Accepted Manuscript

Functionalized chitosan based nano-filter membranes for pH-controlled sepa‐

ration of amino acids

Tina Chakrabarty, Amit K. Thakur, Vinod K. Shahi

PII: S1383-5866(13)00076-2

DOI: http://dx.doi.org/10.1016/j.seppur.2013.01.054

Reference: SEPPUR 11044

To appear in: Separation and Purification Technology

Received Date: 9 October 2012

Revised Date: 31 January 2013

Accepted Date: 31 January 2013

Please cite this article as: T. Chakrabarty, A.K. Thakur, V.K. Shahi, Functionalized chitosan based nano-filter

membranes for pH-controlled separation of amino acids, Separation and Purification Technology (2013), doi: http://

dx.doi.org/10.1016/j.seppur.2013.01.054

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1

Functionalized chitosan based nano-filter membranes for pH-controlled separation of amino acids Tina Chakrabarty, Amit K. Thakur, Vinod K. Shahi*

Electro-Membrane Processes Division, Central Salt & Marine Chemicals Research Institute, Council of Scientific & Industrial Research (CSIR), G. B. Marg, Bhavnagar-364002 (Gujarat) INDIA Tel: +91-278-2569445; Fax: +91-278-2567562/2566970; E-mail: vkshahi@csmcri.org; vinodshahi1@yahoo.com

2

ABSTRACT

pH-controlled separation of AAs was achieved by electro-membrane cell (EMC) with three

compartments (central, comp. 1 and comp. 2). EMC is based on the principles of

electrodialysis (ED) using indigenous cation-exchange and anion-exchange nanofilter (C-NF

and A-NF, respectively). Water permeabilities (1.1-1.5 mh-1 bar-1) and molecular cut-off

(MWCO) values (400–500 Da) for C-NF and A-NF, were suggested their nanofilter (NF)

nature. Electrochemical properties of these membranes confirmed their suitability for EMC.

Chronopotentiometric studies revealed the electro-transport of positively charged lysine

(LYS+) and negatively charged glutamic acid (GLU-) across C-NF and A-NF, respectively, at

pH: 6.1 under applied voltage. While glycine at its pI (pH: 6.1) remained immobile. Thus,

separation of AAs (ternary mixture) by iso-electric point (pI) focusing of one component was

proposed. Under optimized experimental conditions (i.e. at 4.0V constant applied voltage,

pH: 6.1 for 0.05M equi-molar LYS-GLU-GLY mixture), about 4.0 k.Whr/kg of AA

separated energy consumption, 82% current efficiency and 90% product recovery showed the

economic and technical feasibility of EMC for industrial exploitation.

Keywords: Electro-driven separations; Charged nanofilter membranes, Amino acids;

Chronopotentiometry; Iso-electric point.

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1. Introduction

Chitosan is amino polysaccharide of technological importance and found diversified

applications as molecular biology and biopolymer [1,2]. Because of its hydrophilic nature

(presence of amino and hydroxyl groups), chitosan based membranes are widely reported for

pervaporation, drug delivery, ultrafiltration and bio-separation [3,4]. However, chitosan

membranes are highly swollen in water and losses physical structure. Efforts were rendered

to prepare cross-linked chitosan-polymer or chitosan-inorganic composite membranes, to

control the membrane stability and selectivity [1,5,6]. Further, acidic or basic modification of

chitosan expected to results charged membrane. Preparation of this membrane can be

achieved in aqueous media, while pore dimension can be tailored by gelation in non-aqueous

media.

Amino acids (AAs) are produced from molasses and raw sugar by a fermentation process,

and utilized as an additive in food products, chemical and pharmaceutical industries.

However, separation of AAs (molecular weight ranging between 100-300 Da) in down-

stream processing is challenging [7]. Electrodialysis (ED) was used to separate the AAs by

varying pH either by buffer or water splitting by bipolar membrane [8,9]. Method for

purification of amino acid containing solutions by electrodialysis also has been described in

U.S. Patent 6,551,803 [10]. But, desalting (purification) of AAs was difficult because salt ion

also migrated along with AA. Further, ion-exchange membranes are dense in nature and low

electro-migration of AA across ion-exchange membrane, jeopardize the method. Bazinet et al

reported electrodialysis with ultrafiltration membrane for the separation of peptides, proteins

and chitosan oligomers [11-15]. The cell configuration proposed in this manuscript is also

similar configuration reported by Bazinet et al, but filtration membranes were replaced by

special charged nanofilter membranes (C-NF and A-NF) with 400–500 Da MWCO.

4

To differentiate between AAs based on their molecular size and charge at different pH,

we developed C-NF and A-NF and used them as a replacement of cation- and anion-

exchange membranes (CEM and AEM, respectively) for one cell pair in electrodialysis cell.

In the proposed EMC, modified chitosan based C-NF and A-NF membranes were used to

achieve fractionation/separation of AAs in the three separating channels namely cationic,

anionic and uncharged AAs, simultaneous. Because of two screening parameters (size and

charge) and polarity of electrical driving force, high resolution for AA separation was

achieved. Designed EMC was tested for pH-controlled separation of GLU, LYS, GLY, as a

model case.

2. Working principle of EMC

Iso-electric values (pI) for proteins or AAs have been extensively used for their separation

from fermentation broth and selectivity depends on the net charge of the molecules at pH

other than pI and nature of membranes [14,16,17]. Cell configuration of EMC is similar to

the cell configuration of EDUF proposed by Bazinet et al., but filtration membranes were

replaced by special charged nanofilter membranes (C-NF and A-NF) with 400–500 Da

MWCO. Schematic diagram of EMC for the separation of AAs using A-NF and C-NF

membranes has been depicted in Fig. 1. Commercially available CEM and AEM were used to

separate the electrode chambers, and prevent the AA molecules to approach the electrodes or

denaturation by electrodes reaction.

Thus, at pH>pI; GLU (pI=3.22) existed as negatively charged (GLU―) because of

deprotonation, and at pH<pI it was as positively charged (GLU+) due to protonation,

according to following scheme.

↔ H3N

+ R COOH H

3N

+ R COO

- H

2N R COO

- ↔

Cationic (<pI) Zwetterionic (pI), Anionic (>pI)

5

Similarly, LYS exists as LYS+ at pH below than 9.59 (pI of LYS), while GLY was neutral at

pH: 6.10 (pI). Thus, at pH: 6.10, mixture of these three AAs existed as GLU―, LYS+, and

GLYo in the CC.

At pH: 6.1, equi-molar mixture of GLU, LYS and GLY fed through CC. Under the

influence of applied potential gradient, LYS+ migrated through C-NF towards cathode from

CC to comp. 1. While GLU− migrated towards anode from CC to comp. 2 across A-NF, and

GLYo remained in CC. This fact was validated by the mass balance of different AAs in three

streams. Thus, high-purity of AAs may be obtained by EMC with high resolution, in

continuous manner in the buffer medium.

3. Experimental

3.1. Materials and membrane preparation

Chitosan (100% deacetylation), poly(vinyl alcohol) (PVA; Mw: 125,000 Da), phosphorous

acid (99% purity), tetraethylorthosilicate (TEOS) were received from Sigma Aldrich

chemicals and used without further purification. CH3I, GLU, LYS, GLY,NaCl, HCHO,

glacial CH3COOH, CH3OH, H2SO4, HCl, and NaOH (AR grade, S.D. Fine Chemicals, India)

were used as received.

Synthesis of N-methylene phosphonic chitosan (PC) and quaternized chitosan (QC) were

prepared by the method reported earlier [18]. One part of chitosan solution (2% (w/v) in

glacial acetic acid (1% (v/v)) and 1 part of phosphorous acid (by weight) were drop-wise

added in water under continuous stirring (1 h). Reaction temperature was raised to 70 oC and

1 part of formaldehyde (36.5 wt%) was also added drop-wise under maintained temperature

(6 h). After removal of 75% (v/v) solvent, obtained viscous solution was precipitated with

acetone, and dried under vacuum at 50 oC. For the synthesis of QC, 2.5% (w/v) chitosan was

dissolved in 42% methanol under constant stirring for 1 h at 30 oC. Then 5% NaCl (w/v) and

6

5% (v/v) CH3I was added at intervals of 4 h in 2:1:1 proportion and mixture was stirred at 70

◦C for 12 h. Quaternized chitosan was then precipitated with acetone, washed and dried under

vacuum at 50 oC.

C-NF and A-NF membranes were prepared by dissolving 20 g PVA (50% w/w in all

cases; used as a plasticizer) and 16 g of PC or QC (40% w/w) in 100 cm3 of water. Solution

was stirred for 2 h, then 4 g of TEOS (10% w/w) was drop wise added under constant

stirring. The pH of solution was adjusted to 2.0 using dilute HCl. Resultant clear solution was

again stirred for 1 h and air bubbles were removed under vacuum. Solution was casted as thin

film of desired thickness on the glass plate covered with poly(ethylene) (HDPE) sheet.

Obtained thin film was dried for5.0 h at 30 ◦C. Afterwards, these films were gelated in the

methanol at 30oC. Further, dried thin films were immersed in a solution containing

formaldehyde (54.1 g), sodium sulfate (150.0 g), sulfuric acid (125.0 g), and water (470.0 g)

for1h at 80 oC to affect the membrane cross-linking by formal reaction. Washed and

equilibrated membranes were stored in double distilled water. Schematic structures of C-NF

and A-NF are presented in Fig. S1 (supporting information).

CEM and AEM used in this work were developed by CSIR-CSMCRI, Bhavnagar, India

[19,20] and based on interpolymer of polyethylene and styrene–divinylbenzene copolymer.

Thin films were obtained by blow film extrusion, and converted to the CEM by sulfonation

with chlorosulfonic acid and AEM by subsequently choloromethylation and amination. These

membranes are commercial and their numerous versatile ED applications were demonstrated

because of high ionic conductivity as well as chemical and mechanical stabilities [21].

3.2. Membrane characterizations

Procedures for measuring water content, conductivity, ion-exchange capacity (IEC) and

counter-ion transport numbers have been described in section S1 (supporting information).

Contact angle values for C-NF and A-NF in water were measured with a tensiometer (DCAT

7

21, Data physics, Filderstadt, Germany). The chronopotentiometric responses of C-NF and

A-NF membranes were recorded in equilibrium with NaCl or AAs solutions using Perspex

cell as reported earlier [22]. Cell contained two compartments separated by the circular ion-

exchange membrane (12.5 cm2). A constant current was applied across the membrane using

two dimensionally stable titanium electrodes coated with precious metal oxide, with the help

of potentiostat/galvanostat (Auto Lab, Model PGSTAT 30 (EcoChemie, B.V. Utrecht, The

Netherlands)). Electrolyte or AA solution of known concentration was placed in two

compartments of the cell (50 and 150 cm3, respectively). Variation in potential with time

under static conditions was recorded. In all these studies, the direction of the current was set

in such a way that the counter-ion should move vertically upward from the outer to the inner

compartment with minimal perturbations caused by natural convection. Two saturated

calomel electrodes were used for the measurement of the potential difference across the ion-

exchange membrane under static conditions. The solutions of both the compartments were

vigorously stirred between two successive experiments to ensure the return of equilibrium

conditions in two solution–membrane interfacial zones.

3.3. EMC for separation of amino acids

EMC was made up of poly vinylchloride (PVC), in which C-NF and A-NF were used to

separate the separating cambers: central compartment (CC), comp. 1 and comp. 2 (Fig. 2).

Parallel-cum-series flow arrangement was used for the separation of ternary AAs (GLU,

GLY and LYS) from their mixture. Expanded TiO2 sheets coated with a triple precious metal

oxide (titanium–ruthenium–platinum; 6.0 μm thickness) of 1.5 mm thickness and 8.0×10−3

m2 area (received from Titanium Tantalum Products (TITAN, Chennai, India)) were used as

cathode and anode. Feed of each compartment (500 cm3) was moved in recirculation mode

with a constant flow rate (60 cm3 h−1) with the help of peristaltic pumps. The whole setup

was operated in ambient conditions (303K) without any additional temperature control. The

8

0.01 M Na2SO4 solution was recirculated into the EW chambers. The AA solution (GLU,

GLY or LYS separately or their mixture) of known concentration (0.02–0.06M) in NaAc

buffer (0.01 M) at known pH was fed into the central compartment, while NaAc buffer (0.01

M) was fed into compartments 1 and 2. DC power supply (Aplab, India, model L1285) was

used to apply the constant potential across the electrodes and current variation was recorded

with time using a multimeter. Under the influence of applied potential gradient, AA with net

negative charge (AA-) was transported from CC to comp. 2, across A-NF, while AA with net

positive charge (AA+) was transported from CC to comp. 1. Solution conductivity and pH

changes of each compartment were regularly monitored. Concentration of AA solution was

determined by UV-visible spectrophotometer (Shimadzu, Japan) at λmax208 nm fixed

wavelength. In case AA mixture solution, concentration was analysed by high performance

liquid chromatography (HPLC) after pre-column derivitization using dansyl chloride. A

mixture of acetonitrile and acetate buffer (pH= 4.5; 0.045 M) was used as the mobile phase

flowing through a C18 reverse phase column. The solvent gradient was 20% to 60%

acetonotrile over 30 min. In all cases an equal volume of AAs solution (separately or their

mixture) was taken for simplicity to study the feasibility of their separation [23]. All the data

have been reported as an average value of three successive measurements.

4. Results and discussion

4.1. Membrane properties

The physicochemical and electrochemical properties of CEM and AEM are presented in

Table S1 (Supporting information).These membranes exhibited good water content, IEC and

counter-ion transport number in the membrane phase under operating conditions. Properties

of these membranes are comparable with the best-known ion-exchange membrane. CEM and

AEM showed 2.12×10-2 and 1.13×10-2 S cm-1 conductivity in the equilibration with 0.10 M

NaCl solution, respectively.

9

Both membranes (C-NF and A-NF) were characterized by measuring their water content,

ion-exchange capacity (IEC), contact angle, membrane conductivity and counter-ion

transport numbers in the membrane phase (Table 1). Furthermore, IEC data were used with

advantage for the estimation of surface charge concentration in the membrane matrix [18].

These informations suggested acidic nature for C-NF, and alkaline nature for A-NF. Charged

natures of these membranes were also revealed by membrane conductivity and counter-ion

transport number. Data suggested that C-NF behaved as cation selective while A-NF behaved

as anion selective membrane. Contact angle values (close to 90◦) for C-NF and A-NF

membranes also suggested their hydrophilic nature and suitability for their application in

protein separations.

Water permeabilities of C-NF and A-NF were varied between 1.1-1.5 mh-1bar-1.

Considering the dense nature of these membranes, solvent fluxes were anticipated in

nanofiltration (NF) range [24]. Molecular weight cut-off (MWCO) values, defined as the

MW of probes (sugars or polyethylene glycol) solute that is retained for 90%, of 400–500 Da

further confirmed the NF-nature of C-NF and A-NF membranes (Fig. 3). Based on these

characterizations, charged and nanofilter nature for C-NF and A-NF was concluded [25].

Also, their good conductivity suggested their suitability for use in EMC under AAs

environment.

4.2. Chronopotentiometric responses of C-NF and A-NF

Detailed description of membrane chronopotentiometry analysis is presented in section S2

(supporting information). The chronopotentiometric responses of the C-NF and A-NF in

contact with NaCl solution (0.10 M) were recorded as a function of applied current density

(1.0-1.5 mA cm−2) (Fig. S2 (A and B) supporting information). C-NF and A-NF membranes

showed 3.536, and 3.952, respectively, Iτ1/2 value under equilibration with 0.10 M NaCl

solution (detailed descriptions are included in section S3 (supporting information). Also, Iτ1/2

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values were fairly constant and independent of I at a given concentration of electrolyte. The

Iτ1/2 value was comparatively high for A-NF than C-NF, which confirmed rapid polarization

in the latter case due to easy transport and high mobility of the counter-ion in the membrane

phase.

For the ion-exchange membrane, transport property depends on the interfacial character of

the membrane and electrolytic environment viz. concentration and pH of electrolyte solutions

in which the membrane is operated. In EMC, separation of AAs was achieved at IEP of GLY

(pH: 6.1), and GLU- electro-migrated across A-NF towards anode, while LYS+ across C-NF

towards cathode. Chronopotentiometric responses (Et–t), for C-NF in equilibrium with 0.10

M LYS and A-NF in equilibrium with 0.10 M GLU at pH: 6.1 were recorded at different

current densities (Fig. 4(A&B)). After a potential jump due to the uncompensated Ohmic

resistance, the potential difference reaches a plateau after about 3–6 s (depending upon

applied current densities) for both membranes (C-NF and A-NF). In Fig. 4A, at I = 1.00mA

cm-2, the inflection in 0.10 M GLU (pH: 6.1) was at 6 s, which is turned sharp at higher

current density. The plots further indicated that the initial potential (E0) and height of the

inflection (ΔE) increases with I, for both type of membranes. One of the most striking

features noted that E0/I values for both types of membranes was found to be constant under

AA environment. A subsequent increase in current density altered the potential at the initial

stage across the membrane and shifted the inflection point close to y-axis. This results in the

quick polarization of the membrane, occurs due to depletion of counter-ions (GLU- and

LYS+) due to its electro-migration across A-NF or C-NF, respectively. Thus,

chronopotentiometric studies confirmed electro-migration of GLU- and LYS+, across A-NF

and C-NF at pH: 6.1.

For C-NF in equilibration with LYS+, Iτ1/2 value was 2.502, while GLU- across A-NF

showed 2.634. Reciprocal of Iτ1/2 measures the ionic transport across the membrane in a given

11

electrolytic environment. Iτ1/2 values for membranes in equilibration with AAs solutions were

relatively less in compare with strong electrolyte. At pH: 6.1, GLY remained zwitter-ion

(GLY0) and thus unable to migrate. Thus, C-NF and A-NF, used in EMC are suitable for

electro-transport of GLU-and LYS+.

4.3. Electro-transport of GLU or LYS

Separation of AAs mixture (GLY, LYS and GLU) was achieved in a 5 compartments

EMC, in which electrode chambers were separated by CEM and AEM. AAs transport

occurred through C-NF and A-NF membranes (Fig. 2). The 0.01 M Na2SO4 solution was

recirculated into the electrodechambers. Experiments were carried out at different applied

voltage (3.0-5.0 V) using AAs solutions (GLU, GLY or LYS, separately or their mixture in

NaAc buffer (0.01 M)) as initial feed of CC. While NaAc buffer (0.01 M) was initially fed

into compartments 1 and 2.

Variation in current density with time during electro-transport of LYS+ from CC to comp.

1 across C-NF or migration of GLU- from CC to comp. 2 across A-NF, at 4.0 V applied

voltage have been depicted in Fig. 5, as a representative case. It is important to record that

current density varied with time under similar fashion for different applied voltage (3.0 and

5.0 V). At pH: 6.1, GLYo remained in CC and current was unchanged (Fig. 5). Initially

current was relatively low because absence of AA in Comp. 1or 2 and thus high resistance.

Further, under similar experimental conditions current increased with applied voltage (Figure

5(B).Thus the observed current of the EMC varied due to variation in the electro-migration of

AA. Since, AA was separated from electrodes by IEMs, this avoids their degradation. At high

applied voltage (4 or 5V) intensity of current increased because of enhanced electro-

migration of GLU- or LYS+ across A-NF and C-NF, respectively.

Electro-transport of LYS+ or GLU- across C-NF or A-NF at pH: 6.1were further checked

by regular concentration monitoring by UV-visible spectrophotometer. Possibility of electro-

12

transport of LYS+ to catholyte and GLU- to anolyte was completely ruled out because

presence of AEM adjacent to cathode and CEM adjacent to anode. This was further verified

by absence of and AA in electrode compartments. Under the influence of the applied

potential (below limiting current density), electro-transport of LYS+ from CC to comp. 1 and

GLU- from CC to comp. 2 was also verified by the variation of pH of all three streams (CC,

Comp. 1 and comp. 2) with time (Fig. 6). The pH of CC almost remained constant. While pH

of comp. 2 increased, may be due to enrichment of GLU−. Similarly pH of comp. 1 reduced

because enrichment of LYS+. This phenomenon resulted concentration bulid-up of LYS+ in

comp. 1, and GLU- in comp. 2, while GLYo remained in the CC. As a reference, in EMC

water splitting was checked by feeding NaCl solution (0.1 M) in CC and variation in pH of

comp. 1, 2 and CC, supported water splitting under applied potential (3-5V) same duration as

their other tests.

Separation process in the EMC occurs on the principal of electrodialysis [26]. In the EMC

one cell pair of CEM and AEM has been replaced with C-NF and A-NF with 400–500 Da

MWCO. C-NF and A-NF allowed relatively easy electro-migration of charged AA species

under applied voltage. At pI of GLY (pH: 6.1), GLU exists as GLU- (above pI), and LYS

exists as LYS+ (below pI). Under applied potential gradient GLU- migrated towards anode

across A-NF (from CC to comp. 2), while LYS+ migrated towards cathode across C-NF

(from CC to comp. 1). Respective charged nature of C-NF and A-NF facilitated their

migration under applied voltage. Also amphoteric GLYo remained in CC and this enables the

electro-separation of different electrically charged AA species.

Rate of electro-migration of LYS+ or GLU−was assessed by their concentration change in

comp.1 and 2, respectively, considering negligible mass(water) transport across membranes,

using following equation [26,27]

( )10

t

CC

A

VJ ta

Δ−=

13

WhereC0 and Ct is the initial and final concentration of GLU−/LYS+ in comp. 1 (molm−3), t is

the time allowed (s), Va the total volume of solution in each compartment (0.50 × 10−3 m3),

and A is the effective membrane area. Flux of LYS+ and GLU−at 4.0 V applied voltage,

initially increased linearly and after attending maxima decreased under similar fashion (Fig.

7(A)). Initial concentrations of LYS+/GLU−in comp. 1 or 2, were almost zero. At the start of

the process, the concentrations of LYS+ and GLU−were reduced in CC, and increased in

comp. 1 and 2, respectively. The concentration of LYS+/GLU− in CC approached a minimum,

while enhanced in the comp. 1 and 2, up to 230 min. beyond, respective flux values were

reduced because of less availability of charged AA species in CC. After complete recovery of

GLU- and LYS+ from CC to comp. 2 or 1, respectively, their flux values approached to

minimum. Concentration of GLYo in CC remained almost same with close to 100% recovery

(Fig.7(B)). This confirms immobile nature of GLYo under the influence of electrical gradient

across C-NF or A-NF. Further, flux of GLU- from CC to comp. 2, increased with applied

voltage (Fig. S3, supporting informations). Effect of AAs concentration on process efficiency

of EMC was also explored by varying concentrations in CC and observing flux of desired AA

in comp. 1 or comp. 2. Data suggested that flux was enhanced with increase in feed AA

concentration.

4.4. Process efficiency of EMC for separating AAs mixture

Energy consumption (W) and current efficiency (CE) are also important parameters for

assessing the suitability of any EMC for practical applications. The energy consumption (W,

kWh kg−1 of AA separated) may be obtained as follows:

( ) ( )∫=−t

m

dtIVkWhKgW

0

1 2

The overall current efficiency (CE) is defined as the fraction of current utilized for the

electro-transport of LYS+ or GLU- from CC to comp. 1 or 2:

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Recovery of product (GLU− or LYS+) is an important parameter to examine the economic

feasibility of any process, and may be defined as:

( )3100cov00

×=FF

TPTP

CC

VCeryreAA

Where C and V denote concentration and volume, subscript 0 and T denote initial or at time

T, and subscript P and F denote product and feed. An electrochemical process must not only

be technically feasible, but should also be inexpensive with low energy consumption and high

recovery of product. To evaluate the technical and economic feasibility of the process, W, CE

(%) and recovery (%) data under different experimental conditions are presented in Table 2.

Electro-transport of LYS+ associated with 3.8 kWhr/kg of energy consumption, 96.8% CE

and 99.1% recovery. While electro-transport of GLU- was separately achieved with 3.3

kWhr/kg of energy consumption, 63.4% CE and 94.4% recovery. 98.9% of GLYo remained

in CC was recovered. These data indicate energy efficient EMC with high product recovery.

Further, Sandeaux et al reported about 60-90% CE for extraction of amphoteric amino acids

by electrodialysis [9]. Also, Nijmeijer et al reported about 24% CE and 5.1 kWhr/kg of

energy consumption for the separation of different AAs by electrodialysis with ultrafilter

membrane (EDUF) [28]. Thus, reported efficiency values in this manuscript are comparable

with the data reported in the literature.

EMC processes efficiency data for protein separation were also recorded at relatively high

applied voltage (4.0-6.0 V) (Table 3). Relatively high W and low CE may be explained by: (i)

electrode reaction and water splitting at electrodes; (ii) simultaneously transport of solvent

(water) induced by amino acid ions. Factors (i) and (ii) are responsible for enhanced energy

consumption and reduced CE with applied potential. These parameters, along with the initial

feed concentration of AA in CC, strongly affected the performance of EMC.

Also, after several successive experiments, the electrochemical properties of C-NF and A-

NF showed about ±1.0% deterioration in terms of membrane properties and process

15

efficiency. Thus any type of fouling was completely ruled out. Based on these observations, it

can be concluded that it is possible to separate LYS+, GLU- and GLYo at pH: 6.1in NaAC

buffer solution by EMC.

The separation of LYS, GLU and GLY (equi-molar, each) from their mixture was carried

out using the EMC at constant applied voltage (4.0 V) at pH: 6.1. Flux values for LYS+ and

GLU- from CC to comp. 1and comp. 2, respectively (Fig. S3, supporting information) were

depicted as a function time. Recovery of different AA under experimental conditions (Fig. 8)

was close to 100% (depended on AA mixture feed concentration). Electro-migration of LYS+

and GLU-across C-NF and A-NF towards cathode and anode, respectively, strongly depend

on their charged nature (pH), charge on membranes and applied voltage.

Proposed EMC is an important tool for separating AAs with close molecular weights by

focusing on their iso-electric values. In this process no appreciable membrane fouling or AAs

denaturation was observed. Because of different charged nature (pI) of AAs or other

biomolecules, their efficient separation can be obtained. In this particular case the difference

in the pI values of GLU and LYS was quite high, but the method could be applied in cases

where pI values are very similar, by very careful control of pH. In case of complex medium

with many AAs or peptides, reported process will be suitable for separation or extraction of

AA based on their pI values. Due to very low MWCO values for C-NF and A-NF, these

membranes are not suitable for the separation of molecules with high molecular weights

(such as peptides). Furthermore, reported process can be used with advantage for the

separating AAs from peptides.

5. Conclusions

EMC has been designed using indigenous charged NF membranes (C-NF and A-NF), for

the separation of AAs with different pI values but similar molecular weights. Water

permeabilities of C-NF and A-NF were found to be vary between (1.1-1.5 mh-1bar-1) and their

16

MWCO values (400–500 Da) suggested charged NF nature. Electrochemical properties of

these membranes suggested their suitability for EMC based on iso-electric focusing. Electro-

transport of LYS+ and GLU− across C-NF and A-NF respectively was studied under given

experimental conditions. Electrostatic interaction between the zwitterionic AA molecules and

membrane surface charge density plays important. Simultaneous electro-transport of LYS+

across C-NF and GLU−across A-NF was observed, under experimental conditions. Also,

GLYo, because of its zwitterionic nature, was immobile under applied electrical gradient and

remained in the feed compartment (CC). Thus, it was possible to separate the AA mixture by

iso-electric focusing. Also, due to relatively low energy consumption (4.0kWhr/kg of AA

separated) high CE (82%), separation of AAs (LYS-GLU-GLY) is technically feasible.

Under optimized experimental conditions (i.e. at4.0V constant applied voltage, pH: 6.1 for

0.05 M equi-molar LYS-GLU-GLY mixture), close to 90% product recovery showed the

economic feasibility of EPMC for industrial exploitation.

Also from data it is clearly evident AAs separation can be efficiently achieved by EMC

due to difference in their p Ivalues, in which applied voltage facilitates separation because the

pH of the solution does not have to change but only the applied voltage, which can be

controlled well. In conclusion it is considered that EMC offers a more techno-economically

efficient method for the production of chemicals. Further, the proposed route offers the

possibility to couple production with separation by electrodialysis.

Acknowledgments

We acknowledge the Analytical Science Division, CSMCRI, Bhavnagar for instrumental

support.

17

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21

Nomenclature:

T Absolute temperature (K)

i Applied current density ( mA cm-2)

V Applied potential (V)

ρ Cross-linking density

I Current (A)

η Current efficiency

m Weight (kg)

A Effective membrane area ( m2)

W Energy consumption (K Wh Kg-1)

φ Front factor

ilim1 First limiting current density (mA cm-2)

F Faraday’s constant (96,500 Cmol−1)

R Gas constant (8.314 Joule mole-1 K-1)

Ct, C0 Co and Ct are the initial and final concentration of

acid in CC (mol m-3)

(ΔE) The height of the inflection

A Membrane area ( m2)

d Membrane density

Δx Membrane thickness (cm)

κm Membrane conductivity (S cm-1)

Ps Membrane permselectivity

J Water dissociation flux (mol m-2 s-1)

n Stoichiometric number (n = 1 in this case)

Δt Time allowed for electro-membrane process (s)

Va Total volume of solution in each compartment

22

Table 1

ameasured in the

equilibration with 0.001 mol dm-3NaCl solution.

b Estimated from membrane potential measurements using NaCl solutions of0.01/0.001 mol/dm3 concentration across the membrane.

Membranes Properties

C-NF A-NF

Membrane thickness (mm) 0.20 0.20

Water content (%) 42.76 24.85

IEC (mequiv./gm) 0.202 0.199

Contact angle 76.70o 77.34o

Membrane conductivity (mS cm-1) a 2.28 2.13

Counter-ion transport number 0.86 0.83

Surface charge concentration

(m mol dm-3)

0.169 0.147

23

Table 2

AA CE, % W, kWh kg-1 of AA electro-transported

Recovery, %

LYS+ 96.8 3.8 99.1

GLY0 --- --- 98.9

GLU- 63.4 3.3 94.4

24

Table 3

Recovery, %

Applied voltage, V.

CE, %

W, kWh kg-1 of AA electro-transported

LYS+ GLU- GLY0

4.0 87.4 3.8 99.1 94.4 98.9

5.0 82.3 4.0 99.1 95.2 99.0

6.0 79.6 4.5 99.2 96.8 99.0

Fig. 1

Fig. 2.

Fig. 3

35

45

55

65

75

85

95

105

0 200 400 600 800 1000 1200

C-NF

A-NF

Molecular weight

% S

olu

tre

reje

ctio

n

Fig. 4.

2.5

3

3.5

4

4.5

5

5.5

0 5 10 15 20 t (s)

E (

V)

1.00 mA cm-2

1.25 mA cm-2

1.50 mA cm-2 A

3

3.5

4

4.5

5

5.5

6

0 4 8 12 16 20 t (s)

E (

V) 1.00 mA cm-2

1.25 mA cm-2

1.50 mA cm-2 B

Fig. 5.

0

10

20

30

40

50

60

50 100 150 200 250

Cu

rren

t, m

A

Time, min

A

GLU

LYS

GLY

0

10

20

30

40

50

60

50 100 150 200 250

Cu

rren

t. m

A

Time, min

B 5.0 V

4.0 V

3.0 V

Fig. 6.

1

3

5

7

9

0 50 100 150 200 250

pH

Time, min

Comp. 1

CC

Comp. 2

Fig. 7.

0

4

8

12

16

0 100 200 300 400

J X

10

-8, m

ol

m-2

s-1

Time, min

GLU

LYS

A

94

95

96

97

98

99

100

0 100 200 300 400

Rec

over

y, %

Time, min

GLY

B

Fig. 8.

35

Table 1. Physicochemical and electrochemical properties of C-NF and A-NF

Table 2. CE, W and % recovery for different AAs separately on EMC, feed solution: 0.05 M,

separately; pH: 6.1, at 4.0V applied voltage

Table 3. CE, W and % recovery values for separation of AAs mixed solution (0.05M each)

(LYS+, GLYo, and GLU-) at pH: 6.1 as feed of CC, by EMC at different applied voltage

Fig. 1. Principle for separation of proteins using C-NF and A-NF membranes under electrical

gradient.

Fig. 2. Schematic presentation of EMC for AAs separations.

Fig. 3. MWCO values obtained from the rejection (more than 90%) curves for sugars and

poly (ethylene glycol) of different molecular weights

Fig. 4. Chronopotentiograms of: (A) A-NF in equilibration 0.10 M GLU; and (B) C-NF, in

equilibration with 0.10 M LYS solution at pH: 6.1 and different applied current density.

Fig. 5. Variation of current with time at: (A) 4.0 V applied voltage and pH: 6.1 during

electro-transport of LYS+ across C-NF; GLU- across A-NF, and CC fed with GLY (0.05 M

each separately); (B) at different applied voltage for electro transport of GLU- (0.05 M)

across A-NF.

Fig. 6. Variation of pH with time (outputof CC, comp. 1 and 2); feed of CC: AAs mixed

solution (LYS, GLU and GLY (0.05 M each) at pH:6.1) at 4.0 V applied voltage

Fig.7. Variation of Flux with time for: (A) LYS and GLU (from central comp. to comp. 1 and

comp. 2, respectively); (B) recovery of GLY in CC, at 0.05 M feed concentration (each), 4.0

V applied voltage, pH: 6.1.

36

Fig. 8. Recovery of AAs for different concentrations of feed mixture at pH: 6.1 and 4.0 V

applied voltage.

1

Highlights

Charge based pH-controlled separation of amino acids.

Indigenously prepared highly selective cationic and anionic charged nano-filter

membranes, used in electro-membrane cell.

Chronopotentiometric studies revealed the electro-transport and selectivity in separation.