Research Article

Received: 9 August 2009 Revised: 5 December 2009 Accepted: 5 December 2009 Published online in Wiley Interscience: 25 January 2010

(www.interscience.wiley.com) DOI 10.1002/jctb.2348

Electro-membrane process for the separation of amino acids by iso-electric focusing

Mahendra Kumar, Bijay P. Tripathi and Vinod K. Shahi
Abstract
BACKGROUND: Amino acids (AAs) are usually produced commercially using chemical, biochemical and microbiological fermentation methods. The product obtained from these methods undergoes various treatments involving extraction and electrodialysis (ED) for salt removal and AA recovery. This paper describes an electro-membrane process (EMP) for the charge based separation of amino acids. RESULTS: Iso-electric separation of AAs (GLULYS) from their mixture, using ion- exchange membranes (IEMs) has been achieved by an efcient and indigenous EMP. It was observed that electro-transport rate (ux) of glutamic acid (GLU) at pH 8.0 (above its pI) was extremely high, while that for lysine (LYS) (pH 9.6) across the anion-exchange membrane (AEM) was very low, under similar experimental conditions. Under optimum experimental conditions, separation of GLU from GLULYS mixture was achieved with moderate energy consumption (12.9 kWh kg1 ), high current efciency (CE) (65%) and 85% recovery of GLU. CONCLUSIONS: On the basis of the electro-transport rate of AA and membrane selectivity, it was concluded that the separation of GLULYS mixture was possible at pH 8.0, because of the oppositely charged nature of the two amino acids due to their different pI values. Moreover, any type of membrane fouling and deterioration in membrane conductivity was ruled out under experimental conditions. This work clearly demonstrates the great potential of EMP for industrial applications. c 2010 Society of Chemical Industry Keywords: amino acid separation; iso-electric focusing; ion-exchange membrane; electro-membrane process

NOTATION
CE tim m Wd Ww R a1 and a2 T F x, area A V0F and Vtp C0F and CtF W V I t m M n Q (J)GLU (J)LYS Current efciency Counter-ion transport number Specic membrane conductivity Weight of dry membrane Weight of wet membrane Gas constant (8.314 J mol1 K1 ) Activities of electrolyte solutions Absolute temperature (K) Faraday constant (96500 coulombs) Thickness of the wet membrane (cm) Electrode area (cm2 ) Initial and nal volume of the FC (cm3 ) Initial and nal concentration of AA in PC (M) Energy consumption (kWh kg1 ) Cell voltage (V) Current (A) Time (s) Weight of GLU or LYS (g) Molecular weight of GLU or LYS, n t Stoichiometric number (n = 1 in this case) Electric quantity passed (Coulombs; A s) Flux of GLU (mol m2 s1 ) Flux of LYS (mol m2 s1 )

INTRODUCTION
With the advance in life science research, numbers of biomolecules with pharmaceutical characteristics have been discovered. In the down-stream process, separation of amino acids (AA) (amphoteric in nature with more than one ionizable group, fundamental constituents of all proteins) was stimulated by an increasing demand for high purity in order to reduce the side effects induced by impurities.1 5 During recent years, a noticeable growth of AA production from molasses and raw sugar by a fermentation process, and their utilization as an additive in food products, chemical and pharmaceutical industries, has occurred.6 10 In particular, glutamic acid (GLU) and lysine (LYS) have found a wide range of applications in the food, pharmaceutical, biotechnology, biochemistry and cosmetic industries.11 13 Various membrane based processes, such as ion-exchange, nanoltration (NF), dialtration, ultraltration (UF), and electrodialysis (ED), were used for the separation of AAs from fermentation broths without precipitation.14 18 Use of acid and base to regenerate resins in the ion-exchange process is a disadvantage because it increases the operational cost. In addition, wastewater produced during resin regeneration

Correspondence to: Vinod K. Shahi, Electro-Membrane Processes Division, Central Salt & Marine Chemicals Research Institute, Council of Scientic & Industrial Research (CSIR), G. B. Marg, Bhavnagar-364002 (Gujarat) India. E-mail: vkshahi@csmcri.org; vinodshahi1@yahoo.com Electro-MembraneProcessesDivision,Central Salt&MarineChemicalsResearch Institute,CouncilofScientic&IndustrialResearch(CSIR),G.B.Marg,Bhavnagar364002 (Gujarat) India

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EMP for the separation of amino acids by iso-electric focusing

LYS

www.soci.org

GLU Electrode Wash

CEM LYS Cathode

AEM

CEM

GLUFeed Comp. (FC) Permeate Comp. (PC)

Electrode Wash 0.1M Na2SO4 GLU + LYS in NaAC Buffer

Figure 1. Schematic of the EMP cell for separation of amino acids.

NaAC Buffer

and washing causes serious environmental pollution. Membrane processes such as NF and UF, are size based separation processes and their separation performance is not high unless the molecular weight (MW) ratio of the two components is larger than 10.17,19 In ED, ions are transported through ion-exchange membranes (IEMs) under the inuence of an applied potential to separate ionic and nonionic constituents.20,21 Recently, bipolar membrane electodialysis (BMED) processes have been developed for the conversion of salts into corresponding acid and base.22 24 BMED processes have also been found to be suitable and cost effective for recovering organic acid or AAs from their respective salt.25 27 Salt diffusion (co-ion leakage) through the bipolar membrane in BMED is a serious problem, which affects the purity of the product and enhances the process cost. There is no ion-exchange membrane with 100% selectivity. There is no electro-membrane process (EMP) based on the principles of ED, used for the efcient separation of amino acids, with close MWs and different iso-electric points.28 31
H
+H N 2

Working principle of the electro-membrane process (EMP) The iso-electric focusing technology has been used extensively in separation of protein or AAs from fermentation broths with great success. The performance is mainly determined by the net charge of molecules at different pH and nature of the membranes.15,32 34 A schematic diagram of the EMP cell used for the separation of AAs from their mixture is illustrated in Fig. 1. The feed and permeate chambers were partitioned by an anion-exchange membrane (AEM) which only allows the passage of anions. Both electrode chambers (catholyte, anolyte) were separated by cation-exchange membranes (CEMs), which prevent the approach of AA molecules to the electrode and further denaturation by electrode reaction. Thus, at pH<pI; GLU (pI = 3.22) existed in GLU+ , while in the negatively charged state (GLU ) above its pI, according to following scheme. Similarly, LYS existed in the LYS+ state below pH 9.59 (pI of LYS), and in the LYS state at pH>pI.
O C O
-

O C OH
+H N 3

H C

Anode

H H2N C

O C O-

(CH2)2 COOH Cationic (<pI)

H
+H N 3

(CH2)2 COOH Zwetterionic (pI), MW:147.13

H COOH2N C COOH2N

(CH2)2 COOH Anionic (>pI)

H C COO-

CH2)4 NH3+ Cationic (<pI)

CH2)4 NH3
+

CH2)4 NH2 Anionic (>pI)

649

Zwetterionic (pI) ,MW:146.19

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www.soci.org At pH 8.0, GLU existed in the GLU state and LYS in the LYS+ state. There was the possibility for migration of LYS+ towards the catholyte through CEM. But, at pH 8.0 (near to its pI), due to the small net positive charge on LYS, its electro-migration from the feed compartment (FC) to the catholyte was not feasible. Further, the presence of LYS+ in the catholyte was checked and found to be negligible. Also, LYS+ remained in the FC due to its electrical polarity and repulsion between positively charged AEM. Also, this observation was validated by the mass balance of LYS in FC before and after the experiments: mass balance was found to be about 95%. GLU passed through the AEM from the FC to the permeate compartment (PC), under the applied electric potential. Thus, high-purity GLU may be obtained in the PC. For high resolution AA separation, a stable and continuous pH gradient with relatively constant conductivity and high buffer capacity is required. Ampholyte was commonly used to provide a stable pH gradient in the traditional iso-electric focusing system due to various advantages, such as high buffer capacity, high solubility, good conductivity at pI ofAAs, and absence of biological effects. Therefore, the purpose of this work is to investigate an EMP, based on the principles of ED to achieve the separation of AAs above or below the pI of one component in spite of their close MW and thus molecular size. Herein, we report an EMP for the separation of AAs from their mixture by iso-electric focusing using CEM and AEM as a separation media. A mixture of GLU and LYS in solution was studied as a model case under similar experimental conditions.

M Kumar, BP Tripathi, VK Shahi

Table 1. Physicochemical and electrochemical properties of the CEM and AEM Properties Thickness1 (m) Water content2 (%) Ion-exchange capacity3 (mequiv./g of dry membrane) Counter-ion transport m numbera ,4 (t ) Specic membrane conductivityb,5 (S cm1 )
a

CEM 150 14.6 1.03

AEM 150 10.8 1.28

0.96 2.05 102

0.91 1.12 102

Measured by membrane potential in equilibration in with 0.01 mol L1 and 0.1 mol L1 NaCl solutions. b Measured in equilibration with 0.1 mol L1 NaCl solution. Uncertainty for measurements: 1: 1.0 m; 2: 0.1%; 3: 0.01 mequiv g1 of dry membrane; 4: 0.01; and 5: 0.01 103 S cm.

tissue paper. The water content was estimated from: Water content (%) = Ww Wd 100 Wd (1)

MATERIALS AND METHODS

Materials Poly (ether sulfone) (PES) was obtained from Sigma-Aldrich Chemicals (Mumbai, India) and all other reagents such as GLU, LYS, H2 SO4 , NaCl, CdCl2 .H2 O, DMF, acetic acid, CH3 COONa (NaAc), ninhydrine, of Analytical grade reagents (AR) grade were obtained from S.D. Fine Chemicals, India, and used without further purication.

For estimation of the ion-exchange capacity (IEC), a piece of the membrane was equilibrated in 1.0 mol L1 HCl or 1.0 mol L1 NaOH solution overnight to convert them into H+ or OH form. The excess acid and base was removed by washing with distilled water. The washed membranes were then equilibrated in 50 mL of 0.50 mol L1 NaCl solutions to exchange the H+ /OH by Na+ /Cl . The amount of H+ or OH ions liberated in solution was determined by acidbase titration.37 tim across the membranes was estimated by membrane potential measurements in equilibration with 0.01 and 0.10 mol L1 NaCl solutions, according to following equation:31
m E m = (2t 1)

RT a1 ln F a2

(2)

Membranes preparation Sulfonation of PES was carried out as reported earlier.35 CEM was prepared using sulfonated poly (ether sulfone) (SPES) in dimethylformamide (DMF) (20%, w/v) and further by casting onto a cleaned glass plate. The prepared membrane was dried in ambient condition for 24 h, then at 60 C for 6 h in a vacuum oven. The AEM used in this work was prepared in the laboratory by a procedure reported earlier and used for various processes.28 31,36 Thus, the membranes obtained (CEM and AEM) were equilibrated with 1.0 mol L1 HCl and 1.0 mol L1 NaOH solution before use. The cleaned and equilibrated membranes were stored in double distilled water.

Electrochemical properties of membranes The thickness of the wet membrane was measured by micrometer with 0.10 m accuracy. The membrane water content was determined from the weight of dry and wet membrane. The weight of dry membrane was recorded after 24 h drying at 60 C, and the weight of the wet membrane was determined after equilibrating the membrane in water for 24 h, with surface water removed using

The physicochemical and electrochemical properties of CEM and AEM are presented in Table 1. Both 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 IEMs.38 The knowledge on membrane conductivity in an actual operating environment is an essential parameter to assess the suitability of membranes for an EMP. Membrane conductivity measurements for CEM and AEM were carried out in equilibration with GLU, LYS and NaCl solution of different concentrations using a potentiostat/galvanostat frequency response analyzer (Auto Lab, Model PGSTAT 30, EcoChemie, B.V. Utrecht, The Netherlands). The membrane was sandwiched between two stainless steel circular electrodes (4 cm2 ). Direct current (dc) and sinusoidal alternating currents (ac) were supplied to the respective electrodes to record the frequency at a scanning rate of 1.0 A s1 within a frequency range 106 to 103 Hz.39 The membrane resistances were obtained from Nyquist plots using a t and simulation method. Membrane conductivity was recorded in equilibration with NaCl, GLU and LYS solutions of different concentrations. The specic membrane

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EMP for the separation of amino acids by iso-electric focusing conductivity ( m ) was estimated by the given equation: m = x ARm (3)

www.soci.org Under the inuence of applied potential, amino acid with negative net charge (AA ; depending on pH) was electrotransported from FC to PC, across AEM. Solution conductivity and pH changes of each compartment were regularly monitored during all the experiments. For individual AA solution, concentration of amino acid (GLU or LYS) was determined by UV-visible spectrophotometer (Shimadzu, Japan) at max 504 nm xed wavelength.41 In a mixture case, AA concentration was analyzed by high performance liquid chromatography (HPLC) after precolumn derivitization using dansyl chloride. A mixture of acetonitrile and acetate buffer (pH= 4.5; 0.045 mol L1 ) was used as the mobile phase owing 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 and to study the feasibility of their separation, above the iso-electric point of the AAs.

The variation of m for both type membranes (CEM and AEM) in equilibration with NaCl, GLU, and LYS solutions of different concentrations are shown in Fig. 2(A, B and C), respectively. m values for both types of IEMs increased with concentration, and were highly dependent on the nature of equilibrating environment and ionic concentration in the membrane/solution interfacial zone. Both membranes exhibited excellent conductivity in equilibration with NaCl solution, but m values were relatively low in equilibration with GLU or LYS solutions of similar concentration. This may be explained due to extremely low dissociation constant of AAs and thus lower ionic concentration.40 Furthermore, conductivities values exhibited by CEM and AEM, suggested their suitability for use in EMP under AAs environment. Experimental procedure for separation of amino acids by iso-electric focusing An experimental cell used for EMP was made up of poly vinyl chloride (PVC), in which two pieces of CEM and one AEM were used to separate the four compartments: two electrode wash chambers (EW) (anolyte and catholyte), FC and PC as depicted in Fig. 1. The parallel-cum-series ow arrangement was used for the separation of AAs (GLU and LYS) from their mixture. Expanded TiO2 sheets coated with a triple precious metal oxide (titaniumrutheniumplatinum; 6.0 m thickness) of 1.5 mm thickness and 8.0 103 m2 area, received from Titanium Tantalum Products (TITAN, Chennai, India) were used as cathode and anode. Space between each electrode and the effective membrane area was 1.10 102 m and 8.0 103 m2 , respectively. Peristaltic pumps were used to feed the AA solution (500 cm3 ) in a recirculation mode into the respective compartment with a constant ow rate (60 cm3 h1 ) to maintain the turbulence. The whole setup was operated in ambient conditions (303 K) without any additional temperature control. The 0.1 mol L1 Na2 SO4 solution was recirculated into the EW chambers. The AA solution (GLU or LYS, separately or their mixture) of known concentration (0.010.05 mol L1 ) in NaAc buffer (0.01 mol L1 ) at known pH was fed into the FC, while NaAc buffer (0.01 mol L1 ) was fed into the PC. A dc power supply (Aplab, India, model L1285) was used to apply constant potential across the electrodes and the resulting current variations was recorded as a function of time using a multimeter.

RESULTS AND DISCUSSION

Electro-transport of GLU or LYS Electro-transport of GLU or LYS (individually) of 0.01 mol L1 concentration in acetate buffer at known pH was carried out in the EMP cell (Fig. 1) at different applied potential (4.06.0 V). AA (GLU or LYS) solution of different concentrations in acetate buffer was initially fed into the FC, while only acetate buffer was fed to the PC. Both EW streams were reconnected and 0.1 mol L1 Na2 SO4 solution was recirculated. Variation in current density with time during electro-transport of AA from FC to PC across AEM is presented in Fig. 3(A) and (B) for GLU and LYS, respectively, as a representative case. Experiments were performed at constant applied potential (4.06.0 V) and resulting variations in current and concentrations of AA in both compartments (FC and PC) were recorded as a function of time. At constant applied potential, current density initially increased and then decreased with time for both AA solutions. Initial feed concentration (0.01 mol L1 AA + 0.01 mol L1 NaAc) of FC and the electrode rinsing solution (0.1 mol L1 Na2 SO4 ) were higher than the concentration of permeate (0.01 mol L1 NaAc). In this case, buffer solution (NaAc) and electrode rinsing solution (0.1 mol L1 Na2 SO4 ) did not inuence the electro-migration of AA because the concentration of NaAc was the same in FC and PC. Migration of Na2 SO4 was prohibited due to the electro-neutral conditions. Thus the observed current of the EMP cell varied due to variation in the electro-migration of AA. A schematic of the EMP for separation of GLU or LYS or their mixture is shown in Fig. 1. Single step separation was achieved by electro-transport of AA from FC to PC through
12 B CEM 6 4 AEM 2 2 0 0 0.02 0.04 0.06 0 0.02 0.04 0.06 Conc. (M) Conc. (M) 0 0 0.02 0.04 0.06 Conc. (M) AEM C CEM 10 8 6 4 AEM CEM

30 A km 10-3 (S cm-1) 25 20 15 10 5 0

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Figure 2. Membrane conductivity ( m ) values for CEM and AEM in equilibration with: (A) NaCl; (B) GLU; and (C) LYS solutions of different concentrations.

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11 Current density (mA cm-2) A 10 14 9 8 7 6 4V 5 30 90 150 Time (min) 210 2 30 90 150 6V 5V 6 4V 10 5V 6V 18 B

M Kumar, BP Tripathi, VK Shahi

210

Figure 3. Variation of current density with time at different applied potential during electro-transport of: (A) GLU (0.01 mol L1 ; pH 8.0) (B) LYS (0.01 mol L1 ; pH 12) solutions, across the AEM.

12 A 10 8 pH FC

14 B 12 10 8 FC

6 6 4 2 0 50 100 Time (min) 150 200 PC 4 2 0 50 100 Time (min) 150 200 PC

Figure 4. pH variations of FC and PC with time in the EMP at 5.0V for (A) GLU (0.01 mol L1 ) (B) LYS (0.01 mol L1 ) solutions as feed for FC.

AEM under the inuence of the applied potential. Since, AA was separated from electrodes by CEMs; electro-migration of AA at specic pH would not occur because of the strongly charged nature of CEM. Electro-transport of AA at a particular pH was further checked by regularly monitoring AA concentration in the catholyte and anolyte using UV-visible spectrophotometer, and it was found to be absent. Under the inuence of the applied potential (below limiting current density), electro-transport of AA from FC to PC was also
12

veried by the variation of pH and solution conductivity for both streams (FC and PC) with time, and is presented in Figs 4 and 5 for GLU and LYS, respectively. The pH of PC decreased due to migration of AA from FC to PC. Increase in the pH of FC may be attributed to depletion of GLU and LYS+ in FC. The pI values of GLU and LYS are 3.22 and 9.59, respectively. At pH 8.0; GLU existed as negatively charged ion (GLU ), while at pH 12; LYS existed in the LYS state. GLU or LYS was electro-transported from FC to PC across AEM under the applied potential. Simultaneously, AA concentration

23

Conductivity (mS)

PC

18

FC

6 FC 3

13 PC

0 0 100 200 300 Time (min)

3 0 50 100 150 200

Figure 5. Variation of conductivity in FC and PC with time at 5.0 V during electro-transport of: (A) GLU (0.01 mol L1 ; pH 8.0) (B) LYS (0.01 mol L1 ; pH 12) solutions.

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EMP for the separation of amino acids by iso-electric focusing in FC decreased, and increased in PC. This phenomenon resulted in an increase in conductivity and AA concentration in PC. The conductivity of FC decreased with the time due to depletion of conducting species (AA ). These observations conrmed the electro-transport of AA across AEM from FC to PC in the EMP. The electro-transport rate can be assessed on the basis of GLU or LYS ux (J) in EMP. J values were estimated from the change of GLU or LYS concentration in FC or PC, considering negligible mass (water) transport through AEM, using the following equation:30,31 V a Ct C0 J= (4) A t where C0 and Ct is the initial and nal concentration of GLU /LYS in compartment 1 (mol m3 ), t is the time allowed for EMP (s), Va the total volume of solution in the permeate compartment (0.50 103 m3 ), and A is the effective membrane area. GLU and LYS ux during electro-transport are presented in Fig. 6(A) and Fig. 7(A), respectively, as a function of electricity passed (i.e. coulombs) at different applied potentials. In both cases at constant applied potential, ux initially increased linearly with electricity passed and then decreased, similarly to the variation of current density (Fig. 3(A)). Initially, concentration of AA in the PC was almost zero. At the start of the process, the concentration of GLU or LYS reduced in FC, and increased in PC with the amount of electricity passed. Over time the concentration of AA in the FC approached a minimum, while in the PC it was enhanced, which
12 10 J 10-5 (mol m-2 s-1) 8 6 4V 4 2 0 800 5 800 Recovery (%) 5V 65

www.soci.org further decreased ux value, similarly to the variation of current density. Thus we can say that the amount of current (charge) passed in the EMP at constant applied potential is also a measure of AA ux across the AEM in the EMP. Here, it is important to note that GLU ux was extremely high, whereas LYS ux across the AEM was very low under similar experimental conditions, in spite of their almost equal molecular weight. Recovery, current efciency (CE) and energy consumption (W) Recovery of product (GLU or LYS ) is an important parameter to examine the economic feasibility of any process, and may be dened as: Ctp Vtp 100 (5) Glu re covery(%) = C0F V0F GLU and LYS recovery (%) at different feed concentrations of AA into PC is presented in Fig. 6(B) and Fig. 7(B), respectively, as a function of electricity passed (coulombs). For both cases, recovery increased with quantity of electricity passed, and decreased with increasing concentration of AA in FC. Recovery of GLU was greater than that of LYS , maybe due to the extremely low migration velocity of LYS across the AEM because of its bulkier size. It was found that at constant applied potential, recovery of GLU decreased with increased concentration in FC because ux becomes independent of feed concentration above a certain minimum concentration.26 Under optimized experimental

6V

85

B 0.02M 0.01M 0.05M

45

25

2400 4000 5600 Electricity passed (Coulombs)

2400

4000

5600

Figure 6. Variation of: (A) ux (J) for GLU (0.01 mol L1 ; pH 8.0) through the AEM (from FC to PC) with electricity passed (coulombs) at different applied potentials; (B) recovery of GLU with electricity passed (coulombs) at 5.0V constant applied potential for different concentrations at pH 8.0; as feed of FC.

10 J 10-6 (mol m-2 s-1)

A 6V

18

B 0.01M

8 Recovery (%) 5V 6 4V 4

15 0.02M 12 0.05M 9

2 800

1800 2800 3800 Electricity passed (Coulombs)

6 800

1800

2800

3800

4800

Figure 7. Variation of (A) ux (J) for LYS solution (0.01 mol L1 ; pH 12) across the AEM with electricity passed (coulombs) at different applied potentials; (B) recovery of LYS with electricity passed (coulombs) at 5.0V constant applied potential for different concentrations at pH 12; as feed of FC.

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www.soci.org conditions, about 85% recovery of GLU during the process reveals the possibility of separating GLU from LYS using AEM in EMP. Energy consumption and current efciency (CE) are also important parameters for assessing the suitability of any EMP for practical applications. The energy consumption (W, kWh kg1 of AA separated) for EMP may be obtained as follows:31 W(kWhkg ) =
0 1 t

M Kumar, BP Tripathi, VK Shahi

Table 2. Energy consumption (W), current efciency (CE), and recovery data for electro-transport of GLU solution (pH 8.0) across the AEM under different experimental conditions GLU Conc. (mol L1 ) 0.01 0.01 0.01 0.02 0.05 Applied voltage (V) 4.0 5.0 6.0 5.0 5.0 W (kWhkg1 GLU separated) 5.4 7.1 8.1 9.4 10.2 CE (%) 37.0 48.7 60.5 39.2 31.6 Recovery (%) 50 72 95 70 38

V Idt m

(6)

The overall current efciency (CE) is dened as the fraction of charge utilized for the electro-migration of GLU from FC to PC: CE(%) = mnF 100 MQ (7)

An electrochemical process must not only be technically feasible, but should also be inexpensive and green in nature. To evaluate the technical and economic feasibility of the electrotransport of GLU or LYS from FC to PC across the AEM in an EMP, W, CE (%) and recovery (%) data under different experimental conditions are presented in Tables 2 and 3, respectively. W, CE (%) and recovery of GLU or LYS increased with applied differential potential. For both cases, with an increase in concentration of AA in FC, W increased, whereas CE (%) and recovery reduced at constant applied potential. At relatively high applied potential, high W and low CE may be explained by the following: (i) electrode reaction and water splitting at electrodes; (ii) simultaneously transport of solvent (water) induced by amino acid ions.26 Factors (i) and (ii) are responsible for an increase in energy consumption and low CE with increase in the applied potential. These parameters, along with the initial feed concentration of GLU/LYS in FC, strongly affected the EMP. With an increase in feed concentration of GLU/LYS, CE(%) and recovery decreased, while W was increased, possibly due to a barrier effect occurring at boundary layers. When the current density exceeds its limiting value, AEM provides the electric current by OH ions. This results in the accumulation of OH ions at the surface of the membrane in the FC, which blocks AA (GLU or LYS). pH decreases at the boundary of AEM and thus variations in pH result in recharging of ions of ampholyte (AA), which blocks their further migration through the membranes.42 45 Under optimum operating conditions, CE and W were found to be 60.5% and 5.38 kWh kg1 , respectively, which correspond to 85% recovery of GLU. Also, after several experiments, the electrochemical properties of

Table 3. Energy consumption (W), current efciency (CE), and recovery data for electro-transport of LYS in solution (pH 12) across the AEM under different experimental conditions LYS conc. (mol L1 ) 0.01 0.01 0.01 0.02 0.05 Applied voltage (V) 4.0 5.0 6.0 5.0 5.0 W (kWhkg1 LYS separated) 19.4 20.4 19.6 22.6 24.6 CE (%) 9.1 10.9 12.7 8.1 7.0 Recovery (%) 12 13 15 13 11

CEM and AEM were measured, showing a 1.0% deterioration. Thus any type of fouling of the IEM was completely ruled out. The electro-transport of LYS is not economically feasible for the separation of GLULYS mixtures due to a relatively high W and low CE and recovery.18 Based on these observations, it can be concluded that it is possible to separate GLU at pH 8.0 from a feed mixture of GLU and LYS in NaAC buffer solution. Effect of pH on electro-transport of GLU or LYS across AEM in EMP The effect of pH on electro-transport of AAs at constant applied potential (5.0 V) is depicted in Fig. 8(A) and (B) for GLU and LYS, respectively. It is obvious from Fig. 8(A), GLU+ ux (below pH 3.22) across AEM from comp. 1 to 2 towards anode was extremely low. The ux value steeply increased beyond pH 3.22 for GLU . Similarly, in Fig. 8(B), LYS+ showed extremely low ux across AEM,
1.6

7 6 J 10-5 (mol m-2 s-1) 5 4

1.2

0.8 3 2 1 0 1 3 5 7 9 pH 11 13 0 1 3 5 7 9 11 13 0.4

Figure 8. Effect of pH feed solution on the ux of amino acids across the AEM during their electro-transport (150 min): (A) GLU (0.01 mol L1 ); (B) LYS (0.01 mol L1 ), solutions at 5.0 V.

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EMP for the separation of amino acids by iso-electric focusing which increased steeply beyond pH 9.59 (pI of LYS) due to the electro-transport of LYS across AEM in the EMP. On the basis of the above observations, it was concluded that separation of a GLULYS mixture was possible at pH 8.0, because of the existence of oppositely charged states due to the difference in their pI values. Separation of amino acid (GLU-LYS) from their mixture in EMP The separation of GLU and LYS (0.01 mol L1 , each) from their mixture was carried out using the EMP cell (Fig. 1) at constant applied potential (5.0 V) and pH 8.0. Flux values for GLU and LYS are presented in Fig. 9(A) as a function of electricity passed (coulombs). GLU ux was high, while LYS ux across the AEM in the EMP was extremely low (less than 1.0 mol m2 s1 ). The separation factor (SF) can be dened as: SF = (J)GLU (J)LYS (8)

www.soci.org from PC to anolyte and catholyte was also monitored and both were found to be negligible. The selective separation of GLU and LYS from their equi-molar mixed solution (0.01 mol L1 ), was carried out at different pH values from 2.011.0 at 5.0 V. The resulting ux variations are presented as a function of pH in Fig. 10(A). Below pH 3.22, both GLU and LYS existed in a positively charged state, and thus their uxes were very low and similar across the AEM under given a electrical polarity. At pH 3.229.59, GLU existed in a negatively charged (GLU ) state, while LYS at pH 8.0 was in a positively charged state (LYS+ ). Thus electro-transport and ux of GLU across the AEM increased progressively, while the ux of LYS+ remained very low due to the diffusion through AEM. At pH 8.0 the difference in the uxes of GLU and LYS+ was high. Beyond pH 9.59, both AAs existed in a negatively charged state (GLU and LYS ), and their ux across the AEM was high but very similar. The SF values are presented as a function of pH in Fig. 10(B), showing the optimum pH 8.0 for selective separation of the AA mixture. The W, CE (%) and recovery (%) data when separating GLU at pH 8.0 from the equi-molar mixture of GLULYS at different feed concentrations are presented in Table 4. Under optimized experimental conditions (i.e. at 5.0 V applied potential, pH 8.0 for 0.01 mol L1 GLU+LYS mixture), 12.9 kWh kg1 energy was consumed for the separation of GLU from the equi-molar mixture of GLULYS, CE (65%) and recovery (85%) of the product, showed the economic feasibility of the process for industrial exploitation. It is clearly evident that separation of GLU and LYS can be efciently
8

Initially the SF value was low, and reached about 7.5 after passage of an appreciable amount of electricity (Fig. 9(B)). The positively charged membrane (AEM) allowed GLU ux under constant applied potential. Thus relatively high SF values reveal the feasibility of separating GLU and LYS at pH 8.0. For AAs GLU and LYS, transmission was strongly dependent on the nature of the charge on the transmitting species (pH), the charge nature of membranes and the electric gradient. Diffusion of LYS+ and GLU
8 J 10-5 (mol m-2 s-1)

A Separation factor (SF) 7 6 5 4

6 Glu 4

2 Lys 0 1000

2000 3000 4000 Electricity passed (Cuolombs)

3 1000

2000

3000

4000

Figure 9. Variation of (A) J; (B) separation factor, with electricity passed (coulombs) at constant applied potential (5.0V) for GLU and LYS mixed solution (0.01 mol L1 each) at pH 8.0 as feed of FC.

7 A 6 5 4 3 2 1 0 0 2 4 6 pH 8 10 12 LYS GLU Separation factor (SF) J 10-5 (mol m-2 s-1)

10 B 8 6 4 2 0 0 2 4 6 pH 8 10 12

Figure 10. Variation of (A) J; (B) separation factor, with pH of GLU and LYS mixed solution (0.01 mol L1 each) as feed into FC, after passage of 4000 coulombs electricity at constant applied potential (5.0 V).

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M Kumar, BP Tripathi, VK Shahi Also from data it is clearly evident that separation of GLU and LYS can be efciently achieved by using AEM under applied potential in EMP due to difference in their pI values, in spite of very close molecular weights. Membrane fouling and deterioration in membrane conductivity was not observed under the experimental conditions tested. This work clearly demonstrates the great potential of EMP for industrial applications.

Table 4. Energy consumption (W), current efciency (CE), and recovery data for electro-transport of GLU from equi-molar GLU+LYS mixed solution (pH 8.0) across the AEM under different experimental conditions GLU+LYS conc. of each (mol L1 ) 0.01 0.02 0.05 Applied voltage (V) 5.0 5.0 5.0 W(kWh kg1 GLU separated) 12.9 16.2 20.5 Recovery of GLU (%) 85 59 44

CE (%) 65.5 40.9 30.5

ACKNOWLEDGEMENTS
The authors thank the Department of Atomic Energy, Government of India for providing nancial assistance by sanctioning project no. 2007/35/35/BRNS. We also acknowledge the services of the Analytical Science Division, CSMCRI, Bhavnagar for instrumental support.

achieved using AEM under a constant applied potential in EMP due to the difference in their pI values, in spite of very close molecular weights and sizes. Thus the proposed EMP using IEM 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, while by focusing one component at its iso-electric point, and the other component in the oppositely charged state (depending on pH), they may be effectively separated. 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.

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CONCLUSIONS
Indigenously prepared CEM and AEM with good physicochemical and electrochemical properties and stabilities were used to develop an EMP for the separation of AAs with different pI values but similar molecular weights. Relatively good membrane conductivity values for both types of IEM in equilibration with NaCl, GLU or LYS solutions suggested their suitability for application in an EMP under the experimental environment. An EMP cell with four compartments (2-EWs, FC and PC) was fabricated, in which electrotransport of GLU and LYS across the AEM towards the anode side was studied to see the feasibility of their electro-transport under given experimental conditions. This is due to the fact that both the pH of AAs solutions and the electrostatic interaction between the zwitterionic AA molecules and membrane surface charge density play important roles in their electro-transport; thus, demonstrating the importance of employing IEMs in the EMP system to obtain AA separation by focusing their iso-electric points with high throughput (ux), purity, recovery and CE. It was observed that during electro-transport, GLU ux was extremely high, whereas LYS uxes across the AEM were very low under similar experimental conditions, in spite of their almost equal molecular weight and similar charged condition. Also, due to the relatively high W and low CE and recovery, electro-transport of LYS is not economically feasible, for the separation of GLULYS mixture. It was concluded that separation of GLU from the mixture of GLULYS is possible at pH 8.0, because of the existence of negatively charged state for both due to the difference in their pI values. Under optimized experimental conditions (i.e. at 5.0V constant applied potential, pH 10 for 0.01 mol L1 GLU+LYS mixture), 12.9 kWh kg1 energy was consumed for the separation of GLU from an equi-molar mixture of GLULYS, CE (65%) and recovery (85%) of the product, showed the economic feasibility of the process for industrial exploitation.

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EMP for the separation of amino acids by iso-electric focusing

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