Separation and Purication Technology 147 (2015) 3243

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Separation and Purication Technology

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

Separation of protein mixtures by an integrated electro-ultraltration

electrodialysis process
Guoqiang Chen a,b, Weijie Song a, Benkun Qi a, Jing Li a, Raja Ghosh c, Yinhua Wan a,
a
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
b
University of Chinese Academy of Sciences, Beijing 100049, China
c
Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4L7, Canada

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

Article history: An integrated process for improving selectivity and permeate ux in ultraltration based protein
Received 24 February 2015 fractionation was developed by combining electrodialysis (ED) with electro-ultraltration (EUF). The per-
Received in revised form 2 April 2015 formance of such a process was rst investigated using individual proteins (bovine serum albumin and
Accepted 4 April 2015
lysozyme) and then with a mixture of the two. The experimental results showed that as with EUF on
Available online 10 April 2015
its own, the build-up of lysozyme concentration polarization near the cation exchange membrane in
the permeate compartment signicantly affected ion migration and led to change in pH and conductivity
Keywords:
of the feed solution during an EUFED process with lysozyme, or bovine serum albumin and lysozyme
Separation
Ultraltration
mixture. As compared with EUF, EUFED of protein mixture resulted in a 20% increase in permeate ux
Electro-ultraltration with the lysozyme transmission remaining the same. The demineralization that occurred during EUFED
Electrodialysis made this process suitable for protein separation from the feed solutions with high conductivity.
Protein 2015 Elsevier B.V. All rights reserved.

1. Introduction of the protein desired in the permeate stream in a given fractiona-

Downstream processing or bioseparation is an extremely
important component in the manufacturing process for proteins
used as food and pharmaceuticals. A protein bioseparation process
should be easy to scale up, give high selectivity, and must be
inexpensive. Chromatography, which is commonly used for protein
purication, gives high selectivity but is expensive to operate and
hard to scale-up. Membrane based techniques are scalable, give
high productivity, can be optimized to give high selectivity of sep-
aration and can be quite cost-effective [14].
Ultraltration, which relies on the sieving properties of porous
membranes, is widely used for concentration and fractionation of
biomacromolecules such as proteins [1,4]. Fouling and concentra-
tion polarization are major problems in ultraltration based sep-
aration processes. The concentration polarization layer adjacent
to a retaining membrane consists mainly of preferentially rejected
species such as high molecular weight proteins. In some cases low
molecular weight proteins may also be co-retained by larger ones,
particularly when these carry opposite charges [57]. Whatever
the mechanism of solute retention, concentration polarization
results in permeate ux decline and the reduction in transmission
Corresponding author. Tel./fax: +86 10 62650673.
E-mail address: yhwan@ipe.ac.cn (Y. Wan).
tion process.
Till date, a number of techniques have been developed to
alleviate the detrimental effects of concentration polarization and
membrane fouling. Electric eld enhanced ultraltration or
electro-ultraltration (EUF) is an effective method for reducing
concentration polarization and its effectiveness has been demon-
strated in protein concentration [8,9], protein fractionation [5],
juice clarication [10], and arsenic removal [11,12] processes. In
EUF processes where the objective is the fractionation of protein
mixture, the electric eld moves the larger proteins away from
membrane surface and thus makes it easier to transmit smaller
proteins through the membrane [5]. However, for this to be effec-
tive, the isoelectric points of the proteins being fractionated should
be quite different, in order to maintain signicant differences in
electric mobility of these proteins. Also, EUF cannot be used very
effectively if the proteins to be separated have like charge at the
operating condition. The choice of solution environment in an
EUF process is very important since the separation relies on elec-
trostatic charge which manifests itself best in a low ionic strength
solution. Not surprisingly, for the feed solutions having high ionic
strength, the efciency of EUF is signicantly reduced, since the
electric eld strength decreases when the current intensity
remains unchanged in this case [8]. The feed solutions with high
ionic strength have to be desalted before treatment by EUF. It is
inconvenient and low efcient. Electrodialysis (ED), which is a

http://dx.doi.org/10.1016/j.seppur.2015.04.003
1383-5866/ 2015 Elsevier B.V. All rights reserved.
 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243
Nomenclature
A electrode area (m2)
AEM anion exchange membrane
CEM cation exchange membrane
Efeed electric eld strength in the feed compartment (V/m)
ED electrodialysis
EUF electro-ultraltration
EUFED electro-ultraltrationelectrodialysis
h height of the feed compartment (m)
I current (A)
Jt permeate ux of the feed solution at the end of each run
(106 m/s)
J0 buffer permeate ux before the experiment (106 m/s)
J1 buffer permeate ux after the experiment (106 m/s)
Qt permeate volumetric ow rate of the feed solution at
the end of each run (m3/s)
Q0 permeate volumetric ow rates of buffer before the
experiment (m3/s)
technique that uses cation and anion exchange membranes placed
within an electric eld is widely used for salt removal [1318].
More recently, the use of ultraltration in conjunction with elec-
trodialysis has been examined for the separation of bioactive pep-
tides [19,20]. If ED is introduced to the EUF process, separation of
molecules and removal of salts, which are both driven by the elec-
tric eld, could be achieved at the same time. Thus, the feed solu-
tions with high ionic strength could be treated by this integrated
method directly and the desalination before the conventional
EUF is removed. The integrated method could also enhance the
separation of molecules in the feed solutions with low ionic
strength by providing higher electric eld strength, since the ionic
strength is further reduced in this process. Therefore, to integrate
ED with EUF could achieve better performance than conventional
EUF for the feed solutions with high or low ionic strength, extend-
ing the application of membrane process driven by electric eld.
Integration of multiple separation technique into one could
potentially improve both separation efciency and product
throughput [2123]. In the present study, the coupling of ED with
EUF for efcient protein fractionation was examined. Bovine serum
albumin (BSA) and lysozyme were used as model proteins and the
fractionation of binary mixtures of the two was performed by the
integrated electro-ultraltrationelectrodialysis (EUFED) process
based on the use of a 30 KDa ultraltration membrane.
The objectives of the present work were:
1. To evaluate the feasibility of using EUFED for protein
fractionation.
2. Assessment of ltration performances using single protein
(either BSA or lysozyme) and binary mixture of the two.
3. Study of the effects of pH and conductivity variations induced
by ion migration on permeate ux during the EUFED process.
2. Materials and methods
2.1. Materials
Bovine serum albumin (BSA, >98%, catalog # A-7030) was pur-
chased from Sigma Chemical, St Louis, MO, USA. Lysozyme (ultra
pure grade, catalog # 0663) was purchased from Amresco Inc.,
Solon, OH, USA. Properties of the two proteins used in this study
above are shown in Table 1. Carbonate buffer (20 mM ionic
strength, pH 10) was prepared by mixing 10 mM solutions of
Na2CO3 with NaHCO3 (both purchased from Beijing Chemical
33
Q1 permeate volumetric ow rates of buffer after the
experiment (m3/s)
Rc concentration polarization resistance (1012/m)
Rf fouling resistance (1012/m)
RfE electrodialysis resistance (1012/m)
Rfeed resistance of the feed compartment (X)
Rm membrane resistance (1012/m)
Rt total resistance (1012/m)
u cross-ow velocity (m/s)
Ufeed electric potential at the feed side (V)
UFM ultraltration membrane
Greek symbols
jfeed conductivity of the feed solution (S/m)
Works, China) to obtain the appropriate pH followed by the addi-
tion of NaCl (purchased from Guangdong Xilong Chemical Co.,
Ltd.) such that its effective concentration in the buffer was
10 mM. Phosphate buffer (50 mM ionic strength, pH 7.4) was pre-
pared by dissolving required quantities of Na2HPO4 with NaH2PO4
to obtain the appropriate pH and ionic strength. Protein solutions
were prepared by slowly adding pre-weighed quantities of protein
powder into buffer solution.
2.2. EUFED rig
The EUFED module was made of polycarbonate and was
fabricated in our laboratory. The arrangement of membranes and
electrodes within the module is shown in Fig. 1b. Both, a DF-120
cation exchange membrane and a DF-120 anion exchange mem-
brane (sourced from Shandong Tianwei Membrane Technology
Company, China) were used in our set-up. The properties of the
ion exchange membranes were listed in Table 2. Therefore it was
signicantly different from a conventional EUF unit (see Fig. 1a
for comparison) which would use two cation or two anion
exchange membranes. Also, unlike in a conventional electrodialy-
sis module, the content of the permeate compartment was not
re-circulated and the trans-membrane pressure was generated on
the feed side. The module consisted of four compartments, sepa-
rated by a cation exchange membrane on the side of cathode, a
30 KDa molecular weight cut-off polyethersolfone membrane
(Shanghai Institute of Applied Physics, China Academy of
Sciences, China) in the middle, and an anion exchange membrane
on the side of anode. The heights of the anodic, feed, permeate
and cathodic compartment were 9, 3, 3, and 3 mm. The effective
membrane area was 35 cm2. The membranes were supported by
polycarbonate plates which were drilled with uniformly dis-
tributed cylindrical pores. Both the anode and cathode consists of
ruthenium coated titanium electrodes (produced by Beijing
Hengli Titanium Industry Co., Ltd., China).
The EUFED rig is shown in Fig. 2. The electrolyte (50 mM
sodium phosphate buffer, pH 7.4) and the feed solution were
Table 1
Properties of proteins used in this study [5].
Property BSA Lysozyme
Molecular weight (kg/mol) 69 14.6
Isoelectric point 4.7 11.0
34 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243

Electrolyte Feed Permeate Electrolyte U feed

Efeed 1
+
H+ h
H
where Ufeed is the electric potential on the feed side and h is the
CO32- CO32- height of the feed compartment. However, due to the different con-
HPO42- ductivities in the four parts of EUFED module, Ufeed could not be
HPO42- obtained directly from the electric potential applied to the module.
HCO3- HCO3-
Anode Therefore, Ufeed was calculated according to Ohms law since the
Cathode
current of four compartments was identical:

H2PO4- h
H2PO4- U feed IRfeed I 2
jfeed A
OH-
Cl - Cl-
where I is the current, Rfeed is the resistance of the feed compart-
Na + Na+
ment, A is the electrode area which is equivalent to the membrane
area and jfeed is the conductivity of the feed solution.
CEM UFM CE M Thus Efeed was calculated as shown below [8,21]:
(a) I
Efeed 3
jfeed A
Electrolyte Feed Permeate Electrolyte In an EUFED process, jfeed decreases gradually with removal of
H+ H+ salts when the pH does not change a lot. As the pH changed signi-
cantly, the trend of jfeed decreasing with time could be different. In
CO32- CO32- either case, the decrease of jfeed leads to the increase of Efeed when
HPO42- I is kept constant.
HPO42- When convective movement towards the membrane surface
HCO3 -
HCO3- was equal to electrophoretic movement caused by electric eld,
Anode Cathode
no protein molecules accumulated on the membrane surface. In
-
H2PO4 this case, the electric eld strength reached its critical value which
-
OH- OH- H2PO4 was called critical electric eld strength.
The membrane resistance during EUFED was determined by
Cl- Cl - the resistance-in-series model following procedure discussed else-
Na +
Na +
Na + where [9].
The permeate ux at the end of each run (Jt) was calculated
using the resistance model as follows:
AEM UFM CEM
DP DP
(b) Jt 4
l Rt lRm Rf Rc
Fig. 1. Schematic diagram for separation: (a) conventional EUF process, and (b)
EUFED process (BSA: negatively charged, lysozyme: positively charged). where DP is the transmembrane pressure, l is the viscosity, Rt is the
total resistance, Rm is the membrane resistance, Rf is the fouling
pumped from their reservoirs and circulated using a BT100-300M resistance, Rc is the concentration polarization resistance.
peristaltic pump (Baoding Longer Precision Pump Co., Ltd., Thus Rc was calculated as follows:
China), and a ZT60-600a peristaltic pump (Baoding Longer
Precision Pump Co., Ltd., China), respectively. The cross-ow rate DP DP DP DP
Rc  Rm  Rf  
in the feed compartment was measured using a rotameter. The lJ t lJ t lJ0 lJ1
trans-membrane pressure was measured using a pressure gauge  
DP DP DP DPA 1 1 1
and was manually maintained constant using a ow control valve.     5
lQ t =A lQ 0 =A lQ 1 =A l Qt Q0 Q1
The electric eld was generated using a potentiostat (DYY-12,
Beijing Liuyi Instrument Factory, China). The two electrolyte where J0 and J1 are the buffer permeate ux before and after the
steams were mixed by tubes in order to reduce the pH change experiment, Qt is the volumetric permeate ow rate at the end of
induced by electrolysis occurring near the electrode. This needed each run, Q0 and Q1 are the volumetric permeate ow rates of buffer
to be done and was very important to keep the ion exchange before and after the experiment.
membrane stable [24]. To avoid the effect of pulsations from the It is important to note that the viscosity of water was used for
peristaltic pumps and to keep a stable pressure, buffer bottles were calculation of Rt, because the concentration of protein solution
added between the pumps and the EUFED module. used in this study was very low; and Qt and Q1 were measured
The electric eld strength in the feed compartment (Efeed) was under electric eld in EUF or EUFED process, while they are mea-
calculated as follows: sured without electric eld in UF process.

Table 2
Properties of ion exchange membranes used in this study.

Membrane Ion exchange capacity (meq/g) Thickness (mm) Water content (%) Area resistance (X cm2) Permselectivity (%)
DF-120 CEM 1.4 0.250.28 4070 4 0.95
DF-120 AEM 1.6 0.30.32 4070 4 0.97

CEM cation exchange membrane, AEM anion exchange membrane

DF-120 membranes exhibit good acid-alkali tolerance.
 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243 35

6 7 6

E
F
P 5
E
8
4

10
11 3

2 3
9 1

Fig. 2. The experimental set-up used in this study.

2.3. Experimental design and procedures Table 4

pH and conductivity of the feed solution at the end of EUF and EUFED experiments
(after 60 min of operation) for different feed solutions.
In single-protein EUFED experiments (i.e., with BSA or lyso-
zyme), the current was maintained constant at 150 mA and protein Operation Feed solution pH and conductivity of the feed solution
concentration used was 1.0 g/L. mode
Initial pH Initial conductivity (104 S/
In EUFED experiments carried out with protein mixture, two 10.0 m) 1840
operating parameters, i.e., the current and feed concentration were EUF Blank buffer 10.1 1853
tested. These are shown in Table 3. The carbonate buffer (with no BSA solution 10.0 1874
protein) was served as control in both EUFED and EUF experi- Lysozyme 11.6 2830
solution
ments in order to interpret effect of different protein characteris-
tics on the pH and conductivity of the feed solution. The results EUFED Blank buffer 5.7 473
BSA solution 6.9 637
are shown in Table 4.
Lysozyme 11.5 1496
In all the experiments, the pressure applied on the membrane solution
was 0.06 MPa and the cross-ow velocity used was 0.01 m/s.
When the electric eld was applied to the module, pH change
took place since the products of electrolysis (i.e., H+ and OH) could
migrate from one compartment to the other. This pH change could with protein solution pumped from the feed tank. After that, the
have a signicant effect on the permeate ux and lysozyme trans- electric eld and pressure were re-applied over the membrane.
mission. Some experiments were carried out with pH adjustment During each experiment, the pH and conductivity of the feed solu-
(using 1 M NaOH or 1 M HCl), and were operated with the carbon- tion were monitored and recorded as specic time interval. The
ate buffer. The results obtained from these experiments were com- experiments were performed using full recycle mode with the
pared with those carried without pH adjustment. Such adjustment retentate and permeate being returned to the feed tank. In order
by titration was carried out when the pH of the feed solution chan- to measure the permeate ux, permeate samples were collected
ged more than 0.3 units from initial pH value. in a 5 mL measuring cylinder, weighed, and returned to the feed
At the beginning of each run, the ultraltration module was tank. The current was kept constant throughout each experiment.
repressurized at 0.08 MPa using deionized water. This transmem- All experiments were carried out for 1 h with the operating tem-
brane pressure was higher than the pressure actually used in the perature being kept at 23 2 C.
EUFED experiments. After that, the two electrolyte compartments After each run, the water permeability was lower than the origi-
were lled with phosphate buffer which was recirculated using nal value and thus a cleaning procedure was carried out as follows:
pump. In the meanwhile, the feed compartment was emptied of (i) the feed compartment was lled with double distilled water and
deionized water, relled with the buffer and hence the permeate this was circulated for half an hour; (ii) the feed compartment was
ux was measured at 0.06 MPa. Since electroosmosis was taken then emptied and relled with detergent solution containing pro-
into account, the permeate ux was measured with an electric eld teolytic enzyme and this was circulated at 50 C for an hour; (iii)
of the same magnitude as that used in the EUFED experiments. the feed compartment was emptied of the detergent solution and
The feed compartment was then emptied of buffer and relled step (i) was repeated three times to ensure that no detergent
was left behind. If necessary, the membrane was soaked in 0.5%
NaOH solution overnight and washed with double distilled water
Table 3
prior to the next use. Before each run, the water permeability
Operating parameters tested in this study.
was measured.
Operating parameters pH was measured using a PHS-2F PH-meter (Shanghai Precision
Current 0 mA Scientic Instrument Co., Ltd). The conductivity of the protein solu-
50 mA tion was measured with a DDS-307A Conductivity Meter (Shanghai
100 mA Precision Scientic Instrument Co., Ltd). The concentration of
150 mA
Feed concentration (BSA to lysozyme ratio) 4.5 g/L:1.0 g/L
lysozyme in permeate was measured using a FPLC system
1.0 g/L:1.0 g/L (Amersham Pharmacia Biotech China Ltd., China) with a
1.0 g/L:0.1 g/L Superose 12 10/300 GL size-exclusion chromatography column
1.0 g/L:0.01 g/L (Amersham Pharmacia Biotech China Ltd., China). The column
36 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243
was operated at a ow rate of 400 lL/min at room temperature
(2025 C) and with 50 mM sodium phosphate buffer containing
150 mM sodium chloride (pH 7.0) as the mobile phase. 100 lL
sample was injected for the chromatographic analysis and concen-
tration of lysozyme or BSA was determined by comparison with a
calibration standard.
3. Results and discussion
3.1. The EUFED process of BSA solution
The evolution of pH, conductivity and Efeed of BSA solution as a
function of time is plotted in Fig. 3ac. It can be seen that the pH
and conductivity of the feed solution decreased with time during
EUFED, while there was nearly no pH and conductivity change
in the EUF process. It could be explained as follows: In EUF experi-
ments of BSA solution, the number of anions in the feed solution
kept unchanged in full recycle mode, since the anions (i.e., OH
ions) migrated from the permeate compartment towards the
anode and were blocked by the cation exchange membrane.
Thus, the pH and conductivity of the feed solution was kept con-
stant. The same result was found in EUF experiments with blank
buffer (Table 4). In EUFED experiments of BSA solution, the anions
in the feed compartment were gradually decreasing and the buffer
capacity of the feed solution was reduced due to removal of the
CO2 
3 ions and HCO3 ions over the duration of the process, because
the anions could migrate from the permeate compartment through
the anion exchange membrane towards the anodic compartment.
As a result, the pH and conductivity of the feed solution decreased
with time. In this case, the ending pH was higher than that in EUF
ED experiments with blank buffer. A possible explanation was as
follows. When the electric eld was applied, more BSA could move
towards the anion exchange membrane leading to the buildup of a
concentration polarization layer. This layer lowered the migration
rate of anions. To keep electro-neutrality, the cations had to pro-
duce from electrolysis and ions leakage at the anion exchange
membrane. Cations leakage was also found by Aider et al. [25] on
the study of chitosan oligomers electroseparation and Labbe
et al. [26] on the study of catechin electromigration. Finally, more
anions such as the OH ions stayed in the feed compartment
resulting in a higher pH. Meanwhile, the demineralization rate
decreased slightly resulting in higher conductivity of the feed solu-
tion than that in EUFED experiments with blank buffer. The dem-
ineralization rate of the feed solution with pH adjustment was
different from that without pH adjustment in EUFED process.
When pH was adjusted by 1 M NaOH in the process to maintain
unchanged, the OH ions neutralized with the H+ ions and the
Na+ ions carried part of current instead of the H+ ions. The differ-
ence of migration rate and conductivity between the Na+ ions
and H+ ions leading to the conductivity distance of the feed solu-
tion between the experiments with and without pH adjustment.
As shown in Fig. 3c, Efeed increased from about 400 V/m to
1150 V/m (EUFED without pH adjustment) and to 700 V/m
(EUFED with pH adjustment), indicating that higher electric eld
strength during EUFED without pH adjustment was obtained due
to the lower conductivity.
The evolution of permeate ux of BSA solution as a function of
time is plotted in Fig. 3d. As shown in Fig. 3d, the steady-state val-
ues in terms of permeate ux could be reached in EUF and conven-
tional UF process. However, the permeate ux during EUFED
improved with time because the electric eld strength was gradu-
ally increasing in this process and hard to build up a steady-state.
Besides the electric eld strength, the charge of BSA determined
the permeate ux. Although the lower negative charge of BSA
was found during EUFED without pH adjustment, higher electric
eld played an important role in improvement of permeate ux
and a higher value was obtained. Whereas, when the charge of
BSA was less enough at the end of the process, it played a more
crucial role in permeate ux than electric eld. As a result, the
permeate ux during EUFED without pH adjustment was caught
up with that during EUFED with pH adjustment in which BSA
was more negatively charged, although a lower electric eld was
applied. It was expected that the permeate ux during EUFED
without pH adjustment would be exceeded by that with pH adjust-
ment if longer operating time was run. And the permeate ux dur-
ing EUFED with pH adjustment could increase with time only if
the electric eld strength was lower than its critical value.
It was observed that during EUFED of BSA solution, the
conductivity and pH which could affect the proteinprotein and
proteinmembrane interactions decreased with time compared
to that during EUF and conventional UF. Two solutions
(CBSA = 1.0 g/L) prepared by NaCl were used to describe effect of
reduction of pH and conductivity on the ltration behavior
(Fig. 4), since the ion composition of the feed solution at the end
of experiment was different from the initial one due to ions migra-
tion. They were 0 mA EUFED NaCl-1 and 0 mA EUFED NaCl-2
with the same conductivity and pH of initial and nal buffer during
150 mA EUFED of BSA solution, respectively. And in this case, no
electric eld was applied. It was found that the permeate ux of
0 mA EUFED NaCl-2 solution was slower than that of 0 mA
EUFED NaCl-1 solution, indicating that the lower pH of 0 mA
EUFED NaCl-2 solution decreased the negative charge of BSA
resulting in reduction of proteinprotein and proteinmembrane
repulsions (the polyethersolfone membrane used in this study
was negatively charged) and as a consequence BSA accumulated
easily on the membrane surface. Reduction of pH played a crucial
role in permeate ux decline although decreasing of conductivity
discharged more charges which enhanced the repulsions of pro-
teinprotein and proteinmembrane. It can be concluded that
when the electric eld was applied, increase of the electric eld
strength induced by decreasing of conductivity was enough to
compensate for the negative effect of reduction of pH on permeate
ux over the duration of the process. However, the pH adjustment
was necessary to keep a high permeate ux in a longer operation
time during EUFED.
Rcs in EUFED and EUF experiments are shown in Table 5. As
shown in Table 5, the higher electric eld strength the rig was
applied with, the lower Rc the process had. This result was in accor-
dance with that of Sarkar et al. [27] working on EUF of synthetic
fruit juice. The 0 mA EUFED NaCl-2 solution had nearly the same
Rc as the 0 mA EUFED NaCl-1 solution indicating that decreasing
of pH and conductivity induced by EUFED almost did not affect
the concentration polarization layer, although in this case BSA
was easy to foul the membrane surface leading to decreasing of
permeate ux. BSA could not pass through the membrane and
the BSA transmission was 0%.
3.2. The EUFED process of lysozyme solution
In contrast with BSA, when the electric eld was applied, lyso-
zyme carrying the positive charge in this study moved toward the
membrane. The evolution of pH, conductivity and Efeed of lysozyme
solution as a function of time is plotted in Fig. 5ac. The pH of the
feed solution increased from 10 to 11.5 during EUFED and to 11.6
during EUF. Meanwhile, the conductivity of the feed solution
decreased slightly during EUFED and increased during EUF. It
can be seen that the variation trends of pH and conductivity were
different from those in EUF and EUFED process of BSA solution or
blank buffer. It could be explained as follows: In EUF experiments
of lysozyme solution, like BSA during EUF, lysozyme in the perme-
ate compartment could also build up a concentration polarization
 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243 37

(a) (c)

(b) (d)
Fig. 3. Variations of parameters in the feed solution with time during EUF and EUFED of BSA solution: (a) pH, (b) conductivity, (c) Efeed and (d) permeate ux. CBSA = 1.0 g/L in
10 mM carbonate buffer (containing 10 mM NaCl), u = 0.01 m/s, P = 0.06 MPa.

Table 5
Concentration polarization resistance (Rc) at the end of EUF and EUFED experiments
of BSA solution.

Operation conditions Rc (1012/m)

0 mA EUF 3.49
150 mA EUF 2.06
150 mA EUFED 0.29
150 mA EUFED pH constant 0.68
0 mA EUFED NaCl-1 3.34
0 mA EUFED NaCl-2 3.6

NaCl-1 and NaCl-2: two solutions prepared by NaCl with the same conductivity and
pH of initial and nal buffer in EUFED or EUF experiments without pH adjustment,
respectively.

at the cation exchange membrane for maintenance of electro-neu-

Fig. 4. Variation of permeate ux with time during UF of BSA solution in different
NaCl solutions. CBSA = 1.0 g/L in NaCl solutions (NaCl-1 and NaCl-2 had the same
conductivity and pH of initial and nal buffer during 150 mA EUFED of BSA
solution, respectively), u = 0.01 m/s, P = 0.06 MPa.
layer near the cation exchange membrane. The migration rate of
cations from the permeate compartment towards the cathodic
compartment decreased resulting in electrolysis and ions leakage
trality. Poulin et al. [20] also observed the same anions leakage on
the study of bioseparation bioactive peptides. As a consequence,
the increment of OH ions led to increase of the pH and conductiv-
ity. In EUFED experiments of lysozyme solution, part of the OH
ions was able to migrate through the anion exchange membrane
towards the anodic compartment resulting in less increase of pH
of the feed solution, compared to that during EUF. In this case,
the lower migration rate of cations decreased the demineralization
38 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243

(a) (c)

(b) (d)
Fig. 5. Variations of parameters in the feed solution with time during EUF and EUFED of lysozyme solution: (a) pH, (b) conductivity, (c) Efeed and (d) permeate ux.
Clysozyme = 1.0 g/L in 10 mM carbonate buffer (containing 10 mM NaCl), u = 0.01 m/s, P = 0.06 MPa.

rate of the feed solution. As a consequence, the conductivity of the
feed solution decreased more slowly than that in EUFED process
of blank buffer. In EUF and EUFED experiments, if pH was
adjusted with 1 M HCl, the H+ ions neutralized with the OH ions
and Cl ions carried part of the current instead of the OH ions.
The difference of migration rate and conductivity between the
Cl and OH ions leading to the conductivity distance of the feed
solution between the experiments with and without pH adjust-
ment. As shown in Fig. 5c, Efeed increased approximately from
400 V/m to 490 V/m (without pH adjustment during EUFED)
and to 550 V/m (with pH adjustment during EUFED). However,
Efeed decreased from 400 V/m to 260 V/m (without pH adjustment
during EUF) and to 285 V/m (with pH adjustment during EUF).
The evolution of permeate ux of lysozyme solution as a func-
tion of time is plotted in Fig. 5d. As shown in Fig. 5d, the permeate
ux during EUF and EUFED without electric eld declined with
time and reached a steady-state value. The permeate ux during
EUF without electric eld was higher than that during EUFED
without electric eld. The possible explanation was as follows.
During EUF without electric eld, the electrostatic attraction
occurred between lysozyme and the cation exchange membrane
because of the opposite charges they have. Therefore, the concen-
tration polarization near the ultraltration membrane surface was
slightly reduced and the permeate ux was higher. It was impor-
tant to note that nearly no difference was found during EUF and
EUFED without electric eld of BSA solution because of the weak
interaction between BSA (negatively charged) and the anion
exchange membrane (positively charged). During EUF and EUF
ED without pH adjustment, the permeate uxes were higher than
those during EUF and EUFED with pH adjustment. In these two
cases without pH adjustment, pH increased from 10 (lysozyme car-
rying positive charge) to higher than 11 (lysozyme carrying slightly
negative charge) and hence the movement of lysozyme towards
the membrane by the electric eld was gradually diminishing
(even away from the membrane surface at the end of process).
At the same time, the electrostatic interaction of lysozyme and
the membrane surface changed from attraction to repulsion. As a
result, the permeate uxes were higher due to reduction of concen-
tration polarization near the membrane surface. The permeate ux
was higher during EUFED with pH adjustment than that during
EUF with pH adjustment because of higher electric eld applied
through the membrane. However, the permeate ux during EUF
ED without pH adjustment was caught up with that during EUF
without pH adjustment in the end with the reason that the high
ionic strength caused lysozyme to transmit the membrane easily.
Whereas, the lysozyme transmission (Table 6) during EUF without
 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243 39

Table 6 transmission than those during EUF and EUFED without pH

Concentration polarization resistance (Rc) and lysozyme transmission at the end of adjustment when the electric eld was applied as a driving force.
EUF and EUFED experiments of lysozyme solution.
In the experiments of lysozyme with NaCl solutions, the permeate
Operation conditions Rc (1012/m) Lysozyme transmission (%) ux of 0 mA EUF NaCl-2 was highest, followed by EUFED NaCl-2,
0 mA EUF 7.25 36.5 EUF NaCl-1 and EUFED NaCl-1 in order. That is because lysozyme
0 mA EUFED 9.93 25.4 could easily pass through membrane in 0 mA EUF NaCl-2 and
150 mA EUF 0.86 62.9 EUFED NaCl-2 (pH of both solutions was near the pI of lysozyme)
150 mA EUFED 1.12 68.1
150 mA EUF pH constant 2.72 72.9
and the high conductivity of 0 mA EUF NaCl-2 further improved
150 mA EUFED pH constant 1.28 75.9 the lysozyme transmission. The experiments of lysozyme with
0 mA EUF NaCl-1 7.73 26.0 NaCl solutions illustrated only the reduction of conductivity
0 mA EUF NaCl-2 2.18 81.2 induced by EUFED process was adverse for improvement of
0 mA EUFED NaCl-1 9.25 25.7
permeate ux and lysozyme transmission, but the EUFED process
0 mA EUFED NaCl-2 16.6 36.4
could have a high permeate ux and lysozyme transmission due to
electric eld applied.

pH adjustment was slightly lower. The possible explanation was

that lysozyme concentration near the membrane surface was
lower due to lower electric eld strength.
The NaCl solutions (the 0 mA EUF NaCl-1 and 0 mA EUFED
NaCl-1 with the same conductivity and pH of initial buffer, the
0 mA EUF NaCl-2 and 0 mA EUFED NaCl-2 with that of nal buffer
during 150 mA EUF or EUFED of lysozyme solution;
Clysozyme = 1.0 g/L) were also prepared to describe the effect of
reduction of pH and conductivity on the ltration behavior
(Fig. 6). It was seen that the permeate ux of 0 mA EUF NaCl-1
was slightly higher than that of 0 mA EUFED NaCl-1 solution, just
as the variation trend with buffer (Fig. 5d). Moreover, the permeate
ux of 0 mA EUF NaCl-2 was higher than that of 0 mA EUF NaCl-1,
since the higher conductivity of 0 mA EUF NaCl-2 shielded part of
charge, compressed the electric double layer, reduced the ltration
resistance and made lysozyme easier to pass through membrane.
In contrast, the permeate ux of EUFED NaCl-2 was lower than
that of EUFED NaCl-1 due to low conductivity.
Rcs and lysozyme transmission in EUFED and EUF experiments
are shown in Table 6. As shown in Table 6 and Fig. 5d, the experi-
ments with higher permeate uxes had lower Rcs. The lysozyme
transmission during EUF without electric eld was slightly higher
than that during EUFED without electric eld. The possible reason
was that the reduction of concentration polarization (as described
above) caused lysozyme easily to migrate through the membrane.
However, it was seen that high concentration polarization layers
near the membrane surface during EUF and EUFED with pH
adjustment (as described above) led to higher lysozyme
Fig. 6. Variation of permeate ux with time during UF of lysozyme solution in
different NaCl solutions. Clysozyme = 1.0 g/L in NaCl solutions (NaCl-1 and NaCl-2 had
the same conductivity and pH of initial and nal buffer during 150 mA EUFED or
EUF of lysozyme solution, respectively), u = 0.01 m/s, P = 0.06 MPa.
3.3. Separation of binary mixtures (BSA and lysozyme) using EUFED
The evolution of pH of protein mixture solution as a function of
time is plotted in Fig. 7a. Like pH change of lysozyme solution
under electric eld, it increased from 9.8 (pH of solution changed
from 10 to 9.8 when BSA and lysozyme were added and it was
not adjusted to keep conductivity constant) to 10.7 during
EUFED and to 11.3 during EUF with the current of 150 mA due
to the concentration polarization layer of lysozyme near the cation
exchange membrane. It was important to note that the concentra-
tion polarization layer of BSA near the anion exchange membrane
(not as compact as that of lysozyme near the cation exchange
membrane because of shear force caused by tangential ow in
the feed solution) during EUFED resulted in lower migration rate
of the anions than that during EUF. Thus, fewer surplus cations
stayed in the permeate compartment inducing formation of a small
amount of the OH ions which caused pH of the protein mixture
solution to increase less. It was found that pH increased during
EUFED and EUF when the current of 100 mA and 50 mA was
applied, and the increment of pH was proportional to the electric
eld strength.
The evolution of conductivity and Efeed of protein mixture solu-
tion as a function of time is plotted in Figs. 7b and c and 8a and b.
As shown in Fig. 7b, the variation trends of conductivity of protein
mixture solution were similar to that of lysozyme solution during
EUF and EUFED. And the higher electric eld strength led to
greater change of the conductivity. As shown in Fig. 8a, if pH was
adjusted, the conductivity of protein mixture solution changed a
little during EUF and EUFED (as described in Section 3.2). In these
experiments above, Efeed is shown in Figs. 7c and 8b. The evolution
of permeate ux of protein mixture solution as a function of time is
plotted in Figs. 7d and 8c. As shown in Fig. 7d, under different elec-
tric eld strengths, the permeate uxes during EUFED without pH
adjustment were higher than those during EUF without pH adjust-
ment because the higher electric eld was applied to this EUFED
process. In carbonate buffer with pH 10, BSA and lysozyme were
negatively and positively charged, respectively. Therefore, the elec-
trostatic attraction between BSA and lysozyme occurred resulting
in formation of BSAlysozyme complex which had a larger size
than any protein (BSA or lysozyme) and carried negative charge
[5]. In this case, the electric eld moved BSA and BSAlysozyme
complex away from the membrane surface enhancing the perme-
ate ux and lysozyme transmission. Since pH in EUF and EUFED
experiments with the current of 50 mA almost unchanged, the
adjustment experiments by 1 M HCl were not carried out. During
EUF and EUFED experiments with the current of 100 mA and
150 mA, the pH adjustment experiments were carried out for com-
parison. However, the permeate ux was not necessarily high
when pH was adjusted. If pH was not adjusted, it increased and
hence the negative charge of BSA and BSAlysozyme complex
was more. Meanwhile, the positive charge of lysozyme was less
40 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243

(a) (c)

(b) (d)
Fig. 7. Variations of parameters in the feed solution with time during EUF and EUFED of protein mixture (BSA and lysozyme): (a) pH, (b) conductivity, (c) Efeed and (d)
permeate ux. CBSA = 1.0 g/L and Clysozyme = 1.0 g/L in 10 mM carbonate buffer (containing 10 mM NaCl), u = 0.01 m/s, P = 0.06 MPa.

and attraction of BSA and lysozyme was weaker. When the electric
eld was applied, the driving force moving BSA and BSAlysozyme
complex away from the membrane surface was stronger, although
the driving force moving lysozyme towards the membrane surface
was weaker. As shown in Fig. 8c, the permeate uxes were slightly
higher during EUFED and EUF with pH adjustment than those
during EUFED and EUF without pH adjustment when the current
of 150 mA was applied. Whereas, when the current of 100 mA was
applied, situation was just the opposite. It indicated that the high
electric eld strength beneted improvement of permeate ux
when the pH was keeping constant in the process, at which the
attraction of BSA and lysozyme was relatively strong. Solution
environment which could reduce the interaction of BSA and lyso-
zyme played a crucial role in improvement of permeate ux in
the presence of lower electric eld strength.
The NaCl solutions (the 0 mA EUF NaCl-1 and 0 mA EUFED
NaCl-1 with the same conductivity and pH of initial buffer; the
0 mA EUF NaCl-2 and 0 mA EUFED NaCl-2 with that of nal buf-
fer) were also prepared to describe the effect of reduction of pH
and conductivity on the ltration behavior (Fig. 9). It was seen that
the permeate ux of 0 mA EUFED NaCl-2 was nearly the same as
the 0 mA EUFED NaCl-1 illustrating that decreasing of conductiv-
ity and pH change during EUFED did not affect the permeate ux.
And the permeate ux of 0 mA EUF NaCl-2 was signicantly higher
than that of 0 mA EUF NaCl-1. It indicated that increasing of the
conductivity during EUF (shielding part of the charge of BSA and
lysozyme) and the pH change from 9.8 to 11.3 (close to the isoelec-
tric point of lysozyme) resulted in reduction of interaction between
BSA and lysozyme.
Rcs in EUFED and EUF experiments are shown in Table 7. As
shown in Table 7, Rc decreased with increase of the current during
EUF and EUFED without pH adjustment. Meanwhile, the lyso-
zyme transmission increased. And in each experiment of mixed
protein during EUFED (with and without pH adjustment), Rc
was lower than that during EUF with the same operating situation,
illustrating that EUFED was a more effective method for reduction
of concentration polarization. At the same time, the lysozyme
transmission was slightly higher during EUFED except that with
the current of 50 mA due to the low electric eld strength. When
pH was adjusted, except the EUF experiment with the current of
100 mA in which the electric eld strength was low, the other
experiments (EUF or EUFED) had nearly the same Rc and lyso-
zyme transmission between these with and without pH adjust-
ment under the same current. It illustrated that pH change had
no signicant effect on Rc. The experiment with the 0 mA EUF
NaCl-2 solution had a lower Rc than that with the 0 mA EUF
 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243 41

(a) Fig. 9. Variation of permeate ux with time during UF of protein mixture in

different NaCl solutions. CBSA = 1.0 g/L and Clysozyme = 1.0 g/L in NaCl solutions
(NaCl-1 and NaCl-2 had the same conductivity and pH of initial and nal buffer
during 150 mA EUFED or EUF of protein mixture, respectively), u = 0.01 m/s,
P = 0.06 MPa.

Table 7
Concentration polarization resistance (Rc) and lysozyme transmission at the end of
EUF and EUFED experiments of protein mixture.

Operation conditions Rc (1012/m) Lysozyme transmission (%)

0 mA EUF 44.21 15.6
50 mA EUF 11.14 57.8
100 mA EUF 5.76 65.6
150 mA EUF 4.4 69.8
100 mA EUF pH constant 6.89 64.8
150 mA EUF pH constant 4.28 68.8
50 mA EUFED 8.23 53.2
100 mA EUFED 5.13 71.9
150 mA EUFED 3.06 72.7
100 mA EUFED pH constant 4.74 66.7
150 mA EUFED pH constant 2.51 72.9
(b) 0 mA EUF NaCl-1 47.08 6.3
0 mA EUF NaCl-2 7.88 70.1
0 mA EUFED NaCl-1 45.72 6.5
0 mA EUFED NaCl-2 42.28 7.2

that with the 0 mA EUFED NaCl-1 solution. Although the reduc-

(c)
Fig. 8. Variations of parameters in the feed solution with time during EUF and EUF
ED of protein mixture (BSA and lysozyme) with and without pH adjustment: (a)
conductivity, (b) Efeed and (c) permeate ux. CBSA = 1.0 g/L and Clysozyme = 1.0 g/L in
10 mM carbonate buffer (containing 10 mM NaCl), u = 0.01 m/s, P = 0.06 MPa.
NaCl-1 solution indicating that increasing of pH and conductivity
induced by EUF beneted improvement of the permeate ux and
lysozyme transmission. Whereas, in the experiment with the
0 mA EUFED NaCl-2 solution, Rc decreased slightly compared to
tion of conductivity itself did not have a remarkable effect on Rc,
the higher electric eld strength caused by reduction of conductiv-
ity during EUFED could further improve the permeate ux and
protein transmission.
Since the mixed protein solution containing 1.0 g/L BSA and
1.0 g/L lysozyme had the same variation trend of pH and conduc-
tivity as 1.0 g/L lysozyme solution in EUF and EUFED process
due to buildup of the concentration polarization layer near the
cation exchange membrane of the cathodic side, the concentration
ratio of lysozyme in mixed protein solution could affect the evolu-
tion of pH and conductivity in EUF and EUFED process of the
mixed protein solution. So the EUF and EUFED experiments at dif-
ferent concentration ratio of BSA and lysozyme were performed
and the results are shown in Fig. 10. As shown in Fig. 10ac, the
evolution of pH, conductivity and Efeed of protein mixture solution
during EUF and EUFED was nearly the same at different concen-
tration ratio of BSA and lysozyme except the concentration ratio
of BSA 1.0 g/L: lysozyme 0.01 g/L. When the concentration ratio
of BSA and lysozyme was 1.0 g/L:0.01 g/L, the variation trends of
conductivity and Efeed were similar with that in EUF and EUFED
process of blank buffer. However, the signicant pH change did
not occur during EUFED. It may be explained by the existence
of few lysozyme in the feed solution. Moreover, a small amount
42 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243

(a) (c)

(b) (d)
Fig. 10. Variations of parameters in the feed solution with time during EUF and EUFED of protein mixture (BSA and lysozyme) solutions at different protein concentration
ratios: (a) pH, (b) conductivity, (c) Efeed and (d) permeate ux. BSA to lysozyme ratios (4.5 g/L:1.0 g/L, 1.0 g/L:1.0 g/L, 1.0 g/L:0.1 g/L, 1.0 g/L:0.01 g/L) in 10 mM carbonate
buffer (containing 10 mM NaCl), u = 0.01 m/s, P = 0.06 MPa.

of lysozyme (more than 0.01 g/L in the feed solution) could affect
the ion migration signicantly during EUF or EUFED, and increas-
ing the amount of lysozyme did not seem to decrease the extent of
ion migration further in this study. As shown in Fig. 10d, in EUFED
experiment with the concentration ratio of BSA 1.0 g/L:lysozyme
0.01 g/L, the permeate ux was highest since the effect of lysozyme
on the ion migration was weak and the protein concentration near
the membrane surface was low. The permeate ux decreased with
the increase of protein concentration during EUFED or EUF con-
sidering that the effect of lysozyme on the ion migration had noth-
ing to do with its concentration when more than 0.01 g/L lysozyme
was in the feed solution (as shown in Fig. 10ac) and the electric
eld strength was nearly the same in EUF or EUFED of protein
mixture at different concentration ratio.
4. Conclusion
EUFED, one stage into which EUF and ED are combined, was
explored for the fractionation of protein mixture of BSA and lyso-
zyme. Compared to EUF, EUFED could improve 20% of the perme-
ate ux and the lysozyme transmission kept the same level due to
a higher electric eld strength caused by demineralization.
Generally speaking, the important conditions to obtain a signi-
cant effect of electric eld were low conductivity of the feed solu-
tion and high charge of higher molecular weight protein for
separation of protein mixture. Therefore, EUFED which was able
to reduce the conductivity of the feed solution by demineralization
had an advantage in treating feed with higher conductivity com-
pared to EUF. Nevertheless, this study was performed on a syn-
thetic solution. For a real solution with more proteins having
different charges and sizes, protein separation by the proposed
method would be still challenging and more work will be done
in the future.
It seemed that only a small amount of lysozyme in the feed
solution could form a concentration polarization layer near the
cation exchange membrane under electric eld. The change of pH
and conductivity subsequently occurred and hence the effect of
electric eld was reduced. Therefore, the rig used in this study
has to be improved for enhancement of the electric eld effect.
It was important to choose an appropriate buffer with the right
pH, ionic strength and buffer capacity during EUF or EUFED.
Keeping pH constant was necessary in a long run, although pH
change hardly affected the permeate ux and lysozyme transmis-
sion in the process of this study (a short duration was explored).
 G. Chen et al. / Separation and Purication Technology 147 (2015) 3243
Acknowledgement
The authors would like to thank the National Natural Science
Foundation of China for the nancial support (Grant No.
20576134).
References
[1] R. van Reis, A. Zydney, Bioprocess membrane technology, J. Membr. Sci. 297
(2007) 1650.
[2] B. Cheang, A.L. Zydney, A two-stage ultraltration process for fractionation of
whey protein isolate, J. Membr. Sci. 231 (2004) 159167.
[3] Y. Wan, R. Ghosh, Z. Cui, High-resolution plasma protein fractionation using
ultraltration, Desalination 144 (2002) 301306.
[4] A. Saxena, B.P. Tripathi, M. Kumar, V.K. Shahi, Membrane-based techniques for
the separation and purication of proteins: an overview, Adv. Colloid Interface
Sci. 145 (2009) 122.
[5] B. Sarkar, S. DasGupta, S. De, Electric eld enhanced fractionation of protein
mixture using ultraltration, J. Membr. Sci. 341 (2009) 1120.
[6] E. Iritani, Y. Mukai, T. Murase, Separation of binary protein mixtures by
ultraltration, Filtr. Sep. 34 (1997) 967973.
[7] S. Saksena, A.L. Zydney, Inuence of proteinprotein interactions on bulk mass
transport during ultraltration, J. Membr. Sci. 125 (1997) 93108.
[8] A.D. Enevoldsen, E.B. Hansen, G. Jonsson, Electro-ultraltration of industrial
enzyme solutions, J. Membr. Sci. 299 (2007) 2837.
[9] W. Song, Y. Su, X. Chen, L. Ding, Y. Wan, Rapid concentration of protein solution
by a crossow electro-ultraltration process, Sep. Purif. Technol. 73 (2010)
310318.
[10] B. Sarkar, S. DasGupta, S. De, Flux decline during electric eld-assisted cross-
ow ultraltration of mosambi (Citrus sinensis (L.) Osbeck) juice, J. Membr. Sci.
331 (2009) 7583.
[11] L.H.C. Hsieh, Y.H. Weng, C.P. Huang, K.C. Li, Removal of arsenic from
groundwater by electro-ultraltration, Desalination 234 (2008) 402408.
[12] Y.H. Weng, L.H. Chaung-Hsieh, H.H. Lee, K.C. Li, C.P. Huang, Removal of arsenic
and humic substances (HSs) by electro-ultraltration (EUF), J. Hazard. Mater.
122 (2005) 171176.
[13] K. Ghyselbrecht, M. Huygebaert, B. Van der Bruggen, R. Ballet, B. Meesschaert,
L. Pinoy, Desalination of an industrial saline water with conventional and
bipolar membrane electrodialysis, Desalination 318 (2013) 918.
43
[14] M.A. Masigol, A. Moheb, A. Mehrabani-Zeinabad, An experimental
investigation into batch electrodialysis process for removal of sodium sulfate
from magnesium stearate aqueous slurry, Desalination 300 (2012) 1218.
[15] T. Sirivedhin, J. McCue, L. Dallbauman, Reclaiming produced water for
benecial use: salt removal by electrodialysis, J. Membr. Sci. 243 (2004)
335343.
[16] H.M.N. AlMadani, Water desalination by solar powered electrodialysis process,
Renew. Energy 28 (2003) 19151924.
[17] K. Kneifel, G. Lhrs, H. Wagner, Nitrate removal by electrodialysis for brewing
water, Desalination 68 (1988) 203209.
[18] J. Shen, J. Duan, Y. Liu, Y. Lixin, X. Xing, Demineralization of glutamine
fermentation broth by electrodialysis, Desalination 172 (2005) 129135.
[19] L. Firdaous, P. Dhulster, J. Amiot, A. Gaudreau, D. Lecouturier, R. Kapel, F. Lutin,
L.-P. Vzina, L. Bazinet, Concentration and selective separation of bioactive
peptides from an alfalfa white protein hydrolysate by electrodialysis with
ultraltration membranes, J. Membr. Sci. 329 (2009) 6067.
[20] J.-F. Poulin, J. Amiot, L. Bazinet, Simultaneous separation of acid and basic
bioactive peptides by electrodialysis with ultraltration membrane, J.
Biotechnol. 123 (2006) 314328.
[21] T. Kppler, C. Posten, Fractionation of proteins with two-sided electro-
ultraltration, J. Biotechnol. 128 (2007) 895907.
[22] R. Ghosh, Separation of human albumin and IgG by a membrane-based
integrated bioseparation technique involving simultaneous precipitation,
microltration and membrane adsorption, J. Membr. Sci. 237 (2004) 109117.
[23] G. Bargeman, J. Houwing, I. Recio, G.H. Koops, C.v.d. Horst, Electro-membrane
ltration for the selective isolation of bioactive peptides from an alpha(s2)-
casein hydrolysate, Biotechnol. Bioeng. 80 (2002) 599609.
[24] A.L. Ahmad, N.A.H. Hairul, Proteinmembrane interactions in forced-ow
electrophoresis of protein solutions: effect of initial pH and initial ionic
strength, Sep. Purif. Technol. 66 (2009) 273278.
[25] M. Aider, S. Brunet, L. Bazinet, Electroseparation of chitosan oligomers by
electrodialysis with ultraltration membrane (EDUF) and impact on
electrodialytic parameters, J. Membr. Sci. 309 (2008) 222232.
[26] D. Labb, M. Araya-Farias, A. Tremblay, L. Bazinet, Electromigration feasibility
of green tea catechins, J. Membr. Sci. 254 (2005) 101109.
[27] B. Sarkar, S. Pal, T.B. Ghosh, S. De, S. DasGupta, A study of electric eld
enhanced ultraltration of synthetic fruit juice and optical quantication of gel
deposition, J. Membr. Sci. 311 (2008) 112120.