−30.
Ministry of Education is gratefully 5. Farhood, H.; Serbina, N.; Huang, L.
acknowledged. PK would like to thank the Biochim. Biophys. Acta 1995, 1235,
Center of Excellence for Innovation in 289−295.
Chemistry (PERCH-CIC) for scholarship. 6. Huang, Z.; Li, W.; Francis, C.; Szoka, J.
Int. J. Pharm. 2012, 427, 64−70.
References 7. Zabner, J.; Fasbender, A. J.; Moninger,
1. Felgner, P. L.; Ringold, G. M. Nature T.; Poellinger, K. A.; Welsh, M. J. J.
1989, 337, 387−388. Biol. Chem. 1995, 270, 18997−19007.
2. Mhashilkar, A.; Chada, S.; Roth, J. A.; 8. Li, S.; Rizzo, M. A.; Bhattacharya, S.;
Ramesh, R. Biotechnol. Adv. 2001, 19, Huang, L. Gene Ther. 1998, 5, 930−937.
279−297.
Phenolic content and antioxidant activity in four edible green algae
from Tha Li district, Loei province
Suwatchai Misuna*, Thananan Machai, Niramol Srichana, Krittipong Choojit,
Bussabavadee Puttanu, Chanchaem Duang-u-pa
Department of Science, Faculty of Science and Technology, Loei Rajabhat University, Loei 42000, Thailand
*E-mail: suwatchai.mis@lru.ac.th
Abstract:
Four edible green algae, Spirogyra sp., Cladophora sp., Nostoc sp. and Wolffia globosa,
were harvested from Tha Li district, Loei province and were extracted with 70% methanol or
absolute ethanol for compound analysis and antioxidant analysis, respectively. Total phenolic
and total flavonoid contents were analysis by spectrophotometric method. Type and content of
phenolic acid were analysed by high performance liquid chromatography technique (HPLC).
Antioxidant activity was determined by DPPH and ABTS assays. The extract from Cladophora
sp. showed the maximum total phenolic contents (594.10 ± 6.04 mg GAE/100 g dry weight),
whereas the maximum total flavonoids contents were found in Spirogyra sp. (317.42 ± 1.12
mg QE/100 g dry weight). Phenolic acid, gallic acid, vanillic acid and ferulic acid were found
in all edible green algae. The highest quercetin contents were found in Cladophora sp. (98.63
± 5.07 µg/100 g dry weight). The extract from Nostoc sp. displayed the strongest antioxidant
activity in both DPPH (IC50 = 68.71 ± 1.11 µg/mL) and ABTS (IC50 = 19.69 ± 1.19 µg/mL)
assays. These findings suggest that some edible green algae from Tha Li district, Loei province
may be a good source of natural bioactive compounds.
1. Introduction antioxidant activity from four edible green

Bioactive compounds such as phenols algae of Tha Li district, Loei Province,
and polyphenols are found in various plants Thailand. Herein, the phenolic content and
and algae, especially marine algae. 1 The antioxidant activity of edible green algae
antioxidant activity of polyphenol extract from Tha Li district are reported for the first
from macroalgae is more potent than the time.
polyphenol extract from terrestrial plants.2
Edible algae have been reported to contain 2. Materials and Methods
bioactive compounds with interesting 2.1 Sample preparation
antioxidant properties.3 Several studies Four edible green algae were
indicated that brown algae showed better harvested from Tha Li district area, Loei
antioxidant activity than green and red algae.4 Province during March 2017. All samples
Edible green algae, Spirogyra sp., were cleaned with distilled water and dried in
Cladophora sp., Nostoc sp. and Wolffia hot air oven at 60 to 65 oC for 48 h. The dried
globosa have been used for traditional food sample was powdered using blender and
in Northeast of Thailand. Nostoc sp., a group stored at -20 oC.
of cyanobacteria, has been reported as a rich 2.2 Crude ethanolic extraction
source of potentially useful bioactive Ten grams of each edible green algae
compounds.5 Several hundred natural were extracted and periodically stirred in 100
products from Nostoc sp. displayed mL of absolute ethanol for 48 h at room
antimicrobial, anti-HIV, anticancer and temperature. Extracts were filtered through
antimalarial activities.5 Whatman No. 4 filter paper and the solvent
The objective of this research was to was removed by using rotary evaporator to
investigate the the phenolic content and yield ethanolic crude extracts.
2.3 Phenolic acid extraction 2.6 HPLC analysis of phenolic acid and
The phenolic acid extraction was flavonoids
performed by using acidic hydrolysis method Chromatographic separation of
with some modification.6 In short, dried individual phenolic acids was carried out
sample of each edible green algae (10 g) was using a SHIMADZU HPLC system, based on
suspended in 70% methanol ( 200 mL) . The matching spectrum and retention times of
mixture was stirred at room temperature for 2 phenolic acid standard. The HPLC system
h and then filtered through the filter paper. consisted of LC- 10AD separation module
The filtrate was concentrated until the final unit, SCL-10A UV-VIS detector and a 250 x
volume was 60 mL by rotary evaporator. 2 M 4. 6 mm, 5 µm, 10 oA, ODS C- 18 column
NaOH (50 mL) was added into the remaining ( GL Science, USA), with 100% acetonitrile
filtrate and stirred continuously for 12 h at ( solvent A) and a gradient system of acetic
room temperature. The mixture was acid in deionized water (solvent B) by
centrifuged at 3,000 rpm about 10 min and increased from 1% to 8% in 5 min and
then filtered through filter paper. The maintaining at 8% for 10 min, then increasing
supernatant was repeatedly extracted three to 20% within 5 min and maintaining at 20%
times with 80 mL of diethyl ether. The for 10 min, followed by returning to initial
combined aqueous phases were acidified condition ( 1% ) in 10 min and keeping for 5
with HCl ( 10 M) until pH 1. 5 and filtered min before next injection. The compounds
through filter paper. The filtrate was further were monitored by the UV- VIS detector at
extracted with diethyl ether (80 mL) for three the wavelength of 280 nm. The amount of
times. The combined diethyl ether layers individual phenolic acid was performed by
were concentrated by rotary evaporator to the standard addition method and consequent
yield a phenolic acid extract. calculation.
2.4 Determination of total phenolic content A portion of ethanolic crude extract
Total phenolic content in ethanolic (0.4 g) was dissolved in 5 mL of 80 %
crude extract of four edible green algae was methanol, filtered through a 0.45 μm
determined by modification of the Folin - micropore membrane before use and was
Ciocalteu assay.7 The absorbance was injected into the HPLC- UV- VIS equipment.
measured by UV- 160A spectrophotometric Type and content of flavonoids were
at 750 nm. Gallic acid was used as a standard analyzed using a HPLC system, with 100%
and the result was calculated as mg Gallic methanol ( solvent A) and a linear gradient
Acid Equivalent ( GAE) per one hundred solvent system of acetic acid in deionized
gram of dry weight. The calibration curve of water (solvent B) by increased from 50% to
gallic acid was Y = 0. 0165X+ 0. 2031, R2 = 75% in 5 min, then increasing to 90 within 5
0.9983. min and increasing to 100% within 5 min
2.5 Determination of total flavonoid followed by returning to initial condition
content (50%) in 10 min and keeping for 5 min before
Total flavonoid content in ethanolic next injection. The compounds were
crude extract of four edible green algae was monitored by the UV- VIS detector at the
determined by modification of the aluminum wavelength of 320 nm. The amount of
chloride colorimetric assay.8 The absorbance individual flavonoids was performed by the
was measured by UV-160A standard addition method and consequent
spectrophotometric at 420 nm. Quercetin was calculation.
used as a standard and the result was 2. 7 Quantification of the phenolic acids
calculated as mg Quercetin Equivalent ( QE) and flavonoids
per one hundred gram of dry weight. The External standardization of phenolic
calibration curve of quercetin was Y = acids or flavonoids (gallic acid, vanillic acid,
0.0049X+0.1375, R2 = 0.9991. syringic acid, p-coumaric acid, ferulic acid,
quercetin and genistein) was used for
quantification of individual phenolic acids or 2.8.2 ABTS radical cation scavenging
flavonoids. Calibration curve were prepared assay
in the range of concentration between 1 and Briefly, a mixture of 7. 4 mM of 2,2’ -
10 µg/mL for determined of phenolic acids or azino- bis( 3- ethylbenzothiazoline- 6- sulpho-
flavonoids. The linearity was estimated by nic acid) ( ABTS) solution and 2. 6 mM of
linear regression analysis applying the least potassium persulfate solution was kept
square method. Limit of detection (LOD) and overnight in the dark. Prior to use in the
limit of quantification were determined based assay, the ABTS radical cation was diluted
on signal-to-noise ratio at 3 and 10, with 50 % methanol for an initial absorbance
respectively. of about 0. 70±0. 02 at 745 nm. Free radical
2.8 In vitro antioxidant activity scavenging activity was assessed by mixing 5
2.8.1 DPPH radical scavenging assay μL of ethanolic crude extract at various
100 µM of DPPH solution was concentrations with 195 μL of ABTS
prepared by dissolving 4 mg of DPPH with working solution. The solution was incubated
100 mL ethanol. A 180 µL of DPPH solution in the dark for 30 min at room temperature.
was mixed with 5 µL of ethanolic crude Then the absorbance was taken by microplate
extract at various concentrations in 96-well reader at 745 nm. Gallic acid and ascorbic
plate. The solution was mixed and incubated acid were used as positive controls. The
in the dark for 10 min at room temperature. scavenging activity was represented as an
Then, the absorbance was taken by a IC50 value.
microplate reader at 515 nm. The control was 2.9 Statistical and data analysis
prepared using the same procedure without Values expressed are means of five
any sample. Gallic acid and ascorbic acid replicate determination ± standard deviation
were used as standard controls. The ( SD) . Values of total phenolic content, total
scavenging activity was represented as 50 flavonoid content, phenolic acid content and
percent inhibition concentration or IC50. 50% inhibition concentration ( IC50) were
statistically analyzed by means of one- way
analysis of variance (ANOVA). Data
correlations were calculated by using
Pearson’s correlation coefficient
Table 1. Total phenolic and total flavonoid contents in four edible green algae
Extraction yields Total phenolic content Total flavonoid content
Edible green algae
(g/100 g dry weight) (mg GAE/100 g dry weight) (mg QE/100 g dry weight)
Spirogyra sp. 4.34 ± 0.22b 123.96 ± 8.80c 317.42 ± 1.12a
Cladophora sp. 4.79 ± 0.07b 594.10 ± 6.04a 182.02 ± 0.37c
Nostoc sp. 5.23 ± 0.17a 566.22 ± 4.74a 258.13 ± 0.37b
Wolffia globosa 4.16 ± 0.35b 219.16 ± 4.35b 156.51 ± 5.34d
Table 2. Type and content of phenolic acids in four edible green algae
Edible green Phenolic acids ( µg/100 g dry weight)
algae Gallic acid Vanillic acid Syringic acid p-Coumaric acid Ferulic acid
Spirogyra sp. 4.47 ± 0.14c 7.43 ± 0.14b N. D. N. D. 3.56 ± 0.01d
Cladophora sp. 7.63 ± 0.58b 5.36 ± 0.57c N. D. N. D. 9.35 ± 0.43b
a a a
Nostoc sp. 15.38 ± 0.67 12.15 ± 0.01 0.77 ± 0.01 2.34 ± 0.24 23.46 ± 0.28a
b b b
Wolffia globosa 8.15 ± 0.32 8.33 ± 0.01 N. D. 0.68 ± 0.01 4.73 ± 0.01c
Values in the same column followed by a different latter ( ) are significantly different (P < 0.05). Each value in
a-d
table is represented as mean ± SD (n = 5), N. D. = not detected
3. Results and Discussion ferulic acid and gallic acid (Table 2) whereas,
3.1 Total phenolic and total flavonoid the ethanolic crude extract from Nostoc sp.
contents showed the lowest on IC50 value ( Table 5) .
The total phenolic contents of four Therefore, the antioxidant activity of Nostoc
edible green algae are shown in Table 1. The sp. might be due to the presence of phenolic
content ranged from 123. 96 to 594. 10 mg acid in the macroalgal extract.
GAE/ 100g dry weight. Cladophora sp. had
highest total phenolic content followed by
Nostoc sp., W. globosa and Spirogyra sp. The
total flavonoid contents ranged from 156. 51
to 317. 42 mg QE/ 100 g dry weight. In
contrast, Spirogyra sp. contained highest
total flavonoid content followed by Nostoc
sp., Cladophora sp. and W. globosa.
3.2 Types of phenolic acids and flavonoids
Types of phenolic acids and
flavonoids in four edible green algae were
identified using HPLC with UV- visible
detection ( Figures 1 and 2) . Gallic acid, Figure 1. HPLC-UV-VIS chromatograms of
vanillic acid, ferulic acid, quercetin and phenolic acids in edible green algae ( Nostoc
genistein were found in all edible green algae sp.). (A) Gallic acid, (B) Vanillic acid, (C)
in difference quantity (Tables 2 and 4) . p- Syringic acid, ( D) p- Coumaric acid, ( E)
Coumaric acid was found in Nostoc sp. and Ferulic acid
W. globosa whereas syringic acid was
detected only in Nostoc sp. (Table 2). Nostoc
sp. had highest content of gallic acid, vanillic
acid and ferulic acid when compared the
other algae (Table 2). The highest amount of
quercetin was found in Cladophora sp.
followed by Nostoc sp. wherese the highest
of genistein was found in Nostoc sp. ( Table
4) . Accordingly, Nostoc sp. had highest
content of both phenolic acid and flavonoids.
Limit of detection ( LOD) and limit of
quantitation ( LOQ) of five phenolic acid
standards and two flavonoid standards are
shown in Table 3.
3.3 Antioxidant activity Figure 2. HPLC-UV-VIS chromatograms of
The antioxidant activity of four edible flavonoid content in edible green algae
green algae was measured by using DPPH (Nostoc sp.)
and ABTS assays and the results are
displayed in Table 5. The ethanolic crude 4. Conclusion
extracts from Nostoc sp. showed the lowest Four edible green algae contained
IC50 value for both assay, indicating the antioxidant compounds, including phenolic
highest antioxidant activity. acids and flavonoids. These polyphenol
In many report, the phenolic acid compounds may play a role in the antioxidant
from plants such as gallic acid, vanillic acid activities observed in algal ethanolic crude
and ferulic acid are natural antioxidant. 2 Our extract.
result showed that Nostoc sp. had highest
content of all phenolic acid particularly
Table 3. Data of phenolic standards and flavonoid standards analyzed by HPLC method
Standards Retention LODa LOQa Regression Correlation
time (µg/ml) (µg/ml) equation coefficient (r)
(min)
Gallic acid 3.743 0.2101 0.5710 Y = 39399x – 4306 0.9979
Vanillic acid 13.142 0.1513 0.5021 Y = 26908x – 654 0.9985
Syringic acid 14.607 0.1569 0.5300 Y = 42062x – 537 0.9984
p-Coumaric acid 21.864 0.1854 0.6161 Y = 76971x – 9075 0.9979
Ferulic acid 24.889 0.1136 0.4434 Y = 37584x + 22518 0.9974
Quercetin 10.695 0.2640 0.8199 Y = 47156x + 10203 0.9962
Genistein 11.367 0.0157 0.0510 Y = 8268x + 2839 0.9982
a
Detection limits were calculated as signal-to-noise ratio of six determinations.
Table 4. Type and content of flavonoids in References

four edible green algae 1. Ambrozova, J. V.; Misurcova, L.; Vicha,
Edible green Flavonoids R.; Machu, L., Samek, D.; Baron, M.;
algae ( µg/100 g dry weight) Mlcek, J.; Sochor, J.; Jurikova, T.
Quercetin Genistein Molecules 2014, 19, 2344-2360.
Spirogyra sp. 16.14 ± 2.40d 3.62 ± 0.78c 2. Fernando, S. I. P.; Kim, M.; Kwang-
Cladophora sp. 98.63 ± 5.07a 7.54 ± 0.32b Tae, S.; Jeong, Y.; You-Jin, J. J. Med.
b
Nostoc sp. 88.86 ± 4.13 11.34 ± 1.59a Food. 2016, 19 (7), 1-14.
c
Wolffia globosa 20.56 ± 7.25 6.17 ± 0.64b
Values in the same column followed by a different 3. Machu, L.; Misurcova, L.; Ambrozova,
latter ( a-c) are significantly different (P < 0.05). Each J. V.; Orsavova, J.; Mlcek, J.; Sochor, J.;
value in table is represented as mean ± SD (n = 5) Jurikova, T. Molecules. 2015, 20, 1118-
1133.
Table 5. Antioxidant activity of four edible 4. Peerapornpisal, Y. J. Fish. Tech. Res.
green algae 2008, 1, 178-189.
Edible green Antioxidant activity 5. Senawong, T.; Misuna, S.; Khaopha, S.;
Algae IC50 ( µg/mL) Nuchdomrong, S. ; Sawatsitang, P. ;
DPPH assay ABTS assay Phaosiri, C. ; Surapaitoon, A. ; Sripa, B.
Spirogyra sp. 142.07 ± 2.06e 33.93 ± 0.88d BMC Comp. & Alte. Med. 2013, 13, 232.
Cladophora sp. 101.44 ± 1.04d 30.17 ± 1.17d
Nostoc sp. 68.71 ± 1.11c 19.69 ± 1.19c
6. Torres, A. M. ; Mau- Lastovicka, T. ;
Wolffia globosa 149.40 ± 1.09e 51.48 ± 1.56e Rezaaiyan, R. J. Agr. Food. Chem. 1987,
Gallic acid 2.34 ± 0.02a 0.64 ± 0.04a 35, 921-925.
b
Ascorbic acid 4.13 ± 0.08 3.95 ± 0.09b 7. Zou, Y.; Lu, Y.; Wei, D. J. Agr. Food.
Values in the same column followed by a different Chem. 2004, 52, 5032-5039.
latter ( a-e) are significantly different (P < 0.05). Each 8. Schierle, J. ; Pietsch, B. ; Ceresa, A. ;
value in table is represented as mean ± SD (n = 5).
Fizet, C. J. AOAC. Int. 2004, 87, 1070-
1082.
Antioxidant and cytotoxic activities and total phenolic content
of Mesua ferrea
Sukanya Keawsa-ard1*, Montakarn Boonyakarn2, Piranchana Phanusan2,
Oranuch Thammasorn3, Samart Kongtaweelert4
1
Department of Basic Science, Faculty of Science, Payap University, Chiang Mai 50000, Thailand
2
Department of Food Science and Technology, Faculty of Science, Payap University,
3
Chiangkham Hospital, Phayao 56110, Thailand
4
Faculty of Science, King Mongkut’s Institute of Technology Ladkrabung 10520, Thailand
*E-mail: sukanya_k40@hotmail.com
Abstract:
Leaves and stem of Mesua ferrea were extracted with n-hexane, dichloromethane and
methanol, respectively. These extracts were evaluated for their antioxidant activity and total
phenolic content. The cytotoxic activity of the leaf extracts was also investigated. The
methanol extract of the stem showed the highest antioxidant activity against DPPH and
ABTS●+ radicals with the IC50 values of 0.150.01 and 0.28±0.01 mg/mL, respectively. Also,
it had the highest total phenolic content of 199.27±6.55 mg GAE/g dry extract. The
dichloromethane and methanol extracts of leaves displayed cytotoxicity against KB cancer
cell line with the IC50 values of 23.70 and 25.14 g/mL, respectively. They also showed
activity against NCI-H187 cell line with the IC50 values of 38.68 and 30.80 g/mL,
respectively whereas the n-hexane extract of leaves exhibited MCF-7 cell line with an IC50
value of 38.12 g/mL. All extracts were non-cytotoxic to Vero cells.
1. Introduction carminative, diuretic, asthma, expectorant,

Nowadays, plants are an important cardiotonic, and antipyretic agent.1,3 Recent
natural source for drug discovery and the scientific studies have been reported on the
crude extracts or isolated compounds of chemical compositions and biological
medicinal plants possess various biological activities of the crude extracts and isolated
activities such as antioxidant, antimalarial, compounds obtained from different parts of
antiulcer, anti-inflammatory, antibacterial, M. ferrea.4 But, there is a few reports in the
antifungal and anticancer activities. Mesua literature mentioned about the antioxidant
ferrea Linn. is commonly known as activity and total phenolic content of leaves
Nagakeshara, Nageshwar (India), Penaga and stem including the cytotoxic activity of
Lilin, Tapis (Malaysia), ironwood (Sri leaves. Therefore, the aim of this study was
Lanka), Bunnak, Saaraphi-doi (Thailand), to evaluate the antioxidant activity and total
and belongs to the family Clusiaceae. It is phenolic content of M. ferrea leaves and
widely distributed in tropical countries like stem in different solvent extracts. Moreover,
India, Sri Lanka, Burma, Thailand, the cytotoxic activity of leaf extracts was
Indonesia, Malaysia, and New Guinea.1,2 M. also investigated. To the best of our
ferrea has been reported as a traditional knowledge, the cytotoxicity of M. ferrea leaf
medicine, the leaves and flowers are used extracts against KB, MCF-7 and NCI-H187
for snake bite and scorpion sting, the bark is cancer cell lines are described here for the
used to treatment of cough, dysentery, first time.
vomiting, sore throat and fever, and the
flowers are used as stomachic, expectorant
and astringent. In Thailand, M. ferrea is a
rare plant and used to treat fever,
2. Materials and Methods persulphate solution in the dark at room
2.1 Plant material temperature for 12 h. The ABTS●+ solution
Leaves and stem of M. ferrea were was diluted with deionized water to an
collected from Payap University, Chiang absorbance of 0.9 at 734 nm. Different
Mai, Thailand. The voucher specimen (code concentrations of the crude extracts were
Sukanya02) was deposited at the Herbarium prepared in ethanol. Each sample solution (1
of Biology Department, Faculty of Science, mL) was mixed with 3 mL of ABTS●+
Chiang Mai University. solution. The reaction mixture was
2.2 Extraction incubated for 5 min in the dark at room
Leaves and stem of M. ferrea were temperature. Then, the absorbance of the
washed with water and dried in a hot air mixture was measured at 734 nm using UV-
oven at 40 °C for 24 and 36 h., respectively, Vis spectrophotometer. (Shimadzu UV2450,
then ground to a fine powder. Each dried Japan). The percentage scavenging activity
powder (100 g) was extracted sequentially and the IC50 value of each solution were also
with 1.5 L of n-hexane, dichloromethane calculated. Trolox and vitamin C were used
(CH2Cl2) and methanol (MeOH) at room as positive controls.
temperature for 7 days. The extracts were 2.5 Total Phenolic Content
concentrated under vacuum using a rotary The total phenolic content of each
evaporator to afford crude extracts. The extract was determined using the Folin-
extracts were kept in the dark at 4 ○C until Ciocalteu reagent7 with some modification.
further investigation. Briefly, a series of concentrations of gallic
2.3 DPPH assay acid (0-0.07 mg/mL) were prepared in 50%
The antioxidant activity of the crude (v/v) methanol and used as standard
extracts was determined by the DPPH assay5 solutions. Then, 1 mL of each extract
with some modification. Different solution (1 mg/mL) was mixed with 5 mL of
concentrations of the extracts were prepared Folin Ciocalteu’s reagent (1:10 v/v). After 8
in ethanol. Then, 20 µL of each the extract min, 1 M Na2CO3 solution (4 mL) was
solution was mixed with 180 µL of added to the mixture and incubated in the
0.004%w/v DPPH solution and incubated in dark at room temperature for 2 h. Then, the
the dark at room temperature for 30 min. absorbance was measured using UV-Vis
The absorbance was measured at 517 nm spectrophotometer at 765 nm (Shimadzu
using spectrophotometer (Multimode UV2450, Japan). Total phenolic contents
detecter, Beckman Coulter DTX880, were expressed in term of gallic acid
U.S.A.). The percentage of the DPPH radical equivalents (mg GAE/g of dry extract).
scavenging activity was calculated as [(Ac- 2.6 Cytotoxic activity
As)/Ac] × 100, where Ac is the absorbance In vitro cytotoxicities of M. ferrea
of the control and As is the absorbance of leaf extracts were performed by the
the extract. The extract concentration Resazurin Microplate assay (REMA)8 using
providing 50% inhibition (IC50) was 3 human cancer cell lines; KB (oral cavity
calculated by plotting inhibition percentages cancer, ATCC CCL-17), MCF-7 (breast
against concentrations of the extract. Trolox cancer, ATCC HTB-22) and NCI-H187
and vitamin C were used as positive (lung cancer, ATCC CRL-5804) cell lines.
controls. In brief, cells at a logarithmic growth phase
2.4 ABTS assay were harvested and diluted to 2.2×104
The ABTS radical scavenging cells/mL for KB and 3.3×104 cells/mL for
activity of all extracts was determined by the MCF7 and NCI-H187 in fresh medium.
method of Re et al.6 with some Then, 5 µL of the extract was diluted in 5 %
modification. The ABTS radical cation DMSO and 45 mL of cell suspension was
(ABTS●+) was prepared by mixing 7 mM added to 384-well plates and then incubated
ABTS solution with 2.4 mM potassium at 37 °C in a 5 % CO2 incubator. After
incubation (3 days for KB and MCF-7 and 5 extracts are given in Table 1. The IC50 value
days for NCI-H187), 12.5 µL of 62.5 µg/mL of each extract was determined by using the
resazurin solution was added to each well, calibration curve which gave a linear
and the plates were incubated at 37 oC for 4 regression equation (R2 > 0.9000). The
h. Fluorescence signal was measured using lower IC50 value indicates a higher
SpectraMax M5 muti-detection microplate antioxidant activity. The MeOH extract of
reader (Molecular Devices, USA) at stem (SM) showed the highest DPPH
excitation and emission wavelengths of 530 activity with the IC50 value of 0.150.01
and 590 nm, respectively. The inhibitory mg/mL, followed by LM, SD, LD, LH and
concentration (IC50) represented the SH, respectively. Similar to the extraction,
concentration that caused 50 % reduction in the more polar extract displayed the best
cancer cell line growth. Ellipticine and antioxidant activity.
doxorubicin were used as positive controls
and 0.5% DMSO was used as negative Table 1. Antioxidant activity by DPPH and
control. The cytotoxicity assay against Vero ABTS methods
cells (African green monkey kidney ATCC Extract/ IC50 ± SD (mg/mL)
CCL-81) was performed using a Green Standard DPPH ABTS
Fluorescent Protein (GFP)-based assay.9 LH 2.86±0.04 0.64±0.01
LD 2.74±0.03 0.54±0.01
LM 0.31±0.01 0.37±0.01
3. Results and Discussion SH 4.57±0.10 0.88±0.02
3.1 Extraction SD 1.50±0.01 0.31±0.00
Leaves and stem of M. ferrea were SM 0.15±0.01 0.28±0.01
extracted sequentially with n-hexane, Trolox 0.10±0.00 0.04±0.00
Vitamin C 0.06±0.00 0.21±0.01
dichloromethane and methanol. The %
yields of the extracts were calculated based
ABTS method measures the relative ability
on dry weight. The results are shown in
of antioxidant to scavenge the ABTS
Figure 1. The extraction yields increased
generated in aqueous phase. This assay is
when the polarity of solvents increased.
based on the discoloration of ABTS●+ by
antioxidant compounds. Reduction of blue-
green ABTS radical colored solution by
electron/hydrogen donating antioxidant is
measured at 734 nm.11 The results of
antioxidant activity of M. ferrea extracts are
presented in Table 1. SM also showed the
highest ABTS activity with the IC50 value of
0.280.01 mg/mL, followed by SD, LM,
LD, LH and SH, respectively. For leaf
extracts, LM also showed the highest
antioxidant activity on ABTS●+ than the
Figure 1. The % yields of the extracts
other extracts. In addition, it was found that
SM exhibited the best ability to scavenge the
3.2 DPPH and ABTS assays
DPPH and ABTS radicals.
DPPH is a stable free radical that
3.3 Total phenolic content
contains an odd electron and accepts an
The total phenolic content of the
electron donated by antioxidant compounds.
extracts was determined using the Folin
The reduction capability of DPPH radical is
Ciocalteu reagent and expressed in the terms
determined by the decrease in its absorbance
of gallic acid equivalent (mg/g dry mass).
at 5l7 nm. It is visually noticeable as a
The absorbances were plotted against the
change in color from purple to yellow.10 The
gallic acid concentrations to obtain a linear
results of antioxidant activity of M. ferrea
calibration curve (0.01-0.07 mg/mL) which LH showed only cytotoxicity against
gave the linear regression equation (y = MCF-7 cell line with the IC50 value of 38.12
13.788x - 0.0131, R2 = 0.9972) for gallic µg/mL. LD inhibited KB and NCI-H187 cell
acid. The equation was used to calculate the lines with the IC50 values of 23.70 and 38.68
total phenolic contents of all extracts. The µg/mL, respectively. LM inhibited KB and
results are shown in Table 2. NCI-H187 cell lines with the IC50 values of
25.14 and 30.80 µg/mL, respectively. All
Table 2. Total phenolic contents of M. extracts were noncytotoxic against Vero
ferrea extracts cells. For the stem extracts, we have recently
Extracts Mean ± SD reported their cytotoxic activities against
(mg/g extract) these three cell lines.3 The IC50 values
LH 31.44±0.23
ranged from 18.01 to 33.54 μg/mL. Based
LD 34.31±0.37
LM 159.40±4.16 on IC50 values, the leaves and stem of M.
SH 6.82±0.73 ferrea showed mild cytotoxic activity
SD 48.82±0.73 against KB, MCF-7 and NCI-H187 cancer
SM 199.27±6.55 cell lines.
Different extraction solvents yield different 4. Conclusion

concentrations of phenolic compounds. SM Leaves and stem of M. ferrea were
gave the highest total phenolic content of extracted sequentially with n-hexane,
199.27±6.55 mgGAE/g dry extract, CH2Cl2 and MeOH. The MeOH was found
followed by the methanol extract of leaves to be the best solvent for the extraction of
with LM, SD, LD, LH and SH, respectively. phenolic compounds than others. The
Phenolic compounds are commonly found in MeOH extracts of stem and leaves possessed
many plants and they have been reported to good antioxidant activity on DPPH and
possess antioxidant activity.12 This study ABTS●+ radicals. Thus, M. ferrea leaves and
indicated that SM showed the highest stem may be sources of natural antioxidants.
phenolic content and exhibited stronger
antioxidant activity on DPPH and ABTS Acknowledgement
assays than other extracts. Thus, M. ferrea The authors would like to express
stem is an important potential source of our sincere thanks to Payap University,
natural antioxidants. Chiang Mai, Thailand for financial support.
3.4 Cytotoxic activity
The cytotoxicities of the leaf extracts References
of M. ferrea were determined by Resazurin 1. Chahar, M. K.; Kumar D. S.; Geetha L.;
Microplate Assay (REMA) using KB, MCF- Lokesh, T.; Manohara, K. P. Afr. J.
7 and NCI-H187 cancer cell lines. Their Pharm. Pharmacol. 2013, 7, 211-219.
cytotoxicities against Vero cell lines were 2. Sharma, A.; Sharma, S.; Naresh, R.;
also carried out. The results are presented in Parashar, B. Sys. Rev. Pharm. 2017, 8,
Table 3. 19-23.
3. Keawsa-ard, S.; Liawruangrath, B.;
Table 3. The cytotoxicities of M. ferrea leaf Kongtaweelert, S. Chiang Mai J. Sci.
extracts 2015, 42, 185-195.
Extracts/ IC50 (g/mL) 4. Asif, M.; Jafari, S. F.; Iqbal, Z.;
standard KB MCF-7 NCI- vero
Revadigar, V.; Oon, C. E.; Majid, A. S.
H187 cells
LH - 38.12 - - A.; Majid, A. M. S. A. J. App. Pharm.
LD 23.70 - 38.68 - Sci. 2017, 7, 242-251.
LM 25.14 - 30.80 - 5. Keawsa-Ard, S.; Liawruangrath, B.;
Ellipticine 0.512 - 0.875 1.335 Liawruangrath, S.; Teerawutgulrag, A.;
Doxorubicin 0.319 0.858 0.050 -
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Free Radical Bio. Med. 1999, 26, 1231- M. A.; June, A. S. Euro. J. Exp. Bio.
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7. McDonald, S.; Prenzler, P. D. Food 11. Shalaby, E. A.; Shanab, S. M. M. Indian
Chem. 2001, 73, 73-84. J. Geo-Mar. Sci. 2013, 42, 556-564.
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Pretreatment of water hyacinth (Eichhornia crassipes (Mart.) Solms) for
biodegradable polyhydroxybutyrate (PHB) production
Napassorn Wititsuwankul*, Phimchanok Jaturapiree
*E-mail: napassorn.wititsuwankul@gmail.com
Abstract:
Water hyacinth (Eichhornia crassipes (Mart.) Solms) can be used as a renewable
resource to produce fermentable sugars. Here, we studied the effect of the ratios of water
hyacinth to acid (1:20 and 1:40 (w/v)) under 130 oC for 2 h using 2% (v/v) sulfuric acid and
the impact of alkaline pretreatment, 2% (w/v) of sodium hydroxide, in the ratio of 1:20 (w/v)
on releasing sugars from the biomass. Alkaline pretreatments were conducted using autoclave
at 121 oC and 15 psi for 30 min. The hydrolysates were adjusted to pH 7.0 - 8.0 using calcium
hydroxide before determining reducing sugars by using HPLC. Water hyacinth treated with
acid in a ratio of 1:20 (w/v) yielded higher total reducing sugar concentration of 11.12 g/L
(0.39 g/L of glucose, 5.14 g/L of xylose, and 5.70 g/L of arabinose). The alkaline
pretreatment showed that 32.93% of hemicellulose was removed from water hyacinth,
compared with unpretreated water hyacinth. These results suggested that water hyacinth
could be used as a renewable carbon source for the synthesis of biodegradable
polyhydroxybutyrate.
1. Introduction bioplastics industry. The search of

Polyhydroxybutyrate (PHB) is a renewable and inexpensive carbon sources
natural polymer with potential as a to reduce production cost is essentially
biodegradable thermoplastic. PHB can be required.7,8
synthesized from various microorganisms in Lignocellulosic biomass, such as
environmental stresses such as nitrogen, agricultural waste materials, has been
phosphate or oxygen limitations1 and considered as a feedstock being most
accumulated as granular inclusions in cell abundant and renewable biomass on the
cytoplasm and the amount might go up to earth.9 Weed biomass is lignocellulosic
90% of the cellular dry weight.2 Its biomass that has been reported as an
structural properties are similar to alternative substrate for PHB production by
polypropylene (PP).3 In recent days, PHB using its hydrolysates. This study employed
has received much attention due to its water hyacinth (Eichhornia crassipes
applications in packaging, medication, and (Mart.) Solms) as a substrate for the PHB
agriculture.4 production. Water hyacinth can grow in vary
However, the cost of PHB environment with rapid growth rates. It is
production is still higher than petroleum- considered as a water weed which causes
based polymers. The PHB production costs water environmental problems in Thailand,
are between 3.1 and 4.4 USD/kg, while the such as irrigation and transport. In order to
cost of PP production is less than 1 use lignocellulosic waste as a substrate, the
USD/kg.5 Moreover, about 50% of the PHB process of raw material conditioning10 is
production cost is attributed to the cost of required to destroy the hardening structure
the substrates, where 70-80% of the total of lignocellulose, then microorganisms can
expense is from the cost of carbon source.6 convert cellulose into reducing sugars.11,12
This is a barrier to the growth of the
The objectives of the present study psi for 30 min, was hydrolyzed using 2%
were to study the effect of the ratios of water (v/v) H2SO4 in the best ratio at 130 oC for 2
hyacinth to acid in converting water h. After hydrolysis, the hydrolysate was
hyacinth into fermentable sugars and to recovered by vacuum filtration. The pH of
study the effect of alkaline pretreatment on the hydrolysate was adjusted to 7.0 - 8.0
the lignocellulosic biomass in bioplastic using calcium hydroxide and filtered by
production. vacuum filtration. The hydrolysate was
determined for reducing sugars by high-
2. Materials and Methods performance liquid chromatography.
2.1 Material 2.4 Determination of reducing sugars by
Water hyacinth (Eichhornia using HPLC
crassipes (Mart.) Solms) was collected from The hydrolysate was filtered through
the waste water canal of Nakhon Pathom a 0.22 µm membrane. The concentrations of
province, Thailand. The feedstock was glucose, xylose, and arabinose were
washed immediately after collected, then cut analyzed by high-performance liquid
to about 2 cm. The feedstock was dried in chromatography (HPLC) (Shimadzu;
hot air oven at 60 oC for 24 h, and milled to LC20AD) using Rezex RNM-Carbohydrate
particle size smaller than 1.0 mm using a column at 45 oC using deionized water as
laboratory hammer mill (Retsch, type SR mobile phase (0.4 ml/min) with refractive
200, Haan, Germany). index detector (Shimadzu; RID10A) and the
2.2 Alkaline pretreatment of water sugar concentrations (glucose, xylose, and
hyacinth arabinose) were quantified by external
For pretreatment, two percentages calibration with standards.
(w/v) of sodium hydroxide (NaOH) was
added to water hyacinth at the ratio of 1:20 3. Results and Discussion
(w/v) and then autoclaved at 121 oC and 15 3.1 Effect of the ratios of water hyacinth
psi for 30 min. The residue was collected by to acid
filtration with muslin cloth and washed The water hyacinth was hydrolyzed
several times with distilled water till using 2% (v/v) H2SO4 in ratios of 1:20 and
obtained the pH between 7 and 8. Then it 1:40 (w/v) for 2 h. The result showed that
was dried in hot air oven at 60 oC for 24 h. the overall reducing sugar concentrations of
2.3 Acid hydrolysis of water hyacinth 1:20 and 1:40 (w/v) were 11.12 and 4.53
2.3.1 Effect of the ratio of water hyacinth g/L, respectively. The concentrations of
to acid glucose, xylose and arabinose are shown in
For hydrolysis, water hyacinth was Table 1. Lower ratio of water hyacinth to
hydrolyzed using 2% (v/v) sulfuric acid sulfuric acid gave higher reducing sugar
(H2SO4) in ratios of 1:20 and 1:40 (w/v) at concentration.
130 oC for 2 h. After hydrolysis, the liquid Likewise, Azizi et al. (2017)
fraction (hydrolysate) was recovered by evaluated the dilute acid hydrolysis of
vacuum filtration with Whatman filter paper brown macroalgae (Sargassum sp.) for
No.1. The pH of the hydrolysate was production of reducing sugars using 10%
adjusted to pH 7.0 - 8.0 using calcium seaweed with 0.15 N of H2SO4 and HCl at
hydroxide and filtered by vacuum filtration. 121 oC for 30 min. The maximum reducing
The hydrolysate was determined for sugars concentrations were 12.01 ± 0.12 and
reducing sugars by high-performance liquid 11.62 ± 0.07 g/L, respectively.
chromatography.
2.3.2 Effect of alkaline pretreatment
The pretreated water hyacinth, 2%
(w/v) of sodium hydroxide in the ratio of
1:20 (w/v) and autoclaved at 121 oC and 15
Table 1. The compositions of water hyacinth hydrolysate (g/L) in ratios of 1:20 and 1:40
(w/v). The results were presented in mean ± SD, n = 3.
Ratio (w/v) Reducing Sugars
Glucose Xylose Arabinose Total
1:20 0.39 ± 0.01 5.14 ± 0.17 5.70 ± 0.16 11.12 ± 0.34
1:40 0.26 ± 0.01 2.22 ± 0.12 2.06 ± 0.04 4.53 ± 0.10
Considering the concentration of glucose concentration was obtained from

hemicellulose fractions in the hydrolysates 0.39 ± 0.01 to 0.40 ± 0.02 g/L (Figure 1).
of water hyacinth in diluted acid The result suggests that diluted NaOH
pretreatment, showed the releasing of 5.14 removed the amount of hemicellulose
g/l of xylose and 5.70 g/L of arabinose. (32.93%) from water hyacinth, diluted
Several reports suggested that xylose, NaOH treatment of lignocellulosic biomass
arabinose and oligosaccharides in the was believed that caused swelling and led to
hydrolysate solution can be used as a an increase in internal surface area,
substrate for the PHB production.14-16 separation of structural linkages between
3.2. Effect of alkaline pretreatment lignin and carbohydrates and disruption of
The alkaline pretreated water the lignin structure.17 The mechanism of
hyacinth, 2% (w/v) of sodium hydroxide in alkaline hydrolysis is also believed as
the ratio of 1:20 (w/v) and autoclaved at 121 saponification of intermolecular ester bonds
o
C and 15 psi for 30 min, was hydrolyzed crosslinking xylan of hemicelluloses and
using 2% (v/v) H2SO4 in the ratio of 1:20 lignin.18
(w/v) at 130 oC for 2 h. The result showed Likewise, several reports were also
that the alkaline pretreatment decreased the showed that the pretreatment with diluted
concentration of xylose and arabinose from NaOH decreased the concentration of
5.14 ± 0.17 to 3.61 ± 0.09 g/L and 5.70 ± hemicelluloses.19 Annamalai and Sivakumar
0.16 to 3.66 ± 0.07 g/L respectively, (2016) reported that the pretreatment of
compared with unpretreated water hyacinth. wheat bran at 121 oC for 30 min with 1%
However, the insignificant increasing of (w/v) NaOH decreases the lignin and
Figure 1. The amount of sugars released from untreated and pretreated water hyacinth
through acid hydrolysis by 2% (v/v) H2SO4. The results were presented in mean ± SD, n =
3.
hemicellulose contents from 20.15 to 8.93% 5. Naranjo, J. M.; Posada, J. A.; Higuita, J.
and 26.34 to 20.25%, respectively.20 C.; Cardona, C. A. Bioresource
Radhika and Murugesan (2012) studied Technol. 2013, 133, 38–44.
alkaline pretreated water hyacinth was 6. Wang, B.; Sharma-Shivappa, R. R.;
hydrolyzed using 72% (v/v) H2SO4 for Olson, J. W.; Khan, S. A. Ind. Crop.
production of reducing sugars and used 2% Prod. 2013, 43, 802–811.
activated charcoal for the detoxification of 7. Khanna, S.; Srivastava, A. K. Process.
acid hydrolysate. Water hyacinth Biochem. 2005, 40, 2173–2182.
hydrolysates used as a sole substrate has the 8. Annamalai, N.; Rajeswari, M. V.;
potential to the PHB production (3.2 ± 0.1 Balasubramanian, T. Biomass.
g/L).21 Bioenerg. 2014, 68, 151–160.
9. Saratale, G. D.; Kshirsagar, S. D.;
4. Conclusion Sampange, V. T.; Saratale, R. G.; Oh, S.
The development and E.; Govindwar, S. P.; Oh, M. K. Appl.
implementation of renewable feedstock for Biochem. Biotechnol. 2014, 174, 2801–
the PHB production have received much 2817.
attention in order to reduce the cost of PHB 10. Yang, B.; Wyman, C. E. Biofuel.
production. The alkaline pretreatment of Bioprod. Bior. 2008, 2, 26–40.
water hyacinth can destroy the hardening 11. Borah, A. J.; Agarwal, M.; Poudyal, M.;
structure of lignin and hemicellulose to Goyal, A.; Moholkar, V. S.
improve the hydrolysis of cellulose to Bioresource. Technol. 2016, 213, 342–
fermentable sugars (glucose). This study is 349.
underway, using cellulase for hydrolysis of 12. Singh, S.; Dikshit, P. K.; Moholkar, V.
cellulose to fermentable sugars (glucose) in S.; Goyal, A. Environ. Prog. Sustain.
order to a substrate for the PHB production 2014, 34, 810–818.
to improve utilization of this lignocellulosic 13. Azizi N.; Najafpour. G.; Younesi, H.
biomass into bioplastics. Int. J. Biol. Macromol. 2017, 101,
1029–1040.
Acknowledgement 14. Sindhu, R.; Silviya, N.; Binod, P.;
This work was supported by the Pandey, A. Biochem. Eng. J. 2013, 78,
Department of Biotechnology, Faculty of 67-72.
Engineering and Industrial Technology, 15. Saratale, G. D.; Oh, M.-K. Int. J. Biol.
Silpakorn University and by The National Macromol. 2015, 80, 627–635.
Research Council of Thailand, Academic 16. Pradhan, S.; Borah, A. J.; Poddar, M.
Year 2018. K.; Dikshit, P. K.; Rohidas, L.;
Moholkar, V. S. Bioresource. Technol,
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Radical scavenging activity and total phenolic content of
Lumnitzera littorea fruit and flower
Luksamee Vittaya1*, Sunanta Khongsai1, Sutkanung Na Ranong1,
Sittichoke Janyong1 Nararak Leesakul2
1
Faculty of Science and Fisheries Technology, Rajamangala University of Technology Srivijaya,
Sikao, Trang 92150, Thailand
2
*E-mail: nokuksamee@hotmail.com
Abstract:
Fruit and flower of Lumnitzera littorea were sequentially extracted with increasing
polarity of four organic solvents: hexane, dichloromethane, ethyl acetate and methanol,
respectively. All eight extracts were evaluated for free radical scavenging activity by using
2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2/-azino-bis-(3-ethylbenzothiazoline-6-
sulfonic acid) diammonium salts (ABTS) methods. Good activity was found in polar solvent
extracts. The total phenolic content was determined by the Folin-Ciocaltue method. The
results showed that the polar extracts exhibited higher total phenolic content than the non-
polar ones. Additionally, free radical scavenging activity of the extracts correlated with
phenolic contents. For L. littorea, further purification of the active crude extract and
characterization of isolated compounds are required to identify bioactive phenolic substances
pesented in ethyl acetate and methanol extracts.
1. Introduction be of therapeutic value in reducing the risk

Lumnitzera littorea is a mangrove of chronic diseases.6 In the literature, several
plant belonging to the COMBRETACEAE mangrove plants presented a good source of
family. L. littorea has red flower and its bark natural antioxidants by scavenging free
is rough with gray to brown color. It is a radicals because of its rich phenolic and
medium to tall tree growing from 10-30 m in flavonoid contents.7-10 Moreover, Saad et al.
tropical and subtropical areas. Mangrove (2011) reported that the hexane, ethyl
plants interestingly grow under stressful acetate and methanol leaf extract of L.
habitat conditions where freshwater and littorea showed antibacterial activity.4
marine systems meet, adapting Antioxidants play an important role
morphologically and surviving steep in preventing pathogenic processes related
temperature gradients and extreme to cancer, aging and other conditions
conditions of salinity. As a result of this, normally resulting from free radicals during
they produce certain chemical compounds body metabolism. However, a few studies of
like phenolics, flavonoids, tannins, alkaloids L. littorea involve its antibacterial activity4
and saponins to protect itself in this but none reported the antioxidant activity of
challenging environment. These compounds L. littorea fruit and flower. Therefore, the
can be extracted by the specific polarity of chemical compounds of L. littorea fruit and
organic solvents1 and show a variety of flower extracted with four organic solvents
application like antioxidant and (hexane, ethyl acetate, dichloro methane and
2,3
antimicrobial activities. The produced methanol) were determined and reported as
natural antioxidants could reactively total phenolic content. Free radical
scavenge the free radicals occurring in many scavenging capacity of both fruit and flower
pathogenic conditions, inhibiting bacterial has been investigated as well.
growth.4,5 Their bioactive components could
2. Materials and Methods mixture was shaken vigorously and allowed
2.1 Standard and reagents to stand at room temperature in the dark for
Folin-Ciocalteu reagent, gallic acid, 30 min. The control was prepared as above
butylhydroxytoluene (BHT), 1,1-diphenyl-2- without extract. The absorbance of the
picrylhydrazyl (DPPH), 2,2/-azino-bis(3- solution was measured at 517 nm against the
ethylbenzothiazoline-6-sulphonic acid) blank. Ascorbic acid was used as a positive
(ABTS) and ascorbic acid were purchased control. The percentage of free radical
from Sigma-Aldrich Co. All other analytical scavenging activity of each concentration
grade chemicals were used without was calculated based on the formula (1 -
purification. (Asample - Asample blank)/Acontrol) x 100 where
2.2 Preparation of the extract Asample is the absorbance of the test sample
Fresh fruit and flower of L. littorea with DPPH solution, Asample blank is the
were collected from Rajamangala mangrove absorbance of the test sample only, and
forest, Rajamangala University of Acontrol is the absorbance of DPPH solution.
Technology Srivijaya, Trang province on The antioxidant activity of the sample was
February 2016. The plant part was dried and expressed as the effective concentration of
macerated for 7 days according to polarity the extract (mg/mL) which was calculated
organic solvents of hexane, dichloro- by plotting inhibition percentages against
methane, ethyl acetate and methanol to sample concentration. Ascorbic acid and
extract bioactive compounds. The resulting butylhydroxytoluene (BHT) were used as
extracts were evaporated to dry residue standards. All measurements were
using a rotary evaporator and refrigerated performed in triplicates and expressed as the
until used to determine the total phenolic average values.
content and antioxidant activity. ABTS scavenging assay
2.3 Determination of total phenolic The 2,2/-azino-bis-3-ethylbenzothi-
content azoline-6-sulphonate radical cation (ABTS+)
The total phenolic contents was deceleration method was investigated as
determined by Folin-Ciocalteu’s method10 described by Re et al.12 ABTS radical cation
using gallic acid as the standard. The (ABTS+) was prepared by mixing 7 mM of
concentration of the samples was fixed at 1 ABTS 5 mL with 140 mM of K2S2O8
mg/mL using only 0.2 mL of each sample (potassium persulfate) 88 L in dark for 16
for adding in 2.5 mL of distilled water. The h at room temperature before use. The
reaction mixture was shaken with 0.2 mL of mixture was diluted with methanol to give
Folin-Ciocalteu reagent. After 1 min, 2.0 an absorbance of 0.700  0.025 units at 734
mL of 7% sodium carbonate solution was nm using spectrophotometer. 0.1 mL of
added to the reaction mixture and then kept sample extract was added to 0.9 mL of
away from light for 60 min. The absorbance diluted ABTS+ solution. The mixture
of all samples was measured at 765 nm reaction was shaken and stood for 6 min at
against the blank. Total phenolic content room temperature. Absorbance was
was expressed as milligrams of gallic acid measured after incubation at 734 nm. The
equivalents (GAE) per gram of dry crude capacity of free radical scavenging was
extract (mg GAE/g crude extract). carried out in triplicates. The ABTS radical
2.4 Antioxidant activity scavenging was calculated using the
DPPH scavenging assay following equation: Scavenging effect (%) =
The free radical scavenging effect on [(Acontrol - Asample)/Acontrol] x 100, where
DPPH radicals was assessed by slightly
modifying the method of Vittaya et al.11 Acontrol is the absorbance of the extract
Briefly, 0.5 mL of each sample (1 mg/mL) without ABTS+ solution, Asample is the
and standards were mixed with 0.5 mL of absorbance of the extract with ABTS+
0.15 mM methanolic DPPH solution. The solution.
2.5 Statistical analysis 3.2 Antioxidant activity
All data obtained were expressed as The antioxidant capacities were
the mean of three values ± standard studied with 1,1-diphenyl-2-picrylhydrazyl
deviation and were subjected to ANOVA (DPPH) and 2,2/-azino-bis-(3-ethylbenzo
test to measure whether there was a thiazoline-6-sulfonic acid)diammonium salts
significant difference in total phenol and (ABTS) methods. The results of these
radical scavenging effects. methods are shown in Figure 2 and the data
are presented in Table 1. In this work, the
3. Results and Discussion scavenging activity of DPPH varied between
Both of fruit and flower of L. littorea 6% and 96% in both fruit and flower
(Figure 1) were successively extracted with extracts (Figure 2). The results showed that
four organic solvents: hexane, the free radical scavenging of the ethyl
dichloromethane, ethyl acetate and acetate and methanolic fruit and flower
methanol, respectively. The phenolic content extracts is higher than that of hexane and
and antioxidant capacity of all extracts were dichloromethane extracts at the same
determined using spectroscopic method. concentration. In the same ABTS method,
the samples exhibited a decrease in
absorption with small to high scavenging
activity, ranging from 2% to 99% in fruit
and flower extracts of L. littorea. Eight
samples of L. littorea showed a high degree
of free radical scavenging activity. From
Table 1, the methanolic extract of fruit of L.
Figure 1. Fruit and flower of L. littorea
littorea showed the highest antioxidant
activity with IC50 21.3  0.2 (DPPH) and
3.1 Determination of total phenolic
contents and flavonoid concentrations 12.9  0.5 g/mL (ABTS), respectively,
The total phenolic content of all while the ethyl acetate extract of flower
extracts was determined by the Folin- showed the highest antioxidant activity with
Ciocalteu method and the data are shown in IC50 4.5  0.2 (DPPH) and 2.3  0.4 g/mL
Table 1. The total phenolic content of these (ABTS), respectively. In comparison, it
extracts was calculated according to the appeared that antioxidant activities of the
equation y = 0.003x + 0.002 (R2 = 0.999) as positive control, BHT (IC50 = 11.6  0.1 and
gallic acid equivalent (GAE, mg/g crude 16.9  1.1 g/mL for DPPH and ABTS) and
extract). The result showed that the total ascorbic acid (IC50 = 4.6  0.1 and 2.4  0.1
phenolic contents in the four extracts were g/mL for DPPH and ABTS) were higher
in the range of 48 – 2920 g GAE/g of the than that of methanolic extract of fruit but
crude extract. In regard to the amount of lower than that of ethyl acetate extract of
phenolic compounds in L. littorea, the ethyl flower. It may be summarized that
acetate and methanolic extracts of both parts methanolic extracts of fruit and ethyl acetate
had higher phenolic content than flower extract were the most active, which
dichloromethane and hexane extracts. It has may well be related to their phenolic
been hypothesized that the presented component. To increase the antioxidant
phenolic compound in all extracts may be effect of methanolic extract of fruit and
associated with its antioxidant activity. ethyl acetate extract of flower from L.
In addtition, the higher amount of littorea, it seems important to analyze and
total phenolic content of L. littorea may play purify their phenolic compounds in further
important role to increase the antioxidant studies.
activity.13
4. Conclusion and ethyl acetate extract of the flower of L.
L. littorea, on successive extraction littorea. Moreover, the total phenolic
with hexane, dichloromethane, ethyl acetate content correlated with antioxidant capacity
and methanol, gave eight extracts. The total determined by both the DPPH and ABTS
phenolic content was carried out and results methods. In addition, the same correlations
revealed that methanolic extract of fruit and were observed in all extracts. Thus, the
ethyl acetate extract of flower exhibited the findings of the present study provide
highest total phenolic content. The evidence of antioxidant properties which
antioxidant capacity of both fruit and flower correspond to the active ingredients in L.
of L. littorea was studied. Good activity was littorea revealed by phenolic content.
exhibited by the methanol fraction of fruit
Figure 2. Percentage of free radical scavenging activity of L. littorea (LI); fruit (F) and
flower (FW); Hexane (H), Dichloromethane (D), Ethyl acetate (E), Methanol (M)
Table 1. Total phenolic content and Free radical scavenging activity of L. littorea extracts
Parts Solvents Total phenolic (g) IC50 (g/mL)
GAE/g CE DPPH ABTS
Fruit Hexane 48.9  2.5 > 1000 >1000
Dichloromethane 271.1  4.2 332.4  4.4 55.8  0.4
Ethyl acetate 827.2  23.3 48.0  0.4 28.7  1.2
Methanol 1041.12.5 21.3  0.2 12.9  0.5
Flower Hexane 73.9  1.0 > 1000 > 1000
Dichloromethane 178.3  15.2 923.5  83.2 115.6  0.6
Ethyl acetate 2916.7  22.4 4.5  0.2 2.3  0.4
Methanol 1241.7  35.9 12.7  0.2 7.4  0.4
BHT - - 11.6  0.1 16.9  1.1
Ascorbic acid - - 4.6  0.1 2.4  0.1
data presented as mean ± SD from analysis of three samples, in triplicate.
Acknowledgements supported this research by providing

The Faculty of Science and Fisheries laboratory facilities and supporting finance.
Technology, Rajamangala University of The authors acknowledge the Center of
Technology Srivijaya, Trang campus Excellence for Innovation in Chemistry
(PERCH-CIC), office of the Higher 6. Vittaya, L.; Chalad C. RMUTSV Res. J.
Education Commission, Ministry of 2016, 8 (1), 31-38.
Education. We would also like to thank Miss 7. Agoramoorthy, G.; Chen, F. A.;
Anna Chatthong for assistance with the Venkatesalu, V.; Kuo, D. H.; Shea, P.C.
English text. Asian J. Chem. 2008, 20, 1311-1322.
8. Vadlapudi, V.; Naidu, K. C. J. Pharm.
References Res. 2009, 2, 1742-1745.
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Tribenzo[a,c,k,l]xanthene:
a novel fluorophore from an intramolecular Scholl reaction
Thikhamporn Uppalabat, Rukkiat Jitchati*
Department of Chemistry, Faculty of Science, Ubon Ratchathani University, Warinchumrap,
*E-mail: rukkiat_j@hotmail.com
Abstract:
The Scholl reaction has been known as a coupling reaction of aromatic
hydrocarbons ( arenes) with the oxidative coupling reagents. The authors have reported the
synthesis of tribenzo[a,c,k,l]xanthene (ARAR3) via Fe-catalyzed intramolecular Scholl
reaction, from the diphenylbenzo[d,e]chromene (ARAR2). The optimization reaction was
quite enough in the case that the yield was satisfied at 75%. The chemical structure was
characterized by 1H and 13C NMR. The photophysical properties were studied by UV-Vis
absorption and emission techniques. The results showed the maximum π-π* absorption at 335
nm and emission at 500 nm. In addition, the highest occupied molecular orbital (HOMO) and
the lowest unoccupied molecular orbital (LUMO) levels were obtained at -5.04 and -2.21 eV,
respectively. The results showed that the ARAR3 was quite promising to be wildly useful
organic material in the future.
1. Introduction molecular properties, such as optical,

Scholl reaction or oxidative thermal, photophysical and electrochemical
dehydrogenative cyclization used for the properties.
construction of new carbon-carbon bonds to
fuse π-conjugated systems has been 2. Materials and Methods
witnessed as a useful chemical strategy for 2.1 General
the synthesis of novel fluorophore.1 Wide All reactants and solvents were
studies on the mechanistic aspects of this purchased from commercial sources and used
reaction have been carried out both without further purification unless otherwise
experimentally and theoretically to better noted. The substrate (ARAR2) was prepared
understand because the unexpected from cyclization reaction. 1H and 13C NMR
cyclization routes are frequently observed. spectra were recorded on a Bruker ARX-300
The intramolecular Scholl reaction2 can be spectrometer. Molecular weight was obtained
proceeded with the oxidant coupling reagents using a Bruker MicroTOF-Q II. UV-visible
as a valuable procedure to access a new absorption and emission spectra were studied
biaryl units.3 with Perkin Elmer spectrophotometer (LS50).
More specifically, the intramolecular The thermal gravimetrical analysis (TGA)
Scholl reaction has been extended the was measured on a Thermo plus TG 8120
formation the six-membered rings.4 Recently, with heating rate of 10 oC/min in nitrogen
Cao et al. investigated a novel compound atmosphere.
containing fused the six-membered rings of 2.2 Synthesis and characterization
oligobenzofuran derivatives by using ferric The synthesis of ARAR2 compound
chloride as an oxidative reagent.5 from cyclization reaction6 was obtained the
In this work, we studied the yellow solid (73%); m.p. 134-138 oC; 1H
intramolecular Scholl reaction of 2,3- NMR (300 MHz, CDCl3) δ 7.38-7.12 (m, J =
diphenylbenzo[d,e]chromene by using the 7.25 Hz, 14H), 6.88, 6.86 (d, J = 6.87 Hz,
oxidative coupling reagent (FeCl3). The 1H), 6.46, 6.44 (d, J = 6.45 Hz, 1H); 13C
authors also performed the study of NMR (75 MHz, CDCl3) δ 152.7, 149.4,
© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) OR1
135.5, 134.8, 134.2, 131.9, 130.9, 129.0, Table 1. The optimization of intramolecular
128.9, 128.3, 127.6, 127.5, 127.4, 123.6, Scholl reactiona
122.9, 119.1, 117.5, 115.6, 106.9; HRMS Entry eq. of FeCl3 Time %yield
calcd for C24H17O (M+H+): 321.1279, found: (min) (ARAR3)
321.1279. 17 15 5 6
The study of the intramolecular 2 30 11
Scholl reaction was obtained ARAR3 3 60 18
48 12 5 12
compound. The mixture of ARAR2 (1 eq.) in 5 30 26
15 ml of dried dichloromethane (CH2Cl2) at 0 6 60 33
o
C and then FeCl3 (6-15 eq.) in 1-3 ml of 7 10 5 11
nitromethane (CH3NO2). The mixture was 8 30 30
stirred under nitrogen atmosphere for 5, 30, 9 60 38
10 6 5 18
60 minutes. After that, the reaction was 11 30 44
quenched by the addition of methanol and 12 60 75
water, respectively. Crude reaction was a
Condition: ARAR2 (0.94 mmol), FeCl3 (6-15 eq.),
extracted with dichloromethane and water. dried CH2Cl2, CH3NO2 at 0 oC under nitrogen
Then the organic part was purified by column atmosphere
chromatography (hexane as a mobile phase)
to obtain the yellow solid (6-75%); m.p. 162- 3.2 Optical property
164 oC; 1H NMR (400 MHz, CDCl3) δ 7.55, The photophysical properties in
7.53 (d, J = 7.54 Hz, 1H), 7.40, 7.38, 7.46 (t, dichloromethane at room temperature of
J = 7.38 Hz, 1H), 7.32-7.09 (m, J = 7.20 Hz, ARAR2 and ARAR3 were studied in UV-
10H), 6.91, 6.89 (d, J = 6.90 Hz, 1H), 6.32, visible region. The results showed that the
6.30 (d, J = 6.31 Hz, 1H); 13C NMR (75 similar absorption spectra of both
MHz, CDCl3) δ 152.6, 149.4, 135.1, 133.8, compounds were obtained at the maximum
131.8, 131.1, 130.9, 129.2, 128.9, 128.6, absorption and shoulder peaks at 335 and
128.4, 127.7, 127.7, 127.1, 123.5, 117.2, 380 nm, respectively. They were assigned to
116.1, 115.6, 108.1. π-π* transition of benzo[d,e]chromene core.
Interestingly, the different maximum
3. Results and Discussion emission wavelengths were obtained at 476
3.1 Intramolecular Scholl reaction study and 500 nm, respectively. They located in
the green emission region in Figure 2. The
results indicated that the red-shifted of
ARAR3 molecule was related to the planar
structure.
The TGA spectra in Figure 3. The
results found that the thermal stability of
Figure 1. The study of intramolecular Scholl ARAR3 exhibited high decomposition
reaction temperature at 269 oC compared with
ARAR2 due to the rigidity.9
From Table 1, the intramolecular The HOMO and LUMO and energy
Scholl reaction of ARAR2 was studied with levels were estimated from the absorption
a varied equivalents of FeCl3. The results spectra and the electrochemical data
found that the entry 12 with 6 equivalents of performance and UV-visible absorption
FeCl3 after 60 minutes showed a good yield spectra.10 From Table 2, the ARAR3 showed
at 75%. It should be noted that the minor the smaller band gap (2.75 eV) compared
products may be the conjugated dimer or with ARAR2 due to a long π-conjugated
trimer or other products from the similar 1H systems which the HOMO and LUMO levels
NMR with ARAR3. located at -5.04 and -2.21 eV, respectively.
Figure 2. Normalized absorption (solid line) Figure 3. TGA spectra of compounds
and emission (dash line) spectra of
compounds in CH2Cl2 solution
Table 2. Photophysical and electrochemical characteristics of compounds

λmax,absa λmax,ema λonseta Egb EHOMOc,d ELUMOe
Compound
(nm) (nm) (nm) (eV) (eV) (eV)
ARAR2 342 476 448 2.77 -5.07 -2.23
ARAR3 335 500 450 2.75 -5.04 -2.21
a
Measured in dichloromethane (1×10-6 M)
b
Estimated from the onset of the absorption spectra (Eg = 1240/onset)
c
Measured in acetonitrile containing 0.1 M n-Bu4NPF6 as a supporting electrolyte at a scan rate of 100 mV/s
d
Calculated from HOMO = -(Eox+4.8eV-Eox(ferocene)); Eox(ferocene) = 0.44 V
e
Calculated from LUMO = HOMO+Eg
4. Conclusion 4. Chai, W.-Y.; Yang, E.-Q.; Zhang,

We studied the intramolecular Y.-L.; Xie, A.-L.; Cao, X.-P.
Scholl reaction to obtain tribenzo[a,c,k,l] Synthesis 2012, 44, 439–445.
xanthene by using FeCl3 as the coupling 5. Tasior, M.; Kim, D.; Singha, S.;
reagent. The product showed the red-shifted Krzeszewski, M.; Ahn, K. H.;
emission as well as high thermal stability. Gryko, D. T. J. Mater. Chem. C 2015,
The results showed that the ARAR3 was 3, 1421–1446.
quite promising to be wildly useful organic 6. Thirunavukkarasu, V. S.; Donati, M.;
material in the future. Ackermann, L. Org. Lett. 2012, 14,
3416–3419.
Acknowledgements 7. Wöhrle, T.; Kirres, J.; Kaller, M.;
The authors would like to Mansueto, M.; Tussetschläger, S.;
acknowledge the financial support from Laschat, S. J. Org. Chem. 2014, 79,
Ubon Ratchathani University and Science 10143–10152.
Achievement Scholarship of Thailand 8. Zhao, K.-Q.; Du, J.-Q.; Long, X.-H.;
(SAST) for Thikhamporn Uppalabat. Jing, M.; Wang, B.-Q.; Hu, P.;
Monobe, H.; Henrich, B.; Donnio,
References B. Dyes and Pigments 2017, 143, 252–
1. Grzybowski, M.; Skonieczny, K.; 260.
Butenschön, H.; Gryko, D. T. 9. Chu, Z.; Wang, D.; Zhang, C.; Fan, X.;
Angew. Chem. Int. Ed. 2013, 52, 9900– Tang, Y.; Chen, L.; Zou, D. Macromol.
9930. Rapid Commun. 2009, 30, 1745–1750.
2. Narita, A.; Wang, X.-Y.; Feng, X.; 10.Keawin, T.; Prachumrak, N.;
Müllen, K. Chem. Soc. Rev. 2015, Namuangruk, S.; Pansay, S.;
44, 6616–6643. Kungwan, N.; Maensiri, S.;
3. Sarhan, A. A.; Bolm, C. Chem. Soc. Rev. Jungsuttiwong, S.; Sudyoadsuk, T.;
2009, 38, 2730-2744. Promarak, V. J. RSC Adv. 2015, 5,
73481–73489.
Photooxidation of organosulfur compounds catalyzed by iodo-BODIPY
derivatives under visible light irradiation
Piyamaporn Tangkasemsamran1, Mongkol Sukwattanasinitt2, Sumrit Wacharasindhu2*
1
Department of Petrochemical and Polymer Sciences, Faculty of Science,
Chulalongkorn University, Phayathai, Bangkok 10330, Thailand
2
Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai, Bangkok 10330, Thailand
*E-mail: sumrit.w@chula.ac.th
Abstract:
In this work, we designed and synthesized three iodo-BODIPY derivatives, I-GB,
3I-GB and I-RB as photocatalysts for oxidation reaction of thiol. Catalysts I-GB and I-RB
were synthesized in good yields from condensation reaction between 4-iodobenzaldehyde
with corresponding pyrroles, followed by DDQ oxidation and complexation with BF3OEt2.
For 3I-GB, beta-iodination of I-GB with iodine monochloride provided the product in 58%
yield. All of iodinated BODIPYs were fully characterized by 1H-NMR, 13C-NMR and high
resolution mass spectrometry. The photocatalytic activities of all iodo-BODIPY derivatives
were studied using Rose Bengal as a reference in an oxidation reaction of 4-chlorothiophenol.
According to 1H-NMR analysis, when irradiating 4-chlorothiophenol under white LED at
room temperature for 60 min, I-GB, 3I-GB and I-RB gave disulfide product in 85, 100 and
100% yields, respectively, while Rose Bengal yields only 54%. From these results, it could
be concluded that these BODIPYs should be able to serve as new photocatalysts for the
oxidation of thiol into disulfide under visible light irradiation.
1. Introduction These methods provide the powerful

Organosulfur such as disulfide is an oxidation reaction in organic synthesis due
important class of compound which is to its effectiveness and ease of handling.
widely used in many applications such as However, those catalysts were difficult to
pharmaceutical science,1 vulcanizing modify the structure and therefore the
2 3
process in rubber industry, role and control improvement of catalytic activity of those
of proteins folding4 in biological system. catalysts is limited. Among organic dye, 4,4-
Typically, the metal catalysts such as difluoro-4-bora-3a,4a-diaza-s-indacene
manganese,5 tellurium,6 gold,7 rhodium, (BODIPY) are class of compound that has
nickel, cobalt,8 aluminum,9 lead and been used as optoelectronic material due to
vanadium10 have been used to oxidize thiol strong good solubility,17 photostability and
into disulfide. Recently, non metal oxidizing short electronic triplet state lifetime
agents such as 2,3-dichloro-5,6- resulting in a good photocatalytic
dicyanobenzoquinone (DDQ),11 halogen activities.18 Moreover, BODIPYs structure
complex,12 enzyme for example laccase13
have been introduced for the oxidation of
thiol. However, those reagents have several
drawbacks such as requirement of
stoichiometric amount, harsh condition, high
toxicity, high cost and high temperature. In a
past decade, the photooxidation under
visible light has been developed using metal
complexes such as TiO2/MoS2, Ru(bpy)3Cl2,
Ir(ppy)3 and organic dyes such as Rose Scheme 1. Photooxidation of 4-
Bengal, Eosin Y, BODIPYs as catalysts14-16. chlorothiophenol (1) into disulfide (2)
could be modified and allowed the dichloromethane (40 mL), treated with DDQ
installation of various functional groups into (292.8 mg, 1.290 mmol) and stirred at room
BODIPY core structure which could lead to temperature under N2 atmosphere for 30
the tuning of photocatalytic activity. Herein, minutes. Then DIPEA (1.590 mL, 9.128
we prepared three BODIPY derivatives mmol) and BF3OEt2 (1.643 mL, 13.04
(Scheme 1) and used them as photocatalyst mmol) were added to the solution and
under visible light in oxidation of thiol (1) stirring was continued at 0 oC under N2
into disulfide (2). atmosphere for additional 16 hours. The
resulting solution was extracted with
2. Materials and Methods saturated NaHCO3 (40 mL), brine (40 mL)
2.1 General and dried over anhydrous sodium sulfate.
All chemicals were purchased from After removal of the solvent, the crude
Sigma-Aldrich, Merck® (Germany) or product was purified by a silica column
Fluka® (Switzerland) and were applied chromatography using 20% ethyl acetate in
without cumulative purification. Analytical hexane as an eluent to give I-RB as a deep
thin-layer chromatography (TLC) was red solid (253.6 mg, 36%). Rf 0.35 (10%
performed on Kieselgel F-254 pre-coated EtOAc/hexane)
plastic TLC plates from EM Science. 4,4-Difluoro-8-(4'-iodophenyl)-
Column chromatography used silica gel (60 1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-
Å, 230-400 mesh) from ICN Silitech. indancene (I-GB). To synthesize I-GB,
Visualization was accomplished with a 254 4-iodobenzaldehyde (300.0 mg, 1.292
nm ultraviolet lamp. The 1H and 13C NMR mmol) and 2,4-dimethylpyrrole (264 μL,
spectra were recorded on a Varian Mercury 2.608 mmol) were dissolved in
NMR spectrometer, working at 400 MHz for dichloromethane (40 mL). Then
1
H and 100 MHz for 13C nuclei (Varian trifluoroacetic acid (0.1 mL, 1.3 mmol) was
Company, CA, USA). White LED reactors added in solution and the mixture was
were operated from a 250 mL beaker lined stirred at 0 oC under N2 atmosphere for 3
with a commercial belt LED (1.5 W 228). hours. The resulting solution was washed
High resolution mass spectra were obtained with DI water (40 mL) and dried over
from electrospray ionization mass anhydrous sodium sulfate. After evaporated
spectrometer (ESI-MS). the solvent, the crude product was purified
2.2 Synthesis of BODIPYs by a silica column chromatography using
4,4-Difluoro-8-(4'-iodophenyl)-3,5- 10% ethyl acetate in hexane as an eluent to
diphenyl-4-bora-3a,4a-diaza-s-indancene give the product as an orange solid. Next,
(I-RB). To synthesize I-RB, the product was subsequently dissolved in
4-iodobenzaldehyde (300.0 mg, 1.293 dichloromethane (40 mL), treated with DDQ
mmol) and 2-phenylpyrrole19 (370.0 mg, (94.4 mg, 0.416 mmol) and stirred at room
2.584 mmol) were dissolved in temperature under N2 atmosphere for 30
dichloromethane (40 mL). Then minutes. Then DIPEA (1.908 mL, 10.96
trifluoroacetic acid (0.1 mL, 1.3 mmol) was mmol) and BF3OEt2 (1.973 mL, 15.657
added in solution and the mixture was mmol) were added to the solution and
stirred at 0 oC under N2 atmosphere for 3 stirring was continued at 0 oC under N2
hours. The resulting solution was washed atmosphere for additional 16 hours. The
with DI water (40 mL) and dried over resulting solution was extracted with
anhydrous sodium sulfate. After evaporated saturated NaHCO3 (40 mL), brine
the solvent, the crude product was purified (40 mL) and dried over anhydrous sodium
by a silica column chromatography using sulfate. After removal of the solvent, the
20% ethyl acetate in hexane as an eluent to crude product was purified by a silica
give the product as a red solid. Next, the column chromatography using
product was subsequently dissolved in dichloromethane and hexane (1:2) as an
eluent to give I-GB as an orange solid [(M+Na)+, M = C19H16BF2I3N2].
(231.0 mg, 39%). Rf 0.50 (40%
CH2Cl2/hexane) 3. Results and Discussion
4,4-Difluoro-2,6-diiodo-8-(4'- 3.1 Synthesis and characterization
iodophenyl)-1,3,5,7-tetramethyl-4-bora- Three BODIPY derivatives were
3a,4a-diaza-s-indancene (3I-GB). In a 100 successfully synthesized as shown in
mL round-bottomed flask, I-GB (60.0 mg, Scheme 2. For I-GB, 2,4-dimethylpyrrole
0.133 mmol) was dissolved in chloroform (3) was condensed with iodobenzaldehyde
(6 mL) and methanol (3 mL). Then iodine followed by oxidization with DDQ to
monochloride (20.0 μL, 0.266 mmol) was provide the corresponding dipyrrolemethane
added in solution. After that the solution was derivative. Then, complexation of
stirred at 70 oC under N2 atmosphere for 16 dipyrrolemethane with BF3OEt2 gave target
hours. The resulting solution was washed I-GB in 20% yield for 3 steps. Moreover,
with saturated sodium thiosulfate solution 3I-GB was synthesized by beta iodination
(20 mL), brine (20 mL) and dried over with iodine monochloride to give 3I-GB in
anhydrous sodium sulfate. After evaporated 58% yield. Similarly, the synthesis of I-RB
the solvent, the crude product was purified was started from pyrrole (4). The imination
by a silica column chromatography using of acetophenone with hydroxylamine
dichloromethane: hexane (1:2) as an eluent hydrochloride gave the corresponding oxime
to give 3I-GB as a red solid (54.3 mg, 58%). in 82% yield followed by [3+2]
Rf 0.50 (30% CH2Cl2/hexane) cycloaddition with calcium carbide in the
2.3 Spectroscopic measurement presence of potassium hydroxide yielded 2-
I-RB phenylpyrrole (4) in 67%. Finally,
1
H-NMR (400 MHz, Chloroform-d) I-RB was successfully synthesized under the
δ 6.64 (d, J = 4.4 Hz, 2H), 6.87 (d, J = 3.6 similar conditions as I-GB in 39% yield
Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.37 – from pyrrole (4).
7.49 (m, 6H), 7.81 – 7.95 (m, 6H); 13C- (a)
NMR (CDCl3, 100 MHz) δ 96.4, 121.0,
128.1, 129.3, 129.4, 130.4, 131.9, 132.3,
133.7, 135.9, 137.4, 142.4, 159.2; HR-ESI-
MS m/z obsd 569.0466 [(M+Na)+], calcd
569.0468 [(M+Na)+, M = C27H18BF2IN2].
I-GB
1
H-NMR (400 MHz, Chloroform-d)
δ 1.41 (s, 6H), 2.55 (s, 6H), 5.99 (s, 2H),
7.04 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, (b)
2H); 13C-NMR (CDCl3, 100 MHz) δ 15.10,
95.17, 121.92, 130.44, 131.61, 135.04,
138.81, 140.54, 143.37, 156.36; HR-ESI-
MS m/z obsd 451.0654 [(M+H)+], calcd
451.0649 [(M+H)+, M = C19H18BF2IN2].
3I-GB
1
H-NMR (400 MHz, Chloroform-d)
δ 1.43 (s, 6H), 2.64 (s, 6H), 7.01 (d, J = 8.4
Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H); 13C-NMR
(CDCl3, 100 MHz) δ 16.06, 17.27, 95.34,
129.74, 131.01, 134.02, 134.29, 138.65, Scheme 2. Synthesis of photocatalysts
139.70, 145.11, 157.24; HR-ESI-MS m/z (a) I-GB and 3I-GB (b) I-RB
obsd 724.8409 [(M+Na)+], calcd 724.8401
All synthesized BODIPYs were fully 3.3 Reaction condition screening
characterized by 1H-NMR, 13C-NMR and The catalytic activities of
mass spectrometry which were consistent synthesized BODIPYs were investigated by
with the previous literature20. oxidative coupling reaction of
3.2 Photophysical properties of BODIPY 4-chlorothiophenol (1) in isopropanol under
derivatives Light Emitting Diodes (LEDs, 1.5W).
To evaluate the potential use of these Firstly, the oxidation of 4-chlorothiophenol
derivatives as photocatalysts, the (1) was performed in the presence of 5
photophysical properties of BODIPYs were mol% of catalysts and compared with
investigated. In table 1, the absorption conventional photocatalyst, Rose Bengal as
maxima of 3I-GB appeared at 557 nm seen in Table 2. All iodo-BODIPY
which was higher than I-GB by 56 nm. This derivatives gave %NMR yield at around 85-
behavior can be explained by the heavy 100 which higher than Rose Bengal (49%).
atom from the additional iodine atoms. In Amongst all iodo-BODIPY derivatives, we
comparison between I-GB and I-RB, the noticed that the increasing of iodine atoms at
red-shifting of absorption maxima was also 2 and 6 positions of I-GB to 3I-GB gave
observed around 34 nm due to the increase more %NMR yield. Moreover, the
of conjugation effect of the extended phenyl additional phenyl groups at 3 and 5 positions
ring at 3 and 5 positions of BODIPY core. of iodo-BODIPY to I-RB could enhance the
These results were also observed in the %NMR yield. With these interesting results
emission spectrum of BODIPYs in the same in hands, we decided to investigate the
manner. The molar absorptivity and relationship of catalytic activity with the
quantum yield of BODIPY dyes were effect of iodine and conjugation.
studied. For extinction coefficient, the
orders from high to low are I-GB, 3I-GB Table 2. The oxidation reaction of
and I-RB, respectively. The decrease of 4-chlorothiophenol with 5 mol% of
molar absorptivity and fluorescence photocatalysta
quantum yield could be explained by the
higher conjugation of the additional phenyl
group when compared between GB and RB
series. This is probably due to the more
Entry Photocatalyst % NMR yield b
complexity of vibrational energy levels of
1 Rose Bengal 49
RB series. Additionally, when the number of
2 I-GB 85
iodine atoms increase, the molar absorptivity
3 3I-GB 100
was lower due to the heavy atom effect20b.
4 I-RB 100
a
Reaction were carried at 0.35 mmol of
Table 1. Photophysical properties of 4-chlorothiophenol with 5 mol% of photocatalyst in
BODIPYsa isopropanol 3.5 mL. bCrude of reaction was
BODIPYs I-GB 3I-GB I-RB
evaporated and evaluated by 1H-NMR.
Ambient
Color of light
solutions Fluorescent
To do so, we therefore reduced the
black light
amount of photocatalyst from 5 to 1 mol%
λmax (nm)
abs
501 557 535
in order to see the catalyst activity of those
λmaxem (nm) 515 592 553
dyes more quantitatively. 3I-GB
 (M-1cm-1) 91127 84461 69300 demonstrated the highest activity among all
synthesized BODIPYs and Rose Bengal
 0.577 0.061 0.104
a (Table 3, entry 3). We thus concluded that
In isopropanol
the addition of iodine atoms provides
stronger effect on the catalytic activity than
the addition of phenyl groups.
Table 3. The oxidation reaction of and Technology, Thailand, through its
4-chlorothiophenol with 1 mol% of program of Center of Excellence Network.
photocatalysta This work is part of the Project for
Establishment of Comprehensive Center for
Innovative Food, Health Products and Agri-
culture supported by the Thai Government
Stimulus Package 2 (TKK2555, SP2), the
Entry Photocatalyst % NMR yield b
Higher Education Research Promotion and
30 min 60 min
National Research University Project of
1 Rose Bengal 54 55
Thailand, Office of the Higher Education
2 I-GB 20 98
Commission (AM1006A-56).
3 3I-GB 96 100
4 I-RB 29 83
a References
Reaction were carried at 0.35 mmol of
4-chlorothiophenol with 1 mol% of photocatalyst in 1. Zottola, M. A.; Beigel, K.; Soni, S.-D.;
isopropanol 3.5 mL. bCrude of reaction was Lawrence, R. Chem. Res. Toxicol. 2009,
evaporated and evaluated by 1H-NMR. 22, 1948–1953.
2. Hosseini, S. M.; Razzaghi-Kashani, M.
4. Conclusion Polymer 2017, 133, 8–19.
In summary, three iodo-BODIPY 3. Popielarski, M.; Ponamarczuk, H.;
derivatives, I-GB, 3I-GB and I-RB were Stasiak, M.; Michalec, L.; Bednarek, R.;
successfully synthesized and their Studzian, M.; Pulaski, L.; Swiatkowska,
photophysical properties were investigated. M. Biochem. Biophys. Res. Commun.
In comparison with Rose Bengal, our 2018, 495, 1635–1641.
synthesized iodo-BODIPYs demonstrated 4. Fan, F.; Zhang, Y.; Wang, S.; Han, Y.;
better photocatalytic activities. It might be Wang, L.; Lu, D. Biochem. Biophys.
caused by Rose Bengal have higher Res. Commun. 2018, 495, 1041–1047.
quantum yield compared to BODIPYs 5. Dharmarathna, S.; King’ondu, C. K.;
which resulting in the decrease of Pahalagedara, L.; Kuo, C.-H.; Zhang,
intersystem crossing probability. This lead Y.; Suib, S. L. Appl. Catal., B 2014,
to a lower singlet oxygen generation under 147, 124–131.
irradiation which is the key oxidizing agent 6. Oba, M.; Tanaka, K.; Nishiyama, K.;
in this work.15 Moreover, the heavy atom Ando, W. J. Org. Chem. 2011, 76,
effect from the additional iodine atoms of 4173–4177.
iodo-BODIPYs provides a higher 7. Corma, A.; Rodenas, T.; Sabater, M. J.
photocatalytic activity than the conjugation Chem. Sci. 2012, 3, 398–404.
effect from the additional phenyl group. 8. Mandal, B.; Basu, B. RSC Adv. 2014, 4,
This study provides basic knowledge design 13854–13881.
the photocatalyst which can be used for the 9. Kim, B.-S.; Kim, B.-T.; Hwang, K.-J., A
oxidation of various organosulfur Practical Method to Cleave Diphenyl
compounds. The further studies on structural Phosphonate Esters to Their
relationship of other BODIPY derivatives as Corresponding Phosphonic Acids in
well as the expansion of the scope in to One Step. 2009; Vol. 30, p 1391–1393.
other organosulfurs will be reported in the 10. Kirihara, M.; Okubo, K.; Uchiyama, T.;
due course. Kato, Y.; Ochiai, Y.; Matsushita, S.;
Hatano, A.; Kanamori, K. Chem.
Acknowledgements Pharm. Bull. 2004, 52, 625–627.
This research is financially supported 11. Vandavasi, J. K.; Hu, W.-P.; Chen, C.-
by the Thailand Research Fund (TRF- Y.; Wang, J.-J. Tetrahedron 2011, 67,
RSA5480004) and Nanotechnology Center 8895–8901.
(NANOTEC), NSTDA, Ministry of Science
12. Zolfigol, M. A.; Chehardoli, G.; J. M.; Ruedas-Rama, M. J.; Orte, A.;
Salehzadeh, S.; Adams, H.; Ward, M. Crovetto, L.; Talavera, E. M.; Alvarez-
D. Tetrahedron Lett. 2007, 48, 7969– Pez, J. M. J. Phy. Chem. A 2012, 116,
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T.; Sukwattanasinitt, M.; 20. (a) Tahtaoui, C.; Thomas, C.; Rohmer,
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72, 788–793. Bonnet, D.; Hibert, M. J. Org. Chem.
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The study of ene-yne-oxazolone intramolecular cyclization using
hydrogen-phosphine oxide
Sirikan Deesiri1, Somsak Ruchirawat1,2, Nopporn Thasana1,2
1
Chemical Biology Program,Chulabhorn Graduate Institute, Chulabhorn Royal Academy,
Laksi, Bangkok 10210, Thailand
2
Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Laksi, Bangkok 10210, Thailand
*E-mail: nopporn@cri or.th
Abstract:
The ene-yne-oxazolone intramolecular cyclization using hydrogen-phosphine oxide
performed under metal-catalyzed conditions yielded phosphorus-containing spirooxazolones.
The conditions resulted in the high-yielding formation of highly regioselective, 5-exo-dig, C-
P and C-C bonds that make up the core structure of spirooxazolindane products. Various ene-
yne-oxazolones were also evaluated for anti-cancer, anti-oxidant, and anti-microbial
activities. Although these spirooxazolindane precursors showed no anti-oxidation or anti-
microbial activities, the spirooxazolindanes themselves may exhibit some biological
activities, which will be assessed later on.
1. Introduction spirooxazolone from ene-yne-oxazolone

Indane has been found as a core using hydrogen-phosphine oxide under
structure in many bioactive natural products transition metal catalysis to optimize the
for some time. They display important yield of phosphine oxide containing
biological activities such as radical- spirooxazolone compound.
scavenging activity which is 20- to 30-times
higher than vitamin E, antibiotic kanamycin 2. Materials and Methods
biosynthesis. Some indanes are also 2.1 General
antagonists of estrogen receptor or inhibitors In our experiments, the 1D NMR
of the enzyme COX-1. Considering the spectra were measured in CDCl3 on a
above biological activities, scientists have Bruker AVANCE-300 and 400 spectro-
been interested in preparing indanes by meters with TMS as the internal standard.
various methods.1 The spiro indane core High resolution mass spectra were obtained
structure has been implicated in its on a Bruker Daltonics MicroTOF instrument
exceptional bioactivity associated with using atmospheric pressure chemical ion
treating pain.2 Moreover, many ionization (APCI) in the positive ion mode.
spirooxazolindanes are effective in treating a Reactions were monitored with Merck silica
variety of diseases such as anxiety, gel (70–230 mesh) thin layer chromato-
depressive disorders, and inappropriate graphy (TLC) and visualized using UV
levels of vasopressin.3 Molecules bearing a light. Silica gel column chromatography was
phosphorus group are also important in carried out on SiliCycle silica gel (70–230
medicinal and pharmaceutical chemistry mesh).
since they have been often used as drugs and 2.2 General synthesis of ene-yne-
as prodrugs to increase the water-solubility oxazolone compounds (3a-3d)
of parental molecules.4 In this work, the ene- A mixture of benzaldehydes 1a-b
yne-oxazolone precursor was prepared in (1.0 equiv.), arylacetylenes (1.2 equiv.),
two steps via the Sonogashira cross- bis(triphenylphosphine)palladium(II) dichlo-
coupling reaction and the Erlenmeyer ride (2 mol%), copper(I) iodide (1 mol%) in
azlactone synthesis. Furthermore, we studied triethylamine (10 mL) was stirred at 50 °C
the synthesis of phosphorus-containing under argon atmosphere. After the complete
consumption of the starting material benz- 2-((4-Fluorophenyl)ethynyl)benz-
aldehyde, the reaction was quenched by aldehyde (2c)7: orange solid, 20% yield; 1H
sat.NH4Cl and extracted with dichloro- NMR (300 MHz, CDCl3) δ (ppm): 10.7 (s,
methane. The combined organic layers were 1H), 8.27 (d, J = 8.8 Hz, 2H), 7.99 (d, J =
washed with brine and dried with 8.0 Hz, 1H), 7.74-7.62 (m, 4H), 7.54 (t, J =
anh.Na2SO4. The dried crude product was 7.2 Hz, 1H); HRMS (microTOF) m/z calcd
purified by silica gel column chroma- for C15H10NO3 [M+H]+: 252.0653, found:
tography (5% ethyl acetate/hexanes) to 252.0653.
obtain ethynylbenzaldehydes 2a-d. After 5-Methoxy-2-(phenylethynyl)benz-
that, the ethynylbenzaldehydes 2a-d (1.0 aldehyde (2d)5: yellow solid, 59 % yield ,
equiv.) were mixed with hippuric acid (1.5 1H NMR (400 MHz, CDCl ) δ (ppm): 10.6
3
equiv.), acetic anhydride (5.0 equiv.) and (s, 1H), 7.58-7.53 (m, 3H), 7.44 (d, J = 2.8
anhydrous sodium acetate (1.1 equiv.) at 100 Hz, 1H), 7.38-7.36 (m, 3H), 7.14 (dd, J =
C to yield the crystalline product. After 2.8, 8.8 Hz, 1H), 3.89 (s, 3H); HRMS
completion, the reaction was quenched by (microTOF) m/z calcd for C16H13O2 [M+H]+:
water and filtered with suction, washed 236.0837, found: 236.0838.
excessively with cold ethanol and water to 2-Phenyl-4-(2-phenylethynyl)benzy-
obtain the ene-yne-oxazolone compounds lidene)-oxazol-5-(4H)-one (3a): yellow
3a-3d. solid, 88 % yield; 1H NMR (300 MHz,
2.3 General synthesis of spirooxazol- CDCl3) δ (ppm): 8.96 (d, J = 7.8 Hz, 1H),
indanes 5 8.18 (d, J = 9.9 Hz, 2H), 7.97 (s, 1H), 7.77-
The ene-yne-oxazolone precursors 7.30 (m, 11H); 13C NMR (75 MHz, CDCl3)
3a-d (0.2 mmol) were stirred with diphenyl- δ (ppm): 167.4, 164.0, 134.5, 133.9 133.4
phosphine oxide 4 (0.2 mmol) in the 132.5 131.9 131.7 (2C), 130.6, 128.9 (3C),
presence of catalyst (5 mol%) and 4Å MS 128.8, 128.6,128.5 (2C), 128.4 (2C), 126.3,
(200 mg) in an appropriate solvent (2 mL) in 125.5 122.6, 96.5, 86.8; HRMS (microTOF)
a sealed tube. After the reaction was m/z calcd for C24H15NNaO2 [M+Na]+:
completed, the reaction was filtered through 372.0994, found: 372.0984.
Celite. After the removal of solvent, the 4-(2-((4-Fluorophenyl)ethynyl)ben-
crude was purified by silica gel column zylidene)-2-phenyloxazol-5-(4H)-one (3b):
chromatography (60% ethyl acetate/ yellow solid, 76% yield; 1H NMR (300
hexanes). MHz, CDCl3) δ (ppm): 8.96 (d, J = 7.8 Hz,
2.4 Spectroscopic characterization
1H), 8.19 (d, J = 7.2 Hz, 2H), 7.94 (s, 1H),
2-(Phenylethynyl)benzaldehyde
5 7.66-7.36 (m, 8H), 7.09 (t, J = 8.7 Hz, 2H);
(2a) : brown oil, 86% yield; 1H NMR (300 13C NMR (75 MHz, CDCl ) δ (ppm): 167.4,
MHz, CDCl3) δ (ppm): 10.7 (s, 1H), 7.99 3
164.1, 162.9 (d, J = 249 Hz), 134.5, 133.9,
(dd, J = 0.9, 7.8 Hz, 1H), 7.66 (td, J = 1.2,
133.7 (2C) (d, J = 8.3 Hz), 133.5, 132.3
7.8 Hz, 1H), 7.57-7.61 (m, 3H), 7.47 (t, J =
131.9, 130.6, 129.0 (2C), 128.8, 128.7,
7.2 Hz, 1H), 7.41 (t, J = 3.0 Hz, 3H);
128.5 (2C), 126.1, 125.5, 118.8 (d, J = 3.4
HRMS (micro-TOF) m/z calcd for
Hz), 115.8 (2CH) (d, J = 22.0 Hz), 95.4,
C15H10NaO [M+Na]+: 229.0623, found:
86.5; HRMS (microTOF) m/z calcd for
229.0622.
C24H15FNO2 [M+H]+: 368.1081, found:
2-((4-Fluorophenyl)ethynyl)benz-
368.1077.
aldehyde (2b)6: pearl yellow solid, 99%
4-(2-((4-Nitrophenyl)ethynyl)benzy-
yield; 1H NMR (400 MHz, CDCl3) δ (ppm):
lidene)-2-phenyloxazol-5-(4H)-one (3c):
10.6 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.60- 1
yellow solid, 56% yield; H NMR (300
7.50 (m, 4H), 7.41 (t, J = 8.0 Hz, 1H), 7.04 MHz, CDCl3) δ (ppm): 9.00 (d, J = 7.6 Hz,
(t, J = 8.8 Hz, 2H); HRMS (microTOF) m/z
1H), 8.27 (d, J = 8.9 Hz, 2H), 8.21 (d, J =
calcd for C15H10FO [M+H]+: 225.0710,
8.4 Hz, 2H), 7.90 (s, 1H), 7.76 (d, J = 8.9,
found: 225.0714.
2H), 7.86-7.60 (m, 2H), 7.60-7.51 (m, 3H), MHz, CDCl3) δ (ppm): 7.83-7.79 (m, 2H),
7.46 (td, J = 7.6, 1.2 Hz, 1H); 13C NMR (75 7.63-7.58 (m, 2H), 7.53 (d, J = 7.5 Hz, 1H),
MHz, CDCl3) δ (ppm): 167.3, 164.4, 147.3, 7.48-7.45 (m, 1H), 7.38-7.21 (m, 11H),
134.9, 134.4, 133.7, 132.8, 132.4 (2C), 7.20-7.14 (m, 3H), 7.04 (d, J = 8.4 Hz, 1H),
132.0, 130.6, 129.6, 129.5, 129.0 (2C) 128.5 6.94 (dd, J = 5.6, 8.4 Hz, 2H), 6.61 (t, J =
(2C), 127.9, 125.4, 124.8, 123.7 (2C), 94.2, 8.8 Hz, 1H), 4.89 (d, J = 14.8 Hz, 1H); 13C
91.8; HRMS (microTOF) m/z calcd for NMR (100 MHz, CDCl3) δ (ppm):178.2 (d,
C24H15N2O4 [M+H]+: 395.1026, found: J = 3.2 Hz), 162.0, 161.9 (d, J = 245.8 Hz),
395.1029. 141.2 (d, J = 2.1 Hz), 139.7 (d, J = 6.5 Hz),
4-(5-Methoxy-2-(phenylethynyl) 137.9 (d, J = 3.1 Hz), 133.0 (d, J = 100.3
benzylidene)-2-phenyloxazol-5-(4H)-one Hz), 132.8, 132.0 (d, J = 9.4 Hz, 2C), 131.7
(3d): orange solid, 32% yield; 1H NMR (d, J = 2.4 Hz), 131.6 (d, J = 2.4 Hz), 131.1
(300 MHz, CDCl3) δ (ppm): 8.58 (d, J = 2.4 (d, J = 9.1 Hz, 2C), 130.8 (d, J = 100.4 Hz),
Hz, 1H), 8.14 (d, J = 7.5 Hz, 2H), 7.93 (s, 130.5 (d, J = 3.5 Hz), 130.2 (d, J = 8.1 Hz,
1H), 7.66-7.47 (m, 6H), 7.43-7.32 (m, 3H), 2C), 129.5, 128.7 (d, J = 0.8 Hz), 128.6 (d, J
6.98 (dd, J = 2.6, 8.7 Hz, 1H), 3.96 (s, 3H); = 11.8 Hz, 2C), 128.3 (d, J = 12.0 Hz, 2C),
13C NMR (75 MHz, CDCl ) δ (ppm): 167.2,
3 128.1 (2C), 128 (3C), 126.1 (d, J = 3.4 Hz),
163.9, 159.4 135.8, 133.9, 133.6, 133.4, 125.0, 124.7, 121.1, 114.9 (d, J = 21.5 Hz),
131.5 (2C), 128.9 (2C), 128.8, 128.42, 76.9 (d, J = 2.8 Hz), 54.9 (d, J = 69.8 Hz);
128.40 (2C), 128.3 (2C), 125.5, 123.0, HRMS (microTOF) m/z calcd for
119.0, 118.0, 115.6, 95.1, 86.8, 55.4; C36H26FNO3P [M+H]+: 570.1628, found:
HRMS (microTOF) m/z calcd for 570.1619.
C25H18NO3 [M+H]+: 380.1281, found: 1-(Diphenylphosphoryl)-3-(4-
380.1284. nitrobenzylidene)-2’-phenyl-1,3-dihydro-
1-Benzylidene-3-(diphenylphos- 5’H-spiro[indane-2,4’-oxazol]-5’-one (5c):
phoryl)-2’-phenyl-1,3-dihydro-5’H-spiro yellow solid, 50% yield; 1H NMR (400
[indane-2,4’-oxazol]-5’-one (5a): yellow MHz, CDCl3) δ (ppm): 7.80 (dd, J = 6.8,
solid, 86 % yield; 1H NMR (300 MHz, 11.6 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.61-
CDCl3) δ (ppm): 7.86-7.79 (m, 2H), 7.62- 7.53 (m, 3H), 7.46-7.41(m, 1H), 7.38-7.10
7.52 (m, 3H), 7.47-7.42 (m, 1H), 6.99-6.91 (m, 15H), 6.95 (d, J = 7.6 Hz, 1H), 4.84 (d,
(m, 5H), 4.89-4.84 (d, J = 15 Hz, 1H); 13C J = 13.6 Hz, 1H); 13C NMR (100 MHz,
NMR (75 MHz, CDCl3) δ (ppm): 178.8 (d, J CDCl3) δ (ppm): 177.6 (d, J = 5.5 Hz),
= 3.6 Hz), 161.9, 140.4 (d, J = 2.8 Hz), 161.9, 146.5, 142.0 (d, J = 19.4 Hz), 139.4
139.8 (d, J = 6.6 Hz), 137.8 (d, J = 3.6 Hz), (d, J = 6.2 Hz), 138.0 (d, J = 4.2 Hz), 133.3,
134.6, 133.0 (d, J = 99.9 Hz), 132.5, 132.0 132.1 (d, J = 99.3 Hz), 131.9 (d, J = 2.4 Hz),
(d, J = 9.4 Hz, 2C), 131.7 (d, J = 2.7 Hz), 131.85, 131.84 (d, J = 1.6 Hz), 131.80 (d, J
131.4 (d, J = 2.6 Hz), 130.9 (d, J = 9.2 Hz, = 9.3 Hz, 2C), 131.2 (d, J = 9.3 Hz, 2C),
2C), 130.6 (d, J = 100.2 Hz), 129.3 (d, J = 130.4 (d, J = 100.6 Hz), 130.0 (d, J = 1.6
1.4 Hz), 128.5 (2C), 128.3 (d, J = 11.9 Hz, Hz), 129.3 (2C), 128.9 (d, J = 1.8 Hz), 128.6
2C), 128.2 (2C), 128.15 (d, J = 11.8 Hz, (d, J = 12.0 Hz, 2C), 128.4 (d, J = 12.0 Hz,
2C), 128.0 (2C), 127.9 (3C), 127.2, 126.0 (d, 2C), 128.2 (2C), 127.7 (2C), 126.4 (d, J =
J = 2.6 Hz), 125.8, 125.1, 120.9, 76.8 (d, J = 2.7 Hz), 124.3, 123.0 (2C), 122.7, 121.3,
2.6 Hz), 55.0 (d, J = 96.5 Hz); HRMS 76.7 (d, J = 2.6 Hz), 54.4 (d, J = 67.9 Hz);
(microTOF) m/z calcd for C36H26NNaO3P HRMS (microTOF) m/z calcd for
[M+Na]+: 574.1445, found: 574.1471. C36H26N2O5P [M+H]+: 597.1573, found:
1-(Diphenylphosphoryl)-3-(4-fluoro- 597.1566.
benzylidene)-2’-phenyl-1,3-dihydro-5’H- 1-Benzylidene-3-(diphenylphospho-
spiro[indane-2,4’-oxazol]-5’-one (5b): ryl)-5-methoxy-2’-phenyl-1,3- dihydro-5’H-
1
yellow solid, 53% yield; H NMR (400 spiro[indane-2,4’-oxazol]-5’-one (5d):
1
yellow solid, 82% yield; H NMR (400
MHz, CDCl3) δ (ppm): 7.86 (dd, J = 8.0, 131.9, 131.2 (d, J = 106.1 Hz), 131.6, 131.1
11.2 Hz, 2H), 7.59 (q, J = 8.0 Hz, 2H), 7.45- (d, J = 9.3 Hz, 2C), 128.5 (d, J = 12.0 Hz,
7.21 (m, 14H), 7.05 (s, 1H), 6.96-6.84 (m, 2C), 128.3 (d, J = 11.9 Hz, 2C), 128.4 (2C),
6H), 6.53 (s, 1H), 4.85 (d, J = 15.2 Hz, 1H), 128.0 (2C), 127.9 (2C), 127.87 (2C), 127.0,
3.58 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 125.1, 123.4, 122.0, 116.7, 109.7, 77.1
(ppm): 178.3 (d, J = 4.4 Hz), 161.9, 160.8, (overlapped), 55.4, 54.9 (d, J = 69.4 Hz);
139.6 (d, J = 2.7 Hz), 139.2 (d, J = 4.0 Hz), HRMS (microTOF) m/z calcd for
134.9, 132.8 (d, J = 6.4 Hz), 132.6, 131.9 (d, C37H29NO4P [M+H]+: 582.1828, found:
J = 100.6 Hz),132.1 (d, J = 9.5 Hz, 2C), 582.1818.
Scheme 1. Synthesis of ene-yne-oxazolones
3. Results and Discussion evaluated by examining the effect of solvent

The synthesis of ene-yne-oxazolone as shown in Table 1. In the experiment, 2-
precursors started with the Sonogashira phenyl-4-(2-(phenylethynyl)benzylidene)
reaction of the corresponding bromobenz- oxazol-5(4H)-one 3a, a model starting
aldehydes to furnish the corresponding material, was reacted with diphynylphos-
ethynylbenzaldehyde compound followed phine oxide 4 in the presence of AgOAc and
by Erlenmeyer-azlactone synthesis (Scheme 4Å MS in DCE at 60 C under argon
1). We initiated our study with the reaction atmosphere; the 1-benzylidene-3-(diphe-
of 2-bromobenzaldehyde 1a and phenylace- nylphosphoryl)-2’-phenyl-1,3-dihy-dro-5’H-
tylene in the presence of bis(triphenylphos- spiro[indene-2,4’-oxazol]-5’-one 5a was
phine)palladium(II) dichloride and copper(I) yielded in 59% (entry 1). When other
iodide in triethylamine at 50 °C under argon solvents were used, the reaction gave the
atmosphere for 5 h which yielded 2-(phenyl- product 5a in the yields of less than 36 %.
ethynyl)benzaldehyde 2a. Other ethynyl- Using 1,4-dioxane as a solvent, the reaction
benzaldehydes 2b-2d were synthesized proceeded to give the product in moderate
using the same method but different yield at 36% (entry 2). Using another
acetylenes and reaction time. Then 2- halogenated solvent DCM gave 32 % yield
(phenylethynyl)benzaldehyde 2a was treated (entry 3). Polar aprotic solvents like DMF,
with hippuric acid, acetic anhydride and MeCN and DMSO gave low to moderate
anhydrous sodium acetate at 100 C for 2.5 yields (entries 4-6 ).
h. to yield 2-phenyl-4-(2-(phenylethy-
nyl)benzylidene)oxazol-5(4H)-one 3a. Other
ene-yne-oxazolones 3b-3d were synthesized
in the same way as previously described
using different ethynylbenz-aldehydes and
reaction time.
The synthesis of phosphorus-
containing spirooxazolone compounds was
Table 1. Effect of solventa electron-donating substituent on the
benzocyclic gave the product 5d in an
excellent yield (82%).
Table 2. Optimization of reactiona

Entries Solvent Time (h) Yield (%)
1 DCE 24 59
2b 1,4-Dioxane 24 36
3 DCM 24 32
4 DMF 18.5 11
5 MeCN 17 16 Entries Catalyst Time (h) Yield (%)
6 DMSO 24 36 1 Pd(OAc)2 24 N/A
a
Reaction condition: 3a (0.2 mmol), 4 (0.4 mmol), 2 Mn(OAc)3•2H2O 24 40
AgOAc (0.3 mmol), 4Å MS (200 mg), solvent (2 3 CuI 20 46
mL), 60 C, Ar 4b AuCl 24 40
b
under reflux 5b AuCl3 24 51
6 AgOAc 24 59
7b AgTFA 22 81
According to the results shown in 8b,c AgTFA 24 16
Table 1, DCE was further used as solvent. 9b,d AgTFA 22 86
Then, we further optimized the reaction a
Reaction condition: 3a (0.2 mmol), 4 (0.4 mmol),
conditions by varying catalyst as shown in catalyst (0.3 mmol), 4Å MS (200 mg), solvent (2
Table 2. Using 3a as the substrate and 4 as mL), 60 C, Ar
b
Catalyst (5 mol%)
the reagent, in the presence of stoichiometric c
At room temperature
amount of Pd(OAc)2, the reaction gave no d
under reflux, 4 (0.2 mmol)
desired product (entry 1). Thus, N/A = no reaction
Mn(OAc)3•2H2O was then used instead and
the product 5a was obtained in 40% yield
(entry 2). When other transition metals were
employed either stoichiometrically (CuI,
entry 3 and AgOAc, entry 6) or catalytically
(AuCl, AuCl3), the product was obtained in
40-81%., which are CuCl, AuCl, AuCl3. At
room temperature, the yield of the reaction
decreased dramatically (entry 8). When we
increased the temperature to reflux and
reduced diphenyl-phosphine oxide from 2 to
1 equiv., the yield increased to 86% (entry Figure 1. Scope of substrates
9); therefore, this reaction required heating
to proceed. 4. Conclusion
With the optimal conditions in hand, In summary, we have demonstrated
we next examined the scope of substrates the synthesis of phosphorus-containing
(Figure 1). An investigation of different ene- spirooxazolone from the cyclization of ene-
yne-oxazolone precursors showed that with yne-oxazolone using hydrogen-phosphine
or without substituent on biarylacetylene oxide and some transition metals as catalyst.
system the reaction gave the corresponding The reactions proceeded regioselectively in
products in moderate to excellent yields. the 5-exo-dig mode to give the
With an electron-withdrawing substituent on corresponding products in moderate to
para-position of the arylacetylene, the effect excellent yields.
of poor electron density on the triple bond
decreased the yields as observed for
products 5b-5c (50-53%). In contrast, the
This work was supported by His 1. Ahmed, N. Stud. Nat. Prod. Chem. 2016,
Majesty the King Honour Celebration 51, 383–434.
Scholarships Project for The Scientists 2. Goehring, R. R.; Kyle, D.; Victory, S.
Development Fund, Chemical Biology WO02/085354 Al, 2002.
Program, Chulabhorn Graduate Institute, 3. Runtz-Schmitt, V.; Schnider, P.;
Chulabhorn Royal Academy, Thailand. My Dolente, C.; Fasching, B. WO
advisor Dr. Nopporn Thasana and my co- 2015/091411 Al, 2015.
advisor Dr. Poonsakdi Ploypradith were 4. Zhou, X. J.; Garner, R. C.; Nicholson, S.;
gratefully appreciated for their invaluable Kissling, C. J.; Mayers, D. J. Clin.
advice and their patience in proofreading Pharmacol. 2009, 49, 1408–1416.
towards the completion of this independent 5. Zhu, S.; Huang, H.; Zhang, Z.; Ma, T.;
study. Furthermore, Dr.Rattana Jiang, H. J. Org. Chem. 2014, 79, 6113–
Worayuthakarn from Chulabhorn Research 6122.
Institute and Thanasan Nilsu, Ph.D 6. Hu, Y.; Huang, H. Org. Lett. 2017, 19,
candidate from Chulabhorn Graduate 5070–5073.
Institute, Chulabhorn Royal Academy, were 7. Korich, A. L.; Hughes, T. S. Org. Lett.
also thankfully acknowledged for their 2008, 10, 5405–5408.
kindness in helping proofreading and
formatting the manuscript.
Anti-cervical cancer activities of 3-(2-hydroxyphenyl)propanoic acid and
3-(2-hydroxyphenyl)acrylic acid derivatives on cervical cancer cell lines
Kraikrit Utama1, Puttinan Meepowpan2, Thiti Janpirom2, Vachira Choommongkol3,
Paitoon Aobchey4, Jiraporn Kantapan5, Padchanee Sangthong2*
1
Master's Degree Program in Biotechnology, Graduate School, Chiang Mai University,
2
Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
3
4
Science and Technology Research institute, Chiang Mai University, Chiang Mai 50200, Thailand
5
Department of Radiologic Technology, Faculty of Associated Medical Sciences, Chiang Mai University,
*E-mail: padchanee.sangthong@cmu.ac.th
Abstract:
Cervical cancer is the second most common cancer that causes death for Thai female
population and Human papillomavirus (HPV) infection is a well-established cause of cervical
cancer. This study aims to synthesize bioactive compounds from commercially available with
3-(2-hydroxyphenyl)propanoic acid and (E)-3-(2-hydroxyphenyl)acrylic acid by chemical
reactions. The structures of the synthesized compounds were characterized using IR and
NMR spectroscopy. Then, (E)-3-(2-hydroxyphenyl)acrylic acid derivatives were evaluated
for cytotoxicity and were found to display inhibitory activity against normal mouse fibroblast
L929 (CCL-1) with IC50 value of 37.77 µM and cervical cancer cell lines (HeLa and SiHa)
with IC50 values of 17.83 and 29.94 µM, respectively. Finally, the active compounds that
show anti-cervical cancer activity will be selected and anti-cervical cancer pathway will be
further investigated using SDS-PAGE and 2-DE. The observed data will be beneficial for
better understanding the anti-cancer pathway in the in vitro model.
1. Introduction which tumor cells can be destroyed via

Cancer is an important global health several pathways such as apoptosis, necrosis
issue which causes population death. In and angiogenesis pathways. In the apoptosis
2012, cervical cancer is the most common pathway, it is known that organic
genital cancer for female with an compounds are involved in the process of
approximation of 14.0% worldwide and of apoptosis induction for cell death or DNA
17.8% for Thai female.1 There are two types damage in tumor cells.4 On the other hand,
of cervical cancer cells including angiogenesis pathway is a process of new
adenocarcinoma and squamous cell blood vessels growth which is related to the
carcinoma and human papillomavirus (HPV) growth of cancer cells. There are several
is a well-established virus that can cause important target factors for cancer therapy in
cervical cancer. It is a double-stranded angiogenesis pathway such as vascular
circular DNA virus which belongs to endothelial growth factor (VEGF), and
Papillomaviridae family. The most common epidermal growth factor (EGF) resulting in
types of cancer-caused HPV are HPV 16 decreasing of blood vessels growth for
and HPV 18.2,3 Although radical surgery in transferring nutrients to cancer cells.5 3-(2-
combination with chemotherapy and/or Hydroxyphenyl)propanoic acid (1) and (E)-
radiation therapy have been commonly used 3-(2-hydroxyphenyl)acrylic acid or 2-
for treatment of cervical cancer, its therapy hydroxycinnamic acid (2) (Figure 1) are
is not satisfactory with poor prognosis of phenolic compounds which are found in
cancer. Chemotherapy is a treatment in medicinal plants such as Cinnamomum
cassia or Cinnamon. These compounds were purchased from Sigma Aldrich (United
investigated for their biological activities Kingdom). The synthesized compounds
including anti-ulcerogenic, anti-bacterial, (Scheme 1) were characterized by means of
anti-viral, anti-hemorrhagic and anti-diabetic spectroscopic techniques. IR spectra were
activities.6,7,8 In recent years, the recorded on a Perkin Elmer FTIR
development of novel anti-cervical cancer Spectrometer-RX I Spectrometer in KBr.
agents derived from synthesized organic Bruker DRX 400 MHz spectrometers and
compounds received much attention. chemical shifts were given in ppm
In the present work, 3-(2- downfield from tetramethylsilane (TMS).
hydroxyphenyl)propanoic acid and 3-(2- 2.2 Synthesis of compound 1b
hydroxyphenyl)acrylic acid derivatives were To a suspension of lithium
synthesized from commercially available 3- aluminium hydride (0.2 g, 6.08 mmol) in
(2-hydroxyphenyl)propanoic acid and 3-(2- THF (40 mL), was added dropwise a
hydroxyphenyl)acrylic acid to furnish the solution of 3-(2-hydroxyphenyl)propanoic
target molecules which were evaluated for acid (1.0 g, 6.02 mmol) in THF (10 mL) and
their anti-cervical cancer activity. The the resulting mixture was brought to
chemical structures were characterized by refluxing temperature for 4 h. The reaction
NMR spectroscopy. All the synthesized was brought to room temperature before it
compounds were evaluated for cytotoxicity was cooled to -78 °C followed by being
against normal mouse fibroblast L929 quenched by slow addition of water. The
(ATCC CCL-1TM) and anti-cervical cancer mixture was neutralized to pH 4-5 by
in cancer cell line model, including HeLa addition of 6 M HCl. The resulting mixture
and SiHa cervical cancer cells. Finally, the was passed through zeolite column and the
compound which contains anti-cervical filtrate was collected and was extracted with
cancer activity will be chosen for further dichloromethane (3x10 mL). The combined
investigation on the anti-cervical cancer organic layers were dried over anhydrous
pathway for better understanding in anti- MgSO4 and analyzed by TLC. The crude
cancer pathway in the in vitro model. products were purified with column
chromatography (4:1 v/v hexanes:ethyl
acetate) to provide the analytically pure
compound 1b.
2.3 Synthesis of compound 1c
Figure 1. The chemical structures of 3-(2- To a solution of 2-(3-
hydroxyphenyl)propionic acid (1) and (E)-3- hydroxypropyl)phenol (1.0 g, 6.58 mmol) in
(2-hydroxyphenyl)acrylic acid (2) dichloromethane (40 mL), was added
montmorillonite K10 (0.10 g). To this
2. Materials and Methods mixture was added dropwise dihydropyran
2.1 Materials and apparatus (0.60 mL, 6.65 mmol) and the resulting
Reactions were performed under mixture was further stirred at room
inert atmosphere (N2 gas). Solvents were temperature for 8 h. The reaction mixture
dried and distilled prior to use. 3-(2- was quenched by adjusting the pH of the
Hydroxyphenyl)propanoic acid (1) and 3-(2- solution to pH 4-5 and followed by
hydroxyphenyl)acrylic acid (2) were extraction using dichloromethane (3 x 10
Scheme 1. Conditions and reagents: a) LiAlH4, dry THF, reflux, 4 h; b) montmorillonite

K10, DHP, dry CH2Cl2, room temperature; c) n-BuLi, trimethylsilyl chloride, 0 oC, 2 h
mL). The combined organic layers were 2.5 Cytotoxicity and anti-cervical cancer
dried over anhydrous MgSO4 and analyzed activity
by TLC. The crude products were purified The cytotoxicity and anti-cervical
with column chromatography (4:1 v/v cancer activity were evaluated against
hexanes : ethyl acetate) to provide the normal mouse fibroblast L929 (ATCC®
analytically pure compound 1c. CCL-1™) and cervical cancer cell line
Compound 1c: pale-yellow oil, IR (HeLa (ATCC® CCL-2™) and SiHa
(CHCl3) (max, cm-1): 3444, 1554, 1455 (ATCC® HTB-35™) using MTT assay. The
1355, 752; 1H NMR (400 MHz, , ppm, normal mouse fibroblast L929 and cervical
J/Hz) δ 7.13-7.09 (m, 2H), 6.88-6.82 (m, cancer cell lines including HeLa and SiHa
2H), 6.65 (s, 1H), 4.66 (dd, J = 4.6, 2.9 Hz, were seeded at concentration of 1x105, 5x104
1H), 3.94-3.88 (m, 1H), 3.79 (ddd, J = 10.1, and 1x105cells/mL in 24-well plates. The
6.4, 4.8 Hz, 1H), 3.56-3.51 (m, 1H), 3.46 cells were incubated at 37 C, 5% CO2
(ddd, J = 10.1, 7.7, 4.7 Hz, 1H), 2.75 (t, J = atmosphere for 24 h. Then, the synthetic
6.8 Hz, 2H), 2.01-1.51 (m, 8H): 13C NMR compounds (0-400 µM) and doxorubicin
(100 MHz, , ppm): δ 154.8, 130.5, 127.7, (cancer drug as a positive control) were
127.5, 120.6, 116.4, 116.4, 99.1, 66.1, 66.0, added to the plates and incubated for 48 h
62.1, 30.7, 30.1, 26.1, 26.1, 25.4, 19.7. and then the culture media was removed.
2.4 Synthesis of compound 2b The cell lines were washed with PBS pH
To a solution of 2-hydroxycinnamic 7.4. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-
acid (1.0 g, 0.61 mmol) in THF (20 mL) was diphenyltetrazolium bromide solution
slowly added a solution of n-BuLi in hexane (MTT) was added in the plates and
(1.12 M, 12.00 mL, 1.34 mmol) at 0 °C and incubated at 37 C, 5% CO2 atmosphere for
the resulting mixture was allowed to stirred 4 h. Finally, the formazan crystals were
at 0 °C for 2 h. The mixture was added dissolved with DMSO, the absorption at 540
dropwise trimethylsilyl chloride (2.00 mL, nm was measured using UV-VIS
1.52 mmol). The resulting mixture was spectrophotometer.
further stirred at 0 °C for 2 h. The reaction
mixture was quenched by adjusting the pH 3. Results and Discussion
of the solution to pH 4-5 and followed by 3.1 Synthesis of active compounds
extraction using dichloromethane (3 x 10 The synthesis of target molecules
mL). The combined organic layers were with anti-cervical cancer activity is shown in
dried over anhydrous MgSO4 and analyzed Scheme 1. 3-(2-Hydroxyphenyl)propanoic
by TLC. The crude products were purified acid (1a) was subjected to reduction reaction
with column chromatography (4:1 v/v using lithium aluminium hydride to furnish
hexanes:ethyl acetate) to provide the 2-(3-hydroxypropyl) phenol (1b) in good
analytically pure compound 2b. yield (85% yield). Then, the primary alcohol
Compound 2b: pale-yellow gum, IR of 1b was protected by dihydropyran to
(CHCl3) (max, cm-1): 3324, 2958 1601, yield 2-(3-((tetrahydro-2H-pyran-2-
1584, 1456, 1638, 1602 752; 1H NMR (400 yl)oxy)propyl)phenol (1c) in good yield
MHz, δ, ppm, J/Hz) δ 8.22 (s, 1H), 7.99 (d, (95% yield). Then, treatment of 2-
J = 16.3 Hz, 1H), 7.50 (dd, J = 7.8,1.6, 1H), hydroxycinnamic (2a) with n-butyllithium
7.27 (td, J=7.69, 1.66 1H), 7.04 (d, J = 16.4 yielded (E)-1-(2-hydroxyphenyl)hept-1-en-
Hz, 1H), 6.99 (br d, J = 8.0, 1H), 6.92 (br t, 3-one (2b) in 10% yield. All compounds
J = 7.6 Hz, 1H), 2.75 (t, J = 7.4 Hz, 2H), were characterized by IR and NMR
1.74-1.67 (m, 2H), 1.48-1.34 (m, 2H), 0.96 spectroscopy and their data were compared
(t, J = 7.3 Hz, 3H): 13C NMR (100 MHz, , with those previously reported in the
ppm): δ 203.9, 156.4, 140.0, 131.9, 129.4, literature.
126.5, 121.6, 120.4, 116.7, 39.9, 26.9, 22.5, Compound 1b was obtained as a pale
13.9. orange oil. The structure of the known
compound was confirmed with 1H and 13C at δ 156.36 (C-OH) and the ortho-carbon of
NMR data with in previously reports.9 From phenol showed a signal at δ 127.68 (C) and
the described data, compound 1b was 121.62 (C) respectively. The HMQC
identified to 2-(3-hydroxypropyl)phenol. spectrum of compound 2b were showed the
Compound 1c was obtained as a protons of trans-olefinic at δ 7.99 and 7.04
pale-yellow oil. The IR spectrum displayed with correlation carbon signals at δ 140.0
a characteristic free hydroxyl as well as an (CH-7) and 116.7 (CH-8). The methylene
H-bonded hydroxyl group bands at 3344 cm- protons exhibited signals at δ 2.75, 1.71 and
1
. The absorption band 1356 cm-1 was 1.41 with correlation carbon signals at δ
attributed to C-O stretching of ether. The 39.9 (10-CH2), 26.9 (11-CH2) and 22.5 (12-
absorption bands at 1554 and 1455 cm-1 CH2). The proton signal of the methyl group
were assigned to benzene ring and the at δ 0.96 showed correlation carbon signal at
absorption band of 752 cm-1 showed meta- δ 13.9 ppm. Therefore, the structure of 2b
substituted of phenyl ring. The 1H NMR was identified to be (E)-1-(2-
spectrum exhibited signals for one hydroxy hydroxyphenyl)hept-1-en-3-one.
proton at δ 6.65 and the methine proton of 3.2 Cytotoxicity and anti-cervical cancer
ether ring at δ 4.66 ppm. The protons of activity
propyls ring showed signal at δ 2.01-1.51 The synthetic compounds were
ppm. The 13C NMR spectra displayed evaluated for their cytotoxicity against
resonance of carbon with a hydroxyl group normal mouse fibroblast L929 (ATCC
at δ 154.8 (C-OH) and the ortho-carbon of CCL-1TM) and anti-cervical cancer activity
phenol at δ 127.7 (C). The compound 1c including HeLa and SiHa cervical cancer
was elucidated by 2D-NMR data including cells which were adenocarcinoma and
heteronuclear multiple-quantum correlation squamous cell carcinoma disease that caused
(HMQC). The HMQC spectrum revealed virus infection of HPV types as 18 and 16,
correlation of the protons and carbons. the respectively (Table 1). (E)-1-(2-
proton of ether showed at δ 4.66 with hydroxyphenyl)hept-1-en-3-one (2b)
correlation carbon with signal at δ 99.1 (CH- showed highest anti-cervical cancer
10). Thereby, the structure of 1c was activities in both HeLa and SiHa cervical
identified to be 2-(3-((tetrahydro-2H-pyran- cancer cell lines with IC50 values of 17.83
2- yl)oxy)propyl)phenol. and 29.94 µM, respectively. The
Compound 2b was obtained as a cytotoxicity of 2b was evaluated with IC50
pale-yellow gum. The IR spectrum value of 37.77 µM. The synthetic
displayed a characteristic free hydroxyl as compounds (1a, 1b, 1c and 2a) showed anti-
well as an H-bonded hydroxyl group at 3324 cervical cancer activities on HeLa cervical
cm-1. The absorption bands at 1638 and cancer cells with IC50 values of 104.30,
1602 cm-1 was assigned to the C=O 83.89, 80.53 and 62.05 µM. In addition,
stretching of a conjugated carbonyl group anti-cervical cancer activities of compounds
and C=C stretching band of alkene 1a, 1b, 1c and 2a on SiHa cervical cancer
respectively. The benzene ring showed cells with IC50 values of 85.85, 54.34,
absorption bands at 1584 and 1456 cm-1. 113.15 and 60.71 µM, respectively.
The absorption band at 752 cm-1 was Moreover, the synthetic compounds (1a, 1b,
assigned to meta-substituted of phenyl ring. 1c and 2a) indicated low cytotoxicity against
The 1H NMR spectrum showed the trans- normal mouse fibroblast L929 cell line with
olefinic protons at δ 7.99 and 7.04, three IC50 values in the range of 37.37-90.74 µM
methylene protons at δ 2.75, 1.71, 1.41 and when compared with doxorubicin.
methyl group at δ 0.96 ppm. The 13C NMR
spectra demonstrated quaternaries of
carbonyl carbon signal at δ 203.95 (C=O).
The carbon of hydroxyl group showed signal
Table 1. The cytotoxicity and anti-cervical References
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Synthetic small molecules for isolation of
Gram-positive bacteria from contaminated samples
Krittapas Jantarug, Sakol Warintaraporn, Chutima Jiarpinitnun*
Department of Chemistry and Center for Innovation in Chemistry (PERCH-CIC),
Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
*E-mail: chutima.jia@mahidol.ac.th
Abstract:
Foodborne bacterial contamination is global health concerns as it is responsible for
bacterial infections via food consumption. Not only bacteria contamination could lead to
serious illness, but also lead to the decline of fresh product consumption, affecting world
economy. Therefore, rapid and reliable method for isolation and identification of bacteria in
food is required. This investigation exploits the endogenous sorting pathway of bacteria in
design of small molecule that could decorate the bacterial cell wall with functional moiety
that could be used for bacteria isolation. In Gram-positive bacteria, covalent assembly of
surface proteins to surface is achieved catalytically via an enzyme called Sortase. Previous
evidences showed that Sortase could catalyze transpeptidation of unnatural substrates that
contained a short peptide substrate motif—LPXTG efficiently. Herein, synthetic analogues of
Sortase substrate were synthesized. Preliminary experiments showed that our synthetic small
molecules could undergo Sortase-catalyzed reaction in bacteria, presenting the functional
moiety of interest on the bacterial surface. Parameters include incubation time as well as
substrate and bacterial concentrations were optimized. Towards bacterial isolation, substrate
motif conjugated to the biotin was successfully synthesized. We anticipated that this
molecule could be used to decorate biotin on bacterial surface, allowing bacterial isolation via
streptavidin-biotin interaction.
1. Introduction Re-engineering of bacterial cell wall

Foodborne pathogen contamination techniques has been developed for utilizing
is considered as one of the worldwide public in several applications including detection
health concerns. Bacterial pathogens often and therapeutic development.2 The
found contaminated in food include biosynthetic pathway for cell wall synthesis
Escherichia coli (E. Coli), Salmonella spp., has been exploited in the design of small
Listeria spp., Vibrio cholera (V. cholera), molecules that could modify bacterial cell
Pseudomonas spp., and Staphylococcus wall. We are particularly interested in the
aureus (S. aureus).1 Bacterial contamination bacterial sorting pathway catalyzed by
could cause serious illness that may be fatal. Sortase enzyme (SrtA). SrtA recognizes a
In addition, it could also lead to the decline pentapeptide LP-X-TG sequence, of which
of products consumption and product recall, X depends on bacterial species. This enzyme
effecting agricultural exports and food functions in the catalysis of transpeptidation
manufacturer. Hence, the rapid and reliable by cleaving substrate peptide bond between
isolation and identification of bacteria in threonine (T) and glycine (G), forming
food sample is absolutely required for the enzyme-substrate complex. Subsequently,
purpose of consumer safety. The rapid lipid II, the intermediate of peptidoglycan
isolation would speed up the identification formation, functions as nucleophile,
of bacteria, which facilitate in finding the attacking and breaking bond between
effective treatment for the infection and enzyme and substrate, yielding part of
minimizing the damage from contamination. substrate attached with lipid II that will
further mature to a peptidoglycan. Hence, a solution of γ-aminobutyric acid 1 was then
chemical moiety that attached to sortase added dropwise to the cooled solution of N-
substrate at N-terminus should undergo (9-Fluorenylmethoxycarbonyloxy)
catalysis and be presented on cell wall.3 succinimide at 0 ºC. The reaction mixture
Here in, we envisioned that a biotin- was stirred at room temperature for 2 hours,
conjugated sortase substrate could be used to forming white precipitates. The reaction
isolate Gram-positive bacteria from a mixture was washed with EtOAc. The clear
contaminated sample. SrtA-catalysis would aqueous layer was collected and cooled with
result in a biotin moiety presented on ice bath. To the cooled aqueous solution,
bacteria. Subsequent treatment with HCl solution was added dropwise until pH
streptavidin bead will allow isolation of reached 2-3 and white precipitates were
bacteria via a tight non-covalent interaction. observed. The reaction mixture was
The non-covalent interactions could be subsequently extracted with EtOAc (3X).
destroyed, releasing isolated bacteria for The combined organic layers were dried
further analysis. over Na2SO4. The solvent was removed
under reduced pressure, affording the crude
2. Materials and Methods product the product 2 in 94% yield.
2.1 General Subsequently, the Fmoc-protected γ-
Commercially available compounds amino butyric acid 2 (1 mmol) was treated
were purchased from suppliers and used with a mixture of N,N,N’,N’-tetramethyl-O-
without purification. Rink amide resin- (1H-benzotiazol-1-yl)uranium hexafluoro
GTEPL-NH2 was custom-synthesized by phosphate (HBTU) (1 mmol), hydroxy
GL-Biochem (China). The reactions were benzotriazole (HOBt) (1 mmol), diisopropyl
conducted under inert nitrogen atmosphere ethylamine (DIEA) (1 mmol) in DMF for
and monitored by thin layer chromatography 20 minutes at room temperature. The
(TLC). reaction mixture was then added to SrtA
All synthetic compounds were substrate appended on Rink amide resins in
identified and characterized by 1H NMR a disposable chromatography column. The
spectroscopy, 13C NMR spectroscopy and reaction was shaken vertically by rotator-
high-resolution mass spectrometry (HRMS). mixer for 16 hours at room temperature.
1
H NMR spectra were obtained by Bruker Subsequently, the resin was filtered and
AscendTM-400 (400 MHz) spectrometers. washed with CH2Cl2 (3X) followed by
13
C NMR spectra were obtained by Bruker MeOH (3X). The solution was removed and
AscendTM-400 (100 MHz), and Bruker the resin was washed again with CH2Cl2
Avance-500 (125 MHz) spectrometers. (3X) and MeOH (3X) and vacuum dried.
Chemical shifts are reported in term of part The efficiency of the conjugation
per million (δ) (DMSO-d6: 1H: δ 2.54, 13C: δ step was determined indirectly by
39.51, CDCl3: 1H: δ 7.26, 13C: δ 77.2, monitoring the detectable absorbance of
CD3OD: 1H: δ 3.31,4.87, 13C: δ 49.1). 1H dibenzofulvene, the resulting product from
NMR data are assumed to be first order with Fmoc cleavage, via UV-Visible
apparent singlet, doublets, triplets and spectroscopy.4 Fmoc protecting group was
multiplets reported as s, d, t and m. removed by treating the resin with 2% v/v
2.2 Synthesis of fluorophore-conjugated of 1,8-diazabicyclo undec-7-ene (DBU) in
Sortase substrate DMF. Washing with CH2Cl2 and MeOH,
γ- Aminobutyric acid 1 (9.70 mmol) yielding NH2-terminal resins 3 in 41%
was dissolved in 9% Na2CO3 solution. The yield.
solution was cooled to 0 ºC with ice bath. The resin (3) was added a solution of
N-(9-Fluorenylmethoxycarbonyloxy) fluorescein isothiocyanate (FITC) (0.0886
succinimide (11.64 mmol) was dissolved in mmol) in DMF. The mixture was agitated in
DMF and cooled to 0 ºC. The cooled a rotator-mixer for 16 hours at room
temperature in the dark to avoid photo- mmol) in 8 mL of DMF was added. The
bleaching. Subsequently, the filtrate was solution was let stirred for few minutes;
removed. The resulting resins were washed then, compound 6 in 2 mL of DMF and 5
with CH2Cl2 (3X) followed by MeOH (3X). mmol of DIEA was added. The reaction was
The washed resins were vacuum dried. conducted in a disposable chromatography
Cleavage solution of trifluoroacetic column. The reaction was shaken vertically
acid (TFA), triisopropylsilane (TIPS), and by rotator-mixer for 16 hours at room
water at ratio 94:3:3 v/v was prepared. The temperature. The yield of the conjugation
cleavage solution was added to resins. The reaction could be determined indirectly by
reaction mixture was stirred for 1.5 hours at monitoring the detectable absorbance of
room temperature. The filtrate was dibenzofulvene, the resulting product from
collected. Subsequently, the solvent was Fmoc cleavage, via UV-Visible
4
removed from the filtrate under reduced spectroscopy. Fmoc protecting group was
pressure, affording a crude fluorescent removed by treating resins with 2% v/v of
product. The crude product was purified by 1,8-diazabicyclo undec-7-ene (DBU) in
high performance liquid chromatography DMF. Washing with CH2Cl2 and MeOH,
(HPLC) using 0.1% TFA in H2O and yielding NH2-terminal resins 7 in 34% yield.
acetonitrile (ACN) as mobile phase. Resins 7 (0.06 mmol) was treated
Fractions that contained the desired with HBTU (0.3 mmol), and HOBt (0.3
products were collected and combined. mmol) in 8 mL of DMF. Subsequently,
Solvents were removed under reduced biotin (0.3 mmol) and 0.6 mmol of DIEA
pressure followed by lyophilization. 82% of was added. The reaction was shaken
purified product was obtained. The product vertically for 16 hours. The reaction was
was characterized using ESI-MS. filtered, yielding biotin-LPXTG on resins.
Compound 2: 1H NMR (400 MHz, The product was cleaved from the resin
CD3OD) δ=1.69-1.78 (m, 2H), 3.19 (t, J = using 95%TFA in CH2Cl2. The crude
6.58 Hz, 2H), 3.48 (t, J = 6.58 Hz, 2H), 4.17 product was purified by HPLC using 0.1%
(t, J = 6.58 Hz, 1H), 4.34 (d, J = 6.88 Hz, of TFA in H2O and ACN as mobile phase.
2H), 7.28 (t, J = 8.20 Hz, 2H), 7.36 (t, J = Solutions that contained products were
7.93 Hz, 2H), 7.60 (d, J = 6.32 Hz, 2H), 7.75 collected and combined. Solvents were
(d, J = 7.85 Hz, 2H) ESI-MS calc for removed under reduced pressure. The
C19H19NO4 [M + Na+]+ = 348.1212, found product was characterized using ESI-MS.
348.1213 Compound 6: 1H NMR (400 MHz,
Compound 4: ESI-MS calc for CDCl3) δ=1.76-1.89 (m, 4H), 2.18 (t, J =
C47H56N8O14S [M + H+]+ = 989.3715, found 7.27 Hz, 2H), 2.36 (t, J = 7.72 Hz, 2H),
989.3727 3.19-3.32 (m, 4H), 3.66 (s,3H), 4.20 (t, J =
2.3 Synthesis of biotin-conjugated Sortase 6.61 Hz, 1H), 4.40 (d, J = 6.61 Hz, 2H), 7.31
substrate (t, J = 7.55 Hz, 2H), 7.40 (t, J = 7.21 Hz,
Fmoc-protected γ-aminobutyric acid 2H), 7.59 (d, J = 7.43 Hz, 2H), 7.76 (d, J =
(2) (1 mmol) in 5 mL of CH2Cl2 was treated 7.43 Hz, 2H)
with methyl 4-aminobutanoate (1.1 mmol), Compound 8: ESI-MS calc for
HBTU (1.1 mmol), and HOBt (1.1 mmol) in C47H56N8O14S [M + H+]+ = 911.4661, found
the presence of DIEA (2 mmol). The 911.4656
reaction was stirred at room temperature for
16 hours. After purification via column 3. Results and Discussion
chromatography, deprotection of 3.1 Design and syntheses of FITC-
methylester by using 3M HCl 10 mL for 3 conjugated SrtA substrate motif
hours yield compound 6 in 32%. The substrate recognition of SrtA is
To the SrtA substrate (LPETG) on considered spacious, allowing the catalysis
resins, HBTU (2.5 mmol), and HOBt (2.5 of structurally diverse protein substrates and
also non-natural substrates containing aureus recognizes substrate motif LPETG.
LPXTG sorting motif.3,5 Hence, we To determine whether the modification of
exploited SrtA- catalyzed transpeptidation N-terminus of LPETG could be covalently
reaction to covalently anchor the chemical anchored on the S. aureus cell wall, we
moiety on the bacterial surface. synthesized a fluorescent moiety conjugated
Based on the SrtA catalytic to N-terminus of LPETG. The inexpensive
mechanism, upon binding of the sorting dye fluorescein isothiocyanate (FITC) was
motif, a catalytic Cys184 of SrtA selectively used. In addition, γ-aminobutyric acid was
cleaves the amide bond between the inserted as a linker to provide flexibility for
threonine (T) and glycine (G) residue of the the compound.
LPXTG motif, forming a semi-stable As shown in Scheme 1, the desired
thioacyl intermediate covalently linked product 4 could be achieved in several steps.
enzyme to substrate.3 The enzyme then Solid phase synthesis was used. The
recognizes lipid II which is the cell wall unprotected carboxylic acid of compound 2
precursor and catalyzes nucleophilic attack was conjugated to the N-terminus of the
of carbonyl in thioacyl bond by the N- sorting motif, LPETG, of which covalently
terminal primary amine group within the attached to the Rink amide resins at its C-
cross-bridge peptide affording the new terminus. The conjugation was performed
covalent bond between lipid precursor and employing standard peptide coupling
protein. The lipid II-linked intermediate is methods. The Fmoc protecting group was
then incorporated into the peptidoglycan removed under base induce -elimination
resulting in the synthesis of bacterial cell (2% DBU). Subsequent treatment with FITC
wall.6 Therefore, to anchor a chemical under slightly basic condition yielded the
moiety of interest to the bacterial cell wall, desired thiourea conjugated FITC adduct on
such chemical moiety should be conjugated resin. The product was cleaved from the
to the N-terminus of the substrate motif. resin under acidic conditions. Purification by
Herein, a pathogenic S. aureus was reverse phase HPLC yield the desired
used as a model of the study. SrtA of S. product 4, the identity of which was
confirmed by ESI-MS.
1. HO
O O
H H
N N
N N N
H H
O O NH2
O HO
O O
H H
HO O N N
O O HBTU, HOBt, DIEA N N N
H H H
NH2 Fmoc-OSu O O N
NHFmoc O NH2
HO HO
2. 2% DBU O
HO O
1 2
3
O O OH
O 1.
HO HOOC DIEA, DMF

O O HOOC O
H H
N N
H2N N N S
H H
O O N NCS
O N N OH
H H 2. 94% TFA
O
HO O
Scheme 1. Synthesis of FITC-LPETG (4)
3.2 Ability of LPETG containing chemical endogenous SrtA-catalyzed cell wall

moiety to present on S. aureus decoration, S. aureus ATCC25923 at 106
To determine whether FITC- CFU/mL was grown at 37°C in the presence
conjugated LPETG 4 could undergo of compound 4 at 1 mM. We monitored
fluorescence intensity when treated at careful washes with PBS to remove unbound
different incubation time ranging from 10 to fluorescent compound 4, the fluorescence
24 hours in order to determine optimal time intensity was measured. The results are
required for expression fluorescent moiety shown in Table 1.
on S. aureus cell wall. Fluorescence
intensity was measured using microplate Table 1. Fluorescence intensity of bacteria
reader with excitation wavelength of 485 nm expressed FITC-conjugated LPETG on its
and emission reading at 535 nm. The results surface in different in amounts of S. aureus
were shown in Figure 1. The results showed Initial amount of Fluorescence intensity of cell
that the optimum time for expressing a S. aureus (CFU) wall -bound compound 4
chemical moiety on the bacterial surface is 0 133 ± 7.2%
1 1117 ± 20%
approximately 18 hours.
10 1607 ± 13%
100 1662 ± 13%
1000 2000 ± 14%
10000 2492 ± 13%
100000 2678 ± 15%
Based on the results shown in Table

1, FITC-conjugated LPETG (at 1 mM)
could fluorescently label S. aureus in the
sample with minimum initial concentration
as low as 1 CFU.
Figure 1. Fluorescence intensity of S. aureus
3.3 Design and syntheses of Biotin-
when treated with compound 4 at different
conjugated SrtA substrate motif for
incubation period
isolating S. aureus from contaminated
sample
In addition, we also observed the
To apply this method for the purpose
plateau when incubated further than 18
of isolating S. aureus from contaminated
hours, indicating that FITC-conjugated
sample, we exploited the uniquely strong
LPETG could be expressed via specific
non-covalent interaction between avidin
pathway and did not bind to pathogen via
protein and biotin (Kd approximately 10-15
non-specific interaction. To exclude the non-
M).7 The strong non-covalent interaction
specific interaction, we also treated
would allow pulling the bound bacteria from
compound 4 with Gram-negative
the contaminated sample. In addition, the
Escherichia coli (E.coli) that lacks of SrtA.
non-covalent bonding interaction could be
The fluorescent intensity observed was
destroyed without perturbing the bacteria,
similar to the control experiment (no
releasing the bacteria for further
bacteria), suggesting that FITC-conjugated
identification or for susceptibility testing. To
LPETG could be expressed on bacteria that
undergo endogenous SrtA-catalysis and
does not contain Srt.
having biotin moiety presented on the S.
We also evaluated the ability of this
aureus cell wall, biotin is conjugated at the
molecule to be express on S. aureus cell
N-terminus of LPETG substrate. As shown
wall at different bacterial concentrations in
in Scheme 2, we lengthened the linker in
order to determine the minimum
order to provide flexibility and space for
concentration of bacterial required for
binding to the targeted avidin. The
isolation. Series of S. aureus at different
unprotected carboxylic acid of linker 6 was
initial colony forming units, ranging from 1
conjugated to the N-terminus of the sorting
CFU to 100,000 CFU, were treated with
motif, LPETG, of which covalently attached
compound 4 (1 mM) for 24 hours. After
Scheme 2. Synthesis of Biotin-LPETG (8)
to the Rink amide resins at its C-terminus. Acknowledgements
The conjugation was performed employing This work was supported in parts by
standard peptide coupling methods. Development and Promotion of Science and
Deprotection of Fmoc group in the presence Technology Talents Project (DPST)
of mildly basic condition yielded terminal scholarship, the Center of Excellence for
amino moiety for further conjugation with Innovation in Chemistry (PERCH-CIC), and
biotin via amide bond formation. The resin the Department of Chemistry, Department of
was then treated with acid to release the Microbiology, Faculty of Science, Mahidol
desired compound 8, which was purified by University. We thank Asst. Prof. Soraya
HPLC and the identity of which was Chaturongakul for providing useful
confirmed by ESI-MS. scientific discussion.
The ability of compounds 8 to isolate
S. aureus from contaminated sample is References
currently under investigation. The results 1. Fu, L.; Valentino, H. R.; Wang, Y.
will be reported in due course. Bacterial Contamination in Food
Production. In Antimicrobial Food
4. Conclusion Packaging, 2016; pp 35–43.
FITC-conjugated LPETG sorting 2. Gautam, S.; Gniadek, T. J.; Kim, T.;
motif was synthesized and tested for its Spiegel, D. A. Trends Biotechnol 2013,
ability to selective re-engineering cell wall S. 31, 258–267.
aureus. The optimum time for the presenting 3. Popp, M. W.; Ploegh, H. L. Angew.
a chemical moiety on bacterial surface via Chem., Int. Ed. 2011, 50, 5024–5032.
endogenous SrtA-transpeptidase catalysis 4. Eissler, S.; Kley, M.; Bachle, D.; Loidl,
was found to be 18 hours. In addition, the G.; Meier, T.; Samson, D. J. Pept. Sci.
minimum initial bacterial concentration 2017, 23, 757–762.
required was 1 CFU. We further applied this 5. Dramsi, S.; Trieu-Cuot, P.; Bierne, H.
methodology for the purpose of bacterial Res. Microbiol. 2005, 156, 289–297.
isolation by synthesizing biotin-LPETG. 6. Nelson, J. W.; Chamessian, A. G.;
The synthesis of the compound was McEnaney, P. J.; Murelli, R. P.;
successfully achieved. Its ability of this Kazmiercak, B. I.; Spiegel, D. A. ACS
molecule to isolate S. aureus from Chem. Biol. 2010, 5, 1147–1155.
contaminated sample is ongoing. 7. Taninaka, A.; Takeuchi, O.; Shigekawa,
H. Phys. Chem. Chem. Phys. 2010, 12,
12578–12583.
Antidiabetic activity of synthetic furofuran lignans containing
multiphenolic groups
Titiruetai Doungwichitrkul, Nantaporn Surachaitanawat, Wisuttaya Worawalai,
Preecha Phuwapraisirisan*
Department of Chemistry, Faculty of Science,
*E-mail: preecha.p@chula.ac.th
Abstract:
Sesamolin (1) and sesamin (2) are major furofuran lignans found abundantly in
sesame (Sesamum indicum) seeds. Compounds 1 and 2 show several intriguing in vivo
biological activities, particularly antidiabetic activity. Current evidences indicated that they
are in fact the proactive precursor, which is subsequently converted by human intestinal
microbes to the true active metabolites. Noticeably, the enhanced bioactivity found in true
active metabolites is possibly caused by the presence of multiphenolic groups, especially
catechol moiety. To prove this postulate, a new series of furofuran lignans containing
multiphenolic groups were synthesized starting from 1 and 2. The synthesized lignans were
evaluated for their antidiabetic activity through -glucosidase inhibition. Furofuran lignan
containing tetraphenolic groups (7) showed the most potent inhibition against maltase and
sucrase with IC50 values of 0.32 and 0.71 mM, respectively, while 1 and 2 showed no
inhibition. The observation suggested -glucosidase inhibition depends on the numbers of
phenolic groups in lignan structure.
1. Introduction Interestingly, 1 and 2 reveal several

Furofuran lignan (2,6-diaryl-3,7- bioactivities that are beneficial to human
dioxabicyclo [3.3.0] octane lignan) is a large health such as anticancer3-7, antioxidative
group of lignan, which is biosynthesized effect8-10 and antidiabetic11-13. Noticeably,
from oxidative coupling of two the aforementioned bioactivities of 1 and 2
phenylpropanoids (2 × C6-C3) to form two were carried out in animal models (in vivo).
fused tetrahydrofuran rings as a central However, they were inactive or showed
core.1,2 weak bioactivity in the in vitro models,
Among all furofuran lignans reported therefore raising the question of true active
so far, sesamolin (1) and sesamin (2), major metabolites when 1 and 2 were orally
lignans in sesame (Sesamum indicum) seed administrated in animal models. Recent
oil, have been most widely investigated.3-13 evidences independently conducted by
Sesamolin (1) and sesamin (2) are closely Peñalvo14 and Liu15 demonstrated that 1 and
related analogues differing only in one 2 are in fact proactive precursors, which are
additional oxygen atom in sesamolin (1) further metabolized by human intestinal
structure (Figure 1). microbes to the corresponding lignans
containing phenolic motifs, together with
enterolactone as a final product. These
observations imply that the presence of free
phenolic motifs would be associated with
enhancing bioactivity. This postulation is
likely consistent with the in vitro -
Figure 1. Structures of sesamolin (1) and glucosidase inhibition of 1 and 2, which
sesamin (2)
were very weak compared with their 2.3 Synthesis of furofuran lignans 3-620
analogue named pinoresinol.16 A mixture of samin (1a, 1 equiv) and
Unlike previous synthetic acetonitrile (1.0 mL) was treated with
17-19
methodologies , Worawalai and phenolics (1.5-2 equiv), Amberlyst-15 (1
coworkers20 synthesized a series of mg/0.005 mmol of samin) and 4 Å
furofuran lignans in two steps, starting from molecular sieves at 70 C for 8-10 h. The
natural sesamolin (1). Of all lignan products reaction mixture was evaporated to dryness
examined, the synthesized lignans having at and purified by flash chromatography.
least one free phenolic motif showed 2.4 Synthesis of lignan 722
enhanced inhibition over those bearing no A mixture of sesamin (2, 0.16 mmol)
free phenolic motif. This observation and lead(IV) tetraacetate (3 equiv) in toluene
supported the postulation that free phenolic was treated at 90 C for 2 h. After cooling,
motif plays a critical role in exerting the reaction mixture was evaporated to
inhibitory effect against -glucosidase and dryness and extracted by ethylacetate/H2O
free radical. (1:1, 3 times) The resulting residue was
Hence, furofuran lignans having treated with acidic resin Amberlyst-15 (1
multiphenolic groups would be expected to mg/0.005 mmol of 2) in acetonitrile/H2O
show more improved inhibitory effect. (9:1, 2 mL) at 70 C for 2 h. The reaction
mixture was evaporated to dryness and
2. Materials and Methods purified by flash chromatography to give 7
2.1 General (42 mg, 80%) as a brown oil.
All experiments were carried out 2.5 -Glucosidase inhibitory activity
under a nitrogen atmosphere. The 1H and -Glucosidase (rat intestinal maltase
13
C NMR spectra were recorded on 400 and sucrase) inhibitory activity was
MHz Bruker AVANCE spectrometers determined according to our previous
(CDCl3 and CD3OD as solvents). Analytical report.23 Briefly, the synthesized lignans (1
thin layer chromatography (TLC) was mg/mL in DMSO, 10 µL) were added to 0.1
performed on pre-coated Merck silica gel 60 M phosphate buffer (pH 6.9, 30 µL), the
F254 plates (0.25 mm thick layer). Column substrate solution (maltose: 10 mM,20 µL;
chromatography was performed on Merck sucrose: 100 mM, 20 µL) in 0.1 M
silica gel 60 (70–230 mesh) and Sephadex phosphate, glucose assay kit (80 µL) and
LH-20. Sucrose, maltose and rat intestinal crude enzyme solution (20 µL). Then, the
acetone powder were obtained from Sigma- reaction mixture was incubated at 3 C for
Aldrich (St.Louis, MO, USA). Glucose 10 min (maltose) and 40 min (sucrose). -
assay kit was obtained from Human Glucosidase activity was measured at 503
Gesellschaft für Biochemica und nm. The assay was performed in triplicate,
Diagnostica mbH (Germany). Acarbose was and acarbose was used as a positive control.
obtained from Bayer (Germany). - The inhibition percentage was calculated
Glucosidase inhibition was measured on a according to [(A0 – A1)/A0] × 100, whereas
BioRed microplate reader model 3550 UV. A is the absorbance without the sample (A0)
2.2 Synthesis of samin (1a)20 and with the sample (A1). The IC50 value
A mixture of sesamolin (1, 0.27 was estimated from a plot of percentage
mmol) and acetonitrile/H2O (9:1, 10 mL) inhibition and sample concentration.
was treated with acidic resin Amberlyst-15 2.6 Spectroscopic data
(1 mg/0.005 mmol of 1) at 70 C for 4-5 h. Samin (1a); Yield: 90%, brown oil;
The reaction mixture was evaporated to (c 0.1, CHCl3); 1H NMR
dryness and purified by flash (CDCl3, 400 MHz)  6.85 (s, 1H), 6.80-6.75
chromatography to give samin (1a, 60 mg, (m, 2H), 5.94 (s, 2H), 5.36 (s, 1H), 4.35 (d, J
90%) as a brown oil. = 8.4 Hz, 2H), 4.16 (dd, J = 9.2, 6.0 Hz,
1H), 3.89 (d, J = 9.2 Hz, 1H), 3.56 (dd, J =
8.8, 7.2 Hz, 1H), 3.25 (brs, 1H), 3.05 (m, 6.77 (m, 2H), 6.67 (d, J = 8.4 Hz, 1H), 6.43
1H), 2.86 (m, 1H); 13C NMR (CDCl3, 100 (d, J = 8.5 Hz, 1H), 5.94 (s, 2H), 4.97 (d, J =
MHz)  148.1, 147.4, 134.7, 119.7, 108.3, 5.4 Hz, 1H), 4.71 (d, J = 4.9 Hz, 1H), 4.25
106.7, 102.4, 101.2, 87.0, 71.4, 69.5, 53.7, (dt, J = 12.6, 8.4 Hz, 2H), 4.12 (dd, J = 14.2,
52.9. The data were consistent with previous 7.1 Hz, 1H), 3.94 (dd, J = 9.0, 3.8 Hz, 1H),
report.21 3.89 (m, 1H), 3.85 (s, 3H), 3.18 (m, 1H),
3; Yield: 6%, orange oil; 1H NMR 3.05 (m, 1H); 13C NMR (100 MHz, CDCl3)
(400 MHz, CDCl3) δ 6.98 (brs, 1H,-OH), δ 148.1, 147.2, 147.1, 142.5, 135.1, 133.6,
6.82 (s, 1H), 6.77 (s, 2H), 5.96 (s, 2H), 5.95 119.6, 119.5, 116.60, 108.3, 106.7, 103.0,
(s, 2H), 5.23 (d, J = 7.7 Hz, 1H), 4.83 (d, J = 101.2, 85.6, 84.4, 72.0, 56.3, 54.1, 53.0.
3.9 Hz, 1H), 4.49 (t, J = 7.7 Hz, 1H), 4.20 6; Yield: 22%, dark brown oil; 1H
(dd, J = 9.4, 2.7 Hz, 1H), 4.03 (dd, J = 9.3, NMR (400 MHz, CDCl3) δ 6.86 (s, 1H),
6.7 Hz, 1H), 3.80 (m, 1H), 3.72 (s, 3H), 3.21 6.82-6.72 (m, 3H), 6.45 (d, J = 8.3 Hz, 1H),
(m, 1H), 3.07 (m, 1H); 13C NMR (100 MHz, 5.93 (s, 2H), 5.00 (d, J = 5.8 Hz, 1H), 4.39
CDCl3) δ 160.7, 156.0, 148.2, 147.3, 134.9, (d, J = 7.0 Hz, 1H), 3.90 (m, 1H), 3.85 (s,
119.5, 108.4, 106.8, 104.1, 101.2, 94.8, 84.4, 3H), 3.81 (s, 1H), 3.45 (m, 1H), 3.35 (m,
83.7, 72.9, 70.7, 55.42, 54.6, 53.7. 1H), 2.87 (dd, J = 15.3, 6.9 Hz, 1H); 13C
4; Yield: 10%, brown oil; 1H NMR NMR (100 MHz, CDCl3) δ 148.0, 147.3,
(400 MHz, CDCl3) δ 6.88 - 6.78 (m, 3H), 146.6, 142.0, 135.1, 133.1, 119.7, 118.9,
5.95 (s, 4H), 5.20 (d, J = 5.8 Hz, 1H), 4.42 116.9, 108.2, 106.7, 103.1, 101.1, 87.6, 81.3,
(d, J = 7.1 Hz, 1H), 4.18 (d, J = 9.7 Hz, 1H), 71.2, 70.0, 56.2, 54.2, 49.5.
3.96 (t, J = 8.3 Hz, 1H), 3.82 (dd, J = 9.7, 7; Yield: 80%, brown oil; 1H NMR
6.3 Hz, 1H), 3.73 (s, 3H), 3.55 (m, 1H), 3.48 (CD3OD, 400 MHz)  6.82-6.68 (m, 6H),
(m, 1H), 2.89 (dd, J = 15.3, 6.7 Hz, 1H); 13C 4.63 (d, J = 4.3, 2H), 4.21 (dd, J = 9.0, 7.1
NMR (100 MHz, CDCl3) δ 160.7, 156.0, Hz, 2H), 3.80 (dd, J = 9.0, 3.5 Hz, 2H), 3.07
148.2, 147.5, 134.8, 119.9, 108.3, 106.8, (m, 2H); 13C NMR (CD3OD, 100 MHz) 
101.2, 101.0, 94.5, 87.6, 81.7, 71.5, 70.3, 145.0, 144.5, 132.4, 116.9, 115.2, 113.5.
55.4, 53.6, 49.6. 84.9, 70.7, 53.5. The data were consistent
5; Yield: 5%, dark brown oil; 1H with previous report.22
NMR (400 MHz, CDCl3) δ 6.84 (s, 1H),
Scheme 1. Preparation of samin (1a)
Scheme 2. Synthesis of furofuran lignans (3-6), arrows indicate the linkage between samin
(1a) and phenolics (ArOH).
Scheme 3. Synthesis of furofuran lignan 7
3. Results and Discussion Moreover, sesamin (2) was further

3.1 Synthesis and characterization oxidized by Pb(OAc)4 followed by acidic
This is the first report of α- hydrolysis to remove the two
glucosidase inhibition of furofuran linans dioxymethylene units. As a result, final
containing multiphenolic groups. At the product 7 with the additional catechol
beginning of this experiment, commercially moiety was obtained (Scheme 3).
available sesame seed oil was used as a
source of sesamolin (1). Sesame seed oil
(150 g) was first saponified by KOH (25 g)
in MeOH (150 mL) to remove fatty acid
(saponifiable matter), yielding
unsaponifiable matter as a mixture of
sesamolin (1, 1.5 g, 1%) and sesamin (2, 3 g,
2%)17. Pure sesamolin (1) was obtained after
purification by column chromatography.
The more reactive samin (1a, 90%) was
synthesized by acid-catalyzed hydrolysis of
1 (Scheme 1). Figure 2. -glucosidase inhibition of
Subsequently, furofuran lignans (3- lignans 1-7
6) were synthesized by replacing the
hydroxy group of samin with two phenolics 3.2 -Glucosidase inhibitory activity
moieties (ArOH) under acidic conditions Of all the products examined,
using Amberlyst-15 and purified by column furofuran lignans containing diphenolic
chromatography (Scheme 2). groups (3-6) slightly improved -
glucosidase inhibition (maltase and sucrase)
in range of 0.68-2.41 mM, while 1 and 2 7. Miyahara, Y.; Hibasami, H.; Katsuzaki,
showed no inhibition (IC50 > 40 mM). H.; Imai, K.; Komiya, T. Int. J. Mol.
Interestingly, lignan 7 having tetraphenolics Med. 2001, 7, 369–371.
revealed most potent inhibition against 8. Hou, R. C.-W.; Wu, C.-C.; Huang, J.-R.;
maltase and sucrase with IC50 values of 0.32 Chen, Y. S.; Jeng, K. C. G. Ann. N. Y.
and 0.71 mM, respectively. This experiment Acad. Sci. 2005, 1042, 279–285.
indicated that the number of free phenolic 9. Hou, R. C. W.; Huang, H. M.; Tzen, J.
groups plays important role in -glucosidase T.; Jeng, K. C. G. J Neurosci Res. 2003,
inhibition. 74, 123–133.
10. Kang, M.-H.; Naito, M.; Tsujihara, N.;
4. Conclusion Osawa, T. J. Nutr. 1998, 128, 1018–
In conclusion, we first synthesized 1022.
furofuran lignans containing 11. Hong, L.; Yi, W.; Liangliang, C.;
multiplephenolics (3-7). Furofuran lignan Juncheng, H.; Qin, W.; Xiaoxiang, Z. J.
containing tetraphenolics (7) showed the Sci. Food Agric. 2013, 93, 1833–1383.
most potent inhibition against maltase and 12. Roghani, D. F.; Roghani, M. Int J
sucrase, while the parent lignans 1 and 2 Endocrinol Metab. 2011, 9, 311–316.
showed no inhibition. The observation 13. Mohammad Shahi, M.; Zakerzadeh, M.;
suggested critical scientific clues that the Zakerkish, M.; Zarei, M.; Saki, A. J Diet
more phenolic groups, the more potent Suppl 2017, 14, 65–75.
inhibition against -glucosidase. 14. Peñalvo, J. L.; Heinonen, S.-M.; Aura,
A.-M.; Adlercreutz, H. J. Nutr. 2005,
Acknowledgements 135, 1056–1062.
This project was funded by the 15. Liu, Z.; Saarinen, N. M.; Thompson, L.
Thailand Research Fund (TRF) and U. J. Nutr. 2006, 136, 906–912.
Chulalongkorn University. TD is grateful to 16. Wikul, A.; Damsud, T.; Kataoka, K.;
the Science Achievement Scholarship of Phuwapraisirisan, P. Bioorg. Med.
Thailand for graduate fellowship. Chem. Lett. 2012, 22, 5215–5217.
17. Hemalatha, S. J Am Oil Chem Soc 2004,
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Vanillic acid-resorcinol esters with potent anti-oxidation and tyrosinase
inhibitory activities
Jiraporn Kara1, Jindaporn Puripattanavong2, Teerapat Nualnoi3, Luelak Lomlim1*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University,
Hat Yai, Songkhla, 90112, Thailand
2
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of
Songkla University, Hat Yai, Songkhla 90112, Thailand
3
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University,
*E-mail: luelak.l@psu.ac.th
Abstract:
Vanillic acid and resorcinol have been known for its antioxidative activity. Esters of
benzoic acids have been reported as tyrosinase inhibitors. In our on-going research, new
resorcinyl esters of vanillic acid (1, 2) were designed, synthesized and evaluated for
antioxidation and tyrosinase inhibitory activities. Both compounds showed potent
antioxidative activity. Compound 2 (EC50=0.0980.01 M) showed approximately 100 times
greater potency than ascorbic acid (EC50=9.1980.03 M) in DPPH assay. Compound 1
(IC50=10.531.19 M) exhibited potent mushroom tyrosinase inhibitory activity as compared
with kojic acid (IC50=31.873.85M). These novel compounds have high potential for
further development as anti-aging and whitening agents for cosmeceuticals.
1. Introduction resorcinyl esters of vanillic acid and

Skin aging and hyperpigmentation like evaluated their antioxidative and tyrosinase
melasma, ephelides, or sunspots are inhibitory activities to determine their
cosmetic problems for population all over potential for cosmeceutical applications.
the world. Skin aging is determined by
predominance of tissue degeneration over 2. Materials and Methods
regeneration. Reactive oxygen species 2.1 General
(ROSs) is one of the key factors involved in Chemicals and reagents used for the
skin aging.1 Natural and synthetic phenolic synthesis of compounds, tyrosinase
compounds exerted antioxidative activity; inhibitory activity assay and 1,1-diphenyl-2-
the phenolic compounds vanillic acid and picrylhydrazyl (DPPH) assay were
resorcinol are well-known for their radical purchased from Fluka and Sigma-Aldrich.
scavenging property.2,3,4 Ester derivatives of FT-IR spectra were recorded using the
vanillic acid have also been reported as Perkin Elmer (PerkinElmer precisely
effective antioxidant in several free radical Spectrum One FT-IR Spectrometer). Fourier
scavenging assays.5 Hyperpigmentation is a Transform Nuclear Magnetic Resonance
skin problem caused by abnormally high Spectrometer (1H NMR and 13C NMR) were
production of the skin pigment melanin. recorded using the Varian Unity Inova 500
Tyrosinase is known to be the key enzyme MHz. Melting point was measured using the
in the rate-limiting step required for Mel-TEMP II Laboratary devices. Mass
melanogenesis; thus, tyrosinase inhibitors spectrometry was performed using the
are effective skin whitening agents. In Thermo Finnigan MAT 95XL. Two
addition to antioxidative activity, vanillic microplate readers, Varioskan LUX from
acid was reported as tyrosinase inhibitors as Thermo Scientific and Biotek Power from
well.6 In this work, we synthesized BioTek Instruments, were used in this study.
2.2 Synthesis
The synthesis pathway of 1 started to afford the carboxylic acid 5. Coupling of
with mono protection of resorcinol by 5 with the phenol 3 gave ester 6. Finally, the
benzylation to obtain benzyl ether 3. The phenolic groups of ester 6 were de-protected
phenolic group of vanillic acid was also via hydrogenolysis to give the desired
protected via benzylation; during this compound 1. Compound 2 was prepared
process the carboxylic group was also from the coupling reaction between
benzylated resulting in the benzyl ester 4. unprotected resorcinol and excess amount of
Hydrolysis of the ester 4 under basic the acid 5, followed by hydrogenolysis
conditions selectively cleaved the ester bond (Figure 1).
i
HO O
HO OH
Resorcinol 3
O O
O
H3CO H3CO
H3CO ii O iii OH
OH
O O
HO
Vanillic acid 4 5
O
O
H3CO
iv O O v H3CO
5+3 O OH
O 1
6 HO
O O
H3CO OCH3
iv O O
5 + Resorcinol
O O
7
O O
H3CO OCH3
O O
HO 2 OH
Figure 1. The general procedure of synthesis of resorcinol esters of vanillic acid: (i) K2CO3,
KI, acetone, benzyl chloride, 48 °C; (ii) DMF, K2CO3, benzyl chloride, room temperature;
(iii) EtOH, KOH, H2O, 85 °C; (iv) DMF, DMAP, EDCI, room temperature; (v) EtOAc,
Pd(OH)2, room temperature
The synthesis of all compounds in and the residue was washed with acetone.
this work is described below. The collected filtrate was concentrated using
3-(Benzyloxy)phenol (3): a rotary evaporator. The residue was
Resorcinol (3.00 g, 27.2 mmol) was purified by silica gel column
dissolved in acetone (27 mL) and then chromatography, using hexane and EtOAc
K2CO3 (5.66 g, 41.0 mmol) and KOH were (7:3) as a mobile phase and then
added. Then, benzyl chloride solution (1.72 recrystallized in MeOH.
g, 13.6 mmol, in 5 mL of acetone) was 4-(Benzyloxy)-3-methoxybenzoate
slowly added into the resorcinol solution. (4): Vanillic acid (3.20 g, 19.0 mmol) was
The reaction mixture was stirred at 48 °C for dissolved in DMF (25 ml). K2CO3 (7.92 g,
45 h. The solid was removed by filtration 57.3 mmol) and benzyl chloride (6.00 ml,
206.6 mmol) were added. The reaction 1,3-Phenylene bis(4-(benzyloxy)-3-
mixture was stirred at room temperature for meth-oxybenzoate) (7): Resorcinol (0.10 g,
22 h. The product was extracted with diethyl 0.9 mmol), 1-ethyl-3-(3-dimethyl-amino-
ether (50 mL) and H2O (100 mL), then re- propyl)carbodiimide (0.35 g, 2.3 mmol),
extracted with diethyl ether twice (100 mL 3,4-dibenzyloxycinnamic acid (0.80 g, 2.3
each). The organic layer was washed with mmol), and 4-dimethylamino-pyridine (0.27
H2O and brine, dried with anhydrous g, 2.3 mmol) were dissolved in 10 mL of
Na2SO4 and evaporated with a rotary DMF. The reaction mixture was stirred at
evaporator. The crude product was purified room temperature for 24 h. The product was
by silica gel column chromatography, using extracted with H2O (50 mL) and EtOAc (3 x
hexane and EtOAc (7:3) as a mobile phase 20 mL). The organic layer was washed with
and then recrystallized in MeOH. water and brine and then dried over
4-(Benzyloxy)-3-methoxybenzoic anhydrous Na2SO4. The crude product was
acid (5): 4-(Benzyloxy)-3-methoxybenzoate collected by filtration and washed with
(2.50 g, 7.2 mmol) was dissolved in 110 mL EtOAc. The organic layer was evaporated
absolute EtOH, followed by adding 27 mL with a rotary evaporator. The crude product
of KOH solution (2.01 g, 0.04 mmol) in was purified by silica gel column
H2O. The reaction mixture was stirred at 85 chromatography, using hexane and EtOAc
°C for 2 h. Then the mixture was extracted (7:3) as a mobile phase.
with diethyl ether (40 mL) and H2O (120 3-Hydroxyphenyl 4-hydroxy-3-
mL). The aqueous phase was acidified with methoxy-benzoate (1): 3-(Benzyloxy)
1 N H2SO4 and then re-extracted with phenyl 4-(benzyloxy)-3-methoxybenzoate
EtOAc (80 mL). The organic layer was (0.1 g, 0.2 mmol) was dissolved in EtOAc
washed with H2O and brine, and dried over (30 mL). 0.006 g of Pd(OH)2 was added.
anhydrous Na2SO4. The solvent then was The reaction mixture was stirred at room
evaporated using a rotary evaporator. The temperature under hydrogen atmosphere for
residue was purified by silica gel column 4 h. The reaction mixture was filtered
chromatography, using hexane and EtOAc through celite and washed with EtOAc. The
(7:3) as a mobile phase. organic layer was evaporated with a rotary
3-(Benzyloxy) phenyl 4- evaporator. The crude product was purified
(benzyloxy)-3-methoxybenzoate (6): 3- by silica gel column chromatography, using
(Benzyloxy) phenol (0.09 g, 0.5 mmol) and CH2Cl2 and MeOH (95:5) as a mobile phase.
1-ethyl-3-(3-dimethylaminopropyl) 1,3-Phenylene bis(4-hydroxy-3-
carbodiimide (0.10 g, 0.7 mmol) were methoxy-benzoate) (2): 1,3-Phenylene bis-
dissolved in DMF (3 mL). Benzyl 4- (4-(benzyloxy)-3-methoxybenzoate) (0.06 g,
(benzyloxy)-3-methoxy-benzoic acid (0.1 g, 0.1 mmol) was dissolved in EtOAc (60 mL)
0.4 mmol), 4-dimethylaminopyridine (0.047 and 0.003 g of Pd(OH)2 was added. The
g, 0.4 mmol) added. The reaction mixture reaction mixture was stirred at room
was stirred at room temperature for 24 h. temperature under H2 atmosphere for 4 h.
The product was extracted with H2O (50 The reaction mixture was filtered through
mL) and EtOAc (3 x 20 mL). The organic celite and washed with EtOAc. The organic
layer was washed with water and brine and layer was evaporated with rotary evapolator.
then dried over anhydrous Na2SO4. The The crude product was purified by silica gel
crude product was collected by filtration and column chromatography, using CH2Cl2 and
washed with EtOAc. The organic layer was MeOH (95:5) as a mobile phase.
evaporated with a rotary evaporator. The
crude product was purified by silica gel 2.3 DPPH assay
column chromatography, using hexane and The assay for DPPH free-radical
EtOAc (8:2) as a mobile phase. scavenging activity was performed as
described by Yamasaki et al.1 The stock
solutions of samples were prepared by triplicate. The percentage of tyrosinase
dissolving the samples in absolute EtOH to a inhibitory activity was calculated as
concentration of 1 mg/mL. The stock followed.
solution of each sample was then diluted to
yield 10 different concentrations (1-10 %inhibition = (ABlank-AInhibitor) x 100
μg/mL). The sample solutions (100 μL) ABlank
were transferred into a microcentrifuge tube
and 100 μL of DPPH (0.6 μM) was added to Ablank = the absorbance of tyrosinase
each tube. The mixture was shaken and solution without sample
incubated at room temperature for 30 min in AInhibitor = the absorbance of tyrosinase
the dark cabinet. After incubation, the solution with sample solution
absorbance at 517 nm was measured using
The IC50 value of each sample was
100 μL absolute EtOH as blank and L-
determined using the same protocol as for
ascorbic acid was used as a reference
the determination of %inhibitory activity;
standard for the assay. Each sample was
only eight different concentrations of the
analyzed in triplicate. The percentage of
tested compounds (ranging from 0.39-300
DPPH scavenging activity was calculated as
μM) were used in the assay. IC50 values
followed.
were determined graphically from log
%inhibition = (Acontrol-Asample) x 100 concentration–inhibition curves using the
Acontrol Software GraphPad Prism
Acontrol = the absorbance of DPPH solution 3. Results and Discussion

without sample 3.1 Chemistry
Asample = the absorbance of DPPH solution 3-(Benzyloxy)phenol (3)7: yield
with sample solution 51% (2.80 g, 14.0 mmol); brown solid, m.p.
54-56 °C. IR: (KBr, cm-1) 3395, 3063, 3032,
The dose-response curve was plotted 2930, 1284, 1147, 695; 1H NMR (ppm,
between %inhibition and concentrations of CDCl3): δ 4.99 (2H, s), 5.02 (1H, s), 6.42-
the compounds. Linear regression analysis 6.56 (3H, m), 7.12 (1H, t, J= 8.2 Hz), 7.29-
was carried out for calculating the effective 7.42 (5H, m).
concentration of sample required to 4-(Benzyloxy)-3-methoxybenzoate
scavenge DPPH radical by 50% (EC50). (4)8: yield 51% (3.40 g, 19.02 mmol);
2.4 Tyrosinase inhibitory activity yellow solid, m.p. 184-185 °C. IR: (KBr,
The tyrosinase inhibitory activity cm-1) 3065, 3032, 2935, 1706, 1649, 1629,
assay was performed by the modified 1270, 1213, 1177, 1131; 1H NMR (ppm,
dopachrome method using L-tyrosine as a
substrate. Each sample was dissolved in 4% CDCl3): : δ 3.85 (3H, s), 5.14 (2H, s), 5.26
DMSO at a concentration of 100 µM. The (2H, s), 6.81 (1H, d, J=8.4 Hz), 7.18-7.37
assay mixture consisting of 140 μL of (10H, m), 7.52 (1H, d, J= 2.0 Hz), 7.56-7.58
phosphate buffer (20 mM, pH 6.8), 20 μL of (1H, dd, J= 8.4, 2.0 Hz).
the sample solution and 20 μL mushroom Synthesis of 4-(benzyloxy)-3-
tyrosinase (30 U/mL) was incubated in a 96- methoxy-benzoic acid (5)9: yield 90% (1.65
well plate at room temperature for 10 min. g, 7.18 mmol); yellow solid, m.p. 160-163
After the incubation, 20 μL of L-DOPA °C. IR (KBr, cm-1): 3432, 3070, 2941, 1676,
(0.85 mM) was added, followed by the 1279, 1233, 1136, 1026; 1H NMR (ppm,
product dopachrome in the reaction mixture DMSO-d6): δ 3.80 (3H, s), 5.15 (2H, s),
then was determined by measuring the 7.13 (2H, d, J=8.4 Hz), 7.31-7.46 (6H, m),
absorbance at 475 nm using microplate 7.53 (1H, dd, J= 8.4, 2.0 Hz), 12.63 (1H, s,
reader. Kojic acid was used as a positive OH).
control. Each sample was analyzed in
Synthesis of 3-(benzyloxy) phenyl J=1.94 Hz), 7.78 (2H, dd, J=8.3, 1.9 Hz);
4-(benzyloxy)-3-methoxybenzoate (6): 13
C NMR (ppm, CDCl3): δ 56.15, 112.12,
yield 77% (0.132 g, 0.30 mmol); pale yellow 114.38, 115.94, 119.15, 121.15, 125.06,
solid, m.p. 140-142 °C. IR: (KBr, cm-1) 129.73, 146.28, 150.74, 151.50, 164.57; HR-
3067, 2929, 1700, 1632, 1262, 1222, 1132, MS: (m/z, [M+1]+) 411.1068 (calcd for
1021; 1H NMR (ppm, CDCl3): δ 3.95 (4H, C22H19O8, 411.1079).
s), 5.24 (3H, s), 6.69-7.47 (15H, m), 7.66 3.2 Biological activity
(1H, d, J=1.9 Hz), 7.76 (1H, dd, J=8.3, 1.9 Compounds 1 and 2 were evaluated
Hz). for their antioxidation and tyrosinase
Synthesis of 1,3-phenylene bis(4- inhibitory activities. In DPPH assay, both
(benzyloxy)-3-methoxybenzoate) (7): yield compounds showed good to excellent
65% (0.35 g, 0.59 mmol); pale yellow solid, antioxidation activity. Compound 1 (EC50
m.p. 140-142 °C. IR: (KBr, cm-1) 3084, =2.34±0.02 µM) exhibited 4 times higher
2927, 1727, 1267, 1208, 1124; 1H NMR potency than ascorbic acid (EC50=
(ppm, CDCl3): δ 3.95 (6H, s), 5.24 (4H, s), 9.198±0.03 µM). Interestingly, compound 2
6.87-7.77 (20H, m). (EC50=0.098±0.01 µM) showed
Synthesis of 3-hydroxyphenyl 4- approximately 100 times greater potency
hydroxy-3-methoxybenzoate (1): yield than the reference compound. Possibly, the
84% (0.05 g, 0.19 mmol, overall high DPPH scavenging activity of these
yield=15%); pale yellow solid, m.p. 156-158 compounds could be a result from their
°C. IR: (KBr, cm-1) 3447, 3120, 2927, 1747, polyphenol character.
1264, 1230, 1121, 1019; 1H NMR (ppm, Tyrosinase inhibitory activity of
compounds 1 and 2 were also determined.
CDCl3): δ 3.94 (3H, s), 5.98 (1H, s), 6.17 Initially, %inhibition of each tested
(1H, s), 6.66-7.24 (5H, m), 7.73 (1H, d, compounds was determined as a screening
J=1.9 Hz), 7.78 (1H, dd, J=8.3, 1.9 Hz); 13C test. At 100 µM, compound 1 demonstrated
NMR (ppm, CDCl3): δ 56.16, 109.45, strong inhibitory activity against tyrosinase
112.16, 113.18, 113. 71, 114.26, 121.26, (98% inhibition) while compound 2
125.10, 130.08, 146.29, 150.73, 151.78, exhibited a weaker inhibitory activity of
156.78, 165.23; HR-MS: (m/z, M+1+) 34% inhibition (Table 1). In addition,
261.0763 (calcd for C14H13O5, 261.0762). compound 1 exhibited approximately 3
Synthesis of 1,3-phenylene bis(4- times stronger tyrosinase inhibitor than kojic
hydroxy-3-methoxybenzoate) (2): yield acid as demonstrated by the IC50 values
66% (0.027 g (0.07 mmol, overall (10.53±1.19 µM and 31.87±3.85 µM,
yield=10%); pale yellow solid, m.p. 161-162 respectively). Due to high lipophilicity,
°C. IR: (KBr, cm-1) 3400, 3082, 2927, 1727, compound 2 precipitated under the assay
1284, 1206, 1125, 1067; 1H NMR (ppm, condition; thus, the IC50 values could not be
CDCl3): δ 3.96 (6H, s), 5.24 (4H, s), 6.15 determined.
(2H, s), 6.98-7.46 (6H, m), 7.65 (2H, d,
Table 1. Antioxidant capacity and tyrosinase inhibitory activity of the synthesized
compounds
Tyrosinase inhibitory activity
Compound DPPH %inhibition IC50

(EC50, M± SEM) (at 100 M) (M±SEM)
O 2.34±0.02 98 10.53±1.19
H3CO
O OH
HO
1
O O
H3CO OCH3
O O 0.098±0.01 34 n.d.
HO OH
Kojic acid n.d. 87 31.87±3.85
Ascorbic acid 9.198±0.03 n.d. n.d.
n.d. = Not determined
4. Conclusion 2. Rice-Evans, C.; Miller N.; Paganga G.

Vanillic acid-resorcinol esters (1 and Trends Plant Sci. 1997, 2, 152–159.
2) have been synthesized from vanillic acid 3. Karamac, M.; Kosiñska1, A.; Pegg,
and resorcinol. Both compounds exhibited R.B. J. Food Nutr. Sci. 2005, 14/55,
more potent radical-scavenging activities as 165–170.
compared with ascorbic acid. Compound 2 4. Bendary, E.; Francis, R.R.; Ali H.M.G.;
showed approximately 100 times greater Sarwat M.I.; Hady S.E. Ann. Agric. Sci.
DPPH radical scavenging activity than 2013, 58, 173–181.
ascorbic acid. Compound 1 showed 5. Tai, A.; Sawano, T.; Ito, H. Biosci.
approximately 3 times greater tyrosinase Biotechnol. Biochem. 2012, 76, 314–
inhibitor than kojic acid. These results 318.
indicated that these novel compounds have 6. Chou, T. H.; Ding, H. Y.; Hung, W. J.;
high potential for further development as Liang, C. H. Origanum vulgare. Exp.
anti-aging and skin whitening agents for Dermatol. 2010, 19, 742–750.
cosmeceuticals. 7. Frlan, R.; Gobec, S.; KiKelj, D.
Tetrahedron 2007, 63, 10698–10708.
Acknowledgements 8. Arnott, E. A.; Crosby, J.; Evans, M. C.;
This work is supported by the Faculty Ford, J. G.; Jones, M. F.; Leslie, K. W.;
of Pharmaceutical Sciences, Prince of Mcfarlane, I. M.; Sependa, G. J. PCT
Songkla University (Grant No. Int. Appl. 200805322, 2008.
PHA590447S). 9. Rao, M. L. N.; Awasthi, D. K.;
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Rev. 2015, 21, 16–29.
N-(2-(Dimethylamino)ethyl)-4-oxo-4H-chromene-2-carboxamide analogues
as acetylcholinesterase inhibitors
Paptawan Suwanhom1, Teerapat Nualnoi2, Vannajan Sanghiran Lee 3, Luelak Lomlim1*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University,
2
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University,
3
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia
*E-mail: luelak.l@psu.ac.th
Abstract:
Chromone derivatives have gained much interest in drug discovery for the treatment
of Alzheimer’s disease. From our current study on the design and synthesis of 4-oxo-4H-
chromene-2-carboxamides as acetylcholinesterase inhibitors (AChEIs), we found N-(2-
(dimethylamino)ethyl)-4-oxo-4H-chromene-2-carboxamide (1) as a moderately active AChEI
(IC50=7.58±0.91 µM). In this study, 7-methoxy- and 6,7-dimethoxy 4-oxo-4H-chromene-2-
carboxamide analogues (2 and 3) were designed and synthesized to explore structure-activity
relationship (SAR). Compounds 1-3 were obtained via two-step synthesis. Chromone ring
formation was achieved by Claisen condensation to yield ethyl-4-oxo-4H-chromene-2-
carboxalate derivatives. Aminolysis of the ester gave the desired products as yellow solid in
good yields. (>60%). Methoxyl group(s) on the chromone ring enhanced AChEI activity.
Compounds 2 (IC50=1.05±0.22 µM) and 3 (IC50=0.14±0.03 µM) were approximately 7 and
54 times more potent than 1. Furthermore, 3 showed acetylcholinesterase inhibitory activity
in the same range as tacrine (IC50=0.13±0.02 µM) but it was less active than donepezil
(IC50=0.006±0.001 µM). Methoxyl substituents also increased selectivity for
acetylcholinesterase (AChE) as compared to butyrylcholinesterase (BChE). Molecular
docking has suggested the interaction and binding affinity of each compound with two target
enzymes which fitted well with the in vitro results.
1. Introduction inhibition of both enzymes has been

Alzheimer’s disease (AD) is a accepted as significant target for the
progressive and fatal neurodegenerative effective management of AD by increasing
disease, clinically characterized by memory of synaptic ACh in the brain3. Donepezil
and cognitive dysfunctions. It is primarily a was approved in 1996 for treatment of mild
disease of old age and is the most prevalent to moderate AD. It has 570 to 1250-fold
cause of dementia in elderly people. 1, 2 selectivity for AChE than for BChE and
Cholinesterase (ChE) inhibitors were the inhibits AChE in dual binding site manner.
first therapies approved in the U.S. for AD The drug molecule interacts with various
and currently be the firstline agents for the amino acid residues both in the catalytic
treatment of AD (e.g. tacrine, galanthamine, anionic site (CAS) and peripheral anionic
rivastigmine and donepezil). The drugs site (PAS) of the enzymes. 4
inhibit breakdown of the neurotransmitter Chromone derivatives have attracted
acetylcholine (ACh) to increase amount of much attention in recent years because of
the cholinergic transmission. ACh can be their diverse pharmacological properties.5
degraded by two types of cholinesterase, Therefore, in the present study, in order to
namely acetylcholinesterase (AChE) and enhance the pharmacological potential of
butyrylcholinesterase (BChE). The this scaffold, N-(2-(dimethylamino)ethyl)-4-
oxo-4H-chromene-2-carbo xamide ml) and extracted with diethyl ether (3x80
analogues were designed, synthesized and ml). The organic layer was concentrated by
evaluated as anti-AD. Molecular docking O
R1 OH O R1 O
study was performed by Autodock VINA H3C O
O CH3 (i) OEt
1.1.2 to investigate the binding mode of R2

O
O R2
O
interaction. a, R1=R2= H
Diethyloxalate d, R1=R2= H
b, R1=OCH3, R2= H e, R1=OCH3, R2= H
c, R1=R2= OCH3 f, R1=R2= OCH3
2. Materials and Methods O O CH3
R1 O R1 O N
2.1 General OEt (ii) N
H
CH3
All chemicals and reagents used in R2

O
R2
O
synthesis, AChE, BChE, 5,5'-dithiobis-(2- d, R1=R2= H 1, R1=R2= H
e, R1=OCH3, R2= H 2, R1=OCH3, R2= H
nitrobenzoic acid (DTNB), acetylthiocho- f, R1=R2= OCH3 3, R1=R2= OCH3
line iodide (ATCI), butyrylthiocholine Figure 1. Synthesis of chromone derivatives
iodide (BTCI), tacrine and donepezil were (1-3): (i) NaH/THF, rt; (ii) N1,N1-
purchased from Sigma-Aldrich. FT-IR dimethylethane-1,2-diamine/CH2Cl2 /reflux,
spectra were recorded using the Perkin then CH3COOH, 70 °C
Elmer precisely. 1H-NMR and 13C-NMR
spectra were recorded using the Varian rotary evaporator to give a yellow solid. The
Unity Inova 500 MHz. Melting Point crude products were purified by column
Apparatus were recorded using the Mel- chromatography employing EtOAc:Hexanes
TEMP II LABORATORY DEVICES. USA. (40:60) as a mobile phase followed by
ESI-MS spectra were recorded on a Thermo crystallization from ethylacetate.
Finnigan MAT 95XL and Microplate reader 2.2.1.1 Ethyl-4-oxo-4H-chromene-2-car-
was recorded using the Power Wave x, boxylate (d) Pale yellow solid (82% yield),
Biotele. m.p. 100-102 °C; IR (KBr):(cm-1) 3448,
2.2 Synthesis of target compounds 1-3 and 2984, 1740, 1658, 1465, 1258, 772. 1H-
characterization. NMR: (DMSO-d6, ppm) 1.32 (t, J=7.1 Hz,
Three chromone derivatives (1-3) 3H), 4.37 (q, J=7.1 Hz, 2H,), 6.92 (s, 1H,),
were designed and synthesized as shown in 7.51 (ddd, J=8.1, 7.2, 1.0 Hz, 1H), 7.71 (m,
Figure 1. Ethyl-4-oxo-4H-chromene-2-car 1H), 7.85 (ddd, J=8.6, 7.2, 1.7 Hz, 1H), 8.02
boxylate derivatives (d-f) were prepared (dd, J=8.1, 1.6 Hz, 1H).
from Claisen condensation between 2.2.1.2 Ethyl 7-methoxy-4-oxo-4H-chro-
substituted 2-hydroxyacetophenone (a-c) mene-2-carboxylate (e) Pale yellow solid
and diethyloxalate. Chromone-2-carboxa (78% yield), m.p. 113-115 °C; IR (KBr)
midoalkylamine derivatives (1-3) were :(cm-1) 3462, 2919, 1743, 1663, 1439,
prepared by aminolysis of ethyl-4-oxo-4H - 1257, 778. 1H-NMR: (DMSO-d6, ppm) 1.34
chromene-2-carboxalate derivatives (d-f) (t, J= 7.1 Hz, 3H), 3.92 (s, 3H), 4.39 (quin,
with the corresponding N-alkyl-dimethyl J= 7.1 Hz, 2H), 6.88 (s, 1H), 7.10 (dd, J=
amines to give chromone derivatives (1-3). 8.9, 2.4 Hz, 1H), 7.23 (d, J= 2.4 Hz, 1H),
2.2.1 General procedures for the 7.94 (d, J= 8.9 Hz, 1H).
preparation of ethyl 4-oxo-4H-chro mene- 2.2.1.3 Ethyl-6, 7-dimethoxy-4-oxo-4H-
2-carboxylate derivatives (d, e, f) chromene-2-carboxylate (f) Pale yellow
To a slurry of sodium hydride (65 solid (86% yield), m.p. 116-117 °C; IR
mmol) stirred in THF (10 ml), a mixture of (KBr):(cm-1) 3606, 2983, 1742, 1632,
substituted 2-hydroxyacetophenone (15 1510, 1255, 865. 1H-NMR: (DMSO-d6,
mmol) and diethyloxalate (35 mmol) in THF ppm) 1.34 (t, J= 7.1 Hz, 3H), 3.86 (s, 3H),
(20 ml) were added and the reaction mixture 3.94 (s, 3H), 4.39 (quin, J= 7.1 Hz, 2H),
was stirred overnight at room temperature 6.89 (s, 1H), 7.29 (s, 1H), 7.35 (s, 1H).
under nitrogen atmosphere. It was then
poured into ice-water (70 ml), HCl (6N, 20
1
2.2.2 General procedures for the H-NMR: (DMSO-d6, ppm) 2.22 (s, 6H),
preparation of chromone-2-carbox 2.50 (m, 2H), 3.41 (m, 2H), 3.86 (s, 3H),
amido-alkylamine derivatives (1-3) 3.93 (s, 3H), 6.75 (s, 1H), 7.23 (s, 1H), 7.34
Ethyl-4-oxo-4H-chromene-2-carbo (s, 1H), 9.00 (t, J= 5.7 Hz, 1H). 13C-NMR:
xylate derivatives (d-f, 0.46 mmol) and N, (DMSO-d6, ppm) 37.7, 45.6, 56.3, 56.8,
N-dimethylenediamine (1.38 mmol) were 58.2, 100.9, 103.9, 110.4, 117.4, 148.2,
dissolved in dichloromethane (5 ml) and 151.5, 155.2, 155.3, 159.5, 176.5. ESI-MS:
allowed to reflux for 20 minutes, glacial (m/z, [M+H]+) 321.1450 (calcd for
acetic acid (5 ml) was added and the C16H21N2O5, 321.1450).
solution was stirred at 75 °C under nitrogen 2.3 In vitro inhibition studies on AChE
for 24 hours. The mixture was extracted and BChE
with ethylacetate (3x80 ml). The organic In vitro, the AChE and BChE
layer was dried by rotary evaporator to give inhibitory activities of the synthesized
yellow solution. The product was purified compounds were evaluated with a method
by column chromatography technique by modified from Ellman’s method6 by using
using CH2Cl2: CH3OH (80: 20) as mobile AChE from Electrophorus electricus
phase and then recrystallized in ethylacetate. (electric eel) and BChE from horse serum.
2.2.2.1 N-(2-(Dimethylamino)ethyl)-4-oxo- This assay was carried out on a 96-well
4H-chromene-2-carboxamide (1) Pale plate, 25 µL of 100 µM sample dissolved in
yellow solid (64% yield), m.p. 97-100 °C; ethanol, 125 µL of 3 mM 5,5'-dithiobis(2-
IR (KBr):(cm-1) 3851, 3311, 2947, 1652, nitrobenzoic acid) (DTNB), 25 µL of 1.5
1532, 1464, 1387, 1018, 758. 1H-NMR: mM of substrate acetylthiocholine iodide
(DMSO-d6, ppm) 2.21 (s, 6H), 2.48 (m, 2H), (ATCI) and butyrylthiocholine iodide
3.39 (m, 2H), 6.81 (s, 1H), 7.53 (m, 1H), (BCTI), 50 µL of 50 mM Tris/HCl (pH= 8)
7.72 (m, 1H), 7.88 (ddd, J=8.5, 7.1, 1.7 Hz and followed by 25 µL of enzyme. The
1H), 8.04 (dd, J=7.8, 1.9 Hz, 1H), 9.03 (t, J= microplate was then read at 405 nm every
5.5 Hz, 1H). 13C-NMR: (DMSO-d6, ppm) 11s for 2 minutes. Each experiment was
34.4, 45.2, 57.8, 110.6, 118.9, 123.7, 124.9, repeated in triplicate. The result of the
126.1, 135.0, 155.2, 155.8, 159.1, 177.4. measurements showed mean velocity value
ESI-MS: (m/z, [M+H]+) 261.1234 (calcd for which was obtained from graph plotted
C14H17N2O3, 261.1239). between times and absorbance. The
2.2.2.2 N-(2-(Dimethylamino)ethyl)-7-met percentage inhibition of all compounds (1-3)
hoxy-4-oxo-4H-chromene-2-carboxamide for the AChE and BChE inhibitory activity
(2) Pale yellow solid (74% yield), m.p. 117- was calculated using the following formula:
119 °C; IR (KBr):(cm-1) 3315, 2947, 2823,
1649, 1609, 1439, 1386, 1022, 833. 1H- %inhibition = (A-B) x 100
NMR: (DMSO-d6, ppm) 2.25 (s, 6H), 2.52 A
(m, 2H), 3.41 (m, 2H), 3.91 (s, 3H), 6.74 (s,
A = Mean velocity of blank
1H), 7.10 (dd, J= 8.9, 2.4 Hz, 1H), 7.17 (d,
B = Mean velocity of sample
J= 2.4 Hz, 1H), 7.94 (d, J= 8.9 Hz, 1H),
9.01 (t, J= 5.5 Hz, 1H). 13C-NMR: (DMSO-
The IC50 values were determined graphically
d6, ppm) 36.7, 44.6, 56.6, 57.4, 101.4, 111.0,
from inhibition curve (log inhibitor
115.8, 117.9, 126.9, 155.6, 157.4, 159.8,
concentration VS percent of inhibition) and
164.8, 177.0. ESI-MS: (m/z, [M+H]+)
done under the same protocol of inhibitory
291.1338 (calcd for C15H19N2O4, 291.1345).
activity test. Eight different concentrations
2.2.2.3 N-(2-(Dimethylamino)ethyl)-6,7-di
of the inhibitor (50 µM – 1.6 x 10-3 µM)
methoxy-4-oxo-4H-chromene-2-carboxa
were used. The response curve was created
mide (3) Pale yellow solid (64% yield), m.p.
using GraphPad Prism 2.01. Donepezil and
115-118 °C; IR (KBr):(cm-1) 3446, 2923,
tacrine were used as positive controls.
2863, 1642, 1598, 1536, 1273, 1021, 863.
2.4 Molecular modeling study Table 1. An in vitro AChE and BChE
Molecular docking studies inhibitory activities of chromone derivatives
demonstrated interactions between enzyme and reference compounds (cpds)
and ligands by computer program which IC50±SD (µM)
Cpds R1 R2 SI
calculated the interaction from the three A B
dimensional structure. The crystal structure 1 H H 7.58±0.91 22.95±0.04 3.03
of the Torpedo californica 2 OCH3 H 1.05±0.22 17.82±0.92 16.97
3 OCH3 OCH3 0.14±0.03 44.56±0.46 318.29
acetylcholinesterase (TcAChE, code ID:
TAC - - 0.13±0.02 0.01±0.001 0.86
1EVE) and Human butyrylcholinesterase
DPZ - - 0.006±0.01 1.06±0.02 176.67
(HuBChE, code ID: 4BDS) were achieved
TAC = Tacrine
from protein data bank (http://www.pdb.org) DPZ = Donepezil
and the molecular docking studies were A=Acetylcholinesterase enzyme from electric eel
performed using the AutoDock Vina 1.1.27 B=Butyrylcholinesterase enzyme from horse serum
where the binding site was covered the SI=Selectivity index= (IC50 of BChE/IC50 of AChE)
active site (PAS and CAS) of the enzyme.
The grid boxes at the center of active site of While all the target compounds
enzyme were defined. For TcAChE, the showed weak activity against BChE
center of the grid box was placed at the (17.82±0.92-44.56±0.46 µM) and showed
bottom of the active site gorge (x= 2.023, y= good inhibition selectivity (3.03-318.29)
63.295, z= 67.062). The dimensions of the against AChE over BChE. From these
active site box were set at 50 Å x 50 Å x 50 results, methoxyl group(s) on the chromone
Å. For HuBChE, the center of the grid box ring seemed to enhance AChEI activity.
determined at x= 135.117, y= 119.222, z= Compounds 2 (IC50= 1.05 ±0.22 µM) and 3
40.667. The dimensions of the active site (IC50= 0.14±0.03 µM) were approximately 7
box were set at 34 Å x 46 Å x 34 Å. After and 54 times more potent than compound 1.
that, ligands were put in the grid box to Furthermore, compound 3 showed
calculate free energy of binding (G). acetylcholinesterase inhibitory activity in the
Ligands were arranged by the calculated G same range as tacrine (IC50=0.13±0.02 µM)
but it was less active than donepezil (IC50=
value; lower G values correspond to more
0.006±0.001 µM). Methoxyl substituents
desirable ligand binding, while higher G
also increased selectivity for
values are less desirable8. The docking score
acetylcholinesterase as compared to
is the predicted binding affinity in kcal/mol.
butyrylcholinesterase.
3.2 Molecular modeling study
With the aim to understand the
3.1 In vitro inhibition studies on AChE
ligand-protein interaction at atomic level
and BChE
detail, molecular docking studies were
The biological activity profiles of the
performed for the most potent AChE
compounds 1-3 as eeAChE inhibitors were
inhibitor reported in this work compared
assayed in comparison with tacrine and
with BChE inhibitor. The result showed
donepezil as reference compounds and
affinity of binding energy (kcal/mol)
evaluated the inhibitory potency of these
between enzyme and compound as shown in
compounds against BChE from horse serum,
in order to gain insight about inhibitor’s Table 2. Affinity of binding energy of
selectivity against both enzymes. The results compounds 1-3, tacrine and donepezil
are summarized in Table 1. binding affinity (kcal/mol)
From Table 1, the target compounds Cpds
TcAChE HuBChE
exhibited different amounts of inhibitory 1 -7.9 -7.6
activity to AChE with IC50 values ranging 2 -7.9 -7.8
3 -8.0 -6.8
from the submicromolar to micromolar
Tacrine -8.3 -8.4
range (0.14±0.03-7.58±0.91 µM). Donepezil -10.9 -9.1
Table 2. Compound 3-TcAChE (a) showed complex, 4-carbonyl group at the chromone
affinity the binding energy -8.0 kcal/mol. In exhibits hydrogen bonding with Gly113 and
the complex, the chromone ring was Gly114 at distance of 3.02 Å. The long
observed to bind to the PAS via a π-π chain of methylene was observed to bind to
interaction with Trp278 (distance of 3.79 Å) the mid-gorge via a hydrophobic interaction
and 4-carbonyl group at the chromone show with Trp79 and cation-π interaction between
hydrogen bond with Phe287 and Phe288 dimethylamine and Trp425 at the distance of
(distance of 3.19 Å). The long chain of 4.09 Å.
methylene was observed to bind to the mid- Tacrine-TcAChE (b) showed the
gorge via a hydrophobic interaction with binding affinity of -8.3 kcal/mol. This
Phe330 and Tyr333. The dimethylamine was compound bound only with CAS of active
observed to bind to the CAS via a cation-π site of enzyme. The structure of tacrine
interaction with Phe329 (distance of 3.94 Å) showed stacking interaction with Trp83 at
while compound 3-HuBChE (d) showed the the distance of 3.89 Å and Phe329 of active
binding energy of -6.8 kcal/mol. This site of of TcAChE. The nitrogen atom on the
a b c
d e f
Figure 2. Molecular docking study of a, b, c-TcAChE and d, e, f-HuBChE
tetrahydroacridine ring exhibited hydrogen µM) in the same range as tacrine (IC50=
bond with carbonyl oxygen of His439 at the 0.13±0.02 µM) but it was less active than
distance of 3.10 Å. Tacrine-HuBChE (e) had donepezil (IC50= 0.006±0.001 µM).
the binding affinity of -8.4 kcal/mol and Furthermore, molecular docking studies
showed π-π interaction with Trp79 (distance indicated compound 3 is able to bind to both
3.50 Å). CAS and PAS in AChE. These results were
Donepezil-TcAChE (c) showed the consistent with the IC50 values. This
binding affinity of -10.9 kcal/mol. The information is valuable for further development
indanone ring of this complex was observed of new AChEIs with higher potency.
to bind to the PAS via a π-π interaction with
Trp278 at the distance of 3.62 Å. The long Acknowledgements
chain of methylene was observed to bind to This work was supported by Prince
the mid-gorge via a hydrophobic interaction of Songkla University (Grant No.
with Phe330. The piperidine ring was PHA590412S) and Universiti Malaya
observed to bind to the CAS via a cation-π Research Grant (UMRG:RP037D-17AFR).
interaction with Phe329 at the distance of
3.96 Å and benzyl group of compound. It Reference
also can interact with π-π interaction by 1. Skovronsky, D. M.; Lee V. M.-Y.;
Trp83 at the distance of 4.15 Å. Donepezil- Trojanowski, J. Q. Annu. Rev. Pathol.
HuBChE (f.) showed the binding energy Mech. Dis. 2006, 1, 151–170.
affinity of -9.1 kcal/mol. This complex 2. Mayeux, R. Annu. Rev. Neurosci. 2003,
exhibited hydrophobic interaction with 26, 81–104.
Tyr329 at mid-gorge of active site and π-π 3. Greig, N. H.; Lahiri, D. K.; Samba
interaction between the benzyl group and murti, K. Int Psychogeriatr. 2002, 14,
Trp79 at the distance of 3.75 Å as shown in 77–91.
Figure 2. 4. Mayeux, R.; M. D.; Sano, M. N Engl J.
Med. 1999, 341, 1670–1679.
4. Conclusion 5. Liu, Q.; Qiang, X.; Li, Y.; Sang, Z.; Li,
In conclusion, 3 new chromone-2- Y.; Tan, Z.; Deng, Y. Bioorg. Med.
carboxamido-alkylamine derivatives (1-3) Chem. 2015, 23, 911–923.
have been designed, synthesized and 6. Ellman, G. L.; Courtney, K. D.; Andres,
evaluated as cholinesterase (AChE and V.; Feather-stone, R. M. Biochem.
BChE) inhibitors. All of these synthetic Pharm. 1961, 7, 88–95.
compounds showed potent AChE inhibitory 7. Trott, O.; Olson, A. J. J. Comput. Chem.
activity in vitro and high selectivity for 2010, 31, 455–461.
AChE over BChE. Compound 3 exhibited 8. Jacob, R. B.; Andersen, T.; McDougal,
the best inhibitory activity (IC50=0.14±0.03 O. M. PLoS Comput Biol. 2012, 8, 1–5.
Bromodiethylsulfonium bromopentachloroantimonate (BDSB) as a novel
activator for chemical glycosylation
Ladawan Punrum, Panuwat Padungros*
Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University,
*E-mail: panuwat.p@chula.ac.th
Abstract:
Bromodiethylsulfonium bromopentachloroantimonate (BDSB) was recently reported
as a novel source of highly electrophilic bromine. It was used to efficiently initiate cation-π
cyclizations of polyene. Herein, chemical glycosylation of thioglycosides activated by BDSB
is reported. Firstly, glycosyl donor (1), acting as electrophilic monomer, is synthesized over
three steps. Commercially available -D-glucose pentaacetate is subjected to anomeric
displacement with thiol and protecting group manipulations to provide thioglycoside (1) in
26% overall yield. Next, synthesis of glycosyl acceptor (2) is accomplished in 50% overall
yield. Methyl--D-glucopyranoside is selectively protected as benzyl ether at C2, C3, and C4
hydroxyl groups in three steps, leaving primary C6 hydroxyl as nucleophilic moiety. Finally,
chemical glycosylation between glycosyl donor (1) and acceptor (2) activated by BDSB is
examined. Several reaction conditions and parameters are investigated. It is found that
treatment of reaction mixture with 1.5 equivalents of electrophilic BDSB in acetonitrile at
–35 oC to room temperature gives the desired O-linked disaccharide (3) in moderate yield and
selectivity, 49% and 1:5 (:). However, applying BDSB in combination with stoichiometric
silver triflate (AgOTf) provides the disaccharide (3) in 79% yield and exclusive  selectivity
without using neighboring participation group at C2. Further mechanistic study of this novel
BDSB activation and scope of substrates are in progress.
1. Introduction are also powerful activators. The most

Chemical glycosylation is an common thiophilic activators are methyl
important reaction in the synthesis of triflate,7 DMTST [dimethyl(methylthio)-
complex carbohydrate molecules such as sulfonium triflate],8 IDCP (iodonium
oligosaccharides, glycoconjugates, dicollidine perchlorate),9 NBS (N-
glycoproteins, and glycolipids.1-3 bromosuccinimide), 10
and NIS (N-
Glycosylation is a chemical ligation between iodosuccinimide),11 the latter often used
glycosyl donor (electrophile) and glycosyl together with triflic acid or silver triflate.
acceptor (nucleophile) through the formation Moreover, bromodimethylsulfonium
of a new covalent bond. This bond is known bromide (BDMS) in a combination with
as a glycosidic linkage.4 Thioglycosides are silver triflate was reported as an efficient
extensively studied as useful glycosyl activator for thioglycosides (Figure 1a).
donors which is activated by Lewis acids or However, BDMS alone was not powerful
thiophilic reagents.5 Ferrier and co-workers enough to activate thioglycosides.12
first introduced mercury salt, HgSO4, as an Recently, a new brominating reagent,
activator of the glycosylation of bromodiethylsulfonium bromopentachloro
6
thioglycosides. Since then several kinds of antimonate (BDSB) was first introduced by
Lewis acids such as AgOTf, Cu(OTf)2, Snyder and co-workers (Figure 1a).13 It was
Yb(OTf)3, La(OTf)3, and Eu(OTf)3 were used as electrophilic brominating source for
examined as activators. Thiophilic reagents the cation-π cyclizations of polyene.14,15
Hence, this could indicate higher reactivity quenched by addition of saturated aqueous
of BDSB. Due to lower cost and structural Na2SO3 and saturated aqueous NaHCO3.
similarity of BDSB compared to that of Saturated sodium potassium tartrate was
BDMS, herein we report an investigation of later added to reduce the aggregation of
BDSB as a novel activator for glycoslylation salts, the mixture was stirred vigorously for
of thioglycosides (Figure 1b). 1 h and then extracted with ethyl acetate 3
times. The combined organic layer was
washed with water and brine, dried over
NaSO4, and concentrated by rotary
evaporation. The crude was purified by flash
column chromatography on silica gel using
hexanes/ethyl acetate as eluent.
Disaccharide 3 was obtained as white solid
(84 mg, 79%). The NMR analysis indicated
the product is anomers. Methyl 2,3,4-tri-
Figure 1. (a) Chemical structure of BDMS and O-benzyl-6-O-(2,3,4,6-tetra-O-benzyl-D-
BDSB; (b) Glycosylation of thioglycosides
activated by BDSB
glucopyranosyl)--D-glucopyranoside (3),20
1
H NMR (400 MHz, CDCl3) δ 7.44 – 7.17
(m, 35H), 5.03 (dd, J = 10.9, 4.0 Hz, 2H),
4.96 (d, J = 10.9 Hz, 1H), 4.91 – 4.52 (m,
2.1 General
All chemicals were purchased and 12H), 4.40 (d, J = 7.7 Hz, 1H), 4.24 (d, J =
used without further purification. Dried 10.4 Hz, 1H), 4.05 (t, J = 9.2 Hz, 1H), 3.93
solvents were taken from a solvent – 3.84 (m, 1H), 3.81 – 3.44 (m, 9H), 3.38 (s,
purification system and kept over 4 Å 3H).
molecular sieves prior use. For
glycosylation, all glassware were flame- 3. Results and Discussion
dried under vacuum and backfilled with 3.1 Synthesis of glycosyl donor
argon gas. Reactions were performed under Thioglycosides are widely used
positive pressure by using argon balloon. donors in oligosaccharide synthesis due to
Molecular sieves (3 Å) were used for their ease of preparation and activation.16,17
glycosylations. NMR spectra were recorded Thioglycoside such as ethyl thioglycoside
on Bruker Avance 400 MHz using CDCl3 as and phenyl thioglycoside are commonly
solvent. used in glycosylation.18,19 In this work, we
2.2 General procedure for glycosylation first describe the preparation of ethyl
Glycosyl donor (1a; 0.16 mmol), thioglycoside donors (Figure 2) and
glycosyl acceptor (2; 0.11 mmol), and investigation for the activation by BDSB.
freshly activated molecular sieves (3 Å; 100 Commercially available -D-glucose
mg) were added in acetonitrile (CH3CN; pentaacetate was treated with 1.2
1 mL). The mixture was stirred under argon equivalents of 1-ethanethiol in the presence
atmosphere for 1 h to remove any trace of of BF3.Et2O in dichloromethane at 0 oC, the
moisture. Then, freshly activated silver anomeric mixture of ethyl thioglucoside
triflate (AgOTf; 0.17 mmol) was added to tetraacetate 4a and 4b were obtained in 29%
the reaction mixture. The mixture was and 40% yields, respectively. Both  and 
cooled down to –35 oC using a dry anomer of ethyl thioglucoside tetraacetate
ice/acetonitrile bath for 30 min. Then BDSB were deprotonated by catalytic K2CO3 in
(0.16 mmol) was added. The reaction was methanol at room temperature for 3 h. After
further stirred for 30 min at –35 oC and was removal of methanol, the residue was treated
slowly allowed to warm up to room with 4.8 equivalents of BnBr, 0.1
temperature. After completion of reaction equivalents of TBAI and 8 equivalents of
monitored by TLC, the reaction mixture was NaH in DMF at 0 oC. Perbenzylated
products 1a and 1b were afforded in 91%
and 72% yield, respectively, over 2 steps.
Figure 3. Synthesis of methyl 2,3,4-tri-O-

benzyl--D-glucopyranoside acceptor
Figure 2. Synthesis of glycosyl donor 1a (Table 1, entry 1). When applying BDSB in
and 1b from -D-glucose pentaacetate combination with AgOTf, yield of 3 was
increased to 63% but with poorer selectivity
3.2 Synthesis of glycosyl acceptor (:=1.2:1) (Table 1, entry 2). Next, solvent
Methyl 2,3,4-tri-O-benzyl--D- effect was investigated by screening
gluco-pyranoside (2) is widely used as a different polar solvent such as MeNO2,
common glycosyl acceptor for MeCN and DMF. Using MeNO2 and MeCN,
glycosylation. Even though 2 is the product 3 was obtained in good yields,
commercially available but the cost is 76% and 79%, respectively (Table 1, entries
relatively high. Thus, 2 was synthesized 3 and 4). When MeCN was used, the high -
from cheap precursor through a scalable stereoselectivity was obtained. On the
synthetic route (Figure 3). In this work, contrary, glycosylation with DMF as solvent
methyl -D-glucopyranoside was first proceeded slowly and only small amounts of
protected at primary C6 hydroxyl group by product was detected (Table 1, entry 5).
tritylation with 2 equivalents of TrCl in Thus, MeCN is the solvent of choice for this
pyridine at room temperature for overnight. reaction.
After removal of pyridine, the crude product After several optimizations, we
5 was benzylated at C2, C3 and C4 hydroxyl found that the glycosylation of 1a and 2
group with 4 equivalents of BnBr, 0.1 proceeded smoothly with BDSB/AgOTf in
equivalents of TBAI and 8 equivalents of acetonitrile at –35 oC and allowed to warm
NaH in DMF at 0 oC. After passing through up to room temperature under argon
a plug of silica, trityl group at C6 of the atmosphere, to give disaccharide 3 in good
crude 6 was deprotected with 80% yield and high -selectivity.
AcOH:THF (1:1) in combination with
catalytic amount of 3M H2SO4 at 80 oC. Table 1. Optimization of the glycosylation
Methyl 2,3,4-tri-O-benzyl--D-glucopyrano activated by BDSB
side (2) was obtained in 50% yield over 3
steps.
3.3 Chemical glycosylation by using
BDSB as an activator Entry Activator Solventa Yieldb (:)c
Initial investigation of glycosylation 1 BDSB DCM 41 (1:5)
between ethyl thioglycoside 1a and glycosyl 2 BDSB+AgOTf DCM 63 (1.2:1)
3 BDSB+AgOTf MeNO2 76 (1:1.7)
acceptor 2 were performed using BDSB at 4 BDSB+AgOTf MeCN 79 (0:1)
low temperature under different reaction 5 BDSB+AgOTf DMF trace
conditions. The result indicated that the a
Reaction temperature; DCM at –78 oC, MeNO2 at –
glycosylation of 1a and 2 in CH2Cl2 20 oC, MeCN at –35 oC, and DMF at –35 oC;
provided the desired disaccharide 3 in 41%
b
Isolated yields; c: Ratio determined by 1H NMR
integration except for entry 4 which is isolated yield
yield with moderate selectivity (:=1:5)
3.4 Scope and limitation of glycosylation 4. Conclusion
using BDSB and AgOTf The glycosylation of thioglycoside
The scope and limitations of the and glycosyl acceptor activated by BDSB
glycosylation method were also investigated provides the O-linked glycosides in
using several alcohols (Table 2). First, moderate yield. However, using BDSB in
glycosylation with glycosyl alcohol 3 was combination with AgOTf in acetonitrile at
obtained in 79% and excellent-selectivity low temperature produces O-linked
without using neighboring participation glycosides in moderate to high yield with
group at C2. good  selectivity. Further mechanistic
study of this novel BDSB activation and
Table 2. Glycosylation activated by BDSB scope of substrates are in progress.
and AgOTf
Acknowledgements
This research is financially supported
by PETROMAT and Department of
Chemistry, Faculty of Science,
Chulalongkorn University.
References
1. Lee, B. H.; Bartram, B.; Schmezer, P.;
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2. Yang, Y. Y.; Ficht, S.; Brik, A.; Wong,
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4. Sugimura, H.; Natsui, Y. Tetrahedron
Lett. 2003, 44, 4729–4732.
Several alcohols were tested as 5. Sanders, W. J.; Kiessling, L. L.
acceptors. Primary alcohols such as benzyl Tetrahedron Lett. 1994, 35, 7335–7338.
alcohol and methanol, secondary alcohol 6. Ferrier, R. J.; Hay, R. W.; Vethaviyasar,
such as isopropanol and cyclohexanol were N. Carbohydr. Res. 1973, 27, 55–61.
subjected to the glycosylation. The results 7. Liao, L.; Ausanneau, F. I. J. Org. Chem.
indicated that glycosylations were proceeded 2005, 70, 6265–6273.
smoothly to provide the O-linked glycosides 8. Kulkarni, S. S.; Liu, Y. H.; Hung, H. C.
7-10 in moderate to high yields with J. Org. Chem. 2005, 70, 2808–2811.
9. Fraser-Reid, B.; Wu, Z.; Udodong, U.
selectivity (Table 2). Using 1-adamantanol
E.; Ottosson, H. J. Org. Chem. 1990,
as tertiary alcohol, the glycosylation
55, 6068–6070.
proceeded slowly and gave low yield of
10. Mitsudo, K.; Matsuda, W.; Miyahara, S.;
product 11 in 27% yield. When cholesterol
Tanaka, H. Tetrahedron Lett. 2006, 47,
was used as acceptor, only trace amount of
5147–5150.
the desired product 12 was observed.
11. Smith, R.; Müller-Bunz, H.; Zhu, X.
Several unidentified by-products were
Org. Lett. 2016, 18, 3578-3581.
detected probably due to bromination of
12. Xiong, D. C.; Zhang, L. H.; Ye, X. S.
olefin group on cholesterol occurred during
Adv. Synth. Catal. 2008, 350, 1696–
the reaction.
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13. Snyder, S. A.; Treitler, D. S. Org. Synth. 17. Smith, R.; Müller-Bunz, H.; Zhu, X.
2011, 88, 54–69. Org. Lett. 2016, 18, 3578−3581.
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A new method for the preparation of aryl halides from phenols bearing
electron-withdrawing groups using PPh3/halogenating agent
Korakot Saowalak1, Parinthorn Temyarasilp2, Anchulee Pengsook1,
Wanchai Pluempanupat1*
1
Department of Chemistry and Centre of Excellence for Innovation in Chemistry, Faculty of Science, and
Special Research Unit for Advanced Magnetic Resonance, Kasetsart University, Bangkok 10900, Thailand
2
Chemistry Program, Faculty of Science and Technology, Valaya Alongkorn Rajabhat University under the
Royal Patronage, Pathum Thani 13180, Thailand
*E-mail: fsciwcp@ku.ac.th
Abstract:
A new and convenient method for the conversion of electron-withdrawing group
substituted phenols into aryl halides through nucleophilic aromatic substitution using
PPh3/halogenating agent is described. Electron-poor phenol derivatives were transformed into
the corresponding aryl halides in moderate to high yields. PPh3/Cl3CCN and PPh3/CBr4 were
the most effective reagents for the chlorination and bromination, respectively.
1. Introduction the other versatile halogenating agents such

Deoxygenation is an important as Cl3CCONH2,4,5 Cl3CCN,6
chemical reaction involving the removal of Br3CCOCBr32,7, and Br3CSO2Ph2 on the
an oxygen atom from organic compounds. chlorination and bromination of alcohols
One applicable method involves the use of and carboxylic acids. In this work, we
PPh3 and halogenating agent (X3CR).1-7 extend our investigation to the
These coupling reagents are important since deoxygenation of phenol derivatives into
the replacement of hydroxyl group by a aryl halides using PPh3 and halogenating
halogen could be performed efficiently agent.10 Herein, we wish to report the use of
under mild and acid-free conditions. The the coupling reagents for the conversion of
reaction of alcohols,2-4 carboxylic acids5 and electron-withdrawing group substituted
sulfonic acids6 with the coupling reagents phenols into the corresponding aryl halides
proceeded via phosphonium salt through nucleophilic aromatic substitution
intermediates which were then converted (SNAr).
into the corresponding halides and
triphenylphosphine oxide.4 2. Materials and Methods
Aryl halides are often utilized as 2.1 General
valuable precursors for the formation of All reagent-grade chemicals and
carbon-carbon bonds via cross-coupling solvents were purchased from commercial
reactions in organic synthesis and suppliers (Aldrich, Acros, and TCI). 1H and
13
pharmaceutical science.8 Although aryl C NMR spectra were recorded on a Bruker
halides could be prepared from various 400 MHz AVANCE III HD spectrometer
starting materials, the simple protocols operating at 400 MHz (1H) and 100 MHz
generally involve the use of aromatic (13C). High-resolution mass spectra (HRMS)
hydroxy compounds, which are were recorded on a MAXIS (Bruker).
commercially available, using halogenating 2.2 General procedure for the
agents such as SOCl2, PCl3, PCl5 or PBr3.9 preparation of aryl halides
However, these reagents are difficult to To a stirred solution of phenols (0.25
handle because of moisture sensitivity and mmol) and PPh3 in dry toluene (0.5-1 M)
also generation of toxic gases such as SO2, was added halogenating agent and stirred at
HCl or HBr. We have recently introduced a refluxing temperature under N2
atmosphere for 6 hours. Then, the mixture CDCl3): δ 163.8, 146.3, 140.9, 140.7, 132.4,
was worked-up with CH2Cl2 and water. The 131.5, 128.7, 128.5, 126.9, 126.7, 126.3,
combined organic layers were dried over 66.0, 32.4, 30.2. HRMS (ESI) calcd for
Na2SO4 and concentrated under reduced C16H14ClO4Na 342.0509 [M+Na]+, found
pressure. The residue was purified by 342.0516 [M+Na]+.
preparative TLC on silica gel (hexane/ 3-Phenylpropyl 4-chloro-3-nitrobenzoate
EtOAc) to afford the desired product. (8b) colorless oil: 1H NMR (400 MHz,
1-Chloro-2,4-dinitrobenzene (1b)11 color- CDCl3):  8.40 (d, J = 4.0 Hz, 1H), 8.10 (dd,
less oil: 1H NMR (400 MHz, CDCl3):  8.73 J = 8.0, 4.0 Hz, 1H), 7.62 (d, J = 8.0 Hz,
(d, J = 4.0 Hz, 1H), 8.39 (dd, J = 8.0, 4.0 1H), 7.31 – 7.17 (m, 5H), 4.40 (t, J = 8.0
Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H). 13C NMR Hz, 2H), 2.79 (t, J = 8.0 Hz, 2H), 2.15 (quin,
(100 MHz, CDCl3): δ 147.7, 146.7, 133.8, J = 8.0 Hz, 2H). 13C NMR (100 MHz,
133.6, 121.6, 121.3. CDCl3): δ 163.8, 148.0, 141.0, 133.6, 132.2,
1-Bromo-2,4-dinitrobenzene (1c)11 color- 131.6, 130.3, 128.6, 128.5, 126.5, 126.2,
less oil: 1H NMR (400 MHz, CDCl3):  8.73 65.7, 32.5, 30.0. HRMS (ESI) calcd for
(d, J = 4.0 Hz, 1H), 8.29 (dd, J = 8.0, 4.0 C16H14ClO4Na 342.0509 [M+Na]+, found
Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H). 13C NMR 342.0505 [M+Na]+.
(100 MHz, CDCl3): δ 147.0, 146.8, 136.4,
127.0, 121.9, 121.5. 3. Results and Discussion
Ethyl 2-chloro-5-nitrobenzoate (5b)12 In this study, phenol containing nitro
colorless oil: 1H NMR (400 MHz, CDCl3):  substituent group was used as a model
8.68 (d, J = 4.0 Hz, 1H), 8.26 (dd, J = 8.0, substrate because the non-electron-deficient
4.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 4.45 aromatic rings are intrinsically reluctant to
(q, J = 8.0 Hz, 2H), 1.43 (t, J = 8.0 Hz, 3H). participate in SNAr reaction. Firstly,
13
C NMR (100 MHz, CDCl3): δ 163.7, screening of various chlorinating and
146.3, 140.8, 132.4, 131.6, 126.8, 126.6, brominating agents was performed using
62.6, 14.3. 2,4-dinitrophenol (1a) to form the
Ethyl 4-chloro-3-nitrobenzoate (6b)13 corresponding aryl chloride 1b and bromide
colorless oil: 1H NMR (400 MHz, CDCl3):  1c at the optimal stoichiometric ratio (3
8.49 (d, J = 2.0 Hz, 1H), 8.16 (dd, J = 8.0, equiv) of substrate and reagent previously
4.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 4.42 reported in the literature.10 The results are
(q, J = 8.0 Hz, 2H), 1.41 (t, J = 8.0 Hz, 3H). shown in Table 1. The use of Cl3CCN or
13
C NMR (100 MHz, CDCl3): δ 163.8, Cl3CCOCCl3 afforded aryl chloride 1b in
148.2, 133.7, 132.2, 131.6, 130.6, 126.7, high yields (entries 3-4) whereas
62.3, 14.4. Cl3CCOOH and Cl3CCONH2 gave the
Ethyl 4-bromo-3-nitrobenzoate (6c)14 desired product in low yields (entries 1-2).
white solid: 1H NMR (400 MHz, CDCl3):  For bromination, Br3CSO2Ph and 2-
8.45 (d, J = 1.9 Hz, 1H), 8.07 (dd, J = 4.4, (tribromomethyl)- quinoline provided aryl
1.88 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 4.42 bromide 1c in low to moderate yields
(q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). (entries 6-7), while Br3CCOOH gave a poor
13
C NMR (100 MHz, CDCl3): δ 164.8, yield of the desired product (entry 5).
150.1, 135.5, 133.6, 131.3, 126.6, 119.5, However, 1c was obtained in high yield in
62.4, 14.4. the presence of CBr4. The case of
3-Phenylpropyl 2-chloro-5-nitrobenzoate Cl3CCOOH and Br3CCOOH was probably
(7b) colorless oil: 1H NMR (400 MHz, because of its carboxyl group forming an
CDCl3):  8.66 (d, J = 2.8 Hz, 1H), 8.27 (dd, undesired reaction with the coupling
J = 8.0, 4.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, reagent. From the results, Cl3CCN and CBr4
1H), 7.32 – 7.16 (m, 5H), 4.42 (t, J = 8.0 were promising candidates in terms of
Hz, 2H), 2.80 (t, J = 8.0 Hz, 2H), 2.15 (quin, chlorinating and brominating agents.
J = 8.0 Hz, 2H). 13C NMR (100 MHz,
Table 1. Effect of halogenating agents various electron-poor phenol derivatives
using PPh3/Cl3CCN and PPh3/CBr4
(Scheme 1).
Entry Halogenating agent Yielda (%)

1 Cl3CCOOH 1b 14
2 Cl3CCONH2 1b 21
3 Cl3CCOCCl3 1b 93
4 Cl3CCN 1b 94, 77b
5 Br3CCOOH 1c 9
6 Br3CSO2Ph 1c 21
7 1c 58
8 CBr4 1c 67
a
Isolated yield.
b
CH2Cl2 was used as a solvent.
The suitable ratio of PPh3 and

halogenating agent was further optimized
for the conversion of 1a into 1b and 1c
(Table 2). Decreasing the amount of
PPh3/Cl3CCN from 3/3 to 2/1.5 equivalents
still provided the desired chloride 1b in high
yield within 6 hours (entries 1-4). Similarly,
aryl bromide 1c was still obtained in the
highest yield when the amount of PPh3
/CBr4 was decreased from 3/3 to 2/2
equivalents (entries 6-8).
Table 2. Effect of PPh3 and halogenating

agent ratio
Scheme 1. Chlorination and bromination of

electron-withdrawing group substituted
phenols
Entry PPh3 Cl3CCN CBr4 Yielda
(eq) (eq) (eq) (%)
1 3.0 3.0 - 1b 94 On refluxing in toluene for 6 hours,
2 2.5 2.5 - 1b 86 4-nitrophenol did not lead to 2b and 2c.
3 2.0 2.0 - 1b 79 Similar results were obtained when phenols
4 2.0 1.5 - 1b 93, 94b containing carboxyl substituent on the
5 1.5 1.5 - 1b 54 aromatic ring were examined (3b-4b and 3c-
6 3.0 - 3.0 1c 67
7 2.5 - 2.5 1c 49 4c). It may be because of a side reaction that
8 2.0 - 2.0 1c 72 the carboxyl group could react with the
9 2.0 - 1.5 1c 35 coupling reagents to produce the undesired
a
Isolated yield. acid chloride or bromide. However, the
b
The reaction time was 9 hours. desired products were obtained in moderate
With the optimized conditions in hand, we to high yields (5b-8b and 6c) when the
then performed the developed reaction on carboxyl group of starting materials was
protected as alkyl ester except for the case of Jang, D. O.; Chavasiri, W. Tetrahedron
5c. In addtion, this reaction mechanism was Lett. 2007, 48, 223–226.
proposed to proceed via SNAr displacement 3. Pluempanupat, W.; Chavasiri, W.
(Scheme 2). PPh3 reacts with X3CR to give a Tetrahedron Lett. 2006, 47, 6821–6823.
phosphonium salt, which subsequently 4. 28. Chaysripongkul, S.; Pluempanupat,
reacts with phenol derivatives yielding an W.; Jang, D. O.; Chavasiri, W. Bull.
aryloxyphosphonium intermediate, which Korean Chem. Soc. 2009, 30, 2066–
then transforms to the corresponding aryl 2070.
halide and triphenylphosphine oxide. 5. Chantarasriwong, O.; Jang, D. O.;
Chavasiri, W. Tetrahedron Lett. 2006,
47, 7489–7492.
6. Kara, M. J.; Isabel L.-S. Tetrahedron
Lett. 2011, 52, 13–16.
7. (a) Stille, J. K. Pure Appl. Chem. 1985,
57, 1771–1780; (b) Miyaura, N.; Suzuki,
A. Chem. Rev. 1995, 95, 2457–2483; (c)
Scheme 2. Proposed mechanism Meijere, A.; Meyer, F. E. Angew. Chem.,
Int. Ed. Engl. 1994, 33, 2379–2411.
4. Conclusion 8. (a) Vanlaer, S.; Voet, A.; Gielens, C.; De
In summary, we have described a Maeyer, M.; Compernolle, F. Eur. J.
new and alternative method for the Org. Chem. 2009, 643–654; Morgentin,
conversion of electron-withdrawing group R.; Jung, F.; Lamorlette, M.; Maudet,
substituted phenols into the corresponding M.; Menard, M.; Plé, P.; Pasquet, G.;
aryl chlorides and bromides by SNAr using Renaud, F. Tetrahedron 2009, 65, 757–
the efficient coupling reagents as 764; (c) Quallich, G. J.; Fox, D. E.;
PPh3/Cl3CCN and PPh3/CBr4, respectively. Friedmann, R. C.; Murtiashaw, C. W. J.
Org. Chem. 1992, 57, 761–764; (d) Sato,
Acknowledgements N.; Narita, N. J. Heterocycl. Chem.
This work was supported by the 1999, 36, 783–786.
Center of Excellence for Innovation in 9. Kijrungphaiboon, W.; Chantarasriwong,
Chemistry (PERCH-CIC), Office of the O.; Chavasiri, W. Tetrahedron Lett.
Higher Education Commission, Ministry of 2012, 53, 647–677.
Education. The Kasetsart University 10. Barbero, M.; Degani, I.; Dugherz, S.;
Research and Development Institute Fochi, R. J. Org. Chem. 1999, 64, 3448–
(KURDI) and the Faculty of Science and the 3453.
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Hybrid molecule: combination between sulfonamides and trimethoprim to
inhibit growth of methicillin-resistant Staphylococcus aureus (MRSA)
Taechin Nowwarat, Napon Nilchan, Chutima Jiarpinitnun*
Department of Chemistry and Center for Innovation in Chemistry (PERCH-CIC), Faculty of Science,
Mahidol University, Rama VI Road, Bangkok 10400, Thailand
*E-mail: chutima.jia@mahidol.ac.th
Abstract:
The pathogenic Gram-positive bacteria Staphylococcus aureus (S. aureus) causes
global health concerns due to its rapid development of resistance towards antibiotics used.
The emergence of antibiotic resistance has complicated the treatment of S. aureus infections,
especially, methicillin-resistant S. aureus (MRSA) that acquired resistance to beta-lactam
antibiotics such as penicillin. Sulfonamide antibiotics have been used to treat S. aureus and
MRSA infections. Sulfa-drug can be used as single-entity drug or in combination with
trimethoprim. However, the rise of sulfa-resistance and trimethoprim-resistance has led to a
decrease in clinical uses. Several studies have exploited the dimerization concept to enhance
efficacy and to overcome bacterial resistance. Here in, we reported the rationally design and
synthesis of a sulfonamide-trimethoprim hybrid. The hybrid ligand was simply designed to
covalently linked trimethoprim and sulfonamide adducts via C-C bond formation. The hybrid
molecule was evaluated for their efficacy against S. aureus control strains and globally
epidemic MRSA strain SF8300. Based on Kirby-Bauer disk diffusion assay, our results
promisingly indicated that the hybrid molecule could successfully inhibit growth of all S.
aureus strains, suggesting that the hybrid compound was effective against antibiotic resistant
strains of S. aureus.
1. Introduction bacterial agents in combination. The

Staphylococcus aureus (S. aureus) is successful combination therapy to treat S.
a pathogenic Gram-positive bacteria aureus and MRSA infections is
responsible for wide range illnesses—from sulfamethoxazole (SMZ) and trimethoprim
common skin infections to life-threatening (TMP). TMP and SMZ are potent and
diseases. S. aureus is also one of the leading selective inhibitors of two essential enzymes
causes of bovine mastitis, affecting dairy in the folate biosynthetic pathway, namely
product quality. S. aureus infections have dihydrofolate reductase (DHFR) and
resulted in tremendous impacts to our dihydropteroate synthase (DHPS),
3-4
economy with regards to healthcare respectively. These two antibiotics
expenses and also indirect impact to food function synergistically, resulting in
and agricultural industries.1-2 dramatic enhancement in anti-bacterial
S. aureus can rapidly develop genetic activity.5 However, rises of sulfa-resistance,
resistance toward antibiotics. The antibiotic trimethoprim resistance, and resistance to
resistance has complicated the treatment of the combination therapy have called for new
infections, especially methicillin-resistant S. antibacterial agents.6-7
aureus (MRSA) that acquired resistance to The simplicity in chemical structures
commonly used β-lactam antibiotics such as and highly potent antibacterial activities of
penicillin. One method to overcome the sulfonamide and TMP have drawn us into
resistance is to explore the possibility of the investigation of these small molecules,
using anti- particularly how to improve their growth
inhibition activity to overcome resistances.
Recent studies have highlighted the use of Then, 6 M HCl was slowly added and stirred
dimerization concept to enhance efficacy for 30 minutes. The reaction was extracted
and overcome bacterial resistance.8-10 Here with CH2Cl2 (3X), followed by washing
in, we incorporated the dimerization concept with saturated NaCl (3X) and dried over
in to the design of hybrid molecule based on anhydrous Na2SO4. The organic layers were
the clinically used antibacterial agents— combined and concentrated under vacuum to
SMZ and TMP. The hybrid ligand was obtain the crude product in 99%. 1H NMR
subjected to determination of its CDCl3 (400 MHz): δ 9.78 (s, 1H), 7.87 (s,
susceptibility against S. aureus and global 1H, J = 1.4 HZ), 7.43 (s, 1H, J = 1.4 HZ),
epidemic MRSA strains. The susceptibility 3.95 (s, 3H), 3.94 (s, 3H). 13C NMR CDCl3
was compared to its monomeric counterparts (100 MHz): δ 189.7, 154.2, 153.0, 134.7,
to evaluate the benefits of dimerization. 133.9, 111.0, 92.1, 60.7, 56.1 ESI calc. for
C9H9IO3 [M + H]+ = 292.9670, found
2. Materials and Methods 292.9762, [M + Na]+ = 314.9489, found
2.1 General 314.9538.
For the syntheses, all materials were 2.2.2 3-morpholinopropanenitrile (3)
purchased from commercial suppliers and Morpholine (58 mmol) and
used as provided. Non-aqueous reactions acrylonitrile (63 mmol) was mixed in 75 mL
were conducted under an inert atmosphere H2O. The reaction was stirred at room
of nitrogen. The reactions were monitored temperature and monitored by TLC with
by thin layer chromatography (TLC). 1H until reaction was completed (3 hours).
NMR and 13C NMR spectra were recorded Then, the reaction was extracted with
on either Bruker Avance-400 or Bruker EtOAc (3X) and dried over anhydrous
Avance-500 NMR spectrometers. High- Na2SO4. Combine and concentrated organic
resolution mass spectra (HRMS) were layers under high vacuum to obtain colorless
obtained on a Bruker micro TOF oil in 99 % yield. 1H NMR CDCl3 (400
spectrometer in the ESI mode. MHz): δ 3.72 (t, 4H, J = 4.7 Hz), 2.68 (t,
For susceptibility testing, Muller- 2H, J = 6.8 Hz), 2.52 (t, 2H, J = 7.0 Hz),
Hinton broth, Tryptic soy broth and Bacto 2.50 (t, 4H, J = 4.7 Hz). 13C NMR CDCl3
Agar were purchased from BD (Sparks, (125 MHz): δ 118.4, 66.2, 53.1, 53.0, 15.1.
MD). Oxacillin and trimethoprim were from ESI calc. for C7H12N2O [M + H]+ =
Sigma-Aldrich. The trimethoprim- 141.1023, found 141.1052, [M + Na]+ =
sulfamethoxazole (SXT) disk was purchased 163.0842, found 163.0865.
from Oxoid (Hampshire, UK). S. aureus 2.2.3 5-(3-iodo-4,5-dimethoxybenzyl)pyri
ATCC 25923 was generously given by midine-2,4-diamine (4)
Cornell University Food Safety Lab. MRSA Compound 3 (7.40 mmol) in 5 mL
strain SF8300 was kindly provided by Dr. DMSO was added with NaOMe (0.74
Binh Diep, University of California at San mmol). The reaction mixture was heated at
Francisco. 80oC for 30 minutes. Then, the solution of 2
2.2 Synthesis of iodo-TMP (4) (5.71 mmol) in DMSO (5 mL) was slowly
2.2.1 3-iodo-4,5-dimethoxybenzaldehyde added with continuous heating for 2 hours.
(2) The reaction mixture was cooled in ice bath
Compound 1, prepared as described and added 40 mL of ice-cold water. The
in a reported procedure,11 (10 mmol) was mixture was extracted with CH2Cl2 (3X) and
dissolved in 100 mL CH2Cl2. Sodium washed with saturated NaCl (3X), followed
hydroxide (10 mmol) and tetrabutyl by drying over MgSO4. The filtered organic
ammonium iodide (TBAI) (15 mmol) were layer was concentrated under reduced
added. Subsequently, iodomethane (MeI) pressure. Purification of the crude product
(11 mmol) was added. The reaction was by column chromatography gave (3-iodo-
stirred at room temperature for 16 hours. 4,5-dimethoxybenzyl)-3-morpholino acrylo
nitrile as a dark red oil. The product was 141.1, 138.4, 136.2 127.9, 127.1, 126.4,
carried on to the next step. 124.6, 116.6, 95.2, 13.8. ESI calc. for
The product was dissolved in EtOH C12H12N2O3S [M + H]+ = 265.0642, found
and added aniline hydrochloride (11.57 265.1307, [M + Na]+ = 287.0461, found
mmol). The reaction was refluxed for 30 287.0591.
minutes. Then, guanidine hydrochloride 2.2.5 (E)-4-(5-((2,4-diaminopyrimidin-5-y
(14.80 mmol) and NaOMe (7.40 mmol) l)methyl)-2,3-dimethoxystyryl)-N-(5-meth
were added to the reaction. The reflux was ylisoxazol-3-yl)benzenesulfonamide (6)
continued for additional 2 hours. The Compound 5 (0.18 mmol),
reaction was concentrated under vacuum to compound 4 (0.09 mmol), 5% mol
1/3 volume and cooled in ice bath. Ice-cold PdCl2(PPh3), and 1-ethylpiperidine (0.9
water (40 mL) was added to give mmol) were added in 10 mL DMF. The
precipitates, which were collected by reaction mixture was stirred at 120oC for 16
filtration and washed with cold H2O. hours. The reaction was concentrated and
Recrystallization in EtOH yielded the extracted with CH2Cl2 (3X); the organic
desired product in 60% yield. 1H NMR layer was washed with saturated NaCl and
DMSO-d6 (400 MHz): δ 7.56 (s, 1H), 7.11 dried over anhydrous MgSO4. Concentrate
(d, 1H, J = 1.8 Hz), 6.96 (d, 1H, J = 1.8Hz), under vacuum afforded crude product,
6.14 (br s, 2H), 5.72 (br s, 2H), 3.76 (s, 3H), which was purified by column
3.64(s, 3H), 3.53 (s, 2H). 13C NMR DMSO- chromatography. Recrystallization in 10 %
d6 (125 MHz): δ 162.4, 162.1, 156.0, 152.0, methanol/CH2Cl2 yielded compound 6 in 35
146.4, 138.9, 129.1, 113.8, 105.2, 92.3, 59.8, % yields. 1H NMR CDCl3 (400 MHz): δ
55.8, 31.7. ESI calc. for C13H15IN4O [M + 7.95 (s, 1H), 7.75 -7.420 (m, 4H) 7.34 (d,
H]+ = 387.0313, found 387.0320, [M + Na]+ 1H, J = 5.2 Hz), 7.25 (d, 1H, J = 5.2 Hz)
= 409.1032, found 409.2033. 6.95 - 6.67 (m, 2H), 6.14 (s, 1H), 3.85 (s,
2.2.4 N-(5-methylisoxazol-3-yl)-4-vinylben 3H), 3.83 (s, 3H), 3.50 (s, 2H), 2.83 (s, 2H).
zenesulfonamide (5) ESI calc. for C25H27N6O5S [M + H]+ =
Sodium 4-vinylbenzenesulfonate 523.1759, found 522.6015, [M + Na]+ =
(1.4 mmol) was dissolved in 5 mL DMF. 545.1578 found 544.3826.
The solution was cooled in the ice bath and 2.3 Preparation of bacterial culture
added oxalyl chloride (2.8 mmol). The A single colony of tested strain was
mixture was stirred for 30 minutes. Then, 5- picked and transferred into 5 mL of Tryptic
methylisoxazol-3-amine (2.1 mmol) and soy broth (TSB) in a test tube, which was
triethylamine (4.2 mmol) were added. The then capped and incubated at 37 °C for 15-
reaction was continued stirring for 16 hours 18 hours. The suspension was subsequently
at room temperature. To work-up the diluted in Phosphate Buffer Saline (PBS) to
reaction, 2 M NaOH was slowly added yield the appropriate inoculum density of
while cooling in the ice bath and stirred for approximately 8x107 CFU/mL.
30 minutes. The mixture was extracted with 2.4 Disk diffusion method
CH2Cl2 (3X) and washed with saturated The disk diffusion was performed
NaCl and dried over anhydrous MgSO4. The based on previously reported procedure.12 A
organic layers were combined and 150-µL aliquot of cell suspension described
concentrated under vacuum to afford the above was spread on the surface of Muller-
crude product, which was purified by Hinton agar (MHA). Whatman paper disk
column chromatography to obtain 5 in 87% (6-mm) was placed on the agar plate, and
yield. 1H NMR CDCl3 (400 MHz): δ 7.57 solution of test compound in DMSO was
(m, 2H), 7.34 (m, 2H), 6.57 (dd, 1H J1 = transferred onto each paper disk. After
17.6, J2 = 10.9), 5.42 (d, 1H J = 17.6), 5.21 overnight incubation of the plate at 37 °C,
(d, 1H J = 10.9), 5.16 (s, 1H), 2.30 (s, 3H). the diameter of the inhibition zones was
13
C NMR CDCl3 (100 MHz): δ 170.7, 150.0, measured.
3. Results and Discussion The synthesis of terminal alkene-
3.1 Chemical synthesis appended sulfonamide could be synthesized
Previous structure-activity from sodium vinyl benzene sulfonate.
relationship of TMP derivatives suggested Transformation to the corresponding
that hydrogen bonds between pyrimidine sulfonyl chloride could be achieved with
ring and DHFR are keys for binding, and the oxalyl chloride. Reaction with 5-
methoxy substituent of TMP could be methylisoxazol-3-amine afforded the
modified without perturbing the binding terminal alkene-appended SMZ (5) in 87%
affinity to DHFR.13 Therefore, we replaced yield. The compound 5 and compound 4 was
the methoxy group with halogen for further subjected to Pd-catalyzed Heck coupling
coupling with the monomeric sulfonamide reaction to covalently link the two entities,
synthesized. The hybrid molecule of yielding the target hybrid molecule (6), as
sulfamethoxazole and TMP could be shown in Scheme 2.
achieved by C-C bond formation between 3.2 Susceptibility testing
the monomeric TMP derivative 4 and vinyl The synthetic hybrid molecule and
benzene sulfonamide 5. The synthesis of its monomeric counterparts were subjected
iodo-TMP (4) was based on previously to qualitative susceptibility assay—disk
reported protocol, as shown in Scheme diffusion assay—against methicillin
1.11,14 Iodination of vanillin by treating with susceptible S. aureus (MSSA) ATCC 25923
iodine in the presence of KI and NaHCO3 as a control strain, global predominant CA-
generated 5-iodovanillin (1). Compound 1 MRSA PFGE strain USA300 (SF8300).
was then subjected to O-methylation This assay allowed access to the ability of
reaction. Condensation with 3- the hybrid compound to inhibit growths of
morpholinopropionitrile (3) under basic pathogenic strains of study. The disk
condition yielded alkene product, which diffusion assay was strictly followed the
underwent cyclization upon treating with standard susceptibility protocol
Guanidine to afford the corresponding iodo- recommended by Clinical and Laboratory
TMP (4) in moderate yield. Standards Institute (CLSI).15 The results
were shown in Table 1.
Scheme 1. Synthesis of iodo-TMP (4)
O
NH2 O O N
O O N O
I 10% PdCl2(PPh3)2 , DMF S
N S NH2 N
+ N H
H
H2N N OCH3 o
120 C ,24 h N
OCH3 N H2N N OCH3
OCH3
4 5 6 35%
Scheme 2. Synthesis of hybrid molecule 6
Oxacillin was tested to verify the SF8300. In addition, we noticed enhanced

resistance of the SF8300 strain.12,15 The inhibition zone when 4 and 5 were tested in
results showed that oxacillin at 1 µg could combination. It might perhaps due to the
not inhibit growth of SF8300 while synergistic effect similar to that observed
completely inhibited the control strain with TMP and SMZ. Further study should
ATCC25923, suggesting that SF8300 is be performed to verify the synergistic
resistant to methicillin antibiotics. The behavior. In addition to the combination of 4
clinically used sulfa-drug SMZ at 25 g and 5, the hybrid molecule (6) at 5 g and
could only inhibited MSSA strain but not 25 g could also inhibit growth of both
MRSA strain. Similarly, TMP and even its MSSA and MRSA strain. These results
combination therapy SXT were also not suggested that the hybrid molecule could
effective against global epidemic MRSA potentially overcome the resistance to TMP
strain SF8300. and SMZ observed with MRSA strain
Our synthetic molecules 4-6 were SF8300.
tested. We observed that the iodo-TMP 4 at The qualitative disk diffusion results
5 g could inhibit growth of both MSSA are promising. We plan to further evaluate
and MRSA strain while the SMZ derivative the anti-bacterial activity via quantitative
5 exhibited incomplete inhibition zone when microdilution assay in order to obtain
tested with MRSA strain. However, when 4 minimum inhibitory concentration (MIC)
and 5 were used in combination at the same and minimum bactericidal concentration
ratio as the clinically use combination (MBC). The evaluation of these value could
therapy SXT (1.25 g TMP+ 23.75 g suggest the effectiveness of the dimerization
SMZ), the mixture effectively inhibited in the design of anti-bacterial agent. In
growth of both MSSA and MRSA strain. addition, we are currently investigating the
Even though the disk diffusion only susceptibility of these compounds against
provides qualitative results, the combination hospital-acquired MRSA strain COL and
of 4 and 5 showed promising inhibitory bovine mastitis induced MSSA strain
activity, especially when compared to the RF122. The results will be reported in due
currently used combination therapy SXT time.
that showed ineffective against MRSA strain
Table 1. Complete Inhibition zone in diameter (mm)

Compound ATCC25923 (MSSA) SF8300 (MRSA)
4 (5 g) 18.75 19.87
5 (25 g) 14.35 Incomplete
SMZ (25 g) 17.45 6
TMP (5 g) 19.25 Incomplete
4 (1.25 g)+ 5 (23.75 g) 29.22 31.51
TMP (1.25 g) + SMZ (23.75 g) 30.44 Incomplete
6 (5 g) 11.15 13.62
6 (25 g) 26.40 26.78
Oxacillin (1 g) 19.78 6
4. Conclusion 4. Yun, M. K.; Wu, Y.; Li, Z.; Zhao, Y.;
Iodo-trimethoprim (4), alkene- Waddell, M. B.; Ferreira, A. M.; Lee, R.
appended sulfamethoxazole (5), and the E.; Bashford D.; White S. W. Science
hybrid molecule (6) were synthesized in few 2012, 335, 1110–1114.
steps from commercially available materials. 5. Zander, J.; Besier, S.; Ackermann, H.;
The qualitative disk diffusion was used to Wichelhaus, T. A. Antimicrob. Agents
access the anti-bacterial activity of these Chemother. 2010, 54, 1226–1231.
compounds against MSSA and MRSA 6. Huovinen, P. Clin. Infect. Dis. 2001, 32,
strains. The results suggested that the iodo- 1608–1614.
TMP 4 and its combination with 7. Charpentier, E.; Courvalin, P.
sulfonamide 5 could inhibit growth of both Antimicrob. Agents Chemother. 1997,
strains tested. The hybrid molecule 6 also 41, 1134–1136.
showed promising results as it could also 8. Nieto, M. J.; Lujań Alovero, F. D.;
effectively inhibited growth of both MSSA Manzo, R. H.; Mazzieri, M. R. Eur. J.
and MRSA strain. To compare the efficacy Med. Chem. 1999, 34, 209−214.
of these compounds, we plan to perform 9. Bukvic´, M. K.; Novak, P.; Cindric´, M.;
microdilution assay to access their MIC and Brajsˇ, K.; Dumic´, M.; Kujundzic´, N.
MBC. However, this investigation indicated Eur. J. Med. Chem. 2007, 42, 138–145.
the potential use of the synthetic compound 10. Zani F.; Incerti, M.; Ferretti, R.; Vicini,
4 and 5 in combination or the hybrid P. J. Med. Chem. 2009, 44, 2741–2747.
compound 6 as the alternative treatment of 11. Nammalwar B.; Bunce, R. A.; Berlin, K.
S. aureus infections. D.; Bourne, C. R.; Bourne, P. C.;
Barrow, E. W.; Barrow, W. W. Eur. J.
Acknowledgements Med. Chem. 2012, 54, 387–396.
The authors are grateful to the 12. Schwalbe, R.L.(Eds); Goodwin, A.C.
financial supports provided in part by Antimicrobial susceptibility testing
Department of Chemistry and Center for protocols, CRC Press, Boca Raton,
Innovation in Chemistry (PERCH-CIC), and 2007, pp. 53–74.
Faculty of Science, Mahidol University. T. 13. Bourne, C.R.; Bunce, R.A.; Bourne,
N. is supported by Scientific Achievement P.C.; Berlin K.D.; Barrow, E.W.;
Scholarship of Thailand (SAST). Barrow, W.W. Antimicrob. Agents
Chemothe.r 2009, 53, 3065–3073.
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T.; Barkema, H. W. J. Clin. Microbiol. 345–354.
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3. Foye, W.O.; Lemke, T.L.; DA, W. Clinical and Laboratory Standards
Principles of medicinal chemistry. 6th Institute: Performance Standards for
ed. Philadelphia: Lippincott Williams & Antimicrobial Susceptibility Testing;
Wilkins; 2008. Wayne 2012, 32, 126–150.
Synthesis of cationic lipids with guanidine and cyclic guanidine polar head
Charoen Posa1, Nattisa Niyomtham1, Saowanee Kumpun2, Boon-ek Yingyongnarongkul1*
1
Faculty of Science, Ramkhamhaeng University, Bangkapi, Bangkok 10240, Thailand
2
Department of Chemistry, Faculty of Science and Technology, Suansunandha Rajabhat University,
*E-mail: boonek@ru.ac.th
Abstract:
Eight cationic lipids possessing guanidine and cyclic guanidine polar head with
cholesterol tail have been designed and synthesized. Cationic lipids with guanidine (1-4) and
cyclic guanidine (5–8) polar head were obtained in excellent (82–100%) and moderate (51–
68%) yields, respectively. The structures of the synthesized lipids were characterized by
spectroscopic methods (IR, NMR and MS). Cationic lipid 2 with guanidine polar head
strongly bound to DNA. However, lipid 8 with cyclic guanidine head and long hydrocarbon
chain bound to DNA with strong affinity. The results showed that the type of polar head and
the spacer length had significant effects on DNA binding property of cationic lipid.
1. Introduction and HRESITOFMS spectra were measured

Cationic lipid-mediated transfection with a Finnigan LC-Q and a Bruker
is one of the most promising methods for in micrOTOF II mass spectrometer. Starting
vitro as well as in vivo applications, based materials and reagents were purchased from
on a combination of efficiency, stability and commercial suppliers and used without
lack of toxicity.1 Cationic lipids are small further purification.
molecules which can easily be designed and 2.2 Synthesis of cationic lipids
studied for structure-activity relationships. 2.2.1 Synthesis of cationic lipids 1-4
Since the first application of cationic lipid in To a stirred solution of
DNA delivery,2 numerous cationic lipids diaminoalkane A-D (1.2 equiv) in CH2Cl2 at
have been synthesized and demonstrated the 0 – 5 C was slowly added a solution of
capability of delivering genetic materials cholesteryl chloroformate (1 equiv) in
into various cells.3 Some of cationic CH2Cl2. The reaction mixture was stirred
liposomes-mediated gene transfers have overnight at room temperature. The reaction
been used in gene therapy clinical trials.4 was stopped by water and extract with
Cationic lipids are commonly composed of CH2Cl2. The combined organic layers were
three main parts including polar head, linker dried over anhydrous Na2SO4 and
and lipophilic domain. Amino and guanidine evaporated under reduced pressure to
groups are often used as a hydrophilic polar dryness. The crude product was purified by
head. However, cationic lipid with cyclic silica gel column chromatography using
guanidine polar head has not been reported CH2Cl2 – MeOH as eluting solvents by
on its efficiency. increasing the more polar component to give
compounds 9A-D.5
2. Materials and Methods To a stirred solution of compounds
2.1 General 9A-D (1 equiv) and DIPEA (4 equiv) in
IR spectra were recorded on a CH2Cl2 was added N,N’-bis(tert-
Perkin-Elmer FT-IR spectrometer. 1H NMR butoxycarbonyl)-S-methylisothiourea (1.2
spectra were recorded on a Bruker equiv) at room temperature. The progress of
AVANCE400 spectrometer using a residual reaction was monitor by TLC. The reaction
solvent signal as internal standard. ESMS was quenched with water and extracted with
CH2Cl2. The combined organic layer was 1228, 1156, 1126, 1070 cm-1; 1H-NMR (400
dried with Na2SO4 anhydrous and MHz, CDCl3)  0.63 (s, 3H, H-18), 0.78 (d,
evaporated under reduced pressure to J = 6.3 Hz, 6H, H-26, H-27), 0.87 (d, J = 6.1
dryness. The crude product was purified by Hz, 3H, H-21), 0.96 (s, 3H, H-19), 1.46 (br
silica gel column chromatography using s, 18H, H-Boc), 1.52 (obscure signal, H-2'),
CH2Cl2 – MeOH (95:5) as eluting solvents 3.33 (br s, 2H, H-3'), 3.51 (br s, 2H, H-1'),
to furnish compounds 10A-D. 4.45 (br s, 1H, H-3), 5.33 (br s, 1H, H-6),
Compound 10A-D was added 20% 5.54 (br s, 1H, NH); MS (ES+): m/z: 729.6
TFA in CH2Cl2 and stirred at room ([M+H]+, calcd for C42H72N4O6 + H, 100%).
temperature for 2 h. The organic solvents Compound 10C
were removed under stream of nitrogen to Amorphous solid (62.9 mg, 85%), IR
dryness to obtain compounds 1 – 4. The (KBr): max 3410, 2932, 1741, 1721, 1648,
desired compounds were further dried under 1622, 1518, 1315, 1245, 1145, 1048 cm-1;
reduced pressure to removed trace amount 1
H-NMR (400 MHz, CDCl3)  0.63 (s, 3H,
of solvents. H-18), 0.82 (d, J = 6.2 Hz, 6H, H-26, H-27),
2.2.1 Synthesis of cationic lipids 5-8 0.87 (d, J = 6.3 Hz, 3H, H-21), 0.96 (s, 3H,
To a stirred solution of 9A-D (1 H-19), 1.46 (br s, 18H, H-Boc), 1.52
equiv) Et3N (4 equiv) in CH2Cl2 was added (obscure signal, H-2' และ H-3'), 3.16 (br s,
2-methylthio-2-imidazoline hydrochloride 2H, H-4'), 3.39 (br quin, J = 5.8 Hz, 2H, H-
(1.2 equiv). The reaction mixture was stirred 1'), 4.45 (m, 1H, H-3), 4.84 (br s, 1H, NH),
at room temperature for overnight. The 5.33 (br s, 1H, H-6); 13C-NMR (100 MHz,
organic solvent was removed under reduced
CDCl3)  11.8, 18.6, 19.2, 20.9, 22.5, 22.7,
pressure to dryness. The crude mixture was
23.7, 24.2, 26.2, 27.0, 27.4, 27.9, 28.0, 28.2,
purified by silica gel column
31.8, 35.7, 36.1, 36.5, 36.9, 38.5, 39.4, 39.7,
chromatography using CH2Cl2 – MeOH
40.1, 40.3, 42.2, 49.9, 56.1, 56.6, 74.1, 79.5,
(90:10) as eluting solvent to give
83.2, 122.3, 139.8, 153.2, 155.9, 156,1,
compounds 5-8.
162.9; MS (ES+): m/z: 743.5 ([M+H]+, calcd
Compound 10A
for C43H74N4O6 + H, 100%).
Amorphous solid (83.3 mg,
Compound 10D
quantitative yield), IR (KBr): max 3329, Amorphous solid (120.0 mg, 82%),
2934, 1723, 1646, 1618, 1334, 1238, 1139,
IR (KBr): max 3334, 2934, 1717, 1638,
1017 cm-1; 1
H-NMR (400 MHz,
1614, 1366, 1330, 1130, 1053 cm-1; 1H-
CDCl3+CD3OD)  0.59 (s, 3H, H-18), 0.78
NMR (400 MHz, CDCl3)  0.64 (s, 3H, H-
(d, J = 6.4 Hz, 6H, H-26, H-27), 0.83 (d, J =
18), 0.83 (d, J = 6.5 Hz, 6H, H-26, H-27),
6.5 Hz, 3H, H-21), 0.91 (s, 3H, H-19), 1.42
0.88 (d, J = 7.4 Hz, 3H, H-21), 0.98 (s, 3H,
(s, 18H, H-Boc), 3.09 (m, 2H, H-2'), 3.38 (t,
H-19), 1.58 (br s, 18H, H-Boc), 2.65 (t, J =
J = 5.6 Hz, 2H, H-1'), 4.39 (m, 1H, H-3),
6.9 Hz, 2H, H-6'), 3.13 (br s, 2H, H-1'), 4.46
5.28 (br s, 1H, H-6), 6.18 (br s, 1H, NH),
(br s, 1H, H-3), 4.58 (br s, 1H, NH), 5.34
8.33 (br s, 1H, NH); 13C-NMR (100 MHz,
(br s, 1H, H-6); MS (ES+): m/z: 772.3
CDCl3+CD3OD)  11.6, 18.5, 19.1, 20.9, ([M+H]+, calcd for C45H78N4O6 + H, 100%).
22.3, 22.6, 23.6, 24.1, 27.8, 28.1, 29.8, 31.7,
Compound 1
35.6, 36.0, 36.4, 36.8, 37.0, 37.1, 37.3, 37.4,
38.4, 39.3, 39.6, 42.1, 49.8, 53.5, 56.0, 56.5,
quantitative yield), IR (KBr): max 3325,
73.9, 79.2, 83.2, 122.2, 139.8, 152.8, 156.3,
2931, 1721, 1643, 1615, 1329, 1238, 1136,
156,5, 162.8; MS (ES+): m/z: 715.5
1014 cm-1; 1H-NMR (400 MHz, CDCl3) 
([M+H]+, calcd for C41H70N4O6 + H, 100%).
0.65 (s, 3H, H-18), 0.84 (d, J = 6.4 Hz, 6H,
Compound 10B
H-26, H-27), 0.86 (d, J = 6.5 Hz, 3H, H-21),
0.98 (s, 3H, H-19), 3.30 (m, 2H, H-2'), 3.40
quantitative yield), IR (KBr): max 3329,
(t, J = 5.6 Hz, 2H, H-1'), 4.18 (m, 1H, H-3),
2935, 1715, 1643, 1619, 1505, 1331, 1286,
5.33 (br s, 1H, H-6); MS (ES+): m/z: 515.6 Compound 6
([M+H]+, calcd for C31H54N4O2 + H, 100%). Amorphous solid (110.0 mg, 51%),
Compound 2 IR (KBr): max 3213, 2934, 2867, 1667,
Amorphous solid (56.4 mg, 1465, 1253, 1033 cm-1; 1H-NMR (400 MHz,
quantitative yield), IR (KBr): max 3328, CDCl3)  0.64 (s, 3H, H-18), 0.83 (d, J = 6.2
2932, 1721, 1643, 1615, 1331, 1234, 1135, Hz, 6H, H-26, H-27), 0.88 (d, J = 6.3 Hz,
1013 cm-1; 1H-NMR (400 MHz, CDCl3)  3H, H-21), 0.97 (s, 3H, H-19), 3.22 (m, 2H,
0.65 (s, 3H, H-18), 0.84 (d, J = 6.4 Hz, 6H, H-3'), 3.38 (br s, 2H, H-1'), 3.72 (s, 4H, H-
H-26, H-27), 0.86 (d, J = 6.5 Hz, 3H, H-21), 3'' and H-4''), 4.39 (m, 1H, H-3), 5.32 (br s,
1.09 (s, 3H, H-19), 3.18 (m, 2H, H-3'), 3.41 1H, H-6), 7.85 (br s, 2H, NH); MS (ES+):
(t, J = 5.6 Hz, 2H, H-1'), 4.19 (m, 1H, H-3), m/z: 555.2 ([M+H]+, calcd for C34H58N4O2 +
5.34 (br s, 1H, H-6); MS (ES+): m/z: 529.5 H, 100%).
([M+H]+, calcd for C32H56N4O2 + H, 100%). Compound 7
Compound 3 Amorphous solid (133.6 mg, 57%),
Amorphous solid (62.3 mg, IR (KBr): max 3207, 2934, 2867, 1666,
quantitative yield), IR (KBr): max 3327, 1465, 1253, 1014 cm-1; 1H-NMR (400 MHz,
2930, 1722, 1646, 1616, 1331, 1236, 1135, CDCl3)  0.61 (s, 3H, H-18), 0.80 (d, J = 6.0
1016 cm-1; 1H-NMR (400 MHz, CDCl3)  Hz, 6H, H-26, H-27), 0.85 (d, J = 6.1 Hz,
0.61 (s, 3H, H-18), 0.79 (d, J = 6.4 Hz, 6H, 3H, H-21), 0.95 (s, 3H, H-19), 3.13 (br s,
H-26, H-27), 0.84 (d, J = 6.5 Hz, 3H, H-21), 2H, H-4'), 3.34 (br s, 2H, H-1'), 3.68 (s, 4H,
1.04 (s, 3H, H-19), 3.20 (m, 2H, H-4'), 3.43 H-3'' and H-4''), 4.34 (br s, 1H, H-3), 5.14
(t, J = 5.6 Hz, 2H, H-1'), 4.20 (m, 1H, H-3), (s, 1H, NH), 5.29 (br s, 1H, H-6), 7.62 (br s,
5.36 (br s, 1H, H-6); MS (ES+): m/z: 543.4 1H, NH), 7.73 (br s, 1H, NH); MS (ES+):
([M+H]+, calcd for C33H58N4O2 + H, 100%). m/z: 569.1 ([M+H]+, calcd for C35H60N4O2 +
Compound 4 H, 100%).
Amorphous solid (48.3 mg, Compound 8
quantitative yield), IR (KBr): max 3327, Amorphous solid (159.3 mg, 68%),
2931, 1722, 1646, 1619, 1335, 1237, 1136, IR (KBr): max 3213, 2933, 2866, 1668,
1016 cm-1; 1H-NMR (400 MHz, CDCl3)  1465, 1252, 1012 cm-1; 1H-NMR (400 MHz,
0.62 (s, 3H, H-18), 0.81 (d, J = 6.4 Hz, 6H, CDCl3)  0.65 (s, 3H, H-18), 0.83 (d, J = 6.6
H-26, H-27), 0.84 (d, J = 6.5 Hz, 3H, H-21), Hz, 6H, H-26, H-27), 0.88 (d, J = 6.4 Hz,
1.05 (s, 3H, H-19), 3.19 (m, 2H, H-6'), 3.42 3H, H-21), 0.98 (s, 3H, H-19), 3.11 (quin,
(t, J = 5.6 Hz, 2H, H-1'), 4.21 (m, 1H, H-3), 2H, H-6'), 3.31 (br s, 2H, H-1'), 3.72 (s, 4H,
5.36 (br s, 1H, H-6); MS (ES+): m/z: 571.7 H-3'' and H-4''), 4.39 (br s, 1H, H-3), 4.89
([M+H]+, calcd for C35H62N4O2 + H, 100%). (br s, 1H, NH), 5.33 (br s, 1H, H-6), 7.28
Compound 5 (br s, 1H, NH), 7.76 (br s, 1H, NH); MS
Amorphous solid (61.2 mg, 52%), IR (ES+): m/z: 597.2 ([M+H]+, calcd for
(KBr): max 3380, 2935, 2867, 1666, 1464, C37H64N4O2 + H, 100%).
1267, 1011 cm-1; 1H-NMR (400 MHz, 2.3 Gel retardation assay
CDCl3)  0.65 (s, 3H, H-18), 0.84 (d, J = 6.3 DNA binding affinities of liposomes
Hz, 6H, H-26, H-27), 0.89 (d, J = 6.4 Hz, were measured at liposome/DNA ratios
3H, H-21), 0.99 (s, 3H, H-19), 3.29 (m, 2H, (w/w) of 5, 10 and 20, by gel
H-2'), 3.38 (m, 2H, H-1'), 3.76 (s, 4H, H-3'' electrophoresis. The liposome/DNA
and H-4''), 4.45 (m, 1H, H-3), 5.36 (br s, 1H, complexes were prepared by adding the
H-6), 5.41 (br s, 1H, NH), 7.83 (br s, 1H, liposome solution to the DNA solution (the
NH), 8.62 (br s, 1H, NH); MS (ES+): m/z: amount of DNA was fixed at 0.1 g). The
541.9 ([M+H]+, calcd for C33H56N4O2 + H, mixture was gently mixed by pipetting up
100%). and down for 2-3 times and the mixture was
held at room temperature for 30 min. Each
complex was added gel loading buffer
(13.3% w/v sucrose in water) to get the final methylisothiourea11 in the presence of
volume of 10 L. The complexes solution DIPEA base followed by Boc deprotection
was inverted to mix and each sample (10 furnished lipids 1-4 in excellent yield.
L) was loaded onto a 1% agarose gel Compounds 9A-D were reacted with 2-
(0.5×TBE buffer). The gel was run at 200 V, methylthio-2-imidazoline hydrochloride12 in
400 mA for 2 h. DNA bands were viewed the presence of Et3N gave desired lipids 5-8
under UV light by ethidium bromide in moderate yield (51-68%). The structures
staining. of synthesized lipids were characterized by
spectroscopic methods (IR, NMR and MS).
3. Results and Discussion 3.2 Gel retardation assay
3.1 Synthesis of cationic lipids 1-8 Gel retardation assay was performed
Cationic lipids with guanidine (1-4) to characterize the electrostatic binding
and cyclic guanidine (5-8) polar head were interactions between the plasmid DNA and
synthesized as shown in Scheme 1. liposome.13 Cationic liposome 1-8 were
Cholesteryl chloroformate (9) was reacted mixed with plasmid DNA at weight ratio of
with diamine A-D having different spacer to 5, 10 and 20 and loaded on agarose gel
give compounds 9A-D in quantitative yield. (Figure 1). The results indicated that the
The primary amino group of these optimal spacer of cationic lipid with
compounds was further converted to guanidine polar head for DNA binding was
guanidine and cyclic guanidine. There are at four methylene units. Thus, cationic lipid
numerous reagents could be used to change 2 strongly bound to DNA at all ratios.
amino to guanidine functionality including Cationic lipids 1 and 3 with shorter and
S-alkylisothiouronium salts,6 N,N’- longer spacer, respectively, could bind to
bis(alkoxycarbonyl)-protected S-alkylisothio DNA at high lipid/DNA ratio. In contrast,
urea derivatives,7 pyrazole-1- carboxamidine cationic lipid with cyclic guanidine polar
hydrochloride,8 di(benzotriazol-1-yl)metha head required longer spacer for binding to
nemine,9 and di(imidazol-1-yl)meth DNA with strong affinity. The results
10 showed that cationic lipid 8 with long
anemine.
Thus, 9A-D were reacted with N,N’- hydrocarbon chain bound to DNA at all
bis(tert-butoxycarbonyl)-S- ratios.
NH2 NH2
H2N H2N
A C
O
NH2
Cl O H2N NH2 H2N
B D
9
O NBoc O
b
H2N n N O BocHN N n N O
H H H
9A; n = 0 10A; n = 0
9B; n = 1 10B; n = 1
9C; n = 2 10C; n = 2
9D; n = 4 10D; n = 4
d c
NH O NH O
N N n N O H2N N N O
H H H n H
5; n = 0 1; n = 0
6; n = 1 2; n = 1
7; n = 2 3; n = 2
8; n = 4 4; n = 4
Scheme 1. Reagents and conditions: a) Diamine (A-D), CH2Cl2; b) N,N’-bis(tert-

butoxycarbonyl)-S-methylisothiourea, DIPEA, CH2Cl2; c) 20% TFA/CH2Cl2; d) 2-
methylthio-2-imidazoline hydrochloride, Et3N, CH2Cl2.
(1) (1)
(2) (2)
(3) (3)
(1) (1)
(2) (2)
(3) (3)
(1) (1)
(2) (2)
(3) (3)
(1) (1)
(2) (2)
(3) (3)
(1) (1)
(2) (2)
(3) (3)
(1) (1)
(2) (2)
(3) (3)
(1) (1)
(2) (2)
(3) (3)
(1) (1)
(2) (2)
(3) (3)
Control
GC-2GC-2
GC-2GC-2
GC-2GC-2
GC-3GC-3
GC-3GC-3
GC-3GC-3
GC-4GC-4
GC-4GC-4
GC-4GC-4
GC-6GC-6
GC-6GC-6
GC-6GC-6
AC-2AC-2
AC-2AC-2
AC-2AC-2
AC-3AC-3
AC-3AC-3
AC-3AC-3
AC-4AC-4
AC-4AC-4
AC-4AC-4
AC-6AC-6
AC-6AC-6
AC-6AC-6
for Innovation in Chemistry (PERCH-CIC)
AC-3 (2)
AC-3 (3)
AC-4 (1)
AC-4 (2)
AC-4 (3)
AC-6 (1)
AC-6 (2)
(3)
Control
1:5GC-2
GC-2
GC-2
1:5GC-3
GC-3
GC-3
1:5GC-4
GC-4
GC-4
1:5GC-6
GC-6
GC-6
Control
AC-6
DNA/Cationic liposome (wt/wt ratio) for scholarship.
1:10
1:10
1:20
1:20
1:10
1:20
1:10
1:20
References
1. Cavazzana-Calvo, M.; Hacein-Bey, S.;
de Saint Basile, G.; Gross, F.; Yvon, E.;
6Bb (1)(1) (1)
(2)(2) (2)
6Bb (3)(3) (3)
(1)(1) (1)
GBb (2)(2) (2)
(3)(3) (3)
(1) (1)
(2) (2)
(3) (3)
(1) (1)
(2) (2)
(3) (3)
Nusbaum, P.; Selz, F.; Hue, C.; Certain,
(1) (1)
(2) (2)
(3) (3)
1 2 3 4
(1) (1)
(2) (2)
(3) (3)
(1) (1)
(2) (2)
(3) (3)
Control
cGC-2
1:10cGC-2
cGC-2
1:5 cGC-3
cGC-3
1:20cGC-3
1:5 cGC-4
cGBbcGC-4
1:20cGC-4
1:5 cGC-6
1:10cGC-6
1:20cGC-6
S.; Casanova, J. L.; Bousso, P.; Le Deist,
(2)
cGBbcGBb
cGBbcGBb
cGBbcGBb
cGC-3 (2)
cGC-3 (3)
cGC-4 (1)
cGC-4 (2)
cGC-4 (3)
cGC-6 (1)
cGC-6 (2)
(3)
(1)
(3)
GBbGBb
GBbGBb
GBbGBb
Control
DNA/Cationic liposome (wt/wt ratio)
6Bb6Bb
6Bb6Bb
6Bb6Bb
Control
cGC-2
cGC-2
cGC-2
cGC-3
cGC-3
cGC-3
cGC-4
cGC-4
cGC-4
cGC-6
cGC-6
cGC-6
F.; Fischer, A. Science 2000, 288,
cGC-6
cGBb
cGBb
1:20
1:10
1:10
GBb
GBb
1:5
6Bb
669672.
2. Felgner, P. L.; Gadex, T. R.; Holm, M.;
Roman, R.; Chan, H. W.; Wenz, M.;
Northrop, J. P.; Ringold, G. M.;
5 6 7 8 Danielsen, M. Proc. Natl. Acad. Sci.
Figure 1. Gel retardation assay of cationic USA 1987, 84, 74137417.
lipids/DNA complexes at a weight ratio of 5, 3. Mintzer, M. A.; Simanek, E. E. Chem.
10 and 20, Lanes marked “Control” Rev. 2009, 109, 259302.
contained DNA alone and was used as a 4. Edelstein, M. L.; Abedi, M. R.; Wixon,
control. The presence of a lower band J. J. Gene Med. 2007, 9, 833842.
indicated that DNA has migrated and has 5. Apiratikul, N.; Penglong, T.; Suksen, K.;
not been bound by the cationic lipid. Svasti, S.; Chairoungdua, A.;
Yingyongnarongkul, B. Russ. J. Bioorg.
The results indicated that the type of polar Chem. 2013, 39, 497503.
head and the spacer length had significant 6. Wityak, J.; Gould, S. J.; Hein, S. J.;
effects on DNA binding property of cationic Keszler, P. A. J. Org. Chem. 1987, 52,
lipid. 2179–2183.
7. Moroni, M.; Koksch, B.; Osipov, S. N.;
4. Conclusion Crucianelli, M.; Frigerio, M.; Bravo, P.;
Cholesterol-based cationic lipids Burger. K. J. Org. Chem. 2001, 66, 130–
having guanidine and cyclic guanidine were 133.
successfully synthesized in good overall 8. Exposito, M. A.; Lopez, B.; Fernandez,
yield. The DNA binding affinity of these R.; Vazquez, M.; Debitus, C.; Bravo, P.;
lipids suggests that this class of compound Burger, K. Tetrahedron 1998, 54, 7539 –
has significant potential for gene delivery. 7550.
Thus, transfection efficiency of all 9. Katritzky, A. R.; Rogovoy, B. V.;
synthesized cationic lipids for gene delivery Chassaing, C.; Vvedensky, V. J. Org.
into targeted cells is under investigation and Chem. 2000, 65, 8080–8082.
will report elsewhere. 10. Wu, Y.-Q.; Hamilton, S. K.; Wilkinson,
D. E.; Hamilton. G. S.; J. Org. Chem.
Acknowledgements 2002, 67, 7553–7556.
This work was supported by 11. Yingyongnarongkul, B.; Howarth, M.;
Ramkhamhaeng University. Support from Elliott, T.; Bradley, M. Chem. Eur. J.
The Thailand Research Fund (TRF) and the 2004, 10, 463–473
Center of Excellence for Innovation in 12. Frederic, M.; Scherman, D.; Byk. G.
Chemistry (PERCH-CIC), Office of Higher Tetrahedron Lett. 2000, 41, 675–579.
Education Commission, Ministry of 13. Yingyongnarongkul, B.; Howarth, M.;
Education is gratefully acknowledged. CP Elliott, T.; Bradley, M. J. Comb. Chem.
would like to thank the Center of Excellence 2004, 6, 753–760.
Sulfonamidation for the synthesis of trisubstituted arene intermediates
Pongsun Jhangthong, Pitak Chuawong*
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, and Special
Research Unit for Advanced Magnetic Resonance (AMR), Kasetsart University, Bangkok 10900, Thailand
*E-mail: Pitak.C@ku.ac.th
Abstract:
The sulfonamide group plays a significant role in medicinal chemistry and
pharmacology. The functional group appears in many commercial drugs such as Celebrex,
Azulfidine, and Gantrisin. Various synthetic approaches have been reported to access aryl
sulfonamides such as the palladium-catalyzed sulfonation of aryl iodides, one-pot synthesis
from magnesium sulfonates with DABSO, a reaction between a Grignard reagent and an N-
chloroamine, and the palladium-catalyzed chlorosulfonylation of arylboronic acids. Herein,
we reported a convenient synthesis of N-(4-amino-3-iodophenyl)-4-methylbenzenesulfonami-
de and N-(4-amino-3-iodophenyl)methanesulfonamide from commercially available 4-
nitroaniline in 5 steps with an overall yield of 67%. The initial iodination reaction of 4-
nitroaniline using molecular iodine and silver sulfate afforded trisubstituted aryl iodide
intermediate. Acetylation of the amino group followed by reduction using sodium
borohydride and copper sulfate afforded arylamine intermediate. Finally, sulfonamidation
and deacetylation provided N-(4-amino-3-iodophenyl)-4-methylbenzenesulfonamide and N-
(4-amino-3-iodophenyl)methanesulfonamide in good yield. The trisubstituted arenes are the
key intermediates for the synthesis of biologically active indole derivatives using the Larock
heteroannulation reaction.
1. Introduction sulfonamide analogs in good yields (Scheme

The medical significance of sulfa 1a).3 A palladium-catalyzed coupling
drugs, also called sulfonamide-containing reaction of aryl nonaflates with the primary
drugs, is initially traced to 1932 by Fritz sulfonamide in the presence of electron-rich
Mietzsch and Joseph Klarer, with extensive diphenyl phosphine ligand was achieved by
use in the treatment of several bacterial Shekhar and co-workers in 2011 even
infections.1 Besides, they are also used for though this methodology has a limitation
preventing infection of burns and urinary due to the decomposition of the coupled
tract infections. Most of the sulfa drugs are products during the purification (Scheme
derived from sulfanilamides such as 1b).4 Recently, Willis published a one-pot
sulfamethazine, sulfathiazole, and sulfonamide synthesis using the palladium-
1
sulfadiazine. Some of the remarkable catalyzed sulfination of aryl iodides
sulfonamide drugs are prescribed for the providing sulfonamides in high yields.5 The
treatment of a broad range of infections reaction was straightforward to encompass
(Figure 1).2 However, sulfa drugs result in both primary and secondary amine, amino
allergic reactions including nausea, derivatives, and anilines (Scheme 1c).5
vomiting, skin rashes, and bacterial Herein, we reported a general,
resistance has been reported.2 Up to now, a convenient method for the synthesis of
number of convenient methods have been trisubstituted arenes from a commercially
used to prepare an aromatic sulfonamide. In available 4-nitroaniline. The trisubstituted
1991, Hlasta utilized the sulfonation of the arene products are the key intermediates for
ortho-lithiated benzamides to afford lithium the synthesis of biologically active indole
sulfinate as the intermediate, followed by derivatives using Larock heteroannulation
the oxidative amination to provide reaction.6
Figure 1. Sulfonamide drugs
Scheme 1. Previous syntheses of aromatic sulfonamides
2. Materials and Methods positive ionization mode (ESI+) and

2.1 General atmospheric pressure chemical
Flash column chromatography was ionization (APCI). Mel-Temp electrother-
performed on silica gel 60 (mesh size 40-63 mal apparatus (Bibby Stuart Scientific Ltd.,
m) from silicycle Inc. Proton nuclear United Kingdom) was used for the melting
magnetic resonance (1H NMR) and carbon point (m.p.) determination. Starting
nuclear magnetic resonance (13C NMR) materials, analytical grade solvents and
spectra were recorded on a Bruker 400 MHz reagents for synthesis were obtained from
AVANCE III HD spectrometer. The commercial sources and used directly
chemical shifts () were reported in ppm without further purification.
relative to the solvent residual peaks 2.2 Synthesis of 2-iodo-4-nitroaniline (2)
(CDCl3, 7.26 ppm, (DMSO-d6, 2.50 ppm) 4-nitroaniline (1) (0.55 g, 4.0 mmol)
for 1H and (CDCl3, 77.16 ppm, (DMSO-d6, was added to a mixture of iodine (1.0 g, 4.0
39.52 ppm) for 13C. The coupling constants mmol) and Ag2SO4 (1.2 g, 4 mmol) in EtOH
(J) were reported in Hz. High-resolution (10 mL), and the mixture was stirred at
mass spectra (HRMS) were obtained from room temperature for 1 h. The reaction
Bruker, microTOF (Bruker Daltonics, mixture was filtered, and the filtrate was
Bremen, Germany) in the electrospray evaporated under reduced pressure. The
resulting residue was dissolved in DCM, 61%). mp 122-123 °C; 1H NMR (400 MHz,
washed with H2O, dried over anhydrous CDCl3) δ 8.58 (d, J = 2.6 Hz, 2H), 8.09
Na2SO4, and evaporated to give 2 as a (ddd, J = 9.3, 2.7, 0.6 Hz, 1H), 6.79 (d, J =
yellow solid (1.0 g, 93%). 1H NMR (400 9.3 Hz, 1H), 1.48 (s, 9H); 13C NMR (100
MHz, CDCl3) δ 8.56 (d, J = 2.5 Hz, 1H), MHz, CDCl3) δ 151.0, 135.99, 135.4, 125.8,
8.05 (dd, J = 8.9, 2.5 Hz, 1H), 6.70 (d, J = 109.3, 84.7, 52.7, 29.4.
8.9 Hz, 1H), 4.85 (s, 2H); 13C NMR (100 2.5 Synthesis of 2,2,2-trifluoro-N-(2-iodo-
MHz, CDCl3) δ 152.5, 139.3, 135.6, 135.4, 4-nitrophenyl)acetamide (3c).
125.8, 112.4, 80.6. To a solution of 2-iodo-4-nitroaniline
2.3 Synthesis of N-(2-iodo-4-nitrophenyl) (2) (0.27 g, 1.0 mmol) in THF (2.5 mL) was
acetamide (3a). added Et3N (0.12 g, 1.2 mmol), and the
To a solution of 2-iodo-4-nitroaniline resulting solution was cooled to 0 °C.
(2) (0.58 g, 2.2 mmol) in DCM (3 mL) was Trifluroacetic anhydride (0.25 g, 1.2 mmol)
added 0.2 mL of pyridine. Afterwards, was added dropwise to the reaction mixture,
acetyl chloride (2.2 g, 2.80 mmol) was and the reaction was allowed to warm up to
added dropwise to the stirred mixture at 0 room temperature and stirred for 25 h. The
°C. The reaction was stirred at room reaction was quenched by water, and the
temperature for 1 h. The reaction was product was extracted with EtOAc (3x2
quenched with 3 mL of 1 M HCl and mL). The organic layer was washed with
extracted with DCM (3x5 mL). The brine, dried over anhydrous Na2SO4, and
combined organic layers were washed with evaporated to dryness to obtain 3c as a
brine, dried over anhydrous Na2SO4, filtered yellow solid (0.66 g, 70%). mp 129-130 °C;
and dried under reduced pressure. The crude 1
H NMR (400 MHz, DMSO-d6) δ 11.56 (s,
product was purified by flash 1H), 8.68 (d, J = 2.6 Hz, 1H), 8.31 (dd, J =
chromatography (9:1 DCM/hexane) to 8.7, 2.6 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H);
achieve 3a as pale yellow solid (0.66 g, 13
C NMR (100 MHz, DMSO-d6) δ 155.3,
97%). mp 139-140 °C; 1H NMR (400 MHz, 154.9, 146.7, 143.2, 133.9, 128.8, 124.2,
CDCl3) δ 8.65 (d, J = 2.5 Hz, 1H), 8.54 (d, J 117.3, 98.2.
= 9.2 Hz, 1H), 8.22 (dd, J = 9.2, 2.5 Hz, 2.6 Synthesis of N-(4-amino-2-iodophe-
1H), 7.73 (s, 1H), 2.31 (s, 3H); 13C NMR nyl)acetamide (4).
(100 MHz, CDCl3) δ 168.6, 143.9, 143.6, N-(2-iodo-4-nitrophe-nyl)acetamide
141.8, 134.3, 125.1, 119.8, 87.4, 25.3. (3a) (0.61 g, 2 mmol) was dissolved in
2.4 Synthesis of tert-butyl (2-iodo-4-nitro- EtOH (5 mL). Then, copper sulfate (55.0
phenyl)carbamate (3b). mg, 10% mol) was sequentially added. After
To a solution of 2-iodo-4-nitroaniline the reaction mixture was cooled to 0 °C,
(2) (0.27 g, 1 mmol) in acetonitrile (2.5 mL) NaBH4 (0.38 g, 10 mmol) was added
was sequentially added di-tert- portionwise. Then, the reaction mixture is
butyldicarbonate (0.52 g, 2.4 mmol) and stirred at room temperature for 18 h.
zinc chloride (12.2 mg, 10% mol). The Subsequently, the reaction mixture is then
reaction was then heated at reflux for 36 h. diluted with EtOAc, washed with water,
Then, the reaction was cooled to room dried over anh. Na2SO4 and concentrated to
temperature and evaporated under reduced give the crude product which is purified by
pressure. The resulting residue was column chromatography (1:1 EtOAc/hex-
partitioned between H2O and EtOAc. The ane) to give 4 as a pale yellow solid (0.39 g,
organic layer was then washed with brine, 70%). mp 135-137 °C; 1H NMR (400 MHz,
dried over anhydrous Na2SO4 and DMSO-d6) δ 9.13 (s, 1H), 7.06 (d, J = 2.5
concentrated in vacuum. The crude product Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 6.53 (dd,
was purified by flash column J = 8.5, 2.5 Hz, 1H), 5.22 (s, 2H), 1.95 (s,
chromatography (7:3 DCM/hexane) to 3H); 13C NMR (100 MHz, DMSO-d6) δ
afford 3b as a pale yellow solid (0.58 g, 155.56, 147.08, 143.62, 134.35, 129.25,
124.60, 116.30, 98.62. HRMS (ESI-QTOF) at reflux temperature for 2 h. The reaction
[M+H]+ calcd for C8H10IN2O: 276.9832, mixture was evaporated under reduced
found 276.9845. pressure and purified by flash column
2.7 Synthesis of N-(2-iodo-4-(4-methylphe chromatography (1:1 EtOAc/DCM) to
nylsulfonamido)phenyl)acetamide (5a). provide 6a as a dark green solid (0.18 g,
N-(4-amino-2-iodophenyl)acetamide 99%). mp 182-184 °C; 1H NMR (400 MHz,
(4) (0.1 g, 0.82 mmol) was dissolved in 0.8 DMSO-d6) δ 9.70 (s, 1H), 7.55 (d, J = 8.2
mL of pyridine. To this solution was then Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 7.22 (d, J
added methanesulfonyl chloride (0.12 g, = 2.3 Hz, 1H), 6.82 (dd, J = 8.6, 2.4 Hz,
1.17 mmol). The reaction was stirred at 1H), 6.66 (d, J = 8.6 Hz, 1H), 3.92 (s, 2H),
room temperature for 1 h. The reaction was 2.34 (s, 3H); 13C NMR (100 MHz, DMSO-
quenched by 1 M HCl and extracted with d6) δ 145.0, 144.4, 143.0, 136.5, 132.6,
EtOAc (3x2 mL). A combined organic layer 129.5, 128.0, 127.5, 126.8, 123.9, 114.9,
was washed with water, brine, and dried 83.1, 40.1, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9,
over anhydrous Na2SO4. The organic solvent 21.0; HRMS (ESI-QTOF) [M+Na]+ calcd
was removed under reduced pressure to for C13H13IN2NaO2S: 410.9635, found
afford 5a as an orange solid (0.36 g, 99%). 410.9640.
mp 161-162 °C; 1H NMR (400 MHz, 2.10 Synthesis of N-(4-amino-3-iodophe-
DMSO-d6) δ 10.36 (s, 1H), 9.32 (s, 1H), nyl)methanesulfonamide (6b).
7.66 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 2.2 Hz, This compound was synthesized
1H), 7.36 (d, J = 8.1 Hz, 2H), 7.21 (d, J = according to the method described for 6a
8.6 Hz, 1H), 7.10 (dd, J = 8.6, 2.3 Hz, 1H), using 5b (0.5 mmol) as the reactant to afford
2.34 (s, 3H), 1.99 (s, 3H); 13C NMR (100 6b as a dark green solid (0.16 g, 99%). mp
MHz, DMSO-d6) δ 168.3, 143.6, 136.3, 173-175 °C ; 1H NMR (400 MHz, DMSO-
135.8, 129.9, 129.3, 127.9, 126.7, 119.7, d6) δ 9.64 (s, 1H), 7.57 (d, J = 1.9 Hz, 1H),
97.2, 23.0, 21.0; HRMS (ESI-QTOF) 7.17 – 7.13 (m, 2H), 4.89 (s, 2H), 2.94 (s,
[M+H]+ calcd for C15H16IN2O3S.: 430.9921, 3H); 13C NMR (100 MHz, DMSO-d6) δ
found 430.9909. 143.1, 133.2, 130.9, 124.5, 117.5, 85.5,
2.8 Synthesis of N-(2-iodo-4-(methyl- 39.2; HRMS (ESI-QTOF) [M+Na]+ calcd
(methylsulfo-namido)phenyl)acetamide for
(5b). C7H9IN2NaO2S: 334.9322, found 334.9326.
This compound was synthesized
according to the method described for 5a 3. Results and Discussion
using 4-toluenesulfonyl chloride (0.22 g, Trisubstituted arenes 6a and 6b were
1.17 mmol) instead of methanesulfonyl prepared from commercially available 4-
chloride to provide 5b as a pale orange solid nitroaniline (1) as illustrated in Scheme 2.
(0.23g, 88%). mp 176-178 °C; 1H NMR The initial iodination reaction of 4-
(400 MHz, DMSO-d6) δ 9.82 (s, 1H), 9.40 nitroaniline using molecular iodine and
(s, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.31 (d, J = silver sulfate afforded trisubstituted aryl
8.6 Hz, 1H), 7.22 (dd, J = 8.6, 2.4 Hz, 1H), iodide intermediate 2 in 93% yield.7 The
3.01 (s, 3H), 2.03 (s, 3H); 13C NMR (100 next step is the protection of the amino
MHz, DMSO-d6) δ 168.4, 137.0, 135.8, group 3 using different protecting groups
129.3, 128.1, 119.9, 97.5, 39.4, 23.1; HRMS including acetyl group, tert-butoxycarbonyl
(ESI-QTOF) [M+Na]+ calcd for (N-Boc), and trifluoromet-hylcarbonyl to
C9H11IN2NaO3S: 376.9427, found 376.9409. generate 3a, 3b, and 3c in 97%, 61%, and
2.9 Synthesis of N-(4-amino-3-iodophenyl 70% yields respectively.8-10 Based on our
-4-methylbenzenesulfonamide (6a). study so far, an acetyl group was chosen
A mixture of N-arylacetamide (5a) owing to the highest yield obtained from the
(0.5 mmol) and thionyl chloride (47.4 mg, reaction. Subsequently, the nitro group of 3a
0.4 mmol) was stirred in methanol (1.5 mL) was reduced using sodium borohydride to
afford 4 in 70% yield.11 The amino group of toluenesulfonyl chloride and methanesulf-
4 was subsequently converted to the oyl chloride in 99% and 88% yields,
sulfonamide group in 5a and 5b using 4-
Scheme 2. The synthesis of trisubstituted arenes 6a and 6b
respectively.12 Finally, trisubstituted arenes Acknowledgements

6a and 6b were obtained upon the We thank the Center of Excellent for
deprotection of N-acetyl group of 5a and Innovation in Chemistry (PERCH-CIC), the
5b in the presence of SOCl2 and MeOH Office of the Higher Education, and the
providing arylamines 6a and 6b in 99% Kasetsart University Research and
yield.13 Development Institute (KURDI) for the
financial support.
4. Conclusion
The synthesis of trisubstituted arenes References
has been accomplished starting from 1. Brownlee, G. Nature 1949, 163, 662.
commercially available 4-nitroaniline in 5 2. Smith, C. L.; Powell, K. R. J. Pediatr.
steps with an overall yield of 67% and 2000, 21, 368.
59% respectively. The resulting trisubstitu- 3. Hlasta, D. J.; Court, J. J.; Desai, R. C.
ted arenes are the key intermediates for the Tetrahedron Lett. 1991, 32, 7179–7182.
synthesis of biologically active indole 4. Shekar, S.; Dunn, T. B.; Kotecki, B. J.;
derivatives via the Larock hetero annulation Montavon, D. K.; Cullen, S. C. J. Org.
reaction. Chem. 2011, 76, 4552–4563.
5. Flegeau, E. F.; Harrison, J. M.; Willis,
M. C. Synlett 2016, 27, 101–105.
6. Larock, R. C.; Lu, Y. D. J. Org. Chem. 10. Vojtickova, M.; Dobias, J.; Hanquet, G.;
1993, 58, 2846–2850. Addova, G.; Cetin, R.; Yildirim, D. C.;
7. Chen, C. S.; Tan, C. M.; Huang, C. H.; Bohac, A. Eur. J. Org. Chem. 2015, 103,
Chang, L. C.; Wang, J. P.; Cheng, F. C.; 105–122.
Chen, J. W. Bioorg. Med. Chem. 2010, 11. Yoo, S.; Lee, S. Synlett 1990, 7, 419–
18, 597–604. 420.
8. Benek, O.; Soukup, O.; Pasdiorova, M.; 12. Huang, A.; Chem, Y.; Zhou, Y.; Guo,
Hroch, L.; Sepsova, V.; Jost, P.; W.; Wu, X.; Ma, C. Org. Lett. 2013, 15,
Hrabinova, M.; Jun, D.; Kuca, K.; Zala, 5480–5483.
D.; Ramsay, R. R.; Marco, J.; Musilek, 13. Wang, G.; Wang, L. F.; Li, C. Z.; Sun,
K. ChemMedChem 2016, 11, 1264– J.; Zhou, G. M.; Yang, D. C. Res. Chem.
1269. Intermed. 2011, 38, 77–89.
9. Neuhaus, J. D.; Morrow, S. M.; Brunavs,
M.; Willis, M. C. Org. Lett. 2016, 18,
1562–1565.
Synthesis and cytotoxicity evaluation
of tetrahydro-ß-carboline derivatives
Koonchira Buaban1, Thongchai Taechowisan2, Weerachai Phutdhawong3,
Waya Phutdhawong1*
1
2
Department of Microbiology, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand
3
Department of Science, Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng Sean campus,
*E-mail: waya.sengpracha@gmail.com
Abstract:
Tetrahydro-ß-carbolines (THßCs) are of interest as they possess a variety of
biological activities and are used in novel pharmaceutical applications. Herein, a series of
substituted tetrahydro-ß-carbolines was synthesized via Pictet-Spengler reaction. All
compounds were evaluated for their cytotoxicity against human cancer cell lines in vitro by
MTT assay. The derivative which contain 2,2-bis(2-chlorobenzyl) group as R2 displayed the
highest cytotoxicity against human hepatocyte carcinoma (Hep G2) and human cervix
epitheloid carcinoma (HeLa) with IC50 values of 26.67 and 25.00 µg/mL, respectively.
1. Introduction cytotoxicity against human cancer cell lines

1,2,3,4-Tetrahydro-ß-carbolines in vitro by MTT assay (Figure 2).
(THßCs) are one of the important classes of
indole alkaloids.1-5 (Figure 1). Most THßCs
are associated with pharmaceutical
properties such as antimalarial,6-7 antiviral,8
anticancer,9 antileishmanial,10 anti-
inflammatory and anti-HIV activities.12
11
These biological effects have been linked

with diverse modes of action and targets
such as inhibition of kinesin Eg5,13
inhibition of various enzymes such as Figure 1. Structures of biologically active
topoisomerases I and II,14 CYP45015 and tetrahydro-ß-carboline compounds
phosphodiesterase type 5 (PDE5).16
According to SAR studies, tricyclic THßC 2. Materials and Methods
derivatives with modification on the 1- and 2.1 General
2-positions of the parent nucleus would Melting points were determined on a
make great influence on their antitumor melting point apparatus SMP2 model.
activities.17-19 1
H NMR and 13C NMR spectra were
Thus, this study has been focused on recorded on Bruker AVANCE 300 (300 and
the synthesis of 1- and 2-substituted THßCs 75 MHz, respectively) with CDCl3 and
and evaluation of their cytotoxicity. The CD3OD as solvents and tetramethylsilane
synthesis was started with the condensation (TMS) as an internal standard. HR-ESI mass
of tryptamine with paraformaldehyde via spectra were recorded on mass spectrometer
Pictet-Spengler reaction. The resulted and were supported by Chulabhorn Research
tetrahydro-ß-carbolines were treated with Institute, Thailand. All reagents were
triethylamine to afford the N-2 substituted purchased from commercial suppliers.
THßCs to be investigated for their
2.2 Synthesis mmol) and benzyl chloride (0.02 mL, 0.18
Synthesis of 2,3,4,9-tetrahydro-1H- mmol) to afford 3d as a brown oil.
pyrido[3,4-b]indole (2). (3,4-dihydro-1H-pyrido[3,4-b]indo l-
Tryptamine (0.22 g, 1.4 mmol) was 2(9H)-yl)(phenyl)methanone (3e). The title
dissolved in AcOH:MeOH (5:0.5 mL) then compound was synthesized from (2) (0.12 g,
paraformaldehyde (0.042 g, 1.7 mmol) was 0.70 mmol) and benzoyl chloride (0.13 mL,
added into the solution and refluxed for 1 h. 1.16 mmol) to afford 3e as a brown oil.
After completion of the reaction, the mixture (3,4-dihydro-1H-pyrido[3,4-b]indo l-
was cooled to room temperature and basified 2(9H)-yl)(furan-2-yl)methanone (3f). The
to pH 9-10 using NH4OH. The solution was title compound was synthesized from (2)
extracted with CH2Cl2. The organic layer (0.092 g, 0.54 mmol) and 2-furoyl chloride
was dried over anh. Na2SO4. After (0.11 mL, 1.1 mmol) to afford 3f as a brown
evaporation of the solvent, the crude product oil.
was purified by column chromatography 2,2-bis(2-chlorobenzyl)-2,3,4,9-te
(silica gel, 15% MeOH:CH2Cl2) to afford trahydro-1H-pyrido[3,4-b]indol-2-ium (3g).
2,3,4,9-tetrahy dro-1H-pyrido[3,4-b]indole The title compound was synthesized from
(2) as a yellow solid. (2) (0.17 g, 1.00 mmol) and 2-chlorobenzyl
Preparation of 2-substituted-2,3,4,9-tetra chloride (0.20 mL, 2.00 mmol) to afford 3g
hydro-1H-pyrido[3,4-b]indole (3a-3g) as an orange solid.
A solution of 2,3,4,9-tetrahydro-1H- Cytotoxicity assay
pyrido[3,4-b]indole (2) in CH2Cl2 (1 The cytotoxic activities of the
mL/mmol) were added TEA (3 eq). An acid synthetic compounds against L929, Hep G2
halide or acryloyl halide was added to the and HeLa cells were evaluated by the MTT
solution mixture and stirred at room (3-(4,5-dimethylthiazole-2-yl)-2,5-
temperature for 24 h. The solution was diphenyltetrazolium bromide} colorimetric
extracted with CH2Cl2 and the organic layer assay20 and acridine orange was used as a
was dried over anh. Na2SO4. After positive control. Each cell lines were seeded
evaporation of the solvent under reduced at 1x105 cell/well (100 µL/well) in a 96-well
pressure, the crude product was purified by plate and incubated overnight. Then two-
column chromatography (silica gel, 5% fold serial dilutions of tested compounds
MeOH:CH2Cl2). were added into the cell culture and
2-Acetyl-1,2,3,4-tetrahydro-ß-car incubated for an additional 24 hr. After that,
boline (3a). The title compound was 100 µL MTT solution (400 µg/mL) was
synthesized from (2) (0.12 g, 0.67 mmol) added into each well and incubated at 37 oC
and acetic anhydride (0.32 mL, 3.36 mmol) for 4 hr. The supernatant was removed, 100
to afford 3a as a yellow solid. µL DMSO was added into each well to
1-(3,4-dihydro-1H-pyrido[3,4-b]in dissolve the MTT formazan, and then
dol-2(9H)-yl)propan-1-one (3b). The title immediately analyzed on a microplate
compound was synthesized from (2) (0.11 g, reader (ELISA) at 540 nm. The IC50 was
0.64 mmol) and propionyl chloride (0.28 defined as the concentration that induces
mL, 3.21 mmol) to afford 3b as a brown 50% cellular death in comparison with
solid. untreated controls and calculated based on
1-(3,4-dihydro-1H-pyrido[3,4-b]in the following formular: {[(Absorbancecontrol-
dol-2(9H)-yl)octan-1-one (3c). The title Absorbancetest)]/ Absorbancecontrol}×100.
compound was synthesized from (2) (0.11 g, 2.3 Spectroscopic measurement
0.62 mmol) and octanoyl chloride (0.53 mL, 2,3,4,9-tetrahydro-1H-pyrido[3,4-
3.12 mmol) to afford 3c as a yellow solid. b]indole (2)
2-benzyl-2,3,4,9-tetrahydro-1H-py Yield: 0.20 g (91%). m.p. 185.9-187.5 oC
rido[3,4-b]indole (3d). The title compound (lit. 199-221 oC).21 1H-NMR (300 MHz,
was synthesized from (2) (0.050 g, 0.29 CDCl3) δ 2.75 (t, J = 5.7 Hz, 2H), 3.18 (t, J
= 5.7 Hz, 2H), 4.01 (s, 2H), 7.12 (pd, J = Hz, 1H), 7.46 (d, J = 7.5 Hz, 1H), 7.94 (br,
1.2, 7.1 Hz, 2H), 7.29 (d, J = 7.3 Hz, 1H), 0.3H), 8.40 (br, 0.7H). 13C-NMR (75 MHz,
7.48 (d, J = 7.0 Hz, 1H), 7.86 (br, 1H). 13C- CDCl3) (mixture of rotamers) δ 14.1, 22.1,
NMR (75 MHz, CDCl3) δ 22.5, 43.2, 43.9, 22.6, 25.5, 29.2, 29.5, 31.7, 33.7, 40.5, 43.6,
108.7, 110.7, 117.9, 119.4, 121.5, 127.6, 44.0, 111.0, 117.8, 118.3, 119.5, 119.8,
132.7, 136.6. ESI-MS22 [M+H]+ : 173. 121.7, 122.1, 127.0, 129.0, 130.7, 136.2,
2-Acetyl-1,2,3,4-tetrahydro-ß-car 172.5; HRESI- MS m/z [M+H]+: calcd for
boline (3a). C19H26N2O: 299.2118, found: 299.2116.
Yield: 0.039 g (27%). m.p. 217.2-218.3 oC 2-benzyl-2,3,4,9-tetrahydro-1H-py
(lit. 237-238 oC).23 1H-NMR (300 MHz, rido[3,4-b]indole (3d).
CD3OD) δ (mixture of rotamers) 2.20 (s, Yield: 0.012 g (16%). 1H-NMR (300 MHz,
1H), 2.23 (s,2H), 2.76 (t, J = 5.7 Hz, 0.5H), CDCl3) δ 2.82-2.86 (m, 2H), 2.91-2.95 (m,
2.84 (t, J = 5.7 Hz, 1.5H), 3.83 (t, J = 5.7 2H), 3.66 (s, 2H), 3.78 (s, 2H), 7.07-7.13(m,
Hz, 1.5H), 3.92 (t, J = 5.7 Hz, 0.5H), 4.72 2H), 7.27-7.40 (m, 2H), 7.47 (d, J = 6 Hz,
(s, 0.5H), 4.75 (s, 1.5H), 6.94-7.10 (m, 2H), 1H), 7.66 (s, 1H). 13C-NMR (75 MHz,
7.29 (dd, J = 4.0, 7.8 Hz, 1H), 7.40 (d, J = CDCl3) δ 21.1, 50.2, 50.9, 61.9, 108.4,
7.8 Hz, 1H). 13C-NMR (75 MHz, CD3OD) δ 110.7, 118.0, 119.4, 121.4, 127.3, 128.4,
(mixture of rotamers) 21.4, 21.8, 21.9, 22.7, 129.2, 131.7, 136.0, 138.2. HREI-MS25:
41.4, 45.4, 46.1, 108.2, 109.1, 111.9, 118.5, 262.1456.
118.6, 119.9, 119.9, 122.2, 122.4, 128.2, (3,4-dihydro-1H-pyrido[3,4-b]indo l-
130.8, 131.4, 138.0, 172.4, 172.5. ESI-MS23 2(9H)-yl)(phenyl)methanone (3e).
[M+H]+ : 215.5. Yield: 0.093 g (61%). m.p. 119.2-122.0 oC
1-(3,4-dihydro-1H-pyrido[3,4b]in (lit. 155-156 oC).26 1H-NMR (300 MHz,
dol-2(9H)-yl)propan-1-one (3b). CDCl3) (mixture of rotamers) δ 2.88 (br s,
Yield: 0.16 g (31%). m.p. 203.7-204.1 oC 2H), 3.70 (br s, 1.7H), 4.16 (br s, 0.3), 4.50
(lit. 204-206 oC).24 1H-NMR (300 MHz, (br s, 0.3H), 4.90 (br s, 1.7H), 7.00-7.15 (m,
CDCl3) (mixture of rotamers) δ 1.24 (t, J = 2H), 7.25-7.60 (br s, 7H), 8.32 (br s, 0.2H),
7.5 Hz, 3H), 2.43 (q, J = 7.5 Hz, 0.4H), 2.51 8.95 (br s, 0.8H). 13C-NMR (75 MHz,
(q, J = 7.5 Hz, 1.6H), 2.80-2.88 (m, 2H), CDCl3) (mixture of rotamers) δ 22.2, 41.1,
3.79 (t, J = 5.6 Hz, 1.6H), 3.97 (t, J = 5.6 46.1, 107.8, 111.1, 117.8, 119.6, 121.8,
Hz, 0.4H), 4.66 (s, 0.4H), 4.82 (s, 1.6H), 126.8, 126.9, 128.6, 130.0, 136.0, 136.3,
7.05-7.20 (m, 2H), 7.33 (d, J = 7.7 Hz, 1H), 171.6. HRESI-MS27 [M] : 276.1263.
7.46 (d, J = 7.5 Hz, 1H), 7.95 (br, 0.4H), (3,4-dihydro-1H-pyrido[3,4-b]ind ol-
8.35 (br, 0.6H). 13C-NMR (75 MHz, CDCl3) 2(9H)-yl)(furan-2-yl)methanone (3f).
(mixture of rotamers) δ 9.6, 22.0, 26.8, 40.1, Yield: 0.019 g (13%). 1H-NMR (300 MHz,
40.5, 43.0, 43.8, 107.8, 111.0, 117.8, 119.5, CDCl3) δ 3.05 (s, 1H), 4.08 (t, J = 1.7 Hz,
121.7, 126.8, 130.6, 136.2, 173.2. HRESI- 2H), 4.91 (br s, 1H), 6.26 (d, J = 1.7 Hz,
MS m/z [M+H]+: calcd for C14H16N2O: 1H), 7.05 (s, 1H), 7.06-7.20 (m, 2H), 7.31
229.1335, found: 229.1339. (d, J = 7.7 Hz, 1H), 7.50 (d, J = 7.2Hz, 1H),
1-(3,4-dihydro-1H-pyrido[3,4-b]in 7.53 (s, 1H), 8.40 (br, 1H). 13C-NMR (75
dol-2(9H)-yl)octan-1-one (3c). MHz, CDCl3) δ 8.4, 20.4, 40.2, 43.5, 109.8,
Yield: 0.12 g (41%). m.p. 149.9-150.0 oC. 110.8, 112.7, 114.7, 119.6, 120.7, 127.7,
1
H-NMR (300 MHz, CDCl3) (mixture of 128.1, 134.6, 135.2, 142.2, 146.1, 158.4.
rotamers) δ 0.87 (t, J = 1.5Hz, 3H), 1.20- HRESI-MS m/z [M+H]+: calcd for
1.50 (m, 10H), 1.65-1.80 (m, 2H), 2.41 (t, J C16H14N2O2: 267.1128, found: 267.1128.
= 7.5 Hz, 0.5H), 2.48 (t, J = 7.5 Hz, 1.5H), 2,2-bis(2-chlorobenzyl)-2,3,4,9-te
2.82 (t, J = 5.4 Hz, 0.5H), 2.86 (t, J = 5.4 trahydro-1H-pyrido[3,4-b]indol-2-ium (3g).
Hz, 1.5H), 3.80 (t, J = 5.7 Hz, 1.5H), 3.97 Yield: 0.1441 g (12%). m.p. 125.7-127.0 oC.
(t, J = 5.7 Hz, 0.5H), 4.67 (s,0.5H), 4.82 (s, 1
H-NMR (300 MHz, CDCl3) δ 3.34 (t, J =
1.5H), 7.07-7.20 (m,2H), 7.33 (d, J = 8.1 6.0 Hz, 2H), 3.99 (t, J = 6.0 Hz, 2H), 4.77
(d, J = 13.1 Hz, 2H), 5.13 (s, 1H), 5.24 (d, J
= 13.1 Hz), 7.11 (td, J = 1.0, 8.0 Hz, 1H),
7.21 (td, J = 1.0, 8.0 Hz, 1H), 7.48-7.57 (m,
5H), 7.85-7.90 (m, 1H), 11.15 (br s, 1H).
13
C-NMR (75 MHz, CDCl3) δ 18.31, 53.21,
57.37, 60.63, 103.00, 112.70, 117.81,
120.05, 123.03, 124.44, 125.00, 126.00,
128.86, 130.81, 132.96, 136.58, 137.10.
HRESI-MS [M]: cald for C25H23Cl2N2+:
421.1233, found: 421.1222.

3.1 Chemistry
The synthesis of 2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole (2) by Pictet–
Spengler reaction from tryptamine (1) and
paraformaldehyde was obtained in high
yield (91%) (Fig. 2). N-alkylation or N-
acylation of tetrahydro-ß-carboline (2) by Figure 2. Reagent and conditions: (i) p-
acyloyl halide or alkyl halide in CH2Cl2 formaldehyde, AcOH : dry MeOH (5:0.5), 4
using NEt3 as base furnished 2-substituted- Å MS, 80 oC, 1-4 hr then basified to pH 9-
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole 10 using NH4OH.; (ii) akyl halide or acryl
(3a-3g). All new synthetic products were halide, NEt3, DCM, RT, 24 hr
confirmed of their chemical structures by
using 1H and 13C-NMR spectroscopy and hi- Table 1. IC50 (µg/mL)a values for 1,2,3,4-
resolution mass spectrometry. Tetrahydro-ß-carbolines derivatives
Comp. Hep G2 HeLa L929
3.2 Cytotoxicity activity
IC50 SIb IC50 SIb IC50
The in vitro anticancer activities of
2 122.00 0.43 88.66 0.59 53.00
the synthesized compounds were evaluated 3a >100 N/A >100 N/A 82.00
against human hepatocyte carcinoma (Hep 3b 57.78 N/A 88.61 N/A >100
G2), human cervix epitheloid carcinoma 3c NA* N/A >100 N/A 76.00
(HeLa) and mouse connective tissue 3d >100 N/A >100 N/A 30.00
3e 24.00 1.67 82.04 0.49 40.00
fibroblast (L929) by using the MTT assay.
3f 64.00 0.85 58.67 0.92 54.20
The acridine orange was used as a positive 3g 26.67 0.63 25.00 0.67 16.71
control. The results are shown in Table 1. Acridine
6.36 1.14 4.11 1.76 7.24
The substituent on the nitrogen atom at the orange
2-position exhibited promising antitumor * NA = not active
a
activities, the benzyl amine derivative 3d Human tumor cells were treated with different
concentrations of samples for 24 h. The data
provided moderate activity against cancer represented the mean values of three independent
cell. For aromatic amide, the benzoyl determinations and shown as IC50 (µM, the
derivative 3e displayed the highest concentration of 50% proliferation-inhibitory effect)
b
cytotoxicity against Hep G2 and HeLa cell SI = Selectivity index calculated as the ratio of
lines with half maximal inhibitory CC50 to IC50. N/A: not applicable
concentration (IC50) values of 24.00 and
On the other hand, the presence of
82.04 μg/mL, respectively. In addition,
the aliphatic amide series resulted in a loss
compound 3f bearing 2-furoyl group
of antitumor activity. However, the
exhibited moderate inhibition with
propionyl group 3b showed high
selectivity index of 0.85 and 0.92 for Hep
cytotoxicity against Hep G2 and HeLa cell
G2 and HeLa cell, respectively.
line in the range of 57.78-88.61 μg/mL with
no selectivity index but showing the weakest
activity against non-cancer cell line with 4. Agnusdei, M.; Bandini, M.; Melloni, A.;
IC50 values more than 100 μg/mL. In Umani-Ronchi, A. J. Org. Chem. 2003,
contract, acetylated compound 3a displayed 68, 7126–7129.
moderate cytotoxicity against Hep G2 and 5. Bandini, M.; Melloni, A.; Piccinelli, F.;
HeLa cell lines. Octanoylated compound 3c Sinisi, R.; Tommasi, S.; Umani-
has no activity toward the Hep G2 cell line. Ronchi, A. J. Am. Chem. Soc. 2006,
Among them, the derivative 3g, which 128, 1424–1425.
contains 2,2-bis(2-chlorobenzyl) group 6. Gupta, L.; Srivastava, K.; Singh, S.;
displayed the highest cytotoxicity against Puri, S. K.; Chauhan, P. M. S. Bioorg.
cancer cell lines and non-cancer cell lines, Med. Chem. Lett. 2008, 18, 3306–3309.
ranging from 16.71-26.67 μg/mL. The 7. Liew, L. P. P.; Fleming, J. M.; Longeon,
selectivity index of 3g for Hep G2 and Hela A.; Mouray, E.; Florent, I.; Bourguet-
cell line was 0.63 and 0.67, respectively. Kondracki, M.-L.; Copp, B. R.
Tetrahedron 2014, 70, 4910–4920.
4. Conclusion 8. Van Maarseveen, J. H.; Hermkens, P.
In summary, a series of N- H.; De Clercq, E.; Balzarini, J.;
substituted-2,3,4,9-tetrahydro-1H-pyrido Scheeren, H. W.; Kruse, C. G. J. Med.
[3,4-b]indole have been synthesized by Chem. 1992, 35, 3223–3230.
Pictet-Spengler reaction, followed by N- 9. Mohamed, H. A.; Girgis, N. M.;
alkylation or N-acylation and tested for their Wilcken, R.; Bauer, M. R.; Tinsley, H.
cytotoxic activities. These compounds N.; Gary, B. D.; Piazza, G. A.;
exhibited inhibition of two cancer cell lines Boeckler, F. M.; Abadi, A. H. J. Med.
(Hep G2 and HeLa) and showed moderate Chem. 2011, 54, 495–509.
or weak activities against non-cancer cell 10. Purohit, P.; Pandey, A. K.; Singh, D.;
line (L929) in vitro by MTT assay. Among Chouhan, P. S.; Ramalingam, K.;
all compounds tested, compound 3g, bearing Shukla, M.; Goyal, N.; Lal, J.; Chauhan,
2,2-bis(2-chlorobenzyl) group as R2 P. M. S. Med. Chem. Comm. 2017, 8,
displayed the highest cytotoxicity against 1824–1834.
Hep G2 and HeLa with IC50 values of 26.67 11. Trudell, M. L.; Soerens, D.; Weber, R.
and 25.00 µg/mL, respectively. W.; Hutchins, L.; Grubisha, D.;
Bennett, D.; Cook, J. M. Tetrahedron
Acknowledgements 1992, 48, 1805–1822.
We thank the Department of 12. Ovenden, S. P. B.; Nielson, J. L.;
Chemistry and Department of Microbiology, Liptrot, C. H.; Willis, R. H.; Tapiolas,
Faculty of Science, Silpakorn University for D. M.; Wright, A. D.; Motti, C. A.
the financial support and cytotoxicity assay. Phytochemistry Lett. 2011, 4, 69–71.
We also thank the Chulabhorn Research 13. Barsanti, P. A.; Wang, W.; Ni, Z. J.;
Institute for measurement of HR-ESI mass Duhl, D.; Brammeier, N.; Martin, E.;
spectra. Bussiere, D.; Walter, A. O. Bioorganic
Med. Chem. Lett. 2010, 20, 157–160.
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Synthesis of pyrrole-azepine core of stemoamide
Nantachai Inprung, Sathita Thaosiriwong, Punlop Kuntiyong*
Organic Synthesis Research Laboratory, Department of Chemistry, Faculty of Science,
Silpakorn University, Sanamchandra Palace, Nakhon Pathom 73000, Thailand
*E-mail: kuntiyong_p@su.ac.th
Abstract:
Stemoamide is the simplest member of Stemona alkaloid family which possesses a
wide variety of interesting activities ranging from their usage in folk medicine, herbicide, and
insecticide. In this research, we focused on synthesis of pyrrole-azepine intermediates 1 and 2
which would be our synthetic precursors toward stemoamide. Both pyrrole-azepine
intermediates were synthesized starting from L-asparagine via a chiral N-alkenylsuccinimide.
Reduction of the succinimide provided the corresponding γ-hydroxylactam and subsequent
diastereoselective N-acyliminium ion cyclization gave pyrrole-azepine intermediates 1 and 2.
1. Introduction first asymmetric total synthesis of

Stemona alkaloids are one of the stemoamide reported by Williams was
important classes of natural products which achieved by asymmetric aldol reaction.4
have interesting biological activities and Torssell reported a stereocontrolled total
structures.1 Stemona alkaloids are synthesis starting from commercially
exemplified in Figure 1. Stemoamide is a available (S)-pyroglutaminol.5 Honda
member of Stemona alkaloids which was reported diastereoselective synthesis using
isolated from the roots of Stemona tuberosa an intramolecular samarium diiodide-
Lour. by Xu and co-workers in 1992.2 It promoted 7-exo-trig cyclization of a ketyl
possesses a wide variety of interesting radical generated from the corresponding
activities ranging from usage as Chinese aldehyde.6
traditional medicine, herbicide, and Previously, we synthesized analogs
insecticide.2 Stemoamide contains a tricyclic of crispine A and a tetracyclic core of
core azepine γ-lactone and four stereogenic Erythrina alkaloids from chiral N-
centers. arylethylsuccinimide in which the new
stereogenic center was constructed with
stereocontrol by a dibenzylamino group.7
Herein we report the synthesis of pyrrole-
azepine core of stemoamide via
diastereoselective N-acyliminium ion
cyclization from chiral N-
alkenylsuccinimide prepared from
commercially available L-asparagine.

Figure 1. Stemona alkaloids
2.1 General
Starting materials and reagents were
Syntheses of stemoamide have been
obtained from commercial sources and were
reported in racemic and enantioselective
used without further purification. Solvents
routes. Wang and co-workers synthesized
were dried by distillation from the
stemoamide in racemic mixture using
appropriate drying reagents immediately
iminium ion cyclization and ruthenium-
prior to use. Tetrahydrofuran was distilled
catalyzed CO insertion as key steps.3 The
from sodium and benzophenone under
argon. Toluene and dichloromethane were reduced pressure. The crude product was
distilled from calcium hydride under argon. purified by flash chromatography (silica gel,
Moisture and air-sensitive reactions were 10:1 CH2Cl2/MeOH) to give N,N-dibenzyl-
carried out under an atmosphere of argon. L-asparagine (4) (6.00 g, 54%) as a pale
Reaction flasks and glassware were oven- yellow oil. Rf (9:1 CH2Cl2/MeOH) 0.36; 1H
dried at 105 oC overnight. Unless otherwise NMR (300 MHz, CDCl3)  7.43-7.13 (m,
stated, concentration was performed under 10H), 6.00 (s, 1H), 5.37 (s, 2H), 5.30 (s,
reduced pressure. Analytical thin-layer 2H), 4.08 (d, J = 13.4 Hz, 2H), 4.03 – 3.87
chromatography (TLC) was conducted using (m, 3H), 3.00 (dd, J = 16.3, 6.4 Hz, 1H),
Fluka precoated TLC plates (0.2 mm layer 2.72 (dd, J = 16.3, 8.2 Hz, 1H); 13C NMR
thickness of silica gel 60 F-254). (75 MHz, CDCl3) δ 174.8, 172.3, 135.4,
Compounds were visualized by ultraviolet
129.6, 128.9, 128.3, 59.8, 54.8, 33.4; -
light and/or by heating the plate after
48.8 (c 1.7, CHCl3); max (film) 3192, 2924,
dipping in a 1% solution of vanillin in 0.1 M
2852, 1669, 1495, 1456 cm-1.
sulfuric acid in EtOH. Flash
2.3 Synthesis of methyl N,N-dibenzyl-L-
chromatography was carried out using
asparaginate (5)
Scientific Absorbents Inc. silica gel (40 mm
particle size). Optical rotations were
measured with a Perkin-Elmer 243
polarimeter at ambient temperature using a
0.9998 dm cell with 1 mL capacity. Infrared To a solution of N,N-dibenzyl-L-
(IR) spectra were recorded on a Nicolet asparagine (4) (4.06 g, 12.3 mmol) in
5DXB FT-IR spectrometer. Proton and acetone (50 mL) was added K2CO3 (2.55 g,
carbon nuclear magnetic resonance (NMR) 18.43 mmol) and Me2SO4 (1.75 mL, 18.4
spectra were obtained using a Bruker mmol) and the mixture was stirred at rt
Avance-300 spectrometer. Peak overnight. The mixture was filtered to
multiplicities are indicated as follows: m remove K2CO3 and to the solution was
(multiplet), s (singlet), d (doublet), dd added dropwise sat. aq. NH4Cl (30 mL).
(doublet of doublets), ddd (doublet of Then the mixture was extracted with CH2Cl2
doublet of doublets), dddd (doublet of (3  100 mL). The combined organic layers
doublet of doublet of doublets), ddt (doublet were washed with 5% NaOH (100 mL),
of doublet of triples), dq (doublet of dried over anhydrous Na2SO4, filtered and
quartets), dt (doublet of triplets), dtd concentrated under reduced pressure. The
(doublet of triplet of doublets), t (triplet), tt crude product was purified by flash
(triplet of triplets), q (quartet) and p (pentet). chromatography (silica gel, 2:1
2.2 Synthesis of N,N-dibenzyl-L-aspara hexane/EtOAc) to give methyl N,N-
gine (4) dibenzyl-L-asparaginate (5) (1.97 g, 59%) as
a colorless oil. Rf (2:1 hexane/EtOAc) 0.08;
1
H NMR (300 MHz, CDCl3)  7.42-7.17 (m,
10H), 6.16 (s, 1H), 5.49 (s, 1H), 3.93-3.82
To a solution of L-asparagine (5.00 (m, 3H), 3.80 (s, 3H), 3.57 (d, J = 13.6 Hz,
g, 33.3 mmol) in MeOH and H2O (1:1, 100 2H), 2.68 (dd, J = 15.0, 6.0 Hz, 1H), 2.61
mL) was added NaOH (3.33 g, 83.3 mmol), (dd, J = 15.0, 6.0 Hz, 1H); 13C NMR (75
K2CO3 (11.5 g, 83.8 mmol) and BnCl (15.46 MHz, CDCl3) δ 173.4, 173.4, 172.4, 139.0,
mL, 133 mmol). The mixture was heated to 139.0, 129.0, 128.5, 127.3, 58.3, 54.9, 51.6,
reflux at 95 oC overnight and acidified with 35.6; -103.8 (c 1.6, CHCl3); max
1M HCl. Then the mixture was extracted
(film) 3355, 2951, 2844, 1730, 1672, 1453
with CH2Cl2 (3  100 mL). The combined cm-1.
organic layers were dried over anhydrous
Na2SO4, filtered and concentrated under
2.4 Synthesis of (S)-3-(dibenzylamino) N-alkenylsuccinimide 7 (994 mg,
pyrrolidine-2,5-dione (6) quantitative yield) as a green oil. Rf (4:1
hexane/EtOAc) 0.55; 1H NMR (300 MHz,
CDCl3)  7.49-7.17 (m, 10H), 5.78 (ddt, J =
16.9, 10.2, 6.6 Hz, 1H), 5.08-4.94 (m, 2H),
To a solution of methyl N,N- 3.91 (dd, J = 9.0, 5.3 Hz, 1H), 3.82 (d, J =
dibenzyl-L-asparaginate (5) (1.25 g, 3.83 13.4 Hz, 2H), 3.63 (d, J = 13.4 Hz, 2H),
mmol) in dry THF (10 mL) under argon 3.50 (t, J = 7.4, 2H), 2.75 (dd, J = 18.6, 9.0
atmosphere at -78 oC was added LDA (4.82 Hz, 1H), 2.60 (dd, J = 18.5, 5.3 Hz, 1H),
mL of 1.59 M solution, 7.67 mmol) and the 2.10-2.00 (m, 2H), 1.65 (p, J = 7.4 Hz, 2H);
mixture was stirred for 2 hours at -78 oC. 13
C NMR (75 MHz, CDCl3) δ 177.3, 175.2,
To this mixture was added dropwise sat. aq. 138.2, 137.2, 128.8, 128.5, 127.5, 115.4,
NH4Cl (20 mL) and the mixture was 57.3, 54.6, 38.1, 32.2, 31.0, 26.9; -
extracted with CH2Cl2 (3  20 mL). The 25.6 (c 1.6, CHCl3); max (film) 2925, 2841,
combined organic layers were dried over 1775, 1701, 1494, 1455 cm-1.
anhydrous Na2SO4, filtered and concentrated 2.6 Synthesis of (3S)-3-(dibenzylamino)-5-
under reduced pressure. The crude product hydroxy-1-(pent-4-en-1-yl)pyrrolidin-2-
was purified by flash chromatography (silica one (8) and (4S)-4-(dibenzylamino)-5-
gel, 4:1 hexane/EtOAc) to give imide 6 hydroxy-1-(pent-4-en-1-yl)pyrrolidin-2-
(1.10 g, 98%) as a white crystal. Rf (2:1 one (9)
hexane/EtOAc) 0.38; 1H NMR (300 MHz,
CDCl3)  9.31 (s, 1H), 7.43-7.14 (m, 10H),
3.91 (dd, J = 9.0, 6.0 Hz, 1H), 3.81 (d, J =
13.5 Hz, 2H), 3.61 (d, J = 13.5 Hz, 2H), Method 1; To a solution of chiral N-
2.61 (dd, J = 18.0, 6.0 Hz, 1H), 2.71 (dd, J = alkenylsuccinimide 7 (334.0 mg, 0.92
18.0, 9.0 Hz, 1H); 13C NMR (75 MHz, mmol) in toluene (5 mL) under argon
CDCl3) δ 178.7, 178.6, 176.3, 176.2, 138.3, atmosphere at -78 oC was added DIBALH
128.9, 128.6, 127.6, 58.8, 54.7, 33.1; - (1.84 mL of 1 M solution, 1.84 mmol) and
the mixture was stirred for 2 hours at -78 oC.
25.4 (c 1.6, CHCl3); max (film) 3234, 1782,
To this mixture was added dropwise MeOH
1705, 1494, 1454, 1338 cm-1.
(1 mL) and sat. aq. NaHCO3 (10 mL). Then
2.5 Synthesis of (S)-3-(dibenzylamino)-1-
(pent-4-en-1-yl)pyrrolidine-2,5-dione (7) the mixture was extracted with EtOAc (3 
20 mL). The combined organic layers were
dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The
To a solution of imide 6 (877.1 mg, product was obtained without purification to
2.98 mmol) in DMF (5 mL) under argon give hydroxylactam 8 (320 mg, 95%) as an
atmosphere at rt was added Cs2CO3 (1.16 g, orange oil.
3.58 mmol), KI (59.4 mg, 0.358 mmol) and Method 2; To a solution of imide 7
5-bromo-1-pentene (0.42 mL, 3.58 mmol) (52.8 mg, 0.146 mmol) in dry THF (5 mL)
and the mixture was stirred for 2 hours. To under argon atmosphere at 0 oC was added
this mixture was added water (20 mL) and LiAlH4 (11.1 mg, 0.292 mmol) and the
the mixture was extracted with CH2Cl2 (3  mixture was stirred for 2 hours at 0 oC.
To this mixture was added dropwise sat. aq.
20 mL). The combined organic layers were
NaHCO3 (5 mL). Then the mixture was
washed with water (5  20 mL), dried over
extracted with EtOAc (3  10 mL). The
anhydrous Na2SO4, filtered and concentrated
combined organic layers were dried over
under reduced pressure. The product was anhydrous Na2SO4, filtered and concentrated
obtained without purification to give chiral
under reduced pressure. The product was
obtained without purification to give +44.6 (c 0.3, CHCl3); max (film) 3370,
hydroxylactam 8 (46.8 mg, 88%) as an 2925, 2851, 1673, 1493, 1455 cm-1.
orange oil. 2.7 Synthesis of (2S,9aR)-2-(dibenzylami
Method 3;To a solution of imide 7 no)-1,2,5,6,9,9a-hexahydro-3H-pyrrolo
(201 mg, 0.555 mmol) in EtOH (5 mL) [1,2-a]azepin-3-one (1) and (2S,9aS)-2-
under argon atmosphere at 0 oC was added (dibenzylamino)-1,2,5,6,7,9a-hexahydro-
NaBH4 (42 mg, 1.109 mmol) and the 3H-pyrrolo[1,2-a]azepin-3-one (2)
mixture was stirred for 2 hours at 0 oC.
To this mixture was added dropwise sat. aq.
NaHCO3 (10 mL). Then the mixture was
extracted with EtOAc (3  20 mL). The
combined organic layers were dried over To a solution of hydroxylactam 8
anhydrous Na2SO4, filtered and concentrated (45.1 mg, 0.124 mmol) in dry CH2Cl2 (5
mL) under argon atmosphere at 0 oC was
under reduced pressure. The crude product
added TMSOTf (0.067 mL, 0.372 mmol)
was purified by flash chromatography (silica
and the mixture was stirred for 2 hours at 0
gel, 2:1 hexane/EtOAc) to give o
C to rt. To this mixture was added dropwise
hydroxylactam 8 (45.1 mg, 22%) and
sat. aq. NaHCO3 (5 mL) and the mixture
hydroxylactam 9 (20.7 mg, 10%) as
colorless oils. was extracted with CH2Cl2 (3  10 mL). The
(3S)-3-(dibenzylamino)-5-hydroxy- combined organic layers were dried over
1-(pent-4-en-1-yl)pyrrolidin-2-one (8). Rf anhydrous Na2SO4, filtered and concentrated
(2:1 hexane/EtOAc) 0.43; 1H NMR (300 under reduced pressure. The crude product
MHz, CDCl3)  7.52-7.13 (m, 10H), 5.78 was purified by flash chromatography (silica
gel, 4:1 hexane/EtOAc) to give pyrrole-
(ddt, J = 16.9, 10.3, 6.6 Hz, 1H), 5.11-4.98
azepine 1 (12.1 mg, 28%) and pyrrole-
(m, 3H), 3.90 (d, J = 13.7 Hz, 2H), 3.71 (d,
azepine 2 (5.7 mg, 13 %) as pale-yellow
J = 13.7 Hz, 2H), 3.52 (dd, J = 9.5, 7.5 Hz,
oils.
1H), 3.43 (ddd, J = 13.7, 8.8, 6.8 Hz, 1H),
(2S,9aR)-2-(dibenzylamino)-
3.18 (ddd, J = 13.9, 8.7, 5.7 Hz, 1H), 2.50
1,2,5,6,9,9a-hexahydro-3H-pyrrolo[1,2-
(ddd, J = 14.1, 9.5, 6.8 Hz, 1H), 2.03 (q, J =
a]azepin-3-one (1). Rf (2:1 hexane/EtOAc)
7.2, Hz, 2H), 1.74 (ddd, J = 14.1, 7.5, 4.4
Hz, 1H), 1.71-1.49 (m, 2H); 13C NMR (75 0.35; 1H NMR (300 MHz, CDCl3)  7.49-
MHz, CDCl3) δ 173.1, 139.5, 137.7, 128.8, 7.11 (m, 10H), 5.87 (dddd, J = 11.7, 7.1,
5.1, 2.3 Hz, 1H), 5.72 (dddd, J = 11.2, 7.4,
128.5, 127.0, 115.1, 79.8, 58.9, 54.7, 39.5,
3.9, 1.7 Hz, 1H), 4.07 (ddd, J = 13.4, 5.7,
32.6, 31.2, 26.7; -4.8 (c 1.8, CHCl3); 3.4 Hz, 1H), 3.92 (d, J = 13.8 Hz, 2H), 3.74
max (film) 3334, 2925, 2851, 1667, 1490, (dd, J = 9.4, 8.0 Hz, 1H), 3.65 (d, J = 13.8
1455 cm-1. Hz, 2H), 3.57 (tt, J = 8.7, 3.0 Hz, 1H), 2.84
(4S)-4-(dibenzylamino)-5-hydroxy- (ddd, J = 13.1, 9.5, 3.2 Hz, 1H), 2.28-2.08
1-(pent-4-en-1-yl)pyrrolidin-2-one (9). Rf (m, 6H), 1.77 (ddd, J = 13.6, 9.4, 3.0 Hz,
(2:1 hexane/EtOAc) 0.26; 1H NMR (300 1H); 13C NMR (75 MHz, CDCl3) δ 172.9,
MHz, CDCl3) 7.38-7.22 (m, 10H), 5.80 (ddt, 139.8, 131.7, 128.9, 128.7, 128.4, 128.2,
J = 16.9, 10.2, 6.6 Hz, 1H), 5.13-4.82 (m, 128.2, 126.9, 58.8, 56.0, 54.6, 53.2, 41.6,
3H), 3.76 (d, J = 14.0 Hz, 2H), 3.68 (d, J = 37.5, 30.0, 28.1; -21.2 (c 1.0, CHCl3);
14.0 Hz, 2H), 3.51-3.24 (m, 3H), 2.50 (dd, J max (film) 2922, 2851, 1681, 1493, 1453,
= 16.5, 8.2 Hz, 1H), 2.40 (dd, J = 16.5, 8.0 1432 cm-1.
Hz, 1H), 2.10-1.97 (m, 2H), 1.75-1.51 (m, (2S,9aS)-2-(dibenzylamino)-
2H); 13C NMR  (75 MHz, CDCl3) δ 173.1, 1,2,5,6,7,9a-hexahydro-3H-pyrrolo[1,2-
138.7, 137.6, 128.6, 128.4, 127.3, 115.3, a]azepin-3-one (2). Rf (2:1 hexane/EtOAc)
85.5, 61.7, 54.5, 39.5, 31.8, 31.2, 28.8; 0.30; 1H NMR (300 MHz, CDCl3)  7.50-
7.09 (m, 10H), 5.67 (dtd, J = 11.0, 5.4, 2.6
Hz, 1H), 5.41 (dq, J = 11.1, 1.7 Hz, 1H),
4.26 (dq, J = 9.0, 2.2 Hz, 1H), 4.14 (ddd, J =
14.0, 8.9, 5.4 Hz, 1H), 3.96 (d, J = 13.9 Hz,
2H), 3.76-3.61 (m, 3H), 2.99 (dt, J = 13.6,
5.1 Hz, 1H), 2.31-2.12 (m, 4H), 1.94 (ddd, J
= 12.7, 8.4, 1.9 Hz, 1H), 1.88-1.76 (m, 1H);
13
C NMR (75 MHz, CDCl3) δ 173.3, 139.9,
132.7, 132.4,128.8, 128.7, 128.4, 126.9,
Figure 3. Synthesis of chiral N-
59.0, 54.9, 54.6, 43.0, 30.9, 27.4, 26.2;
alkenylsuccinimide 7
-38.5 (c 0.4, CHCl3); max (film) 2922, 2851,
1682, 1494, 1454, 1346 cm-1.
The results of reduction of N-
alkenylsuccinimide 7 by various reducing
agents and conditions are shown in Table 1.
The synthesis of stemoamide in this
For reduction by DIBALH and LiAlH4,
report focused on construction of new
hydroxylactam 8 was obtained exclusively
stereocenter of pyrrole-azepines 1 and 2
(entries 1-2). Attempts to accomplish
which would be synthesized from
another regioisomer of hydroxylactam were
hydroxylactam 8 via diastereoselective N-
succeeded by reduction with NaBH4 (entry
acyliminium ion cyclization. The
3) which provided hydroxylactam 8 and
hydroxylactam 8 could be prepared from
hydroxylactam 9 in 22% and 10% yields,
selective reduction of succinimide 7 which
respectively.
would be derived from L-asparagine via N-
alkylation and succinimide formation
respectively (Figure 2).
Figure 4. Reduction of chiral N-

alkenylsuccinimide
Table 1. Reduction of succinimide 7

Reducing Temperature Yield of
Entry Solvent
agent (oC) 8/9 (%)
Figure 2. Retrosynthetic analysis of 1 DIBALH toluene -78 95/0
(+)-stemoamide 2 LiAlH4 THF 0 88/0
3 NaBH4 EtOH 0 22/10
The preparation of chiral N-
alkenylsuccinamide 7 was started with To construct pyrrole-azepine core of
benzylation of L-asparagine using benzyl stemoamide, hydroxylactam 8 was treated
chloride in basic conditions to give N,N- with TMSOTf to give the corresponding
dibenzyl-L-asparagine (4). Methylation of regioisomeric pyrrole-azepines 1 and 2 (2.2 :
this compound using Me2SO4 and K2CO3 in 1), each of which is a single diastereomer
acetone provided methyl N,N-dibenzyl-L- (Figure 5). The new sterogenic center was
asparaginate (5) and this was followed by investigated by NOE experiment of pyrrole-
imide formation by LDA to give chiral azepine 1 which showed correlation between
succinimide 6. Then, N-alkylation between the ring junction proton (3.57 ppm) and the
chiral succinimide 6 and 5-bromo-1-pentene benzylic proton (3.92 ppm). Attempts of
furnished N-alkenylsuccinimide 7 in cyclization of hydroxlactam 9 with TMSOTf
quantitative yield (Figure 3). and BF3.OEt2 did not yield the desired
bicyclic product presumably due to steric
hindrance of the dibenzylamino group.
mide prepared from commercially available
L-asparagine. Both pyrrole-azepines 1 and 2
will be used as important synthetic
precursors for the synthesis of
(+)-stemoamide.
Acknowledgements
Figure 5. Synthesis of pyrrole-azepines 1 The funding for this work is
and 2 provided by the Thailand Research Fund
(TRF) grant RSA5780028 and the Faculty of
Our plan for completion of a Science, Silpakorn University SRF-TBG-
synthesis of (+)-stemoamide is shown in 2556-03. The Development and Promotion
Figure 6. Both pyrrole-azepines 1 and 2 of Science and Technology Talents (DPST)
would be converted to tricyclic core 12 in 2 provides a scholarship for NI.
separate routes involving allylic oxidation,
Wittig reaction and iodolactonization for 1 References
and hydroboration, esterification, diazo 1. Pilli, R. A.; Rosso, G. B.; de Oliveira, M.
formation and carbene C-H insertion for 2. da C. F. Nat. Prod. Rep. 2010, 27, 1908.
Then Cope elimination followed by 2. Lin, W. H.; Ye, Y.; Xu, R. S. J. Nat.
hydrogenation would provide (+)- Prod. 1992, 55, 571–576.
stemoamide. 3. Wang, Y.; Zhu, L.; Zhang, Y.; Hong, R.
Angew. Chemie., Int. Ed. 2011, 50, 2787–
2790.
4. Williams, D. R.; Reddy, J. P.; Amato, G.
S. Tetrahedron Lett. 1994, 35, 6417–
6420.
5. Torssell, S.; Wanngren, E.; Somfai, P. J.
Org. Chem. 2007, 72, 4246–4249.
Figure 6. Synthesis plan for (+)-stemoamide 6. Honda, T.; Matsukawa, T.; Takahashi, K.
Org. Biomol. Chem. 2011, 9, 673–675.
4. Conclusion 7. Kuntiyong, P.; Bunrod, P.; Namborisut,
In summary, we have reported the D.; Inprung, N.; Sathongjin, J.; Sae-guay,
synthesis of pyrrole-azepines 1 and 2 via C.; Thongteerapab, S.; Khemthong, P.
diastereoselective N-acyliminium ion Tetrahedron 2017, 73, 4426–4432.
cyclization from chiral N-alkenylsuccini-
Ring opening of epoxides catalyzed by PdCl2 impregnated on
Al2O3-pillared clay
Sauwalak Wassanapadit, Piyarat Trikittiwong*
*E-mail: piyarat.t@sci.kmutnb.ac.th
Abstract:
Al2O3-pillared clay are synthesized by intercalation of aluminium (lll) chloride into
clay interlayers and calcined at 500 °C for 1 h. PdCl2 impregnated on Al2O3-pillared clay
(Pd/Al-PILC) were prepared by impregnated and calcined at 450 °C for 4 h.The modified
clay catalysts are characterized by X-ray diffraction (XRD) and Brunauer-Emmett-Teller
(BET) method, comparing with purified clay and Al2O3-pillared clay. The Pd/Al-PILC
efficiently catalyzed the ring-opening of epoxides to produce 2-methoxy-2-phenylethanol in
excellent yields under extremely mild reaction conditions. The quantitative yield of 2-
methoxy-2-phenylethanol was obtained using 30 wt.% Pd/Al-PILC to styrene oxide at 65 °C
for 30 min.
1. Introduction and non-toxic compound of

Ring opening of epoxides reaction is aluminosilicate.13
a major reaction used in the preparation of Considering the large experience of
intermediates for the synthesis of organic our research group in the use of bentonite
substances in industries such as epoxy clay which is a low-priced natural materials,
resins, paints, polymers, coatings and high surface area and high acidity, we
medicines, etc.1-5 In the production process, interested in development of this material.
catalysts are commonly used. This makes it The bentonite clay is crystalline hydrous
less time consuming and less more aluminosilicates and posses a layered
productive. The single phase catalysts for structure. Each clay layer is constructed
ring opening of epoxides reaction, such as from the combination of two basic types of
zirconium(IV) oxynitrate,6 hydrazine layers: a sheet of silica tetrahedral and a
7
sulphate and alkoxyl-modified chiral salen sheet of alumina octahedral. They are held
Mn(III)8 have been reported. Homogeneous together by sharing apical oxygen atoms.14
catalyst has the disadvantage, a difficulty of The structure of bentonite clay is composed
separation of catalysts from product, and of layers made up of two silica tetrahedral
highly toxic.9 The development and sheet with a central alumina octahedral
synthesis of homogeneous catalyst such as sheet. The distance between the clay-layer is
zeolite10-11 and metal oxides12 have been called the basal spacing which has located
introduced. This makes it easy to separate by additional cations and water.15 Then the
the catalyst from the products resulting in pillared clay prepared by exchanged
reducing production costs and polyoxocations into the interlayer and
environmental pollutant. Heterogeneous calcined, the polyoxocations transform into
catalyst is usually used to support catalyst metal oxide pillars, resulting to the pillared
dispersion. It is more stable in reaction clay. The metal oxide pillared clay has high
conditions and increases the surface area. In thermal stability, surface area and pore
this research, we are interested in the use of volume, which is of great interest because of
bentonite support. It is a simple, inexpensive its potential application as catalysts and
adsorbents in various fields.16-19 Metal
chloride impregnated on metal oxide Na-clay was intercalated with
pillared clay was prepared by impregnation aluminum polyoxocations. First,
and calcinations. The small crystals of metal AlCl3·6H2O (Fluka Chemika) was dissolved
chloride highly distribute insides pillared in water and hydrolysed with 0.2 M NaOH
clay structure and have high thermal (Merck), at an OH−/Al3+ mole ratio equal to
stability. Organic reactions under these 1.9. The resulting solution was left under
catalysts can be taken place very efficiently vigorous stirring for 24 h so that aluminum
using metal chloride impregnated on metal polycation (Al13) was formed, and then
oxide pillared clay catalysts added to the Na-clay dispersion, using a
The clay catalyst in ring opening of molAl3+/kgclay ratio of 3.8. The slurry was
epoxides reaction study is widely reported, aged under stirring for 24 h, and then
for example, Mn(III) salen complex/Al- washed by centrifugation until absence of
PILC,20 Fe/Al-PILC,21 Pd/Al-PILC,22 Fe- chloride. The intercalated solid was
PILC.23-24 These catalysts are highly separated by filtration and dried at 100 °C
effective and selective in the synthesis of for 24 h. Finally, it was heated at 5 °C/min
product. up to 500 °C in an oven and calcined for 1 h.
In this research, PdCl2 impregnated The product is referred as Al-PILC.
on Al2O3-pillared clay (Pd/Al-PILC) has Pd was incorporated by wet
been used as an efficient catalyst in ring impregnation of Al-PILC using solutions of
opening of epoxides reaction. Moreover, we PdCl2 (Fluka Chemika) in ethanol. The
have developed an efficient method for ring resulting wet solid was dried at 80 °C for 24
opening of epoxides reaction by using h and finally calcined for 4 h at 450 °C. The
Pd/Al-PILC under mild reaction conditions. catalyst, with a nominal 2wt.% load, is
referred as Pd/Al-PILC.
2. Materials and Methods 2.2 General catalytic procedure
2.1 Synthesis of Pd/Al-PILC A typical reaction mixture was
The raw clay mineral used in this consisted of styrene oxide (1.00 mmol), 3
work was the bentonite clay. This clay mL MeOH and the catalyst was added (30
mineral was purified by dispersion in water wt.% catalyst/styrene oxide) in a round
and fractionated centrifugation in order to bottom flask connected with a condenser for
remove quartz and other dense impurities. refluxing, The mixture was continuously
Na-clay was prepared by cation exchange. stirred. After that, the catalyst was filtered
Purified clay was suspended in 5 M NaOH out of the reaction mixture. The production
with ratio of clay to NaOH solution of 1 g: was followed by GC (Agilent 6890 GC;
50 mL for 24 h at room temperature. The Optima-1 column, 30 m 0.32 mm) using
hydroxide ions were removed from Na-Clay cyclohexanone as an internal standard. The
using a dialysis membrane. Then the product total pore volume and the mean pore
was dried at 100 °C diameter were calculated by the BET
method.
Table 1. Data from XRD and BET method

Samples d001(Å) SBET(m2/g) Vp(cm3/g) dp(nm)
Purified Clay 11.06 7.54 0.0222 11.7
Al-PILC 17.54 145.50 0.1132 3.9
Pd/Al-PILC 16.41 102.00 0.1414 4.4
d001; basal spacing, SBET; specific surface area, Vp; total pore volume and dp; mean pore diameter.
2.3 Spectroscopic measurement adsorption. The adsorption isotherms for N2

The textural characterization of all at −196 °C were measured by the BEL
samples was accomplished by nitrogen Japan Model BELSORP-mini. The specific
surface area, the total pore volume and the epoxide was performed without a catalyst,
mean pore diameter were calculated by the no product was observed. Thus, the catalyst
BET method. was needed for this reaction. Pd/Al-PILC
The X-ray powder diffraction showed higher activities than Al-PILC and
patterns between 2 and 30° of 2θ were Na-clay, respectively. PdCl2 has a good
measured by a Rigaku, Dmax 2200/utima+ dispersion on the Al-PILC, resulting in high
X-ray powder diffractometer (40 kV, 30 catalytic efficiency.
mA) with a monochromater and Cu Kα
radiation. Table 2. Conversion of 2-methoxy-2-
phenylethanol by different catalysts
3. Results and Discussion Entries Catalyst Yield Recovery
3.1 Catalyst characterization (%) (%)
1 None 0 100
The basal spacing of purified clay,
2 Na-clay 0 100
Al-PILC and Pd/Al-PILC are shown in 3 Al-PILC 25 74
Table 1. The basal spacing increased from 4 Pd/Al-PILC 100 0
11.06 Å in the purified clay to 17.54 Å in Reaction conditions: styrene oxide (1.00 mmol), 3
the Al2O3-pillared clay. The layer structure mL MeOH, catalyst 30 wt.% to styrene oxide at 65°C
of clay was not destroyed during preparation for 30 min
of catalysts. However, the board 001 peak
was shown due to the transformation of 3.2.2 The amount of catalyst
aluminium polyoxocations into rigid The amount of catalyst selected for
aluminium oxide pillars that increased the this reaction was determined by the weight
disordering within interlayer spacing of clay of the substrate. The results were shown in
structure. The PdCl2 impregnated on Al2O3- Table 3. For all reactions, the yields of 2-
pillared clay maintained the layered methoxy-2-phenylethanol were increased
structure, maintaining also the large basal with increasing the amount of the catalysts.
spacing to 16.41 Å Therefore, The optimized condition of this
The BET specific surface of purified reaction was 30 wt.% Pd/Al-PILC for 30
clay, Al-PILC and Pd/Al-PILC are min, giving 2-methoxy-2-phenylethanol in
compared in Table 1. The nitrogen excellent yield.
adsorption-desorption isotherms of those
prepared catalysts presented the distorted Table 3. Amount of catalyst
Entries Amount Yield Recovery
reversible type IV isotherm. As a result, the (%) (%)
specific surface area of Al-PILC is higher 1 None 13 88
than the surface area of purified clay 2 10 % 84 16
because aluminium oxide pillars were 3 20 % 90 11
distributed inside the interlayer of clay. 4 30 % 100 0
Reaction conditions: styrene oxide (1.00 mmol), 3
Pd/Al-PILC showed higher specific surface mL MeOH, Pd/Al-PILC at 65°C for 30 min
area than purified clay but lower than
that of Al-PILC. This could be explained 3.2.3 The effect of reaction temperature
that the incorporation of the crystals of and reaction time
PdCl2 blocked the access of the interlayer The reaction temperature and
microporous region of the support. reaction time on styrene oxide ring opening
catalyzed by Pd/Al-PILC and the results
3.2 Catalytic performance were summarized in Table 4. The yield
3.2.1 Effect of the presence of catalysts could be increased when the reaction time
The catalytic activity of Pd/Al-PILC was increased. For all reactions, compound
is compared to those of Na-clay, Al-PILC was presented as a single product, with the
and without a catalyst. The results are shown amount of recovered styrene oxide in which
in Table 2. When the ring-opening of this made the estimated mass balance of
100%. The data of catalytic activity at two Reaction conditions: styrene oxide (1.00 mmol), 3
temperatures, room temperature and reflux mL MeOH, Pd/Al-PILC at 65°C for 30 min
temperature was also presented. The
reaction was carried out at room 4. Conclusion
temperature, but the yield of 2-methoxy-2- The efficient synthesis of 2-
phenylethanol was moderate. The better methoxy-2-phenylethanol catalyzed by
yield could be accomplished when the Pd/Al-PILC was reported. The catalytic
reaction temperature was increased to 65 °C, activities of Pd/Al-PILC were performed
giving the excellent yield of 99% within 30 comparing to the activities of Na-clay and
min. Al-PILC. Pd/Al-PILC showed the best
activity. The optimized condition was using
Table 4. Ring opening reaction of styrene the styrene oxide (1.00 mmol), 3 mL MeOH,
oxide with MeOH catalyzed by Pd/Al-PILC 30 wt.% Pd/Al-PILC under refluxing
Entries Time Temp. Yield Recovery temperature for 30 min. The excellent yield
(min) (◦C) (%) (%) of 2-methoxy-2-phenylethanol was
1 5 65 43 57 achieved. The general procedure herein
2 10 65 71 30 described can be applied to a variety of
3 15 65 79 22
related reactions.
4 20 65 88 15
5 25 65 90 11
6 30 65 100 0 Acknowledgements
7 30 R.T. 50 50 We gratefully thank the Department
Reaction conditions: styrene oxide (1.00 mmol), 3 of Industrial Chemistry, Faculty of Applied
mL MeOH, catalyst 30 wt.% to styrene oxide Science, KMUTNB, Thailand for the
provision of chemicals, laboratory facilities
The optimized condition was using and financial support.
the styrene oxide (1.00 mmol), 3 mL MeOH,
30 wt.% Pd/Al-PILC under refluxing References
temperature (65 °C) for 30 min. The 1. Baitao, L.; Xiaojing, J.; Yanrun, L. C.;
excellent yield of 2-methoxy-2- Zhenjian, Z.; Xiujun, W, J. Ino. Chi.
phenylethanol was achieved. 2016, 419, 66–72.
2. Marwa, F.; Ilyes, K.; José, M. F., J. Mol.
Cat. 2016, 420, 282–289.
3. Nilamadanthai, A.; Gaffar, I.;
Ahsanulhaq, Q.; Arumugam, P.;
Fig 1. Ring opening of epoxides reaction Shanmugam, M., J. Mic. Mes. Mat.
2017, 247, 190–197.
3.2.4 The effect of reuse of Pd/Al-PILC 4. Patrick, S.; Toby, J. L.; Jennifer, M.D.;
The effect of reuse of Pd/Al-PILC on Loagan, Y.; James, R.V., J. Tet. Let.
styrene oxide ring opening and the results 2017, 58, 3478–3481.
were summarized in Table 5. The yields are 5. Melania, B.; Malgorzata, B.;
slightly reduced probably due to the catalyst Przemysław, K., J. Rea. Fun. Pol. 2017,
loss of the active site used in the reaction 119, 9–19.
and Pillar clay's layer collapses several 6. Sandip, S. S.; Madhukar, S. S.; Trupti,
times over the course of the reaction. B. S.; Pradeep, K., J. Tet. Let. 2015, 56,
Table 5. Reuse of catalysts 5916–5919.
Entries Times Yield Recovery 7. Alcino, J. L.; Jorge, A. R.; Rui, M. A., J.
(%) (%) Tet. Let. 2008, 49, 1694–1697.
1 1 100 0
2 2 95 6
8. Jing, H.; Yan, L.; Jiali, C.; Xiaohong, C.,
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1616–1622. K. B.; Ana, R. S.; Baltazar, C.; Cristina,
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Design, synthesis and antibacterial activities of a series of
bis(indolyl)arylmethanes
Chayamon Chantana1, Onnicha Khaikate1, Nannaphat Chumsri1, Umaporn Thathaisong2,
Jaray Jaratjaroonphong1*
1
Burapha University, Saensook, Chonburi 20131, Thailand
2
Department of Microbiology, Faculty of Science, Burapha University, Saensook, Chonburi 20131, Thailand
*E-mail:jaray@buu.ac.th
Abstract:
3,3-Bis(indolyl)methanes (BIMs) are found in natural products and are known to
exhibit a broad range of biological activities. Although great success has been achieved in the
synthesis of BIMs, some of the reported methods suffer from disadvantages such as the use of
expensive and corrosive reagents, high catalyst loading and low yields of the products. In this
work, FeCl36H2O is shown to be an efficient catalyst for the Friedel-Crafts reaction of
aldehydes with a variety of indoles in methanol or acetonitrile under “open-flask” and mild
condition. In the presence of 15 mol% of FeCl36H2O at room temperature, the reaction
proceeds smoothly to afford some new 3,3-bis(indoyl)arylmethane alkaloids in moderate to
excellent yields. Moreover, all the twelve BIMs synthesized showed promising antibacterial
activity against two Gram-positive bacteria namely, Staphylococcus aureus and Bacillus
subtilis. Structure-activity relationship analysis revealed that hydroxyaryl substituted BIMs
showed potent antibacterial activity. The 4-hydroxyphenyl linked 3,3-bis(5-methoxy-1H-
indol-3-yl)methane 3k (R = 4-HOC6H4, R1 = 5-OMe) was found to exhibit the most potent
antibacterial activities against both S. aureus and B. subtilis. The development of synthetic
protocol will be useful for economical and ecofriendly synthesis of potent antibacterial BIM
class of compounds.
1. Introduction most of the examples reported to date suffer

Indole alkaloids are one of the most from some disadvantages such as the use of
valuable sources of natural product with high toxicity, corrosiveness or expensive
diverse biological activity.1-5 In particular, catalyst, high catalyst loading, elevated
3,3-bis(indolyl)methanes (BIMs) are found temperature, long reaction time, low yields
commonly in both marine and terrestrial of products and special care for moisture/air
organisms. The naturally occurring and sensitive reagents.
synthetic BIMs display a broad range of
OH CF3
biological activities including HIV-1 CH3 OH
integrase inhibitor, antibacterial activity and
growth inhibitory activity on prostate cancer HN NH
Vibrindole A (a metalolite
cell lines (Figure 1).4-7 Methods for the of marine bacterium
HN NH HN NH
V zbrzo par ahaemoly tzcus)
synthesis of BIMs have been developed9-15 antibacterial 6
HIV-1 integrase inhibitor6 cytotoxic7
which mainly centered on Friedel-Crafts Figure 1. Phamacologically active BIMs

alkylation of two molecules of indole with
aldehydes using either protic or Lewis acid As part of our ongoing studies in
catalysts such as I2,8 hexamethylenetetra- developing a simple, convenient and eco-
mine-bromine,9 p-toluenesulfonic acid,10 friendly method for the synthesis of
AlPW12O40,11 In(OTf)3,12 AuCl3,13 FePO4,14 biologically active compounds, we report
and Fe(III) salt in ionic liquids.15 However, herein the design and synthesis of some
novel symmetrical BIMs via the Friedel- 70% EtOAc/30% hexane as eluent) to give
Crafts reaction of aldehydes with a variety the corresponding BIMs 3.
of indoles using FeCl36H2O as catalyst in Spectral data of known compounds 3
methanol or acetonitrile under “open-flask” were in agreement with those in the
and mild conditions (Figure 2). Furthermore, literature. 9-16
the synthesized compounds were evaluated 2.3 Spectroscopic measurement of the
for their antibacterial activity. selected BIMs
2.3.1 4-(Di(1H-indol-3-yl)methyl)-
R1
H R
R1
mil d condi tion
R1 R1
phenol (3f)17
O cat. FeCl3 6H2 O R
H H
CH 3OH or CH 3CN
Dark red solid; Rf = 0.10 (1:4
HN NH
air, rt
HN NH EtOAc/hexane); 1H-NMR (400 MHz,
Figure 2. Synthesis of BIMs CDCl3): δ 7.94 (brs, 2H), 7.41 (d, J = 8.0
Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 7.23-7.17
2. Materials and Methods (m, 4H), 7.02 (t, J = 7.5 Hz, 2H), 6.76 (d, J
2.1 General information = 8.4 Hz, 2H), 6.68 (s, 2H), 5.85 (s, 1H);
The high resolution 400 MHz 1H 13
C-NMR (100 MHz, CDCl3): δ 153.8,
NMR, together with 100 MHz 13C NMR 136.7, 136.3, 129.8, 127.0, 123.5, 121.9,
spectra were performed on Bruker DPX-400 120.0, 119.2, 115.0, 111.0, 39.3; IR (Film):
spectrometers at chemistry depart-ment, νmax 3411, 1611, 1509, 1456, 1417, 1337,
faculty of science, Burapha university and 1169, 1093, 744 cm-1
all spectra were measured in CDCl3 or 2.3.2 4-(Bis(5-methoxy-1H-indol-3-
CD3OD as solvent. The IR spectra were yl)methyl)phenol (3k)17
recorded on a Perkin Elmer System 2000 Dark red solid; Rf = 0.17 (3:7
FT-IR. High resolution mass spectra were EtOAc/hexane); 1H-NMR (400 MHz,
recorded on Finnigan MAT 95. Radial CD3OD): δ 7.21 (d, J = 8.7 Hz, 2H), 7.15 (d,
chromatography on a chromatotron was J = 8.4 Hz, 2H), 6.76-6.71 (m, 6H), 6.64 (s,
performed using Merck silica gel 60 PF254 2H), 5.67 (s, 1H), 3.61 (s, 6H); 13C-NMR
with CaSO4 1/2 H2O and was activated by (100 MHz, CDCl3): δ 155.7, 153.0, 135.7,
heating in an oven at 80 oC for 45 min. Thin 132.3, 129.6, 127.5, 124.6, 118.7, 115.2, 112.4,
layer chromatography (TLC) was performed 110.9, 102.0, 55.7, 39.3 IR (Film): νmax 3410,
with Merck silica gel 60 PF254 aluminium 1582, 1509, 1483, 1209, 1170, 1092 cm-1
plate.
2.2 General procedure for the synthesis of 3. Results and Discussion
symmetrical BIMs 3 3.1 Synthesis
To a mixture of aldehyde (1 mmol), For initial optimization of the
FeCl36H2O (0.15 mmol) and solvent reaction conditions, indole (2 mmol) and
(CH3OH or CH3CN) 5 mL in a round benzaldehyde (1 mmol) were chosen as
bottom flask was added a solution of indole model substrates for the template reaction.
(2 mmol) in the solvent 5 mL at room Gratifyingly, the reaction using 15 mol% of
temperature. After the reaction was stirred FeCl36H2O as catalyst in acetonitrile (10
until completion (TLC analysis), the mL) proceeded smoothly and excellent yield
reaction mixture was quenched with of the desired product 3a was isolated
aqueous NaHCO3 (10 mL) and extracted (Table 1). Other solvent such as methanol, a
with EtOAc (2x10 mL). The combined polar protic solvent, gave the lower yield of
organic layer was washed with brine (10 3a. The scope of this trans-formation in the
mL), dried over anhydrous Na2SO4, and double Friedel-Crafts reactions of indoles
filtered. The filtrate was evaporated and a variety of aldehydes was then
(aspirator then vacuo) to give a crude evaluated (Figure 3).
product, which was purified by radial
chromatography (SiO2, 100% hexane to
R1 R1
O cat. FeCl3 6H 2O R
2 R 1 +
N R H CH 3 OH or CH3CN
H air, rt
HN NH
1 2 3
F Cl Br
HN NH HN NH HN NH HN NH
3a (>99%, CH 3CN, 10 min) 3b (>99%, CH 3 CN, 10 min) 3c (88%, CH 3CN, 30 min) 3d (92%, CH 3CN, 30 min)
(47%, CH 3OH, 10 min) (8%, CH 3 OH, 10 min) (13%, CH 3 OH, 30 min) (17%, CH3 OH, 30 min)
CO2CH 3 OH OCH 3
3e (84%, CH3 CN, 10 min) 3f (72%, CH 3CN, 24h) 3g (82%, CH 3CN, 10 min) 3h (61%, CH3 CN, 5 min)
(82%, CH3 OH, 30 min) (>99%, CH 3 OH, 30 min) (86%, CH3 OH, 30 min) (16%, CH 3OH, 24 h)
OH OH OH OH
Cl Cl Br Br H 3 CO OCH3 O 2N NO 2
3i (78%, CH 3CN, 48h) 3j (75%, CH 3CN, 48h) 3k (12%, CH 3CN, 48h) 3l (45%, CH3 CN, 48h)
(>99%, CH3 OH, 24h) (96%, CH3 OH, 6.5h) (>99%, CH3 OH, 10min) (35%, CH 3OH, 48h)
Figure 3. Synthesis of 3,3-bis(indoyl)methane derivatives by the reaction of indole and

aldehyde using FeCl36H2O as the catalyst
At the beginning of the search for the activated by FeCl36H2O to give

aldehyde substrate scope, when indole was intermediate I. The addition of indole to the
served as a nucleophile partner, a variety activated aldehyde complex affords
ofelectron-deficient and electron-rich intermediate II. Formation of 3,3-BIM 3
aromatic aldehydes as well as could be explained by the addition of a
isobutyraldehyde as a representative of second indole to the intermediate III, which
aliphatic aldehyde, using acetonitrile as a is generated by dehydration of II.
solvent underwent double Friedel-Crafts
reactions to smoothly generate the (III)Fe
O
H
Fe(III) N
corresponding 3,3-BIMs 3b-d and 3h in O
FeCl3 6H 2O
O H
R
good to excellent yields. It should be noted R H R H
HN
that the yields of 3e, 3f, 3g, 3i, 3j and 3k are I II
higher in methanol. As we expected that

substituted indoles, such as 5-chloro-, 5-
bromo-, 5-methoxy and 5-nitroindoles, also R R
N
reacted with 4-hydroxybenzaldehyde in H
methanol to form the corresponding HN NH N
3 H
products 3i-l in moderate to excellent yields. III
The plausible mechanism of the Figure 4. The plausible mechanism for
reaction is shown in Figure 4. In the first BIMs formation
step, the carbonyl group of the aldehyde is
3.2 Antibacterial activity of some novel be made (Table 2). In the case of
BIMs compounds containing unsubstituted indole
Very recently, BIMs were group (3a-h), BIMs with 4-hydroxyphenyl
synthesized by the reaction of indole with group 3f showed strongest activity. These
various substituted aldehydes using results might be explained by the effect of
microwave irradiation under solvent free the phenolic moiety. Antibacterial activity of
conditions and the synthesized compounds BIMs containing 4-hydroxyphenyl group
showed good antibacterial activity against and substituted indoles (3i-3l) were then
Staphylococcus aureus.16 In this paper, some examined. The 4-hydroxyphenyl linked 3,3-
novel BIMs synthesized were evaluated for bis(5-methoxy-1H-indol-3-yl)methane 3k (R
antibacterial activity against Gram-positive = 4-HO-C6H4, R1 = 5-OMe) was found to
bacteria i.e. Staphylococcus aureus ATCC exhibit the most potent antibacterial
25923 and Bacillus subtilis ATCC 6633. activities against both S. aureus and B.
The inhibition zone values are summarized subtilis. Moreover, 3k showed obvious
in Table 1. The results showed that all of the antibacterial activity against S. aureus and
compounds inhibited both S. aureus and B. B. subtilis with MIC values of 40 and 160
subtilis when used at 400 µg/disc µg/ml, respectively (Table 2). Whereas, the
concentration, and four compounds (3f, 3h, MBC values of 3k on the inhibition of S.
3i and 3k) inhibited it by more than 10 mm aureus and B. subtilis are 40 and 160 µg/ml,
of diameter of inhibition zone. When respectively.
investigating the structure-activity
relationship (SAR), some observations can
Table 1. Antibacterial activities evaluation of compounds 3a-3f

compound diameter of inhibition zone (mm) compound diameter of inhibition zone (mm)
B. subtilis S. aureus B. subtilis S. aureus
3a 8 7 3g 8.5 7
3b 8.5 8 3h 11 11
3c 9 10 3i 10.5 11
3d 8 11.5 3j 10 9
3e 7.5 13 3k 13 14
3f 12 13 3l 10 8
Gentamicin 24 23
Table 2. Antibacterial activities evaluation of modified structure

compound diameter of inhibition zone MIC MBC
(mm) (µg/ml) (µg/ml)
B. subtilis S. aureus B. subtilis S. aureus B. subtilis S. aureus
3k 13 14 40 160 40 160
Gentamicin 23.19 23.19 0.5 1 1 2
NZ = no diameter of inhibition zon
4. Conclusion loadings to yield a single-step

In summary, we have demonstrated environmentally friendly synthesis are the
an efficient FeCl36H2O-catalyzed double advantages of the present procedure.
Friedel-Crafts reaction of indoles with a Furthermore, all the BIMs synthesized
wide variety of aromatic and aliphatic showed promising antibacterial activity
aldehydes in acetonitrile or methanol under against two Gram-positive bacteria namely
“open flask” and mild conditions. Typically, S. aureus and B. subtilis and could be
the corresponding BIMs 3 were smoothly promising candidates as antibacterial agents.
obtained in good to excellent yields. Mild
reaction conditions with an inexpensive
commercially available catalyst in low
Acknowledgements 8. Ji, S. J.; Wang, S.; Zhang, Y.; Loh, T.-P.
This work was financially supported Tetrahedron 2004, 60, 2051–2055.
by the Research Grant of Burapha 9. Bandgar, B. P.; Bettigeri, S. V.; Joshi, N.
University through National Research S. Monatsh. Chem. 2004, 60, 1265–
Council of Thailand (Grant nos. 81/2558 1273.
and 99/2559) and the Center of Excellence 10. Pashas, M. A.; Jayashankara, V. P. J.
for Innovation in Chemistry (PERCH-CIC), Pharmacol. Taxicol. 2006, 1, 585–590.
Commission on Higher Education, Ministry 11. Firouzabadi, H.; Iranpoor, N.; Jafari, A.
of Education, Thailand. We also thank the A. J. Mol. Catal. A.: Chem. 2006, 244,
Thailand Research Fund for financial 168–172.
support to J.J. 12. Nagarajan, R.; Perumal, P. T.
Tetrahedron 2002, 58, 1229–1232.
References 13. Nair, V.; Abhilash, K. G.; Vidya, N.
1. Sundber, R. J. The chemistry of indoles, Org. Lett. 2005, 26, 5857–5859.
Academic Press, New York, 1970. 14. Behbahani, F. K.; Sasani, M. J. Serb.
2. Loounasmaa, M.; Tolvanen, A. Nat. Chem. Soc. 2012, 77, 1157–1163.
Prod. Rep. 2000, 17, 175–191. 15. Ji, S.-J; Zhou, M.-F.; Gu, D.-G.; Jiang,
3. Gul, W.; Hamann, M. T. Life Sci. 2005, Z.-Q.; Loh, T.-P. Eur. J. Chem. 2004,
78, 442–453. 1584–1587.
4. Kaushik, N. K.; Kaushik, N.; Attri, P.; 16. Sarva, S.; Harinath, J. S.; Sthanikam, S.
Kumar, N.; Kim, C. H.; Verma, A. K.; P.; Ethiraj, S.; Vaithiyalingam, M.;
Choi, E. H. Molecules 2013, 18, 6620– Cirandur, S. R. Chinese Chem. Lett.
6662. 2016, 27, 16–20.
5. Chadha, N.; Silakari, O. Eur. J. Med. 17. Sandi, B.; Jaideep, B.; Shabana, I.;
Chem. 2017, 134, 159–184. Babu, L.;Melissa, R.; Ramesh, M.;
6. Deb, M. L.; Bhuyan, Pl J. Synthesis Rammohan, R.; Baljinder, S.; Sudip, M.;
2008, 2891–2898. Baldev, S.; Ikhlas, A.; Ram; A. Eur. J.
7. Zhuo, M.-H.; Jiang, Y.-J.; Fan, Y.-S.; Chem. 2013, 63, 435-443.
Gao, Y.; Liu, S.; Zhang, S. Org. Lett.
2014, 16, 1096–1099.
Gold nanoparticles and pyrrolidinyl peptide nucleic acid-based
platform for the detection of RNA
Kriangsak Faikhruea, Tirayut Vilaivan*
*E-mail: vtirayut@chula.ac.th
Abstract:
Following our previous success in the development of a gold nanoparticle and PNA-
based dual-mode fluorescence and colorimetric platform for DNA detection, the applicability
of the system for detection of RNA was further evaluated. This platform consists of citrate-
capped gold nanoparticles (AuNPs) and pyrrolidinyl peptide nucleic acid (acpcPNA) – a
conformationally constrained DNA mimic with neutral-charged backbone consisting of
nucleobase-modified proline and cyclic -amino acid. In fluorescence detection mode, the
electrostatic adsorption of positively-charged lysine-modified fluorescein-labeled PNA on the
AuNPs surface resulted in quenching of the fluorescence signal, which was selectively
restored when the complementary RNA was added. For colorimetric detection mode,
positive-charged single-stranded PNA induced the aggregation of AuNPs leading to a
pronounced color change from red to blue. The aggregation was suppressed in the presence
of the complementary RNA. This platform is therefore a potentially versatile and useful tool
for RNA detection for various application. This was showcased by the detection of miRNA
which is an important biomarker for cancer diagnosis.
1. Introduction determination,6,7 fluorescence probe8,9 and

MicroRNAs (miRNAs) are non- fluorescence imaging.10,11 Recently, peptide
protein-coding, endogenous small RNAs nucleic acid (PNA) - a DNA mimic
with 19-25 nucleotides in length and found consisting of neutral pseudopeptide
in all eukaryotic cells. The detection of backbone - has been used as a probe to
miRNAs has gained much attention because detect miRNA21.12,13 PNAs exhibit higher
the level of miRNAs expression can be used affinity and better discrimination between
as biomarkers for abnormal cellular complementary and mismatched targets than
activities including cancer and solid natural oligonucleotides due to reduced
tumors.1,2 The miRNA21 is one of the electrostatic repulsion.14 Pyrrolidinyl PNA, a
miRNAs that is commonly overexpressed in new generation of PNA with
solid tumors of the lung, breast, stomach, conformationally rigid backbone consisting
prostate, colon, brain, head and neck, of proline and a cyclic -amino acid was
esophagus and pancreas.3 Due to the very recently developed by Vilaivan et al.15-17
low concentration of miRNA21 expression Certain members of the pyrrolidinyl PNA
in the typical cancer cells in range of 0.35- family such as acpcPNA and acbcPNA
1.20 amol/ngRNA4 together with the presence shows excellent chemical and biological
of similar miRNAs, practical methods to stability, high specifically binding to hybrid
detect miRNA21 must possess high with oligonucleotides at low ionic strength
sensitivity, good accuracy and specificity. and powerful mismatch discrimination. This
Previously reported methods to detect system of PNA has been designed to be a
miRNA21 are quantitative polymerase chain probe for many nucleic acid detections.18 In
reaction (PCR),5 electrochemical recent years, metallic nanoparticles such as
gold nanoparticles (AuNPs) have attracted to the deprotected amino terminal at the last
much attention as a reporter for sensing SPPS cycle. Next, side-chain deprotection
applications due to their unique optical was performed by heating the PNA on the
properties in terms of extremely high solid support with 1:1 aqueous ammonia-
extinction coefficients, distance-dependent dioxane at 65 oC overnight. The obtained
color, and outstanding fluorescence PNA was cleaved from resin by
19
quenching ability. Several methods have trifluoroacetic acid (TFA), purified by
been developed based on AuNPs and PNA reverse phase HPLC and characterized by
for detection of biomolecules, but most of MALDI-TOF mass spectrometry.
these involve the multistep and time
consuming covalent immobilization of the Table 1. PNA and RNA sequences for this
PNA or DNA probes onto the AuNPs study
surface.20,21 DNA bioassays based on Sequence (5’3’)
unmodified AuNPs and PNA have also been FAM-PNA21 FAM-AGTCTGATAAGC-
KKKKKNH2
developed. Electrostatic interaction between miRNA21 UAGCUUAUCAGACUGAUG
single stranded positively-charged PNA and UUGA
AuNPs lead to AuNPs aggregation causing a non-target RNA UAGUUGUGACGUACA
distinct color change due to surface plasmon
resonance (SPR) properties of AuNPs.22,23 2.3 Colloidal gold preparation
In contrast, hybridization between the PNA Gold nanoparticles (AuNPs, 12 nm)
probe and complementary nucleic acid was synthesized by citrate-reduction method
target can suppress the aggregation. In this (also known as Turkevich method).24,25 In
study, we developed a new dual-sensing brief, a solution of hydrogen
platform to detect miRNA21 based on tetrachloroaurate (III) (1 mM, 50 mL) was
acpcPNA and AuNPs which can be detected heated to boiling. Then, a trisodium citrate
by both fluorescence and colorimetric solution (40 mM, 1 mL) was rapidly added.
modes. The solution was continuously stirred until a
ruby-red solution was obtained. The mixture
2. Materials and Methods was cooled to room temperature and stored
2.1 General at 4-8 C. The morphology, size, and shape
All the chemicals used in this study of the synthesized gold nanoparticles were
were of analytical grade. The Fmoc- characterized by transmission electron
monomers for PNAs synthesis were microscopy (TEM) (TEM-2100 at Scientific
synthesized in house following a reported and Technological Research Equipment
protocol.17 (5,6)-Carboxyfluorescein NHS Centre (STREC), Thailand) and by UV-
ester (FAM-NHS) was purchased from Visible spectroscopy (Cary 100).
Thermo Scientific. Hydrogen 2.5 Fluorescence study
tetrachloroaurate(III) trihydrate was In all experiments, the fluorescence
obtained from Sigma-aldrich. Tri-Sodium spectroscopic measurement was measured at
citrate was from Riedel-de Haen. Synthetic excitation wavelength of 460 nm with
miRNA21 target used in this study was photomultiplier tube (PMT) detector setting
purchased from Pacific Science Co., Ltd. at 940 V. The PNA solution was prepared at
Thailand. 0.1 μM in 10 mM Tris-HCl buffer (pH 7.4)
2.2 Synthesis of FAM-PNA at final volume = 1 mL. The AuNPs (0.25-
The PNAs were synthesized with the 0.75 μg/mL) was added into the PNA
sequence as shown in Table 1 through Fmoc solution and incubated for 15 minutes. For
solid-phase peptide synthesis (SPPS) the fluorescence restoration experiment,
according to the previously reported 0.12 μM of miRNA21 was added into the
protocol.18After the desired sequence of the mixture of PNA and AuNPs (0.25 μg/mL)
PNA was obtained, FAM-NHS was coupled
and fluorescence signals were measured
every 10 minutes for 1 hour.
2.6 Colorimetric and UV-visible studies
The solution containing 50 μg/mL of
AuNPs was used for miRNA21 detection
experiment. A solution containing 1.0 μM of
the PNA probe was incubated with RNA
(1.2 μM) in 10 mM phosphate buffer (pH
7.0) for 15 minutes before the AuNPs
solution was added at final volume = 20 μL
and incubated for 15 minutes. Figure 2. Absorption spectra of the
The UV-visible spectra were synthesized gold nanoparticles
measured over 200-800 nm. In every case,
20 μg/mL of AuNPs solution was required. 3.2 Fluorescence detection of miRNA21
The 0.1 μM solution of PNA was incubated First, the quantity of AuNPs was
with RNA (in range of 0.001-0.005 μM) in optimized to find the smallest amount of
10 mM phosphate buffer (pH 7.0) for 15 AuNPs that can effectively quench the
minutes. After that, the mixture was added fluorescence signal of FAM-PNA21. As
into the AuNP solution and the final volume shown in Figure 3A, when 0. 25 μg/ mL of
= 1 mL. AuNPs was added to FAM-PNA21 solution
( 0. 1 μM) , the fluorescence signal was
3. Results and Discussion dramatically dropped and no significant
3.1 Synthesis and characterization of change was observed beyond that
AuNPs concentration. As a result, the minimum
The AuNPs was synthesized by amount of AuNPs that can quench the
citrate-reduction of tetrachloroauric(III)
acid. The size of AuNPs was controlled by
ratio of gold to citrate. Transmission
electron microscopy (TEM) image showed
the spherical with an average size of 12 nm
(Figure 1). UV-visible spectrum of the
synthesized AuNPs showed the absorbance
at around 520 nm, which is in good
agreement with the literature (Figure 2).22
20nm
Figure 1. Transmission Electron

Microscopy (TEM) image (left) and size
distribution (right) of synthesized gold Figure 3. Quenching of FAM-PNA21 probe
nanoparticles by AuNPs (A) and restoration of the
fluorescence signal after addition of
miRNA21 as a function of time (B)
Figure 4. Sensitivity of fluorescence assay of miRNA21by the present method
fluorescence of FAM-PNA21 at 0.1 μM was The calibration curve in Figure 4 showed

0. 25 mg/ mL. According to the detection that the limit of detection (LOD) for the
principle, addition of the miRNA21 target fluorescence detection mode was 0.8 nM, as
should release the PNA from the gold calculated from 3(SD)/slope, where the SD
surface due to the specific hybridization was obtained from fluorescence signal of
between the PNA probe and the RNA target PNA+AuNPs mixture (blank).
to form a negatively-charged hybid that can 3.3 Colorimetric detection of miRNA21
no longer be adsorbed on the AuNPs. For the colorimetric detection of
Accordingly, the quenched fluorescence miRNA21, the color change of the AuNPs in
signal should be restored. The results are the presence of the FAM-PNA21 probe
shown in Figure 3B, after addition of the caused by electrostatically-induced
miRNA21 ( 0. 12 μM, 1. 2 equiv) the aggregation was investigated. The color
fluorescence signal was restored to around change of the AuNPs in the presence of
60% of the original fluorescence of the fixed concentration of FAM-PNA21 probe
FAM-PNA21 probe before addition of the ( 1.0 μM) and varying RNA targets was
AuNPs and remained constant after 30 showed in Figure 5A. The results clearly
minutes of incubation. The reasons that the validated the proposed detection principle
restored fluorescence could not reach the and confirmed the specificity of the
original level might be due to the incomplete detection since non-target RNA could not
hybridization/dissociation of the probe or by prevent the aggregation of the AuNPs.
fluorescence quenching of the FAM label From the result under UV-light as shown in
upon hybridization of the probe with the Figure 5B, the fluorescence light was clearly
RNA target. Based on the available data it is observed only PNA+targeted RNA mixture.
not yet possible to specify the exact reason To study the sensitivity of the
at present. Nevertheless, it is clear that the colorimetric detection, the concentration of the
restoration of the fluorescence signal was miRNA target was varied. The naked-eye
specific since the signal was not restored, detection results showed that the shade of
and actually slightly decreased, in the the solution color depended on the quantity
presence of non-target RNA in place of of the miRNA21 added. The color was
miRNA. purple at low miRNA21 and became more
To determine the sensitivity of the reddish at higher miRNA21 ( Figure 6A) .
detection, a linear calibration curve was Moreover, by using a UV-visible
prepared at different miRNA21 spectrophotometer, a linear calibration curve
concentrations ranging from 0.03-0.07 µM.
Figure 5. Naked-eye detection of miRNA21
by AuNPs and PNA probe under A) day-light,
B) UV-light (365 nm)
Conditions: (a) AuNPs, (b) with FAM-PNA Figure 6. Sensitivity of colorimetric assay of
21, (c) with FAM-PNA21+miRNA21 mixture, miRNA by the present method
(d) with FAM-PNA21+non-targeted RNA, (e)
with miRNA21 members of the TV lab for the synthesis of
the PNA monomers.
between the concentration of the miRNA21
target and the absorption ratio at 520 nm and References
650 nm ( A520/ A650) was obtained over 1. Hamidi-Asl, E.; Palchetti, I.;
the concentration range from 0-0. 005 µM. Hasheminejad, E.; Mascini, M. Talanta
From the calibration curve, the limit of 2013, 115, 74–83.
detection for miRNA21 in colorimetric 2. Kumarswamy, R.; Volkmann, I.; Thum,
mode was found to be 0.2 nM. T. RNA Biology. 2011, 8, 706–713.
3. Lu, Z.; Liu, M.; Stribinskis, V.; Klinge,
4. Conclusion C. M.; Ramos, K. S.; Colburn, N. H.; Li,
In this work, a new dual-mode – Y. Oncogene 2008, 27, 4373–4379.
namely fluorescence and colorimetric – 4. Ma, W.; Fu, P.; Sun, M.; Xu L.; Kuang,
detection platform for miRNA21 based on H.; Xu, C. J. Am. Chem. Soc. 2017, 139,
citrate-capped AuNPs and acpcPNA probe 11752–11759.
has been developed. Single-stranded FAM- 5. Ludlow, A. T.; Robin, J. D.;
labeled PNA probe electrostatically Mohammed, S.; Litterst, C. M.; Shelton,
interacted with AuNPs, causing the D. N.; Shay, J. W.; Wright, W. E.
fluorescence quenching and color change Nucleic Acids Res.2014, 42, 104.
from red to blue due to particles 6. Wen, Y.; Liu, G.; Pei, H.; Li, L.; Xu Q.;
aggregation. This interaction is suppressed Liang, W.; Li, Y.; Xu, Li.; Ren, S.; Fan,
by the presence of the miRNA21 target in a C. Methods. 2013, 64, 276–282.
sequence specific fashion. Based on model 7. Yin, H.; Zhou, Y.; Zhang, H.; Meng, X.;
experiments with synthetic RNA targets, this Ai, S. Biosen. Bioelectron. 2012, 33,
platform can be used to specifically detect 247–253.
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specificity. V.; Pompa, P. P.; Fiammengo, R. J. Am.
Chem. Soc.2014, 136, 2264–2267.
Acknowledgements 9. Li, H.; Mu, Y.; Qian, S.; Lu, J.; Wan, Y.;
This work was supported by The Fu, G.; Liu, S. Analyst 2015, 140, 567–
Thailand Research Fund (TRF) grant no. 573.
DPG5780002. We would like to also thank 10. Chan, H. M.; Chan, L. S.; Wong, R. N.
S.; Li, H. W. Anal. Chem. 2010, 82,
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Limbut, W.; Kanatharana, P; Vilaivan, Martín-Gago, J. A.; Morales Mdel, P.;
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Synthesis and optical properties of blue emitters from pyrene derivatives
Tanika Khanasa1*, Vinich Promarak2
1
Kasetsart University Chalermphrakiat Sakon Nakhon Province Campus, Sakon Nakhon 47000, Thailand
2
School of Molecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology,
Rayong 21210, Thailand
*E-mail: tanika.k@ku.th
Abstract:
Four blue emitting materials from pyrene substituted carbazole and phenyl
naphthalene-1-amine moieties (PyCars 1-3 and PyNap) were synthesized by Ullmann
coupling and Suzuki coupling reactions. The structures of materials were characterized by
spectroscopic methods. The absorption maxima of PyCars 1-3 and PyNap in
dichloromethane solution were 347 ( 19,764 M-1cm-1), 342 ( 40,166 M-1cm-1), 358 (
24,845 M-1cm-1) and 355 ( 21,426 M-1cm-1) nm, respectively, whereas the emissions were
found to be 457, 487, 472 and 468 nm. The stroke shifts of the four emitters were ranged
between 110-145 nm. The remarkably large stroke shifts indicate that the pyrene series
exhibits pull-push characteristics due to their strong arylamine donor and pyrene accepter in
molecules.
1. Introduction PyNap was connected to pyrene for tuning

Development of highly efficient blue color of the materials due to its strong
emitting materials from small organic electron donating ability compared to
molecules is still considered as an important carbazole. Based on these ideas, the high
research field due to the cheap, ease of fluorescence intensity of the materials was
synthesis, and color-tunable ability of expected to be obtained.
organic materials.1 Among various types of
organic emitters, pyrene is remarkably
considered as a good fluorophore2 owing to
the large -conjugation and strong -
electron delocalization energy.3 A number of
blue emitting materials from pyrene have
been reported.4-7 However, developing
efficient luminescent material is still
challenging for highly stable and pure blue
emitter. Therefore in this work, the novel
blue emitting materials from pyrene with
electron donor moieties including carbazole
(PyCars 1-3) and phenyl naphthalene-1-
amine (PyNap) were carefully designed as
shown in Figure 1. The bulky electron donor
tricarbazole in PyCar2 was introduced to
pyrene core to suppress the aggregation and
increase thermal stability of the materials
compared to monocarbazole in PyCar1,
whereas thiophene moiety was designed to
extend the conjugation system. The non- Figure 1. Structures of target pyrenes
planar phenyl naphthalene-1-amine donor in
2. Materials and Methods bromosuccinamide were purchased from
2.1 General TCI chemicals. Potassium phosphate,
1,4-dibromobenzene, 2-thiophene sodium carbonate and sodium t-butoxide
boronic acid, pyrene-1-boronic acid, N- were purchased from Sigma Aldrich.
Figure 2. Synthesis of PyCar1 and PyCar3. Reagents and conditions: (i) CuI, K3PO4, ()-
1,2-trans-diaminocyclohexane, toluene, reflux 24 h (ii) Pd(Ph3)4, 2 M Na2CO3, THF, reflux,
12 h (iii) NBS, THF, rt
Figure 3. Synthesis of PyCar2 and PyNap. Reagents and conditions: (i) CuI, K3PO4, ()-
1,2-trans-diaminocyclohexane, toluene, reflux, 24 h (ii) pyrene-1-boronic aci, Pd(Ph3)4, 2 M
Na2CO3, THF, reflux, 12 h (iii) CuI, t-BuONa, , ()-1,2-trans-diaminocyclohexane, toluene,
reflux, 24 h
The chemicals were used without further 5 min. The reaction was heated to reflux for
purification. Toluene and tetrahydrofuran 12 h. The crude was added water (50 ml)
(AR grade) were purchased from Carlo and extracted with DCM (2 x 100 ml). The
Erba. THF was distillated from combined organic phase was washed with
Sodium/benzophenone under N2 water (100 ml), brine solution (100 ml),
atmosphere. The two starting materials 3,6- dried over anhydrous Na2SO4, filtered and
di-tert-butyl-9H-carbazole (1) and 3,3'',6,6''- removal of the solvent to dryness.
tetra-tert-butyl-9'H-9,3':6',9''-tercarbazole Purification by column chromatography
(6) were synthesized according to the over silica gel eluting with a mixture of
literature methods.8 The chemical structures DCM and hexane (1:9) followed by
of all intermediates and final products were recrystallization with a mixture of DCM and
elucidated by spectroscopic techniques methanol afforded a white solid (0.172 g,
including 1H-NMR, 13C-NMR, IR and MS. 69%); 1H-NMR (600 MHz, CDCl3, δ): 1.51
2.2 Synthesis of 9-(4-bromophenyl)-3,6-di- (s, 18H; CH3), 7.53 (d, J = 9.0 Hz, 2H;
tert-butyl-9H-carbazole (3) ArH), 7.55 (d, J = 8.4 Hz, 2H; ArH), 7.76
A mixture of 3,6-di-tert-butyl-9H- (d, J = 8.4 Hz, 2H; ArH), 7.84 (d, J = 7.8
carbazole (1) (0.50 g, 3.0 mmol), 1,4- Hz, 2H; ArH), 8.05 (t, J = 7.2 Hz, 1H; ArH),
dibromobenzene (2) (3.55 g, 15.0 mmol), 8.09 (d, J = 7.8 Hz, 2H; ArH), 8.12 (d, J =
CuI (0.47 g, 1.5 mmol), ()-1,2-trans- 7.8 Hz, 2H; ArH), 8.20 (s, 2H; ArH), 8.22
diaminocyclohexane (0.25 ml, 1.5 mmol), (d, J = 8.4 Hz, 2H; ArH), 8.28 (d, J = 7.8
K3PO4 (1.62 g, 2.5 mmol) in toluene 50 ml Hz, 1H; ArH), 8.32 (d, J = 9.0 Hz, 1H;
was degassed with N2 for 5 min. The ArH). MALDI-TOF (m/z) (M+) calcd for
reaction was heated to reflux for 24 h. The C42H37N: 555.2965, found: 555.5826.
crude was filtered out the solid residue and 2.4 Synthesis of 3,6-di-tert-butyl-9-(4-
the filtrate was evaporated to dryness. The (thiophen-2-yl)phenyl)-9H-carbazole (4)
crude was added water (100 ml) and 4 was prepared in a similar manner
extracted with DCM (2 x 100 ml). The to PyCar from 3 (0.3 g) and 2-
combined organic phase was washed with thiopheneboronic acid (0.12 g) and obtained
water (100 ml), brine solution (100 ml), as white solid (0.23 g, 77%); 1H-NMR (600
dried over anhydrous Na2SO4, filtered and MHz, CDCl3, δ): 1.46 (s, 18H; CH3), 7.30
removal of the solvent to dryness. (d, J = 9.0 Hz, 2H; ArH), 7.44 (d, J = 8.4
Purification by column chromatography Hz, 2H; ArH), 7.46 (d, J = 8.4 Hz, 2H;
over silica gel eluting with a mixture of ArH), 7.69 (d, J = 9 Hz, 2H; ArH), 7.80 (t, J
DCM and hexane (1:9) followed by = 9.1, 8.6 Hz, 1H; ArH), 7.82 (d, J = 9.1 Hz,
recrystallization with a mixture of DCM and 1H; ArH), 7.85 (d, J = 8.7 Hz, 1H; ArH),
methanol afforded a white solid (0.62 g, 8.13 (s, 2H; ArH). MALDI-TOF (m/z) (M+)
81%); 1H-NMR (600 MHz, CDCl3, δ): 1.46 calcd for C30H31NS: 437.2273, found:
(s, 18H; CH3), 7.30 (d, J = 9.0 Hz, 2H; 437.4796.
ArH), 7.44 (d, J = 8.4 Hz, 2H; ArH), 7.46 2.5 Synthesis of 9-(4-(5-bromothiophen-2-
(d, J = 8.4 Hz, 2H; ArH), 7.69 (d, J = 9 Hz, yl)phenyl)-3,6-di-tert-butyl-9H-carbazole
2H; ArH), 8.13 (s, 2H; ArH). MALDI-TOF (5)
(m/z) (M+) calcd for C26H28BrN: 433.1422, N-Bromosuccinamide (0.094 g, 0.53
found: 433.2479. mmol) was added in small portions to a
2.3 Synthesis of 3,6-di-tert-butyl-9-(4- solution of 4 (0.23 g, 0.53 mmol) in THF
(pyren-1-yl)phenyl)-9H-carbazole (20 ml). The mixture was stirred at room
(PyCar1) temperature under N2 for a further 1 h. The
A mixture of 3 (0.20 g, 0.61 mmol), crude was added water (50 ml) and extracted
pyrene-1-boronic acid (0.15 g, 0.61 mmol), with DCM (2 x 50 ml). The combined
Pd(Ph3)4 (0.01 g, 0.012 mmol), 2 M Na2CO3 organic phase was washed with water (50
3 ml in THF 30 ml was degassed with N2 for ml), brine solution (50 ml), dried over
anhydrous Na2SO4, filtered and removal of 2.8 Synthesis of 3,3'',6,6''-tetra-tert-butyl-
the solvent to dryness. Purification by 9'-(4-(pyren-1-yl)phenyl)-9'H-9,3':6',9''-
column chromatography over silica gel tercarbazole (PyCar2)
eluting with a mixture of DCM and hexane PyCar2 was prepared in a similar
(1:9) followed by recrystallization with a maner to PyCar1 from 7 (0.15 g) and
mixture of DCM and methanol afforded a pyrene-1-boronic acid (0.042 g) and
white solid (0.21 g, 85%); 1H-NMR (600 obtained as white solid (0.088 g, 52%); 1H-
MHz, CDCl3, δ): 1.46 (s, 18H; CH3), 7.30 NMR (600 MHz, CDCl3, δ): 1.48 (s, 32H;
(d, J = 9.0 Hz, 2H; ArH), 7.44 (d, J = 8.4 CH3), 7.38 (d, J = 8.4 Hz, 4H; ArH), 7.48
Hz, 2H; ArH), 7.46 (d, J = 8.4 Hz, 2H; (d, J = 8.4 Hz, 4H; ArH), 7.68 (d, J = 8.4
ArH), 7.69 (d, J = 9 Hz, 2H; ArH), 7.80 (d, Hz, 2H; ArH), 7.84 (d, J = 8.4 Hz, 2H;
J = 9.1 Hz, 1H; ArH), 7.82 (d, J = 9.1 Hz, ArH), 7.94 (d, J = 8.4 Hz, 2H; ArH), 7.98
1H; ArH), 8.13 (s, 2H; ArH). MALDI-TOF (d, J = 8.4 Hz, 2H; ArH), 8.05 (t, J = 8.4 Hz,
(m/z) (M+) calcd for C30H30BrNS: 502.1269, 1H; ArH), 8.13 (d, J = 8.4 Hz, 2H; ArH),
found: 502.2727. 8.15 (d, J = 8.4 Hz, 2H; ArH), 8.18 (s, 4H;
2.6 Synthesis of 3,6-di-tert-butyl-9-(4-(5- ArH), 8.24 (t, J = 8.4 Hz, 1H; ArH), 8.30 (s,
(pyren-1-yl)thiophen-2-yl)phenyl)-9H- 2H; ArH), 8.31 (d, J = 9.6 Hz, 1H; ArH),
carbazole (PyCar3) 8.36 (d, J = 9.6 Hz, 1H; ArH). MALDI-TOF
PyCar3 was prepared in a similar (m/z) (M+) calcd for C74H67N3: 997.5324,
manner to PyCar1 from 5 (0.2 g) and found: 997.8101.
pyrene-1-boronic acid (0.095 g) and 2.9 Synthesis of N-(4-bromophenyl)-N-
obtained as white solid (0.138 g, 56%); 1H- phenylnaphthalen-1-amine (9)
NMR (600 MHz, CDCl3, δ): 1.51 (s, 18H; A mixture of phenyl naphthalene-1-
CH3), 7.53 (d, J = 9.0 Hz, 2H; ArH), 7.55 amine (8) (0.20 g, 0.88 mmol), 1,4-
(d, J = 8.4 Hz, 2H; ArH), 7.76 (d, J = 8.4 dibromobenzene (2) (1.08 g, 4.46 mmol),
Hz, 2H; ArH), 7.80 (d, J = 9.1 Hz, 1H; CuI (0.044 g, 0.22 mmol), ()-1,2-trans-
ArH), 7.82 (d, J = 9.1 Hz, 1H; ArH), 7.84 diaminocyclohexane (0.03 ml, 22 mmol), t-
(d, J = 7.8 Hz, 2H; ArH), 8.05 (t, J = 7.2 Hz, BuONa (0.42 g, 2.64 mmol) in toluene 20
1H; ArH), 8.09 (d, J = 7.8 Hz, 2H; ArH), ml was degassed with N2 for 5 min. The
8.12 (d, J = 7.8 Hz, 2H; ArH), 8.20 (s, 2H; reaction was heated to reflux for 24 h. The
ArH), 8.22 (d, J = 8.4 Hz, 2H; ArH), 8.28 crude was filtered out the solid residue and
(d, J = 7.8 Hz, 1H; ArH), 8.32 (d, J = 9.0 the filtrate was evaporated to dryness. The
Hz, 1H; ArH). MALDI-TOF (m/z) (M+) crude was added with water (100 ml) and
calcd for C46H39NS: 637.2817, found: extracted with DCM (2x100 ml). The
637.4566. combined organic phase was washed with
2.7 Synthesis of 9'-(4-bromophenyl)- water (100 ml), brine solution (100 ml),
3,3'',6,6''-tetra-tert-butyl-9'H-9,3':6',9''- dried over anhydrous Na2SO4, filtered and
tercarbazole (7) removal of the solvent to dryness.
7 was prepared in a similar manner Purification by column chromatography
to 3 from 6 (0.4 g) and 2 (0.65 g) and over silica gel eluting with a mixture of
obtained as white solid (0.34 g, 70%); 1H- DCM and hexane (1:9) followed by
NMR (600 MHz, CDCl3, δ): 1.46 (s, 32H; recrystallization with a mixture of DCM and
CH3), 7.32 (d, J = 8.4 Hz, 4H; ArH), 7.45 methanol afforded a white solid (0.17 g,
(d, J = 8.4 Hz, 4H; ArH), 7.61 (d, J = 7.2 48%); 1H-NMR (600 MHz, CDCl3, δ): 6.88
Hz, 4H; ArH), 7.62 (d, J = 8.4 Hz, 2H; (d, J = 9.0 Hz, 2H; ArH), 6.98 (t, J = 7.8 Hz,
ArH), 7.84 (d, J = 8.4 Hz, 2H; ArH), 8.16 (s, 1H; ArH), 7.06 (d, J = 7.8 Hz, 2H; ArH),
4H; ArH), 8.24 (s, 2H; ArH). MALDI-TOF 7.21 (t, J = 7.8 Hz, 2H; ArH), 7.28 (t, J =
(m/z) (M+) calcd for C58H58BrN3: 875.3866, 7.8 Hz, 2H; ArH), 7.33 (d, J = 7.8 Hz, 1H;
found: 875.5788. ArH), 7.39 (t, J = 7.8 Hz, 2H; ArH), 7.79 (d,
J = 8.4 Hz, 1H; ArH), 7.90 (d, J = 8.4 Hz,
2H; ArH). MALDI-TOF (m/z) (M+) calcd to give 5 in high yield. Finally, final
for C22H16BrN: 373.0522, found: 373.1772. products PyCar1 PyCar2 PyCar3 and
2.10 Synthesis of 3,3'',6,6''-tetra-tert- PyNap were successfully prepared by Pd-
butyl-9'-(4-(pyren-1-yl)phenyl)-9'H- catalyzed Suzuki coupling reaction of
9,3':6',9''-tercarbazole (PyNap) corresponding aryl bromides 3, 7, 5 and 9
PyNap was prepared in a similar with pyrene-1-boronic acid under the same
manner to PyCar1 from 9 (0.17 g) and systems of Pd(Ph3)4 as a catalyst, 2 M
pyrene-1-boronic acid (0.11 g) and obtained Na2CO3 as a base in aqueous THF to give
as white solid (0.054 g, 24%); 1H-NMR PyCar1 PyCar2 PyCar3 and PyNap in 72,
(600 MHz, CDCl3, δ): 6.88 (d, J = 9.0 Hz, 52, 56 and 24% yields, respectively. The
2H; ArH), 6.98 (t, J = 7.8 Hz, 1H; ArH), overall yields of PyCar1, PyCar2, PyCar3
7.06 (d, J = 7.8 Hz, 2H; ArH), 7.21 (t, J = and PyNap were found to be 56, 16, 37 and
7.8 Hz, 2H; ArH), 7.28 (t, J = 7.8 Hz, 2H; 11%. The lowest yield in PyNaP found in
ArH), 7.33 (d, J = 7.8 Hz, 1H; ArH), 7.39 (t, both Ullmann and Suzuki coupling reactions
J = 7.8 Hz, 2H; ArH), 7.79 (d, J = 8.4 Hz, lead to very low overall yield. Lower overall
1H; ArH), 7.90 (d, J = 8.4 Hz, 2H; ArH), yield in PyCar2 than that of PyCar3 even if
7.94 (d, J = 8.4 Hz, 2H; ArH), 7.98 (d, J = PyCar3 has shorter synthetic pathway
8.4 Hz, 2H; ArH), 8.05 (t, J = 8.4 Hz, 1H; possibly due to its steric hindrance.
ArH), 8.13 (d, J = 8.4 Hz, 2H; ArH), 8.15 However, due to the relatively short
(d, J = 8.4 Hz, 2H; ArH). MALDI-TOF preparation process, PyCar1 can be easily
(m/z) (M+) calcd for C38H25N: 495.2087, prepared and manufactured in large scale.
found: 495.3422.
A
1.0
Normalized Abs Intensity (a.u.)
PyCar1
PyCar2
3.1 Synthesis 0.8 PyCar3
The blue emitting materials from PyNap
pyrene derivatives PyCars 1-3 and PyNap 0.6
were successfully synthesized by Cu-

0.4
catalyzed Ullmann coupling and Pd-
catalyzed Suzuki coupling reactions as 0.2
outlined in Figures 2 and 3. First step, a
stoichiometric Ullmann coupling reaction 0.0
300 350 400 450 500
between carbazole starting compounds (1 wavelength (nm)
and 6) or phenyl naphthalene-1-amine (1
equivalent) and 1,4-dibromobenzene 2 (5 B
1.0
equivalent) under catalytic system of copper PyNap (ex 355 nm)
Normalized PL Intensity (a.u.)
PyCar1 (ex 347 nm)

iodide as a catalyst, potassium phosphate or 0.8 PyCar2 (ex 342 nm)
sodium t-butoxide as a base and ()-1,2- PyCar3 (ex 358 nm)
trans-diaminocyclohexane as a co-catalyst 0.6
gave aryl bromide intermediates 3 (81%), 7

0.4
(70%) and 9 (48%). Moderate yields in case
of phenyl naphthylamine series correspond 0.2
to the lower electron donating ability of
naphthylamine compare to phenylamine ring 0.0
400 450 500 550 600 650
in carbazole.9 Intermediate 5 with extended wavelength (nm)
conjugation system by introducing
thiophene unit was prepared by Suzuki Figure 4. Absorption (A) and emission
coupling between 3 and 2-thiopheneboronic spectra (B) of PyCar1-3 and PyNap in
acid to give 4 in good yield followed by DCM solution
bromination of 4 with N-bromosuccinamide
Table 1. Optical properties of PyCar1-3 and PyNap in DCM solution
Pyrene Derivatives abs (nm) log  (M-1cm-1) em (nm)a Stroke shift (nm)
PyCar1 347 4.30 457 110
PyCar2 342 4.60 487 145
PyCar3 358 4.39 472 114
PyNap 355 4.33 468 113
a
The emission spectra collected by excitation at absorption maxima for each compounds.
3.2 Optical properties Acknowledgements

Absorption and emission spectra of This work was financially supported
PyCar1-3 and PyNap in DCM solution are by The Thailand Research Fund (TRG-
shown in Figure 4. All compound show one 5880249).
strong absorption band located around 300-
450 nm, which are attributed to localized - References
* transitions of the entire molecules 1. Karuppusamy, A.; Kannan, P. J. J. of
without the Intramolecular Charge Transfer Luminescence 2018, 194, 718–728.
(ICT) bands of accepter pyrene. PyCar2 2. Jayabharathi, J.; Jeeva, P.;
shows the most red shift in absorption due to Thanikachalam, V.; Panimozhi, S. J.
its longest conjugation system. Surprisingly, Photochem. Photobiol. 2017, 346, 296–
PyNap exhibits more red shift in absorption 310.
than both of PyCar1 and PyCar2 although 3. Islam, A.; Wang, Q.; Zhang, L.; Lei, T.;
it has shorter conjugation length. PyCar2 Hong, L.; Yang, R.; Liu, Z.; Peng, R.;
shows highest molar extinction coefficient Liao, L.-S.; Ge, Z. Dyes and Pigments
due to its huge chromophore in three 2017, 142, 499–506.
carbazole units. In emission spectra (Figure 4. Feng, X.; Hu, J.-Y.; Wei, X.-F.;
4B), PyCar2 shows most red shift which is Redshaw, C.; Yamato, T. J. Mol. Struct.
attributed to the steric hindrance of three 2018, 1086, 216–222.
carbazoles. The stroke shifts of the four 5. Li, Y.-F.; Xie, X.; Gong, X.-J.; Liu, M.-
emitters were ranged between 110-145 nm. L.; Chen, R.-F.; Gao, D.-Q.; Huang, W.
The remarkably large stroke shifts indicate Dyes and Pigments 2017, 145, 43–53.
that the pyrene series exhibits pull-push 6. Zhang, R.; Zhang, T.; Xu, L.; Han, F.;
characteristics due to their strong arylamine Zhao, Y.; Ni, Z. J. Mol. Struct. 2017,
donor and pyrene accepter in molecules. 1127, 237–246.
7. Melgoza-Ramírez, M. Gonz alez-Juarez,
4. Conclusion E.; Güizado-Rodríguez, M.; Barba, V.;
In summary, we have successfully Rodríguez, M.; Ramos-Ortíz, G.;
synthesized a series of pyrene derivatives by Maldonado, J.-L. J. Mol. Struct. 2016,
key step Ullmann coupling and Suzuki 1103, 25–34.
coupling reactions. All compounds show 8. Khanasa, T.; Prachumrak, N.;
strong absorption and emission in blue Rattanawan, R.; Jungsuttiwong, S.;
region around 300-480 nm, which indicate Keawin, T.; Sudyoadsuk, T.; Tuntulani,
that they can be used for blue emitting T.; Promarak, V. Chem. Commun. 2013,
materials in OLED. 49, 3401−3403.
9. Louie, J.; Driver, M. S.; Hamann, B. C.;
Hartwig, J. F. J. Org. Chem. 1997, 62,
1268–127.
Molecular docking investigation on antimicrobial mechanism of plumbagin
derivatives
Issaree Charoenpinyoying, Boworn Sriyaphai, Angsina Kongdee,
Luckhana Lawtrakul*
*E-mail: luckhana@siit.tu.ac.th
Abstract:
Plumbagin or 5-hydroxy-2-methyl-1,4-naphthoquinone (C11H8O3) is a novel inhibitor
of the growth of M. tuberculosis, the main causative agent of tuberculosis. However, the
inhibition mechanism of this compound is still not clearly clarified. Therefore, quantum
chemistry and molecular docking calculations are used to study the antimicrobial mechanism
of plumbagin and its derivatives. Plumbagin has a naphthoquinone skeleton, which is analog
to one of the intermediate in the menaquinone synthetic pathway. Menaquinone is an
essential compound that mycobacteria used for respiration and electron transportation.
Therefore, the inhibition of this bacteria specific metabolic pathway could be the
antimicrobial mechanism of plumbagin compounds. The chemical structures of plumbagin
derivatives (drug candidates) are constructed and optimized at B3LYP/6-31G** level by
GaussView6 and Gaussian16 programs, respectively. AutoDock 4.2 is used to predict how
substrates and drug candidates, bind to a receptor of known 3D structure. The inhibition
constant (Ki) and the binding energy (B.E.) are used in evaluating drug-protein interactions.
1. Introduction anti-fungal.3-5 Moreover, some literature

Tuberculosis (TB) has been a serious reveals that PL and its derivatives show an
infectious disease for a long time and still effective mycobacteria inhibition.
remain a major problem. TB is one of the
top 10 causes of death worldwide. Reported
by the World Health Organization (WHO),
10.4 million people were infected with TB
and 1.7 million of them died from the
disease in 2016.1 The resurgence of TB is
mostly caused by the increasing of the
multidrug-resistant (MDR) TB and
extensively drug-resistant (XDR) TB strains Figure 1. Chemical structure of Plumbagin
in Mycobacterium tuberculosis (Mtb), the
main causative agent of TB. Hence, there is PL has a similar naphthoquinone
an urgent need to develop new anti-TB structure with 1,4-Dihydroxy-2-naphthoate
drugs. (DHNA), the fifth intermediate product of
Plants and other natural products the menaquinone biosynthesis pathway in
have been used as a useful source of drug Mtb.6 Menaquinone (vitamin K2) is the only
design for many diseases. Plumbagin [PL, type of quinone available in mycobacteria.7-9
(5-hydroxy-2-methyl-1,4-naphthoquinone)] These necessary compounds have a role to
(Figure 1) is an organic compound which is shuttle electron between the membrane-
extracted from the roots of Plumbago bound protein moieties which are important
zeylanica L.2 Many studies have proved that for the respiratory system in Mtb.10 So, this
PL has a vast variety of properties including essential process is a desired target for
anti-microbial, anti-malarial, anti-tumor and developing the new anti-TB compounds.
© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) PH1
Figure 2. Prenylation of DHNA to DMK-8 in Menaquinone biosynthesis pathway
The compounds that are structurally calculations (DFT) at B3LYP/6-31G(d,p)
analog to the substrates in the biosynthesis level.12,13
pathways are widely expressed their
inhibition activities toward those Table 1. Chemical structure and biological
11
pathways. This knowledge benefited us to activity of plumbagin and its derivatives
analyze the antimycobial mechanism of PL
and its derivatives.
The prenylation of DHNA to
Demethylmenaquinone-8 (DMK-8) is
catalyzing by 1,4-dihydroxy-2-naphthoate
prenyltransferase (Men A) as shown in
Figure 2. DHNA is a substrate for Men A
enzyme for this reaction. So, PL is predicted
to inhibit the menaquinone biosynthesis
pathway by being a competitive inhibitor
MIC
with the substrate DHNA. Compound R-group
(μM)
Table 1 shows the chemical structure Plumbagin H 21.3
of PL and its derivatives and also their (HNQ)
minimal inhibitory concentration (MIC) on Acetyl PL CO-CH3 34.8
Mtb.6 This information has confirmed that (AHNQ)
Benzoate PL CO-Ph 27.4
PL and its derivatives can be used as the (BHNQ)
inhibitors for Mtb but, the problem is that Butyrate PL CO-CH2-CH2-CH3 77.4
the mechanism is still unknown. (ByHNQ)
Here, anti-TB mechanism of PL and Cinnamate PL CO-CH=CH-Ph -a
its derivatives are studied. Molecular (CHNQ)
Crotonate PL CO-CH=CH-CH3 15.6
docking is used to investigate the binding (CrHNQ)
possibility between the Men A enzyme and Levulinoate PL CO-CH2-CH2-CO- -
PL derivatives in comparison with the (LHNQ) CH3
original substrate DHNA. The binding Propionate PL CO-CH2-CH3 16.4
energy (B.E.) and the inhibitory constant (PHNQ)
a
- refers to nonavailable information
(Ki) are used for evaluating the results.
2.2 Host preparation and verification
The 3D structure of Men A enzyme
2.1 Ligand preparation and optimization
is constructed by the homology modelling of
The structure of plumbagin and its
the exist DNA sequence in Phyre2
seven derivatives (Table 1) are constructed
server.14,15 The long DNA sequences will be
and optimized by GaussView 6.0 and
broken down and then each of them will be
Gaussian16 program with density functional
matched with the exist 3D structure in the
server. Then the small pieces of the model
will be connected to finally create the full
3D structure of the protein. The obtained 3D
crystal structures of the protein are verified
the accuracy by the superimposition of each
obtained structures (from same DNA
sequence) in Discovery Studio 4 program
package.16
2.3 Molecular docking calculations
Molecular dockings were performed
by using Autodock 4.2 program package to
study the binding possibility of the ligands
and Men A host.17 The Gasteiger charges Figure 3. The 3D structure of Men A and
were assigned for all molecules and the ligands binding pocket
Lamarckian Genetic Algorithm is used with
100 docking runs. The dockings are based The binding pocket of the ligands is
on the fixed-host and flexible-ligands located near the DNA sequence from
method. From the literature, the binding Gly119 to Leu144 on helix VI and VII. The
region of DHNA on Men A enzyme is molecular docking results of each ligands in
suspected to be on a sequence from Leu51 the binding pocket are presented in Table 2.
to Asp69 and also another review predicted
that the substrate would interact with Mg2+ Table 2. Molecular docking results in the
ion that is paired with Asp residues.18,19 binding region
Therefore, the grid box size of 68 x 68 x 86 Mean
Ki Ki,avg
points with the center point at 38.307, Ligand B.E. Fa
(μM) (μM)
(kcal/mol)
59.963, and 25.844 in x, y, and z-dimension
DHNA -5.07 10 139.69 191.99
respectively is being used. HNQ -5.52 81 87.71 89.82
AHNQ -6.39 84 15.77 20.68
3. Results and Discussion BHNQ -7.58 60 2.19 2.77
3.1 Host and ligands preparation results ByHNQ -6.42 44 12.69 19.66
CHNQ -7.34 30 2.54 4.16
For the ligands, after the
CrHNQ -6.67 56 9.30 12.89
optimization, the most stable conformation LHNQ -6.51 34 11.52 16.89
of each ligands is obtained and is further PHNQ -6.45 63 13.06 18.69
used for docking. The host is selected from a
F refers to frequency
the structure set that has the root-mean-
square-deviation (RMSD) value of zero. Overall, the mean binding energy of
3.2 Molecular docking results all ligands and substrate are in the same
The chemical conformation of the level. The binding energy (B.E.) of
ligands is selected based on the value of the plumbagin and DHNA are almost the same,
mean binding energy and the frequency or the difference is just about 0.5 kcal/mol. For
the number of conformations per cluster. the plumbagin derivatives, the obtained B.E.
Also, the lower the binding energy, the more values are slightly lower than the substrate
stable the binding interaction it is and the which the difference does not exceed 3
higher the frequency means that more kcal/mol. Thus, all ligands are able to bind
structures will lie in that conformation. As a inside the binding region. The detail of the
result, binding area of the ligands can be binding interactions is discussed in the next
obtained in the region as shown in Figure 3. sub-part.
There are nine helices of Men A and each of
them are name in roman number.
Figure 4. Binding interactions between host and ligands
3.3 Binding interactions of each ligands This result is related to the past studies of
Figure 4 shows the binding the suspected binding region to be on a
interactions between the host and ligands in sequence from Leu51 to Asp69 which is
the binding region. Overall, pi bonds are located on the helix VIII.19 Therefore, there
formed between Ala139 and the aromatic is a high possibility that the PL and its
rings of all ligands. Despite ByHNQ, all derivatives will bind with Men A host to
ligands including the substrate form inhibit TB by the interruption of the
hydrogen bonds with Tyr129. Also, menaquinone biosynthesis pathway in Mtb.
hydrogen bond interactions are presented
between Asn59 and all of the ligands except Acknowledgements
BHNQ and CHNQ. Since all of the ligands The authors gratefully acknowledge
have similar aromatic rings skeleton, the the financial support provided by
attach R-group will make each ligand has a Thammasat University Research Fund under
different interaction. the TU Research Scholar, Contract
No. 2/21/2559. The authors also would like
4. Conclusion to thank to Dr. Pimonluck Sittikornpaiboon
The molecular docking investigation for her help on the analysis of 3D-structure
between Men A enzyme and nine ligands; of Men A.
DHNA, HNQ, AHNQ, BHNQ, ByHNQ,
CHNQ, CrHNQ, LHNQ, and PHNQ reveals References
that all of the ligands have a possibility to 1. World Health Organization, Global
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Determination of ammonia using Pt/PANI ES nanocomposite: a DFT study
Duangporn Ngampring, Chinapong Kritayakornupong*
Department of Chemistry, Faculty of Science, King Mongkut’s University of Technology Thonburi,
*E-mail: chinapong.kri@kmutt.ac.th
Abstract:
Due to the conductive properties of polyaniline emeraldine salt (PANI ES), it has
been used as a sensor for detecting toxic volatile organic compounds. Doping PANI ES by
nanoparticle is found to be an effective method for increasing conductive properties and
sensing ability of PANI ES. In this work, structural and electronic properties of platinum
doped polyaniline emeraldine salt (Pt/PANI ES) sensing with NH3 were studied using the
B3LYP/6-31G(d) level of theory with LANL2DZ ECP basis set for platinum atom. The result
exhibited that NH3 molecule was adsorbed at the Pt atom with the bond distance and
chemical adsorption energy of 2.126 Å and -44.44 kcal/mol respectively, which was stronger
than that evaluated from the pristine PANI ES structure. In addition, percentage change in
HOMO-LUMO gap (Eg) corresponding to sensitivity of Pt/PANI ES sensor is significantly
higher than that determined for the parent. Our calculated results revealed that the Pt/PANI
ES nanocomposite is a potential sensing material for detecting NH3.
1. Introduction material can be improved by several

It is well known that ammonia (NH3) methods including combinations with other
is widely used in many industries; such as, polymers, addition of substituents and doped
food, fertilisers, textiles, motor vehicles, with metals. Platinum is one popular metal
chemical technology, medical diagnoses, for doping with other materials; such as,
environmental protection and fire power carbon nanotubes (CNTs), graphene (PGs),
plants. However, NH3 as a toxic volatile boron nitride nanotubes (BNNTs), and so
organic compound (VOC) is harmful to the on.4-6 The results all the studies found that
human body; for example, it causes irritation doped platinum on materials could enhance
to the eyes, nose, mouth and skin.1,2 the sensitivity and selectivity for sensing
Nowadays, Polyaniline (PANI) has received small gas molecules. Therefore, doped
some interests and both of theoretically and platinum on PANI ES is a good choice for
experimentally studied. The electronic improving the sensing ability of PANI. In
property of PANI depends on the chemical this work, we studied the structural and
structure; such as, the emeraldine salt (ES; electronic properties; such as, bond
semiconductors), leucoemeraldine base distances, bond angles, dihedral angles,
(LE; insulator), pernigraniline base (PB; interaction energies, charges transfer,
insulator) and emeraldine base (EB; HOMO, LUMO and HOMO-LUMO gaps of
insulator). For NH3 sensing, pure sensing ammonia using dimer, tetramer and
polyaniline emeraldine salt (PANI ES) was hexamer polyaniline emeraldine salt (PANI
utilised at the UB3LYP/6-31G(d) level.3 The ES) doped with platinum that were
result showed that the interaction between calculated by the density functional theory
NH3 and pure PANI ES was a consequence (DFT).
of physisorption and after the interaction
with the NH3, electronic property was a 2. Methods
slight change. Therefore, pure PANI ES has All geometry optimization were
a low sensing ability. The limitation of the performed using Gaussian 03 program.7
Geometry optimizations were calculated at ES, 2PANI ES/NH3, Pt/2PANI ES and
B3LYP/6-31G(d) level of theory on Pt/2PANI ES/NH3 complexes are displayed
PtnPANI ES/NH3 oligomer (n = 2, 4 and 6) in Figure 1.The intermolecular bond
and using LANL2DZ ECP for platinum distance (dN4-H7) of the pure 2PANI ES,
atom. The adsorption energy (Eads) is 4PANI ES and 6PANI ES were 1.015 Å,
defined as: 1.013 Å and 1.012 Å, respectively. The
Eads (1)  E( nPANIES / NH 3 )  E( nPANIES )  E( NH 3 ) inter-ring angles (∠C3N4C5) of 2PANI ES,
4PANI ES, 6PANI ES and 8PANI ES were
Eads (2)  E( Pt / nPANIES )  E( nPANIES )  E( Pt )
130.9º, 130.6º and 130.0º, respectively. For
Eads (3)  E( Pt / nPANIES / NH 3 )  E( Pt / nPANIES )  E( NH 3 ) the pure nPANI ES, the N5-H8 bond
where Eads (1) , Eads (2) and Eads (3) are distance and C3N5C6 angle decreased along
with the number of the oligomer. The
adsorption energy of nPANI ES/NH3, dihedral angle C2C3N4C5 and C6C5N4C3 of
Pt/nPANI ES and Pt/nPANI ES/NH3 while, dimer, tetramer and hexamer of pure PANI
E( nPANIES / NH 3 ) , E( nPANIES ) , E( NH 3 ) , E( Pt / nPANIES ) , ES were 22.4˚ and 22.4˚, 22.7˚ and 22.7˚
E( Pt ) and E( Pt / PANIES / NH 3 ) are the total energy and 24.1˚ and 24.1˚, respectively. The
of nPANI ES/NH3, nPANI ES, NH3, Pt and dihedral angles developed with the
nPANI ES/NH3 (n =2, 4 and 6), increased number of repetitive units leading
respectively. to a reduction of the conjugation. In the
case of nPANI ES/NH3, NH3 is adsorbed
3. Results and Discussion at the top site of the N 4-H7 bond. The
3.1 Structural properties distance between the pure 2PANI ES,
The optimized geometrics of the pure 4PANI ES and 6PANI ES and NH 3 (dH8-
N10 ) was 1.895, 1.930 and 1.953 Å,
nPANI ES, nPANI ES/NH3, Pt/nPANI ES
and Pt/nPANI ES/NH3 (n = 2, 4 and 6) were respectively. It could be seen that the size
calculated using the B3LYP/6-31G(d) and of the oligomer increases as the H8-N10
B3LYP/6-31G(d)∪LANL2DZ methods and distances increased.
their structural parameters are listed in Table 1.
The optimized structures of the pure 2PANI
Table 1. Structural parameter such as inter molecular bond distance, inter ring angles,
dihedral angle (in Å and degree units)
Complexes dN4-H7 dH7-N9 dC1-Pt8 dPt8-N9 ∠C3N4C5 ∠C2C3N4C5 ∠C6C5N4C3
2PANI ES 1.015 - - - 130.9 22.4 22.4
4PANI ES 1.013 - - - 130.6 22.7 22.7
6PANI ES 1.012 - - - 130.0 24.1 24.1
2PANI ES/NH3 1.044 1.895 - - 128.7 24.2 24.3
4PANI ES/NH3 1.038 1.930 - - 128.9 24.2 23.4
6PANI ES/NH3 1.035 1.953 - - 128.6 24.7 24.2
Pt/2PANI ES 1.015 - 2.076 - 132.4 12.5 12.5
Pt/4PANI ES 1.014 - 2.079 - 132.4 15.0 17.7
Pt/6PANI ES 1.014 - 2.083 - 132.2 16.2 18.0
Pt/2PANI ES/NH3 1.015 - 2.125 2.128 128.7 9.90 26.4
Pt/4PANI ES/NH3 1.014 - 2.128 2.127 128.9 12.2 23.7
Pt/6PANI ES/NH3 1.014 - 2.132 2.126 128.6 13.9 22.7
a)
7 1.015 Å Pt/2PANI ES, 1.014Å in Pt/4PANI ES and
4 1.014Å in Pt/6PANI ES. The dihedral angle
3 5 of Pt/nPANI ES ranged from 12.5º to 18.0º.
1 2 6 The dihedral angle of Pt/nPANI ES was
lower than the pure nPANI ES, meaning that
the degree of planarity in the Pt/nPANI ES
b)
complex was higher than the pure nPANI
9 ES. In all cases of the adsorption of NH3
1.895 Å
using Pt/nPANI ES, the adsorption site was
7 1.044 Å the top of the Pt atom. The distance between
4
3 5 the Pt atom and N atom of NH3 (dPt8-N9) was
2.128Å in Pt /2PANI ES, 2.127 Å in
1 2 6 Pt/4PANI ES, and 2.126 Å in Pt/6PANI ES.
The increase of the bond distance with the
c) size of the oligomer was caused by the
bulkiness of the oligoaniline chain.
8 3.2 Adsorption energies
Adsorption energies (Eads) are a key
2.076 Å 6
1 2 4 5 factor for explaining interaction between the
polymer and NH3. The calculated values of
the adsorption energy of such complexes are
d) listed in Table 2. In the case of nPANI
ES/NH3, the adsorption energies of dimer,
9 tetramer and hexamer PANI ES/NH3 were -
2.128 Å
15.72, -13.29 and -10.76 kcal/mol,
8 respectively. This suggested that the
2.125 Å 6 interaction between the pure nPANI ES and
1 2 4 5 NH3 was a result of physisorption. For
Pt/nPANI ES, the highest adsorption energy
Figure 1. The optimized structure of (a) of -31.33 kcal/mol was observed for
pure 2PANI ES (b) 2PANI ES/NH3 (c) Pt/2PANI ES, which was higher than those
Pt/2PANI ES and (d) Pt/2PANI ES/NH3; C, obtained from Pt/4PANI ES and Pt/6PANI
N, H and Pt atom were represented by grey, ES (-22.95 and -14.31 kcal/mol,
blue,white and cerulean colors, respectively respectively). The interaction of the large-
sized Pt/PANI ES oligomers was weaker
The results were consistent with the than those of small-sized oligomers
further extension of the N5-H8 bond reflecting the effect of the bulkiness.
distances of the nPANI ES/NH3 complexes Considering the Pt/nPANI ES/NH3, the
compared to the pure nPANI ES, which interactions between NH3 and Pt/nPANI ES
indicated a dedoping process. By doping were a result of chemisorption with the
with Pt (Pt/nPANI ES), the Pt atom was interaction energy of -51.56, -45.66 and -
adsorbed at the bridge site of the C1-C2 44.44 kcal/mol for Pt/2PANI ES/NH3,
bond. The distance between Pt and PANI ES Pt/4PANI ES/NH3 and Pt/6PANI ES/NH3,
(dC1-Pt8) of 2.076 Å was shorter than the respectively. This indicated that the higher
values of 2.079 and 2.083 Å for the Pt/PANI adsorption energy of Pt/nPANI ES/NH3
ES dimer, tetramer and hexamer, compared to nPANI ES/NH3.
respectively. After doping with Pt, the dN4-H7
showed a slight extension of 1.015Å in
Table 2. Adsorption energy (in kcal/mol unit), Mulliken charge analysis (in e- unit), LUMO,
HOMO and HOMO-LUMO gap (in eV unit) and percent change of HOMO-LUMO gap
Complexes Eads QPt QNH3 EHOMO ELUMO Eg %ΔEg
2PANI ES - - - -8.27 -3.70 4.57 -
4PANI ES - - - -7.05 -2.99 4.06 -
6PANI ES - - - -6.39 -2.56 3.83 -
2PANI ES/NH3 -15.72 - +0.078 -7.89 -3.40 4.49 1.79%
4PANI ES/NH3 -13.29 - +0.067 -6.88 -2.83 4.05 0.00%
6PANI ES/NH3 -10.76 - +0.063 -6.29 -2.48 3.81 0.71%
Pt/2PANI ES -31.33 +0.137 - -7.95 -5.63 2.32 -
Pt/4PANI ES -22.95 +0.091 - -6.94 -5.01 1.93 -
Pt/6PANI ES -14.31 +0.062 - -6.34 -4.60 1.74 -
Pt/2PANI ES/NH3 -51.56 - +0.237 -7.56 -3.76 3.80 64.94%
Pt/4PANI ES/NH3 -45.66 - +0.231 -6.69 -3.21 3.48 80.31%
Pt/6PANI ES/NH3 -44.44 - +0.227 -6.81 -2.94 3.24 86.21%
3.3 Charge analysis semiconductor. The percentage change in

The amount of charge transfer the HOMO-LUMO gap (%ΔEg) was 1.79%,
between the polymer and NH3 was also 0.00% and 0.71% for 2PANI ES/NH3,
calculated using the B3LYP/6-31G(d) and 4PANI ES/NH3 and 6PANI ES/NH3,
B3LYP/6-31G(d)∪LANL2DZ methods and respectively. It could be seen that the
are listed in Table 2. For the nPANI ES/NH3 HOMO-LUMO gaps had some slightly
complexes, the electron migration from NH3 changes. After the interaction with Pt, Pt
to PANI ES dimer, tetramer and hexamer gave the largest amount of electron transfer
showed the amounts of +0.078, +0.067 and to nPANI ES resulting in the LUMO energy
+0.063 e-, respectively. By doping with Pt, level and HOMO energy level to show
the net charge transfer was positive showing significant change. The conductive
that electron migrations from Pt to nPANI properties of the Pt/nPANI ES complexes
ES reflected the n-type doping process. The were the semiconductor with low HOMO-
highest net charge transfer was obtained LUMO gaps of 2.31, 1.93 and 1.74 eV for
from Pt/2PANI ES Pt/4PANI ES and Pt/2PANI ES, Pt/4PANI ES and Pt/6PANI
Pt/6PANI ES, respectively. In the case of ES, respectively. In all cases of Pt/nPANI
Pt/nPANI ES/NH3, the electron migrations ES/NH3, the HOMO-LUMO gap was lower
from Pt to dimer, tetramer and hexamer than nPANI ES/NH3. The value of the
Pt/PANI ES were 0.237, 0.231 and 0.227 e-, HOMO-LUMO gap had a decreased flow in
respectively. It could be seen that, a much the size of the oligomer with the value 3.81
higher charge transfer in the case of eV in Pt /2PANI ES/NH3, 3.48 eV in
Pt/nPANI ES/NH3 than nPANI ES/NH3 was Pt/4PANI ES/NH3 and 3.24 eV in the
in agreement with the results of the Pt/6PANI ES/NH3 complexes. Moreover,
structural properties and adsorption energy. after sensing with NH3, the HOMO-LUMO
3.4 Electronic properties gap displayed changes of 64.95%, 80.31%
The electronic properties of the and 87.28% for Pt/2PANI ES, Pt/4PANI ES
nPANI ES/NH3 complexes are listed in and Pt/6PANI ES, respectively. It was found
Table 2. In the case of PANI ES/NH3, the that adsorption of NH3 on PANI ES/Pt had a
HOMO- LUMO gaps value of 4.49, 4.05 higher sensitivity than the pure nPANI ES.
and 3.81 eV for 2PANI ES/NH3, 4PANI
ES/NH3 and 6PANI ES/NH3 indicated that 4. Conclusion
the electronic properties of the nPANI The B3LYP/6-31G(d)∪LANL2DZ
ES/NH3 complexes were the insulator and method was performed for the NH3 sensing
using the Pt/nPANI ES complexes (n = 2, 4 6. Ganjia, M. D.; Sharifib, N.; Ardjmandb,
and 6). The adsorption of NH3 using M.; Ahangaric, M. G. Appl. Surf. Sci.
Pt/nPANI ES showed a shorter distance 2012, 261, 697–704.
between Pt/nPANI ES and NH3, higher net 7. Frisch, M. J.; Trucks, G. W.; Schlegel,
charge transfer, and higher binding energy H. B.; Scuseria, G. E.; Robb, M. A.;
more than the pure nPANI ES. In addition, Cheeseman, J. R.; Montgomery, Jr., J.
after sensing with ammonia, the HOMO- A.; Vreven, T.; Kudin, K. N.; Burant, J.
LUMO displayed significant changes. C.; Millam, J. M.; Iyengar, S. S.;
Therefore, the doping platinum on PANI ES Tomasi, J.; Barone, V.; Mennucci, B.;
could enhance the sensing ability. Thus, the Cossi, M.; Scalmani, G.; Rega, N.;
researchers showed that the Pt/PANI ES Petersson, G. A.; Nakatsuji, H.; Hada,
could be used for NH3 sensor devices. M.; Ehara, M.; Toyota, K.; Fukuda, R.;
Hasegawa, J.; Ishida, M.; Nakajima, T.;
Acknowledgements Honda, Y.; Kitao, O.; Nakai, H.; Klene,
Computational chemistry research M.; Li, X.; Knox, J. E.; Hratchian, H.
group at department of chemistry and P.; Cross, J. B.; Bakken, V.; Adamo, C.;
KMUTT are gratefully acknowledgment for Jaramillo, J.; Gomperts, R.; Stratmann,
all computer resources and facilities. This R. E.; Yazyev, O.; Austin, A. J.;
work was also supported by the Higher Cammi, R.; Pomelli, C.; Ochterski, J.
Education Research Promotion and National W.; Ayala, P. Y.; Morokuma, K.; Voth,
Research University Project of Thailand G. A.; Salvador, P.; Dannenberg, J. J.;
(NRU). Zakrzewski, V. G.; Dapprich, S.;
Daniels, A. D.; Strain, M. C.; Farkas,
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Optical and electronic properties of Ag/PANI ES interacting with ammonia :
a theoretical investigation
Taksaphorn Nopkaew, Chinapong Kritayakornupong*
Department of Chemistry, Faculty of Science, King Mongkut’s University of Technology Thonburi,
*E-mail: chinapong.kri@kmutt.ac.th
Abstract:
Ammonia is a volatile organic compound which is used in industrial and cause of
problem on human activity. Several materials have been applied for ammonia detection. Among
them, polyaniline emeraldine salt (PANI ES) is a candidate material for measuring ammonia.
Ag/PANI ES nanocomposite is an interesting material for increasing sensing ability of such
compound. In the present study, the B3LYP and TD-B3LYP methods were performed to
examine electronic and optical properties of PANI ES and Ag/PANI ES with and without
ammonia molecule. The LANL2DZ ECP basis set was used for silver atom, while the 6-31G(d)
basis set was applied for carbon, nitrogen and hydrogen atoms. It was found that silver enhanced
the conductivity of the PANI ES sensor, showing the HOMO-LUMO gap of 0.77 eV. For
sensing with NH3, the conductivity of Ag/PANI ES was decreased. The calculated UV-vis-NIR
spectrum of PANI ES is in good agreement with the experimental observation. The polaron peak
of Ag/PANI ES was red-shifted compared to PANI ES. Interestingly, polaron peak of Ag/PANI
ES is disappeared after sensing with ammonia. Our results demonstrated that Ag/PANI ES is a
candidate optical sensing material for ammonia detection.
1. Introduction and copolymer.5 For the sensing ability of the

At present, polyaniline is widely used toxic gas sensor, conductivity is an important
as sensing material, since there are many factor, and changing the structural and
advantages; such as, light weight, cheap, low electronic properties has an effect on the
band gap and easy synthesis.1 Structural conductivity. Ammonia has also received
polyaniline (PANI) has several chemical some interest, as it is the most used compound
structures; such as, leucoemeraldine base in industry and the cause of health problems
(LB), pernigraniline base (PNB), emeraldine in humans. Therefore, it has been developed
base (EB) and emeraldine salt (ES). It appears by using polyaniline doped with silver
that the ES form is the only structure, which nanoparticles for sensing toxic gas because
can produce conductivity. In general, the silver nanoparticles have catalytic, electrical
electronic properties of polyaniline depend on and optical properties. Polyaniline and
the type of polymers, synthesis methods and polyaniline doped with silver nanoparticles
doping processes.2 The changing structure of studied for their optical and electronic
polyaniline is obtained from the oxidation- properties showed that polyaniline doped with
reduction reaction or protonation. Although, silver nanoparticles was more stable than pure
polyaniline has many advantages, it has lower polyaniline at a high temperature and
conductivity than metal oxides and low increased the conductivity compared with the
solubility in solvents.3,4 This disadvantages parent.6 This represents that the doping
can be improved by doping process, process is an alternative method for the
modification of the precursor in the synthesis explication of sensing material. In this work,
density functional theory DFT) calculations were 4.57, 4.06 and 3.85 eV of the pure PANI
were performed to investigate the electronic ES and 4.49, 4.06 and 3.82 eV of PANI ES
and optical properties of a representative with the ammonia molecule.
model of pure polyaniline emeraldine salt
(PANIES) and polyaniline doped with silver Table 1. HOMO, LUMO and HOMO-LUMO
nanoparticles (Ag/PANI ES) before and after gaps (EH-L) of systems in eV calculated at
interaction with the ammonia molecule to be B3LYP/6-31G(d) and B3LYP/6-31G(d)
applied as sensor material. LANL2DZ levels of theory
HOMO LUMO EH-L
Complexes
2. Computational Methods (eV) (eV) (eV)
All calculations were performed using
2PANI ES -8.28 -3.72 4.56
the Gaussian03 program.7 Electronic
properties of pure polyaniline (nPANI ES; n = 4PANI ES -7.07 -3.01 4.06
2, 4, 6 mers) and polyaniline doped with silver 6PANI ES -6.41 -2.57 3.84
nanoparticles (Ag/nPANI ES) without Ag/2PANI ES -8.37 -6.24 2.13
ammonia molecule, where n is number of
Ag/4PANI ES -7.17 -5.79 1.38
repeating units were evaluated by B3LYP
with 6-31G(d) basis set for all atoms, except Ag/6PANI ES -6.35 -5.58 0.77
LANL2DZ ECP basis set for silver atom. The 2PANI ES/NH3 -7.90 -3.41 4.49
UV-vis-NIR spectra of all complexes were 4PANI ES/NH3 -6.89 -2.84 4.05
simulated by time-dependent density
6PANI ES/NH3 -6.29 -2.48 3.81
functional theory ( TD-DFT) with the same
level of theory as mentioned above. Ag/2PANI ES/NH3 -7.90 -4.73 3.17
Ag/4PANI ES/NH3 -7.08 -4.36 2.71
3. Result and Discussion Ag/6PANI ES/NH3 -6.37 -4.20 2.17
3.1 Electronic properties
The electronic properties of nPANI
ES and Ag/nPANI ES (n = 2, 4 and 6) and In the case of PANI ES doped with
without the ammonia molecule elucidated by silver nanoparticles (Ag/PANI ES), the
the B3LYP/6-31G(d) and B3LYP /6-31G(d) calculation showed that the HOMO-LUMO
LANL2DZ methods, respectively are shown gaps were decreased compared to the parent,
in Table 1, and the optimized structure of the referring to the increased conductivity of
complexes are depicted in Figure 1. The about 2.13, 1.38 and 0.77 eV of 2, 4 and 6
results showed that the HOMO-LUMO gaps oligomers of Ag/PANI ES, respectively.
of 4.56, 4.06 and 3.84 eV were estimated for Therefore, silver could enhance the
2PANI ES, 4PANI ES and 6PANI ES, conductivity of PANIES. Moreover,
respectively, indicating that the nPANI ES decreasing the conductivity in the case of
was a semiconductor. In addition, the HOMO- Ag/PANI ES after sensing with ammonia
LUMO gaps of polyaniline sensing with the (Ag/PANI/NH3) was consistent with the large
ammonia molecules (nPANI ES/NH3) was HOMO-LUMO gaps. However, the difference
slightly decreased when compared with the in the HOMO-LUMO gaps value of Ag/PANI
pure PANI ES of about 4.49, 4.05 and 3.81 eV ES with and without ammonia was greater
of 2PANI ES/NH3, 4PANI ES/NH3 and than PANI ES. This means that Ag/PANI ES
6PANI ES/NH3, respectively. The results has a higher sensitivity to sensing with the
resembled the values calculated of other ammonia molecules.
research8 in which the HOMO-LUMO gaps
a) PANI ES b) PANI ES/NH3
c) Ag/PANI ES d) Ag/PANI ES/NH3

Figure 1. Optimized structure of complexes a) PANI ES b) PANI ES/NH3 c) Ag/PANI ES
and d) Ag/PANI ES/NH3 obtained with B3LYP/6-31G(d) and B3LYP/6-31G(d)
LANL2DZ methods
3.2 Optical properties data, which are 355, 430 and 815-825 nm.9
The TD-B3LYP/6-31G(d) and TD- Therefore, 6PANI ES was used to be the
B3LYP/6-31G(d) LANL2DZ methods representative for explaining the optical
were used to clarify the UV-vis-NIR spectra properties in this work. By doping silver on
of the pure nPANI ES and Ag/nPANI ES PANI ES, the UV-vis-NIR spectrum of
sensing with ammonia, respectively. The UV- Ag/6PANI ES was red-shifted to 2142 nm of
vis-NIR spectra of the complexes are shown the π→polaron according to the high
in Figure 2 and 3, and calculated the conductivity. In the case of 6PANI ES sensing
wavelengths (λ), excitation energies (Eex), with ammonia, the wavelength (1857 nm)
oscillator strengths of the π-polaron state and displayed a blue shift that referred to the
molecular orbitals are given in Table 2. For dedoping process. Moreover, the UV-vis-NIR
the pure nPANI ES, the UV-vis-NIR spectrum spectrum of the Ag/6PANI ES sensing with
of 2PANI ES was peaked at 266, 336 and 750 ammonia disappeared at the π→polaron peak,
nm of π→π*, polaron→π*, and π→polaron, according to highest sensitivity for sensing
respectively. For 4PANI ES, two peaks were ammonia. This can be explained by electron
proposed at 390 nm (polaron→π*) and 1349 migration from NH3 to Ag/6PANI ES. The
nm (π→polaron). Similarly, 6PANI ES results indicated that Ag/PANI ES was
provided two peaks at 351 nm (polaron→π*) appropriate to use for optical sensing
and 2078 nm (π→polaron). The results were ammonia material.
consistent with the experimental reported
Table 2. Wavelength (λ), excitation energy (Eex), oscillator strength (f) and transition of MOs of
UV-vis-NIR spectra of nPANI ES and Ag/nPANI ES with and without sensing with ammonia
molecule
Complexes π polaron (nm) Eex (eV) Oscillator strength (f) MOs
2PANI ES 749.71 1.6538 0.3635 5253
Ag/2PANI ES 768.30 1.6137 0.0314 6263
2PANI ES/NH3 721.13 1.7193 0.3419 5859
Ag/2PANI ES/NH3 469.91 2.6385 0.0412 6768
4PANI ES 1349.43 0.9188 0.7799 100101
Ag/4PANI ES 1086.67 1.1410 0.0164 110111
4PANI ES/NH3 1222.16 1.0145 0.7633 105106
Ag/4PANI ES/NH3 548.16 2.2618 0.0055 115116
6PANI ES 2078.10 0.5966 0.9960 148149
Ag/6PANI ES 2141.63 0.5789 0.1935 158159
6PANI ES/NH3 1856.99 0.6677 1.0246 153154
Ag/6PANI ES/NH3 664.75 1.8651 0.0012 163164
1.4 1.4 1.4

2PANI ES 4PANI ES 6PANI ES
1.2 1.2 1.2 351
Oscillator strength (f)

1.0 1.0
2078
1.0
1349
0.8 0.8 0.8
390
0.6 0.6 0.6
336
0.4 750 0.4 0.4
266
0.2 0.2 0.2
0.0 0.0 0.0

0 200 400 600 800 1000 1200 0 500 1000 1500 2000 0 500 1000 1500 2000 2500 3000
Wavelength (nm) Wavelength (nm) Wavelength (nm)
Figure 2. UV-vis-NIR spectra of nPANI ES (n = 2, 4 and 6)

1.8
Ag/6PANI ES/NH3
1.6
Ag/6PANI ES
1.4
344 6PANI ES/NH3
6PANI ES
1.2
351
1857 2078
1.0
403
0.8
328
0.6
0.4
2142
0.2 831
0.0
0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200
Wavelength (nm)
Figure 3. UV-vis-NIR spectra of 6PANI ES and Ag/6PANI ES with and without ammonia
molecule
4. Conclusion 4. MG, M.; AB, P.; HK, H. J. J. Polym. Sci.,
The DFT studied the nPANI ES (n = 2, Part A: Polym. Chem. 1997, 35,
4 and 6) and Ag/nPANI ES with and without 1673−1679.
the ammonia molecule. The results showed 5. Kamran, M.; Ullah, H.; Shah, A.
that the HOMO-LUMO gap of PANI ES was -u.-H. A.; Bilal, S.; Tahir, A. A.;
a semiconductor with 3.84 eV of 6PANI ES. Ayub, K., Polymer 2015, 72, 30−39.
By doping with silver, the HOMO-LUMO gap 6. Gupta, K.; Jana P.C.; Meikap A.K., Synth.
of complexes increased to 0.77 eV obtained Met. 2010, 160, 1566−1573.
from Ag/6PANI ES. In the case of sensing 7. Frisch, M. J.; Trucks, G. W.; Schlegel, H.
with ammonia molecules, the difference of the B.; Scuseria, G. E.; Robb, M. A.;
HOMO-LUMO gaps of Ag/PANI ES with and Cheeseman, J. R.; Montgomery, Jr., J. A.;
without ammonia was greater than the parent. Vreven, T.; Kudin, K. N.; Burant, J. C.;
For calculation of UV-vis-NIR of PANI ES Millam, J. M.; Iyengar, S. S.; Tomasi, J.;
and Ag/PANI ES with and without ammonia, Barone, V.; Mennucci, B.; Cossi, M.;
the wavelength of Ag/6PANI ES displayed a Scalmani, G.; Rega, N.; Petersson, G. A.;
red shift compared with a parent referring to Nakatsuji, H.; Hada, M.; Ehara, M.;
high conductivity. By sensing ammonia, the π Toyota, K.; Fukuda, R.; Hasegawa, J.;
→ polaron peak of 6PANI ES showed a blue Ishida, M.; Nakajima, T.; Honda, Y.;
shift that indicated the dedoping process. Kitao, O.; Nakai, H.; Klene, M.; Li, X.;
Furthermore, the π → polaron peak of Knox, J. E.; Hratchian, H. P.; Cross, J. B.;
Ag/6PANI ES disappeared after sensing with Bakken, V.; Adamo, C.; Jaramillo, J.;
ammonia. This referred that silver could Gomperts, R.; Stratmann, R. E.; Yazyev,
enhance the conductivity and optical O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
properties for sensing ammonia. These results Ochterski, J. W.; Ayala, P. Y.;
suggested that doping silver could improve the Morokuma, K.; Voth, G. A.; Salvador, P.;
sensitivity of polyaniline and be applied to use Dannenberg, J. J.; Zakrzewski, V. G.;
for sensing ammonia material. Dapprich, S.; Daniels, A. D.; Strain, M.
C.; Farkas, O.; Malick, D. K.; Rabuck, A.
Acknowledgements D.; Raghavachari, K.; Foresman, J. B.;
Computational chemistry research Ortiz, J. V.; Cui, Q.; Baboul, A. G.;
group at department of chemistry and Clifford, S.; Cioslowski, J.; Stefanov, B.
KMUTT are gratefully acknowledgment for B.; Liu, G.; Liashenko, A.; Piskorz, P.;
all computer resources and facilities. This Komaromi, I.; Martin, R. L.; Fox, D. J.;
work was also supported by the Higher Keith, T.; Al-Laham, M. A.; Peng, C. Y.;
Education Research Promotion and National Nanayakkara, A.; Challacombe, M.; Gill,
Research University Project of Thailand P. M. W.; Johnson, B.; Chen, W.; Wong,
(NRU). M. W.; Gonzalez, C.; and Pople, J. A.;
Gaussian 03. Gaussian, Inc., Wallingford
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2. Kumar, D.; Sharma, R. C. Eur. Polym. J. 23701−23711.
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B. F.; Okutan, M. Mater. Sci. Semicond.
Process.2016, 56, 357−361.
Theoretical study of photoinduced intramolecular electron transfer in
Ru(II)-Re(I) mixed-valence complex
Rangsiman Ketkaew1*, Jen-Shiang K. Yu2, Yuthana Tantirungrotechai1*
1
Computational Chemistry Research Unit, Department of Chemistry, Faculty of Science and Technology,
2
Institute of Bioinformatics and Systems Biology, and Department of Biological Science and Technology,
National Chiao Tung University, Hsinchu 300, Taiwan
*E-mail: rangsiman1993@gmail.com, yt203y@gmail.com
Abstract:
We study the kinetics of photoinduced intramolecular electron transfer (PIET) of
[Ru(II)(dmb)2-(BL)-Re(I)(CO)3Cl]2+ complex, where BL is bridging ligand. The rate constant
of ET (kET) between donor and acceptor was determined following Marcus theory. The inner-
sphere reorganization energy (λinner) and electronics coupling matrix element (HDA) of
complex were computed in the framework of density functional theory (DFT). In this work,
we proposed two computational schemes for computing kET, comprised of (i) a truncated
model, where donor and acceptor are separate, and (ii) a full complex model, where the
constrained density functional theory (CDFT) was specifically used to calculate charged-
constrained state. It is shown that CDFT can be used to access the diabatic state, in which
λinner and HDA have been computed directly. The role of BL in the efficiency of forward ET of
Ru-Re complex was systematically investigated. It indicates that the length of BL and
electron delocalization in π-conjugated linker play an important role in electronic
communication between donor and acceptor. Our study demonstrates that the low
reorganization energy is necessary for fast forward ET in Ru-Re complex.
1. Introduction properties. Combining two different metal

The mixed-valence binuclear complexes as a synergistic photocatalyst,
complex has widely received significant such as ruthenium and rhenium, has been
attention in materials science due to their achieved.7 The attention of binuclear
many applications such as photoexcitation complex has been further extended to the
upon optoelectronics properties and Ru-Re mixed-valence complex, where they
photosensitizer catalyst for reduction of were linked by bridging ligand (BL).
small gases.1–3 Exploration in cheap Sato et al. synthesized the high
photocatalysts turning carbon dioxide (CO2) efficient Ru(II)-BL-Re(I) binuclear complex
and sunlight to carbon monoxide (CO) is a for CO2 reduction and compared the
challenge. The transition metal photocatalytic activity of different ligand
photocatalysts have been used to speed up Ru(I) complex.8 It was shown that using
the CO2 reduction. Previous studies have only Re catalyst cannot drive this reaction
reported the use of an electrochemical because Re cannot absorb the irradiated
ruthenium (Ru) catalyst to yield the CO as light. The following study by Nakada et al.
well as Rhenium (Re) complex also has shown that the efficiency of Ru(II)-BL-Re(I)
been extensively used.4,5 Suntharalingam et catalysis depends on the rate of
al. studied the homogeneous metal complex intramolecular electron transfer. Figure 1
and investigated mono- and bimetallic shows the simple mechanism of electron
terpyridine complexes in DNA binding.6 transfer (ET) of Ru(II)-BL-Re(I) driven by
They found that using two transition metals light. [Ru(dmb)3]2+ specie was reduced by
are better than one metal because its strong one electron from BI(CO2-)H reductant. It
corresponding to the photoexcitation was experimentally observed that the strong
photoinduced intramolecular electron diabatic state. Caution of structural
transfer (PIET) controls the catalytic activity optimization using CDFT must be taken in
of Ru(II) specie on CO2 reduction. relating real situation of electron transfer to
Remarkably, electron transferring between the diabatic state of system through the
Ru and Re metal complexes via bridging charged localization approach.
ligand obviously refers to the electronics In this study, we investigated the
communication, which is described by PIET of Ru(II)-(CH2CH2)n-Re(I), where n =
Marcus theory. 1-4. The role of length of bridging ligand,
which is π-conjugated system, in electron
transfer was theoretically studied and
analyzed using DFT and CDFT calculations.
The two independent models were presented
for PIET. The first is a truncated model,
where donor and acceptor are separate,
which addresses system to be an adiabatic
state. This model was directly calculated
using a conventional DFT. On the other
hand, for diabatic state, it was presented by a
Figure 1. Photoinduced intramolecular
full complex model, where donor and
electron transfer process in Ru(II)-Re(I)
acceptor are connected. This model
complex in water phase
represents the picture of the PIET. The
energy profile of the two-state is shown in
At this stage, where the limit of
Figure 2. We extracted the internal
experiments has been reached, to gain the
reorganization energy and electronics
understanding and to solve the problem,
coupling energy from diabatic two-state of
using theoretical investigation is mandatory.
system using CDFT calculation to
Density functional theory (DFT) has been
investigate the efficiency of PIET.
notably used to analyze the electronics
structure of molecule. One can use DFT to
calculate the adiabatic state of studied
system. When we deal with the
photoinduced intramolecular electron
transfer, which refers to diabatic state, the
different situation of ET needed to be
distinguished. DFT fails to address diabatic
state for Ru(II)-BL-Re(I) hybrid complex.
Wu and Van Voorhis developed the
constrained density functional theory
(CDFT) to consider system with an arbitrary
on the charged localization.9 With CDFT
method, for diabatic state, we are able to
calculate the coupling energy at the crossing
point of two states.10–12 They also used
CDFT calculation to compute the electron
transfer matrix element (HDA) of hybrid Figure 2. Potential energy profile for a
complex, where donor and accepted are simple two-state adiabatic and diabatic
connected. Ding et al. applied CDFT to system of Ru(II)-BL-Re(I)
determine the HDA parameter of the bridged-
two ferrocene intervalence.13 Their work
revealed that CDFT typically make an
exaggeration in energy of charge localized
2. Theory and Methodology electron transfer, carefully examine the
2.1 Marcus theory for electron transfer frontier molecular orbitals (MOs) for
Photoinduced intramolecular reaction and product state of system. Figure
electron transfer (PIET) was addressed by 3 and Figure 4 show the proposed model of
combining CDFT and Marcus theory. The PIET at the diabatic state.
probability of ET between different sites,
from donor to acceptor, during an excited
state decreased with an increasing the
distance between these two sites. The rate of
electron transfer (kET) can be determined
according to Marcus equation (equation 1).
The significant keys that control the kinetics
involved in a unimolecular ET are identified
by three terms of energy: the nuclear Figure 3. (A) Simple mechanism of PIET;
reorganization energy (λ); electron transfer stepwise and direct transfer. (B) 2 and 3-
matrix element or electronics coupling fragments separation for full complex model
energy (HDA) between donor and acceptor;
and the standard reaction Gibbs energy
(ΔG°).
(1)
(2)
where λ is the energy required to change the Figure 4. Truncated Ru(II) fragment (donor)
structure of complex resulting from the and truncated Re(I) fragment (acceptor). The
molecular rearrangement that occurs as the other side of such fragment was replaced by
charge is distributed throughout the hydrogen.
complex. HDA indicates the ability of
electronics communication when electron 2.3 Computational details
travel from donor to acceptor, which can be Ground state geometry optimization
determined using the equation 2. The Gibbs of Ru(II)-BL-Re(I) complex for a truncated
energy of activation (ΔG*) is determined by model and a full complex model was carried
ΔG° and the nuclear reorganization energy out with different method using density
(λ). functional theory (DFT) framework. For a
2.2 Proposed model of electron transfer truncated model, the Ru(II) and Re(I)
for Ru(II)-BL-Re(I) complex fragments were separately computed by
The electronics state optimization of conventional DFT using Gaussian09
Ru(II)-BL-Re(I) hybrid complex must program.14 For a full complex model, we
correspond to the constrained state. In this used the constrained density functional
work, the studied reaction is Ru(II)1+-BL0- theory (CDFT), which was implemented in
Re(I)0 → Ru(II)2+-BL0-Re(I)1-, the total net NWChem 6.6 open-source program, to
charge has not. The one-electron-doped calculate the electronics diabatic state of
Ru(II)-BL-Re(I) complex was considered Ru(II)-BL-Re(I) complex.15 CDFT and the
for PIET. The transferring electron was method of corresponding orbital
localized on Ru in the reactant state and was transformation were utilized to determine
localized on Re in the product state. To the λ and HDA. All calculations employed
verify the existing localization of the the B3LYP functional with the mixed basis
electron in the diabatic state and to set: 6-31G(d) for the light atoms and SDD-
determine which orbitals are involved in the ECP for Ru and Re atoms. The 1st excited
state properties of complex were predicted Table 1 reports the computed λ and HDA of
using the CAM-B3LYP/6-31G(d)/SDD- all Ru(II)-BL-Re(I) systems. The λ of
ECP at level of calculation. The COSMO truncated model was much higher than both
solvation model used with water (Ɛ=88) at 2- and 3-fragments full complex models. For
273.15 K was applied for all calculations. full complex model, the 3-fragment model
MOs were used to analyze the localized yields the λ a little lower than the other.
electron density of Ru(II)-BL-Re(I) Ru(II)-BL2-Re(I) complex gives the lowest
complex. λ, which means that this complex requires a
little energy to change the geometry of
3. Results and Discussion complex from reactant state to product state.
We computed the Marcus equation Senevirathna et al. reported that a little inner
elements; the inner nuclear reorganization reorganization energy is desirable for high
energy (λ) and electron transfer matrix efficiency of photoinduced electron
element (HDA). For a truncated model, one transfer.16
can simply use the Nelson's four-point Not only the low λ is needed for fast
method to compute λ of system for adiabatic forward electron transfer, but we also looked
state. at the HDA, which implies to an electronics
(3) communication between two sides of
system. We were not able to compute HDA
(4)
for a truncated model because the
(5) calculation of charged localization was not
be accomplished using a conventional DFT.
where and are the hole and electron
Therefore, the other choice for considering
reorganization energies, and E is single
the charge localization was a full complex
point energy, and Q is an optimized
model. To our knowledge and the Marcus
geometry with the subscripts 0, +, and –
theory, the distance dependence of electron
denote neutral, cationic, and anionic states,
transfer was taken into account of the π-
respectively. For a full complex model,
conjugated bridge. The computed HDA of all
where donor and acceptor were connected,
Ru(II)-BL-Ru(I) complexes were found to
we proposed the direct equation to calculate
be around 300 – 700 in meV. The 2-
the inner λ by combining the and
fragment model has HDA higher than that
(equation 6). computed by 3-fragment model. This result
shown that the electron can directly travel
from Ru(II) fragment to Re(I) fragment.
(6)
Moreover, the Ru(II)-BL2-Re(I) complex
where is an hole/electron reorganization yields the highest computed HDA compared
energy computed at charge localized/neutral to other complexes. From electron transfer
geometry using CDFT method. theory, the rate of PIET is increased when
the low reorganization energy and the high
HDA were together received.
Table 1. Computed inner nuclear reorganization energy (λ) and electron transfer matrix
element (HDA) of Ru(II)-BL(Re(I) complex for different electron transfer model
λ (meV) HDA (meV)
Complex Truncated Full complex model Full complex model
model 2-fragments 3-fragments 2-fragments 3-fragments
BL1 269 15.4 15.0 643 426
BL2 225 14.4 14.9 749 436
BL3 258 17.1 16.0 555 353
BL4 273 18.2 18.0 500 344
Figure 5. 2D structures of Ru(II)-BLn-Re(I) complex, computed singly occupied molecular
orbital (SOMO), and highest occupied molecular orbital (HOMO) at excited state for 3-
fragments full complex model
To analyze charged localization of geometries. Either method could be applied

the studied complex for before and after at an arbitrary nuclear geometry and, as
PIET, the frontier molecular orbitals computational result, may be used to
including HOMO and SOMO were calculate λ and HDA of PIET. The series of
computed as shown in Figure 5. HOMO π-conjugated diene bridging ligand,
reveals the electron density of system before corresponding HDA as a function of distance
electron transferring. When the Ru(II)-BL- between donor and acceptor sites, were
Re(I) complex was excited, SOMO was then observed for reactant and product state. A
properly used to describe the role of π- full complex model yields a natural
conjugated diene in PIET. The energy gap definition of electron transfer distance, in
between HOMO and SOMO of all contrast to a truncated model, which
complexes are almost the same, represented an independent against to the
corresponding an internal electronic PIET. The 3-fragments model yields HDA
communication. Ru(II)-C4H4-Re(I) complex less than 2-fragments model. The estimation
or BL2 system was found that it can be a of distance generally based on molecular
promising efficient electron transfer mixed- structure data. Our computational results
valence complex. also reveal that the low inner nuclear
reorganization energy is required for further
4. Conclusion development of an efficient electron transfer
We studied the photoinduced hybrid complex.
intramolecular electron transfer (PIET) of
covalent Ru(II)-BL-Re(I) complex. The Acknowledgements
picture of diabatic state of system was Authors acknowledged the
studied using charged localization approach. CHALAWAN cluster, supported by
We proposed a truncated model and full National Astronomical Research Institute of
complex model, in which nuclear Thailand (NARIT), for computing resources.
reorganization energy (λ) and electron R.K. thanks the NWChem developers for
transfer matrix element (HDA) were useful suggestion in CDFT calculation.
computed by DFT and CDFT. Generally
good agreement between the two models
was obtained over a range of complex
Supporting Information 13. Gaussian 09, Revision E.01, Frisch, M.
DFT and CDFT geometry coordinate J.; Trucks, G. W.; Schlegel, H. B.;
for all Ru(II)-BL-Re(I) complexes can be Scuseria, G. E.; Robb, M. A.;
found at http://bit.ly/2018-ET-RK-project. Cheeseman, J. R.; Scalmani, G.;
Barone, V.; Mennucci, B.; Petersson, G.
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6046.
DFT calculation, biological activity, anion sensing application studies and
crystal structure of (E)-4-methoxy-2-((4-methylbenzo[d]thiazol-2-
ylimino)methyl)phenol
Hüseyin Unver1*, Mustafa Yıldız2, Nuray Yıldırım3, Gökhan Alpaslan4,
Bahadir Boyacioglu5, Ayhan Elmali6
1
Department of Physics, Faculty of Science, Ankara University, Beşevler-Ankara 06100, Turkey
2
Department of Chemistry, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University,
Çanakkale TR-17100, Turkey
3
Health Services Vocational School, Çanakkale Onsekiz Mart University, Çanakkale TR-17100, Turkey
4
Department of Medical Services and Techniques, Vocational School of Health Services,
Giresun University, Giresun 28200, Turkey
5
Vocational School of Health Services, Ankara University, Kecioren-Ankara TR- 06290, Turkey
6
Department of Physics Engineering, Faculty of Engineering, Ankara University, Beşevler TR-06100, Turkey
*E-mail: huseyin.unver@ankara.edu.tr
Abstract:
(E)-4-methoxy-2-((4-methylbenzo[d]thiazol-2-ylimino)methyl)phenol was synthesi-
zed from the reaction of 4-methylbenzo[d]thiazol-2-amine with 2-hydroxy-5-
methoxybenzaldehyde. The structure of compound has been investigated by using FT-IR,
UV-VIS, 1H-NMR, 13C-NMR spectroscopic and X-ray crystallographic techniques. The
molecular structure, HOMO-LUMO analysis, molecular electrostatic potential (MEP) and
nonlinear optical (NLO) effects of the compound were investigated by using DFT
calculations. Additionally, the title compound was tested for its biological activity. UV-Vis
spectroscopic studies of the interactions between the Schiff base and calf thymus DNA (CT-
DNA) showed that the compound interacts with CT-DNA. The colorimetric response of the
compound receptors was investigated before and after the addition of an equivalent amount
of each anion to evaluate anion recognition properties.
1. Introduction materials are useful as they provide visual

Schiff bases and their metal information more easily. Various studies
complexes have attracted curiosity due to have been carried out in order to develop
DNA binding and cleavage properties.1,2 these materials.7,8
Applications of them as chemical nucleases
are the focus of current research. It has been 2. Materials and Methods
demonstrated that they can be used in some 2.1 General
studies as sequence specific DNA binding 2-hydroxy-5-ethoxybenzaldehyde, 4-
agents, and diagnostic agents in medicinal methylbenzo[d]thiazol-2-amine, EtOH,
applications and for genomic research.3,4 Ethidium bromide (EB), calf thymus DNA
Schiff bases are important to synthesize (CT-DNA) and DMSO were purchased from
sensors for recognizing and sensing anions, Aldrich. The Tris–HCl buffer solution was
and to enlarge their areas of use. The prepared with triple-distilled water. CT-
intramolecular hydrogen bond and proton DNA stock solution was prepared by
transfer explained above is thought to affect diluting DNA to Tris–HCl/NaCl buffer (5
the anion-sensory properties.5,6 The research mM Tris–HCl, 50 mM NaCl, pH 7.2), and
of highly selective and sensitive kept at 4 °C for no longer than two days.
chromogenic chemo-sensors for the sensing 2.2 Synthesis of compound
of ions is a very important field in research. 4-Methylbenzo[d]thiazol-2-amine
Undoubtedly, the colorimetric anion sensors (0.872 g, 5.32 mmol) was added to EtOH
are of more importance, because such (100 mL) solution of 2-hydroxy-5-
methoxybenzaldehyde (0.808 g, 5.32 mmol). Computational procedures
The mixture was stirred and refluxed for 1 h. All theoretical computations were
Compound was obtained from the done by using Gauss-View5 molecular
evaporation of EtOH. It was crystallized visualization and Gaussian 09W program
from CHCl3 : n-hexane (3:2) as a orange package.13,14 For calculation of the molecule
crystal, m.p. 139-140 °C, 1.81 g (87%) geometry was used the obtained atomic
yield. Found: C, 64.05; H, 4.71; 9.39. Calc. coordinates from X-ray geometry. Geometry
For C16H14N2O2S; C, 64.41; H, 4.69; N, 9.39 optimization of the molecule was performed
%. IR(KBr) /cm-1 O-H; 3416, Ar-H; 3058, by using DFT method with Becke’s three
C=N; 1636, C=C; 1569, C-N; 1490, C-O; parameters hybrid exchange-correlation
1351. 1H-NMR (400.1 MHz, DMSO) δ functional (B3LYP) at 6-311++G(d,p) basis
10.19 (s, 1H, Ar-OH); 9.32 (s, 1H, Ar- set.15,16 Molecular electrostatic potential
CH=N-); 7.79-6.91 (m, 6H, Ar-H), δ 3.73 (s, surface was created by using B3LYP/6-
3H, OCH3); δ 2.56 (s, 3H, -CH3). 13C-NMR 311++G(d,p) method. The HOMO-1,
(100.6 MHz, DMSO) δ 191.43, 170.15, HOMO, LUMO and LUMO+1 energy
165.55, 155.77, 152.64, 150.91, 134.02, values and their shapes were calculated and
132.74, 127.82, 126.04, 123.97, 120.11, simulated by using the same basis set.
118.65, 112.09, 56.48 and 18.63.
2.3 Spectroscopic measurement 3. Results and Discussion
The 1H and 13C NMR spectra were 3.1 Description of the crystal structure
recorded on a Bruker AVANCE- 500 The title compound crystallizes in
spectrometer operating at 400 and 101,6 the monoclinic space group P21/n with Z = 4
MHz. Infrared absorption spectra were in the unit cell. The structures derived from
obtained from a Perkin Elmer BX II o-hydroxy Schiff bases can exist two types
spectrometer in KBr discs and were reported of intramolecular hydrogen bonds which
in cm-1 units. The UV-VIS spectra were belong to O–H···N in phenol-imine form
measured using a SHIMADZU 1800 series and N–H···O in keto-amine one. As seen
spectrometer. Elementary analyses were from X-ray results, the molecular structure
performed on a Vario EL III CHNS adopts the phenol-imine tautomeric form,
elemental analyzer. Melting points were because the structure have intramolecular
measured with an Electro Thermal IA 9100 hydrogen bond between the o-hydroxy
apparatus using a capillary tube. group and the imine nitrogen atom. While
X-Ray crystallography the O1–C11 bond length is found at 1.351
A suitable single crystal of the (4) Å, the N2–C9 bond length is found as
compound was mounted on goniometer and 1.281 (4) Å. The benzothiazole system is
data were collected at 296 K on a Bruker essentially planar [maximum deviation of
Kappa APEXII CCD diffractometer using 0.031 (9) Å for the C6 atom]. The dihedral
graphite monochromated MoKα radiation angle between the phenyl ring and
(λ=0.71073 Å). Cell parameters of the benzothiazole ring is 19.96 and molecular
compound were determined by using structure is not planar. The molecular
SAINT software.9 Absorption correction ( structure is stabilized by an intramolecular
= 0.23 mm-1) was obtained by the multi-scan O1–H1···N2 hydrogen bond (Figure 1). The
method via SADAPS V2012/1 software.10 details of the hydrogen bonds are
The compound was solved by a dual-space summarized in Table 2.
algorithm using SHELXT-2014 and refined 3.2 Optimized structure
with SHELXL-2017.11 The molecular figure Optimized structure of the compound
is prepared by the help of ORTEP3 program was performed by using DFT/B3LYP
packages.12 Details of the data collection method with 6-311++G(d,p) basis set. The
conditions and the parameters of the biggest differences between experimental
refinement process are given in Table 1. and calculated bond lengths and angles as
0.031 Å in C8–S1 bond and are 1.6 in C8– Figure 3, the maximum positive region of
N1–C6 and C9–N2–C8 angle, respectively. the compound was observed around methyl
According to crystallographic studies, the hydrogen atoms of methoxy group. These
dihedral angle between the C10-C15 and region may have nucleophilic feature. In
benzothiazole is 19.96, while this angle has addition, the most negative V(r) value is -
been calculated at 0.02° for optimized 0.039 a.u. for O1 atom. These values give
structure. The essential reason of this information concerning the region from
different is resulted from crystal packing in where the compound can have ligand
solid phase of the molecule. The C8–N2 and bindings and intermolecular interactions.
C9–C10 bond lengths which are formed by
N2 and C9 atoms connected of C8 and C10 Table 1. Crystal data and refinement for the
atoms in phenyl and benzothiazole ring are title compound
found as 1.401(4)/1.382 Å and1.452(4)/ Cheical formula C16H14N2O2S
1.441 Å (exp./cal.), respectively. In order to Crystal shape/color Block/Orange
compare the theoretical results with the Formula weight 298.35
experimental values, root mean square error Crystal system Monoclinic
Space group P21/n
(RMSE) is used. The calculated RMSE for Unit cell parameters a = 6.0861(8) Å
bond lengths and bond angles are 0.012 Å b = 19.093(3) Å
and 0.58, respectively. Low values of c = 12.7311(18) Å
RMSE can be attributed to the absence of  = 103.83(3)o
effective intermolecular interactions. Volume 1436.5(3) Å3
Chemical reactivity descriptors Z 4
Dx (Mg cm-3) 1.380
The highest occupied molecular
 (mm-1) 0.23
orbital (HOMO) represents the outermost F000 624
orbital filled by electrons and behaves as an Crystal size (mm3) 0.20  0.17  0.16
electron donor, while the lowest unoccupied Tmin; Tmax 0.628, 0.746
molecular orbital (LUMO) considers as the No. of measured, independent 28727, 3585, 2427
first empty innermost orbital unfilled by and observed reflections
Criterion for observed I > 2σ(I)
electron and behaves as an electron
reflections
acceptor. In our study, HOMO-1, HOMO, Rint 0.065
LUMO and LUMO+1 energies and their R[F2 > 2σ (F2)], wR, S 0.071, 0.195, 1.07
shapes are shown in Figure 2. As seen from No. of reflection 3585
Figure 2, HOMO and LUMO electrons are No. of parameters 190
Weighting scheme w=1/[σ2(F02)+(0.0766
almost localized over the whole molecule.
P)2 + 1.31893P]; P=(
Using HOMO and LUMO energy values for F02+2Fc2)/3
molecule, we can be determined to chemical ∆ max, ∆ min (e Å-3) 0.37, -0.32
reactivity descriptors such as I (ionization
potential), A (electron affinity), χ Table 2. Hydrogen-bond geometry (Å)
(electronegativity), η (chemical hardness), ζ D–H···A D–H H···A D···A D–H···A
(softness) and ψ (electrophilicity index)
O1–H1···N2 0.82 1.93 2.646 (4) 146
parameters of molecule. These values are
given in Table 3.
Molecular electrostatic potential surface 3.3 DNA-Binding
We calculated MEP of the optimized The absorption spectra of Schiff base
molecular structure at the B3LYP/6- in the absence and presence of CT-DNA at
311++G(d,p) level. The red color parts different concentrations. With the
represent the negative electrostatic potential concentration of CT-DNA increased,
regions or electrophilic reactivity, while blue hyperchromism of 86.9%-133.5% and
ones represent the positive ones or higher wavelength of 1-19 nm are observed
nucleophilic reactivity. As can be seen in at 278 nm, while hypochromism of 1.0%–
46.0% and red shift of 1 nm are observed at
310 nm. In addition, the maximum
absorption shifted to red indicates a decrease
in energy between the HOMO and LUMO.
The extent of the hypochromism and
hyperchromism are commonly consistent
with the strength of intercalative and
electrostatic interaction (Figure4).
3.4 Colorimetric anion-sensing Figure 1. ORTEP-3 drawing of the
The binding ability of the sensor compound with displacement ellipsoids
compound (5x10-6 M) was studied using plotted at 30% probability level;
naked eye detection in DMSO. In natural intramolecular hydrogen bonds are
light, the observed color changes were from represented by dashed lines
yellow to violet, purple, whitish-maroon and
orange for F- , CN-, AcO- and OH-. There
was discernible fluorescence change under
the hand held long wave UV lamp as
maroon for F- and AcO-, and blue for CN-
(Figure 5).
Table 3. The calculated quantum molecular

descriptors of the compound
Parameters B3LYP
EHOMO (eV) -5.900
ELUMO (eV) -2.637
EHOMO–ELUMO 3.263
Ionization potential [I = −EHOMO] (eV) 5.900
Electron affinity [A = −E LUMO] (eV) 2.637
Chemical hardness [η =(I − A)/2] (eV) 1.631
Electronegativity [χ = − (I + A)/2] (eV) -4.268
Softness [ζ = 1/2 η] (eV) 0.306
Electrophilicity index [ψ = χ /2 η ] (eV)
2
5.584
4. Conclusion
In this study, (E)-4-methoxy-2-((4-
methylbenzo[d]thiazol-2-ylimino)methyl)- Figure 2. The molecular orbital surfaces and
phenol is reported. The molecular structure Figure 2. The molecular orbital surfaces and
their energy values of the title compound
of the compound was confirmed using X-ray their energy values of the title compound
crystallographic, elemental analysis, UV-
Vis, NMR and FTIR studies. Theoretical
calculations were employed to determine the
molecular structure, spectroscopic values,
HOMOs, and LUMOs levels of the
compound. The theoretical calculations were
found to be in good agreement with the
experimental data. The ligand was found to
act as DNA binding and anion sensor agents.
Figure 3. Molecular electrostatic potential map calculated at B3LYP/6-311++G(d,p) level
Figure 4. UV-Vis spectra of compound in the absence and presence of increasing amounts of
CT-DNA at room temperature in Tris–HCl/NaCl buffer (pH 7.2)
Figure 5. The color changes of the compound (1 equiv) upon addition of various anions
under (a) normal light hand (b) hand held UV lamp (λ =365 nm)
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In-silico modification of recombinant interleukin-18 for binding affinity
prediction towards its binding receptor
Kaweeyut Poonkaeo1, Surapong Chatpun1, Surasak Sangkhathat2,3, Varomyalin Tipmanee2*
1
Institute of Biomedical Engineering, Faculty of Medicine, Prince of Songkla University,
2
Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University,
3
Department of Surgery, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand
*E-mail: tvaromya@medicine.psu.ac.th
Abstract:
Cancer is a common cause of death worldwide. Various methods have been
introduced as efficient treatments including immunotherapy. Immunotherapy deals with an
application of functional protein to boost immune system for cancer elimination. An
immunotherapeutic protein called interleukin-18 (IL18) has caught our interest due to its
efficiency for natural killer cell and cytotoxic T lymphocyte promotion as well as metastasis
inhibition. In this work we have used molecular dynamics simulation (MD) and protein-
protein docking to predict potential mutation point. Since IL18 binds interleukin-18 receptor
alpha (IL18a) to perform an action, the alteration of amino acid in IL18 could play a key
point. Some mutations such as N91K and N111K were selected based on a structure of IL18-
IL18Ra (PDB code 3WO3). We also chose our previously published recombinant E6K-T63A
and wild type protein (WT) as comparative controls. MD simulation with AMBER16 force
field with 310 K (37 C) and 1 atm of IL18 in 0.15M NaCl solution was modelled to mimic
cellular environment. We found E6K-T63A and N111K provide better binding to IL18Ra,
compared to WT-IL18. It implied the higher affinity of N111K. This can lead to novel
alteration of interleukin-18 and shorten time and budget for further improvement.
1. Introduction Among the interleukin-1 family

Cancer is a large group of diseases members, interleukin-18 (IL-18) is one of
that occurred from uncontrolled growth of the cytokines that related to immunotherapy.
abnormal cells. It can spread to many parts IL-18 has anti-cancer properties through a
of the body. WHO has reported that cancer stimulation of natural killer cells (NK cells)5
is a common cause of death globally, with and Cytotoxic T lymphocytes,6 including the
approximately 8.8 million deaths in 2015.1 inhibition of cancer cell growth and
In 2017, the American Cancer Society metastasis.7 In the action of IL-18, the IL-18
estimated that 1,688,780 new cancer cases binds to a specific receptor on the surface of
and 600,920 cancer deaths were occurred in target cell. IL-18 receptor binding sites
the United States.2 Currently, there are many compose of the IL-18 receptor α chain (IL-
methods of cancer therapy and control. 18Rα) and the IL-18 receptor β chain (IL-
Immunotherapy is an alternative method of 18Rβ). The binding sites of IL-18 are
cancer therapies which include monoclonal divided into two parts. First part is a site I
antibody, cancer vaccines, T-cell therapy, and II that is specific to IL-18Rα and second
oncolytic virus therapy and non-specific part is site III that is specific to IL-18Rβ.8
immunotherapy.3 It is reported that non- Previously, there was research that
specific immunotherapy involves indirect engineered IL-18 by altering the binding
action to eliminate cancer cells by residues to improve its activity using single
stimulating immune system through some point mutagenesis. This research found that
cytokines.4 the combination of E6K and T63A
mutations showed highest activity.9 Either sodium (Na+) or chloride (Cl-) ion
However, in vitro study to synthesis and was added to neutralize the IL-18 mutants.
characterize new antibody or protein is very Then the protein-solution system was
time-consuming and cost related. In silico equilibrated at 310 K (37°C). The systems
molecular modeling and simulation have were simulated at temperature of 310 K and
contributed greatly to the understanding of pressure of 1.013 bar (1 atm) to mimic an in
protein structure-function relationships. This vivo environment via Berendsen algorithm.
computational technique cuts down material AMBER16ieq force field was applied for
and overhead cost and reduces time to spend protein structure along with TIP3P water
for bench tests. box in the simulation with the size of about
In this study, we focus on the 120 x 120 x 110 Å3. The system was
modification of amino acids sequence and equilibrated in the periodic boundary
the enhancement of the binding affinity of condition with Particle Mesh Ewald and a
IL-18 by using in silico study. cutoff of 16 Å. The simulation package in
this study was based on PMEMD
2. Materials and Methods implementation in AMBER16 package. The
2.1 Mutagenesis simulation was carried out for 3 ns with
The x-ray crystallographic structure NVT ensemble using a 1 fs time step, and
of human mature IL-18 (157 amino acids) 150 ns NPT simulation with 2fs-time step.
was obtained from RCSB protein data bank The first 100 ns trajectory was treated as an
(www.rcsb.org) with PDB code 3WO3.10 equilibrated phase and 20 structures from
Visual Molecular Dynamics (VMD) version the last 50 ns trajectory were used for
1.9.311 was used for the replacement of protein-protein docking.
amino acids in the protein sequence. Figure 2.3 Structure analysis
1 illustrates amino acid residues in the MD results of the IL-18 wild-type
vicinity of IL-18 receptors. and mutants were analyzed in terms of root-
To modify amino acid sequences, mean-square displacement (RMSD) module,
amino acid residues were selected based on root-mean-square fluctuation (RMSF) using
the distance between the amino acid within VMD package. In addition, protein-protein
the IL-18 structure and amino acid within docking was performed to determine the
the receptor structure of IL-18. This distance binding affinity of IL-18 mutants to IL-18
must not exceed 10 angstroms because the receptor and compared with binding affinity
ionic bond occurs when the distance of IL-18 wild type to IL-18 receptor. In the
between two amino acids is less than 10 later part, ZDOCK webtools15 was used.
angstroms. In this study, asparagine 111
(Asp111) was replaced to be a lysine and
arginine respectively, since the binding site
suggested the negatively charged residue
nearby this amino acid residue. After
obtaining the amino acid position, the amino
acids were modified by Rosetta backrub
webtools (see Table 1).12
2.2 Molecular dynamic simulation
After structure modification, the
protonation state was determined at pH7
using PROPKA webtools.13 The missing Figure 1. Structures of Interleukin-18
hydrogen atoms were added to the model of (red) and Interleukin-18 receptor (blue).
the IL-18 mutants using AMBER 16.14 The In the black frame, there contains IL-18
IL-18 mutants were solvated by TIP3P water amino acid residues in the vicinity of
molecules along with NaCl (0.15mol dm-3). modified IL-18 receptors.
Table 1. Modified amino acid sequences of Interleukin-18
Single point mutation Meaning
Wild type Amino acid sequence was not replaced
E6K+T63Aa Glutamic acid (Glu) was replaced by lysine (Lys) at the 6th amino acid sequence
Threonine (Thr) was replaced by alanine (Ala) at the 63rd amino acid sequence
N111K Asparagine (Asn) was replaced by lysine (Lys) at the 111st amino acid sequence
N111R Asparagine (Asn) was replaced by arginine (Arg) at the 111st amino acid sequence
a
Positive control derived from Saetang, J. et.al. 2016.9
3. Results and Discussion N111K and N111R modification influence

3.1 Root-mean-square displacement the structural conservation of the IL-18. To
To verify the effect of a point confirm this result, 3D structures of wild-
mutation on the IL-18 structure, RMSD type IL-18 and its mutants were visualized
measurement was carried out. For N111K by VMD (Figure 3). The results of 3D
and N111R, as show in Figure 2, RMSD structure reveal that there are no differences
results indicate closer similarity to between the structures of mutation.
E6K+T63A and wild type IL-18 and the Therefore, the mutation has no effect on
trends of the graph are not more than 3 structural alteration of IL-1
angstroms. RMSD results suggest that
Figure 2. Root-mean-square displacement (RMSD) from the molecular dynamic simulation

of all IL-18 mutations
Figure 3. (A) IL18-IL18Rα complex (B) Structure of wild type IL-18 (C) Structure of
E6K+T63A IL-18 (D) Structure of N111K IL-18 (E) Structure of N111R IL-18
3.2 Root-mean-square fluctuation for IL-18), with reference to the starting

Apart from the conformational template (3wo3.pdb). The entire mutation
conservation, structural flexibility (Figure 4) has a similar RMSF pattern in all IL-18
was also assessed in order to observe the mutant. This result confirms that mutation
dynamic behavior of IL-18 structure. The has an impact on flexible conservation of
flexibility was evaluated by RMSF plotted IL-18.
against protein residue order (residues 1-159
Figure 4. Root-mean-square fluctuation (RMSF) in Angstrom units from MD simulations of

all IL-18 mutation
Table 2. Docking score of wild type IL-18 and its mutants
Mutation Score Binding affinity
Wild type 1280.047 -
E6K+T63A 1358.759 Increase
N111K 1328.901 Increase
N111R 1189.898 Decrease
3.3 Protein-protein docking modification is an interesting option for
To evaluate the biological activity of developing interleukin-18 proteins to be
IL-18 mutations and IL-18 wild-type, more effective. Structural visualization was
Protein-Protein docking was used to used to confirm the Protein-Protein docking
measure the ability of the IL-18 to bind IL- results. According to Figure 5A, the lysine
18 receptor. Compared with the wild type, side chain moves closer to the receptor than
the results show that binding score of the side chain of the asparagine to form a
E6K+T63A (positive control) and N111K hydrogen bond. On a contrary, the side
had a higher score than N111R. The results chain of the arginine slightly shifts out of the
also suggest that replacing the residue 111 to receptor compared to the side chain of the
lysine can enhance the binding affinity of asparagine (Figure 5B). For this reason, the
IL-18 when compared with the wild type. binding affinity increases for the N111K
On the other hand, replacing residue 111 to mutation but decreases for the N111R
arginine cannot enhance the binding affinity mutation compared to the wild type.
of IL-18. That means N111K amino acid
Figure 5. Structure comparison between wild-type IL-18 and two mutants (A) closer
distance of lysine in N111K (yellow) to a receptor responsible to better binding affinity
compared to asparagine in the wild-type (red). (B) similar orientations of asparagine in
N111R (green) and in the wild-type (red) with the distance of asparagine in the wild-type
slightly closer to a receptor, indicating lower affinity of N111R
4. Conclusions 2. Siegel, L.; Miller, D.; Jemal, A. CA.

In summary, we applied molecular Cancer J. Clin. 2017, 67, 7–30.
dynamics simulations and molecular 3. Farkona, S.; Diamandis, P.; Blasutig,
docking to evaluate structure and dynamic M. BMC Med. 2016, 14:73. pp 1–18.
properties of three mutants of interleukin-18. 4. Nelson, D.; Fisher, S.; Robinson, B. J.
Preliminarily, we have found that the single Immunol. Res. 2014, 2014, 789069.
point mutation could potentially affect the 5. Chaix, J. J. Immunol. Baltim. Md 1950.
structure and the binding affinity of IL-18 2008, 181, 1627–1631.
anti-cancer target. 6. Okamoto, I.; Kohno, K.; Tanimoto, T.;
Ikegami, H.; Kurimoto, M. J. Immunol.
Acknowledgements Baltim. Md 1950. 1999, 162, 3202–
This work was supported by Faculty 3211.
of Medicine, Prince of Songkla University. 7. Cao, R.; Farnebo, J.; Kurimoto, M.;
Cao, Y. FASEB J. Off. Publ. Fed. Am.
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12. Davis, W.; Arendall, B.; Richardson, H.; Vreven, T.; Weng, Z.
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265–274.
Adsorption study of chloroform and hexane by using silver nanoparticles
supported on reduced graphene oxide
Nattawadee Wisitruangsakul1*, Kanyapat Teekayupak2, Tiwaporn Kruk1
1
Department of Chemistry, Faculty of Science, King Monkut’s University of Technology Thonburi,
2
*E-mail: nattawadee.wis@kmutt.ac.th
Abstract:
Two volatile organic compounds, chloroform and hexane, were adsorbed on reduced
graphene oxide supported with silver nanoparticles (Ag@GRO). The adsorption study was
performed by means of UV-visible spectroscopy and Fourier transform infrared (FTIR)
spectroscopy. When chloroform was adsorbed on Ag@RGO, UV-visible absorption spectrum
revealed a red-shifted and blue-shifted of maximum absorption peaks at 263 and 400 nm to
268 and 392 nm, respectively. When hexane was adsorbed on Ag@RGO, The spectrum
revealed a red-shifted of maximum absorption peaks at 263 and 400 nm to 270 and 408 nm,
respectively. These were due to changes of    * transition of the C-C bonds in aromatic
ring. FTIR spectra also showed that when adsorption of chloroform took place, the absorption
peaks of bending of aromatic C=C bonds (802 cm-1) and stretching of aromatic C-C bonds
(1022 cm-1) were shifted to 799 and 1027 cm-1, respectively. For adsorption of hexane, shifts
of these two vibrational bonds were also found. Moreover S/N of FTIR spectra for adsorption
of chloroform and hexane were improved when RGO was supported with silver nanoparticles
in comparison to those adsorption on the pristine RGO.
1. Introduction palladium-gold nanoparticles has been used

Volatile organic compounds (VOCs) as a catalyst for oxygen reduction.5
are organic compounds those can be easily Detection of ammonia has been reported by
evaporated at room temperature and harmful using nanocomposite between polypyrole
to human health as well as environment. and RGO.6 Presently, low cost and fast
VOCs have been utilized in various monitoring of VOCs is still that of interest.
applications, such as solvents, fuel in This project is to study the adsorption of
transportation, components in daily products VOCs on RGO decorated with silver
(paint, smoke, bleach, insecticide, nanoparticles in order to gain their
contaminated in air, drink and food).1 GO enhancement in FTIR signal
and RGO have also been reported to be used
in various applications including VOCs 2. Materials and Methods
monitoring due to their advantages of simple 2.1 Chemicals
and low cost management.1,2 Improvement Chloroform(CHCl3), Sodium
in absorption efficiency of GO and RGO by borohydride (NaBH4), Graphite, Silver
decorating with other nonmetallic has been nitrate (AgNO3), citric acid (C6H8O7), conc.
reported. Doping AgNPs on RGO by Sulfuric (conc. H2SO4), Potassium
refluxing method has been studied by permanganate (KMnO4), 30%
Chettria et al.3 Detection of ammonia by Hydrogenperoxide (H2O2), and Hexane
using GO decorated with silver (C6H14) were purchased from Sigma-
nanoparticles, which was reduced by using Aldrich. All the chemicals were analytical
vitamin C, has been reported by grade and used as received. All the aqueous
Kavinkumar et al.4 GO supported with solutions were prepared with twice distilled
water throughout the whole experiments. pronounce absorption peak at 420 nm,
2.2 Preparation of RGO supported with which due to its localized surface Plasmon
AgNPs resonance. A strong optical absorption in
In this work, RGO was prepared UV-Visible region is also found for RGO at
from graphite powder based on Hummer’s 270 nm due to the p-p* transition of
method. Silver nanoparticles (AgNPs) was aromatic C-C bonds. After decorating RGO
chemically prepared by using citric acid as a with AgNPs, the absorption peaks are
reducing agent.7,8 Decoration of AgNPs onto shifted to 400 and 263 nm, respectively.
RGO was performed by ultra-sonicating the This implying the electron transfer between
RGO suspension and AgNPs solution AgNPs and RGO.
together to get Ag@RGO suspension. Then
the suspension was dried at room
temperature till the powder form was
achieved.
2.3 Adsorption of VOCs on Ag@RGO
Ag@RGO powder was placed on a
small glass slide. The sample of VOCs,
chloroform and hexane, were sprayed
separately into a small bottle with 2 cm in
diameter and 1.5 cm height. After that,
immediately turned the bottle upside down
to completely cover the Ag@RGO powder. Wavelength (nm)
Then waiting for 1 hour before further
Figure 1. UV-Visible spectra of AgNPs,
characterization.
RGO and Ag@RGO
Structural characterization and FTIR spectra of RGO and Ag@RGO
investigation were performed using typical are shown in Figure 2. For FTIR spectrum
UV-Visible and FTIR spectroscopic of RGO, strong bending vibration of
techniques. UV-Visible absorption spectra aromatic C-C bonds is found at 803 cm-1.
were recorded on a UV-Visible spectrometer Another two strong absorption peaks at 1025
(LAMBDA35/FIAS300, Perkin Elmer Co. and 1094 cm-1 have been assigned to
Ltd.). The aqueous suspensions of grahene stretching vibration of C-O bonds. After
oxide or reduced graphene oxide were used decorated with AgNPs, two latter peaks are
as the UV-Visible samples, and the pure shifted to 1022 and 1096 cm-1, respectively.
water was used as reference. The FT-IR This implies the structural changes of RGO
spectra were obtained on a Fourier transform when decorated with AgNPs. FTIR
infrared spectrometer (Nicolet 6700, spectrum of Ag@RGO when absorbed
Thermo Fisher Scientific) at room chloroform (Figure 3) shows the bending
temperature over a frequency range of 4000- vibrational peak of aromatic C-C bonds at
400 cm-1. The samples for FT-IR 799 cm-1 and a pair of stretching vibrational
measurement were prepared by grinding the peaks of C-O bonds at 1027 and 1095 cm-1.
dried powder of reduced graphene oxide or The FTIR results show that both RGO and
graphene oxide with KBr, and compressed Ag@RGO can absorb chloroform as
into thin pellets. represented by shifts of the vibrational
absorption peaks. However, structural
3. Results and Discussion changes of absorbent are more pronounced
Characterizations of AgNPs, RGO, when using Ag@RGO compared to using
andAg@RGO through the UV-Visible the pristine RGO. FTIR spectrum of
spectroscopic method are shown in Figure 1. Ag@RGO when absorbed hexane (Figure 4)
A UV-Visible spectrum of AgNPs reveals a shows that bending vibrational peak of
aromatic C-C bonds is shifted to 799 cm-1.
And the stretching vibrational peaks of C-O
bonds are shifted to the position at 1025 and
1099 cm-1, respectively. In comparison with
FTIR spectra of RGO, stretching vibrational
peaks of C-O bonds (1025 and 1094 cm-1)
are no significant changes implying the
weak absorption of hexane on RGO.
Therefore, changes in FTIR absorption
peaks of hexane on Ag@RGO are found due
to the absorption of hexane on Ag@RGO is
predominant.
Figure 4. FTIR spectra of Ag@RGO,

C6H14-Ag@RGO, C6H14-RGO9 and RGO
structural changes upon absorption is

improved when using Ag@RGO. For
absorption of hexane, no significant change
is observed when absorbed on RGO.
Nevertheless, changes of FTIR absorption
spectrum of hexane on Ag@RGO can be
monitored.
Figure 2. FTIR spectra of Ag@RGO and
RGO. References
1. Baria, C.; Mati, I. R.; Romeo, I.;
Garrido, J.; Laguna, M. J. Appl. Phys.
Lett. 2000, 77, 2274–2276.
2. Ma, L.; Z, J. M.; X, K. W.; J, V. Appl.
Surf. Sci. 2015, 343, 121–127.
3. Chettri, P.; Vendamani, V. S.; Tripathi,
A.; Singh, M. K.; Pathak, A. P.; Tiwari,
A. Appl. Surf. Sci. 2017, 406, 312–318.
4. Kavinkumar, T.; Manivannan, S.
Ceramics Int. 2016, 42, 1769–1776.
5. Zheng, J. N.; Li, S.S.; Ma, X.; Chen, F.
Y.; Wang, A. J.; Chen, J. R.; Feng, J. J.
J. Power Sources. 2014, 262, 270–278.
6. Tiwari, D. C.; Atri, P.; Sharma, R. Syn.
Figure 3. FTIR spectra of Ag@RGO, Met. 2015, 203, 228–234.
CHCl3-Ag@RGO, CHCl3-RGO9 and 7. Hummer, W. S.; Offeman, R. E. J. Am.
RGO Chem. Soc. 1958, 80, 1339–1344.
8. Lee, P. C.; Meisel, D. J. Phys. Chem.
4. Conclusion 1982, 86, 3391–3395.
FTIR results show that chloroform 9. Prasobsab, W. BSc’s Degree Thesis,
could be absorbed on both RGO King Mongkut’s University of
and Ag@RGO. However, monitoring of the Technology Thonburi, 2016.
Theoretical study of twisted intramolecular charge transfer and
intermolecular double proton transfer in the excited state of
N,N’-bis(salicylidene)-p-phenylenediamine Schiff base
Nichakan Miengmern1,2, Supa Hannongbua1,2, Songwut Suramitr1,2*
1
2
Center for Advanced Studies in Nanotechnology for Chemical, Food and Agricultural Industries,
KU Institute for Advanced Studies, Kasetsart University, Bangkok 10900, Thailand
*E-mail: fsciswsm@ku.ac.th
Abstract:
Density functional theory (DFT) and time-dependent density functional theory method at
the CAM-B3LYP/6-311G(d,p) level was applied to investigate the intramolecular and
intermolecular hydrogen bonding dynamics in the first excited (S1) state of N,N’-
bis(salicylidene)-p-phenylenediamine (BSP) and in acetonitrile solution. In the BSP single
molecule, we demonstrated that the intramolecular charge transfer (ICT) takes place in the S 1
state. This excited state ICT process was followed by intramolecular proton transfer. For the
hydrogen-bonded BSP-H2O complex, it was demonstrated that the intermolecular hydrogen
bonds can induce the formation of the twisted intramolecular charge transfer (TICT) state and the
conformational twisting is along the C–C bond. Moreover, the intermolecular hydrogen bonds
can also facilitate the intermolecular double proton transfer in the TICT state. This work
successfully presents the stepwise excited state intramolecular double proton transfer (ESDPT)
process of BSP. Therefore, the intermolecular hydrogen bonds can alter the mechanism of
intramolecular charge transfer and proton transfer in the excited state for the BSP molecule.
1. Introduction (K*) via an extremely fast and irreversible

The aromatic internally hydrogen ESIPT process occurring in the sub-
bonded Schiff bases with excited-state picosecond timescales. The excited keto (K*)
intramolecular proton transfer (ESIPT) could be recognized by fluorescence spectrum
characteristics have been drawing abundant largely shifted to longer wavelengths
attention recently due to their unusual optical (normally the significant Stokes shift can be
properties.1 The ESIPT is a process in which as large as 6,000-12,000 cm−1).2 Based on this
photo-excited molecules relax their energy radiation channel coupling with radiationless
through tautomerization by transfers of paths, it relaxes to the ground-state keto
protons, commonly involves the transfer of a tautomer. Then, a fastback proton transfer
hydroxyl proton to an acceptor such as imine process goes from keto to enol closing this
nitrogen. As seen in Figure 1, upon the reaction cycle, restoring the initial species,
photoexcitation process, the stable ground stable in the ground state. The deprotonated
state structure as enol (E) form can be excited intermediate in the interconversion of the two
to the corresponding excited state being a forms, referred to as enol-keto form, is highly
normal excited-state form (E*). In the process thermodynamically driven. This peculiar
of ESIPT, the excited enol (E*) optical property has various practical
tautomerization into the excited photoproduct applications, including probes for solvation
dynamics and biological environments, time-dependent density functional theory
photochromic materials, chemosensors, method. The calculated results show that the
organic light-emitting diodes devices, cell optimized geometry can well reproduce the
imaging and so forth.3-5 The hydrogen bond is structural parameters. On the calculation of
one of the important weak interactions, it has the electronic absorption spectra, TD-DFT
attracted more attention owing to its calculations were carried out in solution
directional character in many photophysical phase. The energetic behavior and the frontier
and photochemical processes, especially in the molecular orbitals (MOs) of the ligands have
ESIPT process, so that the mechanism of been examined in solvent media using the
ESIPT reaction has been proposed and widely CAM-B3LYP method with the 6-311G(d,p)
studied. basis sets. Therefore, the main aim of the
present study is to find out the detailed
mechanism and the reliable ESIPT dynamics
process of BSP containing two intramolecular
hydrogen bonds.

2.1 Computational methods
Quantum chemical calculations based
on density functional theory (DFT) were
carried out using a Gaussian09 program.6 The
Figure 1. Characteristic four-level photocycle geometry optimizations (S0) were used to
scheme of the ESIPT process determine the minimum energy structures of
N,N’-bis(salicylidene)-p-phenylenediamine
Particularly interesting are molecules (BSP) Schiff base ligand (Figure 2) were
with hydrogen bond donating and accepting performed using hybrid exchange-correlation
groups that can form a dimer with dual functional, the Coulomb-attenuated method
intermolecular hydrogen bonds. In such based on the B3LYP functional (CAM-
systems, excited state double proton transfer B3LYP) with 6-31G(d,p) basis set.7 The
(ESDPT) can take place, resulting in the theoretical excitation energies of BSP were
formation of an excited tautomeric dimer calculated within the framework of TD-DFT
(EE* → KK*). The extent of dimerization approach for the S1 state with the same hybrid
depends on the concentration, the temperature, functional at the basis set level of 6-
and the solvent. Under certain conditions, 311G(d,p), were then performed by single
mixtures of monomer and dimer may be point energy calculations at the optimized
present, which makes spectroscopic studies of geometries of the ground state. This method is
such systems more complex. As a whole, the based on the polarizable continuum model
investigations of double proton transfer (PCM) to obtain a valid approximation of
process are very important, which could help solvent effects.
researchers to have a deeper understanding of
multiple protons transfer mechanism. 3. Results and Discussion
In this work, the Schiff base ligand, 3.1 Geometries of the ground and excited
N,N’-bis(salicylidene)-p-phenylenediamine states
(BSP), both single molecule and BSP-H2O The dienol, enol- keto and diketo
complex have been theoretically investigated tautomeric forms of N,N’-bis(salicylidene)-p-
by the density functional theory (DFT) and phenylenediamine (BSP) Schiff base ligand
(Figure 2) were calculated in acetonitrile
employing the CAM-B3LYP/6-31G(d,p) level
of theory. All the optimizations for attempting
to locate such a minimum lead directly to its
diketo form, which commonly implies that the
ESIPT reaction from the dienol to diketo
forms will be a barrierless process. In Figure
3, the relative energies of the three tautomeric
forms of BSP were calculated at the same
level of theory. The present calculations show
that, among the three tautomers of BSP, Figure 3. Relative Energy (eV) of the S0 of
dienol form is the most stable in all media by BSP (black line) and BSP-H2O complex (red
8.73 kcal mol-1. The values are in the order of line) for isomerization calculated by
dienol > enol-keto > diketo. On the other DFT/TDDFT(CAM-B3LYP)
hand, the relative energies of the three
tautomeric forms of BSP-H2O complex As shown in Figure 2 and Table 1, all
demonstrated that diketo form is more stable the optimized structures exhibit two
by 4.82 kcal mol-1 with respect to the least intramolecular hydrogen bonds of BSP
stable dienol form. In an aggregation system, whereas BSP-H2O complex demonstrated
the lone pairs will be involved in hydrogen both intramolecular and intramolecular
bonding with the water, making them less hydrogen bonds, respectively. Clearly, there is
available to hydrogen bond with the enol a significant strain on the structure due to the
form. migration of the hydrogen atom. For BSP,
dienol form is noteworthy that our theoretical
bond distances of O1-H2 and O4-H5 are
lengthened, and those of H2···N3 and H5···N6
are shortened in the S1 state with the
enlargement of bond angles δ(O1-H2-N3) and
δ(O4-H5-N6). A similar trend has been also
found in enol-keto form. It can be seen that
both intramolecular hydrogen bonds become
stronger in the S1 state. Generally, a stronger
intramolecular hydrogen bond interaction in
the excited-state will lead to a smaller
structure adjustment and a smaller energy
barrier in the ESIPT process, thus facilitating
the occurrence of ESIPT reaction. While in
diketo form, the distances of hydrogen bonds
H-N are 1.049 Å, about 0.002 Å larger than
that of S0. The increase of the distance of
hydrogen bonds in S1 is expected to make the
reverse proton transfer from S1 less favorable
Figure 2. The optimized geometries of (a) compared with the one in S0 state from the
dienol, (b) keto–enol and (c) diketo tautomeric view of hydrogen bond strength. In BSP-H2O
form of BSP at PCM-CAM-B3LYP/6- complex, the distances of O1-H2, H4-N5, O6-
31G(d,p) theoretical level H7 and H9-N10 are increased.
Table 1. Primary bond lengths (Å) and bond angles, δ (deg) of dienol, enol-keto and diketo
tautomeric form of BSP and BSP-H2O complex structures in the acetonitrile solvent
dienol enol-keto diketo
Parameters
S0 S1 S0 S1 S0 S1
BSP ligand
O1-H2 0.998 1.010 0.997 1.001 1.652 1.658
H2-N3 1.697 1.642 1.700 1.680 1.047 1.049
O4-H5 0.998 1.010 1.656 1.705 1.653 1.658
H5-N6 1.697 1.642 1.047 1.043 1.047 1.049
1(C4-C5-C6-N3) 0.142 0.142 0.118 0.186 0.012 0.002
2(C6-N3-C7-C8) 35.736 2.950 35.518 13.945 17.128 0.020
3(C'4-C'5-C'6-N'3) 0.133 0.142 0.010 0.079 0.026 0.003
4(C'6-N'3-C'7-C'8) 35.654 2.950 18.149 0.305 17.295 0.020
BSP-H2O complex
1(C4-C5-C6-N3) 15.472 11.511 15.976 16.079 0.211 0.051
2(C6-N3-C7-C8) 39.899 19.215 40.530 29.965 22.964 0.794
3(C'4-C'5-C'6-N'3) 15.543 11.524 0.208 2.721 7.644 0.041
4(C'6-N'3-C'7-C'8) 39.892 19.233 22.728 1.892 33.657 0.762
On the other hand, distance of the 311G(d,p) level of theory are summarized in
other hydrogen bonds is decreased. It was Table 2. The calculations show that electrons
demonstrated that water molecule can induce are spread out all over the big molecule. In the
the intermolecular hydrogen bonds between first excited state, electrons are excited from
BSP and water. In addition, the intermolecular the highest occupied molecular orbital
hydrogen bonds can induce the formation of (HOMO) to the lowest unoccupied molecular
the twisted intramolecular charge transfer orbital (LUMO), exhibiting the ππ*-type
(TICT) state and the conformational twisting character. These results demonstrated
is along the C-C bond. Moreover, the intramolecular charge transfer (ICT).
intermolecular hydrogen bonds can also As can be seen from Table 2, the S0 →
facilitate the intermolecular double proton S1 absorption wavelength of BSP arising from
transfer in the TICT state. Differently, in BSP, its most stable dienol form is calculated to be
hydrogen bonds H···N is shorter than those in 335 nm corresponding to the  transition,
BSP- H2O complex, which not only shows with an oscillator strength of 1.4306, in
that surrounding solvents have effects on agreement with the experimental peak at 367
target molecule, but also suggests that nm observed in acetonitrile. In addition, we
hydrogen bonds could be stronger in BSP. have calculated the emission wavelengths of
3.2 Absorption spectra of tautomers of BSP BSP shows a broad band located at 450 nm
The optimized geometries were used corresponding to an intramolecular charge
to calculate the absorption and emission transfer transition (ICT). For the other forms,
spectra of the three tautomers of BSP and the calculated vertical excitation energies and
BSP-H2O complex. The vertical wavelength vertical emission energies also observed
of all three tautomers of BSP both in the emission spectra arise from the S1 state,
present and absent water were calculated which apparently supports the occurrence of
using TD-DFT with the CAM-B3LYP/6- the fully ESIPT reaction. To gain insight into
the emission properties, density functional 4. Conclusion
theory (DFT) computation at the CAM- In summary, we have carried out
B3LYP/6-311G level were carried out by detailed theoretical studies on ESIPT process
using Gaussian09 software, our calculated of BSP molecules, both single and BSP-H2O
frontier molecular orbitals (MOs) of BSP complex in acetonitrile using DFT and TD-
structure are displayed in Figure 4. It can be DFT methods. Our theoretical results indicate
clearly seen that the dominant ππ*-type that the CAM-B3LYP/6-311G(d,p) method
transition could be assigned with the can provide a good description of the ground
composition from HOMO to LUMO. The state geometries and the absorption and
calculated HOMO and LUMO energies of emission spectrum of BSP. The
BSP are -7.13 and -0.82 eV for dienol form, intermolecular hydrogen bonds were
-6.77 and -1.82 eV for enol-keto form and demonstrated to be significantly strengthened
-6.66 and -1.24 eV for diketo form, the energy in the electronically excited state, which
gaps (△Eg) amount to 6.31, 5.68 and 5.42 eV, provides a driving force and facilitates the
respectively. The above results confirm that ESDPT reaction. Meanwhile, the plotted
the frontier molecular orbitals involved in the frontier MOs clearly reveals that the
lowest energy transitions and optical rearrangement of electronic density is a very
properties of the molecular system can be important positive factor for the proton
tuned by the tautomerization. transfer. In theoretical results suggest that the
ESDPT of compound tends to take place
stepwise.
Table 2. Absorption and fluorescence spectra (λ, in nm) of BSP and BSP-H2O complex
structures in the acetonitrile solvent obtained by the TD-CAMB3LYP/6-311G(d,p) calculations
Orbital
Compound Transition λ (nm) f
transitions
BSP ligand
Absorption
dienol S0 → S1 H > L (90.5%) 335 1.4306
enol-keto S0 → S1 H > L (89.3%) 391 0.9020
diketo S0 → S1 H > L (82.2%) 410 1.2370
Fluorescence
dienol S1 → S0 H > L (96.3%) 450 1.8093
enol-keto S1 → S0 H > L (92.2%) 450 1.1276
diketo S1 → S0 H > L (88.8%) 463 1.4207
BSP-H2O complex
Absorption
dienol S0 → S1 H > L (91.1%) 323 1.4405
enol-keto S0 → S1 H > L (90.7%) 384 0.7584
diketo S0 → S1 H > L (71.0%) 396 1.1205
Fluorescence
dienol S1 → S0 H > L (100.0%) 455 1.5841
enol-keto S1 → S0 H > L (93.7%) 443 0.9313
diketo S1 → S0 H > L (89.0%) 463 1.4105
Figure 4. Optimized structures, HOMO and LUMO of three tautomeric forms of BSP
Acknowledgements 2. Tseng, H.-W.; Liu, J.-Q.; Chen, Y.-A.;

This research was supported by the Chao, C.-M.; Liu, K.-M.; Chen, C.-L.;
Center of Nanotechnology Kasetsart Lin, T.-C.; Hung, C.-H.; Chou, Y.-L.;
University, Kasetsart University Research and Lin, T.-C. J. Phys. Chem. Lett. 2015, 6,
Development Institute (KURDI), National 1477−1486.
Research University Project of Thailand 3. Park, S.; Kim, S.; Seo, J.; Park, S. Y.
(NRU), Research Assistant Scholarship Macromol. Res. 2008, 16, 385−395.
(Center for Advanced Studies in 4. An, B.; Feng, S.; Wen, K.; Wu, W.;
Nanotechnology for Chemical, Food, and Yuan, H.; Zhu, Q.; Guo, X.; Zhang, J.
Agricultural Industries) and Department of Org. Electron. 2017, 45, 1−8.
Chemistry, Faculty of Science, Kasetsart 5. Chen, K.-Y.; Tsai, H.-Y. Int. J. Mol. Sci.
University for financial support. 2014, 15, 18706−18724.
6. Scalmani, G.; Frisch, M. J. J. Chem.
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Substituent effect on photophysical properties of salicylidene Schiff base
and its derivatives as colorimetric dyes: a TD-DFT study
Amonchat Koonwong1,2, Suwannee Sriyab1,2, Supa Hannongbua1,2, Songwut Suramitr1,2*
1
2
Center for Advanced Studies in Nanotechnology for Chemical, Food and Agricultural Industries,
KU Institute for Advanced Studies, Kasetsart University, Bangkok 10900, Thailand
*E-mail: fsciswsm@ku.ac.th
Abstract:
The effects of substitution groups on the photophysical properties and excited-state
intramolecular proton transfer (ESIPT) of the salicylidene Schiff base derivatives (SA1, SA2,
SA3 and SA4) were considered using density functional theory (DFT) and time-dependent
DFT (TD-DFT) calculations at CAM-B3LYP/6-311G(d,p) level. Geometries and absorption
spectra of all derivatives were calculated at the same level of theory. The substitution of the
salicylidene Schiff base derivatives resulted in the red shift spectra, which implies that the
electron donor substituted has the effect on the π-conjugated system. In addition, results of
molecular orbital analysis show that vertical S0→S1 transition of these molecules corresponds
essentially excitable from HOMO (π) to LUMO (π*). The potential energy curves (PECs) of
selected derivatives were used to investigate the occurrence of ESIPT. The substitution of
salicylidene Schiff base derivatives could easily facilitate the ESIPT process, because the
electron donor substitution makes O-H bond of selected salicylidene Schiff base derivatives
weaker than those of other derivatives and the hydrogen bond in N-H becomes stronger in the
excited state, resulting in lower PT barrier. The obtained information of the electronic
structure, the photophysical property and the chance of ESIPT of salicylidene Schiff base
derivatives molecules is useful for molecular design of colorimetric molecular probes.
1. Introduction quantum chemical calculations is an

The excited-state intramolecular accepted method in the field of chemical
proton-transfer (ESIPT) reaction, a fast enol- calculation research and can provide
to-keto (E-to-K) proton transfer occurring in accurate information understand molecular
the excited states of intramolecularly properties. That can be predicted the trend of
hydrogen-bonded molecules, has been the structural and electronic properties.
extensively investigated because of its basic Theoretical design of ESIPT molecules
interest and potential applications.1 Because structural prerequisite for ESIPT is an
ESIPT molecules are normally more stable intramolecular hydrogen (H) bond that is
in the E form in the ground state than in the built with acidic proton donor and basic
K* form in the excited state, photoexcitation proton acceptor (X–H---Y). In general,
of such molecules is immediately followed proton donor is hydroxyl group (X=O) and
by four-level cyclic proton-transfer reactions proton acceptor (Y) is basic nitrogen atom in
(E→E*→K*→K→E) mediated by intra- heteroaromatic rings.1
molecular hydrogen bonds (see Scheme 1). In this work, we selected amine as a
It is characteristically observed, therefore, proton accepting heteroaromatic part. It is
that the absorption from E and emission well known that those rings form acid-base
from K* result in an anomalously large complex and thus show processability in
Stokes shift without self-absorption, finally acidic solvents because nitrogen atom in
providing ideal motives for four-level lasing, these heteroaromatic rings is able to be
UV photostabilizers, and solar energy protonated. This proton acceptability in turn,
concentrators.2-4 Theoretical studies by means that they can be employed for excited
state intramolecular proton transfer (ESIPT) electronic excited state (S1) were optimized
when combined with intramolecular by the TD-CAM-B3LYP approach and with
hydrogen (H) bond. On this basis, we 6-311G (d,p) basis set.7,8 In addition, the
designed class of ESIPT the salicylidene absorption spectra was also explored by the
Schiff base derivatives, they are SA1 TD-CAM-B3LYP/6-311G (d,p) calculations
(without substituent), electron donating under the S0 and S1 minimum structures,
groups (methyl) substituted SA2, electron respectively. The polarizable continuum
withdrawing groups (chlorine) substituted model (PCM) using the integral equation
SA3 and electron donating groups (tert- formalism variant was considered with
butyl) substituted SA4 (see Figure 1). Due to acetonitrile as solvent throughout the whole
their six-membered intramolecular H-bonds, theoretical calculations unless otherwise
they are expected to have four-level stated. All these calculations were
photocyclic scheme driven by ESIPT, as performed with the Gaussian 09 package.9-11
shown in Scheme 1.
2. Computational Details
Geometries of the electronic ground
state (S0) were calculated using the DFT
method. One of the most commonly used
versions is B3LYP density functional, which
Becke's three-parameter hybrid exchange
functional with Lee-Yang-Parr gradient-
corrected correlation and the 6-31G (d,p)
basis set was used for all atoms.5,6 Figure 1. Structures and partial atom
Frequency calculations were also done at the numberings of the salicylidene Schiff base
same level to ensure that the optimized derivatives (SA1, SA2, SA3 and SA4)
structure was the minimum on the potential studied in this work
energy surface (PES). Structures of the first
Scheme 1. Modified Jablonski diagram for the photophysical processes in the salicylidene
Schiff base derivatives
Table 1. Selected structural parameters of the salicylidene Schiff base derivatives (bond
distance in Å and dihedral in degree)
Parameters SA1 SA2 SA3 SA4
Bond distance (Å)
r(H1-O1)
0.998 0.998 1.005 1.000
r(O1-C)
1.340 1.343 1.329 1.344
r(C=N)
1.287 1.288 1.284 1.287
r(N-H1)
1.741 1.737 1.710 1.704
r(N-C)
1.411 1.410 1.411 1.410
r(C-O2)
1.367 1.366 1.365 1.366
r(O2-H2)
0.967 0.966 0.966 0.966
Dihedral angle (degree)
(O1-C-C-C) 0.201 0.267 0.200 0.364
(N-C-C-O2) -2.250 -2.262 -1.747 -2.305
3. Results and Discussion and SA4. The enol-form ground state the H-
3.1 Structures property bond in SA3 is stronger than that SA1 and
All the calculated structures exhibit a SA2. This presented SA3 (withdrawing
six-membered-ring intramolecular H-bond. groups) stable structure in keto-form.
The selected structural parameters of SA1, According in Figure 2, the order in the
SA2, SA3 and SA4 calculated as a result in relative stability of the keto-form is SA3 >
Table 1, for the O1-H1 and N--H1 bond SA4 > SA2 > SA1. These results shown that
distance, in both enol-form ground state, the all four compound indicate the introduction
O1-H1 distances in SA3 is longer than in of different substitutions can effectively
SA1, SA2 and SA4, while N--H1 distances adjust the stable of the salicylidene Schiff
in the former is shorter than in SA1, SA2 base derivatives.
Figure 2. Comparison relative energies between stabilize of the salicylidene Schiff base
derivatives
3.2 Vertical excitation energy salicylidene Schiff base derivatives

To theoretically elaborate the molecules mainly arise from the electron
absorption spectra, we have calculated the excitation from the highest occupied
vertical excitation energy in acetonitrile, molecular orbital (HOMO) to the lowest
which are listed in Table 2, together with the unoccupied molecular orbital (LUMO),
available experimental values. Our showing the ππ* -type character in Table 3.
calculations show that the S1 states of the For of the salicylidene Schiff base
derivatives, HOMO and LUMO are similar. similar, and that of compound SA4 is a little
HOMO is mainly populated on phenol and larger. This can be explained by HOMO-
imine, and LUMO is mainly contributed by LUMO energy gap (see Table 3). When R
phenol and imine. It should be mentioned group is electron donating group, such as, H,
that R group contributes much to HOMO CH3, C (CH3)3, the HOMO and LUMO
and LUMO. As a result, the transition from energy levels changes only a little. When R
HOMO to LUMO of the salicylidene Schiff group is strong electron withdrawing group
base derivatives molecules all shows Cl (SA3), both HOMO and LUMO energy
obviously intramolecular charge transfer levels decrease obviously, with a more
character. Furthermore, in SA3, upon dramatic effect on the former, resulting in a
electronic excitation from HOMO to much higher energy gap.
LUMO, electron density on the O atom
increases and that of N atom decreases
dramatically.
As can be seen from Table 2, the
S0→S1 absorption wavelength of the
salicylidene Schiff base derivatives
(SA1,SA2, SA3 and SA4) is calculated to be
321, 327, 329 and 326 nm, in good
agreement with the experimental peak at
334, 343 349 and 350 nm, respectively.
According to the calculated vertical
excitation energies, the absorption spectra is
simulated, which are shown in Figure 3.
Furthermore, from Table 2 one can Figure 3. The simulated electronic
see that the calculated vertical excitation absorption spectra
energies of compounds SA1, SA2, SA3 are
Table 2. Physical properties of the salicylidene Schiff base derivatives and oscillator strength
(f) obtained by the TD-CAM-B3LYP/6-311G (d,p) calculations, together with the available
experimental values
State Transition eV λcal (nm) f Transition composition λexpt (nm)
Enol-form
SA1 S0→S1 3.87 321 0.563 HOMO→LUMO (91%) 334
SA2 S0→S1 3.79 327 0.502 HOMO→LUMO (94%) 343
SA3 S0→S1 3.77 329 0.521 HOMO→LUMO (89%) 349
SA4 S0→S1 3.81 326 0.518 HOMO→LUMO (94%) 350
Keto-form
SA1 S0→S1 3.36 368 0.395 HOMO→LUMO (97%) 380
SA2 S0→S1 3.24 382 0.365 HOMO→LUMO (98%) 398
SA3 S0→S1 3.23 383 0.392 HOMO→LUMO (97%) 400
SA4 S0→S1 3.20 387 0.358 HOMO→LUMO (98%) 397
Table 3 HOMO-LUMO energy levels and gap (in eV) of the salicylidene Schiff base
derivatives (SA1, SA2, SA3 and SA4)
Compounds HOMO LUMO ΔEg
SA1 0.103
-0.323 -0.220
SA2 0.103
-0.321 -0.218
SA3 0.091
-0.310 -0.219
SA4 0.104
-0.321 -0.217
3.3 Calculated potential energy curves little energy barrier to achieve the ESIPT
To give a deep insight into the reaction from E-S1 to K-S1. The ESIPT
ESIPT process, the constrained potential process is barrier less, indicating that the
energy curves (PECs) were constructed by ESIPT reaction in compound SA4 is the
performing calculations with CAM-B3LYP fastest. It should be mentioned that the
for S0 and TD-CAM-B3LYP for S1. energy barriers are in the order SA1 > SA4
Eventually, the calculated potential energy > SA2 > SA3, which is in substantially
curves involving the S0 and S1 electronic consistent with the N--H distance order in
states are depicted in Table 4. When the E-S1 state. So the shorter the H-bond in
compound SA1 is photo-excited into the S1 the E-S1 state, the faster the ESIPT takes
excited state, it only needs to overcome a place.
Table 4. Calculated relative energies (ΔER, in eV) and energy difference between the K*S1 and
E*S1 forms (ΔEes, in eV) of the salicylidene Schiff base derivatives
ΔER
Compounds ΔEES
E*(S1) K*(S1)
SA1 3.87 3.36 -0.51
SA2 3.79 3.24 -0.55
SA3 3.77 3.23 -0.54
SA4 3.81 3.20 -0.61
4. Conclusion 5. Becke, A. D. J. Chem. Phys. 1993, 98,
The TD-DFT method was used to 5648–5652.
explore the excited-state intramolecular 6. Hehre, W. J.; Ditchfield, R.; Pople, J. A.
proton transfer (ESIPT) tautomerization J. Chem. Phy. 1972, 56 (5), 2257–2261.
mechanism of a series of O-H--N hydrogen 7. Van Caillie, C.; Amos, R. D. Chem.
bonding compounds. All the calculated Phys. Lett. 2000, 317 (1), 159–164.
structures exhibit a six-membered ring 8. Pedone, A. c J. Chem. Theory Comput.
intramolecular hydrogen bond. Among the 2013, 9 (9), 4087–4096.
four systems considered here, the enol-form 9. Miertuš, S.; Scrocco, E.; Tomasi, J. s.
is more stable in the ground state while the Chem. Phys. 1981, 55 (1), 117–129.
keto- form is more stable in the first excited 10. Cances, E.; Mennucci, B.; Tomasi, J. J.
state. The potential energy curves of the Chem. Phy. 1997, 107 (8), 3032–3041.
ground state and the first excited state along 11. Frisch, M. J.; Trucks, G. W.; Schlegel,
the proton transfer direction were calculated H. B.; Scuseria, G. E.; Robb, M. A.;
to elucidate the ESIPT process. It is found Cheeseman, J. R.; Scalmani, G.;
that the shorter the hydrogen bond in the E- Barone, V.; Mennucci, B.; Petersson, G.
S1 state, the lower the energy barrier in the A.; Nakatsuji, H.; Caricato, M.; Li, X.;
ESIPT process, and the easier the ESIPT Hratchian, H. P.; Izmaylov, A. F.;
takes place. Bloino, J.; Zheng, G.; Sonnenberg, J.
L.; Hada, M.; Ehara, M.; Toyota, K.;
Acknowledgements Fukuda, R.; Hasegawa, J.; Ishida, M.;
This work was supported by the Nakajima, T.; Honda, Y.; Kitao, O.;
Center for Advanced Studies in Nakai, H.; Vreven, T.; Montgomery, J.
Nanotechnology for Chemical, Food and A., Jr.; Peralta, J. E.; Ogliaro, F.;
Agricultural Industries, KU Institute for Bearpark, M.; Heyd, J. J.; Brothers, E.;
Advanced Studies, Kasetsart University, Kudin, K. N.; Staroverov, V. N.;
Bangkok, Thailand. Kobayashi, R.; Normand, J.;
Raghavachari, K.; Rendell, A.; Burant,
References J. C.; Iyengar, S. S.; Tomasi, J.; Cossi,
1. Chou, P. T., J. Chin. Chem. Soc. 2001, M.; Rega, N.; Millam, J. M.; Klene, M.;
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3. Luiz, M.; Biasutti, A.; Soltermann, A. Morokuma, K.; Zakrzewski, V. G.;
T.; Garcia, N. A. Polym. Degrad. Stab. Voth, G. A.; Salvador, P.; Dannenberg,
1999, 63 (3), 447–453. J. J.; Dapprich, S.; Daniels, A. D.;
4. Vollmer, F.; Rettig, W. J. Photochem. Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.;
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In-silico aided inhibitor design of benzoimidazole-containing derivatives as
novel direct InhA inhibitors
Pharit Kamsri1*, Auradee Punkvang1, Patchreenart Saparpakorn2, Supa Hannongbua2,
Sanya Sureram3, Prasat Kittakoop3, Nitima Suttipanta4, Khomson Suttisintong5,
Adrian Mulholland6, James Spencer7, Potjanee Srimanote8, Pornpan Pungpo9
1
2
3
Chulabhorn Research Institute, Bangkok 10210, Thailand
4
Faculty of Pharmaceutical Science, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand
5
National Nanotechnology Center, NSTDA, Pathum Thani 12120, Thailand
6
Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TD, UK
7
School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK
8
Graduate Program, Faculty of Allied Health Sciences, Thammasat University, Pathum Thani 12121, Thailand
9
*E-mail: pharit.kamsri@npu.ac.th
Abstract:
Benzimidazole derivative has been identified as new potential lead InhA inhibitors
based on virtual screening and biological assay evaluations. This compound exhibited the
good enzyme assay (IC50) and mycobacterial assay (MIC). To develop novel benzimidazole-
containing derivatives based InhA inhibitors, in silico based virtual screening was performed
on ChemBridge database. Our finding indicated that the 5928640 is a potential candidate to
be a novel InhA inhibitor benzimidazole-containing hit for further design. Therefore, novel
InhA inhibitors based on 5928640 derivatives were designed using bioisostere replacement
combined with ADMET prediction and molecular docking calculations. MD simulations and
waterswap calculations were applied to gain insight into the binding mode and key
interactions for binding of 5928640 in InhA binding pocket. The obtained results revealed
that hydrogen bond interaction between benzimidazole core with Tyr158 is the key
interactions of new benzimidazole-containing derivatives. In addition, hydrophobic
interactions could be considered as the contribution for stabilizing the InhA-ligand complex.
Accordingly, the obtained results from this work aid to rational design of new benzimidazole-
containing derivatives as new potential InhA inhibitors for mycobacterial drug development.
1. Introduction by the mutations effect of amino acid

Enoyl-acyl carrier protein (ACP) residues in KatG and InhA binding pockets,
reductase or InhA of Mycobacterium leading to the drug resistant of M.
tuberculosis (M. tuberculosis) is potential tuberculosis. However, it is still a valuable
attractive target for anti-tuberculosis drugs target to identify potential and selective
development. The InhA has been identified inhibitor of InhA.1-5 To overcome the drug
as the primary target of isoniazid (INH), the resistant of M. tuberculosis, new inhibitors
first-line drug of tuberculosis treatment. A that directly bind with the InhA were
prodrug, INH is activated to get the active developed.6-8 However, low inhibitory
radical by M. tuberculosis catalase activity based on mycobacterial whole assay
peroxidase (KatG). Then, the active radical of these lead compounds were reported.
is covalently bound with nicotinamide Benzoimidazole derivatives have
adenine dinucleotide (NAD+) at the position been developed as anti-tuberculosis agents
4, producing an active INH-NAD adduct. with high effective against mycobacterial
However, the inhibitory activity is reduced whole cell.9-11 Therefore, we aim to identify
new InhA hits with highly specific with 2.2 MD simulations
target enzyme and highly active The AMBER16 package was used
mycobacterial whole cell based on for MD simulations to determine the
benzoimidazole derivatives. Substructure structural stability and potential binding
search, ADMET predictions and molecular mode of InhA-hit complex. The ff14SB
docking were employed to identify new force field was used as parameter for InhA
specific InhA inhibitors. In addition, the and General amber force field (GAFF) was
structural stability of hit compounds in InhA applied as parameters for ligand and NAD+
binding site was investigated using cofactor. All missing hydrogen atoms of
molecular dynamics (MD) simulations. InhA were added by LEaP module. Atomic
Bioisostere replacement combined with partial ESP charges for ligand were obtained
ADMET prediction were applied for rational from the HF/6-31G(d) calculations.
design of new potent InhA inhibitors. The Complex structure was solvated by TIP3P
obtained results from this work aided to waters. To neutralize the charge of solvated
identify new InhA inhibitors based on system, Na+ ions were added. To reduce
benzoimidazole derivatives. The potential the bad steric interactions of water
hits compounds of InhA inhibitors were molecules and ions, the system was first
proposed for InhA enzyme assay and minimized with atomic positions of solute
mycobacterial whole cell assay to further species. Then, the whole system was
develop new potent anti-tuberculosis agents. optimized. Thereafter, the system was
gradually warmed up from 0 to 300 K. The
2. Materials and Methods solute species were restrained to their initial
2.1 Virtual screening approach coordinate structures. This was followed by
Ligand based virtual screening based the position-restrained dynamics simulation
on substructure search and ADMET under an isobaric condition. Finally, 30 ns
predictions combined with molecular MD simulations without any restraints were
docking calculations were applied to performed using the same conditions.
identify novel InhA inhibitors based on 2.3 Design of novel InhA inhibitors based
benzoimidazole-containing derivatives. The on benzoimidazole-containing derivatives
small molecules from commercial database Based on MD simulations, novel
(ChemBridge database) were selected as InhA inhibitors based on benzoimidazole-
ligand library for this virtual screening. containing derivatives were design based on
ChemBridge database contained 1,300,000 Bioisostere replacement that obtained from
compounds. InhA complexed with diphenyl SwissBioisostere. ADMET prediction using
ether (PDB code: 2X23) was downloaded SwissADME was used to predict the
from Protein databank and used as receptor ADMET properties of new designed
for virtual screening.12 To select the compounds.13
potential hit compounds of InhA inhibitors
with good lead-like properties, ADMET 3. Results and Discussion
prediction using SwissADME was 3.1 Virtual screening of new InhA
employed.13 Lead-likeness and no PAINS inhibitors
violation compounds were selected for In this work, three steps of virtual
molecular docking calculations. Molecular screening were performed to identify novel
docking calculations using GOLD program InhA inhibitors based on benzoimidazole-
with Goldscore was used to predict the containing derivatives. First, substructure
docking score and binging mode of small search based on benzoimidazole core
benzoimidazole-containing molecules in structure was applied to obtain 9,420
InhA binding site. derivatives of benzimidazole-containing
from Chembridge database. To get good
drug properties and good candidate for new
InhA inhibitors, ADMET prediction was InhA inhibitors. In addition, this compound
applied. Using two criteria’s,168 compounds might be easily to modify as high BBB
were achieved based on lead-like properties permeation. Therefore, to obtain the novel
and no PAINS violation. Molecular docking InhA inhibitors based on benzoimidazole-
calculations were applied to predict binding containing derivatives with highly specific
mode and Goldscore of lead-like to InhA binding site, MD simulations of
compounds. Based on Goldscore, the 5928640-InhA complexed was applied to
estimate binding affinity in InhA binding investigate the structural stability and
site, four compounds with Goldscore higher binding mode in InhA binding site.
than 80 were selected as hit compounds
(Table 1). Two series of benzoimidazole-
containing hit compounds, 2-(1H-benzimi
dazol-2-ylthio)acetamide (three compounds)
and (1H-Benzimidazol-2-yl) methyl (one
compound) derivatives were classified.
Based on ADMET predictions as shown in
Figure 2, we found that hit compounds are
high GI absorption (Gastrointestinal
Absorption), except 5914960.
Unfortunately, low blood–brain
barrier (BBB) permeation of all hits were
obtained. 5928640 (2-(1H-benzimidazol-2-
ylthio)-N-(4-methoxyphenyl)acetamide), the Figure 1. Virtual screening workflow
highest Goldscore with good ADMET
prediction was considered as potential hit of
Table 1. Hit compounds based on benzoimidazole-containing derivative

ID Goldscore Structure
5928640 82.90
6533900 81.33
6659051 80.86
5914960 80.04
Figure 2. ADMET prediction of hit Figure 4. Binding mode and binding
compounds interaction of 5928640 in InhA binding site
3.2 Binding mode of InhA-5928640 5928640 is favorable to bind with

complexed InhA binding site via hydrogen bond
To gain more insight into the binding interactions. Three hydrogen bond
characteristic of 5928640 in InhA binding interactions were observed between
site, MD simulations was carried out over 5928640 with NAD+ cofactor and amino
30ns of simulations time as showed in acid residues. Nitrogen atom and NH of
Figure 3. Based on RMSD plotted, the benzoimidazole core formed hydrogen bond
binding mode and binding interaction of interactions with hydroxyl group of Tyr158
5928640 complexed with InhA were derived and an oxygen carbonyl on nicotinamide
from MD simulations as displayed in Figure ring of NAD+, respectively. In addition, an
4. oxygen of methoxyl substituent on phenyl
ring interacted with a NH amide sidechain
of Gln214. The binding affinity of 5928640
was increased due to the pi-pi interaction of
benzoimidazole ring with nicotinamide ring
of NAD+. Hydrophobic interactions between
5928640 with Ala198, Met199, Ile202,
Val203 and Leu218 sidechain were
observed.
3.3 Rational design of 5928640 derivatives
To obtain the novel InhA inhibitors
Figure 3. RMSD plotted of 5928640 based on benzoimidazole-containing
complexed with InhA derivatives, the integrated results from MD
simulations with bioisostere replacement
were considered. The 4-methoxyphenyl of
5928640 was located at hydrophobic
binding site of InhA. This fragment was
Table 2. ADMET prediction of 5928640 derivatives
GI BBB Pgp CYP inhibitors
Molecule TPSA WLOGP
absorption permeant substrate 1A2 2C19 2C9 2D6 3A4
Triclosan 29.46 5.14 High Yes No Yes Yes Yes No No
Diphenyl ether 29.46 6.09 High No No Yes Yes Yes Yes No
D01 83.08 3.13 High No No Yes Yes Yes Yes No
D02 83.08 2.88 High No No Yes Yes No Yes No
D03 83.08 2.55 High No No Yes No No No No
D04 83.08 2.35 High No No Yes No No No No
5928640 92.31 3.11 High No No Yes Yes Yes Yes Yes
selected as replacement region of 5928640. Phanom University, Faculty of Science,
Therefore, 592 compounds of 5928640 Ubon Ratchathani University, Kasetsart
derivatives were designed. Based on University, Blue Crystal 4 (BC4), University
ADMET prediction, 110 compounds with of Bristol, and NECTEC are gratefully
high GI absorption and lead-like properties acknowledged for supporting this research.
were obtained. Four compounds of 5928640
derivatives as shown in Table 2 were good References
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EPSRC Council Global Challenges
Research fund. Faculty of Science, Nakhon
Molecular modeling of InhA inhibitors in the class of benzimidazole
derivatives as a novel anti-tuberculosis agents
Bunrat Tharat1, Bongkochawan Pakanwong1, Paptawan Thongdee1,
Kotchakorn Yotyiamkrae1, Bundit Khamsri1, Thitima Chuansupak1, Naruedon Phusi1,
Chayanin Hanwarinroj1, Kampanart Chayajaras1, Thimpika Pornprom1, Nitima Suttipanta2,
Pharit Kamsri3, Auradee Punkvang3, Patchreenart Saparpakorn4, Supa Hannongbua4,
Prasat Kittakoop5, Khomson Suttisintong6, Adrian Mulholland7, James Spencer8,
Pornpan Pungpo1*
1
2
3
4
5
6
National Nanotechnology Center, NSTDA, 111 Thailand Science Park, Klong Luang, Pathum Thani, Thailand
7
Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol, BS8 1TS, UK
8
School of Cellular and Molecular Medicine, Biomedical Sciences Building, University of Bristol,
Bristol, BS8 1TD, UK
Abstract:
Benzimidazole derivatives are good candidates for anti-tuberculosis agents that
display a promising potency for inhibition of InhA. In this work, molecular docking
calculations combined with bioisosteres were applied to get new potential structures of this
series. This derivative has the same general structure but different in R substituent. Cpd05
was the highest active compound. Cpd32 and Cpd46 shown moderate active compounds.
Cpd55 was less active compound. These compounds were classified based on the predicted
binding affinity. To gain more details of binding interactions of new designed compounds,
molecular dynamic simulations (MD) were performed on the designed inhibitors. The
interactions of Cpd 05, Cpd32, Cpd46 and Cpd55 with amino acid surrounding the InhA
binding pocket were analyzed. The hydrogen bond interactions were found as the crucial
interactions for binding in the InhA binding site. These results lead to the synthesis of new
series of benzimidazole derivatives.
1. Introduction enzyme of the type II fatty acid synthesis

Tuberculosis (TB) is one of the most system. It is involved in the production of
common infectious diseases caused by mycolic acid and is a known target for
Mycobacterium tuberculosis, TB kills two isoniazid, an effective antibiotic for
million people every year and it continues to tuberculosis treatment.2
be a major cause of morbidity and mortality Antibiotics for TB treatment are
all over the world. About one-third of classified into two classes, first-line drugs
the world's population is currently infected and second-line drugs. First-line drugs are
with TB. If the present trend continues, mainly bactericidal and combine a high
tuberculosis is likely to claim more than 30 degree of efficacy with a relatively low
million lives within the next decade. toxicity to the patient during treatment.
Five percent of all TB cases are now These drugs include isoniazid, rifampicin,
estimated to be multi-drug-resistant TB.1 streptomycin, ethambutol, pyrazinamide,
The enoyl-acyl carrier protein reductase of and fluoroquinolones. Second-line drugs
Mycobacterium tuberculosis is a key are mainly bacteriostatic, which have
a lower efficacy and are usually more toxic. 3. Results and Discussion
These drugs include para-aminosalicylic 3.1 Validation of the molecular docking
acid, ethionamide, and cycloserine.3 calculations
Isoniazid (INH), a frontline drug for over Molecular docking calculations were
640 years used in the treatment of TB. As a employed to predict the potential binding
prodrug, INH must be first activated by mode of benzimidazole derivative in the
KatG, a catalase-peroxidase that oxidizes InhA binding pocket. To ensure that the
INH to an acyl-radical, which binds binding modes of InhA inhibitors obtained
covalently to NAD+, the co-substrate for from molecular docking calculations are
InhA. The INH-NAD adduct functions as a reliable, the docking parameters in
potent inhibitor of InhA.4 INH drug resistant Autodock 4.2 program were validated. The
tuberculosis caused by KatG mutation. superimposition between the docked
Hence, there is an urgent need to conformation and the X-ray crystal structure
discover and develop new anti-TB agents of diphenyl ether derivative is shown in
that target novel biochemical pathways and Figure 1. The docked conformation of
to treat drug-resistant forms of the disease. diphenyl ether derivative is close to the
Recently, molecular docking and molecular binding mode found in the X-ray crystal
dynamics simulations have been performed structure with rmsd of 0.61 Å, indicating
to study the binding of benzimidazoles that the docking parameters are reasonable
derivative onto the active site of InhA in an to generate the binding mode of diphenyl
attempt to address the mycobacterial ether derivative in the InhA binding pocket.
resistance against the drug. The integrated Therefore, molecular docking calculations
results should aid in the rational design of could be extended to search the binding
InhA inhibitors with high potential anti- modes of benzimidazole derivatives in the
tubercular activity. data set.

The four chemical structure, docking
score and binding energy of benzimidazole
derivatives were shown in Table 1.The X-
ray structure of InhA enzyme complexed
with diphenyl ether was taken from Protein
Data Bank (PDB code: 2X23). Chemical
structures of these inhibitors were
constructed using the standard tools Figure 1. Superimposition of the X-ray
available in GaussView 5.0.8 program and crystal structure (carbon atoms colored by
were then fully optimized using the ab initio purple) and docked conformation (yellow)
(M062X/6-31G*) method implemented in of diphenyl ether derivative in the InhA
Gaussian program. Molecular docking binding pocket
calculations was applied to obtain the
binding mode of benzimidazoles derivatives
using Autodock 4.2 program. Moreover,
molecular dynamics simulations of Cpd05,
Cpd33, Cpd46 and Cpd55 were performed
for 30 ns to elucidate the binding mode and
binding interactions into the InhA binding
site.
Table 1. The chemical structures, docking score and binding energy of benzimidazoles
derivatives
Compound R Docking score Binding energy

kcal/mol kcal/mol
05 -11.97 -8.78
32 -11.46 -9.31
46 -11.33 -8.40
55 -11.21 -7.47
3.2. Molecular docking analysis of Cpd05, Cpd46 were investigated. The obtained
the highest active compound results show that Cpd32 formed hydrogen
Cpd05, the highest active compound bond interaction between oxygen atom and
from docking score (-11.97 kcal/mol) was nitrogen atom of nicotinamide ribose
selected to analyze the crucial interactions of cofactor (NAD+) with hydroxyl group of
benzimidazole derivative in InhA binding ligand. In addition, hydrogen bond
site. The obtained results show that Cpd05 interactions between oxygen atoms of NAD+
formed hydrogen bond interaction between with hydrogen atom of ligand at R position
hydroxyl groups of ligand with oxygen atom are shown in Figure 3. Moreover,
of nicotinamide ribose cofactor (NAD+). In hydrophobic interactions between hydrogen
addition, cation- interactions between atom of ligand with hydrogen atom of
aromatic ring of Cpd05 with hydrogen atom Phe97, Met98, Met103, Ala157, Tyr158,
of Lys165 residue and sigma-Pi interaction Pro193, Ala198, Val203 and Leu218
between aromatics ring of ligand with residues were founded.
hydrogen atom of Met161 residue is shown For Cpd46, hydrogen bond
in Figure 2. Moreover, hydrophobic interactions between oxygen atom of NAD+
interaction between hydrogen atom of ligand with hydrogen atom of ligand at R position
with hydrogen atom of Phe97, Met98, and hydrogen bond interactions between
Met103, Ala157, Tyr158, Pro193, Val203 oxygen atom of NAD+ with hydroxyl group
and Leu218 residues were founded. of ligand were found as crucial interaction
3.3. Molecular docking analysis of Cpd32 for binding in InhA binding site. In addition,
and Cpd46, moderate active compounds Cation-Pi interactions between aromatic ring
The binding mode and binding of ligand with atom hydrogen of Lys165
interactions of moderate InhA inhibitors of residue and - interaction between
benzimidazole derivatives, Cpd32 and aromatic ring of ligand with hydrogen atom
of Met161 residue is shown in Figure 4. 3.4. Molecular docking analysis of Cpd55,
Moreover, hydrophobic interactions between the less active compound
hydrogen atom of ligand with hydrogen The lowest docking score (-11.21
atom of Phe97, Met98, Met103, Phe149, kcal/mol) was classified as the less active
Tyr158, Pro193, Ala198, Ile202, Val203 and compound of Cpd55. The obtained results
Leu218 residues were founded. show that Cpd55 formed hydrogen bond
interactions between hydroxyl group on R
substitute of ligand with Tyr158 and Pro156
residues. In addition, hydrogen bond
interactions between oxygen atoms of NAD+
with hydrogen atom of ligand were found as
shown in Figure 5. Moreover, hydrophobic
interactions between hydrogen atom of
ligand with hydrogen atom of Phe97, Met98,
Met103, Tyr158, Ile202, Val203 and
Leu218 residues were founded.
Figure 2. The binding mode of Cpd05 in

InhA binding pocket obtained from
molecular docking calculations

InhA binding pocket obtained from
molecular docking calculations.
3.5. System equilibration from molecular
Figure 3. The binding mode of Cpd32 in dynamics simulations
InhA binding pocket obtained from MD simulations of Cpd05, Cpd32,
molecular docking calculations Cpd46, and Cpd55 bound with InhA were
performed for 30 ns to evaluate the
structural stability of the complexes and
their binding strength. The RMSDs for all
atoms of three different solute species
(InhA, NAD+, and ligand) over the 30 ns of
simulation times were analyzed and plotted
in Figure 6. The plateau characteristic of the
RMSD plot over the simulation time is the
criteria to indicate the equilibrium state of
each solute species. Figure 6 showed that
NAD+ reaches the equilibrium state at the
early time. However, RMSDs of all
compounds are more fluctuated, particularly
Figure 4. The binding mode of Cpd46 in Cpd32. For Cpd05 and Cpd46 reach the
InhA binding pocket obtained from equilibrium state after 5 ns (Figure 6a and
molecular docking calculations 6c) Cpd32 reach the equilibrium state after
15 ns (Figure 6b) and Cpd55 reach the with hydrogen atom of ligand (Figure 7e).
equilibrium state after 15 ns. (Figure 6d) Moreover, hydrophobic interactions between
3.6. The binding modes of benzimidazole hydrogen atom of ligand Cpd05 with
derivatives in InhA hydrogen atom of Met199, Ile202 and
The binding modes of Cpd05, Leu207 residues, Cpd32 was found
Cpd32, Cpd46 and Cpd55 bound with InhA hydrogen bond interaction between oxygen
pocket observed from the simulations are atoms of Met98 residue with hydroxyl group
illustrated in Figure 7. Cpd05 was found of ligand. In addition, hydrogen bond
hydrogen bond interaction between interactions between oxygen atoms of
hydrogen atom of Tyr158 residue with NAD+ with hydrogen atom of ligand and
nitrogen atom of ligand. In addition, Cation-Pi interaction between aromatic ring
hydrogen bond interactions between oxygen of ligand with nitrogen atom of Gly98
atoms of NAD+ residue (Figure 7f).
(a) (b)
(c) (d)
Figure 6. RMSD plots of compounds 05 (a), 32 (b), 46 (c), and 55 (d) complexes with InhA
In addition, hydrophobic interactions hydrogen atom of Phe149 and Ala198

between hydrogen atom of ligand with residues, Cpd55 was found hydrogen bond
hydrogen atom of Ala1 57 , Met9 8, Met1 0 3 interaction between hydrogen atoms of
and Met1 6 1 residues were founded, For Phe149 residue with oxygen atom at
Cpd46 Hydrogen bond interaction between hydroxyl group of ligand. In addition,
hydrogen atoms of Pro193 residue with hydrogen bond interactions between oxygen
hydroxyl group of ligand was found. In atoms of NAD+ with hydrogen atom of
addition, hydrogen bond interactions ligand. ( Figure 7 h) Moreover, hydrophobic
between hydrogen atoms of NAD+ with interactions between hydrogen atom of
nitrogen atom of ligand. The - interaction ligand with hydrogen atom of Met155,
between aromatic ring of ligand with Leu207, Met199 and Ile202 residues were
aromatic ring of NAD+ was founded (Figure founded.
7g). Moreover, hydrophobic interactions
between hydrogen atom of ligand with
3
4. Conclusion as the crucial interactions for binding.

Molecular docking calculations and Moreover, the hydrophobic interaction, Pi-Pi
molecular dynamics simulations were interaction, cation- interaction, -
performed on a series of benzimidazole interaction, and hydrophilic interactions of
derivatives to achieve a better understanding ligands with amino acid in InhA binding
of the crucial interactions for binding pocket were observed. This information
affinity of these derivatives in the InhA leads to the design benzimidazole
binding pocket. The obtained results show derivatives with effective in inhibiting InhA.
the hydrogen bond interactions were found
(e) (f)
(g) (h)
Figure 7. The binding mode of Cpd05 (e), Cpd32 (f), Cpd46 (g) and Cpd55 (h) in InhA
binding pocket obtained from molecular dynamics simulations
This research was supported by the 1. Theivendren, P.; Arumugam, S.;
Thailand Research Fund (RSA5980057), Subramanian A.; Shrinivas, D. J. Drug.
RGJ Advanced Programme (RAP60K0009) Discov. Ther. 2016, 10, 188–194.
and the National Research Council of 2. Yuya, I.; Shiho, A.; Yuji, K.; Masato,
Thailand. The financial support from Young K.; Shunsuke, A. Eur. J. Med. Chem.
Scientist and Technologist Program (YSTP) 2011, 46, 1849–1856.
(SCA-CO-2560-4687-TH) to B. Tharat is 3. Auradee, P.; Patchreenart, S.; Supa, H.;
gratefully acknowledged. Faculty of Peter, W.; Pornpan, P. Molecules 2010,
Science, Ubon Ratchathani University, 15, 2791–2813.
NANOTEC, Blue Crystal 4 (BC4), 4. Shrinivas, D.; J.; Uttam, A.; M.;
University of Bristol and NECTEC are Deepshikha, K.; Manoj, S.; K.;
gratefully acknowledged for supporting this Mallikarjuna, N.; N.; Tejraj, M.; A.
research. Bioorg. Chem. 2015, 59, 151–1670.
Key structural basis of nitrothiazolyl carboxamide analogues as DNA
gyrase subunit B inhibitors for anti-tuberculosis agents:
molecular docking calculations
Thitima Chuansupak1, Bongkochawan Pakamwong1, Paptawan Thongdee1,
Kotchakorn Yotyiamkrae1, Bunrat Tharat1, Bundit Khamsri1, Naruedon Phusi1,
Chayanin Hanwarinroj1, Nitima Suttipanta2, Pharit Kamsri3, Auradee Punkvang3,
Patchreenart Saparpakorn4, Supa Hannongbua4, Prasat Kittakoop5, Khomson Suttisintong6,
Ubolsree Leartsakulpanich7, Adrian Mulholland8, James Spencer9, Pornpan Pungpo1*
1
2
Faculty of Pharmaceutical Science, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand
3
4
5
6
National Nanotechnology Center, NSTDA, 111 Thailand Science Park,
7
National Center for Genetic Engineering and Biotechnology (BIOTEC), NSTDA, 111 Thailand Science Park,
8
9
Bristol BS8 1TD, UK
Abstract:
DNA gyrase has been identified as an attractive target for anti-tuberculosis agents.
This enzyme is the target of fluoroquinolone that can inhibit M. tuberculosis. However, these
drugs fail to inhibit mycobacterium due to mutation of fluoroquinolone binding pocket.
Therefore, new inhibitors which can inhibit DNA gyrase at ATPase binding site have been
developed. Herein, molecular docking calculations were applied to investigate the key
structural basis for DNA gyrase subunit B inhibitions in a series of nitrothiazolyl
carboxamide derivatives. The results demonstrate that that hydrogen bond interaction
between ligand and Thr162 residues is the crucial interaction of this analog. This leads to a
better understanding of the key structural basis of nitrothiazolyl carboxamide derivatives to
design novel potential DNA gyrase inhibitors.
1. Introduction have made quite a successful progress of the

Tuberculosis (TB) is caused by TB drug discovery pipeline, with
mycobacteria namely Mycobacterium gatifloxacin and moxifloxacin currently
tuberculosis (MTB) that most often affect undergoing phase III clinical trials for the
the lungs. TB is curable and preventable. treatment of TB. In MTB, DNA gyrase is
Also, it is critical that a good supply of new the sole type II topoisomerase that ensures
chemical entities are forthcoming in order to the regulation of DNA topology and has
overcome the high rates of attrition been genetically demonstrated to be a
encountered with the drug discovery bactericidal drug target. The clinical success
process. DNA gyrase is an essential enzyme of fluoroquinolones had not only validated
that introduces negative supercoils into the durability of DNA gyrase as a potential
DNA and regulates the super-helical state of anti-tubercular target but has also shown that
the bacterial chromosomes. Quinoline drugs inhibitors of this enzyme could also be
preferably targeted the GyrA subunit and active against non-replicating, persistent
mycobacteria, and might reduce the duration Table 1. In vitro biological evolution of the
of TB therapy. The ATPase activity of synthesized compounds.
bacterial DNA gyrase that resides in the
GyrB subunit remains pharmaceutically
under-exploited even though it is an
Compound R GyrB assay
effective target of coumarins and related (IC50)(μM)
compounds. GyrB has been genetically
demonstrated to be a bactericidal drug target 44 0.1496
for MTB but there has not been any
effective therapeutics developed against this
target for TB1-2. This work, we aim to 22 2.6
better understand the binding mode and key
F
interaction for binding of nitrothiazolyl 49 5.4
O
carboxamide derivative. The obtained
43 9.25
results from this study will be guide us for
rational design new and more potent GyrB
inhibitors as anti-TB agents.
46 O CH
3
17.42
Structures and biological activities of 57 52.9
S
selected compounds of nitrothiazolyl O O
carboxamide derivatives were summarized
in Table 1.1,2 All derivatives were
constructed by standard tools in Gaussview
5.0.8 program and then fully optimized by
M062X/6-31G* method using Gaussian 09
program. Molecular docking calculations
using the Autodock 4.2 program was
performed to predict the potent inhibitors in
the GyrB binding pocket.
3. Results and Discussion Figure 1. Superimposition of the X-ray

3.1 Molecular docking calculations crystal structure (blue) and docked
The results from molecular docking conformation (green) of the 4B6C adduct in
calculations indicate that the molecular the GyrB binding pocket
docking calculations are reliable to predict
the binding mode. The binding interactions mode and interaction of the highest active
with RMSD of 1 angstrom are shown in compound (compound 44) as shown in
Figure 1. Therefore, the nitrothiazolyl Figure 2 displays that the molecule forms
carboxamide derivatives were docked into cation- interaction with primary amine
GyrB binding site by molecular docking group of Arg75 and Arg433. Sigma-Pi
approache with the same docking parameter interaction with Asn122 is also observed.
with docking calculations of X-ray crystal There are four hydrogen bond interactions
ligand. between compound 44 and amino acid
3.2 Binding mode of the highest active residues surrounding their binding pocket.
compounds First hydrogen bond interaction is between
The docking studies of these primary amide and the carbonyl group of the
compounds reveal that some important Asn45. The second and third hydrogen bond
differences among their binding patterns at interactions are the nitrogen atom of nitro
the GyrB active-site are observed. A binding group with Arg75 and Gly76. The fourth
hydrogen bond is p-fluoro benzyl group
interacted with the amino group of Asn122.
Figure 4. Binding modes and crucial

interactions of moderate activity compound
22
Figure 2. The binding mode and crucial
interactions of highest active compound 44 Figure 5 shows binding modes and
crucial interactions of moderate activity
3.3 The binding mode of moderate compound (compound 49). This compound
compounds formed a - interaction between the
Figure 3 shows binding modes and benzene ring with Tyr143 sidechain. Arg75
crucial interactions of moderate activity and Arg433 sidechains form electrostatic
compound (compound 43). There is a and cation-Pi interactions with nitro
cation interaction between the primary functional group and thiazole ring,
amine group of Arg75 residue with phenyl respectively. In addition, fluorine atom on
ring. The hydrogen atom of NH group phenyl ring interacts with NH of Asn122
formed the hydrogen bond interaction with sidechain via hydrogen bond interaction.
Asp72. The nitro functional group on
thiazole ring interacts with a hydrogen atom
of Asn122.
Figure 5. Binding modes and crucial

interactions of moderate activity compound
Figure 3. Binding modes and crucial 49
interactions of compound 43
3.4 The binding mode of low active
Figure 4 shows binding modes and
compounds
crucial interactions of moderate activity Figure 6 shows binding modes and
compound 22. There is a cation crucial interactions of low active compound
interaction formed between Arg433 side 46 (compoumd 46). An interaction between
chain and thiazole ring. Arg75 sidechain the primary amine group of Arg75 residue
forms electrostatic interaction with the nitro
and phenyl ring occurs via a cation-
functional group.
interaction. Hydrogen bondings are formed
between NH and Asp72 and the oxygen
atom of the nitro functional group and the 4. Conclusion
NH sidechain of Asn122. The binding mode and binding
interactions of a series of compounds of
nitrothiazolyl carboxamide in the GyrB
binding site were investigated by molecular
docking calculations. Hydrogen bond
interactions, - interaction and cation-
interaction are crucial for binding of
nitrothiazolyl carboxamide in the GyrB
binding pocket.
Acknowledgements
This research was supported by the
Figure 6. Binding modes of low active Health Systems Research Institute
compound 46 (HSRI.60.083), the National Research
Council of Thailand and the Center of
Figure 7 shows binding modes and Excellence for Innovation in Chemistry
crucial interactions of compound 57. There (PERCH-CIC). The Faculty of Science,
is a interaction between the hydrogen Ubon Ratchathani University, Faculty of
atom of the piperidine ring and His82 Science, Kasetsart University, NANOTEC,
sidechain. Hydrogen bondings are formed and NECTEC are gratefully acknowledged
between the nitrogen atom of thiazole ring for supporting this research.
and the NH group of His82 sidechain and
between sulphanilamide and an oxygen References
atom of Tyr149. Additional interaction 1. Jeankumar, V. U.; Renuka, J.; Kotagiri,
between the nitro functional group and S.; Saxena, S.; Kakan, S. S.; Sridevi, J.
Arg433 occurs via electrostatics. P.; Yellanki, S.; Kulkarni, P.;
Yogeeswari, P.; Sriram, D. ChemMed
Chem 2014, 9, 1850–1859.
2. Jeankumar, V. U.; Kotagiri, S.;
Janupally, R.; Suryadevara, P.; Sridevi,
J. P.; Medishetti, R.; Kulkarni, P.;
Yogeeswari, P.; Sriram, D. J.
Bioorganic Med. Chem. 2015, 23, 588–
601.
Figure 7. Binding modes of low active

compound 57
Structure based drug design of 2-(1H-benzo[d]imidazol-2-yl)-2-cyanovinyl
as DNA gyrase subunit B inhibitors for anti-tuberculosis agents
Kotchakorn Yotyiamkrae1, Bongkochawan Pakanwong1, Paptawan Thongdee1,
Bundit Khamsri1, Thitima Chuansupak1, Naruedon Phusi1, Chayanin Hanwarinroj1,
Thimpika Pornprom1, Pharit Kamsri2, Auradee Punkvang2, Patchreenart Saparpakorn3,
Supa Hannongbua3, Prasat Kittakoop4, Khomson Suttisintong5, Nitima Suttipanta8,
Adrian Mulholland6, James Spencer7, Pornpan Pungpo1*
1
2
3
4
5
6
Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, UK
7
Bristol BS8 1TD, UK
8
Department of Pharmaceutical, Faculty of Science, Ubon Ratchathani University,
Abstract:
This work, we aim to investigate the key interactions of 2-(1H-benzo[d]imidazol-2-
yl)-2-cyanovinyl derivatives in ATPase domain of DNA gyrase (GyrB) using molecular
docking calculations. The obtained results showed that hydrogen bond interactions are the
important for binding of these inhibitors. The most effective compounds showed hydrogen
bond interactions with Asn45, Thr94 and Ser119 with 2-(1H-benzo[d]imidazol-2-yl)-2-
cyanovinyl scaffold. Phenyl thiophene structure formed hydrogen bond interactions with
Ile89, Asp90 and Val92. Hydrophobic interactions could improve the binding affinity of
these inhibitors. In addition, the effect of substituents on the binding interactions and
biological activity were investigated. Therefore, the obtained results in this work aid to
rational design of 2-(1H-benzo[d]imidazol-2-yl)-2-cyanovinyl derivatives as highly effective
DNA gyrase subunit B inhibitors.
1. Introduction introduces negative supercoils into DNA. In

Tuberculosis (TB) is contagious, but bacteria, topoisomerase II consists of two
it’s not easier to catch. The chances of polypeptide subunits, GyrA and GyrB,
catching TB from someone you live or work which form a heterotetramer: (A2B2).2 In
with are much higher than from a stranger. most eukaryotes, topoisomerase II consists
Most people with active TB who have of a single polypeptide, where the N- and C-
received appropriate treatment for at least terminal regions correspond to GyrB and
two weeks are no longer contagious.1 GyrA, respectively.3
DNA topoisomerases regulate the Since antibiotics began to be used to
number of topological links between two fight TB, some strains have become resistant
DNA strands (i.e. change the number of to drugs. Multidrug-resistant TB (MDR-TB)
superhelical turns) by catalysing transient arises when an antibiotic fails to kill all of
single- or double-strand breaks, crossing the the bacteria, with the surviving bacteria
strands through one another, and then developing resistance to that antibiotic and
resealing the breaks. Topoisomerase II often others at the same time.3 The field of
(called gyrase in bacteria) primarily structure-based drug design is a rapidly
growing area in which many successes have are trusty in predicting binding mode and
occurred in recent years. The breaking of the superimposition between the docked
proteomic and structural information has conformation and the X-ray crystal structure
provided hundreds of new targets and with RMSD at 1.00 angstrom as shown in
opportunities for future drug discovery. In Figure 1.
summarizes the process of structure-based
drug design and includes, primarily, the
choice of a target, the evaluation of a
structure of that target, the pivotal questions
to consider in choosing a method for drug
discovery, and evaluation of the drug leads.
Key principles in the field of structure-based
drug design will be illustrated through a case
study that explores drug design for DNA
gyrase subunit B.4

The structure and biological activity
of seven selected 2-(1H-benzo[d]imidazol-
2-yl)-2-cyanovinyl derivative were
summarized in Table 1.2 The first step is all
compound constructed using GAUSSVIEW
5.0.8 program then optimized at the
M062X/6-31G* level using the GAUSSIAN
09 program. The 3D structure of the receptor
should be considered which can be
downloaded from Protein Data Bank (PDB Figure 1. Superimposition of the X-ray
code: 4B6C). Molecular docking crystal structure (blue) and docked
calculations were applied as selected gadget conformation (purple) of the 4B6C in the
to obtain new structures of 2-(1H- Gyrase B binding pocket
benzo[d]imidazol-2-yl)-2-cyanovinyl
derivatives and using Autodock 4.2 program 3.2. Molecular docking analysis of Cpd22
was performed to predict the binding modes and Cpd16, high active compounds
and binding interactions in DNA gyrase The results show interaction of
subunit B binding pockets. Cpd22 and Cpd16 were find out more
interaction. For Cpd22, show hydrogen
3. Results and Discussion bond interaction between 1H-
3.1 Validations of the molecular docking benzo[d]imidazole group with Asn45,
calculations Asp90, Thr94 and Ser119. In addition, the
Molecular docking calculations were hydrogen bond interaction between
employed to predict the potential binding thiophene ring and benzene ring of ligand
mode of 2-(1H-benzo[d]imidazol-2-yl)-2- with Ile89, Asp89 and Val92 as show in
cyanovinyl derivative adducts in DNA Figure 2. Moreover, the results show more
gyrase subunit B binding pocket. To ensure hydrophobic interaction between hydrogen
that the binding modes of adduct atom of ligand with hydrogen atom of amino
inhibitors obtained from molecular docking acid, Val42, Asn45, Thr94, Ser119, Val121,
calculations are reliable the docking Asn122, Leu128, Ile89, Asp90, Val91 and
parameters in Autodock 4.2 program were Val92 were observed.
validated. Molecular docking calculations
Table 1. The chemical structures of 2-(1H-benzo[d]imidazol-2-yl)-2-cyanovinyl
Name R X R1 R2 R3 IC50 (µM)

Cpd4 H S COOH H H 1.8
Cpd10 Cl S COOH H H 5.9
Cpd16 NO2 S COOH H H 0.7
Cpd19 CH3 O COOH H H 1.0
Cpd22 CH3 S COOH H H 0.3
Cpd23 CH3 S H COOH H 1.1
Cpd24 CH3 S COOH H COOH 0.9
For Cpd16, the results were found

hydrogen bond interactions between 1H-
benzo[d]imidazole group with Ser119,
Val121, Asn122, Leu128 and Ile164. The
thiophene ring and benzene ring founded
hydrogen bond interaction with Asp90 and
Thr94 as show in Figure 3. Moreover, the
results show more hydrophobic interaction
between hydrogen atom of ligand with
hydrogen atom of amino acid, Ile89, Asp90
and Asn45 were observed.
Figure 2. The binding mode of Cpd22 in 3.3. Molecular docking analysis of Cpd19
DNA gyrase subunit B binding pocket Cpd23 and Cpd24, moderate active
obtained from molecular docking compounds
calculations The results show interaction of
Cpd19 was found hydrogen bond interaction
between 1H-benzo[d]imidazole group with
Glu49, Asp72, Gly76, Ile77, Pro78, Gln436
and Glu437 as show in Figure 4. Moreover,
the results show more hydrophobic
interaction between hydrogen of ligand with
hydrogen of Ala46, Glu49, Asp72, Gly76,
Ile77, Pro78, Thr162, Ile164, Gln436,
Glu437, Phe440 and Arg75 were observed.
For Cpd23, the obtained results
show hydrogen bond interactions between
1H-benzo[d]imidazole group with Val92,
Thr94 and Ser119. The thiophene ring and
benzene ring were hydrogen bond
DNA gyrase subunit B binding pocket
interaction with Asp90 and Val92 as show
obtained from molecular docking
in Figure 5. Moreover, the results show
calculations
more hydrophobic interaction between
hydrogen atom of ligand with hydrogen
atom of amino acid, Val42, Val92, Val121, 1H-benzo[d]imidazole group with Thr94
Leu128, Ile164, Ile89 and Asp90 were and Ser119. The thiophene ring and benzene
observed. ring were found hydrogen bond interaction
with Gln436 and Asp90 as shown in Figure
6. Moreover, the results showed that
hydrophobic interaction between hydrogen
atom of ligand with hydrogen atom of amino
acid, Asn45, Val121, Asn122, Leu128 and
Asp90 were observed.
3.4. Molecular docking analysis of Cpd4
and Cpd10, low active compounds
The results show interaction of Cpd4
Figure 4. The binding mode of Cpd19 in was found hydrogen bond interactions
DNA gyrase subunit B binding pocket between 1H-benzo[d]imidazole group with
obtained from molecular docking Ser119. The thiophene ring and benzene
calculations ring were found hydrogen bond with Ile89,
Asp90, Val92 and Thr94 as show in Figure
7. Moreover, the results show more
hydrophobic interaction between hydrogen
atom of ligand with hydrogen atom of amino
acid, Val42, Val121, Ile89, Asp90, Val92
and Thr94 were observed.
showed hydrogen bond interactions between
1H-benzo[d]imidazole group with Val121
and Leu128. The thiophene ring and
Figure 5. The binding mode of Cpd23 in benzene ring were found hydrogen bond
DNA gyrase subunit B binding pocket interaction with Asp90, Val92 and Thr94 as
obtained from molecular docking show in Figure 8. Moreover, the results
calculations show more hydrophobic interaction between
hydrogen atom of ligand with hydrogen
atom of amino acid, Asn45, Ile89, Asp90,
Val92 and Thr94 were observed.

DNA gyrase subunit B binding pocket
Figure 6. The binding mode of Cpd24 in obtained from molecular docking
DNA gyrase subunit B binding pocket calculations
obtained from molecular docking
calculations

show hydrogen bond interactions between
Acknowledgements
Health Systems Research Institute
(HSRI.60.083), the National Research
(PERCH-CIC). The Faculty of Science,
Ubon Ratchathani University, Faculty of
Science, Kasetsart University, NANOTEC,
and NECTEC are gratefully acknowledged
for supporting this research.
DNA gyrase subunit B binding pocket References
obtained from molecular docking 1. Lewin C. S.; Howard M. A.; Smith J. T.
calculations J. Med. Microbiol. 1991, 35, 358-362.
2. Variam. U. J.; Shalini S.; Rahul V.;
4. Conclusion Rudraraju S. R.; Renuka J.; Pushkar K.;
Molecular docking calculation was Perumal Y.; Dharmarajan, S.
successfully to predict the binding mode ChemMedChem. 2016, 11, 539-548.
and binding interactions of 2-(1H-benzo[d] 3. http://www.ebi.ac.uk/interpro/entry/IPR
imidazol-2-yl)-2-cyanovinyl derivatives in 011557?q=DNA%20gyrase (accessed
DNA gyrase subunit B binding pocket. data 29/01/2018)
Hydrogen bond interaction and hydrophobic 4. http://www.ebi.ac.uk/interpro/search?q=
interaction between 1H-benzo[d]imidazole DNA+gyrase&submit1=1 (accessed data
group, thiophene ring amino acid residue 29/01/2018)
surrounding in DNA gyrase subunit B
binding pocket were founded. The obtained
results aid to rational design novel DNA
gyrase subunit B inhibitors as anti-
tuberculosis agents.
Elucidating structural basis of 4-aminoquinoline derivatives as novel
M. tuberculosis DNA GyrB inhibiters: molecular docking study
Bandit Khamsri1, Bongkochawan Pakamwong1, Bunrat Tharat1, Kotchakorn Yotyiamkrae1,
Paptawan Thongdee1, Thitima Chuansupak1, Naruedon Phusi1, Chayanin Hanwarinroj1,
Kampanart Chayajarus1, Pharit Kamsri2, Auradee Punkvang2, Nitima Suttipanta3,
Patchreenart Saparpakorn4, Supa Hannongbua4, Khomson Suttisintong5, Prasat Kittakoop6,
James Spencer7, Adrian Mulholland8, Pornpan Pungpo1*
1
2
3
4
5
6
7
Bristol BS8 1TD, UK
8
Abstract:
Tuberculosis is caused by Mycobacterium tuberculosis (M. tuberculosis) infection
which still remains serious health problem of death worldwide. With an increase in the
emergence of multidrug resistant M. tuberculosis and extensively drug resistant tuberculosis
(XDR-TB) there is an urgent need to develop new anti-mycobacterial agents. Herein, we
applied molecular docking study to investigate the key structural basis of DNA GryB
inhibitors. Molecular docking calculations using Autodock 4.2 program were used to study the
binding interactions between 2-methyl-4-(1-piperidyl)quinoline derivatives and DNA GyrB
binding site. The crucial interactions of these inhibitors in DNA GyrB binding pocket are van
der Waals interactions, hydrogen bond interactions,  - and -cation interactions with DNA
GyrB. Therefore, our results obtained from molecular docking study aid to better understanding
of structural basis 2-methyl-4-(1-piperidyl)quinoline derivatives for rational design with more
potent DNA GyrB inhibitors as potential anti-tuberculosis agents.
1. Introduction program. Second-line drugs are mainly

Tuberculosis is caused by M. bacteriostatic, which have a lower efficacy
tuberculosis. In 2016, it was estimated by the and usually more toxic than first-line drugs
World Health Organization that one-third of including ciprofloxacin, para-aminosalicylic
the world population was infected with latent acid, ethionamide, and cycloserine.2 Drug
tuberculosis, approximately 10.4 million resistant tuberculosis is a significant and
people fell ill with tuberculosis, and 1.7 growing public health threat. Consequently,
million died of tuberculosis.1 Drugs the design of more potent anti-tuberculosis
resistances for tuberculosis were classified drugs for the management of drug-resistant
into two classes, first-line drugs and second- tuberculosis is imperative. DNA GyrB
line drugs. First line drugs include isoniazid, (topoisomerase type II) of M. tuberculosis
rifampicin, streptomycin, ethambutol, and can be an attractive target in this prospect due
pyrazinamide. These are the tuberculosis to the uniqueness of the M. tuberculosis
drugs that generally have the greatest activity genome which codes for only two types of
against tuberculosis bacteria and they are topoisomerases (type I and II) unlike other
core to any tuberculosis drug treatment pathogens. DNA GyrB, a crucial enzyme,
causes negative supercoiling of DNA which crystal structure with RMSD of 1 Å,
relieves strain during the DNA unwinding. indicating that the docking parameters are
Functional DNA GyrB usually exists as a reasonable to generate the binding mode of 4-
heterotetramer with two A subunits and two aminoquinoline derivatives in the DNA GyrB
B subunits. Fluoroquinolones which target binding pocket.
DNA GyrA subunit have been facing a major X
hurdle of their resistance developed by M. HN N

R
tuberculosis which makes DNA GyrB H
subunit a drug able target for discovery of

potent anti-tubercular agents. DNA GyrB N
subunit is involved in the process of ATP R1
hydrolysis which in turn provides energy to
DNA GyrA subunit for maintaining the DNA N
topological state.3,4
Several compounds have been 1-24 X=O
reported as DNA GyrB inhibitors which 25-48 X=S
include benzimidazole ureas, pyrazinamides Figure 1. The general chemical structures of
and triazolopyridine ureas.4 In the present 4-aminoquinoline derivatives
study, structural optimization of the reported
lead, aminopyrazinamide derivative by 3. Results and Discussion
Computer Aided Drug Design (CADD) Molecular docking calculations were
approaches including docking calculations used to study the crucial binding interactions
would be applied to 4-aminoquinoline between 4-aminoquinoline derivatives. 48
derivatives of DNA GyrB inhibitors. compounds were docked to the DNA GyrB
Molecular docking calculations were domain of M. tuberculosis. The obtain results
performed to investigate the crucial binding are compared with the biological activities
interactions of DNA GyrB inhibitors. from literatures3. The results of molecular
Moreover, the obtained results should be docking calculation is showed as follows.
provide the new concept for designing new 3.1 Molecular docking calculations
compounds effective against M. tuberculosis The binding mode of highest active
with highly potential and low toxic anti- compound 33 (IC50 0.62 µM) in DNA GyrB
tuberculosis activity. binding pocket is presented in Figure 2. The
crucial binding interactions were found
2. Materials and Methods hydrogen bond interaction between N of
Structures and biological activities of amine group with H atom of Gly76 and
4-aminoquinoline derivatives inhibitors Ser119 residues, H atom of methyl group
against M. tuberculosis were taken from with Asp72 residue, H atom of amine group
literatures.3 The general structure of with O atom of Asn45, Val118 and Tyr143
4-aminoquinoline derivatives is shown in residues. Moreover, - and -cation of
Figure 1. All Structures and biological aromatic ring with Pro78 and Arg433
activities were presented in Table 1. The X- residues were found. The binding mode of
ray crystallographic structure of DNA GyrB less active compound 37 (IC50 39.44 µM) is
is obtained from the Protein Data Bank (PDB showed in Figure 3. The crucial binding
code 4B6C). The molecular docking interactions were found hydrogen bond
calculations were performed to find crucial interaction between N atom of amine with H
interactions and binding modes of 4- atom of Ile77, Pro78 and Ile164 residues, H
aminoquinoline derivatives in DNA GyrB atom of methyl group with O atom of Gly76
binding pocket using Autodock 4.2 program. and Ser119 residues. - and -cation of
The docked conformation of 4-amino- aromatic ring with Pro78 and Arg433
quinoline derivatives was found in the X-ray residues, - of phenyl groub with Asn122
Table 1. The chemical structures and biological activity of 4-aminoquinoline derivatives
Compound R1 R MTB (IC50)(µM)
1 H Phenyl 9.79
2 H 4-Fluorophenyl 19.74
3 H 4-Chlorophenyl 8.20
4 H 4-Nitrophenyl 10.63
5 H p-Tolyl 14.82
6 H 4-Methoxyphenyl 18.54
7 OCH3 Phenyl 9.32
8 OCH3 4-Fluorophenyl 14.80
9 OCH3 4-Chlorophenyl 14.33
10 OCH3 4-Nitrophenyl 5.33
11 OCH3 p-Tolyl 8.63
12 OCH3 4-Methox phenyl 5.86
13 F Phenyl 9.84
14 F 4-Fluorophenyl 0.78
15 F 4-Chlorophenyl 29.63
16 F 4-Nitrophenyl 2.14
17 F p-Tolyl 8.83
18 F 4-Methoxyphenyl 7.62
19 CF3 Phenyl 8.30
20 CF3 4-Fluorophenyl 11.80
21 CF3 4-Chlorophenyl 17.30
22 CF3 4-Nitrophenyl 22.30
23 CF3 p-Tolyl 9.02
24 CF3 4-Methoxyphenyl 19.80
25 H Phenyl 9.40
26 H 4-Fluorophenyl 16.72
27 H 4-Chlorophenyl 8.12
28 H 4-Nitrophenyl 1.21
29 H p-Tolyl 2.66
30 H 4-Methoxyphenyl 13.51
31 OCH3 Phenyl 0.91
32 OCH3 4-Fluorophenyl 2.31
33 OCH3 4-Chlorophenyl 0.62
34 OCH3 4-Nitrophenyl 0.88
35 OCH3 p-Tolyl 7.94
36 OCH3 4-Methoxyphenyl 6.17
37 F Phenyl 39.44
38 F 4-Fluorophenyl 23.54
39 F 4-Chlorophenyl 21.50
40 F 4-Nitrophenyl 25.50
41 F p-Tolyl 25.41
42 F 4-Methoxyphenyl 17.84
43 CF3 Phenyl 29.40
44 CF3 4-Fluorophenyl 22.84
45 CF3 4-Chlorophenyl 4.12
46 CF3 4-Nitrophenyl 7.54
47 CF3 p-Tolyl 5.10
48 CF3 4-Methoxyphenyl 34.50
residue were found. Finally, the binding H atom of methyl group with O atom of
modes of moderate active compound 39 (IC50 Asp72 residue, H atom of methyl group with
21.50 µM) is showed in Figure 4. The crucial N atom of Gly76 residue, were found. - of
binding interactions including hydrogen aromatic ring with Pro78 residue and
bond interaction between N atom of amine −cation of aromatic ring with Arg134 and
with H atom of Gly76 residue, H atom of Arg433 residues were observed. The
amine group with O atom of Val118 residue, obtained results clearly reveal the crucial
interactions for binding in DNA GyrB
binding pocket of 4-aminoquinoline
derivatives.
Figure 4. The crucial interaction of medium

active compound 39 in the binding pocket of
DNA GyrB of M.tuberculosis
Figure 2. The crucial interaction of the 3.2 Effect of R position from molecular
highest active compound 33 in the binding docking calculations
pocket of DNA GyrB of M.tuberculosis R position of 4-fluorophenyl group
(Compound 14) is compared with R position
of 4-chlorophenyl group (Compound 15). All
the results were shown in Figure 5. The
crucial binding interactions of compound 14
(IC50 0.78 µM) including hydrogen bond
interaction between H atom of aromatic with
N atom of Gly76 and Arg134 residues, H
atom of aromatic with O atom of Asp72
residue, oxygen group with His82 and
Val118 residues were observed. Moreover,
Pi-Cation of aromatic ring with Arg75 and
Arg433 residues were found. For the crucial
binding interactions of compound 15 (IC50
29.63 µM), hydrogen bond interaction
between N atom of amine with H atom of
Val121 and Thr162 residues, oxygen group
with H atom of Gly76 and Thr162 residues.
Figure 3. The crucial interaction of the - and -Cation of 4-chlorophenyl group
lowest active compound 37,in the binding with Pro78 and Arg75 residues were found.
pocket of DNA GyrB of M.tuberculosis
Figure 5. Binding orientation of compounds Figure 6. Binding orientation of compounds
14 and 15 in the binding pocket of DNA 9 and 15 in the binding pocket of DNA GyrB
GyrB of M.tuberculosis obtained from of M.tuberculosis obtained from molecular
molecular docking calculations docking calculations
3.3 Effect of R1 position from molecular 3.4 Effect of X position from molecular
docking calculations docking calculations
R1 position of methoxy group X position of oxygen group
(Compound 9) is compared with R1 position (Compound 9) is compared with X position
of F group (Compound 15). The binding of sulfur group (Compound 33). The
orientation results obtained from molecular molecular docking results is shown in Figure
docking calculations are presented in Figure 7. The crucial binding interactions of
6. The binding mode of compound 9 (IC50 compound 9 were hydrogen bond interaction
14.33 µM) reveal the crucial interactions between O atom of methoxy group with H
including hydrogen bond interaction atom of His82 residue, H atom of amine
between O atom of methoxy group with H group with N atom and O atom of Gly76
atom of His82 residue, H atom of amine residue, N atom of amine with H atom of
group with N atom of Gly76 residue, H atom Thr162 residue. - and -cation of 4-chloro
of 4-chlorophenyl group with O atom of phenyl group with Pro78, Arg75 and Arg433
Gly76 residue, N atom of amine group with residues. Finally, for crucial binding
H atom of Thr162 residue. Moreover, - interactions of compound 33 in DNA GyrB
and -cation of 4-chlorophenyl group with binding pocket, hydrogen bond interaction
Pro78, Arg75 and Arg433 residues were between N atom of amine group with H atom
found. For the crucial binding interactions of of Gly76 and Ser119 residues, H atom of
compound 15, hydrogen bond interaction amine group with O atom of Asn45, Asp72,
between H atom of amine group with N atom Val118 and Tyr143 residues, H atom of
of Gly76 and Arg134 residues, H atom of methyl group with O atom of Asp72 residue.
amine group with O atom of Asp72 residue, Moreover, - and -cation of aromatic ring
oxygen group with His82 and Val118 with Pro78 and Arg433 residues were
residues were observed. Moreover, -cation founded. Therefore, sulfur group of
of aromatic ring with Arg75 and Arg433 compound 33 showed higher important for
residues were found. DNA GyrB inhibition than oxygen atom of
compound 9.
obtained results aid to rational design for new
and more potent DNA GyrB inhibitors as
highly potential anti-tuberculosis agents.
Acknowledgements
Health Systems Research Institute
(HSRI.60.083), the National Research
(PERCH-CIC). The financial support from
Royal Golden Jubilee (RGJ) Ph.D. Program
(PHD/0132/2559) to B. Khamsri is gratefully
acknowledged. The Faculty of Science, Ubon
Ratchathani University, Faculty of Science,
Kasetsart University, NANOTEC and
Figure 7. Binding orientation of compounds NECTEC are gratefully acknowledged for
9 and 33 in the binding pocket of DNA GyrB supporting this research.
of M.tuberculosis obtained from molecular
docking calculations References
1. http://www.who.int/tb/en/.
4. Conclusion 2. http://dx.doi.org/10.5772/54960.
Molecular docking calculations were 3. Medapi, B.; Suryadevara, P.; Renuka, J.;
carried out to identify the crucial interactions, Sridevi, J.P.; Yogeeswari, P.; Sriram, D.
favored the binding affinity of 4- Eur. J. Med. Chem. 2015,103,1–16.
aminoquinoline derivatives for DNA GyrB 4. Cabral, J.H.M.; Jackson, A.P.; Smith,
inhibition. The crucial interactions of these C.V.; Shikotra, N.; Maxwell, A.;
inhibitors in the binding pocket including Liddington, R.C. Nature, 1997, 388, 903–
hydrogen bond interactions with Asn45, 906.
Asp72, Gly76, Val118 and Thr162 residues.
- and -cation were observed. These
Computer aided molecular design of ATPase inhibitors in a series of
7-chloro-4-piperazinoquinoline derivatives: molecular docking calculations
Bongkochawan Pakamwong1, Kotchakorn Yotyiamkrae1, Paptawan Thongdee1,
Bunrat Tharat1, Bandit Khamsri1, Thitima Chuansupak1, Naruedon Phusi1,
Chayanin Hanwarinroj1, Kampanart Chayajarus1, Pharit Kamsri2, Auradee Punkvang2,
Nitima Suttipanta3, Patchreenart Saparpakorn4, Supa Hannongbua4, Prasat Kittakoop5,
Ubonsree Leartsakulpanich6, Khomson Suttisintong7, James Spencer8, Adrian Mulholland9,
Pornpan Pungpo1*
1
2
3
4
5
6
National Center for Genetic Engineering and Biotechnology, NSTDA, 111 Thailand Science Park,
Klong Luang, Pathum Thani 12120, Thailand
7
National Nanotechnology Center, NSTDA, 111 Thailand Science Park,
Klong Luang, Pathum Thani 12120, Thailand
8
Bristol BS8 1TD, UK
9
Abstract:
Mutations of fluoroquinolone drug binding site of DNA gyrase are the majority
problems of fluoroquinolone drug resistant. Therefore, new inhibitors that they can inhibit
ATPase domain of DNA gyrase have been developed to overcome this drug resistant
problem. 7-chloro-4-piperazinoquinoline derivatives were developed as new ATPase
inhibitors for anti-tuberculosis agents. To improve the ATPase activity of this inhibitors,
molecular docking calculations were applied. The obtained results revealed that hydrogen
bond interactions of ligand with Asn45, Ile89, Val91, Val92, Met93, Thr94 and Gln436
residue were observed. The integrated results obtained provide key structural features for
rational design of new and more potent ATPase inhibitors as novel anti-tuberculosis agents.
1. Introduction supercoiling. As gyrase is essential in

Tuberculosis (TB) caused by prokaryotes, it is a good target for
Mycobacterium tuberculosis (MTB) is the antibacterial agents.3
first infectious disease declared by the DNA topoisomerase maintain the
World Health Organization (WHO) as a DNA topology during replication,
global health emergency.1 The bacteria that transcription and recombination. Among
cause tuberculosis are spread from one many topoisomerase, DNA gyrase is the
person to another through tiny droplets sole topoisomerase II enzyme present in
released into the air via coughs and sneezes.2 MTB. It mainly consists of GyrA and GyrB
The drug ability of DNA Gyrase has domains, in the holoenzyme complex as
been well established clinically and its A2B2. While the GyrA subunit mainly
inhibition has shown catastrophic effect on interacts with the DNA and possesses the
bacterial cell growth and survival. DNA active-site tyrosine responsible for DNA
gyrase is a remarkable enzyme, catalyzing cleavage and relegation, the GyrB subunit
the seemingly complex reaction of DNA helps in the ATP hydrolysis, thus acting as a
catalytic site for the enzyme. This enzyme is Molecular docking calculations
absent in the eukaryotic organisms, though a approach using Autodock 4.2 program were
less homologous enzyme does exist. Thus, it performed to study crucial interactions
seems to be an attractive target for between 7-chloro-4-piperazinoquinoline
developing novel drugs against TB. derivatives and DNA gyrase subunit B.
Research efforts both from industry and Firstly, docking parameters were validated
academia on the development of DNA GyrB to get high accuracy prediction of docking
inhibitors. Potent GyrB inhibitors have been calculation parameters. The obtained results
identified belonging to the chemical classes showed that the molecular docking
of pyrrolamides.4 calculations was reliable to predict the
The ATPase domain of GyrB makes binding mode and binding interactions of
the other half of functional gyrase hetero 4B6C ligand in GyrB binding site with
tetramer complex. This domain promotes RMSD value 1 Å Åas shown in Figure 1.
ATP hydrolysis, providing sufficient amount
of energy for the DNA super coiling
activity. Depriving the enzyme, the source
of energy via inhibition of GyrB domain
should still exert the same phenotypic effect
on the bacterial viability to the one exhibited
by fluoroquinolones, that inhibits the GyrA
domain. Moreover, mutations in gyrase that
confer resistance to fluoroquinolones are
outside the 43 kDa N-terminal domain that
is required for ATPase activity.5
There is an urgent need to discover
and develop new anti-TB agents that target
novel biochemical pathways and to treat
drug-resistant forms of the disease. In this
work, molecular docking calculation has
been performed to study the binding of 7- Figure 1. Ligand superimposition of the
chloro-4-piperazinoquinoline derivatives for X-ray crystal structure (green) and docked
rational design of novel potent anti-TB conformation (pink) of the 4B6C in the
agents against drug resistant TB. GyrB binding pocket
2. Materials and Methods 3.2. Molecular docking analysis of the

Structure and biological activities of highest active compound (compound 29)
six selected 7-chloro-4-piperazinoquinoline The interactions of compound 29
derivatives were summarized in Table 1.5 (IC50 of 2.5 μM) with of amino acids
All derivatives were constructed by standard surrounding the binding pocket of GyrB
tools in Gaussview 5.0.8 program and then enzyme were observed. Hydrogen bond
fully optimized by M062X/6-31G* method interactions were found as the crucial
using Gaussian 09 program. Molecular interactions for binding in GyrB binding
docking calculations using the Autodock 4.2 site. At sulfur atom (S) of X substituent,
program was performed to predict the hydrogen bond interaction of this substituent
binding orientation of potent inhibitors in with Pro78 and His82 residues in the GyrB
the GyrB binding pocket. binding pocket were observed. Moreover,
hydrogen bond interaction could form
3. Results and Discussion between chlorine atom (Cl) with Ala46 and
3.1 Validation of the molecular docking Val70 residues, hydrogen atom (H) with
Asp72 and Val 45 residues and nitrogen
calculations
atom (N) with Val42 residue. Van der Waals Ile164 residues were shown in Figure 2.
interaction with Val42, His82, Val118 and
Table 1. Six chemical structures and their biological activities of 7-chloro-4-

piperazinoquinoline derivatives5
compound1, 4 compound 29, 30, 34 compound 42

Subset 1 Subset 2
Subset 1
Compound R1 GyrB assay (IC50) (μM)
1 Cl 12.2
4 OCH3 51.6
Subset 2
Compound X R2 GyrB assay (IC50) (μM)
29 S F 2.5
30 S Cl 10.7
34 S COCH3 27
42 S C2H5 40.6
residues surrounding the binding pocket of

GyrB enzyme were investigated. Hydrogen
bond interactions were found as the crucial
interactions for binding in GyrB binding
site. Hydrogen bond interaction could be
formed by carbonyl (C=O) group and
nitrogen atom (N) of ligand (Compound1)
with Arg433 and Val122 residues,
respectively. In addition, hydrogen bond
interaction between hydrogen atom with
Asn45, Asp72 and Val118 residues in the
GyrB binding pocket were observed. Van
der Waals interaction of inhibitor with
Val122, Leu128, Tyr143, Thr162 and Ile164
Figure 2. The binding mode of compound residues were show in Figure 3.
29 in the GyrB binding pocket obtained The interactions of compound 30
from molecular docking calculations (IC50 of 10.7 μM) with of amino acid
residues surrounding the binding pocket of
3.3. Molecular docking analysis of GyrB enzyme were elucidated. Hydrogen
moderate active compounds (compound 1 bond interactions were found as the crucial
and 30) interactions for binding in GyrB binding
The interactions of compound 1 site. Sulfur atom (S) of X substituent formed
(IC50 of 12.2 μM) with of amino acid hydrogen bond interactions with Pro78 and
His82 residues. Moreover, hydrogen bond chain nitrogen atom (N) of ligand
interaction could be formed by side chain (compound 4) with Asn122 residue and
nitrogen atom (N) with Val42 residue, hydrogen atom (H) with Asp72 residue were
hydrogen atom with Asn45 and Asp72 show in Figure 5.
residues in the GyrB binding pocket. Van The important interactions of
der Waals interactions with Val42, Asn45, compound 34 (IC50 of 27 μM) and
Ile77 and Val118 residues were observed as compound 42 (IC50 of 40.6 μM) were
shown in Figure 4. hydrogen bond interactions. For Compound
34, hydrogen bond interaction between
sulfur atom of X substituent with Pro78 and
His82 residues were observed. Compound
42 formed hydrogen bond interactions
between sulfur atom at R2 with Leu128
residues. In addition, hydrogen bond
interaction of nitrogen atom (N) at R2 with
Ser119 residues could be found as shown in
Figure 6 and 7.
Compound 29 was the highest active
compound. Whereas, Compound4 was the
less active compound. At different R2
position, fluorine atom of compound29 was
electron withdrawing group and methoxy
Figure 3. The binding mode of compound 1 group of compound 4 was electron donating
in the GyrB binding pocket obtained from group. Based on the obtained result of
molecular docking calculations docking calculations, the crucial interaction
of compound 29 was hydrogen bond
interaction. At X substituent, sulfur atom (S)
formed hydrogen bond interaction with
Pro78 and His82 residues. In addition,
hydrogen bond interaction could from
between chlorine atom (Cl) with Ala46 and
Val70 residues, hydrogen atom (H) with
Asp72 and Val 45 residues and nitrogen
atom (N) with Val42 residues. The
interaction of compound 4 was hydrogen
bond interaction between carbonyl group
with Arg433 residue. Moreover, hydrogen
bond interactions between side chain
nitrogen atom (N) with Asn122 residue,
Figure 4. The binding mode of compound hydrogen atom (H) with Asp72 residue were
30 in the GyrB binding pocket obtained observed.
from molecular docking calculations The result can be concluded that the
number of hydrogen bond interaction of
3.4. Molecular docking analysis of less compound 4 was deceased as compared with
active compounds (compound 4, 34 and hydrogen bond interaction of compound 29.
42) Therefore, more hydrogen bond interactions
Hydrogen bond interaction between affected to the highest active compound of
carbonyl group of compound 4 with Arg433 compound 29.
residue was found. Moreover, hydrogen
bond interaction could be formed by side
4. Conclusion
Molecular docking calculations of
7-chloro-4-piperazinoquinoline derivatives
was successfully to achieve a better
understanding of the crucial interactions for
binding affinity of these derivatives in the
GyrB binding pocket. The obtained results
showed that the hydrogen bond interactions
were found as the crucial interactions for
binding in the GyrB binding site. This
information leads to get better understanding
for design 7-chloro-4-piperazinoquinoline
derivatives with effective in inhibiting
GyrB.
Figure 5. The binding mode of compound 4
in the GyrB binding pocket obtained from Acknowledgements
molecular docking calculations This research was supported by the
Thailand Research Fund (RSA5980057) the
National Research Council of Thailand and
Higher Education Research Promotion. The
Faculty of Science, Ubon Ratchathani
University, NANOTEC and NECTEC are
gratefully acknowledged for supporting this
research.
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Figure 6. The binding mode of compound V.; Sriram, D., Bioorg. Med. Chem.
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conditions/tuberculosis/diagnosis-
treatment/drc-20351256
3. Collin, F.; Karkare, S.; Maxwell, A.,
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5. Jeankumar, V. U.; Reshma, R. S.; Vats,
Figure 7. The binding mode of compound R.; Janupally, R.; Saxena, S.;
42 in the GyrB binding pocket obtained Yogeeswari, P.; Sriram, D., Eur. J.
from molecular docking calculations Med. Chem. 2016, 1
Rational design of PknB inhibitors in class of 4-oxo-crotonic acid
derivatives as highly potent anti-tuberculosis agents
Chayanin Hanwarinroj1, Naruedon Phusi1, Kampanart Chayajarus1, Pharit Kamsri2,
Auradee Punkvang2, Nitima Suttipanta3, Patchreenart Saparpakorn4, Supa Hannongbua4,
Khomson Suttisintong5, Prasat Kittakoop6, James Spencer7, Adrian Mulholland8,
Pornpan Pungpo1*
1
2
3
4
5
6
7
Bristol BS8 1TD, UK
8
Abstract:
Protein serine/threonine kinase B (PknB) is an essential serine/threonine kinase of M.
tuberculosis, plays an important role in a number of signalling pathways involved in cell
division and metabolism. However, PknB inhibitors show less potency against whole cell.
To develop protein kinase B inhibitors as new potential anti-tuberculosis agents in class of
4-oxo-crotonic acid derivatives, molecular docking calculations and molecular dynamic
simulations (MD simulations) were applied to understand the crucial interactions, binding
mode and binding energy of selected 4-oxo-crotonic acid derivatives. The results obtained
from molecular docking calculations indicated that highly active compound form hydrogen
bond interactions with back bone of Leu17, Val95, and Asp156 in the PknB binding pocket.
Moreover, van der Waals interactions with Tyr94, Val95, and Met145 in the PknB binding
pocket were crucial for binding affinity. The binding mode and binding energy of highly
active compound obtained from MD simulations were employed to gain insight into the
complex structures of the protein kinase B inhibitors and their binding energetics. The results
can be helpful for further design a novel anti-tuberculosis drug design.
1. Introduction reductase (InhA) is an attractive target for

Tuberculosis (TB) caused by M. designing novel antibacterial agents because
tuberculosis remains as a major world health this enzyme is not found in the mammalian
problem. It is second only to HIV/AIDS as system.2,3 However, this target developed
the biggest killer worldwide. In 2016, 10.4 the resistance to isoniazid. Intensive
million people worldwide suffered from TB research efforts are directed toward
and an estimated 1.7 million people death identification and development of novel
from TB.1 In particular, standard regimens InhA inhibitors, but most of the newly
using existing drugs such as isoniazid discovered InhA inhibitors show poor
(INH), the main drug for TB treatment, are cellular activity against M. tuberculosis.4
ineffective because of increasing prevalence Accordingly, finding new drugs to treat TB
of drug-resistant tuberculosis such as as well as identifying new targets are an
multidrug-resistant tuberculosis (MDR-TB) urgent priority. Protein kinase B (PknB) is a
and extensively drug-resistant tuberculosis Ser/Thr protein kinase (STPK) and
(XDR-TB). The 2-trans-enoyl-ACP represents a signaling element involved in
mycobacterial physiology and virulence of Table 1. Structure and biological activity of
M. tuberculosis. Protein kinase B has been 4-oxocrotonic acid derivatives as PknB
considered as an attractive target for inhibitors
O
development of anti-TB agents because this O
enzyme is essential for bacterial growth and R

R' R
for survival of the pathogen within the host. O

O H3CO
A large number of PknB inhibitors have
(a) cpd. 55 and cpd. 85 (b) cpd. 02
been screened and identified as potential
anti-TB agents. Lead PknB inhibitors show cpd. R R’ %
promising enzyme potency and cellular inhibition
at 20 µM
activity against M. tuberculosis. Moreover, 02 OCH3 - 24.7±1.5
PknB inhibitors are able to target PknB 55 isopropyl 56.2±5.0
outside of the M. tuberculosis cell, avoiding
the problem of poor cell wall permeability.5 85
OCH3
2-[(tert- 87.9±5.3
Because of poor cell wall permeability, Butoxycarbo
many anti-TB agents with promising O2N
nyl)amino]
enzyme potency show weak or no cellular ethyl
activity against M. tuberculosis nowadays,
substituted aminopyrimidine compounds complexes in this study. The available X-ray
have been synthesized as a new class of structure of PknB in a complex with 1,4-
PknB inhibitor. Some of these compounds dihydroxy-5,8-bis({2[(hydroxyethyl)amino]
show the nanomolar enzyme binding affinity ethyl}amino)-9,10-anthracenedione (PDB
and micromolar minimum inhibitory code 2FUM) was used as an initial structure
concentrations against M. tuberculosis.6 of receptor for molecular docking
However, the detailed structure basis for calculations. The number of Genetic
binding of 4-oxo-crotonic acid compounds Algorithm (GA) runs was set to 200 run per
as PknB inhibitor is not well understood. ligand with the default search algorithm
Accordingly, molecular docking calculation parameters. The docking protocols were
and molecular dynamics simulations (MD validated based on the root-mean-square
simulation) were employed in this work to deviation (RMSD) value between the
model the complex structures of substituted docked and observed X-ray conformations.
4-oxo-crotonic acid in PknB inhibitors. The 2.3 Molecular dynamic simulations
integrated results could be helpful for further Molecular dynamic simulation (MD
design a novel and more potent anti- simulations) were performed to predict the
tuberculosis agents. inhibitors in the PknB binding pocket.
TIP3P water model and Na+ were chosen to
2. Materials and Methods represent water for salvation and ions for
2.1 Biological data neutralize system. To reduce the bad steric
Structures and biological activities of interactions of solvate water molecules and
ninety 4-oxocrotonic acid derivatives were Na+ ions of each system, the inhibitor-PknB
complex was first minimized by 1,000 steps
obtained from literature.6 All derivatives
with atomic positions of solute species
were constructed by standard tools in restraint with using force constant of 500
Gaussview 5.08 program and then fully kcal/mol Å2. Non-bonded cut-off was set to
optimized by M06-2X/6-31G* method using 8 Å. The threshold value of the energy-
Gaussian09 program. gradient for the convergence was set as
2.2 Molecular docking calculations 0.001 kcal/mol/ Å. Then, the whole system
Molecular docking using the was minimized by 1,500 steps as the same
Autodock 4.2 program was employed with conditions of water and ions minimization
the aims of generating inhibitor enzyme without restraining condition. Next, the
systems were gradually warmed up from 0 Waals interactions were obtained between
to 300 K in the first 20 ps followed by side chain of cpd. 55 with side chain of
maintaining the temperature at 300 K in the Val72, Gly97, Met145, Met155 and Asp156
last 10 ps with 2 fs time simulation steps in a residues, respectively. For less active
constant volume boundary. The solute compound, the crucial interactions are
species were restrained to their initial hydrogen bond interaction between C-O of
coordinate structures with a weak force R substituent with side chain of Leu17
constant of 10 kcal/mol Å2 during the residue. In addition, van der Waals
temperature warming. This was followed by interactions were found between core
70 ps of the position-restrained dynamics structure and side chain of Val72, Val95 and
simulation with a restrain weight of 2 Met145 residues, respectively. The obtained
kcal/mol Å2 at 300 K under an isobaric results suggested the crucial interactions of
condition. Finally, 10 ns MD simulations 4-oxocrotonic acid in PknB binding pocket
without any restraints were performed using could be helpful for rational design new
the same conditions. The root-mean square 4-oxocrotonic acid derivatives.
deviations (RMSDs) of the PknB enzyme 3.2 Molecular dynamic simulations
and the inhibitors, and binding free energies 3.2.1 Structural stability during MD
were calculated to evaluate the binding simulations
affinities of inhibitors in PknB binding In order to calculate structural
pocket using the Molecular Mechanics stability during MD simulation of cpd. 85 in
Poisson-Boltzmann Surface Area (MM- PknB binding pocket, The RMSDs for all
PBSA) methods.

3.1 Molecular docking calculations
3.1.1 Crucial interaction of selected
inhibitors
High accuracy prediction of docking
parameter in Autodock 4.2 program was
validated based on the RMSD value between
X-ray and docked structures. RMSD value is
1.09 Å. Therefore, binding mode of selected
compounds in this study was predicted as
the same parameters. The binding mode of Figure 1. Crucial interactions of highest
highest active compound is cpd. 85 in PknB active compound (cpd. 85)
binding pocket is showed in Figure 1.
The crucial binding interactions are
hydrogen bond interaction between side
chain of R substituent with backbone of
Leu17, Val72, Tyr94 and Met155 residues,
respectively. In addition, van der Waals
interaction between side chain of R
substituent and side chain of Val25, Lys40,
Met92, Val95 and Asp156 residues were
founded. The binding mode of moderate
active compound is cpd. 55 is showed in
Figure 2. The crucial binding interactions
are hydrogen bond interaction between
carbonyl group of core structure with side Figure 2. Crucial interactions of moderate
chain of Met92 residue. Moreover, van der active compound (cpd. 55)
Figure 4. RMSD plots for the cpd. 85 in
PknB binding pocket during MD simulation
Figure 3. Crucial interactions of less active

compound (cpd. 02)
atoms of solute species relative to the initial

structure over the 10 ns of simulation times
were calculated and plotted in Figure 4.
The RMSD plots indicate that cpd. 85
reached equilibrium state after 5 ns. Figure 5. Crucial interactions of highest
Therefore, the result obtained from MD active compound (cpd. 85) obtained from
simulations of cpd. 85 was reasonable for MD simulations
further analyze in more detail of binding
free energy, binding mode and binding
interaction. The binding free energy
calculations were calculated by MM-PBSA
method the result indicated that the
calculated binding free energy of cpd. 85
was -13.29 kcal/mol (the binding energy
obtained from experimental was -12.58
kcal/mol). The obtained result indicated that
binding free energy calculations based on
MM-PBSA method provide good value and
closed to experimental binding free energy.
3.2.2 Binding mode and binding Figure 6. Superimposition of docked ligand
interactions of cpd. 85 (yellow) and the structure obtained from
The binding mode of cpd. 85 MD simulations (Cyan)
obtained from MD simulations formed four
hydrogen bond interactions between 4. Conclusion
hydrogen atom of R substituent with Molecular docking calculations and
carbonyl group of Glu59, Glu93 residue and Molecular dynamic simulations were
carbonyl group of core structure with side successfully to predict the binding mode
chain of Met145 residue, respectively. binding interactions and binding free energy
Moreover the hydrogen bond interaction of 4-oxocrotonic acid derivatives as PknB
between carbonyl group of R substituent inhibitors. Hydrogen bond interaction with
with side chain of Asp156 residue was Glu59, Glu98, Met145 and Asp156 are key
founded. The van der Waals interaction amino acid residue for improve the
between side chain of R substituent and side biological activity of novel 4-oxocrotonic
chain of Val25 and Val95 residues were acid as anti M. tuberculosis agents.
founded.
Acknowledgements 3. Heath, R.J.; Rock, C.O. Curr Opin
This research was supported by the Investig Drugs. 2004, 5, 146–153.
National Research Council of Thailand, the 4. White, S.W.; Zheng, J.; Zhang, Y.M.;
Thailand Research Fund (RSA5980057), the Rock, C.O. Annu Rev Biochem. 2005,
Thailand Graduate Institute of Science and 74, 791–831.
Technology (TGIST) (SCA-CO-2560- 5. Lougheed, K.E.; Osborne, S.A.; Saxty,
4375TH) to C. Hanwarinroj. Faculty of V.L.; Kettleborough, C.A.; Bryans, J.S.;
Science, Ubon Ratchathani University and Taylor, D.L.; Smerdon, S.J.; Buxton,
NECTEC are gratefully acknowledged for R.S. Chugh, J.; Nott, T.J.; Patel, D.;
supporting this research. Spivey,B.; Whalley, D.; Chapman, T.;
Bouloc, N. Tuberculosis 2011, 91, 277–
References 286.
1. Global Tuberculosis Report 2014; World 6. Changliang, X.; Xiaoguang, B.; Jian, X.;
Health Organization: Geneva, Jinfeng, R.; Yun, X.; Ziqiang, L. Juxian,
Switzerland, 2016. W.; Jingjing, S.; Liyan, Y.; Yucheng, W.
2. Campbell, J.W.; Cronan, J.E. Annu Rev RSC Advances 2017, 7, 4763–4775.
Microbiol. 2001, 55, 305–332.
Molecular docking study of pyrrolyl benzohydrazide derivatives to explore
the structure requirements of InhA inhibitors
Paptawan Thongdee1, Bongkochawan Pakamwong1, Kotchakorn Yotyiamkrae1,
Bunrat Tharat1, Bundit Khamsri1, Thitima Chuansupak1, Naruedon Phusi1,
Chayanin Hanwarinroj1, Kampanart Chayajarus1, Pharit Kamsri2, Auradee Punkvang2,
Nitima Suttipanta3, Patchreenart Saparpakorn4, Supa Hannongbua4, Prasat Kittakoop5,
Ubonsree Leartsakulpanich6, Khomson Suttisintong7, James Spencer8, Adrian Mulholland9,
Pornpan Pungpo1*
1
2
3
4
5
6
National Center for Genetic Engineering and Biotechnology, NSTDA, 111 Thailand Science Park,
7
8
Bristol BS8 1TD, UK
9
Abstract:
Enoyl ACP reductase, InhA has been identified as promising target for anti-
tuberculosis development. This enzyme is the binding pocket of isoniazid, the first line drug
for anti-tuberculosis agents. However, the potency of isoniazid is decreased due to the
mutation of activation enzyme, KatG. Pyrrolyl benzohydrazide derivatives were designed as
new InhA inhibitors with moderate activities against InhA and mycobacterial cell. Herein, we
aim to develop new highly potent InhA inhibitors based on pyrrolyl benzohydrazide
derivatives. Molecular docking calculations were used to elucidate the key interactions for
binding of these inhibitors in InhA binding pocket. The obtained results showed that
hydrogen bond interaction between ligand and Pro156, Tyr158, Ala198, Val203 and NAD+
cofactor. Moreover, pi-pi interaction was improved the binding affinity of inhibitors in InhA
binding site. The integrated results from docking in this work aided to rational design new
and more potent InhA inhibitors in a series of pyrrolyl benzohydrazide as novel anti-
tuberculosis agents.
1. Introduction in lungs, called pulmonary TB. The disease

Enoyl-ACP reductase (ENR) can also spread to other parts of the body,
catalyzes the last step of the elongation referred to as extra-pulmonary TB that may
cycle in the synthesis of fatty acids. Fatty be latent or active. M. tuberculosis has been
acid biosynthesis is essential for survival is the major public health threat that remains to
mammals, plants, fungi and bacteria (the be the leading cause of mortality with
archaea make isoprenoid-based lipids).1 malaria and AIDS, and infecting nearly
ENR also known as InhA, it is enzyme of eight million people worldwide, probably
Mycobacterium tuberculosis (M. tuber- killing more than three million yearly2.
culosis) caused causative agent for Inhibition of the InhA is one of the most
tuberculosis (TB), an infection caused by the effective treatment of killing M.
slow growing bacteria that frequently found tuberculosis, as clinically demonstrated by
isoniazid, the most potent TB drug. pocket are H-bond interaction and -
Unfortunately, both multi-drug and interaction. In this literatures review, we
extensively drug resistant (XDR) of M. study the structure and inhibition values of
tuberculosis isolates are resistant to InhA at 50 µM. Table 1 shows the chemical
isoniazid, predominantly due to mutations in structures and % inhibition value of
KatG, the catalase peroxidase involved in compounds 1, 6, 51 and 52, respectively.
the activation process of isoniazid. This Compounds 1, 51 and 52 show inhibition
problem led to extensive efforts to identify values are higher inhibitory activity than
direct InhA inhibitors.3 Pyrrolyl compound 6. Figure 1 shows the binding
benzohydrazide derivatives were designed interactions of compounds 1, 6, 51 and 52.
as new InhA inhibitors with moderate As depicted in figure 1a, the six H-
activities against InhA and mycobacterial bond interactions green line) between
cell. Herein, we aim to develop new highly compound 1 with Gly96, Val203, Leu218
potent InhA inhibitors based on pyrrolyl residues and NAD+ cofactor. In binding
benzohydrazide derivatives. Presently, mode of compound 51, formed six H-bond
molecular docking calculations were used to interactions with Met103, Tyr158, Met161,
elucidate the key interactions for binding of Ala198 residues and NAD+ cofactor in
these inhibitors in InhA binding pocket. figure 1b. Figure 1c shows the results of
docking, all seven H-bond interactions
2. Methods between compound 52 and Phe97, Pro156,
Fifty-three structures and biological Met161, Ala198, Ile202 residues and NAD+
activities of inhibitors were obtained from cofactor. Moreover, - interaction between
literatures review.2 The 3D structures were ligand with Tyr158 residue was found
generated using GaussView 5. 0. 8 program (Figure 1d). Figure 1e, compound 6 showed
(Gview). This results 3D structure from the H-bond interaction with ligand formed
Gview leads to optimize by M062X/6-31G* Tyr158, Met161, Ala198 residues and
method using Gaussian09 program. NAD+ cofactor.
2.1 Molecular docking studies 3.2 Effect of R substituent from molecular
Molecular docking was done to docking calculations
clarify the binding mode of the compounds Compounds 1 and 6 have the same
to provide straight forward information for general structures. Only R structure of
further structural optimization.2 Molecular compound 6 contained bromine atoms at R
docking calculations using Autodock 4. 2 substituent, according to Table 1. The
program was performed to predict the potent inhibition value of compound 1 higher than
inhibitors in the InhA binding pocket. The the inhibition value compound 6. On the
X-ray crystal structure (PDB code: other hand, the results of molecular docking
InhA2x23) was used as receptor for this of compound 6 was formed H-bond
calculation. interaction more than compound 1.
Compound 6 contained Br atoms at R
3. Results and Discussion substituent as compared to compound 1,
3.1 Interactions for binding of these these atoms must be affected to inhibitory
Pyrrolyl benzohydrazide derivatives in activity. Compound 1 contained hydrogen
InhA binding pocket by Molecular atoms. Small size of compound 1 helped to
docking calculations recognize the orientation InhA binding site
Molecular docking calculations were ( Figure 1a) . As compared to compound 6,
used to elucidate the key interactions for the orientation of this compound is different
binding of these inhibitors in InhA binding ( Figure 1e) as compared with compound 1.
pocket. The obtained results showed that the This result suggested that R substituent
important interaction between ligand, amino should be low electron density and size.
acid and NAD+ cofactor in InhA binding The - interaction of compound 52 was
found as the crucial interaction to bind with Therefore, the important interaction
InhA binding pocket. This interaction between ligand, NAD+ cofactor and amino
effected to inhibitory activity of InhA. So, acid is H-bond interaction and pi-pi
inhibition may be related to the position and interaction. Moreover, the substituent of
between ligand and amino acid and new design compounds should be electron
electrostatic interaction of bromine withdrawing group like F and Cl at R
substituents. substituent and electron donating group such
as CH3 group at pyrrolyl.
Figure 1. Docked mode of ( a) compound 1 formed H-bond, ( b) compound 51 formed

H-bond, ( c) compound 52 formed H-bond, ( d) compound 52 formed - interaction and
(e) compound 6 formed H-bond with inhibitors
Table 1. The chemical structures and % inhibition value
R
O
N OCH2R N
R'
1-47 R
48-53
Compound R R´ % Inhibition of InhA at 50 µM
O
HN
N
1 - 63
O O
O Br
Br
HN
N
6 Br - 11
O O Br
O Br
Br
HN
N
51 CH3 Br 75
O Br
O Cl
Cl
HN
52 CH3 N 76
Cl
O Cl
Structure based drug design using 1. 1. Massengo-Tiasse, R. P.; Cronan, J. E.
molecular docking calculations was Cell. Mol. Life Sci. 2009, 66 ,
successfully elucidated to predict InhA 1507−1517.
inhibitors based on pyrrolyl benzohydrazide 2. Joshi, S. D.; More, U. A.; Dixit, S. R.;
derivatives in InhA binding site. The results Balmi, S. V.; Kulkarni, B. G.;
showed that selected compounds formed H- Ullagaddi, G.; Lherbet, C.;
bond interactions with Gly96, Phe97, Aminabhavi, T. M. Bioorg. Chem.
Met103, Pro156, Tyr158, Met161, Ala198, 2017, 75, 181−200.
Ile202, Val203, Leu218 residues and NAD+ 3. Manjunatha, U. H.; Rao, S. P. S.;
Kondreddi, R. R.; Noble, C. G.;
cofactor. The - interaction was found as
Camacho, L. R.; Tan, B. H.; Ng, S. H.;
important interaction of InhA inhibitor. Ng, P. S.; Ma, N. L.; Lakshminarayana,
Accordingly, the obtained results from this S. B.; Herve, M.; Barnes, S. W.; Yu,
work aid to rational design new pyrrolyl W. X.; Kuhen, K.; Blasco, F.; Beer, D.;
benzohydrazide derivatives as highly potent Walker, J. R.; Tonge, P. J.; Glynne, R.;
InhA inhibitors. Smith, P. W.; Diagana, T.T. Sci.
Transl. Med. 2015, 7, 1−9.
Acknowledgement 4. Shrinivas, D. J.; Sheshagiri R. D.;
This research was supported by Pradeepkumar, M.; Kulkarni, V. H.
the Thailand Research Fund (RSA5980057) Indo Am. J. Pharm. Res. 2016, 6,
and the National Research Council of 4925−4933.
Thailand. Faculty of Science, Ubon 5. Xing, D.; Yi, L.; Yuan L. X.; Shi, M.
Ratchathani University, Faculty of Science, A.; Jing, L.; Peng, S.; Xing L. J.; Shu
Q. L. Int. J. Mol. Sci. 2016, 17, 144.
Kasetsart University and NECTEC are
gratefully acknowledged for supporting this
research.
Determination of antioxidant properties and total phenolic compound of
various pigmented Thai rice varieties grown of Thailand
Sudarat Chanchoie1, Chayanin Hanwarinroj1, Naruedon Phusi1,Duangdao Sattayakul1,
Jitlada Dechatiwong1,Saisamorn Lumlong1,Kampanart Chayajaras1,
Somjintana Taweepanich1, Janpen Intaraprasert1, Nuchanaporn Pijarn1, Pharit Kamsri2,
Nitima Suttipanta3, Pornpan Pungpo1*
1
2
3
Faculty of Pharmaceutical Science,UbonRatchathani University, Ubon Ratchathani 34190,Thailand
*E-mail:pornpan_ubu@yahoo.com
Abstract:
In this study, six types of pigmented Thai rice varieties grown in Northeastern region
of Thailand including Hommali germinated brown rice, Hommali brown rice105, Hommali
normal rice, organic Homnil brown rice, organic riceberry rice and organic brown rice were
selected for determination of antioxidant properties and total phenolic compound.These Thai
rice varieties were extracted by methanol.The antioxidant activity using DPPH Radical
Scavenging Activity and Ferric Reducing Antioxidant Power (FRAP) were investigated.
Moreover, Folin-Ciocalteucolorimetric method was used to determine total phenolic
compounds of rice crude extracts. The methanol crude extract of rice shows highest
antioxidant activity obtained from DPPH Radical Scavenging Activity, Ferric Reducing
Antioxidant Power (FRAP) and Folin-Ciocalteu colorimetric methods. For DPPH assay the
antioxidant activity of organic riceberry rice and Hommali germinated brown rice are 95.16 ±
1.28% and 82.86 ± 1.63%, respectively. For FRAP assay the antioxidant activity of Hommali
germinated brown rice and Organic brown rice are 91.78 ± 3.83 mg and 70.6 ± 11.03 mg,
respectively. For Folin-Ciocalteu colorimetric method the antioxidant activity of Hommali
germinated brown rice is 109.68 ± 0.23 mg. Based on the obtained results pigmented Thai
rice varieties are highly potential sources for health-promoting compounds.
1. Introduction pigment of rice, and responsible for the anti-

Nowadays, the consumer's appetite oxidants.3-10 In this work, we aim to evaluate
for more valuable meals has become well- the antioxidant activity and total phenolic
recognized. However, most people still often content of Thai rice grown in Northeastern
seek for supplements such as vitamins. Rice region of Thailand.
is a good choice for consumers who care
about their own health. Because the world 2. Materials and Methods
has changed the way people live. Rice is 2.1 Rice samples and crude extractions
very useful and valuable to human life. It is Six types of Thai rice varieties
the main food that is important to the world grown in Northeastern region of Thailand
population.1,2 As Thais, we have been were selected in this study. Rice extraction
consuming rice as a main food. It is a source process for this study was summarized in
of carbohydrates that provide energy and Figure 1. Briefly, rice samples were
warmth. It is also used to make candy, extracted by a reflux in a bottle containing
fertilizers, toys, jewelry and medicine. In methanol at 120°C for 3 hrs. Each crude
addition, rice contains other important and extract was dried under reduced pressure.
beneficial components to the human body. The concentrated extracted samples were
The campaign material was found in the kept in a refrigerator (4-5°C) for antioxidant
activity assays. The antioxidant activities of samples were dissolved in methanol to get
rice were tested by DPPH radical rice crude solutions. These solutions were
scavenging and FRAP assays. Moreover, reacted with Folin-Ciocalteu reagent (1 N),
total phenolic compounds were evaluated. and then neutralized with of 20 % sodium
carbonate. Distilled water was added to the
mixing solutions. These solutions were
incubated for 40 minutes at room
temperature, the absorbances of the mixing
solutions were measured at 725 nm using
UV-visible spectrophotometer. Tannic acid
used as a standard compound in this work.
The obtained results were expressed as mg
tannic acid (TAE)/g of dye weight rice
sample.

3.1 DPPH assay
The antioxidant percentages
evaluated by DPPH method of methanol
Figure 1. Flow chart for extraction and extraction were summarized in Figure 2.
antioxidant assays The results reveal that percentage of
antioxidants of rice samples are in the range
2.2 2-diphenyl-2-picrylhydrazyl( DPPH) from 18.82 ± 6.43% to 89.14 ± 1.12%.
assay These results (Figure 2) suggests that the
The radical-scavenging activity of antioxidant activity of pigmented rice,
rice samples was evaluated using DPPH organic riceberry (95.16  1.28%) is higher
radical-scavenging method. DPPH working than brown rice and Hommali normal rice.
solution in MeOH was added to the rice In addition, germinated rice such as
extracted solutions. Mixing solutions were Hommali germinated brown rice (82.86 
left at room temperature for 1 h in the dark. 1.63%) has higher antioxidant activity than
A decrease in absorbance of rice extracts Hommali brown rice (46.22  0.35%) and
solution was measured at 517 nm using UV- Hommali normal rice (18.82  6.43%). The
visible spectrophotometer and compared to two highest antioxidant activities of the
that of the control (without the rice extracted crude extract were obtained from organic
sample). % DPPH activity of Trolox® was riceberry rice and Hommali germinated
used as the standard reference compound. brown rice with the value of 89.14 ± 1.12%
2.3 Ferric reducing Antioxidant power and of 82.86 ± 1.63%, respectively.
(FRAP) assay 3.2 Ferric Reducing Activity Power
Total anti-oxidant capacities of rice
Value (FRAP assay)
extracts were determined using FRAP assay. Figure 3 shows the effect on
Briefly, rice extracted solution was added to antioxidant activity of methanol crude
working FRAP reagent. The mixing extracts from FRAP assay. The antioxidant
solutions were measured at 593 nm using activities are ranging from 16.29 ± 2.47 mg
UV-visible spectrophotometer. L-ascorbic to 109.68 ± 3.83 mg. This is similar trend
acid was used as a standard to compare the with DPPH assay with the correlation
total anti-oxidant capacity. coefficient of 0.67. Germinated rice,
2.4 Total phenolic compound
Hommali germinated brown rice (109.68 
The total phenolic content of rice
3.83 mg) has higher antioxidant activity than
crude samples was measured using the
Hommali brown rice (42.77  2.80 mg) and
Folin-Ciocalteu reagent. The rice extracted
Hommali normal
Figure 2. The percentage of DPPH Figure 3. FRAP Value of the rice samples
scavenging activity of the rice samples
rice (16.29  2.47 mg), respectively. The

two highest antioxidant activities of the
crude extracts are from Hommali
germinated brown rice and Organic brown
rice with the values of 109.68 ± 3.83 mg and
of 83.18 ± 11.03 mg, respectively.
3.3 Total phenolic compounds
Figure 4 shows the phenolic content
of the methanol extraction. The total content
of phenolic compounds ranged from 37.77 ±
3.57 to 91.78 ± 0.52 mg. Rices with the two
highest total phenolic contents of crude Figure 4. Total Phenolic Content of the rice
methanol extract are Hommali germinated
samples
brown rice with the value of 91.78 ± 0.52
mg and Hommali normal rice with the value high antioxidant activity with the value of
of 70.60 ± 26.61 mg. 95.36 ± 1.28% and of 82.86 ± 1.63%,
3.4 The potential rice respectively. For FRAP assay, Hommali
Based on our study, Hommali germinated brown rice and Organic brown
germinated brown rice is the most potential rice have two relatively high values of
rice with high antioxidant activities (82.86  109.68 ± 3.83 mg and of 83.18 ± 11.03 mg,
1.63 for DPPH and 109.68  3.83 for FRAP) respectively. For Total Phenolic content,
and total phenolic content (91.78 ± 0.52 Hommali germinated brown rice has the
mg). These results beneficially aid highest value of 91.78 ± 0.52 mg. The
customers to consider the alternatives from results of the present study suggest that the
natural food with high antioxidant. six Thai rices have a high potential to be
natural antioxidant sources for food.
4. Conclusion
The antioxidant activities of six Acknowledgements
types of rice were tested by using DPPH, This research was supported by the
Ferric reducing antioxidant power (FRAP) National Research Council of Thailand, the
assays and total phenolic compound. The Thailand Research Fund, Faculty of Science,
antioxidant activities obtained from DPPH Ubon Ratchathani University and Faculty of
assay suggested that organic Riceberry rice Science, Nakhon Phanom University are
and Hommali germinated brown rice show
gratefully acknowledged for supporting this 6. Chaiyasut, C.; Sivamaruthi, B. S.;
research. Pengkumsri, N.; Keapai, W.; Kesika, P.;
Saelee, M.; Tojing, P.; Sirilun, S.;
References Chaiyasut, K.; Peerajan, S.; Lailerd, N.,
1. Muthayya, S.; Sugimoto, J. D.; Pharmaceuticals (Basel), 2016, 10 (1),
Montgomery, S.; Maberly, G. F. Ann. pii: E3.
N. Y. Acad. Sci. 2014, 1324, 7–14. 7. Sinthorn, W.; Chatuphonprasert, W.;
2. Ghasemzadeh, A.; Karbalaii, M. T.; Chulasiri, M.; Jarukamjorn, K. Pharm.
Jaafar, H. Z. E; Rahmat, A. Chem Cent Biol. 2016, 54(5), 770–779.
J.2018, 12 (1), 17. 8. Chatthongpisut, R.; Schwartz, S.J.;
3. Uttama, S.; Itharat, A.; Rattarom, R.; Yongsawatdigul, J. Food. Chem., 2015,
Makchuchit, S.; Panthong, S.; 188, 99–105.
Sakpakdeejaroen, I. J. Med. Assoc. 9. Srisawat, U.; Panunto, W.; Kaendee, N.;
Thai., 2014, 97, Suppl 8, S125–132. Tanuchit, S.; Itharat, A.;
4. Insuan, O.; Chariyakornkul, A.; Lerdvuthisopon, N.; Hansakul, P. J.
Rungrote, Y.; Wongpoomchai, R. J. Med. Assoc. Thai. 2010, 93, Suppl 7,
Cancer. Prev.,2017, 22 (2), 89–97. S83–91.
5. Maksup, S.; Pongpakpian, S.; 10. Muntana, N.; Prasong, S.; Pak. J. Biol.
Roytrakul, S.; Cha-Um, S.; Sci. 2010, 13 (4):170–174.
Supaibulwatana, K. J. Sci. Food. Agric.
2018, 98 (2), 566–573.
Studies of sildenafil and phosphodiesterase type 5 binding properties by
using computational methods
Bunpot Intarathat, Autchara Namkhaw, Supawadee Srinimnuan, Supa Hannongbua
Patchreenart Saparpakorn*
*E-mail: fscipnsk@ku.ac.th
Abstract:
Phosphodiesterase type 5 (PDE5) is one of significant eleven isozymes of
phosphodiesterases (PDEs) involving hypertension and erectile dysfunction (ED) disease.
The treatment of these disorder has been affected via the use of strong PDE selective drugs.
In this work, the crystallographic data were complexed between sildenafil and PDE5 was
chosen to study the physical movement in the dynamic conditions using molecular dynamics
(MD). The simulations were performed for 100 ns using GROMACs program with
Amber99SB forcefield. From MD simulations, there are important residues forming
hydrogen bond (TYR664), - interactions (PHE820) and H– (VAL782 and LEU804)
interactions. The equilibrated complex structure was further investigated using quantum
mechanical (QM) calculations to study binding interactions between ligand and protein in the
binding pocket. The results indicate important interactions between sildenafil and PDE5 and
provide insights for designing new potent PDE5 inhibitors.
1. Introduction Potassium channels influence the basal tone

Phosphodiesterases (PDEs) of cavernous smooth muscle, the initiation
hydrolyze two of the most essential and termination of contractile events, and
signaling molecules in cells, cyclic relaxation because of their ability to
adenosine monophosphate (cAMP) and hyperpolarize the cell2. The aim of this work
cyclic guanosine monophosphate (cGMP). is to study specifically the interaction
The total of PDEs are grouped into 11 between sildenafil and amino acids in the
families, called PDE1-PDE11.The main of binding pocket. Information from the study
the PDE families (PDE1, 2, 3, 10 and 11) can indicate the structural effect of ligand
are dual-substrate and can hydrolyze both during the binding and can use for further
cAMP and cGMP. PDE4, 7 and 8 are analysis in order to improve the activity of
specific hydrolyzing cAMP while PDE5, 6, this derivative.
and 9 only break down cGMP1. Viagra is the
commercial drug of the prescription as 2. Materials and Methods
medicine sildenafil citrate. It is used to treat For starting conformation, the crystal
erectile dysfunction (ED) in men that works structure of sildenafil in complex with
by increasing blood flow to the penis. In this Phosphodiesterase type 5 (PDB code: 2H42)
research, pharmacomechanical (receptor- was taken from Protein Data Bank3. The
mediated) mechanisms that raise either molecular dynamics simulations for 100 ns
cGMP or cAMP, causing reduce Ca2+ in were then performed by using GROMACs
cells, are of potential use in treating ED. program. Dodecahedron box, TIP3P water
Furthermore, drugs acting through models and the Amber99SB protein, nucleic
electromechanical mechanisms (voltage- AMBER94 force field were used.
gated channels) can relax cavernous smooth
muscle and trigger penile erection.
Figure 4. RMSF of backbone residues in
PDE5
Figure 1. Crystallographic structure of
sildenafil Root mean square fluctuation
(RMSF) of backbone amino acids in MD
3. Results and Discussion trajectories is investigated for the fluctuation
3.1 Structural stability of amino acids, as shown in Figures 4. Most
Root-mean-square deviation of amino acids reveal low fluctuation with
(RMSD) of MD trajectories is investigated the RMSF less than 1 Å along MD
in order to evaluate the structural stability. simulations, except the amino acids in the
After the 20 ns MD trajectories, the low loop between the helices. (Figure 5.)
fluctuation with the RMSD value of
backbone atoms and inhibitor is found with
the RMSD value lower than 1 Å, as shown
in Figures 2-3. Therefore, trajectories during
20-100 ns are selected for further study.
Figure 5. Amino acids showing high

fluctuations are labeled in yellow (residue
667-678) and blue (residue 796-798)
Figure 2. RMSD of backbone atoms of 100
ns MD trajectories 3.2 Binding investigation
The 20-100 ns of MD trajectories
were clusterized using cluster analysis using
RMSD cut off of 1.5 Å. In the first cluster,
about 81 % of total conformations are found.
The representative structure from this cluster
was selected for investigating the binding
mode as shown in Figure 6. Then,
conformations of 20-100 ns MD trajectories
were further analyzed conformations and
investigated the number of hydrogen bonds
Figure 3. RMSD of inhibitor of 100 ns MD between inhibitor and amino acids in the
trajectories binding pocket.
Table 1. Interaction energy of amino acids
within 4 Å in the binding pocket
Residue Interaction Energy
(kcal/mol)
TYR612 0.01
HIS613 -0.05
ASN661 -0.54
ASN662 -1.23
SER663 0.12
TYR664 -2.84
ILE665 -1.04
Figure 6. Selected conformation from LEU725 -0.24
cluster analysis
LEU765 -0.30
ALA767 0.29
ILE768 0.08
ALA779 -1.38
VAL782 -3.15
ALA783 -0.69
PHE786 -1.70
LEU804 -2.76
ILE813 -0.12
Figure 7. Number of hydrogen bonds MET816 0.86
between sildenafil and PDE5 GLN817 0.43
From the results, hydrogen bond PHE820 -8.53

interactions between sildenafil and PDE5
are found. The number of hydrogen bond
interactions are found between 1-3 H-bonds
as shown in Figure 7. To confirm the
interactions between sildenafil and amino
acids in the binding pocket, quantum
chemical calculations were selected to study
the binding energies to each amino acid.
Residual interaction energies were
investigated by using M062X/ 6-31g(d)
level of method.
Figure 8. Important amino acids from QM
From the result of QM calculations
calculations
as shown in Table 1, there are 4 amino acids
(TYR664, VAL782, LEU804 and PHE820)
4. Conclusion
with the interaction energies lower than -2
In summary, molecular dynamics
kcal/mol. Hydrogen bond, interaction
simulations and quantum chemical
and H-pi interactions were found to be key
calculations are able to explain structural
interactions with inhibitor.
stability and interaction energies between
amino acids and inhibitor in the binding
pocket. Twenty amino acids are found
within 4 Å from sildenafil. The key amino
acids form H-bond interaction (TYR664), 2. William, D. S. Rev Cardiovasc Med.
- interaction (PHE820) and H– 2002, 4, 17–25.
interaction (VAL782 and LEU804) to 3. Monalisa, F. A.; Fabio, R. F.; Eirini, B.,
sildenafil. The results reveal the structural Anelia, H.; Isaac, L.; Rodrigo, B. de
effect of sildenafil which will be useful to Alexandre.; Faiyaz, A.; Vincent, M.;
improve the activity of this derivative. Constantine, A. S. Endocr Rev. 2014,
35, 195–233.
Acknowledgements 4. Huanchen, W.; Yudong, L.; Qing, H.;
This work was supported by Jiwen, C.; Roya, Z.; Sharron, H. F.;
Department of Chemistry, Faculty of Jackie, D. C.; Howard, R.; Zhongcheng,
Science, Kasetsart University, Kasetsart X.; Guiting L.; Hengming, J. Biol.
University Research and Development Chem. 2006, 281, 21469–21479.
Institute (KURDI), Science Achievement 5. Yi-You, H.; Zhe, L.; Ying-Hong, C.;
Scholarship of Thailand (SAST) and Ling-Jun, F.; Yinuo, W.; Xingshu, L.;
Thailand research fund (DBG6080005). Hai-Bin, L. J. Chem. Inf. Model. 2013,
53, 3044−3053.
References 6. Boolell, M.; Gepi-Attee, S.; Gingell, C.
1. Michael, P. D. Med. Chem. Lett. 2012, J.; ALLEN, J. M. Br J Urol. 1996, 78,
22, 6794–6800. 257–261.
Molecular docking study of the alkaloids from Tilicora triandra against
acetylcholinesterase for the treatment of Alzheimer’s disease
Pathumwadee Yotmanee*, Kanokporn Lehboon, Apichart Suksamrarn
Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand.
*E-mail: pathumwadeei@gmail.com
Abstract:
Alzheimer's disease (AD), a neurodegenerative disorder of the central nervous
system, is believed to be a multifactorial syndrome. Cholinergic hypothesis is one of the most
important theory for currently available drug therapies for AD. This cholinergic deficit is due
to acetylcholinesterase (AChE) enzyme action, which hydrolyses the acetylcholine (Ach) in
the synaptic process. It can be reversibly enhanced by inhibiting the activities of AChE
enzymes. Tiliacora triandra (Colebr.) Diels or Ya-nang in Thai belongs to the family of
Menispermaceae. It is used as medicine in traditional folklore including anti-alzheimer. This
study was carried out to determine the effect of bisbenzylisoquinoline alkaloid from root of
Tilicora triandra against AD involving AChE inhibitory properties. The experimental data
showed alkaliod analogue, i.e., O-methyl analogue of Tilliacorinine (compound 079) is
significantly better anti-AChE than galantamine activity. The molecular docking study helps
to understand the bisbenzylisoquinoline derivatives and human AChE enzyme interaction to
reveal the molecular basis for their binding to AChE. It was found that compound 079 was a
strongest binding with hAChE. There are hydrophobic and electrostatic interactions with only
peripheral anionic site where the hydrogen bonding with Thr75, Tyr72 and Ser293 as well as
the cation-pi with Trp286 residues were found.
1. Introduction neuropathological point of view, AD is

The elderly related diseases become characterized by cholinergic deficit that is
increasingly prominent with the aging of the due to acetylcholinesterase (AChE) enzyme
population. The incidence of Alzheimer’s action, which hydrolyses the ACh in the
disease (AD) in scenario shows the elevated synaptic process. Therefore, acetyl-
trend year by year. AD affects about 30 cholinesterase (AChE) has been considered
million people worldwide and estimates will as an effective target for the treatment of
be rising to 107 million people in 2050.1 AD AD, and hence acetylcholinesterase inhibitor
is a kind of neurodegenerative disorder (AChEI) was also regarded as an important
related to progressive cognitive impairments agent for AD therapy.8
of the patients such as memory loss, relative Five drugs have been approved by
decline in language skill and guidance.2,3 the US Food and Drug Administration
It causes a huge impact for the (FDA) for AD treatment such as tacrine,
social, economic, and political areas. AD donepezil, rivastigmine, galantamine and
multifactorial syndrome with several target memantine.10 However, because of the
proteins contributing to its etiology.4,5 There bioavailability problems and side effects of
are many hypotheses about its pathogenesis, current pharmacotherapy, therefore more
such as free-radical injury hypothesis, effective AChEIs to treat AD are urgently
amyloid peptide hypothesis, cholinergic required.11 In recent years, several effective
hypothesis and Tau hyper-phosphorylation AChEIs such as Huperzine-A, Galantamine
hypothesis.6-9 Cholinergic hypothesis is and Physostigmine derived from medicinal
one of the most important theory for plants showed that they possessed the
currently available drug therapies.8 From a advantages of higher inhibitory activity and
insignificant toxicity and side effects.12-14 against hAChE was almost 10 times greater
Tiliacora triandra (Colebr.) Diels (Ya-nang than that of 073. Since their inhibitory
in Thai) belonging to the family of mechanisms against hAChE are still unclear,
Menispermaceae was used as food and it is of great interest to investigate the
medicine in traditional folklore. It has been binding mechanisms of these inhibitors by
applied for anti-bacterial, anti-malarial, anti- molecular docking calculation. This study
cancer, anti-inflammation, anti-oxidant, anti- can be provided a molecular basis for
fever, alcohol detoxification, and anti- understanding on the structural modification
alzheimer. of alkaloid analogues influence their binding
The 3D structure of AChE was found affinities.
to exist in multiple forms.15,16 The available
X-ray crystal structures of Torpedo 2. Materials and Methods
californica AChE (TcAChE) reveal that the Molecular docking of bis-benzyl-
active site is located at the bottom of a isoquinoline analogues was performed using
narrow cavity about 20 Å deep termed the Autodock 4.2 along with AutoDockTools
aromatic gorge. The active site of the AChE (ADT).18 The compounds were built by
consists of a catalytic triad (CT) composed using ACDLab and energy optimization was
of Ser200···His440···Glu327 and a catalytic performed with HF/6-31G. Crystal structure
anionic site (CAS) composed of Trp84, of hAChE in complex with galantamine was
Phe330, and Glu199. Additionally, there is a obtained from Protein Data Bank with PDB
peripheral anionic site (PAS) composed of ID: 4EY6.19 This protein was edited using
Asp72, Tyr70, Tyr121, Trp279, and Phe290, ADT to remove all water molecules and to
located at the gorge entrance as shown in generate all hydrogen atoms of the
Figure 1.17 molecule. Non-polar hydrogens and lone
pairs were then merged and each atom was
assigned with Gasteiger partial charges. A
grid box of 80 × 80 × 80 points with a
spacing of 0.375 Ǻ was positioned at the
center of active site gorge. For each docking
experiment, 250 independent dockings were
carried out. The lowest docked energy of
each conformation in the most populated
cluster was selected. Analysis and
visualization of the docking results were
done using Acceryls Discovery Studio 2.5
Figure 1. Active site of A) TcAChE PDB (Acceryls, Inc., San Diego, CA, USA).
code: 1EVE17 and B) human
acetylcholinesterase (hAChE) PDB code: 3. Results and Discussion
4EY6 3.1 Evaluation of docking performance
and accuracy into hAChE binding site
In the current work, four T. triandra For selecting the suitable docking
derivatives, Tiliacorinine (073), Tiliacorine parameter of ADT in this study, the native
(074), 2’-Nortiliacorinine (075) and galantamine drug was docked back to its
synthesized O-methyl analogue of binding pocket of hAChE protein (PDB
Tiliacorinine (079), have been discovered code: 4EY6). The root–mean–square
with strong or moderate activity against deviation (RMSD) of overlaying docking
human AChE (hAChE) (Table 1). Although pose on the X-ray pose was 0.4846 Å
073 and 079 analogues have quite similar (Figure 2). The RMSD of the native ligand
structures, the experimental data in this docking conformation less than 2.0 Å from
study show that the inhibitory effect of 079 the experimental one can be accepted to
validation of using the calculating scoring
function.20 Then, this proper setting para-
meter was used to investigate the orientation
of bis-benzylisoquinoline analogues in the
hAChE binding gorge.
3.2 Docking study of the bis-
benzylisoquinoline analogues
Figure 2. Active site of hAChE (4EY6).
Table 1. Inhibitory activity IC50 (μM) of RMSD values for re-docking of
bis-benzylisoquinoline analogues galantamine.
Compound Structure IC50 (μM)
To provide insight into the
mechanism of inhibition, the molecular
docking was performed to gain the
073 2.33
functional and structural aspect of the ligand
within the enzyme’s active site. The detail
interactions of bis-benzylisoquinoline
analogues (compounds 073, 074, 075 and
079) at the active site of the enzymes are
given in Figures 3. The bisbenzyl-
isoquinoline analogue is symmetrically
074 1.17
structured, each consists of three rings
where were labeled as A, B and C
followed by the numbers of 1 and 2 in the
left- and right-hand sides, respectively.
The connecting ring between the two sides
of the structure was labeled as D. As a
075 0.74
docking result, the carriage of bis-
benzylisoquinoline is at the mouth of the
hAChE because of the large size of the
inhibitor molecule. This makes it possible to
place only on the PAS region compared to
the smaller galantamine that can reach up to
the catalytic triad (Figure 3).
079 0.22
The order of the stability of binding
free energies is 073 < 074  075 < 079
which exhibited the Gbind values of -9.65 , -
9.97, -9.66 and -10.23 kcal/mol,
respectively, that is relatively corresponding
with IC50 values. All four inhibitors has
hydrophobic and electrostatic interactions
Galantamine21 1.45 with the residues in the regions of PAS
(green colored residues). The docking
calculation results show the less number of
interactions between 073 and hAChE. There
are only two hydrogen bonds between -
OCH3 on A1 ring and -OH on C2 ring with
Thr75 and Ser293, respectively (Figure 3C),
which corresponds to the highest IC50
values obtained from the bioactivity data.
Figure 3. Binding orientations and interactions of bis-benzylisoquinoline analogues with
protein residues at the active site of hAChE. A) Bis-benzylisoquinoline labeled structure, B)
galantamine, C) 073 analogue, D) 074 analogue, E) 075 analogue and F) 079 analogue
The 074 analogue in Figure 3D is the ring and Tyr72 as well as cation-pi
R-configuration of 073 by changing the H at interaction between N16 on B2 ring and
position C19 on B1 ring resulting to turn it Trp286 were additionally observed. The
out since the steric hindrance between its –OCH3 moiety on C1 side was still
amine group and Trp286. Then, the B2 ring stabilized by hydrogen bond and
was turned back in the same place to hydrophobic interactions from S293 and
preserve the similar interaction with 073 V365 residues, respectively.
analogue by forming the hydrogen bond As a result, among bis-
interactions between –OCH3 on the A1 and benzylisoquinoline analogues, the 079 is the
C1 rings with Thr75 and Ser293, best for hAChE inhibition. Additionally, it
respectively. Additionally, it made another demonstrates the higher absolute binding
hydrogen bond interactions between O9 on free energy than galantamine (Gbing of
the D ring with Tyr72 and cation- -8.91 kcal/mol). Even if the galantamine has
interaction between the amine group on B2 the amount of 3 hydrogen bonds that interact
ring and Trp286. The –OCH3 substitute on to Glu202, Ser203 and Tyr337 residues
the C1 ring is also stabilized by hydrophobic locating in the catalytic triad and anionic
interaction from Val365. This result makes a regions, but the smaller molecular size
more stable attachment of 074 than 073 of galantamine causes the decreasing
analogue to hAChE. The difference between of nonbonding interaction compared to 079
073 and 075 (Fig. 3E) analogues is the analogue (Figure 3B).
substitute of an amine group on the B2 ring
(changed from the –N(CH3)3 to -NH3). The 4. Conclusion
smaller amine group leads to the orientation The present study investigated hAChE
of 075 as 074 pattern that provides the inhibitory activities of the bis-
interaction and stabilization to hAChE as benzylisoquinoline analogues using
strong as 074 analogue. Structural molecular docking technique. All four
modification of high quantity but low inhibitors (compounds 073, 074, 075 and
efficiency substance from natural product 079) have hydrophobic and electrostatic
has been recently more powerful. As it was
interactions with only the region of
found that the modification of 073 to 079
peripheral anionic site (PAS). The O-methyl
structure by replacing –OH group at C37
position on the C2 ring with O-methyl group analogue of 073 (compound 079) has a
leading to increase the hAChE inhibitory highest binding affinity into binding site of
activity greater than 073 about 10 fold. As the hAChE, in which it forms the hydrogen
shown in Figure 3F, the different orientation bonding with Thr75, Tyr72 and Ser293 as
of 079 and 073 analogues in the PAS region well as the cation-pi interaction with Trp286
of hAChE was observed due to the –OCH3 residues. Additionally, the hydrophobic
moiety on C1 or C2 ring that can be fit well interactions between -OCH3 groups on C1
with the Leu289’s hydrophobicity. The 073 and C2 rings were built with Val365 and
molecule is positioned by arranging the - Leu289, respectively. The absolute Gbind
OCH3 moiety on the C1 ring to the Leu289 order between these four analogues with
residue while the 079 has the –OCH3 moiety
hAChE (079 > 075  074 > 073) was
both on the C1 and C2 sides, it can be
possibly to select either –OCH3 substitute on reasonably in good agreement with the
both rings to Leu289. However, the experimentally derived IC50 values.
selection of C2 side can be increase the
interaction with hAChE more than 073 Acknowledgements
analogue, which has 3 hydrogen bonds, 1 P.Y. thanks The Thailand Research
cation- interaction and 2 hydrophobic Fund (DBG5980003). K.L. thanks the
interactions. The hydrogen bond of O9 on D financial support from the Center of
Excellence for Innovation in Chemistry 10. http://www.fda.gov/NewsEvents/Testim
(PERCH-CIC), Office of the Higher ony/ucm110879.htm.
Education Commission, Ministry of 11. Shen, L. L.; Liu, G. X.; Tang, Y. Acta.
Education. The research facilities, software Pharmacol. Sin. 2007, 28, 2053–2063.
packages and computing times from 12. Wang, H.; Tang, X. C. Zhongguo. Yao.
Computational Chemistry Unit Cell, Faculty Li. Xue. Bao. 1998, 19, 27 – 30.
of Science, Chulalongkorn University is 13. Scott, L. J.; Goa, K. L. Drugs 2000, 60,
gratefully acknowledged. 1095–1122.
14. Yoshida, S.; Suzuki, N. Eur. J.
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Graham, K.; Arrighi, H. M. Alzheimers 16. Dvir, H.; Harel, M.; Bon, S.; Liu, W.
Dement. 2007, 3, 186–191. Q.; Vidal, M.; Garbay, C.; Sussman, J.
3. Francis, P. T.; Palmer, A. M.; Snape, L.; Massoulié, J.; Silman, I. Embo. J.,
M.; Wilcock, G. K. J. Neurol. 2004, 23, 4394–4405.
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2011, 12, 543–543. Martin, E.; Mitnitski, A.; Geula, C. J.
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6. Harris, M. E.; Hensley, K.; Butterfield, 19. Cheung, J.; Rudolph, M. J.; Burshteyn,
D. A.; Leedle, R. A.; Carney, J. M. Exp. F.; Cassidy, M. S.; Gary, E. N.; Love,
Neurol. 1995, 131, 193–202. J.; Franklin, M. C.; Height, J. J. J. Med.
7. Hardy, J.; Selkoe, D. J.; Science 2002, Chem. 2012, 55, 10282–10286.
297, 353–356. 20. Wang, R.; Lu, Y. J. Med. Chem. 2003,
8. Bartus, R. T.; Dean, R. L. 3rd.; Beer, B.; 46, 2287–2303.
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Comparative study on two polymerization methods for synthesis of
copper(II) ion imprinted polymers
Angkana Chaipuang1,2, Chalida Phungpanya1,2, Chalermporn Thongpoon3,
Kanchana Watla-iad1,2, Orawan Suwantong1,2, Prachak Inkaew1,2, Theeraphan Machan1,2*
1
Program of Applied Chemistry, School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
2
Center of Chemical Innovation for Sustainability, Mae Fah Luang University, Chiang Rai 57100, Thailand
3
Program of Chemistry, Faculty of Science and Technology, Pibulsongkram Rajabhat University,
*E-mail: theeraphan.mac@mfu.ac.th
Abstract:
The comparative between two methods of suspension and bulk polymerizations for
synthesis of copper (II) ion imprinted polymers (Cu-IIP) was prepared in the ratio of 1:4:8
(template ion: mixed functional monomers: crosslinker) under the condition of suspension
polymerization and the ratio of 1:4:20 (template ion: functional monomer: crosslinker) in the
condition of bulk polymerization. The morphologies of the obtained polymers were
characterized by scanning electron microscopy and Fourier transform infrared spectroscopy.
The binding kinetics of the polymers were also evaluated by using %bound. They were found
that Cu-IIP materials exhibited a good recognition by giving a high %bound recognition and
adsorption kinetic studies.
1. Introduction higher concentration levels, it is toxic and

The general procedure of ion may cause different diseases5 as damage the
imprinted polymers (IIP) materials consists nervous system and behavior problems. The
of the preparation of metal ligand complex concentration of copper in many samples is
and the copolymerization between the low, so sensitive, and selective method were
functional monomer and crosslinker to required for determination of copper in
create polymer materials that has the many sample such as food, environmental,
specific cavities in three dimensional water, and biological samples.6
networks. IIP materials are usually prepared Normally, flame atomic absorption
by three steps. The first one, the template of spectrometry (FAAS),7 ion chromatography
metal ion and polymerizable ligand or (IC),8 and UV-Vis spectroscopy (UV-Vis)9
functional monomer are mixed to form the have been applying as the analytical method
self-assembly. Secondly, the mixed solution for determination of copper ion. They are
was poly-merized by using crosslinker and often determined of copper at millimolar
the last, the metal was removed by (mM), exhibiting the advantages such as
extraction with acidic solution.1,2 Nowadays, ease to use, high precision and inexpensive
IIP has become very popular because of technique. However, they are requiring large
their ease of synthesis, high environmental amount of sample solution and complicating
stability and high ability to be reused, as process when preparation of the analytical
well as broad applicability and high samples. The aim of this work is to develop
selectivity towards the target metal ion due the good efficiency ion imprinted polymer
to a recognition effect resulting from their for high effective properties and deter-
preparation process.3,4 Copper is the type mination of copper(II) ion.10 Cu-IIP was
trace elements that are very important for prepared for separation of Cu(II) ion by
the human body and plays an essential role using a coordination bond between the
in manifold physiologic processes. The selected ligands. It is the most frequently
efficiency is a symptom of anemia, but at used method to prepare imprinting polymer
© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) PO1
due to its simplicity. The morphological bath for 24 h. This condition is named Cu-
characterization of the polymer particles was IIP02.12
evaluated by scanning electron microscope After the polymerizations were
(SEM) and Fourier Transform Infrared completed the obtained polymers were
Spectroscopy (FTIR).11 In addition, %bound grinded in motar and they were washed with
recognition and adsorption kinetic studies 1 M HCl 50 mL followed by washing with
were qualified to assess their high mixture of acetone/DI water (50 %, v/v for 1
adsorption capacity for copper(II) ion. h. These processes were repeated 4 times for
complete removal of all Cu(II) ion template.
2. Materials and Methods The polymers were finalized washing with
2.1 Reagents and chemicals MeOH and drying in hot air oven at 60 °C.
Copper(II) sulphate pentahydrate In part of control experiment, non-imprinted
(CuSO4.5H2O) was obtained from Quality polymers (NIP) each methods have also
Reagent Chemical. Methacrylic acid been synthesized in the same procedure but
(MAA), 4-vinyl pyridine (4-VP), itaconic without addition of the template Cu(II)
acid (ITA), ethylene glycol dimethacrylate ion.12
(EDMA).and.1,1'azobiscyclohexanecarbonit 2.3 The morphologies of Cu-IIP and NIP
rile (ACBN) were supplied by Sigma- The surface morphologies of the
Aldrich. Methanol (MeOH) and toluene polymer were observed using SEM (LEO
were obtained from Laboratory reagents & 1450 VP LEO) with measured at
fine chemicals. 36.5-38.0% of Hydrochloric magnification 5.00 KX. Cu-IIP and NIP
acid (HCl) was supplied by J.T. Baker. were dried under vacuum. The copper(II)
2.2 Polymer preparation ion imprinted polymers were coated with the
The suspension polymerization of electrically conductive material with gold
Cu(II) ion imprinted polymer was carried sputtering machine. The collecting of static
out by preparing CuSO4.5H2O 1 mmol and 4 electric fields at the specimen due to the
mmol of MAA and 4-VP as functional electron irradiation desired during imaging.
monomer and dissolved in 10 mL of DI 2.4 FTIR spectrometer (Fourier transform
water. After stirring the mixture solution for infrared spectrometer)
15 min, 8 mmol of crosslinker as EDMA FTIR-Spectra were characterized by
and 195 mg initiator ACBN were added and using FTIR spectrometer (Perkin Elmer
stirring for a further 15 min followed by model Spectrum GX). The spectrum was
adding 10 mL of toluene and 2%wt of HEC scan 32 numbers and samples can be ground
5 mL. Then the solution was purged with into a fine powder and mixed in a powder
nitrogen gas for 15 min. The mixture matrix of potassium bromide (KBr) by
solution was polymerized at 70 °C in an oil diffuse sample preparation technique. The
bath for 24 h. This condition is named Cu- wavenumber was controlled from 400 to
IIP01. 4,000 cm-1.
The bulk polymerization of Cu(II) 2.5 %Bound recognition experiments
ion imprinted polymer prepared by mixing %Bound recognition was studied by
CuSO4.5H2O 1 mmol and 2 mmol of ITA as adding 20 mg Cu-IIP and NIP of into 1 mL
functional monomer then dissolving in 30 of the initial copper(II) ion in methanol
ml of MeOH. After stirring the mixture solution at concentration 8.0 mM and then
solution for 15 min, 20 mmol of crosslinker stirring at 180 rpm 30 °C for 1.5 h. The
ethylene glycol dimethacrylate (EDMA) and solution in microtube was centrifuged for 10
100 mg initiator ACBN were added and min at 18000 rpm before removal of the
stirring for a further 15 min. The solution supernatant. Afterwards, 0.2 mL at different
was saturated with nitrogen gas for 15 concentration was pipetted into 96-well
minutes then polymerized at 70 °C in an oil plate before measuring at 750 nm by using
the spectrophotometer. The %bound recog- 1:4:8 and Cu-IIP02 has been synthesized in
nition was calculated using Eq. (1) and (2). methanol which is polar protic organic
Eq. (1) solvent and good porogenic solvent for
dissolving CuSO4.5H2O under ultrason-
Where Ci (mM) is the initial ication. The mixture of CuSO4.5H2O:ITA:
concentration of CuSO4.5H2O solution; Cf EDMA in the ratio of 1:2:20. Then, 1,1'-
(mM) is the final concentration of azobiscyclohexanecarbonitrile as initiator
CuSO4.5H2O solution; V (L) is the volume was also added into the solution. The
of solution; and W (g) is the weight of the solution mixture was then heated to 70 °C
polymers used. under nitrogen atmosphere. Cu-IIP01 in the
first step, copper ion interact with oxygen
Eq. (2) atom of the carboxyl from two MAA
Where Q bound is the amount of molecules and the nitrogen atom from 4-VP
analyte bound to the polymer after loading; molecules and Cu-IIP02 in the first part
and Q initial is the initial amount of analyte copper ion interact with oxygen atom of the
before loading to the polymer.13 carboxyl from four ITA molecules14 via
coordinate bonding to form prepolymeriza-
IF = QMIP/QNIP Eq. (3) tion complex in porogens. EDMA is the
Where QMIP and QNIP represent the crosslinker to link all MAA, 4-VP and ITA
binding of polymers with template molecule functional monomers. After these complete
of MIP and NIP, respectively. self-assembly processes, the radical initiator
2.6 Adsorption kinetic experiments ABCN was added to generate the free
Analysis of the adsorption kinetics of radical polymerization. In the last synthetic
copper ion solution on the imprinted step, the template Cu(II) ion is removed
polymer was carried out in a batch process. from the obtained imprinted polymer by
In each test, 20 mg of Cu-IIP and NIP were washing with acidic solution to produce high
placed in 1 mL of 8.0 mM methanolic recognized binding sites of copper(II) ion in
CuSO4.5H2O solution and then stirred at 180 the polymer.
rpm 30 °C. The solution added in microtube 3.2 Fourier transform infrared spectroscopic
was centrifuged for 10 min at 18000 rpm measurements
before removal of the supernatant. After- Figure 1 exhibits FTIR spectra of
wards, 0.2 mL of the solution at different Cu-IIP01-02 after removal of copper(II) ion
time intervals, of 1, 3, 7, 9, and 24 hour was and NIP01-02. All of FTIR spectrum signals
pipetted and placed into 96-well plate and are similar spectra that display the FTIR
measured the absorption at the wavelength spectrum in the range of 4000-400 cm-1. The
of 750 nm for copper ion solution broaden peak at 3655 cm-1 and 3612 cm-1
(SPECTRO star Nano BMG LabTech). The are the stretching vibration of O-H in metha-
observed absorptions were used to calculate crylic acid and itaconic acid, respectively.
the adsorption capacity (Q) by using Eq. (1), The IR band at 3000 cm-1 is the C-H stretch-
(2) and (3). ing of methacrylic acid, at 2990 cm-1 is the
stretching of C-H in itaconic acid and
3. Results and Discussion ethylene glycol dimethacrylate. The C=O
3.1 Cu-IIP preparations stretching vibration is also observed at 1750
Cu-IIP01 has been synthesized in DI cm-1 which also shows the character of
water and toluene which are polar protic polymers. The band of C=N stretching
solvent and non-polar aromatic solvent as a vibration at 1645 cm-1 displays that 4-vinyl-
good porogens for dissolving CuSO4.5H2O, pyridine is polymerized and become the
functional monomer and crosslinker under polymerized matrix. The C=C stretching
ultrasonication. The mixture of CuSO4. vibration at 1488 cm-1 is from the part of
5H2O:MAA&4-VP:EDMA in the ratio of molecular structure of 4-VP and the broaden
spectra at 1188 cm-1 and 1256 cm-1 were the
band of C-O bond stretching of the metha-
crylic acid, itaconic acid and ethylene glycol
a
dimethacrylate which are polymerized to
form the polymeric matrix. This result
confirms the same functional group of
polymers so that they exhibit the homo-
logous cavity.
Figure 1. FTIR spectra of Cu-IIP01-02 and

NIP01-02
3.3 Characterization of polymers

The morphological characterization
of the polymers was shown in Figure 2. Cu-
IIP01-02 and NIP01-02 showed the same
size of polymer particles. The suspension
polymerization method can get the small
drop of polymer particles so that they show
small porous and rough surfaces. The porous
structure probably formed during polymer-
ization of the copper(II) polymer complex
from the polymerization solutions in DI
water, toluene and methanol which were
used as porogenic solvents during polymer
particle formations.
Figure 2. SEM photographs of the polymer

particles Cu-IIP01 (a), NIP01 (b), Cu-IIP02
(c), and NIP02 (d)
IIP02 is 1.70 and 1.21 respectively. The time
is very important to optimize that confirm
the suitable time of polymer particles to
recognize with copper(II) ion.
4. Conclusion
The Cu-IIP01 and Cu-IIP02 were
prepared by suspension and bulk polymeri-
zation, respectively, with using copper(II)
ion as the template molecule, MAA, 4-VP
and ITA as the functional monomer, EDMA
as the crosslinker and ACBN as the initiator
Figure 3. %Bound recognition studies of dissolved in DI water, toluene and methanol,
polymers respectively. The Cu-IIP materials showed
the good efficiency and small polymer
3.4 %Bound recognition studies particles when comparing to NIP. Moreover,
%Bound recognition showed specific Cu-IIP materials display more effective and
% bound with template ion in range from recognize abilities to the template Cu(II) ion
1.38-5.52 % of Cu-IIP01 and 0.42-2.75 % of than the NIP at 9 h that is suitable time to
Cu-IIP02 when the time at 1.5 h. The NIP apply with the real sample analysis.
showed %bound recognition less than Cu-
IIP. This obtaining results show the recog- Acknowledgements
nition property of the synthesized polymers. We would like to acknowledge the
The recognition is very important to selec- scientific and Technological Instrument
tive adsorption performance of the polymers Center (STIC), office of postgraduate
as shown in Figure 3. studies of Mae Fah Luang University for
FTIR, SEM, Microplate reader and financial
support from Science Achievement
Scholarship of Thailand (SAST) and Mae
Fah Luang University (MFU).
References
1. Roushani, M.; Abbasi, S.; Khani, H.;
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2. Mafu, L. D.; Msagati, T. A.; Mamba, B.
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Mater. Chem. A 2015, 3 (26), 13598-
3.5 Kinetic adsorption studies 13627.
Figure 4 shows kinetic curves of 4. Jiang, Y.; Kim, D. Chem. Eng. J. 2011,
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Cu-IIP01 and 4.24-6.44 % of Cu-IIP02 as Spectrochim. Acta A 2016, 153, 45-52.
the time increased. The adsorption tended to 6. Abbasi, S.; Roushani, M.; Khani, H.;
saturation after 3-5 h. NIP showed line Sahraei, R.; Mansouri, G. Spectrochim.
graph likewise trend to Cu-IIP but lower Acta A 2015, 140, 534-543.
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imprinting factor (IF) of Cu-IIP01 and Cu-
7. Luo, X.; Huang, Y.; Deng, F.; Luo, S.; 12. Chaipuang, A.; Phuangpanya, C.;
Zhan, Y.; Shu, H.; Tu, X. Microchim. Machan, T. The International Polymer
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93-111.
The removal of cationic dye from wastewater with
polymethacrylic acid grafted silica gel
Praethong Laopa1*, Duangruthai Puksachat2
1
Department of General Science, Faculty of Science and Engineering, Kasetsart University,
Chalermphrakiat Sakon Nakhon Province Campus, Sakon Nakhon 47000, Thailand
2
Bachelor of Science (Applied Chemistry), Department of General Science, Faculty of Science and Engineering,
Chalermphrakiat Sakon Nakhon Province Campus, Sakon Nakhon 47000, Thailand
*E-mail: paethong.s@ku.th
Abstract:
This research focuses on the surface modification of silica gel with polymethacrylic
acid (PMAA) by ARGET ATRP for removal of cationic dye from aqueous solution. The
PMAA grafted silica gel was successfully prepared by functionalized (3-aminopropyl)
trimethoxysilane (APTMS) onto silica surface, previously modified with 2-bromoisobutyryl
bromide which known to be an efficient initiator for ATRP, and subsequently the polymer
was then directly grown via ARGET ATRP in aquatic condition. The resulting PMAA
grafted silica gel was analyzed by FT-IR and presents clear evidence that the PMAA is
grafted onto the silica surface. Various experimental factors for removal cationic dyes such as
contact time, initial dye concentration, adsorbent dosage, and pH of dye solution were
examined. According to the experiments, the optimum condition for adsorption of gentian
violet was by using 0.3 g of the adsorbent material in dye solution at pH 9 with 60 minutes
contact time. The experimental data were fitted nicely to the Langmuir isotherm with R-
square of 0.9947. The recycling efficiency of the PMAA grafted silica gel after five cycles by
stripping with 0.1 M acetic acid was reduced only 10% from 98%. The results indicated that
this modified material is a multiple-use adsorbent.
1. Introduction polymethacrylic acid (PMAA) by surface-

Nowadays, pollution of dyes initiated activators regenerated by electron
wastewater is becoming increasingly transfer-atom transfer radical polymerization
alarming. The source of dyes wastewater is (SI-ARGET ATRP) for the removal of
mainly coming from textile, plastic, paper, cationic dye, namely crystal violet (CV),
food, and cosmetic industries. Adsorption from aqueous solutions has been
methods are a very effective separation investigated. This polymer is rich in
technique for the removal of dyes from carboxylic functional groups, which may
wastewater because of its easy operation and bind cationic dye molecules in aqueous
versatility.1,2 Silica (SiO2) is one of the most solution. ARGET ATRP has been
extensively used adsorbents for eliminating extensively attempted for the grafting of
dyes from wastewater because it is an inert polymers onto SiO2. This method uses low
and non-toxic material with high specific the concentration of the catalyst (several
surface area (~800 m2/g), relatively large parts per million) by using an excess amount
pores and high mechanical stability.3,4 of reducing agent to regenerate CuI from
However, the surface of silica contains CuII and maintaining appropriate CuI/CuII
acidic silanol (Si-OH) which causes a strong balance, allowing the reaction in the
and often irreversible non-specific presence of oxygen.7,8 The study consists in
adsorption. Thus, surfaces of silica have the characterization of each modified silica
been modified with the organic compound to and the determination of the factors
improve properties in recent years.5,6 In the affecting the adsorption, including the pH of
present study, silica modified with
Figure 1. Schematic representation of the synthesis of polymethacrylic acid grafted silica gel
by surface-initiated ARGET ATRP
the solution, adsorbent dosage, dye rinsed with acetone and finally dried in an
concentration, contact time and reusability. oven at 110 °C for 1.0 h.
2.3 Preparation of 2-bromoisobutyryl
2. Materials and Methods amide immobilized silica gel (SiO2-BIB)
2.1 Chemical and materials 10 g of the SiO2-APTMS was
The silica gel for column immersed in 30 mL of dichloromethane.
chromatography was obtained from Merk, Then, 12 mL (85.0 mmol) of Et3N was
with a nominally 0.063-0.200 mm particle added to the reaction mixture and stirred for
diameter. Methacrylic acid (Aldrich, >98%), 10 min. After that, 3 mL (9.5 mmol) of BIB
(3-aminopropyl) trimethoxysilane (APTMS) was added and the reaction was allowed to
(Aldrich, >98%), 2-bromoisobutyryl proceed for 16 h at ambient temperature (27-
bromide (BIB) (acros, 98%), 2,2-dipyridyl 33 ºC). After the obtained SiO2-BIB was
(Across, 99+%), copper(II) bromide filtered and washed with ethanol and
(Across, 99+%), 2,20-bipyridine (BiPy) acetone, it was dried by air at room
(Across, 99%), L-ascorbic acid (AA) temperature prior to further modifications.
(Aldrich, 99%), triethylamine (Et3N) 2.4 Preparation of polymethacrylic acid
(Across, 99+%), dimethylaminopyridine grafted silica gel (SiO2-PMAA) via
(DMAP) (fluka, 98%), were used as ARGET ATRP
received. Solvents and other reagents were Initially, SiO2-BIB (10 g) was added
analytical reagent grades and used without to the 250 mL of the round bottom flask
further purification. Ultrapure water, which containing the 100 mL of 3.3 M MA
had a resistivity greater than 18 M cm−1 monomer solution. The MA solution was
was used throughout this work. prepared by dissolving of MA (27.98 mL,
2.2 Preparation of (3-aminopropyl) 0.32 mol) in 60 mL ultrapure water and
trimethoxysilane (APTMS) functionalized then, the solution was adjusted to pH 9 by
silica gel (SiO2-APTMS) adding 2 M NaOH. The mixture was made
The silica gel (30 g) was cleaned in to a final volume of 100 mL with ultrapure
concentrated HCl (100 mL) under water. The solution of BiPy (0.059 g, 0.38
continuous stirred at 90 ºC for 4.0 h. mmol), and CuBr2 (0.040 g, 0.18 mmol) in
Subsequently, the acid-treated silica was 10 mL of ethanol was slowly added to the
rinsed with a large volume of ultrapure reaction mixture. After that, 0.0176 g of AA
water, and adjust to neutral by using 1 M was added and then the flask of the reaction
NaOH. The derivatized silica was filtered was sealed and the reaction was allowed to
and finally dried in an oven at 110 °C for 18 stir at room temperature for 6 h.9 The SiO2-
h. Next, the silica and 4.57 mL of APTMS PMAA particles were collected with filtered
were added to a 250 mL flask containing and washed with ultrapure water and
100 mL of toluene. Then the flask was acetone. The FTIR measurements were
sealed and the reaction allowed to proceed collected on the ATR mode of Perkin-Elmer
for 2 h under continuous stirring. The system 2000 with 32 scans at a resolution of
obtained SiO 2 -APTMS was filtered and 4 cm-1 with a frequency range of 400-4000
cm-1.
2.5 Adsorption test for 24 h to reuse in the reaction cycle with
The basic dye, crystal violet (CV), the fresh CV dye solution.
was selected for adsorption tests obtained
from AJAX Chemical. A stock solution with 3. Results and Discussion
the concentration of 5,000 ppm was 3.1 Preparation and characterization of
prepared and the solutions for adsorption modified silica gel
tests were prepared from the stock solution Figure 1 shows schematically the
to the desired concentrations (100-2,000 three-step approach to the immobilization of
mg/L). Fresh dilutions of the desired dye the PMAA on the silica gel surface. Initially,
concentrations were made at the start of the hydroxyl groups on the silica gel surface
each experiment. In adsorption experiments, were generated by heating the substrate with
0.3 g of modified and unmodified silica gels concentrated HCl. The hydroxyl groups on
were added into 50 mL of CV dye solutions the silica gel surface were reacted with
at the desired concentration and pH. The APTMS to produce the corresponding SiO2-
flasks were shaken on the orbital shaker at APTMS. The ATRP initiators were formed
150 rpm at ambient temperature (25±1 ◦C) on the silica gel surface by the reaction of
for 60 min to ensure the establishment of the 2-bromoisobutyryl bromide with the
equilibrium. After reaching the adsorption amino groups on SiO2-APTMS surfaces.
equilibrium, the solid was filtered, the Subsequently, PMAA was directly grown on
concentration of dye in the solution was the surface of silica gel by surface-initiated
measured by a spectronic 200 genesis via ARGET ATRP in the presence of CuBr2
spectrophotometer (thermo, USA) at the and bipy (mole ratio CuBr2/BiPy = 1/2) as
wavelength (λ) = 585 nm. The percentage of the catalyst in aquatic solution for 6 h
dye adsorption was calculated using the Eq. according to previously reported.9 The
1:10 chemical composition of all modified silica
(C  Ct ) gel was determined by ATR FTIR (Figure
%adsorption  0  100 eq. 1
C0 2). All spectra showed the strong and broad
Where C0 and Ct are the concentrations of peak around 900-1200 cm−1 indicate the
dye in solution (mg/L) at time t = 0 and t = t, asymmetric stretching of Si-O-Si and
respectively. The adsorption capacity (q, symmetric stretching of Si-OH, respectively.
mg·g-1) of adsorbents was computed using The typical peaks at 780 cm−1 and 628 cm-1
Eq. 2:10 were assigned to the Si-O vibrations. The IR
qe  (C0  Ct ) 
V eq. 2 spectra of SiO2-APTMS and SiO2-BIB did
W not show any difference to silica without
V is the volume of the solution added (L), modification because APTMS and BIB
and W is the amount of adsorbent (g). All groups were formed onto silica surface as
experiments were performed at least three selfassembled monolayers (SAMs) which
times, and all the results were reported as are not thick enough to measure by FTIR
average values. on ATR mode. The IR spectrum of the
2.6 Recycle experiment SiO2-PMAA surface (Figure 2d) shows new
The adsorption-desorption recycle peaks at the 1538 cm−1 and the 3400-3600
experiments were carried out by adding 0.3 cm−1 regions, arising from the stretching of
g of modified and unmodified silica gel in the carboxyl group and the OH group,
50 mL of CV dye solution (100 mg/L, pH 9) respectively. These indicated that the PMAA
at 25 ◦C, and the suspensions were shaken chains have been successfully grafted on the
for 60 min. The supernatants were separated surfaces of the SiO2-BIB.
by filtration and analyzed for remaining dye
content. Then, the solids were recovered by
incubating in 50 mL of 0.1 M acetic acid for
10 min and following 1 L of deionized water
between the cationic dye and the positively
charged surface obtained from the amine
group of APTMS moieties. The adsorption
efficiency has increased when the surfaces
of SiO2-APTMS changed to SiO2-BIB
because of the hydrophobic interaction
between the dye and the adsorbent. This
result confirmed that APTMS, BIB, and
PMAA were successfully grafted on the
surface of silica. Since the optimum pH for
Figure 2. IR spectra of a) SiO2, b) SiO2- adsorption of CV dye by SiO2 and SiO2-
APTMS, c) SiO2-BIB, and d) SiO2-PMAA PMAA was found to be 9, this pH was used
for further studies.
3.2 Dye adsorption by modified silica gel
3.2.1 Effect of pH and sorbent types on
dye sorption
The adsorption efficiency of the
SiO2, SiO2-APTMS, SiO2-BIB, and SiO2-
PMAA was investigated in batch systems.
The results show in Figure 3. As seen from
this figure, the percentage adsorption of
cationic dye by silica was low at the acidic
solution because the surface of silica was
positively charged, resulting in electrostatic
repulsion from the positive charge of the dye
molecule. However, it was negatively
charged at high pH, resulting in enhanced Figure 3. Effect of adsorbents (0.3 g) types
adsorption capacity through increased cation on the adsorption of crystal violet (100
exchange capabilities. The adsorption mg/L) at pH 3, 5, 7, 9
efficiency of the cationic dye increased
significantly at both acidic and basic
solutions when the silica has been modified
with PMAA. This behavior can be attributed
that the lower the pKa (4.462 ± 0.002) of
the PMAA moieties on the silica surface,12
the carboxylic groups were neutrally
charged. Therefore, this adsorption is
controlled by a hydrogen bonding between
cationic dyes and PMAA on the silica
surface. The percentage adsorptions of CV
dye by SiO2-PMAA at pH 5, 7, and 9
were high and slightly different because of
adsorbate uptake at the higher the pKa Figure 4. Effect of contact time on the
which the carboxylic groups of PMAA on adsorption of CV (100 mg/L) at pH 9 by
silica surface were negatively charged due to SiO2 and SiO2-PMAA
enhance the adsorption of cationic dye
through electrostatic attraction. The lowest
3.2.2 Effect of contact time
dye adsorption was observed with SiO2- The effect of contact time for the
APTMS because of the ion repulsion removal of CV by SiO2 and SiO2-PMAA is
shown in Figure 4. The adsorption processes
were fast in the first 10 min and then slow.
After that, the dye removal stayed stable in
the >50 min, which was indicative of
equilibrium. Nevertheless, the contact time
of all experiments was performed for 60 min
to ensure adsorption equilibrium.
3.2.3 Effect of adsorbent dosage and
adsorption isotherms
The effect of initial dye
concentration was investigated in the
concentration range 100 to 2000 mg/L. The Figure 5. Effect of adsorbent dosage on the
results in Figure 5 show that the adsorption adsorption of CV (100 mg/L) at pH 9 by
of cationic dye by two adsorbents increased SiO2 and SiO2-PMAA
with the increasing concentration.
Figure 6. Adsorption isotherms of adsorbents, the fitting curves of the Langmuir model of
CV on (a) SiO2 and (b) SiO2-PMAA, the fitting curves of the Freundlich model of (c) SiO2
and (d) SiO2-PMAA at 25 ◦C, respectively
The equilibrium adsorption data adsorbed (mg/g) at equilibrium. In the

were analyzed by fitting them to Langmuir Langmuir equation, qm is the maximum
and Freundlich models. The linear form of amount of adsorption (mg/g) and b is the
the Langmuir and Freundlich models is adsorption equilibrium constant (L/mg).
given in Eq. 3 and 4, respectively.11 Freundlich treatment gives the parameters,
Ce 1 C eq. 3 1/n, indicative of heterogeneity factors
  e
qe qmb qm related to binding strength and KF related to
1
log qe  log k F  log Ce eq. 4 the absorption capacity. The linearized
n Langmuir and Freundlich isotherms of dye
Where Ce is the equilibrium concentration of are shown in Figure 6. The obtained data of
the dye (mg/L), qe is the amounts of dye modified and unmodified silica gel were
well fitted with the Langmuir models (R2 = 4. Conclusion
0.9850 and R2 = 0.9947). The results The silica gel modified with PMAA
implied that the adsorption behavior of CV was successfully prepared by SI-ARGET
onto SiO2 and SiO2-PMAA were the ATRP. The results of adsorption
monolayer adsorption. Similar phenomena experiments indicated that SiO2-PMAA
have been observed for CV adsorption on showed high efficiency for removing the CV
coniferous pinus bark powder (CPBP)13 and dye from water solution. The reusability of
semi-interpenetrated networks hydrogels the SiO2-PMAA after five cycles was
consisted of poly(acrylic acid-acrylamide- reduced only 10% from 98%.
methacrylate) and amylose.11 According to
the Langmuir equation, the maximum Acknowledgement
adsorption capacity (qm) of SiO2 and SiO2- The author is grateful to KURDI for
PMAA were 172 and 250 mg/L, the financial support.
respectively. The results indicated that the
adsorption efficiency of SiO2-PMAA is a References
higher than unmodified silica gel. 1. El-Sayed, G. O. Desalination 2011, 272
(1), 225-232.
3.2.4 Recycle of the adsorbent
2. Wang, S.; Li, H.; Xu, L. J. Colloid
In order to compare the stability and
Interface Sci. 2006, 295 (1), 71-78.
reusability of the modified and unmodified
3. Karadag, E.; Saraydin, D.; Aydin, F.
silica gel, desorption processes were
Turk. J. Chem. 1998, 22, 227-236.
demonstrated by using the aqueous solution
4. Krysztafkiewicz, A.; Binkowski, S.;
of 0.1 M acetic acid. The results show that
Jesionowski, T. Appl. Surf. Sci. 2002,
about 88% of adsorption efficiency was
199 (1), 31-39.
achieved even after six cycles (Figure 7).
5. Chen, M.; Chen, Y.; Diao, G. J. Chem.
However, the adsorption efficiency of silica
Eng. Data 2010, 55 (11), 5109-5116.
gel decreased to only 52% after six cycles.
6. Chen, Y.; Gao, J.; Wen, X.; Wu, W.
This phenomenon demonstrated that the
RSC Adv. 2016, 6 (22), 18340-18347.
SiO2-PMAA could effectively remove
7. Laopa, P. S.; Vilaivan, T.; Hoven, V. P.
cationic dye from aqueous solution and
Analyst 2013, 138 (1), 269-277.
shows reusability and high separation
8. Tugulu, S.; Barbey, R.; Marc Harms,
efficiency.
M.; Fricke, M.; Volkmer, D.; Rossi, A.;
Klok, H. Macromolecules 2007, 40 (2),
168-177.
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Ghorbal, A. Euro-Mediterr. J. Environ.
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PMAA for the adsorption of CV (100 mg/L) 767-773.
at pH 9
PLA foams for scaffold application
Pasuta Sungsee, Varaporn Tanrattanakul*
*E-mail: varaporn.t@psu.ac.th
Abstract:
Poly(lactic acid), PLA, is a biodegradable biomaterial used as a scaffold in tissue
engineering application. PLA scaffold can be normally processed by many methods such as
electrospinning, 3D printing, polymer phase separation, melt processing etc. Cellular
structure or foam is a required structure of materials for the scaffold application. The present
work is a preliminary study that focuses on a typical foam formation by using
azodicarbonamide (AZDC) and zinc oxide (ZnO) as a chemical blowing agent and
accelerator, respectively. PLA foam was prepared by compression molding. The objective of
the present study is to evaluate the influence of ZnO content on properties of PLA foam
including mechanical, thermal degradation, and in-vitro degradation properties. This is
because ZnO content is one of important factors controlling the cellular structure of PLA
foam. It was found that PLA foam presented closed cell morphology. The mechanical
properties including notched Izod impact strength, tensile and flexural properties decreased as
ZnO content increased. The in-vitro degradation study of PLA foam was determined in term
of %weight loss in 2 months and demonstrated that an increment in ZnO content increased
the %weight loss of PLA foam due to an increase of its average cell size.
1. Introduction Preparation of PLA foam by melting

Scaffold is artificial structure with process and chemical blowing agent are an
cellular form which is suitably cultured with alternative choice because of easy operation,
many types of cells in order to induce cell low cost and simple equipment. Chemical
proliferation, differentiation and tissue blowing agents (CBA) are divided into
formation in vitro and invivo.1 Biomaterials endothermic and exothermic blowing
mainly used to fabricate scaffold is divided agents. Azodicarbonamide (AZDC) is a
into 3 types which are ceramic, natural common chemical blowing agent and has
polymer and synthetic polymer. Poly(lactic been used in PLA foam preparation.6,7
acid) (PLA) is a hydrophobic aliphatic Normally, zinc oxide (ZnO) is used as an
polyester and is widely used in biomedical accelerator of AZDC to decrease foaming
applications. It is easy to process which temperature. Lou et al.8 found that
allows PLA to use in medical application in AZDC/ZnO contents and mold opening
various shapes including sutures, nano- temperature affected the average cell sizes,
particles, micelles rods films and scaffold.2 void fraction (%VF), mechanical properties
PLA scaffold has been prepared by various and degradability of PLA foam prepared by
techniques such as solvent casting/porogen melt blending in an internal mixer followed
leaching, thermal phase separation (TIPS), by compression molding. However, the
electrospining, 3D printing, and melting effect of ZnO contents on PLA foam
process. PLA foam prepared by melting prepared by compression molding and
process using physical blowing agent have opportunity to utilize the obtained foam as a
been reported.3-5 PLA foam prepared by scaffold in tissue engineering application
physical foaming method (supercritical CO2) have not been reported. The objective of the
contains the microcellular structure but it is present study was to determine the effect of
required expensive special equipment. ZnO content in PLA foam in terms of cell
morphology, thermal degradation, in-vitro 5 °C/min from 30 °C to 500 °C under
degradation and mechanical properties. nitrogen atmosphere. Fourier Transform
Infrared (FTIR) analysis was carried out
2. Materials and Methods using a Bruker® TENSOR 27 spectrometer
2.1 Materials in ATR mode. FT-IR spectra of the samples
PLA 4042D was produced by Nature were recorded in the range 4000-400 cm-1
Works LLC (USA). Azodicarbonamide with resolution of 4 cm-1 and 132 scans.
(AZDC) was purchased from Greatchem 2.4 Mechanical properties testing
and Supply Pty., Ltd. (Thailand) and Zinc The notched Izod impact strength
Oxide (ZnO) was purchased from Kit and tensile properties were determined
Phaibun Chemistry Ltd., Part (Thailand). according to ASTM 256 and ASTM D412
2.2 PLA foam preparation die C, respectively. The crosshead speed
PLA, AZDC, and ZnO were mixed was a 5 mm/min for tensile testing. Six
together and then melt blending was carried specimens were used for every sample. An
out in a twin screw extruder at 150 °C with a average value with standard deviations was
screw speed of 150 rpm. It was necessary to reported.
avoid AZDC decomposition during 2.5 In-vitro degradation testing
extrusion. The compounded PLA was finally A phosphated-buffered solution
pressed in a closed mold (130 x 130 x 2 (PBS) at a concentration of 0.1 M and pH
mm3) with a temperature of 150 °C under a 7.4 was in-house prepared according to
pressure of 150 kg/cm2 for 10 min to obtain Rakmae et al..9 In-vitro hydrolytic
the foam samples. The AZDC/ZnO weight degradation of PLA foam was determined
ratio were varied into 3/0, 3/0.1, 3/0.5, and by soaking a specimen in PBS. Three
3/1 wt/wt and total content of AZDC/ZnO specimens (2 x 10 x 60 mm3) from each
was 2wt%. The obtained PLA foams were sample were firstly weight to obtain an
named PLA0, PLA0.3, PLA0.5, and initial weight (Wo) and were then vertically
PLA1.0, respectively. placed in a 100 mL test tube filled with 35
2.3 PLA foam characterizations mL PBS solution. The immersed specimens
The density of PLA foam (ρf) was were incubated at 37 °C for 8 weeks. The
measured by buoyancy method (ASTM PBS in all test tubes was weekly replaced by
D792). Three specimens were weighted in the fresh one. At the end of each period, the
air and in n-hexane on analytical balance. specimens were removed from PBS and
The average densities were calculated. The wiped with a filter paper to remove surface
void fraction (VF) was calculated according water. After that, these specimens were
to equation (1) rinsed by distilled water for 3 times and
vacuum dried at a temperature of 65 °C to a
%VF = [1- (ρf /ρs)] x 100 (1) constant weight (Wd). The percentage of
weight loss of the specimen during immer-
where ρs is a density of unfoamed PLA sion in PBS solution was calculated by
(1.223 g/cm3) and ρf is a density of foamed equation (2).9
PLA.8 The morphology of PLA foam was
investigated by using SEM-FEI Quanta W  Wd 
400. All specimens were immersed in liquid %Weight loss   0   100 (2)
nitrogen and then immediately fractured  W0 
before coating with gold. The longest
diameter of each pore was measured from 3. Results and Discussion
SEM micrographs and average cell sizes 3.1 PLA foam characterizations
were calculated. Thermogravimetric analysis PLA foam was successfully prepared
(TGA) was carried out using STA449F3 by chemical compression molding method.
Jupitor® (NETZSCH) with a heating rate of The closed cell morphology observed by
Figure 1. SEM micrograph of PLA foams with different AZDC/ZnO ratios: (a) 3/0.3,
(b) 3/0.5, and (c) 3/1.0
SEM was found for all foam samples The thermal degradation temperatures are
(Figure 1). The foam properties including in listed in Table 2. The thermal stability of
Table 1. It was found that PLA foam PLA foams decreased as ZnO content
without ZnO (PLA0) showed the highest increased. The PLA pellet exhibited only
density. As ZnO content increased, the one transition temperature (Td2) at 359 °C
average cell size and %VF increased whereas PLA foams exhibited 2 transition
whereas the densities decreased. This was temperatures. The very small peak (Td1)
because an increase in ZnO content expected to be a decomposition of remaining
increased an efficiency of AZDC decom- AZDC and the sharp peak (Td2) corres-
position.8 ponded to decomposition of PLA foams.
-24
Table 1. Properties of PLA foams 100 217.9 C
Density Average cell -20

Sample %VF
(g/cm3) size (µm) 80
Deriv. weight (%/min)

-16
PLA0 0.815 33.4 N/A
Weight (%)
60
PLA0.3 0.695 43.1 794 ± 104 -12
PLA0.5 0.652 46.7 914 ± 195

40 -8
PLA1.0 0.628 48.7 1224 ± 63 242.4 C
315.7 C
-4
20
Thermal stability of AZDC and PLA 0
foams with different ZnO content was 0
50 100 150 200 250 300 350 400 450 500
determined from TGA results. The TGA and Temperature ( c)
DTG thermograms of AZDC are displayed Figure 2. TGA and DTG thermograms of
in Figure 2. The decomposition of AZDC AZDC
occurs through the competition of at least 3
exothermic reactions producing solids and Table 2. Thermal degradation temperature
gaseous mixtures of nitrogen, carbon of PLA foams
monoxide, cyanic acid and ammonia.10 In Foam Td onset1 Td onset2 Td1 Td2
the present study, AZDC displayed three name (°C) (°C) (°C) (°C)
decomposition temperature (Td): 217.9 °C, PLA pellet - 344 - 359
PLA0 201 334 213 356
242.4 °C, and 315.7 °C; corresponding to PLA0.3 196 329 206 342
three different reactions of AZDC PLA0.5 188 317 201 346
decomposition.10 PLA1.0 182 314 197 338
Figure 3 displayed TGA and DTG
thermograms of PLA pellet and PLA foams.
100
(a) decomposition rate of AZDC which was
consistent with the previous work.8
80 PLA pellet 3.2 Mechanical properties
104 PLA0
Weight (%)
60 101
PLA0.3 The notched Izod impact strength of
98
PLA0.5
PLA1.0
PLA significantly decreased with the
40
95 addition of ZnO, and ZnO content showed
150 200 250 300 350
20
slightly effect on the impact strength (Figure
5). The impact strength related to the
0
50 100 150 200 250 300 350 400 450 500
density. The lower density showed the lower
Temperature ( C) impact strength. ZnO content also affected
5
(b)
the tensile and flexural properties as shown
-5
in Figures 6 and 7, respectively.
PLA0.5
Deriv. weight (%/min)
0.6
0.3 PLA0 4.0 1.0
-15
Notched Izod impact strength (kJ/cm2)

0.0 PLA1.0 PLA0.3
0.815
-0.3 0.8
-25
-0.6 3.0 0.695
2.7
Density (g/cm3)
170 190 210 230 0.652 0.628
-35 0.6
PLA pellet
2.0
-45 0.4
50 100 150 200 250 300 350 400 450 500 1.3 1.2
1.1
Temperature ( C)
1.0
0.2
Figure 3. (a) TGA and (b) DTG
thermograms of PLA pellet and PLA foams 0.0 0.0
PLA0 PLA0.3 PLA0.5 PLA1.0
PLA pellet Figure 5. Effect of ZnO content on density

AZDC/ZnO (3/1)
and notched Izod impact strength of PLA
foams
Tranmittance (%)
1115 cm-1 800

(a)
PLA1.0 foam 1364 cm-1
585
600
506
1361cm-1
1127 cm-1
E(MPa)
400 333 321

4000 3400 2800 2200 1600 1000 400
Wavenumber (cm-1)
Figure 4. FTIR spectra of PLA pellet, 200
AZDC/ZnO (3/1 wt/wt) and PLA1.0 foam

0
The remaining AZDC could be

confirmed by FTIR results. The shifted (b)
40 7.9 8
characteristic peaks of AZDC/ZnO (3/1)
Stress at break (MPa)
were observed in the spectra of PLA1.0 5.9

Strain at break (%)
30 6
foam as showed in Figure 4. The shifted 25.4
characteristic peak from 1115 to 1127 cm-1 20

19.9
3.4
4
3.3
assigned to the stretching vibration of C=O 10.2
10 2
and N-C of AZDC. The shifted 8.1
characteristic peak from 1364 to 1361 cm-1 0 0

assigned to the bending vibration of N-C=O PLA0 PLA0.3 PLA0.5 PLA1.0
of AZDC.11 Furthermore, it was found that Figure 6. Effect of ZnO content on tensile
Td onset1 and Td1 of PLA foam decreased as properties of PLA foams: (a) Young’s
ZnO content increased (Table 2). This modulus and (b) stress at break and strain at
indicated that ZnO decreased the break
decomposition temperature and activated
2500
(a) 3.3 In-vitro degradation
2000
2017 In-vitro degradation testing in term
of %weight loss was used to initially
Flexural modulus (MPa)
1682 1630
1469
1500 determine the opportunity to utilize obtained
PLA foam as a scaffold in tissue
1000
engineering. Because the scaffold needs
500 appropriate time for degradation and
resorption until the tissue engineered
0
transplant is completely remodeled by the
host tissue. Thus, in-vitro degradation
40 37.4
(b) testing is important.14 Figure 8 displays the
variations of %weight loss of PLA foam
Flexural stress (MPa)
30 specimens after immersion in PBS for 8

weeks. ZnO content strongly affected the
17.8
20 15.6 degradation behavior. In the first week, rate
of degradation of all samples was similar.
10
7.1
Since the second week, the higher ZnO
0
content provided the higher %weight loss
and degradation rate. Degradation rate of all
samples decreased after 4 weeks and higher
Figure 7. Effect of ZnO content on flexural
ZnO content still provided the higher
properties of PLA foams: (a) flexural
%weight loss and degradation rate. This was
modulus and (b) flexural stress
because the higher ZnO content yielded the
larger cell size in foams that enhanced PBS
As mentioned above, increasing of
penetration. 9 The %weight loss of the foam
ZnO content increased the efficiency of the
samples after the end of the experiment was
decomposition of AZDC attributing to an
in the range of 0.66–1.73% which were
increase in the average cell size and a
lower than that of PLA scaffold prepared by
decrease in the density of PLA foam. In
the solvent casting and particular leaching
general, the larger cell size of foams causes
method.15
the lower mechanical properties. Therefore,
the increase in ZnO content decreased over 2.0
PLA0
all mechanical properties of PLA foams. PLA0.3
PLA1.0
Furthermore, %VF and the cell morphology 1.6
of foam affected the mechanical properties.

Weight loss (%)
1.2
The %VF of the PLA foams was in the
range of 33.4-48.7% corresponding to those 0.8
of human cancellous bone (30-90%).12 The

0.4
tensile modulus and stress at break of the
PLA foams were in the range of 321-585 0.0
MPa and 8.1-25.4 MPa, respectively, which 0 1 2 3 4 5
Immersion time (weeks)
6 7 8
were also close to those of human Figure 8. Effect of ZnO content on %weight
cancellous bone. The tensile strength and loss of PLA foams
modulus of cancellous bone are 400 MPa
and 7-20 MPa, respectively.13 However, the 4. Conclusion
PLA foams in the present study exhibited This study showed the success in
closed cell morphology whereas scaffold for preparation of PLA foams through
bone tissues engineering requires the inter- compression molding using AZDC and ZnO
connected cell morphology. as blowing agent and accelerator,
respectively. The obtained foams exhibited
closed cell morphology. The thermal 6. Zimmermann, M. V. G.; Brambilla, V.
stability of PLA foam decreased as ZnO C.; Bradalise, R. N.; Zattera, A. J. J.
content increased. The remaining AZDC Mater. Res. 2013, 16 (6), 1266–1273.
was found in PLA foams confirmed by TGA 7. Najafi, N.; Heuzey, M-C.; Carreau, P.
and FTIR results. In addition, an increase of J.; Therriault, D.; Park, C. B. J. Euro.
ZnO content decreased the density, the Polym. J. 2015, 73, 455–465.
mechanical properties and the in-vitro 8. Luo, Y.; Zhang, J.; Qi, R.; Lu, J.; Jiang,
degradation of PLA foam owing to increase P. J. Appl. Polym. Sci. 2013, 330–337.
of its average cell size. 9. Rakmea, S.; Ruksakulpiwat, Y.;
Sutapan, W.; Suooakarn, N. Meter. Sci.
Acknowledgements Eng. C. 2012, 32, 1428–1436.
This work was financial supported 10. Reyes-L., J. A.; Marcilla, A. J. Appl.
by Prince of Songkla University, the grant Polym. Sci. 2007, DOI
number SCI5901465. 10.1002/app.26922
11. Xie, Y.; Li, P.; Zhang, J.; Wang, H.;
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Poly(lactic acid)/epoxidized natural rubber blends:
effect of processing condition
Jenjira Klinkajorn, Varaporn Tanrattanakul*, Ekwipoo Kalkornsurapranee
*E-mail: varaporn.t@psu.ac.th
Abstract:
The objective of this work was to evaluate the effect of processing condition on the
mechanical and thermal properties and morphology of poly(lactic acid) blended with
epoxidized natural rubber (PLA/ENR blend) and PLA blended with ENR grafted with maleic
anhydride (PLA/ENR-g-MA blend). ENR contains 25mol% epoxide. ENR-g-MA was
prepared by melt mixing with 0.5 phr of MA. Polymer blends were prepared by melt
blending in a twin screw extruder (TWSE) and an internal mixer. The blend ratio of
PLA:rubber was 90:10. A ternary blend (PLA/ENR/ENR-g-MA) was also prepared at a
similar condition at a ratio of 90:5:5. The blending temperature was 170 C for both
instruments. The blending speeds were 100 rpm for TWSE and 60 rpm for internal mixer. It
was found that the impact strength of all blends prepared by the internal mixer was higher
than that of the blends prepared by TWSE whereas the tensile properties were similar, except
the strain at break. Glass transition temperature (Tg) of PLA phase shifted to a lower
temperature and Tg of ENR-g-MA was lower than that of ENR. The rubber phase showed
spherical particles. The processing instrument show significant effect on the thermal
properties and particle size of rubber in the blends.
1. Introduction and synthesis graft copolymers.4-7

Recently, biodegradable polymers Epoxidized natural rubber (ENR) was also
have been used to solve the problem from used for toughening PLA because it is more
plastic waste disposal on the environment. polar than NR.8,9 The graft copolymer of
The most promising polymer in this regard ENR may provide more compatibility than
is poly(lactic acid) (PLA). It is derived from the graft copolymer of NR. Tanrattanakul
lactic acid monomer that obtained from and co-workers succeeded to enhance
fermentation of starch and sugar derived compatibility and mechanical properties of
from vegetable sources such as corn, wheat, PLA/NR blend by addition NR grafted with
cassava starch or rice. PLA has many maleic anhydride (NR-g-MA) into the
interesting properties to use as a material in blends.3 Thus, we expected that ENR-g-MA
packaging applications including good could be more compatible with PLA than
mechanical properties, thermal stability and ENR. The compatibility and mechanical
low environmental impact. A major properties of polymer blends also related
disadvantage of PLA is high brittleness. with morphology, which is affected by the
Natural rubber (NR) has been used to processing parameters in the melt state such
increase toughness of PLA in terms of block as temperature, shear force and blending
copolymerization1,2 and polymer blending.3-6 time.10
The difference of polarity and molecular The objective of this research was to
weight of PLA and NR attributes to low determine the effect of blending equipment
miscibility of PLA/NR blends. There are on the mechanical and thermal properties of
many researches to enhance compatibility of PLA/ENR and PLA/ENR-g-MA blends.
PLA/NR blend by modification NR such as Blend morphology has been investigated as
reduction molecular weight by mastication well.
2. Materials and Methods and the average values with the standard
2.1 Materials deviation were reported.
PLA 4043D was purchased from 2.5 Blend characterization
Naturework Co. Ltd., USA. ENR was The morphology of the blends was
purchased from Muang Mai Guthrie Public characterized by using Quanta® 400 FEI
Co. Ltd., Thailand (Mooney viscosity: ML Scanning electron microscope (SEM). All
(1+4) 100 °C = 77.7 and %epoxidation = 25 specimens were fractured in liquid nitrogen.
mol%). Maleic anhydride was purchased The fractured surfaces were etched with
from Sigma-Aldrich. petroleum ether for 12 h in order to remove
2.2 Synthesis of ENR-g-MA rubber particles and dried in a vacuum oven
ENR and MA were dried in a for 4 h before coated with gold.
vacuum oven at 40 °C for 24 h. ENR was Approximately 200 particles selected
masticated in an internal mixer at 135 °C randomly were measured particle diameter
with a rotor speed of 60 rpm for 2 min and and reported as a number-average diameter.
then 0.5 phr of MA was added. Mixing time Glass transition temperature (Tg) and
was 8 min. crystallization behavior of the blends were
2.3 Polymer blend preparation characterized with Perkin Elmer® DSC 7 at
PLA was dried at 105 °C for 12 h. a heating scan of 10 °C/min from 20 °C to
ENR and ENR-g-MA were cut into small 200 °C. After first heating scan the sample
pieces. The blend ratio of PLA:rubber was was cooled at a cooling scan of 100°C/min
90:10. A ternary blend (PLA/ENR/ENR-g- and then heated again. The heat of fusion of
MA) was also prepared at a similar pure crystalline PLLA (∆Hc) is 93 J/g.7
condition at a ratio of 90:5:5. Polymer Dynamic mechanical thermal analysis
blends were prepared by melt blending in a (DMTA) was investigated in a dual
twin screw extruder (Prism® TSE16TC) and cantilever mode by using METTLER
an internal mixer (Brabender® Mixer350E). Toledo® DMA 1, under the following
The temperature profile of TWSE was 140 condition: -80 °C to 120 °C, frequency 1 Hz,
and 170 °C for screw and die, respectively. heating rate 3 °C/min and strain control
Because of short screw (screw length = 27.3 0.05%.
cm), the extrusion was repeated to obtain
homogeneous blend. The screw speed of the 3. Results and Discussion
1st extrusion was 100 rpm and the 2nd one 3.1 Mechanical properties
was 150 rpm in order to decrease residence The Izod and Charpy impact strength
time of melt in the extruder to avoid thermal of PLA/ENR/ENR-g-MA blends at a ratio of
degradation. For the internal mixer, the 90/10/0, 90/5/5 and 90/0/10 prepared by the
blending temperature was set at 170 °C and twin screw extruder (TWSE) and internal
rotor speed 60 rpm. A 2-mm thick sheet was mixer at same blending temperature (170
prepared by compression molding at 150°C °C) are shown in Figure 1 and Figure 2,
under pressure of 200 MPa for 13 min. The respectively. The impact strength of the
blends were stored in a desiccator before notched specimens may not relate to that of
testing. the un-notched samples because the tip of v-
2.4 Mechanical properties testing notch is a stress concentrator leading to
The tensile properties of the blends fracture at this position whereas a crack in
were determined by using a universal testing the un-notched specimen can occur at any
machine, according to ASTM D638. The position. The Izod and Charpy mode are
Izod and Chapy impact strength of v- different due to a position of testing sample,
notched and un-notched specimens were resulting in different impact strength. Thus,
determined by using an impact testing it is not necessary to compare results
machine, according to ASTM D256. At least between both modes. The effect of
8 specimens of every sample were tested processing method on the impact resistance
can be obviously seen from the notched-Izod also increased the stress at break and the
impact strength. The PLA/ENR blend elongation at break of PLA/ENR blend.
(90/10/0) prepared by the internal mixer 35
TWSE (a)
Un-notched Charpy impact strength

showed the higher value than that by TWSE. 30
Internal mixer 24.35
25 21.88
The addition of ENR-g-MA increased the 20
20.58 19.68
(kJ/m2)
impact strength of the binary blend (90/0/10) 15
10.09
and ternary blend (90/5/5). This result 10
4.67
6.04
indicated that the ENR-g-MA could be used 5
0
directly or mixed with ENR to enhance the PLA/ENR/ENR-g-MA
toughness of PLA. ENR-g-MA improved
7
the notch sensitivity of PLA. The notched TWSE 5.49 (b)
Notched Charpy impact strength

6 Internal mixer
impact strength of 90/0/10 blend was higher 5 4.44

3.83
than that of PLA. In Figures 1-3, virgin PLA 4
3.50
(kJ/m2)
2.67
was used to prepare testing specimens; thus 3

1.45 1.92
it showed high mechanical properties due to 2
no thermal processing in the blending 0
equipment. PLA/ENR/ENR-g-MA
45
(a)
Figure 2. Effect of processing method on
Un-notched Izod impact strength
TWSE
40
35
Internal mixer
34.18 the Charpy impact strength: (a) un-notched
30 25.28 specimens and (b) notched specimens
(kJ/m2)
25.18
25
18.42
20 2500 TWSE
15.14 (a)
Internal mixer
15
4.63 2000 1759

10
5.14
5 1307
1500 1323 1267
1187
E (MPa)
0
1091
PLA/ENR/ENR-g-MA 1000
619
8 500
TWSE (b)
7
Notched Izod impact strength
Internal mixer 6.03

5.60 0
6
4.87
5 PLA/ENR/ENR-g-MA
4.03
(kJ/m2)
4.10
4
80
TWSE (b)
3 2.42 Internal mixer
70
1.82
2
Stress at break (MPa)
60
50.60
1
50
0
40
PLA/ENR/ENR-g-MA 30 22.53
16.36
13.60 17.86
Figure 1. Effect of processing method on 20
10
7.10 7.92
the Izod impact strength: (a) un-notched 0
specimens and (b) notched specimens PLA/ENR/ENR-g-MA
6
TWSE (c)
The tensile properties of the blends 5 Internal mixer

Elongation at break (%)
4.25
prepared by TWSE and internal mixer were 4 3.50
also compared as shown in Figure 3. The 3

2.17
1.88
2.25
2.00
effect of processing method can be 2

1.29
1
obviously seen in the tensile results of
0
PLA/ENR-g-MA blend (90/0/10). The PLA/ENR/ENR-g-MA
Young’s modulus of the blend prepared by Figure 3. Effect of processing conditions on

internal mixer was lower than that of by the tensile properties: (a) Young’s modulus,
TWSE. In contrast, the stress at break and (b) stress at break and (c) elongation at
elongation at break of the blend was break
opposite. The addition of ENR-g-MA was
Mixing of two immiscible polymers
in the melt state induced a phase separation
that depends on processing conditions. The ENR which may be attributed to a lower
addition of compatibilizer affects the phase modulus. The stress at break of the ternary
stability and morphology of polymer blends. blend was highest which might due to
The SEM micrographs of the polymer combination of miscibility and molecular
blends prepared with the internal mixer and weight. The strain at break showed strong
the TWSE are shown in Figure 4. All blends relation with the particle size of rubber. The
were immiscible and showed dispersed smaller particle the higher strain at break
spherical rubber particles in continuous was observed.
phase PLA. The average particle diameter of DMTA was used as a tool to identify
all blends is listed in Table 1. It was found miscibility between PLA and the ENR-g-
that all the blends prepared by internal mixer MA in the blend. It was found that the
showed the smaller rubber particle size temperature of maximum tan δ of the blends
diameter than that of prepared by TWSE. were decreased with increasing ENR-g-MA
This might be due to longer time and higher content, as shown in Figure 5. The 
shear of blending in the internal mixer. The transition temperatures of the blends are
presence of MA decreased the rubber listed in Table 2. The lower temperature in
particle size of the blends. The average the range of -46 °C to -43 °C represented Tg
particle diameter could be ranked in the of ENR phase and the higher one in the
following order: 90/10/0>90/5/5>90/0/10, range of 58 °C to 65 °C represented Tg of
which observed in both processing methods. PLA phase.
Theoretically, the size of the dispersed phase
implies miscibility between the continuous 90/10/0 (Internal mixer) PLA
2
and the dispersed phase. High immiscibility 90/5/5 (Internal mixer)
90/10/0 (TWSE)
induces coalescence of the dispersed phase
Tan delta
1.5
90/0/10 (Internal mixer) 90/5/5 (TWSE)
because phase separation is preferred in the 90/0/10 (TWSE)
blend. The decrease in dispersed rubber 1
diameter in the blends containing MA 0.5
indicated an increase in miscibility.

0
-80 -50 -20 10 40 70 100
Table 1. Average diameter of ENR particles Temperature ºC
in the blends Figure 5. The tan δ – temperature curves of

Average diameter PLA and blends
Sample (m)
PLA/ENR/ENR-g-MA Internal Table 2. Phase transition temperatures of
TWSE
mixer
PLA blends
90/10/0 1.64 ± 0.35 1.04 ± 0.21
90/5/5 1.18 ± 0.23 0.78 ± 0.15 α Transition
90/0/10 0.69 ± 0.14 0.47 ± 0.11 temperature (°C)
Sample
ENR PLA
The smaller diameter of rubber phase phase
seemed to have a positive effect on the 100/0/0(PLA pellet) - 64.1
90/10/0(TWSE) -46.0 62.3
impact strength of the blends. The particle 90/10/0(Internal mixer) -45.0 60.7
size should be submicrons (< 1 m) in order 90/5/5(TWSE) -45.4 61.0
to obtain higher impact strength. Effect of 90/5/5(Internal mixer) -43.5 59.5
particle size on the tensile properties is 90/0/10(TWSE) -43.8 59.6
complicated, based on each property. ENR- 90/0/10(Internal mixer) -43.0 58.3
g-MA had lower molecular weight than
Figure 4. SEM micrograph of PLA/ENR25 blends and PLA/ENR-g-MA blends with
different processing conditions: (a) PLA/ENR25 blends with TWSE, (b) PLA/ENR25 blends
with internal mixer, (c) PLA/ENR-g-MA blends with TWSE, and (d) PLA/ENR-g-MA
blends with internal mixer
Obviously, the  transition in the present study. In contrast, all blends

temperatures of PLA and ENR in 90/0/10 showed Tcc which depended on blending
prepared by internal mixer strongly shifted method and blend composition. ENR and
to lower and higher temperature, ENR-g-MA acted as the nucleating agents
respectively, which indicated the reactive that induced cold crystallization of PLA and
blend. The  transition temperature of PLA higher ENR-g-MA content provided the
was shifted by 6 °C by adding ENR-g-MA. lower Tcc.
The transition temperatures of the blends 154 ºC
60 ºC 100/0/0(PLA)
prepared in TWSE tended to slightly higher 151ºC
90/10/0
58ºC
than those prepared in internal mixer. (TWSE)
Heat flow Endo Up W/g
148 ºC 90/10/0
DMTA results agreed with SEM 56 ºC 104ºC (Internal mixer)
152ºC 90/5/5
micrographs and supported the assumption 57 ºC
130 ºC
(TWSE)
that MA increased miscibility of PLA/ENR 55 ºC

101 ºC 149 ºC 90/5/5
(Internal mixer)
blend leading to higher compatibility as the 55 ºC

129ºC 155 ºC
90/0/10
(TWSE)
mechanical properties increased and MA 90ºC 150 ºC 90/0/10
54 ºC (Internal mixer)
reacted as an emulsifier by decreasing the 123 ºC
interfacial tension and increasing the 25 50 75 100 125 150 175 200
Temperature C
interfacial adhesion between PLA and ENR
attributed to a reduction in dispersed particle Figure 6. DSC thermograms of PLA and
size. blends
Effect of processing method on
thermal properties of the blends is exhibited Table 3. Crystallization properties of PLA
in Figure 6. DSC results were obtained from and PLA/ENR/ENR-g-MA blends prepared
the second heating scan in order to by TWSE and Internal mixer
Sample ∆Hm Xc (%)
eliminated different thermal history. Table 3
100/0/0(PLA) 27.4 29.5
represents heat of fusion at melting peak
90/10/0(TWSE) 28.1 30.2
(∆Hm) and degree of crystallinity of PLA in 90/10/0(Internal mixer) 7.5 8.1
the blend (Xc). The glass transition 90/5/5(TWSE) 29.4 31.6
temperature (Tg) of PLA in the blends 90/5/5(Internal mixer) 7.8 8.4
showed a similar result as that observed in 90/0/10(TWSE) 33.1 35.6
DMTA. The presence of MA decreased Tg 90/0/10(Internal mixer) 17.3 18.6
of PLA and preparation in TWSE provided Xc = [∆Hm x 100]/93
slightly higher Tg than internal mixer. The
90/0/10 blend showed the lowest Tg. The As we know, lower Tcc initiates
addition of ENR and ENR-g-MA affected faster crystallization. Therefore, the degree
cold crystallization temperature (Tcc) of of crystallinity (Xc) of the polymer blends
PLA in the blends. Generally, PLA has low prepared by TWSE was higher than that by
crystallization rate and no Tcc was observed internal mixer and the higher ENR-g-MA
content provided the higher Xc of the blends. Acknowledgement
The occurrence of Tcc also induces the This research has been supported by
double melting peak which is common for Natural Rubber Innovation Research Center,
polyester. The lower melting peak belongs Prince of Songkla University (Grant No.
to the crystalline initiated during heating SCI600382S).
scan in DSC. In the present study, the
double melting peak was noticeable in the References
blends prepared by TWSE. In contrast, the 1. Chumeka, W.; Pasetto, P.; Pilard, J. F.;
blends prepared from the internal mixer Tanrattanakul V. J. Appl. Polym. Sci.
showed a broad melting peak. This related to 2015, 132, 4126-4135.
the broad peak at Tcc. The 90/0/10 blend 2. Chumeka, W.; Pasetto, P.; Pilard, J. F.;
(TWSE) also showed a broad Tcc peak and Tanrattanakul V. Polym. 2014, 55, 4478-
very small peak of the lower melting 4487.
temperature. Blending in the internal mixer 3. Juliwanlee W.; Tanrattanakul V. Pure
took longer residence time leading to more and Applied Chemistry International
thermal degradation than blending in TWSE. Conference 2016, Bangkok, Thailand,
As a result, chain molecules of PLA became Feb 9-11, 2016.
smaller contributing to insignificant 4. Deng, Y.; Thomas, N. L. Eur. Polym. J.
difference in crystal size between 2015, 71, 534–546.
crystallization during compression molding 5. Chumeka, W.; Pasetto, P.; Pilard, J. F.;
and during heating scan in DSC. The Tanrattanakul V. J. Polym. Environ.
difference in melting behavior of PLA was 2013, 21, 450–460.
not a scope in the present study. 6. Juntuek, P.; Ruksakulpiwat, C.;
Chumsamrong, P.; Ruksakulpiwat, Y. J.
4. Conclusion Appl. Polym. Sci. 2012, 125, 745–754.
Effect of processing methods on the 7. Jaratrotkamjorn, R.; Khaokong, C.;
mechanical properties of PLA blended with Tanrattanakul V. J. Appl. Polym. Sci.
ENR and ENR-g-MA was studied. It was 2012, 124, 5027-5036.
8. Zhang, C.; Wang, W.; Huang, Y.; Pan
shown that the blends prepared by the
Y.; Jiang, L.; Dan, Y.; Luo, Y.; Peng, Z.
internal mixer had higher impact strength Mater. Des. 2013, 45, 198–205.
and elongation at break than that prepared 9. Pongtanayut, K.; Thongpin C.;
by TWSE. The addition of ENR-g-MA Santawitee, O. Energy Procedia 2013,
provided more compatible blend. The 34, 888–897.
morphology of the blends indicated that MA 10. Favis, B. D. Polymer Blends: Paul, D.
in ENR-g-MA acted as a compatibilizer to R., Bucknall, C. B., Eds.; Wiley: New
York, 2000; Vol. 1, Chapter 16, p 501.
reduce rubber particle size and enhanced
compatibility between PLA and ENR. The
processing method show effect on the
thermal properties and blend morphology.
Novel unsaturated polyester copolymers based on poly(butylene succinate)
for using as toughening agent for PLA
Vachiravit Chalermpanaphan, Chantiga Choochottiros*
Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
*E-mail: chantiga.c@ku.ac.th
Abstract:
Polylactide (PLA) are biodegradable plastic which have excellent properties
comparable to some petroleum-based polymers. This work, we focused on synthesis of novel
unsaturated polyester copolymers which consist of rubbery segment (poly(butene succinate))
to provide flexibility and lactide segment to enhance compatibility between toughening agent
and PLA. The poly(butene succinate) (dPBS) was synthesized via polycondensation and
copolymerization with lactide via ring-opening polymerization to obtain (dPBS-PLA). The
chemical structure and composition of the copolymers were confirmed by nuclear magnetic
resonance spectroscopy (1H-NMR), infrared spectroscopy (IR) and gel permeation
chromatography (GPC). Thermal properties of dPBS-PLA copolymers were investigated by
using differential scanning calorimetric (DSC) and thermogravimetric analysis (TGA). Glass
transition temperature (Tg) of dPBS was -35 ºC and increased to -16 ºC after copolymerized
with lactide. The blending of dPBS-PLA and PLA were done by film casting and
composition of dPBS-PLA/PLA were varied. Films have thickness around 0.05 mm.
Mechanical properties of the films were determined by tensile testing. The result showed that
Young’s modulus of the blends increased comparing to neat PLA. This implied that the
unsaturated bond on dPBS-PLA created cross-linking structure.
1. Introduction Polylactide (PLA) or poly(lactic

The invention of synthetic plastics acid), is key thermoplastic polymers that can
over the past hundred years has led to huge solve the issue in both of these categories,
evolution of development in sociality and since it originate from starch of plant and
technology. Plastic industry manufacturing ultimately break into biologically and/or
polyethylene, polypropylene, and related environmentally benign by-products by
resins established enormous revenue in the fermentation.2
United States, and these productions are Applications of PLA, a polymer
alternatively utilized to replace glass and constructed from lactic acid (2-hydroxy
metal materials ranging from drink bottles to propionic acid) have been diversely
automobile industry.1 The point, which contributed from ordinary packaging to
continuously challenged scientists in field of advance utilization such as medical
polymer, is the question in sustainability and materials.3-7
environmental impact. Enlargement of Poly(L-lactide) (PLLA) has impact
“green” chemical processes encourage the strength of 26 J m-1, which relative to brittle
concentration on improvement and polymeric polystyrene.8 In addition,
consumption of non-environmentally polylactide has comparable tensile strength
destructive polymeric materials. The and elastic modulus to polyethylene
classification to describe green polymers terephthalate. Limitation in brittleness of
starts from considering their original source, PLA results in restrictive applications to
i.e. polymer derived from agricultural apply this material to tough and high elastic
products, to their ultimate degradation into requirment products.9 The toughening
environmentally benign by-products. schemes and improved composites are
required to adjust endurance and resilience into the flask. Then, polycondensation was
of PLA products. carried out at 220 ºC under vacuum of 7
Poly(butylene succinate) (PBS) is mbar for 1 h. The obtained product (dPBS)
biodegradable polyester which has excellent was purified by dissolving in chloroform
in flexibility.10 There have some research and then precipitated in excessive methanol.
showing blending of PLA and PBS to The powder product was dried in vacuum
enhance the flexibility of PLA.11 However, oven at 40 ºC.
the miscibility between the two component 2.3 Synthesis of copolymer dPBS-PLA
need to be concerned. The dPBS (1.75 mmol) and L-lactide
Here, we challenged to develop new (0.1 mol) were placed in a three-neck round
copolymers which not only initated bottom flask and heated to 140 ºC under
chemical structure of PBS but also nitrogen gas until a clear mixture was
composed of unsaturated bond along their obtained. Sn(Oct)2 (0.34 mL, 1.05 mmol in 2
chains to provide crosslinking functional mL of toluene) was added. Polymerization
group. We expected that it could enhance was carried out for 1 h and the product was
intermolecular interaction between precipitated in n-hexane. The polymer was
copolymers and PLA to improve washed with methanol and dried overnight
compatibility and mechanical properties. in a vacuum oven at 40 ºC.
The new copolymers consisted units of 2.4 Preparation of PLA/dPBS films
poly(butylene succinate), dPBS as flexible In order to study the properties of
segment and polylactide as compatible PLA/dPBS, series of PLA casting films with
segment to PLA. The dPBS will be varies dPBS content were prepared
synthesized via polycondensation (Scheme following ASTM D882 with the thickness of
1) and copolymerization with lactide via 0.05 mm. PLA (9-11 g), varies dPBS content
ring-opening polymerization. (1-10 phr) were dissolved in chloroform and
mixed for 3 h. The solution was casted into a
2. Materials and Methods petridish, and evaporated in vacumn oven at
2.1 General 40 ºC for 24 h and heated up to 80 ºC for 24
Cis-2-butene-1,4-diol (CBD), h to obtain PLA/dPBS film. In case of
succinic acid (SA), and 1,4-butanediol were thermal crosslinking films, benzoyl peroxide
obtained from Tokyo Chemical Industry which is thermal initiator was added (0.8
Co., Ltd. Benzoyl peroxide (BP) was wt%) during blending.
purchased from MERCK. Titanium 2.5 Characterization
butoxide, tin 2-ethyl hexanoate, and 2.5.1 Chemical structure
deuterated chloroform (CDCl3) was bought Nuclear magnetic resonance (1H-
from Sigma-Aldrich Pte., Ltd. Analytical- NMR) spectra were recorded on a Bruker
grade chloroform, HPLC-grade chloroform, 400 MHz AVANCE III HD spectrometer
and analytical-grade methanol were supplied operating at 400 MHz, using chloroform-d
from RCI Labscan, Thailand. PLA pellets and tetramethylsilane (TMS) as the
(IngeoTM Biopolymer 2003D) were a gift corresponding solvent and internal chemical
from PTT Public Company Limited, shift standard, respectively. The Fourier
Thailand. transform infrared (FTIR) spectra were
2.2 Synthesis of poly(butene succinate); obtained from a Bruker Vertex 70
dPBS spectrophotometer with 32 scan at a
1.2 mol of CBD and 1 mol of SA resolution of 4 cm-1. Molecular weight and
were added into three-neck round-bottom polydispersity index were determined by a
flask equipped with condenser and nitrogen Shimadzu Class-VP size-exclusion
inlet balloon. The mixture was stirred for 4 h chromatograph (SEC) equipped with a
at 190 ºC. Then, a suitable amount of MIXED-D column 300×7.5 mm (polymer
catalyst tetrabutyl titanate was introduced Laboratories, Varian Inc.) and a refractive
Scheme 1. (a) Polycondensation of CBD and SA, (b) trans-esterification of dPBS
index detector. Chloroform was used as an 3. Results and Discussion

eluent at a flow rate of 1.0 mL min-1. 3.1 Synthesis of dPBS and dPBS-PLA
2.5.2 Thermal properties Polycondensation between CBD and
Differential scanning calorimetry SA was successfully synthesized to obtain
(DSC) was conducted using Mettler-Toledo dPBS. The chemical structure of dPBS was
equipment (Schwerzenbach) which nitrogen confirmed by 1H-NMR and FTIR. The
gas was purged into the DSC cell at a flow characteristic peaks of dPBS showed at 2.58
rate of 50 mL min-1. The samples were first (CH2, c), 4.63 ppm (CH2, a), and 5.68 ppm
heated from -50 ºC to 170 ºC at a heating (CH, b) (Figure 1a). In addition, qualitative
rate of 10 ºC min-1 and were annealed for 10 analysis by 1H-NMR was done to calculate
min at 170 ºC to erase any previous thermal degree of polymerization (DPn) which was
history, followed by cooling to -50 ºC at a 46 (Table 1). FTIR spectrum (Figure 2) also
rate of 10 ºC min-1. The second heating was showed characteristic peaks of dPBS such as
subsequently made to 170 ºC at heating rate 3524 cm-1 (ν OH), 2941 cm-1 (ν CH), 1719
of 10 ºC min-1. cm-1 (ν C=O), 1148 cm-1 (ν C-O-C), and 967
2.5.3 Mechanical properties (=CH bending).
The tensile testing were measured The dPBS was used as macro
using a HOUNSFIELD H50KS machine. initiator to copolymerize with lactide
The rectangle-shaped samples for tensile through ring-opening polymerization
testing were cut from the dual blade shear reaction. Sn(Oct)2 was used as catalyst. The
cutter, and dimension of the sample was producted that obtained was named as
100×10×0.05 mm3 (ASTM D882-12). The dPBS-PLA. The confirmation of
samples were moved apart at a constant copolymerization of lactide chain on dPBS
speed of 3 mm min-1 until they fractured at was done by using 1H-NMR. The additional
room temperature. At least three samples characteristic peaks of PLA showing at 1.56
were tested for each sample and calculated ppm (CH3, a) and 4.34 ppm (CH, c) (Figure
as: 1b).
The molecular weight of dPBS and
Tensile strength (MPa) = F/A (1) dPBS-PLA were investigated by GPC as
shown in Table 1.
Elongation at break (%) = (ΔL/Lo)×100 (2)
Table 1. Molecular weight of dPBS and
Where F and A are the maximum force ( N) dPBS-PLA
to break specimen and area that affect the GPC DPn*
sample
Mw Mn PDI dPBS PLA
force (mm2), respectively. Where ΔL = L-Lo; dPBS 7997 15773 1.97 46 -
L is final length and Lo is initial length. dPBS-PLA 9367 20022 2.13 46 37
*DPn is degree of polymerization and was calculated from
1H-NMR and GPC results
DSC technique was used to
characterize thermal properties of dPBS and
dPBS-PLA. The results showed in Figure 3.
Glass transition temperature (Tg) and
melting temperature (Tm) were observed. Tg
of dPBS was ca. -35 ºC. Compare Tg of
dPBS to PBS, the PBS has Tg ca. -18 ºC.11
The lower Tg of dPBS might be due to
present of unsatureated bond on dPBS
affected to lower Tg. After copolymerization
with lactide, the Tg of dPBS-PLA was
increased to -16 ºC. This may be due to PLA
segment interrupted the chain mobility.
After blending of dPBS-PLA at 1-10 phr to
PLA, Tg of the blends decreased to 45-54
ºC (Figure 4) comparing to neat PLA (Tg ≈
60 ºC). After thermal crosslinked, Tg shifted
to higher temperature (50 ºC) but still lower
than neat PLA. These results refered to
plastized PLA by dPBS-PLA.
Figure 1. 1H-NMR spectra of (a) dPBS and

(b) dPBS-PLA in CDCl3
Figure 3. DSC thermosgrams of dPBS and

dPBS-PLA
Figure 2. FTIR spectra of CBD, SA, and

dPBS
Figure 4. DSC thermosgrams of
PLA/dPBS-PLA blend without crosslinked
(Figure 5). In case of thermal crosslinking
by adding benzoyl peroxide during film
casting, TGA thermogram showed single
step degradation and Td was 280 ºC (Figure
6). This result implied formation of network
structure.
Figure 5. TGA thermogram of dPBS-PLA
The degradation temperature of 5%

weight loss (Td) of compounds were
investigated by using TGA. Neat PLA
showed single step degradation and Td was
270 ºC. After blending with dPBS-PLA,
TGA thermogram showed two steps
Figure 6. TGA thermogram of crosslinked
degradation and Td were 186 ºC and 250 ºC
PLA/dPBS-PLA film
Table 2 Mechanical properties of neat PLA, PLA/dPBS before and after thermal crosslinking
Sample Young’s modulus (MPa) Elongation at break (%)
Neat PLA 153.0±12.0 2.58±0.2
Content (phr)
PLA/dPBS-PLA 1 5 10 1 5 10
161.4±12.4 153.4±5.7 125.0±23.2 3.89±0.2 2.81±0.2 1.82±0.2
After thermal crosslinking
Sample Young’s modulus (MPa) Elongation at break (%)
Neat PLA 209.9±21.8 2.45±0.1
Content (phr)
PLA/dPBS-PLA 1 5 10 1 5 10
215.5±6.8 107.7±15.2 103.5±3.3 2.51±0.2 5.28±0.6 1.63±0.2
3.3 Mechanical properties elongation at break, blending with 1 phr of

Film casting was done with thickness dPBS-PLA performed increasing to 3.89%
of 0.05 mm. Here, we investigated effect of comparing to neat PLA and slightly
dPBS-PLA to PLA comparing between decreased as dPBS-PLA increased. This
before and after thermal crosslinking. The related results of Young’s modulus.
results of tensile testing are showed in Table Here, the crosslinking reaction was
2. First, we considered effect of dPBS-PLA done by adding thermal initiator, benzoyl
without crosslinking. The neat PLA had peroxide. The crosslinked films showed
Young’s modulus of 153 MPa. After increasing of Young’s modulus compared to
blending with dPBS-PLA, Young’s modulus neat PLA. The increasing of Young’s
were not significantly changed when adding modulus of crosslinked films may be due to
dPBS-PLA to PLA for 1 and 5 phr. This network structure formation. In case of 1
might be due to small interfacial interaction and 5 phr of dPBS-PLA, 1 phr showed
between PLA and dPBS-PLA. So, Young’s slightly increasing of modulus. But 5 phr of
modulus were slightly changed. In case of dPBS-PLA showed decreasing of modulus.
10 phr dPBS-PLA, the result showed Because increasing of rubbery phase content
decreasing of Young’s modulus. This might to PLA, it could impact to strength of
be due to high content of dPBS-PLA and overall plastics. However, the elongation at
leaded to agglomeration. In case of
break of crosslinked film at 5 phr of dPBS- References
PLA showed highest elongation at break. 1. http://www.americanchemistry.com
2. Gruber, P.; O’Brien, M. in Biopolymer:
4. Conclusion Polyester III. Applications and
The dPBS-PLA copolymers which Commercial Product; Steinbüchel, A.,
consisted of unsaturated bond in main chain Doi, Y., Eds.; Wiley-VCH: Weinheim,
have been successfully to prepare. The 2002; pp 235-250.
results showed that dPBS-PLA performed as 3. Tsuji, H.; Sumida, K. J. Appl. Polym.
plasticizers by decreased Tg of the blends. In Sci. 2001, 79, 1582-1589.
addition, the blends showed indifferent in 4. Bleach, N. C.; Tanner, K. E.; Kellomaki,
Young’s modulus after blending with dPBS- M.; Tormala, P. J. Mater. Sci. Mater.
PLA but increased after crosslinking. So, Med. 2001, 12, 911-915.
this dPBS-PLA could perform as plasticizer 5. Dorgan, J. R.; Lehermeier, H. J.; Palade,
and network builder. L. I.; Cicero, J. Macromol. Symp. 2001,
175, 55-66.
Acknowledgements 6. Lehermeier, H. J.; Dorgan, J. R.; Way,
This work was supported by D. J. Membr. Sci. 2001, 190, 243-251.
Research University Network (RUN) and 7. Ouchi, T.; Ohya. Y. J. Polym. Sci. Pol.
Development and Promotion of Science and Chem. 2004, 42, 453-462.
Technology Talents Project (DPST). The 8. Auras, R.; Harte, B; Selke, S. Macromol.
author would like to thank Department of Biosci. 2004, 4, 835-864.
Materials Science, Faculty of Science, 9. Grijpma, D. W. , Nijenhuis, A. J.; van
Kasetsart University and Prof. Suwabun Wijk, P. G. T.; Pennings, A. J. Polym.
Chirachanchai for supported analytical Bull. 1992, 29, 571-578.
instrument. Finally, the author would like to 10. Park, J. W.; Im, S. S. J. Appl. Polym.
thank Dr. Puripong Totsatitpaisan and PTT Sci. 2002, 86, 647-655.
Global Chemical Public Company Limited 11. Xu, J.; Guo, B. H. Biotechnol. J. 2010,
for PLA resin supported. 5, 1149-1163.
High recovery stress obtained in benzoxazine-epoxy shape memory
polymers reinforced with carbon fiber
Jutamart Plylaharn, Manunya Okhawilai, Sarawut Rimdusit*
Polymer Engineering Laboratory, Department of Chemical Engineering,
*E-mail: sarawut.r@chula.ac.th
Abstract:
Shape memory polymers (SMPs) are materials that possess an ability to recover their
original shape upon receiving an external stimulation. In this research, shape memory
behavior of benzoxazine (BA-a)/epoxy binary systems reinforced with woven carbon fiber
were investigated. The contents of the woven carbon fiber were varied from 23, 47, and
57vol%. Shape fixity, shape recovery, and recovery stress of BA-a/epoxy reinforced woven
carbon fiber were observed by a universal testing machine equipped with an environmental
chamber. The results revealed that shape fixity of the composite slightly decreased with
increasing carbon fiber content; however, the shape fixity values were greater than 95%. No
significant difference in shape recovery was noticed with various carbon fiber content by
providing the values to be up to 100%. Interestingly, the recovery stress of the composite
having 47vol% of carbon fiber was as high as 28.9 MPa which was 10 folds greater than the
BA-a/epoxy alloy i.e. 3.4 MPa.
1. Introduction metal alloys. Furthermore, the properties of

Shape memory polymers (SMPs) are thermoset-type SMPs (TS-SMPs) were
materials which possess an ability to recover reported to provide greater recovery stress
their original shape upon receiving an compared to thermoplastics-type SMPs (TP-
external stimulation, including temperature SMPs) in addition to its inherent higher
change, electricity, light induction, chemical resistance, a greater storage
mechanical force, magnetic field, and pH modulus, as well as higher glass transition
variation.1,2 Generally, the SMPs are temperature.4 TS-SMPs are thus the main
stimulated by heat. The shape of SMPs focus of this research.
which is stimulated using heat can be Polybenzoxazine (PBA-a) is a new
deformed when the material was heated class of thermosetting phenolic resin. The
above its transition temperature (Ttrans). It PBA-a has excellent properties, such as high
can remain in a temporary shape when the thermal stability, ease of processing, no by-
temperature is lower than the transition product upon curing, low water absorption,
temperature (Tg). When, the temperature almost near zero shrinkage and high glass
was over its Ttrans, the material can transition temperature.5 The benzoxazine
spontaneously turn to its primary shape.2 resin (BA-a) can also be used as a curing
SMPs possess several advantages compared agent of epoxy.6 Moreover, copolymers
to shape memory metal alloys including, from BA-a and epoxy were reported to
low density, high percentage of strain, low provide an improvement in thermal and
cost, ease of processing, simple design and mechanical properties of the resulting
good manufacturability. In the other hand, copolymers.7
SMPs usually have relatively low modulus Recently, Rimdusit et al.8 developed
and strength while it is in the rubbery state shape memory copolymers based on
i.e. the recovering state.3 This clearly results benzoxazine resin (BA-a), aliphatic-
in the rather low recovery stress of the aromatic epoxy mixture using Jeffamine
material compared to that of shape memory D230 as the epoxy curing agent. Enhanced
thermo-mechanical performance of the Company, respectively. Aliphatic epoxy was
epoxy SMPs was achieved by the addition neopentyl glycol diglycidyl ether (NGDE)
of the BA-a resin. The addition of the BA-a and was supported by Adiya Birla Chemical
in epoxy enhanced the storage modulus in (Thailand).
the glassy state with increasing crosslink 2.2 Synthesis of benzoxazine resin
density, flexural strength and flexural BA-a resin was synthesized by a
modulus at room temperature of the solventless technique.5 In the reactor,
resulting polymers. Furthermore, the bisphenol-A, para-formaldehyde and aniline
recovery stress of the obtained BA-a/epoxy were mixed at the 1:4:2 molar ratio at room
shape memory alloys increased up to 38 kPa temperature. The mixture was then stirred
with increasing BA-a content compared to continuously at 110 ºC until it became
the value of 20.4 kPa of the unmodified transparent yellow liquid (around 1 hours).
shape memory epoxy (no benzoxazine After that, the resulting benzoxazine
addition). More recently, Tanpitaksit et al.6 monomer was cooled at room temperature to
studied SMPs from binary mixtures based yield yellow solid which was then ground
on benzoxazine-modified aliphatic epoxy. into powder for further use.
These binary polymer alloys provided 2.3 Composite preparation
relatively high thermo-mechanical Firstly, the BA-a monomer was
properties with good shape memory mixed with aliphatic epoxy at a fixed
properties. The highest value of the recovery volume ratio of 50:50 at a temperature of 80
º
stress of these binary benzoxazine-epoxy C and stirred for 30 minutes. Secondly, the
mixtures was reported to be as high as 1.59 mixture was heated in an oven at a
MPa at 50mol% of BA-a (which is the ratio temperature of 130 ºC for 12 hours, 140 ºC
of maximum recovery stress).6 In addition, for 4 hours. Then, the BA-a/epoxy mixture
Bin Hong et al.9 studied woven carbon fiber- was pre-impreged with woven carbon fiber
reinforced shape memory polymer having various volume percentages
composites. This report suggested the high including 23, 47, and 57vol% by hand lay-
recovery stress (RS) of the composites to be up technique. The 4-ply of the obtained
substantially enhanced with an incorporation prepreg was heated in an oven at a
of reinforcing woven carbon fiber. The temperature of 150 ºC for 1 hour. Finally,
recovery stress value as high as 4 MPa was the 4-ply of the obtained prepreg was then
reported. compressed employing a compression
This research aims to improve shape molder at a temperature of 170 ºC and a
recovery stress of SMPs based on pressure of 10 MPa for 3 hours.
benzoxazine-epoxy polymer matrix by 2.4 Specimen characterizations
utilizing woven carbon fiber reinforcement. Thermogravimetric analysis was
It is expected that woven carbon fiber operated on thermogravimetric analyzer
should significantly improve recovery stress (model TGA1 Module) from Mettler-
of the starting SMPs as carbon fiber has Toledo. The testing temperature program
been reported to be a good reinforcement.10 was ramped at a heating rate of 20 ºC/min
from 30 to 850 ºC under nitrogen
2. Materials and Methods atmosphere with a constant flow rate of 50
2.1 Materials ml/min. The sample mass used was
Benzoxazine resin (BA-a) is based measured to be approximately 10-15 mg.
on bisphenol-A, para-formaldehyde and The degradation temperature (Td) and solid
aniline. Bisphenol-A (AR grade) was residue of the samples were reported at their
supported by PTT Phenol Company Limited. 5% weight loss and at 800 ºC, respectively.
Paraformaldehyde (AR grade) and aniline Shape memory properties including
(AR grade) were purchased from Merck shape fixity, shape recovery and recovery
Company and Panreac Quimica SA stress of the BA-a/ECF4 were obtained
using a universal testing machine equipped 100
with an environmental chamber. The
Residual weight (%)

experimental procedure of the recovery 80
stress was schematically shown in Figure 1.

60
Primarily, a fixed shape was performed by
applying a flexural force on a specimen (10 40
mm × 50 mm × 1.2 mm) at its Tg+20 ºC i.e. CF
BA-a/Epoxy
20 23vol%
160 ºC then the sample was cooled down to 47vol%
57vol%
room temperature. The force was then 0

0 100 200 300 400 500 600 700 800
removed completely, and the temporary Temperature ( oC)
shape was observed. The deflection after
unloading was controlled to be the same for Figure 2. Thermogravimatric curve of BA-
each specimen i.e. 5% strain. Afterwards, a/epoxy resins reinforced woven carbon
the specimen was heated up to its Tg+20 ºC fiber
and the recovery stress during heating was
measured. 800 °C were expectedly found to increase
with increasing woven carbon fiber contents,
that is, from 24.6wt% of the sample without
woven carbon fiber to 73.7wt% of the
sample with 57vol% of carbon fiber. The
char yield at 800 ºC of all specimens were
summarized in Table 1.
Table 1. Char yield of the BA-a/epoxy

reinforced with woven carbon fiber
composite at various woven carbon fiber
contents
Figure 1. A photograph of experimental Woven carbon fiber content Char yield (%)
procedure for shape recovery stress (vol%)
determination 0 24.6
23 45.6
3. Results and Discussion 47 63.5
57 73.7
3.1 Thermal stability of BA-a/epoxy 100 95.4
Thermogravimetric analysis was
3.2 Shape fixity behaviors of BA-a/epoxy
used to study the thermal stability of the
BA-a/epoxy reinforced with woven carbon
Shape fixity of BA-a/epoxy
fiber shape memory polymer composites
reinforced with woven carbon fiber SMPCs
(SMPCs) as can be seen in Figure 2.
at varied contents of carbon fibers from 23,
The result indicated that the Td5 of
47, and 57vol% were investigated by a
the BA-a/epoxy reinforced with woven
universal testing machine equipped with an
carbon fiber increased with an increase in
environmental chamber. The results are
woven carbon fiber content i.e. the values
shown in Figure 3.
were 289, 314, and 323 ºC for the BA-
The result shows that an increase in
a/epoxy reinforced woven carbon fiber with
volume fraction of the woven carbon fiber in
23, 47, and 57vol% of carbon fiber content,
the BA-a/epoxy resulted in a slight decrease
respectively. Moreover, the solid residues at
in the composite’s shape fixity. However,
the shape fixities of all specimens were
observed to be greater than 95% which were
high enough for typical shape memory
performance and significantly higher than
that of some other shape memory
composites.11 Epoxy reinforced with woven
glass fiber exhibited the shape fixity value
of 81%.
100
90
Shape fixity (%)
80 Figure 5. Series of images showing shape

recovery of BA-a/epoxy reinforced with
70
woven carbon fiber SMPCs at 47vol% of
woven carbon fiber content
60
50
3.4 Recovery stress of BA-a/epoxy
0 10 20 30 40 50 60 reinforced with woven carbon fiber
Woven carbon fiber content (vol%) The recovery stress versus time
Figure 3. Shape fixity of BA-a/epoxy resins under bending mode of our SMPCs is shown
reinforced with woven carbon fiber in Table 2. From this figure, the maximum
recovery stresses of the SMPCs samples
3.3 Shape recovery of BA-a/epoxy were observed to be 9.5, 28.9, and 13.5 MPa
reinforced with woven carbon fiber at the woven carbon fiber contents of 23, 47
Shape recovery behaviors of BA- and 57vol%, respectively which were higher
a/epoxy reinforced with woven carbon fiber than that of the neat BA-a/epoxy i.e.3.4
are presented in Figure 4. Shape recovery of MPa.12 It can be seen that a maximum
BA-a/epoxy reinforced with various carbon recovery stress was obtained from the
fiber contents exhibited relatively high composite having 47vol% of carbon fiber. It
shape recovery of about 99%. Figure 5 might because carbon fiber reinforcement
shows image series of shape recovery could enhance elastic modulus and stiffen
determination of the BA-a/epoxy reinforced the stable network of the composite.10
with 47vol% of carbon fiber.
Table 2. Recovery stress versus time of BA-
a/epoxy reinforced with woven carbon fiber
100
SMPCs
Woven carbon fiber content Recovery Stress
Shape recovery (%)
80
(vol%) (MPa)
60 23 9.5
47 28.9
40 57 13.5
20
4. Conclusion
0 The recovery stress of BA-a/epoxy
20 25 30 35 40 45 50 55 60 binary systems reinforced with carbon fiber
Woven carbon fiber content (vol%)
have been successfully developed from 3.4
Figure 4. Effects of carbon fiber volume MPa12 of the BA-a/epoxy without
fraction on shape recovery of BA-a/epoxy reinforcing fiber to 28.9 MPa of the
reinforced woven carbon fiber composite with 47vol% of carbon fiber. An
increase in carbon fiber volume content
resulted in a slight decrease in shape fixity
value where no difference in shape recovery
was observed in the composite with various 2. Leng, J.; Lan, X.; Liu, Y.; Du, S. Prog.
carbon fiber content. Finally, the Mater. Sci. 2011, 56, 1077-1135.
degradation temperature of the composite 3. Liu, Y.; Du, H.; Liu, L.; Leng, J. Smart
also was improved by an addition of the Mater. Struct. 2014, 23.
woven carbon fiber. The BA-a/epoxy 4. Xiao, X.; Kong, D.; Qiu, X.; Zhang, W.;
reinforced with carbon fiber is the promising Liu, Y.; Zhang, S.; Zhang, F.; Hu, Y.;
material to be apply as shape memory Leng, J. Sci. Rep. 2015, 5, 14137.
polymer composites. 5. Rimdusit, S.; Tiptipakorn, S.; Jubsilp,
C.; Takeichi, T. React. Funct. Polym.
Acknowledgements 2013, 73, 369-380.
This research has been supported by 6. Tanpitaksit, T.; Jubsilp, C.; Rimdusit, S.
the Ratchadaphiseksomphot Endowment Express Polym. Lett. 2015, 9, 824-837.
Fund of Chulalongkorn University, the 7. Allen D. J.; Ishida H. Polymer 1996, 37,
Institutional Research Grant (The Thailand 4487-4495.
Research Fund), IRG 5780014, Department 8. Rimdusit, S.; Lohwerathama, M.;
of Chemical Engineering, Chulalongkorn Hemvichian, K.; Kasemsiri, P.;
University,Contract No.RES_57_411_21_ Dueramae, I. Smart Mater. Struct. 2013,
076 and Neopentyl glycol diglycidyl ether 22.
(NGDE) was supported by Aditya Birla 9. Rousseau, I. A. Polym. Eng. Sci. 2008,
Chemical (Thailand). 48, 2075-2089.
10. Fejős, M.; Romhány, G.; Karger-
References Kocsis, J. J. Reinf. Plast. Compos. 2012,
1. Wei, Z. G.; Sandstrom, R. J. Mater. Sci. 31, 1532-1537.
1998, 33, 3743-3762.
Tensile behaviors of multi-walled carbon nanotube filled
polybenzoxazine/urethane alloys reinforced with aramid fiber
Jusmin Daungkumsawat, Manunya Okhawilai, Tewarak Parnklang, Sarawut Rimdusit*
Polymer Engineering Laboratory, Department of Chemical Engineering, Faculty of Engineering,
Abstract:
In this research, effects of multi-walled carbon nanotubes (MWCNTs) mass
concentrations on properties of MWCNTs filled aramid fabric reinforced polybenzoxazine
(PBA)/polyurethane (PU) alloys were evaluated. The polymer matrixes were prepared by
blending of MWCNTs at various mass concentrations ranging from 0.0–2.0wt% in PBA/PU
(80/20) alloy and hand lay-up onto aramid fabric followed by a compression molding. The
obtained composites provided outstandingly high tensile properties with an addition of
MWCNTs. The tensile strength of the aramid fabric reinforced PBA/PU (80/20) composites
increased from 296.7 MPa to 415.5 MPa with an addition of 1.0wt% MWCNTs and the
maximum modulus and strain at maximum stress were measured to be 18.22 GPa and 5.18%,
respectively. The highest glass transition temperature (Tg) of the composites was observed at
1.0wt% MWCNTs. The degradation temperature and char yield also increased with
increasing MWCNT contents, indicating that MWCNT can substantially improve the thermal
stability of the polymer matrixes. Our results suggested that the addition of 1.0wt% MWCNT
in aramid fabric reinforced PBA/PU composites is an optimal for high strength and low
deformation applications such as ballistic helmets.
1. Introduction process, high thermal stability, good

Fiber reinforced polymer composites mechanical properties and can be alloyed
are composite materials that made of with various types of resin for a wide range
polymer matrixes and embedded reinforcing of applications.4 Rimdusit et al. reported that
fibers in which desirable performances are the toughness of the polybenzoxazine were
achieved.1 Fiber reinforced polymer compo- enhanced when it was alloyed with urethane
sites exhibited high mechanical and thermal elastomer (PU).5 Flexural strength of the
properties2 and they have been increasingly alloys exhibited the synergistic behavior at
employed in many engineering applications 10-20wt% of PU. Moreover, cross-linked
such as aerospace, automotive, and military densities and glass transition temperatures
communities such as armors and light (Tg) was unexpectedly observed to increase
weight ballistic helmets. Aramid fiber is one with increasing mass concentrations of PU.
type of high-performance reinforcing fibers While, the degradation temperature at 5%
possessing outstanding properties such as weight loss (Td,5) of PBA/ PU alloys was
high tensile properties, high modulus, low higher than Td,5 of the neat polybenzoxazine
density, good heat and flame resistance.3 (330 oC). Therefore, alloying PBA with PU
Whereas the polymer matrixes utilized in can effectively improve thermal stability of
fiber reinforced composites are usually the polybenzoxazine. Okhawilai and
epoxy, vinyl ester, and phenolic resins. Rimdusit studied mechanical properties and
Polybenzoxazine ( PBA) , as a new kind of ballistic performances of aramid fabric
thermoset resin developed from phenolic reinforced PBA/ PU alloys for soft armor
resin has possesses many outstanding application.4 The highest flexural strain at
properties including low viscosity, near-zero maximum stress about 1.35±0.17% and the
shrinkage, no by-product gained from curing highest energy absorption about 399±14 J of
aramid fiber-reinforced PBA/PU (80/20) following the patented solvent-less tech-
composites were achieved because of a nology.10 The benzoxazine monomer is a
proper adhesion between the polymer matrix light yellow solid at room temperature. A
and fibers in the composite. solid monomer was crushed into a fine
Nanocomposites involving multi- powder and kept in a refrigerator for future
walled carbon nanotubes ( MWCNTs) has use. Polyurethane (PU) was prepared by
been utilized to modify mechanical mixing toluene diisocyanate and poly-
properties of polymer matrix.6-8 MWCNTs propylene glycol having molecular weight
exhibit remarkable properties due to its low of 2000 g/mol in a four-necked round-
density and high aspect ratio. MWCNTs bottomed flask under a nitrogen stream at a
also possessed exceptional mechanical temperature of 70 ºC for 40 minutes to yield
properties such as extremely high modulus a light yellow prepolymer. The urethane
and stiffness6 Taraghi et al. reported that an prepolymer was kept in a nitrogen-purged
addition of small amounts of MWCNTs closed container and stored in a freezer.
enhanced the Young’s modulus up to 20% 2.3 Specimen preparation
in comparison with the woven Kevlar The polymer matrices were prepared
fabric/epoxy system without MWCNTs.9 by adding MWCNTs at desired mass
As a consequence, in this research, concentrations into PBA/ PU (80/20) alloys
effects of MWCNTs contents on the tensile at 110 oC. The prepared MWCNT molding
behavior of PBA/ PU alloys reinforced with compound was subsequently coated on
aramid fabrics were evaluated. In addition, aramid fabrics. The mass concentration of
influence of MWCNTs on glass transition the aramid fabrics in the composites was
temperatures, thermal degradation approximately 80wt%. The prepregs were
temperatures, and char yields of PBA/PU obtained by using a hand lay-up method
alloys without reinforcing fabric is also followed by a compression molding at a
investigated. temperature of 200 oC and a pressure of 100
bar for 2 hours.
2. Materials and Methods 2.4 Characterization
2.1 Materials The tensile tests of 8-ply aramid
Benzoxazine resin is based on bis- fabric reinforced PBA/PU (80/20)
phenol A, aniline and para-formaldehyde. composites were performed based on ASTM
Bisphenol A (polycarbonate grade) was D3039 standard. The specimen dimension
supported by PTT Phenol Co., Ltd. Aniline was 150×25×3.15 mm3. The test specimens
(AR grade) and para-formaldehyde (AR were performed with a cross head speed of 2
grade) were purchased from Panreac mm/min using a universal testing machine
Quimica SA Company and Merck (8872, Instron Co., Ltd., Thailand).
Company, respectively. Toluene diisocy- Thermogravimetric analysis (model
anate and polyether polyol with a molecular TGA1 Module) from Mettler-Toledo was
weight of 2000 g/mol were supported by used to analyze thermal stability of the
IRPC Public Company Limited. Aramid polymer matrices without aramid fabrics.
fabrics having areal densities of 340 g/m2 The sample was ground into small pieces
were purchased from Thai Polyadd Limited with a mass of 5-10 mg. The sample was
Partnership (Thailand). Whereas MWCNTs heated from 25 oC to 800 oC with a heating
were purchased from Nano Generation rate of 20oC/min under nitrogen atmosphere
Company Limited. at a flow rate of 50 mL/min. The
2.2 Resin preparation degradation temperature at 5% weight loss
Benzoxazine resin was synthesized (Td,5) and the char yield at 800 oC were
from bisphenol-A, para-formaldehyde and reported for each specimen.
aniline at a molar ratio of 142 at a Differential scanning calorimetry
temperature of 110 °C for 40 minutes (model DSC 2910) from Mettler-Toledo was
used to measure glass transition 3.2 Thermal stabilities of MWCNT filled
temperatures of the polymer matrices PBA/PU alloys
without aramid fabrics. A sample mass of 5– Degradation temperature at 5%
10 mg was sealed in an aluminum pan with weight loss (Td,5) and char yields at 800 oC
lid and heated from 30 oC to 300 oC with a of all samples are summarized in Table 1.
heating rate of 10 oC/min under a nitrogen
purging at a gas flow rate of 50 mL/min. Table 1. Degradation temperatures at 5%
weight loss and char yields of the alloys at
3. Results and Discussion various MWCNT mass concentrations
MWCNT Td,5 (oC) Char yield
3.1 Glass transition temperatures (Tg) of (wt%) (%)
MWCNT filled PBA/PU alloys
0.0 337 24.9
Effects of MWCNT mass 0.5 341 25.6
concentrations on glass transition 1.0 342 25.8
temperatures (Tg) of MWCNT filled 2.0 344 26.1
PBA/PU composites is shown in Figure 1. 100 - 96.8
215
The degradation temperature at 5%
210 weight loss (Td,5) and char yields at 800 oC
were measured for evaluating thermal
205 stabilities of the alloys. Td,5 of the PBA/PU
alloy without MWCNTs was about 337oC.
T ( C)
200
o
g
While Td,5 of PBA/PU alloys at MWCNT

195 mass concentrations of 0.5wt%, 1.0wt%, and
190 2.0wt% were 341 oC, 342 oC, and 344 oC,
respectively. Therefore, an addition of
185
0.0 0.4 0.8 1.2 1.6 2.0 MWCNTs improved the thermal stabilities
MWCNT Contents (wt%) of the polymer matrix. The char yields or the
Figure 1. Glass transition temperatures of residual weights at 800 oC of the polymer
PBA/PU filled with MWCNTs composite at alloys were found to increase with
various mass concentrations increasing MWCNT mass concentrations as
expected. The char yield at 800oC of the
At the MWCNT mass concentrations PBA/PU alloy without MWCNTs was
of 1.0wt%, the Tg of the alloys shifted determined to be 24.9wt%. Whereas the char
towards higher temperature (from 195 oC to yields at 800 oC of PBA/PU alloys at
205 oC). However, the Tg of the MWCNT MWCNT mass concentrations of 0.5wt%,
filled PBA/PU alloys decreased when the 1.0wt%, and 2.0wt% were 25.6%, 25.8%,
MWCNT mass concentration was 2.0wt% and 26.1%, respectively. Increasing
(i.e. 200 oC). The reason for the increasing MWCNT mass concentrations enhanced the
of Tg was attributed to the polymer chain char yield of the alloy because MWCNT
confinement effect at the vicinity of exhibited extremely high char yield about
MWCNTs. Aggregation and uneven 96.8% which help increase thermal stab-
dispersion of MWCNTs at high mass ilities of the polymer matrixes.
concentrations of i.e. 2.0wt% attributed to 3.3 Effects of MWCNT mass concentrations
the decrease of Tg of the alloys as also on tensile behaviors of PBA/PU alloys
observed and reported in previous reinforced with aramid fabrics
literatures.6,11 Figure 2 shows the specimen
dimension of PBA/PU alloys reinforced
with aramid fabrics before testing.
Tensile Strength (MPa)
(a)
Young's Modulus (GPa)
500 20
400
Young's Modulus (GPa)

Tensile Strength (MPa)
15
300
10
200
(a) (b) (c) (d) 5
100
Figure 2. The specimen of PBA/PU alloys
reinforced with aramid fabrics at (a) 0 0
0.0wt%, (b) 0.5wt%, (c) 1.0wt%, (d) 0.0 0.5 1.0 2.0
MWCNT Contents (wt%)
2.0wt% MWCNTs before tensile testing
(b)
The tensile strength and Young’s
6.0
modulus of the samples are shown in Figure
Strain at Maximum Stress (%)
3. At MWCNT mass concentrations in the 5.5
range of 0.0–1.0wt%, an addition of
MWCNTs into PBA/PU alloys resulted in 5.0
an increase of tensile strengths and Young’s
modulus values of the composites. At 4.5
1.0wt% MWCNTs, the tensile strength of
4.0
the composites reached 415.5 MPa, which
was higher than the tensile strength of the 3.5
aramid fabric reinforced PBA/PU comp-
osites without MWCNTs (296.7 MPa). In 3.0
0.0 0.4 0.8 1.2 1.6 2.0
addition, the strain at maximum stress was
MWCNT Contents (wt%)
raised to 5.18% by adding 1.0wt%
MWCNTs. It was generally accepted that Figure 3. Tensile strength and Young’s
the adamantine MWCNTs with substantial modulus (a) and strain at maximum stress
adhesion to the matrix can restrict the (b) of PBA/PU reinforced with aramid
mobility of the polymer chain and resulted fabrics at various MWCNT mass
in the higher tensile strength and modulus of concentrations
the obtained composites. Therefore,
MWCNTs acted as an effective 4. Conclusion
reinforcement of our polybenzoxazine- The effects of MWCNT mass
urethane alloys with relatively good concentrations on the tensile behavior of the
interfacial inter-action between the matrix PBA/PU composites reinforced with aramid
and the reinforcement. However, at 2.0wt% fabric and the thermal properties of the
MWCNTs, the tensile strength, the modulus, PBA/PU alloys were investigated. The high
the strain at maximum stress tended to tensile properties of aramid fabric reinforced
decrease likely due to the aggregation of PBA/PU composites at 1.0wt% MWCNTs
MWCNTs. suggested an optimum filler content. The
glass transition temperatures and
degradation temperature of the PBA/PU
alloys also increased with increasing
MWCNT mass concentrations. The resulting
composites were suitable for high strength
and low deformation applications such as
light weight ballistic helmet.
Acknowledgement 4. Okhawilai, M.; Rimdusit, S. Advanced
This research has been supported by and Emerging Polybenzoxazine Science
the Ratchadaphiseksomphot Endowment and Technology; Froimowicz, P. Eds.
Fund of Chulalongkorn University and the Elsevier: Amsterdam, 2017; pp 699-
Institutional Research Grant (The Thailand 723.
Research Fund), IRG 5780014, Department 5. Rimdusit, S.; Mongkhonsi, T.;
of Chemical Engineering, Chulalongkorn Kamonchaivanich, P.; Sujirote, K.;
University, Contract No.RES_57_411_21_ Thiptipakorn, S. Polym. Eng. Sci. 2008,
076 and Bisphenol-A was supported by PTT 48, 2238-2246.
Phenol Company Limited. 6. Chen, Q.; Xu, R.; Yu, D. Polym. J.
2006, 47, 7711-7719.
References 7. Laurenzi, S.; Pastore, R.; Giannini, G.;
1. Bai, J. in Advanced Fibre-Reinforced Marchetti, M. Compos. Struct. 2013, 99,
Polymer (FRP) Composites for 62-68.
Structural Applications; Campbell, M. 8. Xiong, J.; Zheng, Z.; Qin, X.; Li, M.;
Eds. Woodhead Publishing: Oxford, Li, H.; Wang, X. Carbon 2006, 44,
2013; pp 1-4. 2701-2707.
2. GangaRao, H. in Applied Plastics 9. Taraghi, I.; Fereidoon, A.; Mohyeddin,
Engineering Handbook; William, A. A. Steel. Compos. Struct. 2014, 17, 825-
Eds. Elsevier: Oxford, 2017; pp 675- 834.
695. 10. Ishida, H. Process for preparation of
3. Akato, K.; Bhat, G. in Structure and benzoxazine compounds in solventless
Properties of High-Performance Fibers; systems. U.S. Patent 5,543,516, 1996.
Gajanan, B. Eds. Woodhead Publishing: 11. Kaleemullah, M.; Khan, S. U.; Kim, J.
Oxford, 2017; pp 245-266. K. Compos. Sci. Technol. 2012, 72,
1968-1976.
Characterization of novel shape memory polymer from
green-polybenzoxazine/epoxy alloy
Phakakrong Hombunma, Manunya Okhawilai, Sarawut Rimdusit*
Polymer Engineering Laboratory, Department of Chemical Engineering,
Abstract:
Shape memory polymer (SMP) is a smart material which can maintain deformed
temporary shape and can recover to its original shape upon an external stimulus such as
temperature, moisture and pH. In this work, a renewable natural resource based on eugenol
and furfurylamine was used to substitute phenol and amine from petroleum based reactants to
provide green polybenzoxazine. Mechanical, thermal and shape memory properties of the
green polybenzoxazine/epoxy alloys were evaluated by a dynamic mechanical analyzer,
thermogravimetric analyzer and a shape recovery test. It was observed that the content of
benzoxazine (E-fa) in aliphatic epoxy (NGDE) to exhibit good shape memory performance
was in a range of 30 to 50wt%. With an increase in E-fa content, glass transition temperature,
degradation temperature at 5% weight loss, and storage modulus of the E-fa/NGDE alloy
increased providing the greatest values of 51 °C, 283 °C, and 4.84 GPa, respectively. The
poly(E-fa)/NGDE alloy SMP also exhibited a high shape fixity and recovery ratio of greater
than 90%. Such results indicated that the poly(E-fa)/NGDE alloy is a good candidate for a
low stimulating temperature shape-memory polymer.
1. Introduction are used to substitute petroleum-based

Shape memory polymers (SMPs) are polybenzoxazine.
smart polymer materials to possess an ability Recently, P. Thirukumaran et al.4
to return from their temporary shapes to successfully synthesized two types of bio-
their permanent shapes as induced by based benzoxazine resins i.e. eugenol-
external stimuli such as light, humidity, furfurylamine based benzoxazine (F-Bz) and
solvents, electric or magnetic fields, ionic eugenol-stearylamine based benzoxazine (S-
strength, pH and temperature1 which the Bz) which were synthesized via a
main stimulus for general SMPs. SMPs can solventless method. The PF-Bz showed the
be widely used in many areas such as maximum thermal stability whereas the
intelligent medical devices, self-deployable enhancement in thermal properties was
structure, automobile and morphing obtained from the copolymer of those types
structures etc..2 of benzoxazine. In this research, the SMPs
S. Rimdusit et al.3 studied and based on eugenol and furfurylamine based
developed SMP based on benzoxazine (BA- green polybenzoxazine/aliphatic epoxy
a) alloyed with epoxy resin. The obtained (NGDE) alloy was prepared. The relevant
SMP alloy retained good thermos- properties of the alloy including thermal,
mechanical properties, excellent shape thermomechanical and shape memory
memory properties with additional ease of behavior were investigated.
sample preparation.
In recent years, the benzoxazine 2. Materials and Methods
research communities have shifted toward 2.1 Materials
incorporating natural renewable resources Eugenol, furfurylamine, and parafor-
into synthesis green polybenzoxazine which maldehyde (AR grade) were purchased from
Sigma-Aldrich Pte. Ltd., Tokyo Chemical degradation temperatures (Td) and char
Industry CO., Ltd., and Merck Company, yields of the samples were reported at their
respectively where neopentyl glycol digly- 5% weight loss and at 800 °C, respectively.
cidyl ether (NGDE) aliphatic epoxy was
supported by Aditya Birla Chemical
(Thailand).
2.2 Resin preparation
Eugenol-furfurylamine based benzo-
xazine monomer (E-fa) was synthesized
from eugenol, furfurylamine and parafor-
maldehyde at a 1:1:2 molar ratio based on a
solventless method.5 The mixture was
heated at a temperature of 100 °C and stirred
Figure 1. The set-up schematic for the shape
for 2 h. The resulting benzoxazine monomer
recovery test
was pale yellow solid at room temperature.
2.3 Benzoxazine-epoxy alloy preparation Shape recovery test of the sample
The as synthesized E-fa was blended upon bending load was performed as shown
with the NGDE which was varied 30 to 50% in Figure 1. The procedure for the thermo-
by weight of E-fa. The mixture was heated mechanical bending cycling of the sample
to a temperature of 90 °C in an aluminum included the following steps. Firstly, the
mold and stirred until a homogeneous original shape of SMPs were kept in an oven
mixture was obtained. The molten resin at Tg + 20 °C; then the sample was bent to a
mixture was thermally cured in an oven and
storage angle θ 0 in a ‘U’ shape, after that the
finally the samples were cooled down to
room temperature. The cured poly(E-fa)/ sample was kept at below Tg – 20 °C; the
NGDE were removed from mold before sample fixed to the apparatus is then put in
testing. an oven at Tg + 20 °C, and then it recovers
2.4 Characterization methods to an angle θ N .6 The shape recovery ratio
Thermomechanical properties of (RN) can be calculated by Equation (1).
poly(E-fa)/NGDE alloys were obtained by a
dynamic mechanical analyzer (DMA, model 0   N
RN  100% (1)
DMA242, Netzsch, Germany). The sample, 0
with a dimension of 10 mm ×50 mm × 2
mm, was tested using a three point bending Shape fixity of poly(E-fa)/NGDE
mode at a heating rate of 2 oC/min from -50 shape memory samples were studied by a
o
C to 100 oC. A test frequency of 1 Hz and universal testing machine under three point
strain amplitude of 5 μm under nitrogen bending. A fixed shape was formed by
atmosphere was used in this experiment. applying a flexural force on a sample (10
Glass transition temperature (Tg) of each mm × 50 mm × 2 mm) at Tg + 20 °C and
sample was obtained from the maximum cooled down to room temperature. The force
peak on the loss tangent curve. was then removed completely to obtain a
Thermogravimetric analysis was temporary shape. The deflection after
studied using a thermogravimetric analyzer unloading was then measured, and shape
(model TGA1 Module) from Mettler-Toledo fixity (Rf) of each sample was determined.
(Thailand). The testing temperature program
was ramped at a heating rate of 20 °C/min 3. Results and Discussion
from 25 to 800 °C under nitrogen 3.1 Thermomechanical properties of poly(E-
atmosphere with a constant N2 purge gas fa)/NGDE alloys
flow rate of 50 mL/min. The sample mass The storage modulus of the poly(E-
used was approximately 5 to 10 mg. The fa)/NGDE alloys at their glassy state (-50
°C) are shown in Figure 2. From the results, was attributed to the more rigidity segments
the storage moduli of the samples increased of the E-fa impeded chain movement of the
with an increase in the contents of the E-fa. polymer.7
The storage modulus of poly(E-fa)/NGDE 3.2 Thermal stability of poly(E-fa)/NGDE
alloys having 30, 40, and 50wt% of E-fa alloys
contents were 4.33, 4.64, and 4.84 GPa,
respectively. It was due to the more rigidity 120
of the E-fa.
100
10
Residual weight (%)

80
60
Storage modulus (GPa)
1
40
20
0.1
0
0 100 200 300 400 500 600 700 800
o
Temperature ( C)
Figure 4. TGA thermograms of poly(E-fa)/

0.01 NGDE alloys at various weight percents of
-50 0 50 100
o
Temperature ( C) E-fa:( ) neat NGDE, ( ) 30wt%, ( )40wt%,
Figure 2. Storage modulus of poly(E-fa)/ ( ) 50wt%, ( ) neat poly(E-fa)
NGDE alloys at various weight percents of
E-fa: ( ) 30wt%, ( ) 40wt%, ( ) 50wt% Thermogravimetric analysis was
used to study the thermal stability of the
1 poly(E-fa)/NGDE alloys which can be seen
in Figure 4. From the figure, the Td5 of the
0.8
alloy increased with an increase in E-fa
content i.e. the values were 275, 276, and
283 °C for the alloys with 30, 40, and
Loss tangent
0.6
50wt% of E-fa content, respectively.
The char yield of the alloys at 800
0.4
°C in nitrogen atmosphere at 30, 40, and
50wt% of E-fa contents were 18, 22, and
0.2 28%, respectively.
3.3 Shape memory behaviors of poly(E-
0 fa)/NGDE alloys
-50 0 50 100
Temperature ( C)
o The shape fixity values of the
Figure 3. Loss tangent of poly(E-fa)/NGDE samples under bending mode are shown in
alloys at various weight percents of E-fa: Figure 5. The result indicated that the
( ) 30wt%, ( ) 40wt%, ( ) 50wt% poly(E-fa)/NGDE alloy could fix in the
temporary shape almost 100% and no
The Tg of the samples was evaluated significant difference in shape fixity was
from the peak of the loss tangent (tan) observed with an increase in E-fa content.
curves as shown in Figure 3. It can be seen Figure 6 shows the shape recovery
that the addition of E-fa promoted a greater behavior of poly(E-fa)/NGDE alloy with 30,
Tg of the samples which the values were 26, 40, and 50wt% of E-fa content at Tg + 20 °C
35, and 51 °C for the samples having 30, 40, i.e. 46, 55, and 71 °C. It can be seen that the
and 50wt% of E-fa content, respectively. It
sample can fully recover to its original shape 4. Conclusion
thus the shape recovery value was 100%. Green polybenzoxazine based on
eugenol and furfurylamine has been
100 successfully synthesized and was applied as
hard segments of shape memory polymers.
The Tg of poly(E-fa)/NGDE SMPs were
95
relatively low which allows ease of shape
Shape fixity (%)
memory performance at low temperature.

90
The addition of E-fa content resulted in an
increase in the Tg and the Td5. Moreover, the
shape memory properties of the alloy
85 including shape fixity and recovery were
almost 100%. It can be concluded from the
results that the poly(E-fa)/NGDE alloys
80
30 35 40 45 50
showed good characteristics of shape
E-fa Content (%wt) memory polymers for low stimulating
Figure 5. Shape fixity of poly(E-fa)/ NGDE temperature.
SMPs at various contents of E-fa
Figure 6. Series of images showing shape recovery of poly(E-fa)/NGDE SMPs
Acknowledgement 2. Leng, J.; Lan, X.; Liu, Y.; Du, S. Prog.

This research has been supported by Mater Sci. 2011, 56, 1077-1135.
the Ratchadaphiseksomphot Endowment 3. Rimdusit, S.; Lohwerathama, M.;
Fund of Chulalongkorn University, the Hemvichian, K.; Kasemsiri, P.;
Institutional Research Grant (The Thailand Dueramae, I. Smart Mater. Struct. 2013,
Research Fund), IRG 5780014, Department 22, 075033.
of Chemical Engineering, Chulalongkorn 4. Thirukumaran, P.; Shakila Parveen, A.;
University,Contract No.RES_57_411_21_07 Sarojadevi, M. ACS Sustainable Chem.
6 and Neopentyl glycol diglycidyl ether Eng. 2014, 2, 2790-2801.
(NGDE) was supported by Aditya Birla 5. Ishida, H. Process for preparation of
Chemical (Thailand). benzoxazine compounds in solventless
systems. U.S. Patents 5,543,516, 1996.
References 6. Xin, L.; Yanju, L.; Haibao, L.; Xiaohua,
1. Meng, Q.; Hu, J. Composites Part A. W.; Jinsong, L.; Shanyi, D. Smart Mater.
2009, 40, 1661-1672. Struct. 2009, 18, 024002/1–024002/6.
7. Tobushi, H.; Hayashi, S.; Hoshio, K.; 8. Rimdusit, S.; Pirstpindvong, S.;
Makino, Y.; Miwa, N. J. Intell. Master. Tanthapanichakoon, W.; Damrongsakkul,
Syst. Struct. 2016, 17, 1075-1081. S. Polym. Eng. Sci. 2005, 45, 288-296.
Supramolecular self-assembling for enhanced oil recovery
Saran Banthong1, Chutima Kongvarhodom1, Laura Romero-Zerón2*
1
Department of Chemical Engineering, Faculty of Engineering, King Mongkut’s University of Technology
Thonburi, Thoongkru, Bangkok 10140, Thailand
2
Department of Chemical Engineering, Faculty of Engineering, University of New Brunswick, Fredericton,
New Brunswick E3B 5A3, Canada
*E-mail: laurarz@unb.ca
Abstract:
Supramolecular self-assemblies are spontaneous organization of molecules formed by
noncovalent interactions. The focus of this research was to formulate and evaluate the self-
assembly capacity of polymer blends displaying improved viscoelasticity and stability for
applications in polymer flooding for Enhanced Oil Recovery (EOR). In this work, an optimum
self-assembly system (SAP) derived from the combination of a partially hydrolyzed
polyacrylamide (HPAM), xanthan polymer (XP), and an associative polymer (AP) was
formulated. This optimum SAP formulation demonstrated enhanced viscoelastic properties,
appropriate salinity tolerance, and increased mechanical and thermal stability compared to the
baseline polymers. As a mobility control agent, the optimum SAP system offers a high effective
viscosity in porous media and therefore enhanced oil displacement efficiency during polymer
flooding of heavy oil at brine salinity concentration of 8.4wt%.
1. Introduction
Crude oil production occurs in three are the most important parameters during the design
phases, which are primary recovery, of polymer flooding.3 Partially hydrolyzed
secondary recovery, and tertiary recovery or polyacrylamide (HPAM) is a commonly used
Enhanced Oil Recovery (EOR) methods. polymer for large-scale applications in most oil
Primary and secondary oil processes recover field projects because of its availability and
around 25% of the original oil in place low manufacturing costs.1,4,5
(OOIP). Therefore, enhanced oil recovery Associating polymers (AP) contain
methods are applied to maximize the hydrophobic groups that offer intermolecular
recovery of the remaining 75% of the oil left associations in aqueous solutions providing increased
behind in the reservoir. EOR methods consist viscosity. AP polymers are expensive due to
in the injection of fluids, such as steam, elevated manufacturing costs.4,6 Xanthan
polymers, surfactants, microbial solutions and so gum (B) is a biopolymer characterized for its low
on. Chemical EOR methods are usually applied manufacturing costs and stability to mechanical shear
because they are operationally less demanding degradation.4
compared to other EOR methods. 1 The most Each type of polymer displays
commonly implemented chemical EOR method is advantages and downsides. Hence, this work
polymer flooding after water flooding. In this aims to formulate a polymer blend system
method, polymer solution is injected into the derived from HPAM, AP, and B that could
reservoir to increase the viscosity of water to drive provide improved viscoelastic properties and
oil to the production wells. Advantages of polymer incremental oil recovery.
flooding are: it requires the injection of lower
volumes of water and produces less water 2. Materials and Methods
lowering the costs of produced water Heavy oil samples were provided by Husky
treatments to comply with environmental Energy Inc. (Calgary, AB, Canada). The viscosity of
regulations.2 the heavy oil was adjusted to 3000 cP at 25
Polymer selection and solution concentration ºC by dilution with natural condensate. The
density of the diluted crude oil was 0.9 g/ml range previously determined from the
at 25 ºC. HPAM with a degree of hydrolysis amplitude sweep.
ranging from 5 to 10 mole% provided by 2.2 Salinity tolerance
Geltech (Midland, Texas, USA), biopolymer (B) The effect of brine salinity levels on
acquired from Groupe Maison Cannelle Inc. the performance of the optimum SAP system
(Richmond, Quebec, Canada), and associating was determined at the following brine
polymer (AP) of low anionicity and low concentrations: 2.1, 4.2, and 8.4wt%.
molecular weight was provided by SNF 2.3 Thermal stability
Holding Co. (Riceboro, Georgia, USA). Polymer The thermal stability of the optimum
solutions and polymer blends were prepared in SAP systems and the corresponding baselines
synthetic reservoir brine. Different concentrations of was studied at 90 ºC for a period of 8 weeks.
synthetic brine were used. Table 1 presents the The optimum SAP systems and
compositions of the different brines. corresponding baselines were prepared in 2.1
Table 1. Synthetic brine compositions and 8.4 wt% brine. All samples were
Concentration (wt% or TDS) subjected to rheology measurements every 2
Components
2.1* 4.21* 8.41* weeks The experimental results are shown as
NaCl 1.72 3.45 6.90 G”(ω) and G’(ω).
MgCl2 0.04 0.09 0.18
CaCl2 0.33 0.65 1.30 2.4 Mechanical degradation
Na2SO4 0.01 0.02 0.04 The ionic and structural strengths of
*
Total brine concentration the SAP systems and corresponding baselines
2.1 Formulation of the self - assembling were investigated via thixotropic analysis. A
polymeric (SAP) system material is thixotropic if it exhibits a decrease of
In this experimental phase the SAP systems the initial structural strength under high-shear
were formulated and the effect of polymer process followed by their complete structural
concentration on the optimum SAP formulation was regeneration in the subsequent state of rest.7
determined. Rheology analysis was used to In these experiments, the optimum polymer
characterize the SAP systems in terms of the viscous blend and the baseline polymers were
(G”) and elastic (G’) moduli as a function of angular prepared in 2.1 and 8.4wt% brine. Shear
frequency (ω). Table 2 presents the experimental stability was evaluated through rheological
design of the polymer blends. analysis by the application of three steps as
follows: Step 1: 20% strain for 16 minutes
Table 2. Experimental design: SAP formulations (within the LVE range), Step 2: 1000% strain
Concentration (wt%) for 8 minutes (outside LVE range), and Step
SAP formulations
B HPAM AP
B-HPAM-AP 0.2 0.2 0.1 3: 20% strain for 16 minutes (within the LVE
B-HPAM 0.2 0.2 0 range). The experimental results are shown as
B-AP 0.2 0 0.1 G”(ω), G’(ω), and a percentage (%) of
HPAM-AP 0 0.2 0.1 regeneration.
B 0.2 0 0
HPAM 0 0.2 0 2.5 Heavy oil recovery
AP 0 0 0.1 Four heavy oil displacement tests
were conducted by a sand-pack system of the
A Bohlin Gemini HR 150 Nano following dimensions: L = 15 cm and cross-
rheometer manufactured by Malvern sectional area = 47.76 cm at 25 ºC. The
(Worcestershire, UK) was used for the rubber sleeve was packed using play sand
rheological analysis. First, an amplitude (100% quartz) and placed into the core
sweep was performed by varying the strain holder. A synthetic brine concentration of
from 1% to 1000% at a constant frequency to 8.4wt% was used during the water flooding
determine the limit of the linear viscoelastic step. The polymer samples were also
(LVE) range at 25 ºC. The frequency sweep prepared in 8.4wt% brine. The overburden
was then run varying the frequency range pressure around the sand-pack was kept
from 0.1 to 100 rad/s within the limit of LVE stable at 500 psi. Figure 1 displays a
simplified schematic of the sand-pack ΔP post waterflooding
RRF  (2)
experimental set-up. The experimental procedure ΔP water flooding
was as follows: (1) The brine was injected into As shown in Table 3, the porosity and
the sand pack and the basic properties of the permeability of the sand-pack systems are
porous media were determined as shown in Table different from each other. Thus, to make the results
3. (2) The diluted heavy oil was then injected comparable with one another, the capillary bundle
into the sand pack until water production stopped. parameter was used to normalize the data for
(3) Next, waterflooding was initiated at a flow rate porosity and permeability.10 The capillary bundle
of 1 ft/day until oil production stopped. (4) After parameter equation is shown below:
that, 1 PV of the polymer solution was injected as (1 -  ) A
EOR polymer flooding. (5) Finally, brine injection Capillary bundle parameter  u  t   (3)
was re-started into the sand pack as a post-polymer φk 0.5
PV
water flooding to recover additional oil. where u is flux (ft/day), t is injection time (day),  is
porosity, k is permeability (mD), A is cross-sectional
area of the sand-pack (ft2), and PV is pore volume
(ft3)

3.1 Formulation of the optimum self-
assembling polymeric (SAP) system
The rheological properties of the baseline
Figure 1. Sand-pack experimental set-up polymers (HPAM, B, and AP) were evaluated in
Table 3. Properties of the porous media terms of elastic and viscous moduli vs. angular
Pore frequency as shown in Figures 2 and 3.
Porosity Permeability
Test # volume
[%] [mD]
[cm3]
Baseline # 1 208 29 10049
Baseline # 2 206 29 8667
SAP # 1 204 29 8109
SAP # 2 187 26 4700
The differential pressure data along the

sand-pack and injection time were recorded and
analyzed. The results were presented as a Figure 2. G’ vs.  and polymer type
resistance factor (RF) and residual resistance
factor (RRF). RF indicates the effective viscosity
of the polymer solution (polymer injection) in the
porous media relative to water (water flooding),
while RRF tells the permeability reduction due to
polymer retention.8,9 RF and RRF equations are
shown below; where P is the differential pressure
along the sand-pack. Ppolymer flooding is the
differential pressure during the polymer flooding
stage. P post-waterflooding is the differential pressure Figure 3. G” vs.  and polymer type
during the water flooding step that follows the Figures 2 and 3 indicate that polymer B
polymer flooding. P waterflooding is the differential exhibits the maximum elastic and viscous moduli
pressure during the water flooding. in the range of angular frequencies evaluated.
ΔP polymerflooding
Moreover, its elasticity dramatically escalates with
RF  (1) the increasing of angular frequency. Polymer B
ΔP water flooding provides excellent viscoelastic properties and in
this work, it was considered as the most effective Table 4 presents the experimental design
polymer compared to the HPAM and AP. After followed to establish the effect of polymer
that, the viscoelasticity of the combinations of concentration (B and AP) on the properties of the SAP
polymers was investigated through rheology system.
analysis as shown in Figures 4 and 5. Figures 6 and 7 show that the G’- and G”-
values of the optimum blends increase with the
increasing concentration of B. Besides, AP also
improves the elasticity of the blend, which could be
due to the hydrophobicity of AP resulting in a higher
number of physical interactions. From these results,
the optimum polymer blend was selected to be: B (0.3
Figure 4. G’ vs. and polymer blend
wt%)-HPAM-AP.
Figure 6. G’ vs.  and B concentration in SAP
Figure 5. G” vs. and polymer blend

The polymer blend HPAM-AP was
neglected in this study because of its very low
viscoelasticity, compared to the other blends. Figures
4 and 5 present that the blend of B-HPAM-AP
provides the highest values of G’- and G”-values in
the entire frequency range. It seems that a self-
Figure 7. G” vs.  and B concentration in SAP
associating system has been formed through
noncovalent interactions in this blend, such as 3.2 Salinity tolerance
hydrogen bonding and hydrophobic interactions, as Figures 8 and 9 display the effect of brine
previous research has demonstrated 1,11. These salinity (2.1, 4.2 and 8.4 wt%) on the viscoelasticity of
interactions enhance the structural strength of the the optimum blends and baseline polymers.
system and improve its rheological properties. Figures 8 and 9 show that the G’-and G”-
Therefore, this blend was selected as the optimum curves of the optimum blends are higher than those of
blend or self-assembling system. the baselines polymers. Also, the G’- and G”-values of
the optimum blends increase with increasing brine
Table 4. Composition of the SAP formulations
salinity. It is possible that stronger hydrophobic
SAP formulations
Concentration (wt%) interactions take place as salinity increases.11 Likewise,
B HPAM AP at high salinities and hardness concentrations the
B (0.1 wt%)-HPAM 0.1 0.2 0
0.2 0.2 0
strength of other physical interactions such hydrogen
B (0.2 wt%)-HPAM
B (0.3 wt%)-HPAM 0.3 0.2 0 bonding, Ca2+ bridges, and inter and/or intra polymer
B (0.1 wt%)-HPAM-AP 0.1 0.2 0.1 molecular entanglements might take place aiding the
B (0.2 wt%)-HPAM-AP 0.2 0.2 0.1 formation of a self-assembly supramolecular
B (0.3 wt%)-HPAM-AP 0.3 0.2 0.1
system.12,-14 From these observations, it can be
concluded that the optimum blend is suitable
for applications in high salinity
environments.
Figure 11. G’ vs. time in 8.4wt% brine

3.4 Thermal stability
Figure 8. G’ vs.  and brine concentration Figures 12 and 13 show the thermal
stability (G’(ω)-value) of the optimum blend
and baseline in 2.1 and 8.4wt% brine at 90 ºC
for 8 weeks.
Figure 9. G” vs.  and brine concentration

3.3 Mechanical stability
Figure 10 illustrates the effect of
shear and ionic strength on the shear stability Figure 12. G’ vs.  and time in 2.1wt% brine
of the optimum blend and baseline polymer
in terms of the time-dependent function of G’ as
suggested by Mezger.7
Figure 13. G’ vs.  and time in 8.4wt% brine

Figure 12 shows that the optimum blend
and baseline were gradually degraded after 2
Figure 10. G’ vs. time in 2.1wt% brine weeks of testing in 2.1 wt% brine concentration.
After 4 weeks, the optimum blend was degraded
faster than the baseline. However, Figure 13
The optimum blend shows a higher structural
indicates that the optimum blend prepared in 8.4
strength than the baseline (Figure 10) with a
wt% brine provides higher thermal stability
regeneration of G’ > 100%. Figure 11 displays the
throughout the 8 weeks compared to the baseline.
shear stability of the polymer samples in 8.4 wt%
Obviously, the optimum blend is suitable for
brine. Again, the optimum blend provides a higher
applications involving elevated temperatures and
structural regeneration (> 100%) than the baseline
high salinity environments.
(96.07%). It is evident that the optimum blend can be
3.5 Heavy oil recovery
used in applications involving high mechanical shear
Figures 14 and 15 illustrate the RF-and
and elevated salinity concentrations.
RRF-curves of the optimum blend and the
baseline polymer, respectively. The optimum 4. Conclusion
blend provides higher RF (i.e. higher effective A self-assembling system based on a
viscosity) than the baseline throughout the tests. polymer blend was formulated in this work. The
Thus, the optimum SAP system provides a better SAP formulation displays suitable viscoelastic
mobility control.15 Figure 15 indicates that the end properties, improved salinity tolerance, thermal
RRF-values for the optimum blend (RRF=2.8) stability, and shear stability compared to the baseline
and the baseline (RRF=1.3) are similar. This polymers. The optimum SAP system also offers
indicates that the SAP system shows higher higher heavy oil recovery due to its enhanced
retention due to adsorption and/or mechanical mobility control properties. Therefore, this optimum
retention within the porous media compared to the SAP system is an interesting alternative as a
baseline. Nevertheless, this degree of retention did viscosifying fluid for EOR processes.
not affect the appropriate propagation of the SAP
Acknowledgements
acknowledge Husky Energy Inc., Geltech,
and SNF Holding Co. for providing heavy oil
and polymer samples.
References
1. Sheng, J.J.; Leonhardt, B.; Azri, N. J.
Can. Pet. Tchnol. 2015, 54, 116-126.
2. Romero-Zeron, L. Advances in Enhanced
system through the sand-pack. Oil Recovery Processes, InTech, Rijeka,
Figure 14. RF vs volume of fluid 2012.
3. Farouq Ali, S. M.; Thomas, S. J. Can.
Pet. Tchnol. 1996, 35, 57-63.
4. Wei, B. Advances in Polymer Flooding,
Viscoelastic and Viscoelastic Materials,
InTech, 2016, DOI: 10.5772/64069.
5. Seright, R.S.; Seheult, Talashek, T. SPE
Reservoir Eval. Eng. 2001, 4, 187-194.
6. Wei, B; Romero-Zeron, L.; Rodrigueb, D.
Adv. Polym. Technol. 2014, 33, 21413 (1-12).
Figure 15. RRF vs volume of fluid 7. Mezger, T.; The Rheology handbook, 3rd
Revised ed., Vincentz Network, 2011.
Figure 16 presents the cumulative of oil 8. Lake, L.W., Petroleum Engineering Handbook,
recovery as a percentage (%) of the original oil in 2007, V, 1149-1260.
place (OOIP) for each injection stage for the SAP 9. Da Silva, I. P. G.; Lucas, E. F. SPE J.2017.
and baseline samples. The optimum polymer 10. Stahl, G.; Schulz, D. S. L. Springer Science &
blend offers an incremental oil recovery of 28% Business Media, 2012.
relative to the baseline. 11. Perttamo, E. K. Master's Degree Thesis,
The university of Bergen, 2013.
12. Wever, D.A.Z.; Picchioni, F.; Broekhuis, A.A.,
Prog. Polym. Sci. 2011, 36, 1558-1268.
Ind. Eng. Chem. Res. 2014, 53, 116 (00-11).
14. Lee, Y.S.; Self-assembly and nanotechnology:
a force balance approach, Wiley & Sons,
2008; pp. 3-8.
Figure 16. Oil recovery vs volume of fluid J. Petrol. Explor. Prod. Technol. 2013.
Effect of drinking water treatment sludge on the properties of
rubber compounds
Wasana Khongwong*, Nittaya Keawprak, Punthinee Somwongsa, Duriyong Tattaporn,
Piyalak Ngernchuklin
Expert Centre of Innovative Materials, Thailand Institute of Scientific and Technological Research (TISTR),
35 Mu 3 Khlong Ha, Khlong Luang, Pathum Thani 12120, Thailand
*E-mail: wasana@tistr.or.th
Abstract:
To investigate the influence of fillers on the physical and mechanical properties of
rubber compounds, two different types of powder fillers, drinking water treatment sludge
(DWTS) and calcium carbonate (CaCO3), were mixed into rubber compound mixtures. The
mixtures were composed of STR20, EPDM, zinc oxide (ZnO), steric acid, paraffin wax,
2-mercaptobenzothiazole (MBT), sulphur, Wingstay L, pigment and filler. The mixtures were
mixed in a Kneader type mixer at temperature of 70 oC and then continuously mixed by a
two-roll mill at temperature of 70 oC. The relationships between type and the amount of filler
versus properties of rubber compounds were demonstrated. The results showed that tensile
and elongation at break of rubber compounds gradually decreased with increasing the amount
of filler. Comparing of size distribution of filler, rubber compounds with narrower size
distribution possessed higher tensile strength and elongation at break than those of wider size
distribution. Both rubber compounds with DWTS and CaCO3 provided good shore A
hardness. Especially, DWTS filler in rubber compounds showed more than 60 shore A
hardness which can be replaced CaCO3 in normal formula.
1. Introduction
Rubber is an elastomeric material1 pollution problems and the production cost
with excellent properties, such as high are still high.7 Therefore, many researchers
electrical insulation, good water resistance have been attempted to seek alternative filler
and corrosion resistance, high tear resistance to replace carbon black and silica.
and excellent elongation. However, pure Drinking water treatment sludge
rubber itself is difficult to encounter the (DWTS) is produced from the service and
industrial application requirements due to its drinking water treatment plant. The large
low strength and poor thermal properties.2 amount of this produced sludge caused
Therefore, many industrial applications, disposal problem, most of them were used
elastomers are mixed with fillers not only to for land filling.8 However, the composition
improve their performance properties but of DWTS contains both organic and
also to enhance the durability of rubber inorganic substances. Typically, it composes
compound and reduce the cost of the final of silica and alumina which is possible to act
product.3 At present, the common reinforced as inexpensive alternative filler for rubber
fillers mainly include carbon black, silica compounds.
(SiO2), calcium carbonate (CaCO3), talc, or In this work, DWTS and CaCO3
glass that are frequency used and also were used as rubber fillers. Through the
mineral filler based on clay minerals such as comparative study of the properties of
kaolin.4,5 However, using the carbon black rubber compounds, the most suitable DWTS
generates air pollution in the rubber- filled in rubber compounds were
processing process,6 using the silica causes investigated.
waste water, waste residue and other
2. Materials and Methods United Kingdom) and the chemical
Two different types of powder composition by X-ray fluorescence (Bruker
fillers, calcium carbonate (CaCO3, S4 explorer, Bruker corporation, Germany).
purchased from GSP Products Co., Ltd., Physical and mechanical properties of
Thailand, white powder) and drinking water vulcanized rubber compounds were
treatment sludge (DWTS, received from conducted according to standard methods.
Metropolitan Waterworks Authority Mooney viscosity was operated according to
(MWA), Thailand, brown sludge) were used ISO 289-1:2005, tensile strength and
as rubber fillers. To study the effect of filler elongation at break were measured
size, thereby the as received DWTS chunks according to ISO 37:2011 (Type 1) and
was prepared, one of them was ground by shore A hardness was performed according
grinding machine (named as DWTS-1) and to ASTM D2240-05.
the other one was ground and then screened
through sieve no. 140 mesh (named as 3. Results and Discussion
DWTS-2) before mixing into rubber Chemical compositions in Table 2
compound mixtures. show that CaCO3 filler had purity more than
The rubber compound mixtures were 98%, but DWTS filler had SiO2 (60%) as a
composed of STR20, EPDM, zinc oxide major component and Al2O3 (28%) as a
(ZnO), steric acid, paraffin wax, 2- minor component together with some other
mercaptobenzothiazole (MBT), sulphur, metal oxides.
Wingstay L, pigment and filler. The rubber The particle size distributions of
compounds were prepared by two-step CaCO3, DWTS-1 and DWTS-2 are shown in
mixing as following. Firstly, the mixing of Figure 1. The mean particle sizes of these
all compositions, except MBT and sulphur fillers were 11.44, 4.99 and 4.95 m,
were performed in the chamber of a Kneader respectively. CaCO3 showed the narrowest
type mixer at temperature of 70 oC. size distribution in the range of 0.2 - 45 m,
Secondly, the mixture was continuously while DWTS-1 showed the broadest size
homogenized by using a two-roll mill at distribution with bimodal types (0.32-100
temperature of 70 oC. The sequence of and 100-2000 m) due to agglomeration of
mixing in a Kneader type mixer was started very small size of DWTS. However, after
from STR20 for 10 min and then EPDM was screening of DWTS-1, particle size
put into the chamber and masticated for 3 distribution of DWTS-2 showed unimodal
min. Next, ZnO and steric acid were filled type with narrow range of 0.32-160 m.
together and crushed for 2 min. Later, Microstructure of fillers is shown as
paraffin wax, Wingstay L, pigment and filler FE-SEM images in Figure 2, the particle of
were slowly loaded and continuously mixed CaCO3 showed the tiny roundness and
for 4 min. After that, the mixture was moved sharpness grains. DWTS-1 and DWTS-2
to a nip gap of two-roll mill mixer and had platelet structure along with the tiny
repeatedly mixed with adding MBT and roundness grains. The tiny primary particles
sulphur simultaneously for 3 min. The seemed to form agglomerate particles which
composition proportions of rubber were corresponding to the size distribution
compounds are shown in Table 1. as described earlier.
Fillers (CaCO3 and DWTS) were Table 3 shows the properties of
characterized morphology using field rubber compounds prepared by different
emission scanning electron microscope types of rubbers. Mooney viscosity
(JSM-6304F, JEOL, Japan), the particle size gradually increased with increasing the
distributions by lazer particle sizer
(Mastersizer 2000, Malvern instruments,
Table 1. The composition proportions of rubber compounds
Ingredients (phr) RC1 RC2 RC3 RC4 RC5 RC6 RC7 RC8 RC9
STR20 50 50 50 50 50 50 50 50 50
EPDM 50 50 50 50 50 50 50 50 50
ZnO 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
Stearic acid 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Paraffin wax 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 5.0
Wingstay L 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Pigment 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Filler: CaCO3 50 100 150 - - - - - -
Filler: DWTS-1 - - - 50 100 150 - - -
Filler: DWTS-2 - - - - - - 100 125 125
MBT 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Sulphur 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Remark: phr = part per hundred rubber (STR20 + EPDM), RC = rubber compound
Table 2. Chemical compositions of fillers

Filler Metal Oxide (%)
CaCO3 SiO2 Al2O3 Fe2O3 CaO MgO TiO2 K2O SO3 MnO
CaCO3 98.55 0.30 0.20 0.10 - 0.80 - - - 0.05
DWTS - 59.25 27.69 8.00 0.64 0.91 0.92 1.20 0.80 0.59
a) CaCO3
b) DWTS-1 c) DWTS-2
Figure 1. Particle size distribution of a) CaCO3, b) DWTS-1, and c) DWTS-2
a) CaCO3 b) DWTS-1 c) DWTS-2
Figure 2. FE-SEM images of a) CaCO3, b) DWTS-1, and c) DWTS-2
Table 3. Effect of CaCO3 and DWTS on the physical and mechanical properties of various
rubber compounds
Name Mooney viscosity Tensile strength Elongation at break Shore A hardness
code (MPa) (%)
Aging Aging Aging
Before /After Before /After Before /After
RC1 48 3.7/3.8 478/417 48/49
RC2 56 3.1/3.5 449/485 54/53
RC3 66 2.8/2.8 450/318 60/61
RC4 56 1.7/1.6 234/230 44/47
RC5 70 1.5/1.5 211/167 52/57
RC6 83 1.3/1.2 115/75 61/62
RC7 58 3.2/3.0 374/297 56/56
RC8 71 2.9/3.1 311/247 57/61
RC9 20 2.4/2.5 444/437 62/62
Remark: Aging at 70 oC for 72 h
number of all three fillers. At the same as paraffin wax was added in rubber
amount of filler, the rubber compounds with compound. Comparison between RC8 (3 phr
CaCO3 possessed lower viscosity than those of paraffin wax) and RC9 (5 phr of paraffin
of the rubber compounds with DWTS. wax), the results showed that Mooney
Trend of tensile strength and elongation at viscosity value of RC9 was obviously
break gradually decreased with increasing decreased and the tensile strength was
the amount of all three fillers. Adding slightly decreased while the elongation at
DWTS-1 affected in tensile strength and break was increased distinctly. Moreover,
elongation at break and provided lower than hardness of RC9 reached to 62 shore A
those adding CaCO3. From these results, it hardness. Through this work, it was found
implied that the narrower the particle size that using DWTS under proper condition, it
distribution, the better the reinforced effect. can act potentially as alternative filler for
However, shore A hardness of rubber rubber compounds. Common filler such as
compounds gently increased with increasing CaCO3 can be replaced by low cost DWTS
the amount of filler. At 150 phr of CaCO3 for filler reinforcement of rubber
(RC3) and DWTS-1 (RC6), before aging of compounds. Moreover, the use of DWTS
rubbers, shore A hardness was resembled filler in rubber compounds is not only for
and rise up to 60 and 61, respectively. value-added but also for cost reduction in
To confirm the effect of filler size rubber process.
distribution, ground DWTS was sieved to
obtain DWTS-2 with a narrower particle 4. Conclusion
size distribution. Remarkably, at 100 phr of The following conclusions were
DWTS-2 (RC7) compared to DWTS-1 obtained based on the current work. (i)
(RC5), it was found that DWTS-2 showed Mooney viscosity of rubber compounds
decreasing of Mooney viscosity and increased with increasing the amount of
increasing in tensile strength, elongation at filler. (ii) Tensile strength and elongation at
break and shore A hardness. break of rubber compounds gradually
Nevertheless, larger amount of decreased with increasing the amount of
DWTS-2 was added into the mixture of filler. (iii) Rubber compounds prepared from
rubber compound (RC8). The result was not narrower size distribution of fillers occupied
satisfaction due to higher viscosity of rubber tensile strength and elongation at break
caused to tensile strength and elongation higher than those from wider size
tendency decreased. This may be because distribution. (iv) Shore A hardness of rubber
flow ability of mixture ingredients was not com-pounds gradually increased with
proficient. Therefore, more plasticizer such increasing the amount of filler. (v) Under
appropriate preparation condition, drinking 4. Domka, L.; Malicka, A.; Stachowiak, N.
water treatment sludge (DWTS) can be used Acta physica polonica A 2008, 114, 413-
as a good filler reinforcement of rubber 420.
compounds. 5. Dai, J.C.; Haung, J. T. Applied clay,
1999, 15, 51-65.
References 6. 6. Han, J. J.; Zhang, Y. D.; Wu, C. F.;
1. Hyo, S. J.; Seung, H. K.; Hyoung, J. C.; Xie, L. S.; Ma, Y. L. J. Macromol. Sci. B
Jae, H. J.; Young G. K. Compos. Struct. 2015, 54, 401-410.
2016, 136, 106-112. 7. Yu, P.; He, H.; Jia, Y.C.; Tian, S. H.;
2. Lorena, M. A.; Silva, D. A.; Elenilson Chen, J.; Jia, D. M.; Luo Y. F. Polym.
Fabiana, G. A.; Filho, R. M.; Marcos, Test. 2016, 54, 176-185.
R.A.; Tiago Eduardo, V. Fuel 2016, 186, 8. Larpkiattaworn, S.; Khongwong, W.;
50-57. Tong-on, S.; Eamchotchawalit, C.;
3. Pajtášová, M.; Mičicová, Z.; Ondrušová, Vorapeboonpong, C. Key Eng. Mater.
D.; Pecušová, B.; Feriancová, A.; Raník, 2015, 659, 69-73.
L.; Domčeková, S. Procedia
Engineering 2017, 177, 470-475.
Relationship between crosslink structures and tensile properties of
sulphur-vulcanized natural rubbers
Watcharin Sainumsai
Program in Rubber and Polymer Technology, Faculty of Science and Technology,
Songkhla Rajabhat University, Songkhla 90000, Thailand
E-mail: watcharin.sa@skru.ac.th
Abstract:
Types of sulphur crosslink were determined by using the chemical probes in
conjunction with the determination of the total crosslink density based upon the chemical
reactivity of the particular chemical probe. The natural rubber (NR) vulcanizate, cured with
the conventional vulcanizing system (CV), only an average of 9.68% crosslinks were
disulphides (-C-S-S-C-), while most of the crosslinks were polysulphides (-C-S-Sx-S-C-,
x=1-6). On the other hand, for the NR vulcanizate cured with the efficient vulcanizing system
(EV) contains mostly monosulphidic (-C-S-C-) crosslinks (an average of 52.17%), while only
an average of 21.02% crosslinks were polysulphides, and 26.81% crosslinks were
disulphides. NR vulcanizate cured with the semi-efficient vulcanizing system (SEV) contains
a more balanced structure with 0.98% of monosulphidic crosslinks, 44.77% disulphidic
crosslinks and 54.25% polysulphidic crosslinks. Polysulphides rich vulcanized natural rubber
shows higher tensile strength and larger elongation at break than monosulphide rich and
disulphides rich compounds. The stresses required to deform NR vulcanizates were not
different for the three samples studied at the % elongation lower than 450.
1. Introduction determining the type of crosslink formed.

The vulcanization of natural rubber High sulphur levels, e.g. 2 to 3.5 phr (parts
(NR) by sulphur in presence of organic per hundred parts of rubber by weight), and
accelerator is a complicated process.1 The low levels of accelerator, 0.5 to 1 phr, are
mechanism of vulcanization and its called conventional systems, and at optimum
acceleration depends on the structure of the cure the vulcanizate contains mostly
rubber, type and concentration of polysulphidic crosslinks with relatively high
accelerators and activators (zinc oxide and level of chain modification. Low sulphur
fatty acid) and on the thermodynamics of levels, 0.25 to 0.7 phr, with high accelerator
each particular reaction. The chemistry of levels, 2.5 to 5 phr, will give mainly
vulcanization is complex and the resulting monosulphidic crosslinks and much less
crosslinks may be mono-, di-, or higher chain modification. They are known as
poly-sulphides,2 with a proportion which is efficient systems.4
among others largely determined by the There is already strong evidence that
vulcanization system, the cure time and the the vulcanization of diene rubbers proceeds
temperature. As it was shown,3 the by the sequence of many reactions. The
vulcanization rate and crosslink active sulphurating agent is produced by a
concentration increase if the amount of multistep reaction. The first step involves
sulphenamide accelerator is increased while the reaction of zinc oxide, fatty acid and
the sulphur is kept constant. The accelerator accelerator to produce the polysulphidic
increases the rate of cure and the efficiency species. Campbell and Wise5 have found
with which sulphur is used in crosslinking that the vulcanization of diene rubbers with
compared to side reactions. The Sulphur sulphenamide accelerated sulphur system,
/accelerator ratio is particularly important in the sulphenamide concentration decreased
rapidly in the induction period and 2. Materials and Methods
crosslinking did not occur until the 2.1 Materials
sulphenamide was consumed. They Commercial grade of Standard Thai
concluded that the sulphenamide was not the Natural Rubber Light (STR-5L) was used.
effective accelerator, but the products The compositions and cure time of
derived from it were the active sulphurating compounds are shown in Table 1. Rubber
agents, such as the 2-bis-benzothiazole-2,2- compounds were prepared by using a
polysulphide (Bt-Sx-NR2, where Bt = laboratory-sized two-roll mill (model
benzothiazole group). Also, Coran6 LRM150, Lab Tech Engineering) at the set
proposed the kinetic study of NR with temperature of 40 C. Rubber was
MBTS or sulphenamide accelerators, that masticated for 2 minutes, stearic acid, zinc
the initial reaction takes place between the oxide, an accelerator (CBS) were mixed for
accelerator and sulphur to produce a 2 minutes. Then, sulphur was added and
polysulphidic species, that could then react mixed for 2 minutes. Finally, passing the
with the rubber chain to form pendent rolled batch endwise through the mill six
groups. Once the sulphenamide accelerator times and pass the batch four times through
is consumed, the crosslinking process is the mill, folding it back on itself each time.
rapid due to the high concentrations of The total mixing time was 8 minutes. The
pendent groups. rubber compounds were left at room
It is clearly demonstrated that many temperature for 24 hours before testing.
factors determine the tensile strength in an Vulcanization was carried out in an
unfilled compound. An important factor is electrically heated hydraulic press at 150 C
the ability of the vulcanizate to crystallise using the optimum cure time (Tc90)
(e.g. the natural rubber).7-14 Other important previously determined with a moving die
factors are the network chain density and the rheometer (TechPro MD+) at 150 C.
chemical nature of the crosslink. Greensmith
et al.15 and Sainumsai et al.16,17 related the Table 1. Composition and preparation
tensile strength with the crosslink density, conditions of the samples
and observed that the tensile strength passes Quantity (phra)
Ingredients
through a maximum value with increasing CV SEV EV
crosslink density. Greensmith et al. also STR 5Lb 100 100 100
reported that the tensile strength of a NR Zinc Oxide 5 5 5
Stearic acid 1 1 1
gum decreased according to the mechanism
CBSc 0.75 1.82 4.60
of vulcanization in the sequence accelerated Sulphur 2.00 0.91 0.46
sulphur > sulphurless > peroxide > high Sulphur/CBS ratio 2.67 0.50 0.10
energy radiation. Cure time (min)d 13 19 28
a
The purpose of this paper is to study Parts (by weight) per hundred parts of rubber
b
the effect of sulphur crosslink type on the Natural rubber
c
N-cyclohexyl-2-benzothiazole sulphenamide
tensile properties of accelerated sulphur- d
Curing temperature 150 C
vulcanized natural rubbers. The sulphur
crosslink type (mono-, di- and poly-
2.2 Determination of crosslink density
sulphide) was controlled by changing the
Equilibrium swelling in toluene was
sulphur to accelerator ratio (2.67, 0.50 and
used to determine the crosslink densities of
0.10) using CBS as accelerator at the similar
the rubber vulcanizates. The crosslink
crosslink density. The compounds are called
density was determined using the method
as CV, SEV and EV, respectively.
described by Cunneen and Russell.18 The
molecular weight of the network chain
between chemical crosslinks for a phantom
network, Mc; is expressed by the Flory–
Rehner relationship:19,20
 2  rVs (Vr1/ 3  Vr / 2) properties of the rubber vulcanizates.24-27
Mc = Depending on the ratio, the sulphur
[ln(1  Vr )  Vr  Vr2
vulcanization can be classified into three
where Vr is the volume fraction of rubber in types: conventional (CV), semi-efficient
the swollen sample, Vs is the molar volume (SEV) and efficient (EV) vulcanizing
of the swelling solvent, ρr is the density of systems.28,29 Each system can result in a
the rubber sample, and  is the rubber- network structure with different
solvent interaction parameter. The values of combinations of polysulphidic, disulphidic
the constant used in the above calculation and monosulphidic crosslink. The network
were Vs = 107 cm3/mole and  = 0.393.4 characteristics of the NR vulcanizates used
The crosslink density () is given by:21 for the present study, including the crosslink
1 density and the type of sulphidic crosslinks,
=
2M c are summarized in Table 2. It can be seen
2.3 Determination of sulphur crosslink that the total crosslink densities of the 3
structures types of NR vulcanizates are comparable
The contents of mono-, di-, and poly- and are in the range of 7.66±0.12 x 10-5
sulphidic crosslink structures were mole/cm3.
determined using the method described by
(a) CV system 9.7% Disulphide
Cunneen and Russell.18,22,23 The
concentration of the polysulphidic crosslink
was evaluated from the equilibrium swelling
in piperidine and propane-2-thiol, the
Polysulphide
concentration of the monosulphidic
90.3%
crosslink was evaluated from the
equilibrium swelling in piperidine and n-
hexanethiol. Finally, the concentration of
disulphidic crosslink was calculated from
(b) SEV system Monosulphide
the total crosslink density, polysulphidic and 1.0%
monosulphidic crosslink densities
Disulphide
previously determined. Polysulphide 44.8%
2.4 Measurements of stress-strain curves 54.2%
and tensile properties

Tensile properties of NR
vulcanizates were measured according to
ASTM D-412 using a universal testing
machine (Instron 5569 series, Norwood,
(c) EV system
USA) at 25 °C. The rate of deformation was Polysulphide
500 mm/min. 21.0%
Monosulphide
Samples were submitted to 52.2%
accelerated ageing in an air-circulating oven Disulphide
at 100 C for 24 hours. After ageing, tensile 26.8%
properties were determined.

3.1 Network characteristics of NR Figure 1. The percentage of monosulphidic,
vulcanizates studied disulphidic and polysulphidic crosslink of
It is well known that the ratio of NR vulcanizates with different vulcaniz

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Proceedings

The Pure and Applied Chemistry International

“Chemistry toward a Sustainable Future”

Editor-in-Chief Vatcharin Rukachaisirikul

ISBN (E-book) 978-616-271-465-8

Prepared by Department of Chemistry, Faculty of Science

1st Edition May 2018 (available in PDF file only)

Editor-in-Chief Vatcharin Rukachaisirikul

AN Analytical Chemistry EE Environmental

MN Material Chemistry and

OR Organic and Medicinal Chemistry PH Physical and Theoretical Chemistry

FA: Food and Agricultural Chemistry

IN: Inorganic Chemistry

NP: Natural Products, Biological Chemistry and Chemical Biology

OR: Organic and Medicinal Chemistry

PO: Polymer Chemistry

Author Index Index 1-10

1. Introduction conductivity, high surface area, and catalytic

Figure 1. Cyclic voltammograms of the 3.0

4.6 condition using amperometry. The obtained

no interfered at ≤ 0.1 mM while the others 0 2000 4000 6000

permselectivity of PoPD membrane on the Figure 4. The linear calibration plot of

1. Introduction the observation in seaweed salad processing,

Extraction efficiency (%)

7.5.11 Mixture solution was purged with high 90

Figure 3. Effect of pH on the extraction

3.4 Effect of contact time 80.0n

The treatment time to remove

performed at 15 to 440 minutes.

60.00 R.; Netten, J.P.V. Sci. Total. Environ.

1. Introduction methods used to detect PAHs e.g., gas

3. Results and Discussion

Figure 2. SERS spectra of thiol-modified

To find a suitable substrate for

Chrysene can be detected by 3 types stretching; 1381 cm-1 peak corresponding to

1. Introduction suffered from poor sensitivity and high

Figure 3. Cyclic voltammograms of

Figure 2. Cyclic voltammograms of (a)

The electrodes show the irreversible

1. Introduction Therefore, the structural information

(d) (e) (f)

1. Introduction obstacles, several absorbent materials have

Figure 1. Schematic representation (a) Scanning electron micrographs of PS-DVB

3. Results and Discussion increasing the stirring speed significantly

Table 1. Analytical performance for

1. Introduction vapor atomic absorption spectrometry (CV-

2.3 Operation of 3D-µPAD for Hg(II) 2.4 Sample preparation

Figure 4. Calibration curve of 3D-µPAD for References

1. Introduction protective effects against cholesterol and

Figure 5. (a) The relationship between pH

3.2.3 Calibration curve and detection

Facile immunoassay for colorimetric detection of parathyroid hormone

1. Introduction determination. There have been both

Schematic 1. Schematic illustration for the

Results in Figure 3 demonstrate that

1. Introduction Nowadays, due to the unique optical

Figure 1. A: Schematic illustration of the

Figure 4. Effect of AuNPs concentration on Table 1. Analytical performances of the

Table 2. Concentrations of creatinine in

1. Introduction 2. Materials and Methods

A circular area of 2000 counts was

The image of detecting paper was

Figure 2. Digitized image of detecting paper