International Journal of Biological Macromolecules 75 (2015) 5866

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Structural characterization of lignin: A potential source of

antioxidants guaiacol and 4-vinylguaiacol
Mohammadali Azadfar , Allan Haiming Gao, Mahesh V. Bule, Shulin Chen
Department of Biological Systems Engineering, Washington State University, Pullman, WA 99164-6120, USA

a r t i c l e i n f o a b s t r a c t

Article history: The structure of lignin obtained from the ozone and soaking aqueous ammonia pretreatment of wheat
Received 10 September 2014 straw has been characterized utilizing chemical analytical methods in order to reveal its antioxidant
Received in revised form 8 November 2014 characteristics, including attenuated total reectance-Fourier transform infrared spectroscopy (ATR-
Accepted 19 December 2014
FTIR), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), pyrolysis/tetramethylammonium
Available online 17 January 2015
hydroxide-gas chromatography/mass spectrometry (Py/TMAH-GC/MS), gel permeation chromatography
(GPC), ultra violetvisible spectroscopy (UVvis), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) antioxidant
Keywords:
evaluation assay. The results demonstrated that the isolated lignin is a -hydroxyphenyl- guaiacyl-
Wheat straw lignin
Hydroxycinnamic acids
syringyl (H-G-S) lignin, with S/G ratio of 0.35 and signicant amounts of phenol 2-methoxy (guaiacol) and
Antioxidant activity phenol 2-methoxy-4-vinyl (4-vinylguaiacol). The Py-GC/MS and Py/TMAH-GC/MS pyrograms indicated
Characterization that the major units in this lignin are derived from hydroxycinnamic acids. The GPC results revealed
the molecular weight of the lignin was considerably low and also the FTIR analysis showed that the
lignin possessed hydroxyl and methoxy functional groups; the factors led to the extracted lignin having a
comparable antioxidant activity to that of currently used commercial antioxidants. The UVvis and DPPH
antioxidant assay results suggested a percentage of inhibition of the DPPH radicals in the following order:
guaiacol (103.6 1.36) > butylated hydroxytoluene (103.3 1) > ferulic acid (102.6 0.79) > pretreated
lignin (86.9 0.34).
2015 Elsevier B.V. All rights reserved.

1. Introduction hydroxyl and methoxy functional groups and a propanoid chain.

Developing valuable products from lignin can improve the eco-
nomics of a biorenery using lignocellulosic biomass. Lignin is
present in signicant amounts in plants, accounting for 1525%
(w/w) of herbaceous biomass. To date however, lignin has little
high-value practical use [1]. As a heteropolymeric aromatic com-
pound, the lignin structure is composed of an aromatic ring with
Abbreviations: ATR-FTIR, attenuated total reectance-Fourier transform
infrared spectroscopy; Py-GC/MS, pyrolysis-gas chromatography/mass spectrome-
try; Py/TMAH-GC-MS, pyrolysis/tetramethylammonium hydroxide-gas chromatog-
raphy/mass spectrometry; GPC, gel permeation chromatography; UVvis, ultra
violetvisible spectroscopy; DPPH, 1,1-diphenyl-2-picrylhydrazyl.
Corresponding author at: LJ Smith Hall, Room 213, Department of Biological
Systems Engineering, Washington State University, Pullman, WA 99164-6120, USA.
Tel.: +1 408 805 2057; fax: +1 509 335 2722.
Corresponding author at: LJ Smith Hall, Room 213, Department of Biological
Systems Engineering, Washington State University, Pullman, WA 99164-6120, USA.
Tel.: +1 509 335 3743; fax: +1 509 335 2722.
E-mail addresses: mohammadali.azadfar@wsu.edu (M. Azadfar),
chens@wsu.edu (S. Chen).
This conguration has many properties that could give rise to
the possibility of using lignin for producing value-added products.
However, the heterogeneity of the lignin structure makes it dif-
cult to break down and isolate into a targeted product. Wheat
straw possesses three mono-lignols (-coumaryl, coniferyl and
sinapyl alcohols) in signicant amounts [1,2]. It has been found
that wheat straw lignin polymer contains, esteried, etheried
and carboncarbon bonds which link guaiacyl (G), syringyl (S), -
hydroxyphenyl (H) units [2]. Due to the complexity of the lignin
structure and the effect of pretreatment processes on it, iden-
tifying and extracting chemicals from lignin requires extensive
characterization to understand the lignins polymeric properties,
linkages, and properties of the functional groups connected to the
aromatic ring. Nonetheless, the features [3] existing in the lignin
macromolecule and its characterization suggests the predominant
presence of antioxidant monomers in the lignin.
Antioxidants are chemicals that inhibit the oxidation process.
The oxidation reaction typically follows three main steps includ-
ing initiation where free radicals are being produced, propagation
where free radicals are created through a chain reaction involv-
ing a series of molecules, and nally termination, during which

http://dx.doi.org/10.1016/j.ijbiomac.2014.12.049
0141-8130/ 2015 Elsevier B.V. All rights reserved.
 M. Azadfar et al. / International Journal of Biological Macromolecules 75 (2015) 5866
two free radicals interact with each other to end the reaction.
When acting as an antioxidant, lignin-derived units have the abil-
ity to break the oxidation propagation reaction through hydrogen
donation which occurs primarily due to the presence of phenolic
hydroxyl groups [4]. Lignin compounds that contain more phe-
nolic hydroxyl groups, fewer aliphatic hydroxyl groups, have a
low molecular weight, and narrow polydispersity are reported
to have higher antioxidant activity [3]. The antioxidant proper-
ties of phenolic compounds from different origins and processes
were reported previously; however, the efcacy of the emersion
of monomers derived from wheat straw by the ozone and soaking
aqueous ammonia (OSAA) pretreatment on the antioxidant activity
of the isolated lignin macromolecule has not yet been studied.
In the present work, we studied the structure of the lignin
isolated by an acidic precipitation method from a basic efu-
ent produced by the OSAA pretreatment of wheat straw in order
to characterize the lignin macromolecule antioxidant character-
istics. In this regard, a series of chemical analytical techniques
were employed, including attenuated total reectance-Fourier
transform infrared spectroscopy (ATR-FTIR), pyrolysis-gas chro-
matography/mass spectrometry (Py-GC/MS) with and without
tetramethylammonium hydroxide (TMAH), ultra violetvisible
spectrophotometer (UVvis), gel permeation chromatography
(GPC), and the radical scavenging activity was measured using 1,1-
diphenyl-2-picrylhydrazyl (DPPH) assay.
The ozone and soaking aqueous ammonia (OSAA) pretreatment
process is a chemical pretreatment that is designed to effectively
remove the lignin of herbaceous biomass [5,6]. Its process requires
only a low temperature and ambient pressure. The process takes
advantage of the synergy inherent in the sequential treatment of
biomass by ozone and ammonia. The ozone treatment increases
the hydrophilicity of the biomass and breaks the physical bar-
rier that prevents diffusion of the ammonia into the biomass. The
ammonia is then able to enter the biomass and cleavage the lignin
linkages and subsequently dissolve the lignin into the efuent.
The ATR-FTIR was employed to study the functionality of lignin
structure isolated from the pretreatment efuent. The Py-GC/MS
and Py/TMAH-GC/MS were used to analyze the structure of lignin-
derived compounds. Pyrolysis combined with gas chromatography
(Py-GC) is a useful tool for synthetic and natural polymer anal-
ysis. The lignin weight-average (Mw ) and number-average (Mn )
Fig. 1. Process owchart.
59
molecular weights was determined using GPC [7]. Eventually, the
UVvis and DPPH assay provided data pertinent to antiradical activ-
ity of lignin in comparison with other commercial antioxidants (i.e.,
guaiacol, butylated hydroxytoluene (BHT), and ferulic acid). The
knowledge obtained on the structural features of the lignin will
aid to establish processes to convert the lignin into value-added
chemicals and provide economics benet for biorenery.
2. Materials and methods
2.1. Materials
The lignin sample was isolated from the ammonia efuent of the
sequential ozone and soaking aqueous ammonia pretreatment of
wheat straw by acetic acid precipitation method. The chemicals
2-methoxy phenol (guaiacol), trans-ferulic acid, 1,1-diphenyl-2-
picrylhydrazyl (DPPH), tetramethylammonium hydroxide (TMAH),
acetic acid, and 2,6-di-tert-butyl-4-methoxyl phenol (BHT) were
purchased from Sigma-Aldrich Inc (Milwaukee, US).
2.2. Ozone and soaking aqueous ammonia (OSAA) pretreatment
The wheat straw was rst soaked in water to bring the moisture
content of the mass up to 45% (w/w) water, and then subjected to
ozone pretreatment. The ozonation reaction was performed with
5.3% ozone concentration (5.3%, w/w) at oxygen ow rate of 2 L/min
for 10 min [5]. Following the ozone pretreatment, the wheat straw
was ushed to soaking aqueous ammonia pretreatment, where 20%
ammonia solution was used to treat the biomass for 4 h at a 1:4
biomass to aqueous ammonia loading, including heating time [6].
The ammonia solution was recovered through the use of a two
step washing process with E-pure water, and the obtained efuent
was gathered for lignin precipitation step. The process owchart is
shown in Fig. 1.
2.3. Precipitation of lignin
The presence of non-lignin components could inuence anti-
radical activity of lignin. Hydrogen bonding between carbohydrate
admixture polar groups and lignin phenolic groups could be formed
and results in a reduction of antioxidant activity [811].
60 M. Azadfar et al. / International Journal of Biological Macromolecules 75 (2015) 5866
Fig. 2. Solid lignin obtained from pretreated wheat straw.
An acetic acid precipitation method which does not contain sul-
fur and uses an environmentally-friendly reagent for precipitating
lignin in efuent was employed [12,13]. Before precipitation, the
ammonium in efuent needs to be removed as much as possible. In
order to evaporate the ammonium hydroxide, the measured efu-
ent was put in a vented oven for 1 h at 75 C. The precipitation of
lignin performed by adding 20 ml acetic acid:water (9:1, v/v) into
efuent (50 ml). Next, the blend was placed in a warm water bath
for 10 min at 80 C, and then the suspension was centrifuged for
5 min at 3500 rpm (MSE, Centaur 2 Sanyo). The precipitate was
water washed in 400 ml E-pure water. This suspension was cen-
trifuged for 5 min with speed of 3500 rpm and the liquid-solid
phases was separated several times. The nal separation tube was
put in a warm water bath for 30 min at 75 C. Then, a centrifuge
step for 10 min was performed also at 3500 rpm. The liquid phase
was separated and the residual lignin was put in a drying oven
at 50 C overnight. This resulted in pure solid lignin for further
characterization (Fig. 2).
2.4. Characterization of lignin structure
The physicochemical structural of the lignin by utilizing the out-
come of the analytical instruments was assessed.
2.4.1. Attenuated total reectance- Fourier transform infrared
spectroscopy (ATR-FTIR)
The FTIR analysis was performed for analysis of functional
groups in the lignin macromolecules. The FTIR spectra were
obtained on an ATR-FTIR spectrophotometer (Shimadzu, Japan)
with 64 scans. The ATR supplement works by measuring the
changes that occur in a totally internally reected infrared beam
when the beam contacts with a sample. Finely powdered of the
lignin were pressed on the crystal surface of the ATR probe. Sam-
ples were analyzed from 4000 to 800 cm1 at a resolution of 4 cm1
[14,15].
2.4.2. Pyrolysis-gas chromatography/mass spectroscopy
(Py-GC/MS)
Pyrolysis of lignin samples was performed with using a CDS
Pyroprobe 5000 connected to an Agilent GC/MS system (7890A
GC, 5975 C mass selective detector) [16,17]. The lignin samples
were loaded into a quartz tube and gently packed with quartz
wool prior to pyrolysis. The pyrolysis was performed at 610 C
(1 min). The inlet temperature was maintained at 250 C, and
the resulting pyrolysis vapors were separated by a (5%-phenyl)-
methyl polysiloxane hydrophobic column with a 30 m 25 mm
inner diameter. Helium was the carrier gas (1 ml min1 ). The gas
ow rate was 1 ml min1 , and the oven was retained at 280 C
for 10 min to prevent any residuals from remaining in the cham-
ber. This gas was then analyzed with a mass spectrometer (Inert
XL MSD, Agilent Technologies), and CO2 was used as the inter-
nal standard. The compounds were identied by comparing their
mass spectra with those of the Wiley and the National Institute of
Standards and Technology (NIST) electronic libraries.
2.4.3. Gel permeation chromatography (GPC)
The molecular weight of extracted lignin was determined by
utilizing gel permeation chromatography (GPC) [18,19]. The molec-
ular weight of the lignin was taken from our previous study [7]:
Isolated lignin was acetylated in pyridine/acetic anhydride (1:1,
v/v) mixture prior to GPC analysis to improve their solubility in THF
solvent. Each 10 mg lignin was suspended in 2 ml of pyridine/acetic
anhydride mixture for 72 h in dark. The supernatant was added
into 48 ml of cold water and stirred at room temperature for 1 h.
The precipitate was water washed, ether washed and freeze dried.
After that, 2 mg of sample was dissolved in 1 ml THF and store at
4 C before analysis. Gel permeation chromatography of the iso-
lated lignin was performed on 1260 innity HPLC system (Agilent
technology) with refractive index detector (RI-G1362A). The col-
umn was PL gel mixed-C (300 7.5 mm, 5 m) and tetrahydrofuran
(THF) was used as eluent. The molecular weight of isolated lignin
was recorded at 25 C.
2.4.4. Lignin antioxidant activity
The antioxidant activity of the lignin in terms of the percentage
of inhibition of 1,1-diphenyl-2-picrylhyrazyl (DPPH) radicals was
determined. In this study, the commercial antioxidants guaiacol,
ferulic acid, and butylated hydroxytoluene (BHT) were utilized as
controls for comparison. An antioxidant activity assay described in
previous studies [9,20] was employed with some modications as
needed for application to our lignin sample. A sample of each test
material (0.1 mg) was mixed with a 4 ml methanolic solution of
DPPH (2.5 mg/100 ml methanol) and after 30 min. The absorbance
at 515 nm of the mixture was measured using a UVvis spec-
trophotometer. The antiradical activity of the test materials was
determined as the decrease in absorbance of the DPPH expressed
as a percentage of the absorbance of a control DPPH solution
without test compounds using a UVvis spectrophotometer (Shi-
madzu, Japan) [21,22]. During the reaction between antioxidants
and the DPPH, reduction of the DPPH radicals is occurred followed
by decreasing in its absorbance at a characteristic wavelength
(515 nm). The absorption disappears by reduction effect of antiox-
idant or a radical species [23]. The percentage of inhibition of the
DPPH radicals was calculated using Eq. (1) [24].
AControl Asample
inhibition % = 100 (1)
Acontrol
where Acontrol is the absorbance in blank probe (antioxidant was
omitted) and Asample is the absorbance in the sample. The results
were presented as mean standard error (SE). All were assessed by
two-factor analysis of variance. All experiments were run triplicate.
3. Results and discussion
It must be stated that all analyses performed in this study rep-
resent the structural features of wheat straw lignin which partly
pretreated by ozone and soaking aqueous ammonia and precipi-
tated from the pretreatment efuent.
3.1. ATR-FTIR analysis
The ATR-FTIR spectroscopy of the pretreated wheat straw lignin
sample is shown in Fig. 3, and the corresponding assignments
of bands are listed in Table 1. Fig. 3 presents the view of the
wavelength region from 600 to 3900 cm1 with bands of interest
identied by their wave number (cm1 ). In Fig. 3 a strong hydrogen
 M. Azadfar et al. / International Journal of Biological Macromolecules 75 (2015) 5866 61

Fig. 3. ATR-FTIR spectra of pretreated wheat straw lignin sample.

band (OH) stretching at 3200 cm1 and CH stretching at 2851,
2921 cm1 in the lignin sample is observed. A strong wide band
between 3500 and 3100 cm1 which is assigned to OH stretch-
ing vibration is an observable characteristic for lignin IR spectra
[25]. The antioxidant activity of phenolic compounds was found
to depend on the number of the hydroxyl groups linked to the
aromatic ring [21,26]. Balasundram et al. [26] stated the antioxi-
dant activity of phenolic compounds increases with an increasing
degree of hydroxylation. It has been reported that the appearance of
another hydroxyl group in the ortho or para position on the phenolic
ring is able to enhance the antiradical activity [27]. The assigned
bands around 2850 and 2925 cm1 are related to the CH (CH3
or CH2 ) stretching vibrations of the methyl and methoxy groups
[28]. The presence of alcoholic and phenolic hydroxyl groups can
also be seen from the strong band in this region. The position
of the methoxy group on the phenolic ring could has positive
effect on radical scavenging activity of lignin. It has been observed
that methoxy substitution in ortho position increased the radical
scavenging efciency of guaiacol as compared to phenol [20,29].
Cuvelier et al. [27] reported the ortho substitution with the electron

Fig. 4. Potential dissociation reaction of ester linkage during the pretreatment of wheat straw lignin. (a) Esteried-linked lignin to hemicellulose; (b) hydroxycinnamic acids
derivative (i.e., ferulic acid); (c) released arabinose-hemicellulose.
62 M. Azadfar et al. / International Journal of Biological Macromolecules 75 (2015) 5866

Table 1 Table 2
Assignment of ATR-FTIR absorption bands of pretreated wheat straw lignin. Identities and relative molar abundances of the phenolic compounds released from
pretreated wheat straw lignin after Py-GC/MS.
Assignment lignin
Label Compound MW Origina
 (cm1 ) T (%)
1 Phenol 94 H
OH stretching vibration of polymer 3200 98.95 2 Phenol, 2-methyl- 108 H
CH stretching vibration of polymer (CH3 and 2921, 2851 98.38, 98.82 3 Phenol, 2-methoxy-(guaiacol) 124 G
CH2 ) 4 Phenol, 2-methoxy-4-[1-propenyl] 138 G
C O stretching vibration- substituted aryl 1653 96.55 5 Phenol, 4-ethyl 2-methoxy 152 G
ketone 6 Phenol, 2-methoxy-4-vinyl 150 G
(16601675 guaiacyl) 7 Phenol, 2,6-dimethoxy- (syringol) 154 S
Aromatic skeletal vibration 1555 91.16 8 Phenol, 2-methoxy-4-[1-propenyl] 164 G
Aromatic skeletal vibration 1411 90.31 (trans-isoeugenol)
G ring breathing with CO stretching 1269 94.94 9 Phenol, 2-methoxy-4-[1-propenyl] 164 G
Aromatic CH in plane deformation (S) 1127 95.73 10 5-tert-Butylpyrogallol 182 S/H
Aromatic CH in plane deformation (G > S) 1044 94.41 11 2-Propanon, 1-[4-hydroxy-3-methoxy phenyl] 180 G
Aromatic CH out-of plane deformation (S + H) 887 97.72 12 2H-1-benzopyran-3,4-diol 316 H
13 Phenol, 2,6-dimethoxy-4-(2-propenyl)- 194 S
14 Phenol, 2,6-dimethoxy-4-(2-propenyl)- 194 S
donor methoxy group is also a factor increasing the stability of the 15 Phenol, 2,6-dimethoxy-4-(2-propenyl)- 194 S
16 Ethanone, 1-(4-hydroxy-3,5-dimethoxyphenyl)- 196 S
phenoxy radical and therefore increases its antioxidative efciency.
17 Estra-1,3,5(10)-trien-17-ol 256 H
The effect of the presence of another methoxy group on the ring as
a
di-methoxy compounds on their antioxidant activity has also been H: H lignin units; G: G lignin units; S: S lignin units.

studied. The studies reported that the 2,6-dimethoxy phenol has

higher antiradical activity than 2, methoxy phenol [8,30]. It has
been reported that there are no absorption bands that are lignin
derived between 2800 and 1800 cm1 wave number range [31].
Some previous work stated that a band around 1720 cm1 is the
characteristic band for ester linkage absorption [6,28,32,33]. How-
ever, it was observed that the ester linkage absorption in the lignin
samples is disappeared. The loss of the 1720 cm1 band absorbance
and reduction in the 1653 cm1 band absorbance in the lignin
indicates that signicant amounts of ester-linked subunits were
released during the pretreatment (Figs. 3 and 4). It has been found
that an alkaline pretreatment selectively cleaves the ester linkages
in lignin structure [34]. A shoulder was observed at 1664 cm1 ,
which is the characteristic band for the carbonyl stretching group in
guaiacyl units. This is related to the ester linkages in lignin structure
and originates from the conjugated carbonyl stretches, possibly
indicating the occurrence of hydroxycinnamic acid derivatives in
the lignin structure [35]. It has been reported that the hydrox-
ycinnamic acids have higher antioxidant activity in compared with
hydroxybenzoic acids [36]. Other work has shown that the vibra-
tion of aromatic ring is assigned to bands around 16001400 cm1
[28,35]. The absorption bands at 1555, 1411 cm1 (T%, 91.16/90.31)
lignin indicate the aromatic skeletal vibration of the lignin, respec-
tively. Monteil-Rivera et al. [25] found that band at 1265 cm1
indicates characteristic of guaiacyl units in wheat straw lignin. The
band at 1269 cm1 indicates the G-ring breathing in lignin samples.
A week band at 1127 cm1 is characteristic band for syringyl units
[31]. Fig. 3 shows low intensity band for syringyl units in lignin
sample. The FTIR spectrum indicates that the content of the gua-
iacyl units are higher than syringyl units in the lignin. The band at
1044 cm1 corresponds to aromatic CH plane stretching of G units.
3.2. Py-GC/MS analysis
The pyrolysis of lignin, released phenolic compounds, which
were observed to be derived from G, S, and H lignin units.
The approximate abundance of phenolic-derived compounds was
65.09%, 23.36%, 11.5% for G, S, and H lignin units, respectively. The
pyrogram revealed that the sample is a -hydroxyphenyl- guaiacyl-
syringyl (H-G-S) lignin. The pyrogram is shown in Fig. 5, and the
identities and relative molar mass of the released phenolic com-
pounds are listed in Table 2. The predominant lignin derivatives
(Fig. 6) included phenol 2-methoxy (3), phenol 2-methoxy-4-vinyl
(6), and 2, 6-dimethoxy phenol (7). The identied phenolic com-
pounds showed that the abundance of G-units was higher than
S-lignin units, with a S/G ratio of 0.35. Moreover, high levels of phe-
nol 2-methoxy-4-vinyl (25.83% of total area of phenolic-derived

Fig. 5. Pyrogram of Py-GC/MS of pretreated wheat straw lignin.

 M. Azadfar et al. / International Journal of Biological Macromolecules 75 (2015) 5866 63

OH Table 3
H3CO H3CO Identities and relative molar abundances of the phenolic compounds released from
OCH3
pretreated wheat straw lignin after Py/TMAH-GC/MS.
OCH3
HO Label Compound MW Origina
OH
(a) (b) (c) 1 Phenol 94 H
2 Phenol, 2,5-dimethyl- 122 H
3 Phenol, 3-methyl- 108 H
Fig. 6. Chemical structures of predominant identied derivatives of pretreated 4 Phenol, 2-methoxy (guaiacol) 124 G
wheat straw lignin by Py-GC/MS: phenol 2-methoxy (a); phenol 2-methoxy- 4-vinyl 5 Phenol, 2-methoxy-4-methyl- 138 G
(b); 2, 6-dimethoxy phenol (c). 6 3,4-Dimethoxytoluene 152 G/H
7 phenol, 4-ethyl-2-methoxy 152 G
8 2-Methoxy-4-vinylphenol 150 G
compounds) was released from the lignin. This evidence might 9 Phenol, 2,6-dimethoxy (syringol) 154 S
demonstrates the presence of hydroxycinnamic acids derivatives in 10 Phenol, 2-methoxy-4-(1-propenyl)-, (E) (isoeugenol) 164 G
solid lignin. It is in agreement with the loss of the 1720 cm1 band 11 2-Propanone, 1-(4-hydroxy-3-methoxyphenyl) 180 G
absorbance and reduction in the 1653 cm1 band absorbance in 12 3-tert-Butyl-4-hydroxyanisole 180 G/H
13 Ethanone, 1-(3,4,5-trimethoxyphenyl) 210 S
the ATR-FTIR spectra; indicates signicant amounts of ester-linked
a
lignin units were cleaved. It has been reported that herbaceous H: H lignin units; G: G lignin units; S: S lignin units.

crops possess hydroxycinnamic acids, ferulic and -coumaric acids,

which make linkages between lignin and hemicellulose [37,38]. Del
Rio and Gutierrez [39] stated that the presence of the 4-vinylphenol
and 4-vinylguaiacol released from Abaca bers after Py-GC/MC was
due to the presence of hydroxycinnamic acids, -coumaric and fer-
ulic acids, respectively. During pyrolysis, decarboxylation of the
hydroxycinnamic acids results in those compounds [39]. In order
to assess, the predominant presence of phenol 2-methoxyl, phenol
2-methoxy-4-vinyl, and 2, 6-dimethoxy phenol, tetramethylam-
monium hydroxide (TMAH) was used as a methylating agent. It
has been reported that pyrolysis in presence of the TMAH helps
to prevent decarboxylation and causes methylation of the pheno-
lic and carboxyl groups [40,41]. The pyrogram is shown in Fig. 7,
and the identities and relative molar mass of the released phe-
nolic compounds are listed in Table 3. Py/TMAH-GC/MS of lignin
demonstrated considerable levels of phenol 2-methoxy-4-vinyl
and phenol 2-methoxy conrming the predominant appearance of
G-lignin units in solid lignin (Fig. 7).
3.3. Lignin molecular weight analysis
The molecular weight of the lignin were 772 (g/mol) and 3735
(g/mol) and the molecular weight of milled wheat straw lignin were
2026 (g/mol) and 3857 (g/mol) for the number-average (Mn ) and
weight-average (Mw ) molecular weight, respectively [7,42]. Lignin
with low molecular weight is more reactive than those with high
molecular weight molecules [43]. Rice-Evans et al. [44] reported
that molecular weight data does provide some useful guide. Their
research showed that small molecular weights are more desirable
for further chemical or catalytic conversion. High molecular weight
increases heterogeneity and polydispersity which are factors that
decrease radical scavenging activity [4]. It has been reported that
Kraft lignin with a higher molecular weight has a lower antirad-
ical activity when compared with the methanol fraction of Kraft
lignin with a lower molecular weight [10]. A similar observation
was reported by Dizhbite et al. [9] that high molecular weight
decreases the radical scavenging activity. The molecular weight of
lignin is also able to affect its functional groups distribution. Ma
et al. [45] stated that alkaline fraction of wheat straw lignin with
lower molecular weight showed higher contents phenolic hydroxyl
group and methoxy functional group; the factor led to the extracted
lignin with the lowest molecular weight (Mn : 1500) having the
highest antioxidant activity as compared to the fraction with higher
molecular weight (Mn : 3000). Low molecular weight results from
extensive depolymerization of lignin, particularly cleavage of ether
linkages, which lead to the formation of new aromatic hydroxyl
groups. In this study, it was observed that the amount of G-lignin
units is signicant when compared to other lignin units (Fig. 6).
According to the presence of free positions in C5 of the aromatic
ring in G-lignin units, these units can also form carboncarbon
bond in comparison to S-lignin units [46]. We inferred that the
low number-average molecular weight of this lignin was essentially
due to the cleavage of ester bonds, which is the predominant reac-
tion that occurs during the alkaline pretreatment of lignocellulosic
biomass [47,48]. However, the carboncarbon linkages in G-units
were not broken during lignin treatment because of their high sta-
bility. Santos et al. [49] also reported that the carboncarbon bonds

Fig. 7. Py/TMAH-GC/MS of pretreated wheat straw lignin.

64 M. Azadfar et al. / International Journal of Biological Macromolecules 75 (2015) 5866
Fig. 8. UVvis spectrum of lignin, and commercial antioxidants ferulic acid, BHT, and guaiacol.
are sensibly stable during alkaline conditions. The amount of the
weight-average molecular weight of the lignin could be related to
the presence of inter-molecular carboncarbon and ether bonds.
Pan et al. [4] studied the importance of the weight-average and
number-average molecular weights of lignin on its antioxidant
activity. Their study revealed that the coefcient of determina-
tion of lignin antioxidant activity versus its Mn and Mw was 0.98
and 0.80, respectively. Their results demonstrated that a reduc-
tion in lignin number-average molecular weight led to a signicant
increase in its antioxidant activity in compared to its weight-
average molecular weight.
3.4. Lignin antioxidant activity analysis
The results of the antioxidant activity test in terms of the
percentage of inhibition of the DPPH radicals were 86.9 0.34,
103.3 1, 102.6 0.79, 103.6 1.36 for the pretreated wheat straw
lignin, and commercial antioxidants butylated hydroxytoluene
(BHT), ferulic acid, guaiacol, respectively. The higher the value the
higher the radical scavenging activity of the compounds tested. The
UVvis spectrum indicated that the high peak (at 515 nm) of DPPH
solution was completely removed by adding the lignin in solution
(Fig. 8). This showed that the DPPH radicals have been trapped
by the lignin phenolic compounds. The lignin as an alkali lignin
showed that it contains hydroxycinnamic acids-derived units,
particularly phenol 2-methoxy-4-vinyl, and guaiacyl derivatives.
Domenek et al. [50] reported that ferulic and -coumaric acids are a
main fraction of free phenolic monomers in alkali lignins and likely
to consider for the high radical scavenging activity. The number
of hydroxyl groups on the aromatic ring and ortho substitutions
with the electron donating methoxy group which increase the
stability of phenoxy radical, determine the antioxidant activity
of hydroxycinnamic acids [51,52]. Fig. 9 shows the potential
reaction between the DPPH and lignin-derived compounds. The
pretreated wheat straw lignin showed a signicant antioxidant
activity when compared with other commercial antioxidants
tested. The antioxidants trapped DPPH radicals from the solution,
and this reaction was conrmed by the color change from deep
violet, DPPH solution or blank probe, to pale yellow color or
colorless, DPPH solution plus antioxidants, which is a typical of
radical scavenging activity assay. Similarly, the lignin also showed
positive antioxidant activity assay results.
The analyses of the analytical data of FTIR, Py-GC/MS,
Py/TMAH-GC/MS, GPC, UVvis, and DPPH assay led us to greater
understanding of the antioxidant characteristics of derived lignin
from wheat straw by ozone and soaking aqueous ammonia
pretreatment. The strong hydrogen band (OH) stretching at
3200 cm1 and CH stretching at 2851, 2921 cm1 and the CH
stretching bands at 2850 and 2925 cm1 (CH3 or CH2 ) perti-
nent to the vibrations of the methyl and methoxy groups conrmed
the presence of phenolic compounds and their methoxy functional
groups in the lignin. It has observed that the content of guaia-
cyl units (1265 cm1 ) were higher than syringyl units (1127 cm1 )
DPPH-H
*
OCH3 OCH3
OCH3
O-H O* O
DPPH*
Fig. 9. Potential reaction between DPPH and lignin-derived compounds (i.e.,
isoeugenol).
 M. Azadfar et al. / International Journal of Biological Macromolecules 75 (2015) 5866
with a S/G ratio of 0.35. We also found that the ester linkages in
the lignin structure were selectively broken (1720 and 1653 cm1 )
during the pretreatment as seen in Figs. 3 and 4. This possi-
bly indicates the occurrence of hydroxycinnamic acids derivatives
in the lignin [35]. Likewise, high levels of phenol 2-methoxy-4-
vinyl (25.83%) from the pyrograms were identied. Also, it has
found that the presence of lignin-derived compounds with low
molecular weight (772 g/mol) induced lignins antioxidant activity
(86.9 0.34).
4. Conclusion
It was inferred that the antioxidant activity of the lignin macro-
molecule was predominantly due to the presence of low molecular
weight monomers derived from hydroxycinnamic acids and gua-
iacyl units of wheat straw by the ozone and soaking aqueous
ammonia pretreatment. We concluded that the lignin has the
potential to be considered as a desirable feedstock for guaiacol and
4-vinylguaiacol as antioxidants.
Competing interest
The authors declare no competing nancial interest.
Acknowledgments
This material is based upon work supported by Washington
State University and the National Science Foundation under Grant
No. 1231085. The authors would like to acknowledge JiJiao Zeng of
University of Florida for providing GPC facility.
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