Separation and Purication Technology 132 (2014) 234243

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Separation and Purication Technology

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

Membrane performance and application of ultraltration

and nanoltration to ethanol/water extract of Eucalyptus bark
Paula C.R. Pinto , Ins F. Mota, Jos M. Loureiro, Alrio E. Rodrigues
LSRE Laboratory of Separation and Reaction Engineering Associate Laboratory LSRE/LCM, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465
Porto, Portugal

a r t i c l e

i n f o

Article history:
Received 7 January 2014
Received in revised form 26 April 2014
Accepted 28 April 2014
Available online 9 May 2014
Keywords:
Ultraltration
Nanoltration
Ethanolic solutions
Bark extract
Polyphenols
Carbohydrates

a b s t r a c t
The aim of this work is to promote the enrichment of an ethanolic extract of Eucalyptus globulus bark in
polyphenolic compounds relatively to other compounds such as carbohydrates. Several at sheet membranes were tested with water and ethanol solutions (52% v/v and 80% v/v) assessing to permeability.
Rejections to gallic and tannic acids and maltose were evaluated for nanoltration membranes and for
the ultraltration membrane of lower cut-off. The dependence of permeability and rejection relative to
ethanol percentage is discussed giving new insights about the membrane performance towards
ethanol/water solutions. Among the tested membranes, two ultraltration (JW 30,000 Da and PLEAIDE
5000 Da) and one nanoltration (SolSep 90801) membranes were selected to the concentration process
of an ethanolic extract of Eucalyptus globulus bark produced at previously optimized conditions. The performance of the three membranes was evaluated concerning polyphenolic compounds and carbohydrate
composition. The volume reduction factor was 1.76. JW membrane revealed the lowest total decrease on
permeability (53%) relative to the initial. All the three membranes showed selective retention of polyphenolic compounds, however JW promoted the highest enrichment of formaldehyde-condensable tannins
(fcT) and proanthocyanidins (Pac) (17% and 28%, respectively). The nal composition of the retentate
(in % weight/dry weight) was: TPC 39%, fcT 46%, Pac 38%, GalT 3.2% and TC 15%. The detailed sugar analysis revealed that some arabinose- and rhamnose-containing oligo/polysaccharides are preferentially
retained, while those with glucose and galacturonic acid moieties are transported through the membrane
to permeate stream. Finally, cleaning performance of membranes was evaluated and 80100% ux
recoveries were attained.
2014 Elsevier B.V. All rights reserved.

1. Introduction
The development of biorenery platforms is currently undergoing rapid expansion. Pulp and paper industries have a privileged
position due to the availability of side-streams lignocellulosic materials usually classied as by-products, such as bark which is a disposal in mill site where the logs are debarked. This is the case of
pulp plants in Portugal which produces about 124,000 tons of Eucalyptus globulus bark per medium size industrial unit. Bark is further
integrated in the mill operation as energy source. The basic chemical
composition of bark and wood is similar concerning the major
macromolecular components: lignin, cellulose and hemicelluloses
[1,2]. However, the extractive and inorganic content is usually
Corresponding author. Address: LSRE Laboratory of Separation and Reaction
Engineering, Faculty of Engineering, Chemical Engineering Department, University
of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal. Tel.: +351 22 041
3606; fax: +351 22 508 1449.
E-mail address: ppinto@fe.up.pt (P.C.R. Pinto).
http://dx.doi.org/10.1016/j.seppur.2014.04.042
1383-5866/ 2014 Elsevier B.V. All rights reserved.

higher in bark than in wood. This is one of the reasons why bark
has not been used for pulp production. Among the undesired
extractive fractions is the polyphenolic fraction. This is composed
by simple phenolics such as gallic and ellagic acids, avonoids,
complex glycosides of phenolic compounds [3,4], hydrolysable tannins, and proanthocyanidins [5,6], often called condensed tannins.
The awareness on these compounds is growing up due to their
properties and biological activities with emerging applications on
cosmetics, nutricosmetic and fortied foods or supplements industries turning it on high added-value additives or active principles
[7,8].
In this perspective, E. globulus bark is a potential raw material to
produce polyphenolic enriched extracts. In our previous work, the
optimum conditions (time, temperature and ethanol %) for the
extraction of polyphenolic compounds from E. globulus bark were
reported. The extract produced at optimum conditions (OC extract)
was obtained in ethanol/water solution (52/48, v/v) and it
demonstrated important biological activity. The yield was 50 g of
material per kg of bark with 1/3 of the extracted material being

P.C.R. Pinto et al. / Separation and Purication Technology 132 (2014) 234243

235

Nomenclature
List of symbols
A
effective membrane area (m2)
Ara
arabinose
Cp
concentration in the permeate (g L1)
Cr
concentration in the retentate (g L1)
Gal
galactose
GalA
galacturonic acid
GalT
gallotannins (% w/w)
Glc
glucose
fcT
formaldehyde-condensable tannins (% w/w)
Jp
volumetric ux through membrane (L m2 h1)
Lp
membrane permeability coefcient (L m2 h1 bar1)
NF
nanoltration
pHPZC
point of zero charge
OC
optimum conditions
Pac
proanthocyanidins (% w/w)
Qp
permeate ow rate (L h1)
Rha
rhamnose

of phenolic nature (assessed by FolinCiocalteu method for

quantication of total phenolic compounds) [1]. Envisaging the
fractionation and the increase of the polyphenolic fraction as the
next step in the valorization process, membrane processing of OC
extract was carried out. Based on its principle, membranes
processing should lead to a fractionation of the polyphenolics
and other components in the extract according to their molecular
weight, hydrodynamic volume (size and shape of the hydrated/
solvated molecule) and solventsolutemembrane interactions.
However, the adsorption and the build-up of a gel layer may act
as a secondary membrane, changing both solute retention and
permeate ux rate [9]. Moreover, the performance of a system
strongly depends on the feed characteristics, operating conditions,
membrane, and system conguration.
Membrane separations have been applied to fractionate and
purify polyphenolic rich streams from several biomass resources
as recently reviewed [10]. Olive mill wastewaters [1114], extracts
of grape seeds [15] and grape pomace [16,17] are the main examples of liquid streams derived from industrial activity processed by
ultraltration (UF) and/or nanoltration (NF) for polyphenols
recovery. Concerning woody bark extracts, only one study for tannins recovery by UF was found [18]. Moreover, most of the studies
in literature deal with aqueous solutions/extracts and just a few
report real streams of organic solvent or binary mixture, namely
ethanol/water [1921].
In this work, seven commercial membranes were characterized
and the impact of solvent composition on membrane performance
was evaluated. Gallic acid (170 g mol1), tannic acid (1701 g
mol1) were used as models for phenolic compounds, and maltose
(342 g mol1) as model for carbohydrates, to test the NF membranes and the UF membrane of lower cut-off. The OC extract
was submitted to UF and NF in concentration mode. The goal
was to evaluate the performance of membrane processing in the
polyphenol enrichment of the E. globulus extract. For this, the ux
declines were evaluated and the compositions of retentates and
permeates were assessed considering total non-volatile solids
(TS), total phenolic compounds (TPC), formaldehyde-condensable
tannins (fcT) quantied as Stiasny number (SN), proanthocyanidins
(Pac), gallotannins (GalT), and sugar composition allowing the
quantication of total carbohydrates (TC).

Man
Rm
Rj
SN
TMP
TPC
TS
TC
UF
Vf
Vr
VRF
Xyl

mannose
membrane resistance coefcient (m1)
apparent solute rejection coefcient
Stiasny number (% w/w)
transmembrane pressure (bar)
total phenolic compounds (% w/w)
total non-volatile solids (g L1)
total carbohydrates (% w/w)
ultraltration
feed volume (L)
retentate volume (L)
volume reduction factor
xylose

Greek letters
l
dynamic viscosity of water/solvent (kg m1 s1)

2. Experimental
2.1. Equipment, membranes and conditioning
Benchtop studies were conducted using a membrane cell system Sepa CF II Med/High Foulant System (GE Osmonics, USA) with
an effective area of 0.014 m2 plus a ow meter, a diaphragm pump
Hydra-Cell, model M-3/G-13, (Wanner Engineering, Inc.) with a
frequency inverter (MC07, MovitracB, SEW Eurodrive), and a
manual hydraulic pump (P19, SPX Corporation, USA). The NF/UF
unit withstands a maximum operating pressure of 69 bar, and a
maximum operating temperature of 177 C. The temperature of
the feed was assured by a Lauda thermostatic bath (Ecoline
Staredition Re 206) and a coil immersed on the feed tank. The feed
temperature was checked by an electronic contact thermometer
(VT-5 S40, VWR).
The UF and NF at sheet membranes studied are listed in
Table 1. Aqueous solutions of ethanol (Panreac) were prepared
on a volume/volume basis using deionized water. All membranes
were preconditioned according to the protocol recommended in
the literature [22]. Prior to use, the membranes were rst rinsed
with water and soaked overnight. Afterwards, the membranes
were soaked with ethanol solutions starting with 10% (v/v) ethanol
and then with increments of 1020% ethanol until 52% or 80% (v/v)
ethanol, depending of the programed assays. For the experiences
with water, membranes were simply soaked with water for three
times and left overnight. The SolSep membranes were directly
washed and conditioned in the working solvent as recommended
by the fabricant. Before operation, each membrane was prepared
by compressing it into the module by means of system hydraulic
pressure (about 1015 bar more than the operating pressure in
the experiments), using water or ethanol/water solutions at a
transmembrane pressure (TMP) of 1 bar for about 30 min to
remove material from the pores. Then, using fresh solution, the
membranes were submitted to compaction with a TMP 12 bar
higher than the operating pressure in the experiments. The permeate ux was measured and usually the time to ensure the steady
state was 1 h.
Ultra-pure water and analytical grade reagents were used for
membrane characterization.

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P.C.R. Pinto et al. / Separation and Purication Technology 132 (2014) 234243

Table 1
Characteristics of the NF/UF membranes.

a
b

Membrane

Rejection to typical solutes/MWCO

Producer

Composition

Operational pH

Tmax (C)

Pmax (bar)

80105
90801
NF270

>99% colorant (500 Da) in ethanol

50% NaCl 90% colorant (350 Da) in ethanol
97% MgSO4

SolSep
SolSep
DowFilmtec

Polyamide derivative
Polyamide derivative
Polyamide thin lm composite

120
80
45

20
20
41

GE
PLEAIDE
JW
EW

1000
5000
30,000
60,000

GE Osmonics
Orelis Environnement
GE Osmonics
GE Osmonics

Polyamide composite
Polyethersulfone (PES)
Polyvinylidene uoride (PVDF)
Polysulfone (PS)

112
114
310a
112b
211
314
111
0.513

50
75

Continuous operations.
Short-term cleaning (30 min).

2.2. Operating procedure

2.2.1. Membrane permeability
After membrane conditioning and compaction, the ux was
measured for water and ethanol solutions 52% and 80% at different
TMP for membrane permeability assessment. Besides the intrinsic
resistance of each membrane, these assays allow evaluating the
irreversible fouling or membrane damage after each experiment
with the extract by comparison with measurements carried out
at the same conditions. For UF membranes the applied TMP were
in the range 19 bar and for NF the range was 130 bar.
Membrane permeability was evaluated in total recirculation
mode (recirculation of permeate and retentate to the feed tank).
The different uxes were monitored over time after a period of stabilization at a feed ow rate of 4.5 0.25 L h1 and 35 C.
2.2.2. Standard rejections
Membrane selectivity is a measure of the membrane ability to
reject a particular solute in detriment of another due to differences
in interactions between solute, nature of membrane and type of
solvent. It is affected by several operating variables such as TMP,
turbulence near membrane surface triggered by tangential ow,
temperature, solute type and concentration, pH, ionic strength, solvent or other factors that can modify the shape and the molecule
conformation.
Feed solutions of 0.6 g L1 of gallic acid (170 g mol1, Acros
organics, 98%), tannic acid (1701 g mol1, Sigma Aldrich, P98%)
and maltose (342 g mol1, Sigma Aldrich, P98%) were prepared
in water and ethanol/water solutions (52% and 80% ethanol, v/v).
The experiments were conducted at constant feed ow rate and
temperature, 4.5 L h1 and 35 C, respectively. TMP was also
constant at 4 bar for GE membrane, 30 bar for NF270 and 14 bar
for the SolSep, 090801 and 080105 membranes.
Membrane selectivity was evaluated in total recirculation
mode. The feed solution was circulated for about 1 h until the
steady state. Afterwards, at preset time intervals, permeate was
collected and further analyzed. Gallic acid and tannic acid concentration was assessed by the FolinCiocalteu assay and the disaccharide was quantied by HPLC, as described in Section 2.4.
Experiments were carried out from the lowest to the highest
solute molecular weight. Each membrane was then washed with
the solvent or cleaning solution until at least 75% of the initial
solvent ux.
2.2.3. Concentration of the E. globulus bark extract
Several UF and NF membranes were initially tested for performance and stability with the ethanolic solutions and, among these,
three membranes were selected to proceed for E. globulus extracts
processing: NF SolSep 090801, UF Pleiade 5 kDa and UF JW 30 kDa.
The selection was based on some important attributes during
membrane characterization such as solvent ux, applied TMP,

solute rejection coefcients, robustness and stability, and cleaning

cycles to retrieve the initial permeability. The point of zero charge
(pHPZC) of the membranes was determined by the pH drift test
described in the literature [23].
E. globulus bark extractions were performed according to the
optimum conditions found in our previous work [1] but using N2
as inert atmosphere. This extract is denoted in this work as
optimum conditions extract (OC extract). Prior to the membrane
separation experiments, the extract was ltered through a polycarbonate membrane (Nucleopore, Whatman) with 10 lm of pore size
to remove small particles of biomass, accounting for 0.08 g L1. The
turbidity and total suspended solids are 411 NTU and 0.50 g L1,
respectively. The TS content of this extract is 7.9 g/L with the
following composition on % weight/dry weight: TPC 37.9%; fcT
36.0%; Pac 32.1%; GalT 3.1%; and TC 16.0%.
The membrane separation operations were carried out in concentration mode, at 35 C and a xed TMP (14 bar for NF membrane
and 4 bar for UF membranes) was applied. In concentration mode
the retentate stream is recycled into the feed tank whereas the
permeate stream is separately collected, resulting in a continuous
volume decline in the feed tank. Volume permeation uxes were
measured up to a volume reduction factor (VRF) of about 1.76.
VRF is the ratio between the initial feed volume and the remaining
volume of retentate at a given operating time. The feed ow rate
was adjusted to 4.5 L h1.
Permeate and retentate samples were collected during each run
for composition analysis regarding the same parameters used for
OC extract characterization: TS, TPC, fcT quantied by SN, Pac, GalT
and TC.

2.3. Cleaning and storage

In this study, immediately after the experiments, the system was
initially ushed with fresh solvent (water, ethanol 52% or ethanol
80%, depending on the experiment) with no TMP. The solution
was discharged and subsequent cleaning cycles (as described below
for standards and for extract experiments) in full recirculation mode
and applying a TMP of 1 bar for 60 min were performed as many
times as necessary to attain at least 75% of the initial feed owrate.
The feed owrate was 4.5 L h1 and the maximum temperature
40 C. Alkaline solutions for cleaning were prepared in water, ethanol 52% or 80% according to each case. Supplier recommendations
were followed: SolSep membranes were cleaned using NaHCO3
0.1 M and for Osmotic and Orelis membranes NaOH 0.1 M was used.
For the experiments using standard compounds (gallic acid,
maltose or tannic acid), after ushing, the rst two cleaning cycles
were performed with the corresponding solvent (water, ethanol
52% or ethanol 80%) used in the experiment. After this, the
permeability was evaluated and, if necessary, subsequent cleaning
cycles with alkaline solution (water, ethanol 52% or 80%, as the
case may be) were performed for 60 min and 1 bar of TMP.

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P.C.R. Pinto et al. / Separation and Purication Technology 132 (2014) 234243

2.4. Analytical methods

For rejection evaluation, gallic acid and tannic acid were quantied by FolinCiocalteu method as described previously [1] using
gallic acid or tannic acid as standards. The disaccharide maltose
was quantied in a HPLC Knauer unit equipped with a Smartline
5000 online degasser, a Smartline 1000 quaternary pump, and a
Smartline 2300 refractive index detector. The analytical column
was SHODEX SC-1011 (300  8.0 mm, 6 lm) using ultra-pure
water as eluent at 0.7 mL min1. Chromatograms were run at
80 C and the volume of injection was 20 lL.
Quantication of TS, TPC, fcT, Pac and TC were performed as
described in our previous work [1]. Briey, TS were quantied by
weighting the dried extract/fractions; TPC quantication was
based on FolinCiocalteu method using gallic acid as standard;
fcT quantied by SN is a comparative parameter which includes
all the monoavonoids, biavonoids and oligomers susceptible to
form methylene linkages and polymerizing through the reaction
with formaldehyde; the value is obtained by the weight of the
produced precipitate [24,25]; Pac was quantied by Bate-Smith
reaction: in this method, proanthocyanidins (also referred as
condensed tannins) are cleaved to yield anthocyanidins [26,27]
and further quantied by absorbance using a mimosa extract as
standard. TC analysis was performed by acid methanolysis
followed by identication and quantication of sugar moieties by
GCMS and GCFID, respectively.
GalT estimation was based on the quantication of gallic acid
methyl ester liberated in the methanolysis of the extract [28]:
dried samples (10 mg) were treated with anhydrous methanolic
HCl (2 mL) 2 M for 17 h at 100 C, the time needed to obtain a
maximum of gallic acid methyl ester from the extract and
fractions. After completion, the solutions were cooled to room temperature, evaporated at reduced pressure, redissolved in methanol
and diluted in methanol/water 90/10 containing 0.1% HCOOH
before injection. Gallic acid methyl ester was quantied using an
analytical column YMC-Park ODS-A in the same HPLC described
above equipped with Smartline UV/DAD Detector 2600 operating
at 280 nm. The separation was performed at room temperature
and eluent owrate 0.4 mL min1 using a gradient composed by
two solutions: A water/methanol: 95/5 (v/v) containing 0.1%
(v/v) formic acid and B 5:95 (v/v) with 0.1% (v/v) formic acid.
The gradient program was 03.30 min 90% A, 6.720.0 min 80%
A, 40.043.3 min 40% A, 43.346.7 min 0% A. The calibration was
performed with gallic acid (P98%, Sigma). Standard solutions
and samples were ltered before injection using a 0.2 lm
disposable lter (Millipore). The quantication data were reported
as gallic acid equivalent. The content on GalT was calculated by
subtracting the content of gallic acid in the extract/fractions to
the value resulting from the methanolysis.

Jp

Qp
A

where Qp is the permeate ow rate (L h1) and A is the effective

membrane area (m2).
Considering negligible the osmotic pressure at membrane surface and in the permeate, Jp is proportional to the differential pressure across membrane or transmembrane pressure (TMP, bar) and
given by the following equation:

Jp

TMP
lRm

J p Lp TMP

The volumetric ux through membrane (Jp, L m2 h1) is

given by

where l is the dynamic viscosity of water/solvent (kg m1 s1), Rm

is the membrane resistance coefcient (m1) and Lp the membrane
permeability coefcient (L m2 h1 bar1).
The volume reduction factor (VRF) is given by:

VRF

Vf
Vr

with Vf representing feed volume (L) and Vr the retentate volume

(L).
The apparent solute rejection coefcient (Rj) for gallic acid,
maltose and tannic acid is dened as,

Rj 1 

Cp
Cr

being, for each compound family, Cp concentration in the permeate

(g L1) and Cr concentration in the retentate (g L1).
3. Results and discussion
3.1. Membranes characterization
Membranes were characterized concerning their Lp in water,
ethanol 52% and 80% at 35 C and constant feed owrate of
4.5 L h1. The purpose of studying water and ethanol 80% was to
provide additional data to evaluate the effect of the ethanol
percentage on the membranes performance. Moreover, in the
previous published work [1], where the optimum of 52% ethanol
for TPC extraction was reported, it was also found that the extract
produced with 80% ethanol presented a high biological activity. If
the fractionation of this extract would be considered in the future,
the characterization of the membranes with the corresponding

250
10

200
5

Water

150
100

Ethanol 52%

0
80105

90801

NF270

GE

Ethanol 80%

50
0

2.5. Membrane parameters and calculations

The membrane permeability coefcient is a common parameter

to evaluate the performance of membranes, representing the liquid
crossing the membrane per time unit, per membrane area unit and
per TMP unit. Experimentally, it is calculated by the slope of Jp vs
TMP for the system:

Lp (L m-2 h-1 bar-1)

In the case of OC extract experiments, after the rst ush, the

system was additionally ushed with the alkaline solution (in
for 10 min without TMP). The cleaning cycles were performed
with alkaline solution of 52% ethanol (0.1 M NaOH or NaHCO3
depending on the membrane). After the alkaline washing, the
system was rst ushed with 52% ethanol followed by a double
washing at TMP of 1 bar for 30 min to remove the residual alkali
of the membrane. Finally, the permeability was evaluated using
fresh solution of 52% ethanol.
After cleaning, each membrane was stored in 0.5% Na2S2O5
aqueous or ethanolic (52% or 80%) solution.

80105

90801

NF270

GE

PLEAIDE

JW

EW

membrane
Fig. 1. Permeability of polymeric membranes for water, ethanol 52% and ethanol
80% at 35 C and feed owrate 4.5 L h1.

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P.C.R. Pinto et al. / Separation and Purication Technology 132 (2014) 234243

solvent would be also valuable. Fig. 1 shows the permeability for

each membrane in water, ethanol 52% and ethanol 80%.
UF membranes were chosen based on supplier reference for
molecular cut-off. The aim was to test a widespread cut-off range
(from 1000 Da to 60,000 Da) and different membrane composition,
as stated in Table 1. Concerning NF membranes, two polyamidebased membranes, SolSep 80105 and 90801, and also the NF270,
a piperazine-based polyamide membrane [29] were tested.
Pure water and aqueous solutions of ethanol 52% (the solvent
composition giving the best results on extraction experiments
[1]) and ethanol 80% were used for permeation studies to evaluate
their effect on membrane performance. Since SolSep 80105 and
90801 are typically membranes for organic solvents, these were
not tested with 100% water. Solvent permeation through membranes is a diffusion process enhanced by the interaction between
the solvent and the hydrophilic and/or hydrophobic domain of the
membrane; the afnity of water to a membrane with hydrophilic
properties would promote the transport, while for ethanol/water
system, in the same membrane, ux would be considerably lower
due to the limited hydrogen bonding capability of the ethanol. In
general, for membrane with dominant hydrophilic properties, the
greater the solvent polarity the highest is the ux and the opposite is observed for membranes with hydrophobic characteristics
[30]. From Fig. 1 it is clear that as the membrane cut-off increases,
the highest is the water permeability due to the decrease of the
resistance offered in uid transport. For all the tested membranes,
a high decrease on Lp was observed from pure water to 52% ethanol, which is in accordance with the increase of viscosity of
uids: from water (0.7202  103 Pa s, 35 C) to ethanol 52%
(1.7896  103 Pa s, 35 C) [31]. However, a Lp decrease was also
observed for ethanol 80%, in spite of the lower viscosity of this
solution (1.436  103 Pa s, 35 C) [31] compared to ethanol 52%.
This clearly shows that solvent viscosity is not the main feature
affecting ux when using ethanol solutions. Other phenomena,
such as the decrease of solvent polarity had impact on Lp for all
above mentioned membranes, except for EW. In this case the Lp
increases when changing 52% to 80% of ethanol. According to
Eq. (2) there is a linear correlation between ux and the inverse
of viscosity if the ux decrease is dominated by solution viscosity
[30,32]. Fig. 2 shows the plot of Jp and 1u for the studied
membranes. EW was the only one showing a linear relationship
demonstrating that the viscosity of the solution is the dominant
parameter affecting solvent ow through membrane. For the
other membranes the effect of other solvent characteristics has
more impact than viscosity alone. The solvent/solvent mixture
characteristics with already reported impact on Lp are surface

tension, molar volume and dielectric constant [33]. Surface tension and dielectric constant decrease in the order water, ethanol
52% and ethanol 80% [31,34,35], while molar volume increases
in this order. Molar volume could, at least, partially explain the
Lp decrease for NF membrane in the order water to ethanol 80%,
while surface tension would have the opposite effect on Lp. However, dielectric constant of the mixture and the related factor,
polarity, could have a practical effect on membrane due to the
interaction (or its absence) with the polymeric material. This
effect depends also on surface energy of the polymer as stated
in the literature [30].
Hence, for membranes with predominant hydrophilic character,
the ux would be considerably lower for ethanol/water mixtures
than for water due to the limited hydrogen bonding capability of
the ethanol delaying and impairing the transport through the
membrane. In accordance, other authors have concluded that the
hydrophilicity and porosity are the most important characteristics
of membranes affecting the uid transport, while viscosity and
polarity are the solvent properties with highest inuence on permeability [36].
From this preliminary study, it was possible to evaluate the
behavior of the different membranes concerning Lp as the net
result of different factors. Among the UF membranes, those of
medium cut-off and simultaneously presenting Lp values higher
than 35 L m2 h1 bar1 were selected for the concentration
assays: membranes JW and PLEAIDE. Among the NF membranes,
the 090801 is the membrane with highest Lp for 52% ethanol
(5.8 L m2 h1 bar1) and, thus, the most promising for the concentration process. In spite of this, a study of rejection coefcients for
three compounds was performed for NF membranes: two phenolic
compounds (gallic acid and tannic acid) and one disaccharide,
maltose, as general representatives of the families of compounds
present in the real extract. In this study, the membrane GE was
included due to its small cut-off. The overall results are presented
in Fig. 3.
Among all membranes, the NF270 presents the highest rejection
for the three compounds. NF 270 is a NF membrane stated to have
high rejection indexes for simple sugars and salts in water (about
90% for glucose in water at 37 C and at 8 bar [37] and more than
97% to MgSO4 (manufacturer data Table 1). However, Restolho
and co-workers [38] have reported rejections of 52% and 34% (in
water, 25 C, 18 bar and 2.0 L min1) for glucose and xylose,
respectively. The values found in this work for rejections are 40%
for gallic acid, 67% for maltose and 95% for tannic acid in 52%
ethanol. The membrane 80105 presented the lowest rejections in
52% ethanol (between 4% and 22%).

1000

EW
JW
Pleaide

-1

Jp (L m h )

750
-2

GE
NF270

500
20

250

GE

15

0
0.0

10

0.5

1.0

1.5

1/ (m s kg-1 )
Ethanol Ethanol
52%
80%

Water

2.0

0
0.0

0.5

1.0

1.5

Fig. 2. Correlation between Jp and l1 for membranes EW, JW, PLEAIDE, GE and NF270 for water, ethanol 52% and ethanol 80% at 35 C and feed owrate 4.5 L h1.

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P.C.R. Pinto et al. / Separation and Purication Technology 132 (2014) 234243

100

Gallic acid

60

Maltose
Tannic acid

40
20
0
80105

90801

NF270

GE

Membrane

100
80

Rf (%)

Ethanol 52%

Gallic acid

60

Maltose
Tannic acid

40
20
0
80105

90801

NF270

Membrane

GE

Ethanol 80%

Fig. 3. Apparent rejection coefcients to gallic acid, tannic acid and maltose in
ethanol 52% (A) and in ethanol 80% (B) for the NF membranes and for the UF
membrane GE at 35 C and feed owrate 4.5 L h1.

In the conditions of this work, the relative rejection in the membranes 90801, NF270 and GE follows the relative molecular weight
order (gallic acid, maltose, tannic acid) in both solvents. However,
for the membrane 80105 a change was observed: the rejection to
gallic acid is higher than to the disaccharide. These observations
can only be explained by different membrane characteristics since
solventsolute interactions are the same as for the other membranes. The polarity differences between the molecules can lead
to a modication of the expected relative rejection based on molecular weight due to polarity itself (interaction with membrane) and
hydrodynamic volume (interaction with the solvent). The transport of the gallic acid relative to the disaccharide (the phenolic
compound is less polar than the disaccharide) was improved by
an additional afnity with this membrane. Among all membranes,
the 80105 seems to be the most hydrophobic. This is in accordance
with the increase of the rejection of the three compounds (but
maintaining the relative rejections) with the increase of ethanol
percentage (52% to 80%) (and consequently, with decrease on solvent polarity) observed for membrane 80105 Fig. 3A and B.
The most relevant difference on rejection between ethanol 52%
and ethanol 80% concerns to tannic acid: for GE and for 90801
membranes, the rejection decreases with the change of the percentage of ethanol from 52% to 80%; this means that the presence
of ethanol promotes the transport of this compound through the
membrane pores. This could be explained based on interaction of
solventmembrane and the solvation of the pores: for membranes
with some hydrophilic character, the hydration of the pore wall
occurs, leading to a decrease on their effective pore size [36]; considering that these membranes are not so effectively solvated by
ethanol as by water, the increase of ethanol percentage led to an
higher effective pore diameter and thus, to a lower hindrance to
the transport of the tannic acid reected in a higher rejection in
52% ethanol than in 80% ethanol. This effect is noticeable for tannic
acid probably due to its higher molecular weight. For membrane
NF270, rejection in 52% and 80% ethanol is similar for all compounds. Although for this membrane no effect was perceptible
from 52% to 80%, the lower rejections observed for this membrane

3.2. Concentration of OC extract from E. globulus bark

The next step was to apply the OC extract in concentration
mode using the selected membranes: JW, PLEAIDE and SolSep
090801. Fig. 4 shows the ux along the concentration experiment
regarding operating time (A) and VRF (B) for each membrane.
The solute build up in the membrane boundary layer
established in laminar ow and in the rst minutes of operation
is known as concentration polarization. It is the main cause for
the ux deviation from the solvent ux. After that, the fouling

1.0
JW

0.8

Jt /J0

Rf (%)

80

(when compared with literature data for water as solvent, as

referred above) is probably due to the introduction of ethanol in
the feed, increasing the effective pore size relative to that obtained
in water.
On the opposite, for membrane 80105, besides the lowest
rejections of all the membranes in 52% ethanol, it was noticed a
high increase on rejection when the solvent is changed to 80% ethanol for all compounds. This could be due to the effect of the higher
afnity for ethanol, occurring an effective solvation of the
pores (reducing the permeability to the compounds) since the
solutesolvent is the same as for the other membranes. Thus,
the character/nature of this membrane should be much less hydrophilic than for the other tested membranes, as was stated before.
These considerations allow a better knowledge about the
behavior of the different NF membranes concerning uxes and
permeability to different solutes in ethanol 52% and ethanol 80%.
Among the NF membranes and GE, the one selected for processing
the OC extract was the SolSep 090801: the ux on 52% ethanol was
the highest with a high rejection to tannic acid (70%) over the
disaccharide (27%) which is favorable in the perspective of a
selective concentration, considering these two standards and
comparing with the other membranes.

PLEAIDE

90801

0.6
0.4
0.2
0.0
0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Operation time (h)

60
50

Jp (L h-1 m-2 )

JW

40

PLEAIDE

90801

30
20
10
0
1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

VCR
Fig. 4. (A) Normalized permeate ux (ratio of instantaneous permeate ux at time
t, Jt, and at initial time, J0) along the operating time. (B) Instantaneous permeate ux
along the VRF for the extract OC. Conditions: concentration mode, 35 C, feed
owrate 4.5 L h1, TMP 4 bar for UF JW and PLEAIDE and 14 bar for NF 90801;
operating time for VRF 1.76: 4.3 h JW; 7.4 h PLEAIDE; 5.6 h 90801.

P.C.R. Pinto et al. / Separation and Purication Technology 132 (2014) 234243

due to solute adsorption onto the membrane surface or membrane pore plugging becomes noticeable and contributes to the
ux drop [9]. In the OC extract processing, the decrease pattern
of permeate uxes is different for the three membranes
(Fig. 4A). 90801 is the membrane with the highest absolute ux
but also with the higher initial decline, notorious up to the VRF
1.37 Fig. 4B which corresponds to about 2.5 h of operation.
From this point onward, the membrane 90801 presents lower
Jp value than the UF membrane JW (Fig. 4B). These differences
suggest that the 90801 membrane is the most susceptible one
to concentration polarization and fouling in the conditions of this
work, thus creating an additional resistance to permeate ux. At
the pH of the OC extract (4.3) the compounds are mainly neutral
and protonated in solution. In the conditions of the processing,
the membrane 090801 (pHPZC 3.3) is negatively charged and
electrostatic attractions should be expected between the surface
and some of the components of the extract. For JW (pHPZC 6.1)
and PLEAIDE (pHPZC 6.4) membranes, electrostatic repulsions are
expected due to the positively charged surface. Thus, considering
this data, fouling should be more severe in the case of PLEAIDE.
However, besides this effect, hydrophobicity of the membranes
would play also an important role in this process. The balance
between electrostatic repulsion and van der Waals interactions
determines the outcomes of membrane fouling, as well as the efciency of the membrane cleaning after processing.
The initial decline of normalized ux Jt/J0 (Fig. 4A) for UF membranes is the same, suggesting that the concentration polarization
effect is similar for both. However, after about 1 h of operation, the
membrane JW holds up higher normalized and absolute ux. At
the end of the concentration stage, for the same concentration
factor in volume, the ux reductions relative to the initial were
53%, 68% and 85% for JW, PLEAIDE and 90801 membranes, respectively. Among these, JW presents simultaneously the lowest
decrease of ux by fouling, reaching the steady state earlier than
the others, and the highest nal ux.

3.3. Retention of polyphenolic compounds: characterization of

retentates
The extract OC contains, as major components, polyphenolic
compounds (polyhydroxy aromatics with an amphiphilic character which is controlled by phenolic- and by carboxyl-groups) as
well as carbohydrates, including pectins and fragments of hemicelluloses and glucans from bark structure [1]. The approach of
this work was to quantify families of compounds for assessing
the composition of the retentates produced in the concentration
process of the E. globulus bark extract. For TS and TPC, as well
as TC, the permeates were also analyzed, allowing to calculate
the respective apparent rejection coefcient Rj depicted in Table 2.
The rejection values for TPC increases in the order JW, PLEAIDE,
9080, following the trend of the membranes cut-off; however,
for TS no signicant difference was found between PLEAIDE and
90801. Lower rejections were found for TC indicating that it

Table 2
Rj for TS, TPC and TC during the concentration of OC extract with the membranes JW,
PLEAIDE and 90801. Conditions: 35 C, feed owrate 4.5 L h1, TMP 4 bar for UF (JW
and PLEAIDE) and 14 bar for NF (90801); operating time for VRF 1.76: 4.3 h JW;
7.4 h PLEAIDE; 5.6 h 90801.
Membrane

TS

TPC

TC

JW
PLEAIDE
90801

0.58
0.77
0.78

0.75
0.85
0.92

0.12
0.17
0.15

50
Feed (OC extract)

40

% w/w

240

JW
PLEAIDE

30

90801

20
10
0
TPC

fcT

Pac

TC

Parameter
Fig. 5. Composition of feed (OC extract) and composition of retentates produced
with membranes JW, PLEAIDE and 90801. The values are represented as % weight/
dry weight of the OC extract (the feed) or dry weight of the retentate obtained for
each membrane. Conditions: concentration mode, 35 C, feed owrate 4.5 L h1,
TMP 4 bar for UF JW and PLEAIDE membranes and 14 bar for NF 90801 membrane;
VRF 1.76.

would be possible the elimination of sugar moieties in the permeate during UF and NF.
Besides TS, TPC and TC, nal retentates were also analyzed for
Pac, fcT and TC. The global results are depicted in Fig. 5.
For the same VRF, the membranes showed different performances as denoted by the composition of the retentate. The
common feature is an accentuated enrichment effect on fcT
(monoavonoids, biavonoids and some oligomers). However,
membranes with lower cut-off (PLEAIDE and 900801) retain more
material carrying phenolic hydroxyl groups, as indicated by TPC in
the respective retentates, probably those compounds of lower
molecular weight. This is not reected on Pac content, assessed
by butanol-acid assay, since this family is mainly composed by avonoid oligomers. In accordance, JW membrane promoted higher
enrichment of this parameter than did for TPC and fcT, probably
due to a noteworthy contribution of compounds with high
molecular weight that responds to the butanol-acid method. On
other side, the Pac content of PLEAIDE and 90801 retentate is similar to that of OC extract (which was not observed for TPC and fcT).
The reason for that could be a preferential entrapment or adsorption of Pac at the membranes contributing for fouling, as assessed
by the mass balance using the content on permeate for PLEAIDE
and 90801 (data not presented). This also occurs for JW, but in a
lower proportion. In accordance, JW showed the lowest ux
decline during the concentration process (Fig. 4).
Hydrolysable tannins (GalT) were quantied by the conventional
methodology: gallic acid analysis before and after methanolysis.
For this, gallic acid present in the extract is previously quantied
and them this value is deducted to the value quantied after acid
methanolysis. Acid methanolysis is the process of cleaving ester
linkages between monosaccharide and gallic acid units composing
GalT, one of the typical structures of hydrolysable tannins. The
results for OC extract, retentates and permeates from each
membrane are displayed in Fig. 6.
The contribution of GalT (estimated as gallic acid) as a parcel of
polyphenolic compounds in the OC extract, permeates and retentates is low (about 3% of the dried extract weigh). This could be
due to a natural low content on hydrolysable tannins in the extract
or due to ester bonds hydrolysis already in the extraction process.
Nevertheless, GalT quantication is a practical indication of the
membrane performance for this type of compounds. The membrane JW presents higher permeability to GalT than 90801 and
PLEAIDE as revealed by the lower content on JW retentate. This
result is in accordance with the differences already referred for
TPC, suggesting that GalT have signicant impact on the TPC
quantication.

P.C.R. Pinto et al. / Separation and Purication Technology 132 (2014) 234243

Finally, the quantication of TC allows concluding that the

membrane processing have only a slight impact on content of carbohydrates: the TC content of the retentates resulting from membranes JW and 90801 is slightly lower than for OC extract, being
more expressive for JW. This observation and the retention effect
on polyphenolic compounds, assessed as condensed (by Pac and
partially by fcT) and hydrolysable tannins, allow concluding that
these membranes could be applied in a concentration process
focused on polyphenolic compounds. Some advantage was noticed
for UF of the extract with JW due to the higher content of Pac in the
retentate than for the other membranes.

3.4. Detailed analysis of carbohydrates

GalT (% w/w)

241

Fig. 6. Estimative of gallic acid liberated from GalT in the OC extract, retentates and
permeates produced with membranes JW, PLEAIDE and 90801. Conditions:
concentration mode, 35 C, feed owrate 4.5 L h1, TMP 4 bar for UF JW and
PLEAIDE membranes and 14 bar for NF 90801 membrane; VRF 1.76.

Ara

JW

PLEAIDE

25

90801
molar fraction
of TC %

molar fraction
of TC %

20

The method used for assessing TC allows analyzing the

individual contributions of each monosaccharide since it involves
the cleavage of the oligo- and/or polysaccharides composing the
extract/retentate. The released monosaccharide units were identied and individually quantied. Taking into account the original
bark composition, it is possible to associate the monosaccharide

15
10
5
0

OC ret per ret per ret per

5
0

OC ret per ret per ret per

6
4
2
0

OC ret per ret per ret per

molar fraction
of TC %

30

Glc

20
10

OC ret per ret per ret per

10

25

20

6
4
2

15
10

OC ret per ret per ret per

GalA

10

5
0

OC ret per ret per ret per

12

molar fraction
of TC %

10

40

molar fraction
of TC %

molar fraction
of TC %
molar fraction
of TC %

Rha

PLEAIDE 90801

15

Gal

10

Xyl

JW

20

JW

PLEAIDE 90801

8
6
4
2
0

Man

OC ret per ret per ret per

JW

PLEAIDE 90801

Fig. 7. Monosaccharide composition (molar fraction) of the OC extract, retentates (ret) and permeates (per) produced with membranes JW, PLEAIDE and 90801. Conditions:
concentration mode, 35 C, feed owrate 4.5 L h1, TMP 4 bar for UF JW and PLEAIDE membranes and 14 bar for NF 90801 membrane; VRF 1.76.

P.C.R. Pinto et al. / Separation and Purication Technology 132 (2014) 234243

composition with the original oligo- and/or polysaccharides. The

aim of this analysis was to evaluate if there exists preferential
rejection for any carbohydrate type, as will be discussed below.
The molar fraction for each monosaccharide is presented in Fig. 7.
Glucose (Glc) and galactose (Gal) are the predominant moieties
in the OC extract accounting, together, to almost 50% of the TC. Glc
is certainly coming from an accessible and amorphous fraction of
cellulose and/or starch of the bark (starch was already found in
E. globulus wood [39]); other sources would be glucomanans
and the sugar moiety of some tannins. The linkage or association
of carbohydrates with polyphenols is also well known in both
hydrolysable [40] and condensed tannins [41] as well as in more
simple phenolics as those identied in E. globulus bark [3]. Gal
would arise from hemicelluloses branching [42] and/or from a
pectin fraction [43]. Arabinose (Ara) and galacturonic acid (GalA)
compose about 25% of the TC. As far as we know, there is no study
in the literature about pectins in E. globulus bark. However, the
composition of pectins in general [43] and in woody materials
[44] as well as data on pectins from bark of other species [45],
suggest that GalA, Gal, Ara and rhamnose (Rha) have arisen from
pectins, which were partially extracted with ethanol/water in the
conditions used in this work. Xylose (Xyl) and mannose (Man)
(810% of TC, each) are probably part of solubilized hemicellulose.
While Xyl is the main residue of xylans in wood [46], Man is usually associated with glucomanans, a minor hemicellulosic fraction.
Interestingly, the content of Man in the extract is higher than Xyl,
which is not in accordance with the relative percentages in the
wood [46] and bark [1].
Concerning the composition of the retentates and permeates:
the molar fractions of Xyl and Man are similar in OC, permeates
and retentates, indicative of a similar distribution within the three
carbohydrate fractions due to a non-selective permeation. However, the molar fractions of Ara, Rha, Man and Gal are lower in
the permeate, particularly for Ara and Rha with about 50% less than
in OC extract and respective retentates. On the opposite, the TC
fraction in permeate became enriched in Glc and particularly in
GalA. For permeate produced by membrane 90801, the GalA
content in the TC increased about 2-fold. The same trend of retention/permeation was observed for the three membranes. However,
NF membrane (90801) stands out by the lowest and highest relative rejection for Rha and GalA, respectively (Fig. 7). Considering
these observations, it is possible to conclude that the membrane
processing of OC extract leads to a modication of the relative
composition of carbohydrate fraction.
3.5. Cleaning
One additional factor taken into account in the membrane
selection is the evaluation of the initial permeability recovery
through cleaning cycles. The colloidal nature of polyphenolic compounds, as well the co-extracted oligomeric or polymeric polysaccharides contributes for the gel layer on membrane surface.
However, this phenomenon is usually reversible by cleaning with
the same solvent of the extract. More difcult to overcome is the
membrane fouling due to adsorption or internal pore plugging.
Usually, this phenomenon is the cause of internal fouling resistance [47] and a chemical treatment is necessary to restore the
membrane characteristics. Alkaline washing was required for all
membranes applied for the OC extract concentration and it was
applied as described in the experimental part. Fig. 8 summarizes
the inuence of cleaning cycles on permeability recovery.
The membrane JW recovered the initial permeability after the
rst cleaning cycle while PLEAIDE with two cleaning cycles
restored 80% of the initial permeability. For the cleaning of NF
membrane, NaOH solutions are not recommended; therefore, a
weaker base, NaHCO3, was applied. After the second cycle, this

100

Flux recovery (%)

242

80
60

JW

40

PLEAIDE
90801

20
0
0

Cleaning cycles
Fig. 8. The inuence of cleaning cycles on permeability recovery of membranes JW,
PLEAIDE and 90801.

membrane has restored 78% of permeability, achieving the initial

permeability with a third cycle (Fig. 8). Using buffered solutions
combined with a detergent would be a good alternative to improve
washing performance of 90801 membranes. The recover obtained
for the three membranes is within the acceptance limits to continue using the membranes, meaning that irreversible fouling
was not signicant. Nevertheless, new OC extract concentrations
must be performed to evaluate the rejection performance and
the productivity in successive experiments.

4. Conclusions
The aim of this work was to test different membranes for the
concentration process of polyphenolic compounds from an ethanolic extract of E. globulus bark. Characterization of the selected
membranes concerning permeability to water, ethanol 52% and
80% and rejection to standards was the rst step. When changing
water to ethanol 52%, the permeability decreased between 47%
(PLEAIDE) and 80% (EW); further increase of ethanol to 80% led
to an additional decrease of permeability for all membranes,
except for EW (UF) and 80105 (NF) membranes. These effects
are related to ethanol/water properties and solvent interaction
with the membranes. 90801 and GE membranes showed a favorable
rejection ratio tannic acid/disaccharide for ethanol 52%, indicative
that some selective enrichment in polyphenolic compounds
would be possible. Among the tested membranes, JW (30 kDa),
PLEAIDE (5 kDa) and 90801 were selected for concentration
process of the bark extract. The best ux performance during
the concentration was found for JW. The three membranes
promoted an enrichment of fcT (2030%) for a VRF of 1.76. JW
promoted the highest concentration of Pac and lower rejection
for TC, what is advantageous considering the purpose of the membrane processing. GalT, the fraction of hydrolysable tannins
detected in the extract, were preferentially rejected by 90801
and PLEAIDE but not by JW membrane. The detailed carbohydrate
analysis showed some selective permeation to glucose and galacturonic acid-containing oligo-/polysaccharides. Higher rejection of
rhamnose and arabinose could be related to the association
of these moieties with polyphenolic compounds. Flux recoveries
of 80100% were attained for all the membranes. However, JW
was the easiest membrane to clean.
Membrane process was successfully applied for concentration
of an ethanolic bark extract achieving, with a VRF of 1.76, an
enrichment of polyphenolic compounds of avonoid nature.
This process could be the primary step in a separation process
envisaging the purication of the Pac fraction of this extract for
high-added value applications.

P.C.R. Pinto et al. / Separation and Purication Technology 132 (2014) 234243

Acknowledgements
This work was carried out under the Project BIIPP No.
11551 Integrated Biorenery in Pulp and Paper Industry funded
by the European Regional Development Fund (ERDF) through the
Operational Programme for Competitiveness Factors (POFC) of
the National Strategic Reference Framework (NSRF). This work
was co-nanced by FCT and FEDER under Programme COMPETE
(Project PEst-C/EQB/LA0020/2013).
Eng. Maria Eduarda Baptista and Dr. Sergio Morales Torres (LA
LSRE/LCM) are acknowledged for support in some of the
permeability and pHPZC assays, respectively.

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