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

64

8

Research ArticleReceived: 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 separationof amino acids by iso-electric focusingMahendra Kumar, Bijay P. Tripathi and Vinod K. Shahi∗

Abstract

BACKGROUND: Amino acids (AAs) are usually produced commercially using chemical, biochemical and microbiologicalfermentation methods. The product obtained from these methods undergoes various treatments involving extraction andelectrodialysis (ED) for salt removal and AA recovery. This paper describes an electro-membrane process (EMP) for the chargebased separation of amino acids.

RESULTS: Iso-electric separation of AAs (GLU–LYS) from their mixture, using ion- exchange membranes (IEMs) has beenachieved by an efficient and indigenous EMP. It was observed that electro-transport rate (flux) 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 GLU–LYS mixturewas achieved with moderate energy consumption (12.9 kWh kg−1), high current efficiency (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 separationof GLU–LYS mixture was possible at pH 8.0, because of the oppositely charged nature of the two amino acids due to theirdifferent pI values. Moreover, any type of membrane fouling and deterioration in membrane conductivity was ruled out underexperimental 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

NOTATIONCE Current efficiency

tmi Counter-ion transport number

κm Specific membrane conductivity

Wd Weight of dry membrane

Ww Weight of wet membrane

R Gas constant (8.314 J mol−1 K−1)

a1 and a2 Activities of electrolyte solutions

T Absolute temperature (K)

F Faraday constant (96500 coulombs)

�x, area Thickness of the wet membrane (cm)

A Electrode area (cm2)

V0F and Vtp Initial and final volume of the FC (cm3)

C0F and CtF Initial and final concentration of AA in PC

(M)

W Energy consumption (kWh kg−1)

V Cell voltage (V)

I Current (A)

t Time (s)

m Weight of GLU or LYS (g)

M Molecular weight of GLU or LYS, n t

n Stoichiometric number (n = 1 in this case)

Q Electric quantity passed (Coulombs; A s)

(J)GLU Flux of GLU (mol m−2 s−1)

(J)LYS Flux of LYS (mol m−2 s−1)

INTRODUCTIONWith the advance in life science research, numbers of biomoleculeswith pharmaceutical characteristics have been discovered. In thedown-stream process, separation of amino acids (AA) (amphotericin nature with more than one ionizable group, fundamentalconstituents of all proteins) was stimulated by an increasingdemand for high purity in order to reduce the side effects inducedby impurities.1 – 5 During recent years, a noticeable growth of AAproduction from molasses and raw sugar by a fermentation pro-cess, and their utilization as an additive in food products, chemicaland pharmaceutical industries, has occurred.6 – 10 In particular,glutamic acid (GLU) and lysine (LYS) have found a wide rangeof applications in the food, pharmaceutical, biotechnology, bio-chemistry and cosmetic industries.11 – 13 Various membrane basedprocesses, such as ion-exchange, nanofiltration (NF), diafiltration,ultrafiltration (UF), and electrodialysis (ED), were used for the sepa-ration of AAs from fermentation broths without precipitation.14 – 18

Use of acid and base to regenerate resins in the ion-exchangeprocess is a disadvantage because it increases the operationalcost. In addition, wastewater produced during resin regeneration

∗ Correspondence to: 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.E-mail: [email protected]; [email protected]

Electro-Membrane Processes Division,Central Salt & Marine Chemicals ResearchInstitute,CouncilofScientific&IndustrialResearch(CSIR),G.B.Marg,Bhavnagar-364002 (Gujarat) India

J Chem Technol Biotechnol 2010; 85: 648–657 www.soci.org c© 2010 Society of Chemical Industry

64

9

EMP for the separation of amino acids by iso-electric focusing www.soci.org

Electrode Wash

0.1M Na2SO4

GLU + LYS

in NaAC Buffer

NaAC Buffer

Electrode WashGLULYS

CEM CEMAEM

GLU-

LYS

FeedComp.(FC)

PermeateComp.(PC)

Ano

de

Cat

hode

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

and washing causes serious environmental pollution. Membraneprocesses such as NF and UF, are size based separation processesand their separation performance is not high unless the molecularweight (MW) ratio of the two components is larger than 10.17,19

In ED, ions are transported through ion-exchange membranes(IEMs) under the influence of an applied potential to separateionic and nonionic constituents.20,21 Recently, bipolar membraneelectodialysis (BMED) processes have been developed for theconversion of salts into corresponding acid and base.22 – 24 BMEDprocesses have also been found to be suitable and cost effective forrecovering organic acid or AAs from their respective salt.25 – 27 Saltdiffusion (co-ion leakage) through the bipolar membrane in BMEDis a serious problem, which affects the purity of the product andenhances the process cost. There is no ion-exchange membranewith 100% selectivity. There is no electro-membrane process (EMP)based on the principles of ED, used for the efficient separation ofamino acids, with close MWs and different iso-electric points.28 – 31

Working principle of the electro-membrane process (EMP)The iso-electric focusing technology has been used extensively inseparation of protein or AAs from fermentation broths with greatsuccess. The performance is mainly determined by the net chargeof molecules at different pH and nature of the membranes.15,32 – 34

A schematic diagram of the EMP cell used for the separation of AAsfrom their mixture is illustrated in Fig. 1. The feed and permeatechambers were partitioned by an anion-exchange membrane(AEM) which only allows the passage of anions. Both electrodechambers (catholyte, anolyte) were separated by cation-exchangemembranes (CEMs), which prevent the approach of AA moleculesto the electrode and further denaturation by electrode reaction.Thus, at pH<pI; GLU (pI = 3.22) existed in GLU+, while inthe negatively charged state (GLU−) above its pI, according tofollowing scheme.

Similarly, LYS existed in the LYS+ state below pH 9.59 (pI of LYS),and in the LYS− state at pH>pI.

+H2N +H3NCC

(CH2)2

O

OH

COOH

H

CC

(CH2)2

O

O-

COOH

H

H2N CC

(CH2)2

O

O-

COOH

H

Cationic (<pI) Zwetterionic (pI), MW:147.13 Anionic (>pI)

C COO-H2N

H

CH2)4

NH3+

C COO-H2N

H

CH2)4

NH2

C COO-+H3N

H

CH2)4

NH3+

Cationic (<pI) Zwetterionic (pI) ,MW:146.19 Anionic (>pI)

J Chem Technol Biotechnol 2010; 85: 648–657 c© 2010 Society of Chemical Industry www.interscience.wiley.com/jctb

65

0

www.soci.org M Kumar, BP Tripathi, VK Shahi

At pH 8.0, GLU existed in the GLU− state and LYS in the LYS+

state. There was the possibility for migration of LYS+ towardsthe catholyte through CEM. But, at pH 8.0 (near to its pI), due tothe small net positive charge on LYS, its electro-migration fromthe feed compartment (FC) to the catholyte was not feasible.Further, the presence of LYS+ in the catholyte was checked andfound to be negligible. Also, LYS+ remained in the FC due toits electrical polarity and repulsion between positively chargedAEM. Also, this observation was validated by the mass balanceof LYS in FC before and after the experiments: mass balance wasfound to be about 95%. GLU− passed through the AEM fromthe FC to the permeate compartment (PC), under the appliedelectric potential. Thus, high-purity GLU may be obtained in thePC. For high resolution AA separation, a stable and continuouspH gradient with relatively constant conductivity and high buffercapacity is required. Ampholyte was commonly used to provide astable pH gradient in the traditional iso-electric focusing systemdue to various advantages, such as high buffer capacity, highsolubility, good conductivity at pI ofAAs, and absence of biologicaleffects.

Therefore, the purpose of this work is to investigate an EMP,based on the principles of ED to achieve the separation of AAsabove or below the pI of one component in spite of their closeMW and thus molecular size. Herein, we report an EMP for theseparation of AAs from their mixture by iso-electric focusing usingCEM and AEM as a separation media. A mixture of GLU and LYS insolution was studied as a model case under similar experimentalconditions.

MATERIALS AND METHODSMaterialsPoly (ether sulfone) (PES) was obtained from Sigma-AldrichChemicals (Mumbai, India) and all other reagents such as GLU,LYS, H2SO4, NaCl, CdCl2.H2O, DMF, acetic acid, CH3COONa(NaAc), ninhydrine, of Analytical grade reagents (AR) grade wereobtained from S.D. Fine Chemicals, India, and used without furtherpurification.

Membranes preparationSulfonation of PES was carried out as reported earlier.35 CEMwas prepared using sulfonated poly (ether sulfone) (SPES) indimethylformamide (DMF) (20%, w/v) and further by casting ontoa cleaned glass plate. The prepared membrane was dried inambient 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 aprocedure reported earlier and used for various processes.28 – 31,36

Thus, the membranes obtained (CEM and AEM) were equilibratedwith 1.0 mol L−1 HCl and 1.0 mol L−1 NaOH solution before use.The cleaned and equilibrated membranes were stored in doubledistilled water.

Electrochemical properties of membranesThe thickness of the wet membrane was measured by micrometerwith 0.10 µm accuracy. The membrane water content wasdetermined from the weight of dry and wet membrane. The weightof dry membrane was recorded after 24 h drying at 60 ◦C, and theweight of the wet membrane was determined after equilibratingthe membrane in water for 24 h, with surface water removed using

Table 1. Physicochemical and electrochemical properties of the CEMand AEM

Properties CEM AEM

Thickness1 (µm) 150 150

Water content2 (%) 14.6 10.8

Ion-exchange capacity3

(mequiv./g of drymembrane)

1.03 1.28

Counter-ion transportnumbera,4 (tm− )

0.96 0.91

Specific membraneconductivityb,5

(S cm−1)

2.05 × 10−2 1.12 × 10−2

a Measured by membrane potential in equilibration in with 0.01 mol L−1

and 0.1 mol L−1 NaCl solutions.b Measured in equilibration with 0.1 mol L−1 NaCl solution.Uncertainty for measurements: 1: 1.0 µm; 2: 0.1%; 3: 0.01 mequiv g−1

of dry membrane; 4: 0.01; and 5: 0.01 × 10−3 S cm.

tissue paper. The water content was estimated from:

Water content (%) = Ww − Wd

Wd× 100 (1)

For estimation of the ion-exchange capacity (IEC), a piece ofthe membrane was equilibrated in 1.0 mol L−1 HCl or 1.0 mol L−1

NaOH solution overnight to convert them into H+ or OH− form.The excess acid and base was removed by washing with distilledwater. The washed membranes were then equilibrated in 50 mLof 0.50 mol L−1 NaCl solutions to exchange the H+/OH− byNa+/Cl−. The amount of H+ or OH− ions liberated in solutionwas determined by acid–base titration.37

tmi across the membranes was estimated by membrane potential

measurements in equilibration with 0.01 and 0.10 mol L−1 NaClsolutions, according to following equation:31

Em = (2tm− − 1)

RT

Fln

a1

a2(2)

The physicochemical and electrochemical properties of CEMand AEM are presented in Table 1. Both membranes exhibitedgood water content, IEC and counter-ion transport number inthe membrane phase under operating conditions. Properties ofthese membranes are comparable with the best-known IEMs.38

The knowledge on membrane conductivity in an actual operatingenvironment is an essential parameter to assess the suitability ofmembranes for an EMP.

Membrane conductivity measurements for CEM and AEM werecarried out in equilibration with GLU, LYS and NaCl solutionof different concentrations using a potentiostat/galvanostatfrequency response analyzer (Auto Lab, Model PGSTAT 30,EcoChemie, B.V. Utrecht, The Netherlands). The membrane wassandwiched between two stainless steel circular electrodes(4 cm2). Direct current (dc) and sinusoidal alternating currents(ac) were supplied to the respective electrodes to record thefrequency at a scanning rate of 1.0 µA s−1 within a frequencyrange 106 to 10−3 Hz.39 The membrane resistances were obtainedfrom Nyquist plots using a fit and simulation method. Membraneconductivity was recorded in equilibration with NaCl, GLU andLYS solutions of different concentrations. The specific membrane

www.interscience.wiley.com/jctb c© 2010 Society of Chemical Industry J Chem Technol Biotechnol 2010; 85: 648–657

65

1


conductivity (κm) was estimated by the given equation:

κm = �x

ARm (3)

The variation of κm for both type membranes (CEM and AEM)in equilibration with NaCl, GLU, and LYS solutions of differentconcentrations are shown in Fig. 2(A, B and C), respectively. κm

values for both types of IEMs increased with concentration,and were highly dependent on the nature of equilibratingenvironment and ionic concentration in the membrane/solutioninterfacial zone. Both membranes exhibited excellent conductivityin equilibration with NaCl solution, but κm values were relativelylow in equilibration with GLU or LYS solutions of similarconcentration. This may be explained due to extremely lowdissociation 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 byiso-electric focusingAn experimental cell used for EMP was made up of poly vinylchloride (PVC), in which two pieces of CEM and one AEMwere used to separate the four compartments: two electrodewash chambers (EW) (anolyte and catholyte), FC and PC asdepicted in Fig. 1. The parallel-cum-series flow arrangementwas used for the separation of AAs (GLU and LYS) from theirmixture. Expanded TiO2 sheets coated with a triple preciousmetal oxide (titanium–ruthenium–platinum; 6.0 µm thickness)of 1.5 mm thickness and 8.0 × 10−3 m2 area, received fromTitanium Tantalum Products (TITAN, Chennai, India) were usedas cathode and anode. Space between each electrode and theeffective membrane area was 1.10 × 10−2 m and 8.0 × 10−3 m2,respectively. Peristaltic pumps were used to feed the AA solution(500 cm3) in a recirculation mode into the respective compartmentwith a constant flow rate (60 cm3 h−1) to maintain the turbulence.The whole setup was operated in ambient conditions (303 K)without any additional temperature control. The 0.1 mol L−1

Na2SO4 solution was recirculated into the EW chambers. TheAA solution (GLU or LYS, separately or their mixture) of knownconcentration (0.01–0.05 mol L−1) in NaAc buffer (0.01 mol L−1)at known pH was fed into the FC, while NaAc buffer (0.01 mol L−1)was fed into the PC. A dc power supply (Aplab, India, model L1285)was used to apply constant potential across the electrodes andthe resulting current variations was recorded as a function of timeusing a multimeter.

Under the influence of applied potential, amino acid withnegative net charge (AA− ; depending on pH) was electro-transported from FC to PC, across AEM. Solution conductivity andpH changes of each compartment were regularly monitored duringall the experiments. For individual AA solution, concentrationof amino acid (GLU or LYS) was determined by UV-visiblespectrophotometer (Shimadzu, Japan) at λmax 504 nm fixedwavelength.41 In a mixture case, AA concentration was analyzed byhigh performance liquid chromatography (HPLC) after precolumnderivitization using dansyl chloride. A mixture of acetonitrile andacetate buffer (pH= 4.5; 0.045 mol L−1) was used as the mobilephase flowing through a C18 reverse phase column. The solventgradient was 20% to 60% acetonotrile over 30 min. In all casesan equal volume of AAs solution (separately or their mixture) wastaken for simplicity and to study the feasibility of their separation,above the iso-electric point of the AAs.

RESULTS AND DISCUSSIONElectro-transport of GLU or LYSElectro-transport of GLU or LYS (individually) of 0.01 mol L−1

concentration in acetate buffer at known pH was carried outin the EMP cell (Fig. 1) at different applied potential (4.0–6.0 V).AA (GLU or LYS) solution of different concentrations in acetatebuffer was initially fed into the FC, while only acetate buffer wasfed to the PC. Both EW streams were reconnected and 0.1 mol L−1

Na2SO4 solution was recirculated. Variation in current density withtime during electro-transport of AA from FC to PC across AEMis presented in Fig. 3(A) and (B) for GLU and LYS, respectively, asa representative case. Experiments were performed at constantapplied potential (4.0–6.0 V) and resulting variations in currentand concentrations of AA in both compartments (FC and PC) wererecorded as a function of time. At constant applied potential,current density initially increased and then decreased with timefor both AA solutions. Initial feed concentration (0.01 mol L−1 AA+ 0.01 mol L−1 NaAc) of FC and the electrode rinsing solution(0.1 mol L−1 Na2SO4) were higher than the concentration ofpermeate (0.01 mol L−1 NaAc). In this case, buffer solution (NaAc)and electrode rinsing solution (0.1 mol L−1 Na2SO4) did notinfluence the electro-migration of AA because the concentrationof NaAc was the same in FC and PC. Migration of Na2SO4

was prohibited due to the electro-neutral conditions. Thus theobserved current of the EMP cell varied due to variation in theelectro-migration of AA. A schematic of the EMP for separation ofGLU or LYS or their mixture is shown in Fig. 1. Single step separationwas achieved by electro-transport of AA− from FC to PC through

0

5

10

15

20

25

30

0 0.02 0.04 0.06

Conc. (M)

0 0.02 0.04 0.06

Conc. (M)

0 0.02 0.04 0.06

Conc. (M)

km ×

10-3

(S

cm

-1)

AEM

CEM

A

0

2

4

6

8

10

12

AEM

CEM

C

0

2

4

6

8

AEM

CEMB

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


65

2


5

6

7

8

9

10

11

30 90 150 210

Time (min)

30 90 150 210

Cur

rent

den

sity

(m

A c

m-2

)

4 V

5 V

6 V

A

2

6

10

14

18

4 V

5 V6 V

B

Figure 3. Variation of current density with time at different applied potential during electro-transport of: (A) GLU (0.01 mol L−1; pH 8.0) (B) LYS(0.01 mol L−1; pH 12) solutions, across the AEM.

2

4

6

8

10

12

14

FC

PC

B

2

4

6

8

10

12

0 50 100 150 200 0 50 100 150 200

Time (min) Time (min)

pH

FC

PC

A

Figure 4. pH variations of FC and PC with time in the EMP at 5.0V for (A) GLU (0.01 mol L−1) (B) LYS (0.01 mol L−1) solutions as feed for FC.

AEM under the influence of the applied potential. Since, AA wasseparated from electrodes by CEMs; electro-migration of AA− atspecific pH would not occur because of the strongly chargednature of CEM. Electro-transport of AA− at a particular pH wasfurther checked by regularly monitoring AA− concentration in thecatholyte and anolyte using UV-visible spectrophotometer, and itwas found to be absent.

Under the influence of the applied potential (below limitingcurrent density), electro-transport of AA− from FC to PC was also

verified by the variation of pH and solution conductivity for bothstreams (FC and PC) with time, and is presented in Figs 4 and 5 forGLU and LYS, respectively. The pH of PC decreased due to migrationof AA− from FC to PC. Increase in the pH of FC may be attributedto depletion of GLU− and LYS+ in FC. The pI values of GLU and LYSare 3.22 and 9.59, respectively. At pH 8.0; GLU existed as negativelycharged ion (GLU−), while at pH 12; LYS existed in the LYS− state.GLU− or LYS− was electro-transported from FC to PC across AEMunder the applied potential. Simultaneously, AA− concentration

0

3

6

9

12

0 100 200 300

Time (min)

0 50 100 150 200

Con

duct

ivity

(m

S)

FC

PC

A

3

8

13

18

23

FC

PC

B

Figure 5. Variation of conductivity in FC and PC with time at 5.0 V during electro-transport of: (A) GLU (0.01 mol L−1; pH 8.0) (B) LYS (0.01 mol L−1; pH 12)solutions.


65

3


in FC decreased, and increased in PC. This phenomenon resultedin an increase in conductivity and AA− concentration in PC. Theconductivity of FC decreased with the time due to depletionof conducting species (AA−). These observations confirmed theelectro-transport of AA− across AEM from FC to PC in the EMP.

The electro-transport rate can be assessed on the basis ofGLU− or LYS− flux (J) in EMP. J values were estimated from thechange of GLU− or LYS− concentration in FC or PC, consideringnegligible mass (water) transport through AEM, using the followingequation:30,31

J = Va

A

Ct − C0

�t(4)

where C0 and Ct is the initial and final concentration of GLU−/LYS−

in compartment 1 (mol m−3), �t is the time allowed for EMP (s),Va the total volume of solution in the permeate compartment(0.50 × 10−3 m3), and A is the effective membrane area. GLU−and LYS− flux 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 constantapplied potential, flux initially increased linearly with electricitypassed and then decreased, similarly to the variation of currentdensity (Fig. 3(A)). Initially, concentration of AA− in the PC wasalmost zero. At the start of the process, the concentration of GLU−

or LYS− reduced in FC, and increased in PC with the amount ofelectricity passed. Over time the concentration of AA− in the FCapproached a minimum, while in the PC it was enhanced, which

further decreased flux value, similarly to the variation of currentdensity. Thus we can say that the amount of current (charge)passed in the EMP at constant applied potential is also a measureof AA− flux across the AEM in the EMP. Here, it is important to notethat GLU− flux was extremely high, whereas LYS flux across theAEM was very low under similar experimental conditions, in spiteof their almost equal molecular weight.

Recovery, current efficiency (CE) and energy consumption (W)Recovery of product (GLU− or LYS−) is an important parameterto examine the economic feasibility of any process, and may bedefined as:

Glu re covery(%) = CtpVtp

C0FV0F× 100 (5)

GLU− and LYS− recovery (%) at different feed concentrations ofAA into PC is presented in Fig. 6(B) and Fig. 7(B), respectively, as afunction of electricity passed (coulombs). For both cases, recoveryincreased with quantity of electricity passed, and decreasedwith increasing concentration of AA in FC. Recovery of GLU−was greater than that of LYS−, maybe due to the extremelylow migration velocity of LYS− across the AEM because of itsbulkier size. It was found that at constant applied potential,recovery of GLU− decreased with increased concentration in FCbecause flux becomes independent of feed concentration above acertain minimum concentration.26 Under optimized experimental

0

2

4

6

8

10

12

800 2400 4000 5600 800 2400 4000 5600

Electricity passed (Coulombs)

J ×

10-5

(m

ol m

-2 s

-1)

4 V

5 V

6 V

5

25

45

65

85

Rec

over

y (%

) 0.01M

0.02M

0.05M

BA

Figure 6. Variation of: (A) flux (J) for GLU− (0.01 mol L−1; pH 8.0) through the AEM (from FC to PC) with electricity passed (coulombs) at different appliedpotentials; (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.

6

9

12

15

18

Rec

over

y (%

)

0.01M

0.02M

0.05M

B

2

4

6

8

10

800 1800 2800 3800

Electricity passed (Coulombs)

800 1800 2800 3800 4800

J ×

10-6

(m

ol m

-2 s

-1)

4 V

5 V

6 VA

Figure 7. Variation of (A) flux (J) for LYS solution (0.01 mol L−1; 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.


65

4


conditions, about 85% recovery of GLU− during the process revealsthe possibility of separating GLU from LYS using AEM in EMP.

Energy consumption and current efficiency (CE) are alsoimportant parameters for assessing the suitability of any EMPfor practical applications. The energy consumption (W , kWh kg−1

of AA separated) for EMP may be obtained as follows:31

W(kWhkg−1) =∫ t

0

V Idt

m(6)

The overall current efficiency (CE) is defined as the fraction ofcharge utilized for the electro-migration of GLU from FC to PC:

CE(%) = m n F

M Q× 100 (7)

An electrochemical process must not only be technicallyfeasible, but should also be inexpensive and green in nature.To evaluate the technical and economic feasibility of the electro-transport of GLU− or LYS− from FC to PC across the AEM in anEMP, W, CE (%) and recovery (%) data under different experimentalconditions are presented in Tables 2 and 3, respectively. W, CE (%)and recovery of GLU− or LYS− increased with applied differentialpotential. For both cases, with an increase in concentration of AA inFC, W increased, whereas CE (%) and recovery reduced at constantapplied potential. At relatively high applied potential, high W andlow CE may be explained by the following: (i) electrode reactionand water splitting at electrodes; (ii) simultaneously transport ofsolvent (water) induced by amino acid ions.26 Factors (i) and (ii) areresponsible for an increase in energy consumption and low CE withincrease in the applied potential. These parameters, along withthe initial feed concentration of GLU/LYS in FC, strongly affectedthe EMP. With an increase in feed concentration of GLU/LYS, CE(%)and recovery decreased, while W was increased, possibly due toa barrier effect occurring at boundary layers. When the currentdensity exceeds its limiting value, AEM provides the electric currentby OH− ions. This results in the accumulation of OH− ions at thesurface of the membrane in the FC, which blocks AA (GLU or LYS).pH decreases at the boundary of AEM and thus variations in pHresult in recharging of ions of ampholyte (AA), which blocks theirfurther migration through the membranes.42 – 45 Under optimumoperating conditions, CE and W were found to be 60.5% and 5.38kWh kg−1, respectively, which correspond to 85% recovery of GLU.Also, after several experiments, the electrochemical properties of

Table 2. Energy consumption (W), current efficiency (CE), andrecovery data for electro-transport of GLU solution (pH 8.0) acrossthe AEM under different experimental conditions

GLU Conc.(mol L−1)

Appliedvoltage (V)

W (kWhkg−1GLUseparated) CE (%)

Recovery(%)

0.01 4.0 5.4 37.0 50

0.01 5.0 7.1 48.7 72

0.01 6.0 8.1 60.5 95

0.02 5.0 9.4 39.2 70

0.05 5.0 10.2 31.6 38

Table 3. Energy consumption (W), current efficiency (CE), andrecovery data for electro-transport of LYS in solution (pH 12) across theAEM under different experimental conditions

LYS conc.(mol L−1)

Appliedvoltage (V)

W (kWhkg−1LYSseparated) CE (%)

Recovery(%)

0.01 4.0 19.4 9.1 12

0.01 5.0 20.4 10.9 13

0.01 6.0 19.6 12.7 15

0.02 5.0 22.6 8.1 13

0.05 5.0 24.6 7.0 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 theseparation of GLU–LYS mixtures due to a relatively high W andlow CE and recovery.18 Based on these observations, it can beconcluded that it is possible to separate GLU at pH 8.0 from a feedmixture of GLU and LYS in NaAC buffer solution.

Effect of pH on electro-transport of GLU or LYS across AEMin EMPThe effect of pH on electro-transport of AAs at constant appliedpotential (5.0 V) is depicted in Fig. 8(A) and (B) for GLU and LYS,respectively. It is obvious from Fig. 8(A), GLU+ flux (below pH3.22) across AEM from comp. 1 to 2 towards anode was extremelylow. The flux value steeply increased beyond pH 3.22 for GLU−.Similarly, in Fig. 8(B), LYS+ showed extremely low flux across AEM,

0

1

2

3

4

5

6

7

1 9753 11 13

pH

1 9753 11 13

J ×

10-5

(m

ol m

-2 s

-1)

A

0

0.4

0.8

1.2

1.6 B

Figure 8. Effect of pH feed solution on the flux of amino acids across the AEM during their electro-transport (150 min): (A) GLU (0.01 mol L−1); (B) LYS(0.01 mol L−1), solutions at 5.0 V.


65

5


which increased steeply beyond pH 9.59 (pI of LYS) due to theelectro-transport of LYS− across AEM in the EMP. On the basisof the above observations, it was concluded that separation of aGLU–LYS mixture was possible at pH 8.0, because of the existenceof oppositely charged states due to the difference in their pI values.

Separation of amino acid (GLU-LYS) from their mixture in EMPThe separation of GLU and LYS (0.01 mol L−1, each) from theirmixture was carried out using the EMP cell (Fig. 1) at constantapplied potential (5.0 V) and pH 8.0. Flux values for GLU andLYS are presented in Fig. 9(A) as a function of electricity passed(coulombs). GLU flux was high, while LYS flux across the AEM in theEMP was extremely low (less than 1.0 mol m2 s−1). The separationfactor (SF) can be defined as:

SF = (J)GLU

/(J)LYS (8)

Initially the SF value was low, and reached about 7.5 afterpassage of an appreciable amount of electricity (Fig. 9(B)). Thepositively charged membrane (AEM) allowed GLU− flux underconstant applied potential. Thus relatively high SF values revealthe feasibility of separating GLU and LYS at pH 8.0. For AAs GLUand LYS, transmission was strongly dependent on the nature ofthe charge on the transmitting species (pH), the charge nature ofmembranes and the electric gradient. Diffusion of LYS+ and GLU−

from PC to anolyte and catholyte was also monitored and bothwere found to be negligible.

The selective separation of GLU and LYS from their equi-molarmixed solution (0.01 mol L−1), was carried out at different pHvalues from 2.0–11.0 at 5.0 V. The resulting flux variations arepresented as a function of pH in Fig. 10(A). Below pH 3.22, bothGLU and LYS existed in a positively charged state, and thus theirfluxes were very low and similar across the AEM under given aelectrical polarity. At pH 3.22–9.59, GLU existed in a negativelycharged (GLU−) state, while LYS at pH 8.0 was in a positivelycharged state (LYS+). Thus electro-transport and flux of GLU−across the AEM increased progressively, while the flux of LYS+remained very low due to the diffusion through AEM. At pH 8.0the difference in the fluxes of GLU− and LYS+ was high. BeyondpH 9.59, both AAs existed in a negatively charged state (GLU− andLYS−), and their flux 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 AAmixture. The W , CE (%) and recovery (%) data when separating GLUat pH 8.0 from the equi-molar mixture of GLU–LYS at differentfeed concentrations are presented in Table 4. Under optimizedexperimental conditions (i.e. at 5.0 V applied potential, pH 8.0for 0.01 mol L−1 GLU+LYS mixture), 12.9 kWh kg−1 energy wasconsumed for the separation of GLU from the equi-molar mixtureof GLU–LYS, CE (65%) and recovery (85%) of the product, showedthe economic feasibility of the process for industrial exploitation. Itis clearly evident that separation of GLU and LYS can be efficiently

0

2

4

6

8

1000 2000 3000 4000

Electricity passed (Cuolombs)

1000 2000 3000 4000

J ×

10-5

(m

ol m

-2 s

-1)

Glu

Lys

A

3

4

5

6

7

8

Sep

arat

ion

fact

or (

SF

)

B

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 L−1 each) at pH 8.0 as feed of FC.

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12

J ×

10-5

(m

ol m

-2 s

-1)

pH

0 2 4 6 8 10 12

pH

GLU

LYS

A

0

2

4

6

8

10

Sep

arat

ion

fact

or (

SF

)

B

Figure 10. Variation of (A) J; (B) separation factor, with pH of GLU and LYS mixed solution (0.01 mol L−1 each) as feed into FC, after passage of 4000coulombs electricity at constant applied potential (5.0 V).


65

6


Table 4. Energy consumption (W), current efficiency (CE), andrecovery data for electro-transport of GLU from equi-molar GLU+LYSmixed solution (pH 8.0) across the AEM under different experimentalconditions

GLU+LYSconc. of each(mol L−1)

Appliedvoltage

(V)W(kWh kg−1GLU

separated) CE (%)Recovery of

GLU (%)

0.01 5.0 12.9 65.5 85

0.02 5.0 16.2 40.9 59

0.05 5.0 20.5 30.5 44

achieved using AEM under a constant applied potential in EMPdue to the difference in their pI values, in spite of very closemolecular weights and sizes. Thus the proposed EMP using IEM isan important tool for separating AAs with close molecular weightsby focusing on their iso-electric values.

In this process no appreciable membrane fouling or AAsdenaturation was observed, while by focusing one componentat its iso-electric point, and the other component in the oppositelycharged state (depending on pH), they may be effectivelyseparated. In this particular case the difference in the pI values ofGLU and LYS was quite high, but the method could be appliedin cases where pI values are very similar, by very careful controlof pH.

CONCLUSIONSIndigenously prepared CEM and AEM with good physicochemicaland electrochemical properties and stabilities were used todevelop an EMP for the separation of AAs with different pIvalues but similar molecular weights. Relatively good membraneconductivity values for both types of IEM in equilibration with NaCl,GLU or LYS solutions suggested their suitability for application inan EMP under the experimental environment. An EMP cell with fourcompartments (2-EWs, FC and PC) was fabricated, in which electro-transport of GLU− and LYS− across the AEM towards the anodeside was studied to see the feasibility of their electro-transportunder given experimental conditions. This is due to the fact thatboth the pH of AAs solutions and the electrostatic interactionbetween the zwitterionic AA molecules and membrane surfacecharge density play important roles in their electro-transport;thus, demonstrating the importance of employing IEMs in theEMP system to obtain AA separation by focusing their iso-electricpoints with high throughput (flux), purity, recovery and CE. It wasobserved that during electro-transport, GLU− flux was extremelyhigh, whereas LYS− fluxes across the AEM were very low undersimilar experimental conditions, in spite of their almost equalmolecular weight and similar charged condition. Also, due to therelatively high W and low CE and recovery, electro-transport ofLYS− is not economically feasible, for the separation of GLU–LYSmixture. It was concluded that separation of GLU from the mixtureof GLU–LYS is possible at pH 8.0, because of the existenceof negatively charged state for both due to the difference intheir pI values. Under optimized experimental conditions (i.e. at5.0V constant applied potential, pH 10 for 0.01 mol L−1 GLU+LYSmixture), 12.9 kWh kg−1 energy was consumed for the separationof GLU from an equi-molar mixture of GLU–LYS, CE (65%) andrecovery (85%) of the product, showed the economic feasibility ofthe process for industrial exploitation.

Also from data it is clearly evident that separation of GLUand LYS can be efficiently achieved by using AEM under appliedpotential in EMP due to difference in their pI values, in spite of veryclose molecular weights. Membrane fouling and deterioration inmembrane conductivity was not observed under the experimentalconditions tested. This work clearly demonstrates the greatpotential of EMP for industrial applications.

ACKNOWLEDGEMENTSThe authors thank the Department of Atomic Energy, Governmentof India for providing financial assistance by sanctioning projectno. 2007/35/35/BRNS. We also acknowledge the services of theAnalytical Science Division, CSMCRI, Bhavnagar for instrumentalsupport.

REFERENCES1 Sato K, Effects of the feed solution concentration on the separation

degree in Donnan dialysis for binary systems of amino acids.J Membr Sci 196:211–220 (2002).

2 Minagawa M and Tanioka A, Leucine transport through cationexchange membranes: effects of HCl concentration on interfacialtransport. J Colloid Interface Sci 198:149–154 (2002).

3 Kang MS, Cho SH, Kim SH, Choi YJ and Moon SH, Electrodialyticseparation characteristics of large molecular organic acid inhighly water-swollen cation-exchange membranes. J Membr Sci222:149–161 (2003).

4 Zydney AL and Pujar NS, Protein transport through porousmembranes: effects of colloidal interactions. Colloid Surface A138:133–143 (1998).

5 Shahi VK, Thampy SK and Rangarajan R, Chronopotentiometricstudies on dialytic properties of glycine across ion-exchangemembranes. J Membr Sci 203:43–51 (2002).

6 Eyal AM and Nadjda CS, Process for the separation of amino acids andtheir salts from an aqueous solution. US Patent No. 6171501 (2001).

7 Kostova A and Bart HJ, Preparative chromatographic separation ofamino acid racemic mixtures: I. adsorption isotherms. Sep PurifTechnol 54:340–348 (2007).

8 Eliseeva TV, Shaposhnik VA, Krisilova EV and Bukhovets AE, Transportof basic amino acids through the ion-exchange membranes andtheir recovery by electrodialysis. Desalination 241:86–90 (2009).

9 Bobreshova O, Novikova L, Kulintsov P and Balavadze E, Amino acidsand water electrotransport through cation-exchange membranes.Desalination 149:363–368 (2002).

10 Fischer A, Martin C and Muller J, Method for purification of aminoacid containing solutions by electrodialysis. US Patent No.6551803 (2003).

11 Kopple JD, Amino acids metabolism in chronic renal failure, in Aminoacids-metabolism and medical applications. ed. by Blackburn GL,Grant JP and Young VR. John Wright, Boston (1983). p. 451.

12 Shikata K, Azuma H, Tachibana T and Ogino K, Synthesis of novel andnon-natural ceramide analogues derived from L-glutamic acid.Tetrahedron 58:5803–5809 (2002).

13 Akagi T, Kaneko T, Kida T and Akashi M, Preparation andcharacterization of biodegradable nanoparticles based on poly(γ -glutamic acid) with l-phenylalanine as a protein carrier. J ControlledRelease 108:226–236 (2005).

14 Monosodium Glutamate Production Factory of Shanghai, TheProduction of Monosodium Glutamate. Light Industry Press, Beijing(1978).

15 Shim Y, Rixey WG and Chellam S, Influence of sorption on removal oftryptophan and phenylalanine during nanofiltration. J Membr Sci323:99–104 (2008).

16 Kuo WS and Chiang BH, Recovery of glutamic acid from fermentationbroth by membrane processing. J Food Sci 52:1401–1404 (1987).

17 Matsuoka Y, Kanda N, Lee YM and Higuchi A, Chiral separation ofphenylalanine in ultrafiltration through DNA-immobilized chitosanmembranes. J Membr Sci 280:116–123 (2006).

18 Chlanda FP and Rockaway NJ, Electrodialytic treatment of aqueoussolutions containing amino acids. US patent No. 5049250 (1991).


65

7


19 Rylatt DB, Napoli M and Ogle D, Electrophoretic transfer of proteinsacross polyacrylamide membranes. J Chromatog A 865:145–153(1999).

20 Cauwenberg V, Peels J, Resbeut S and Pourcelly G, Application ofelectrodialysis within fine chemistry. Sep Purif Technol 22:115–121(2001).

21 Subramanian K, Gomathi KG and Asokan K, Electrosynthesis ofpotassium permanganate in a cation exchange membrane cell.Ind Eng Chem Res 47:8526–8532 (2009).

22 Novalic S, Okwor J and Kulbe KD, The characteristics of citricacid separation using electrodialysis with bipolar membranes.Desalination 105:277–282 (1996).

23 Balster J, Stamatialis DF and Wessling M, Electrocatalytic membranereactors and the development of bipolar membrane technology.Chem Eng Process 43:1115–1127 (2004).

24 Xu TW and Yang WH, Citric acid production by electrodialysis withbipolar membranes. Chem Eng Process 41:519–524 (2002).

25 Strathmann H, Bauer B, Rapp HJ and Bell CM, Theoretical and practicalaspects of preparing bipolar membranes. Desalination 90:303–323(1993).

26 Madzingaidzo L, Danner H and Braun R, Process development andoptimizations of lactic acid purification using electrodialysis.J Biotechnol 96:223–239 (2002).

27 Grib H, Bonnal L, Sandeaux J, Sandeaux R, Govach C and Mameri N,Extraction of amphoteric amino acids by an electromembraneprocess: pH and electrical state control by electrodialysis withbipolar membranes. J Chem Technol Biotechnol 73:64–70 (1998).

28 Khan J, Tripathi BP, Saxena A and Shahi VK, Electrochemicalmembrane reactor: in situ separation and recovery of chromic acidand metal ions. Electrochim Acta 52:6719–6727 (2007).

29 Kumar M, Tripathi BP, Saxena A and Shahi VK, Electrochemicalmembrane reactor: synthesis of quaternary ammonium hydroxidefrom its halide by in situ ion substitution. Electrochim Acta54:1630–1637 (2009).

30 Kumar M, Tripathi BP and Shahi VK, Electro-membrane process forin situ ion substitution and separation of salicylic acid from itssodium salt. Ind Eng Chem Res 48:923–930 (2009).

31 Kumar M, Tripathi BP and Shahi VK, Electro-membrane reactor forseparation and in situ ion substitution of glutamic acid from itssodium salt. Electrochim Acta 54:4880–4887 (2009).

32 Minagawa M, Tanioka A, Ramirez P and Mafe S, Amino acid transportthrough cation exchange membranes: effects of pH on interfacialtransport. J Colloid Interface Sci 188:176–182 (1997).

33 Li S, Li C, Liu Y, Wang X and Cao Z, Separation of L-glutamine fromfermentation broth by nanofiltration. J Membr Sci 222:191–201(2003).

34 Zhai SL, Luo GS and Liu JG, Aqueous two-phase electrophoresis forseparation of amino acids. Sep Purif Technol 21:197–203 (2001).

35 Gohil GS, Nagarale RK, Binsu VV and Shahi VK, Preparation andcharacterization of monovalent cation selective sulfonatedpoly(ether ether ketone) and poly(ether sulfone) compositemembranes. J Colloid Interface Sci 298:845–853 (2006).

36 Narayanan PK, Adhikary SK, Harkare WP and Govindan KP, IndianPatent No. 1,60,880 (1987).

37 Gohil GS, Shahi VK and Rangarajan R, Comparative studieson electrochemical characterization of homogeneous andheterogeneous type of ion-exchange membranes. J Membr Sci240:211–219 (2004).

38 Nagarale RK, Gohil GS and Shahi VK, Recent developments on ion-exchange membranes and electro-membrane processes. AdvColloid Interf Sci 119:97–130 (2006).

39 Robbins BJ, Field ST, Kolaczkowski RW and Lockett AD, Rationalisationof the relationship between proton leakage and water flux throughanion exchange membranes. J Membr Sci 118:101–110 (1996).

40 Shaposhnik VA and Eliseeva TV, Barrier effect during the electrodialysisof ampholytes. J Membr Sci 161:223–228 (1999).

41 Gao Q, Xu W, Xu Y, Wu D, Sun Y, Deng F et al, Amino acid adsorptionon mesoporous materials: influence of types of amino acids,modification of mesoporous materials, and solution conditions.J Phys Chem B 112:2261–2267 (2008).

42 Bobreshova O, Novikova L, Kulintsov P and Balavadze E, Amino acidsand water electrotransport through cation-exchange membranes.Desalination 149:363–368 (2002).

43 Eliseeva TV, Shaposhnik VA and Luschik IG, Demineralization andseparation of amino acids by electrodialysis with ion-exchangemembranes. Desalination 149:405–409 (2002).

44 Kikuchi K, Gotosh T, Takahashi H and Higasino S, Separation of aminoacids by electrodialysis with ion-exchange membranes. J Chem EngJpn 28:103–109 (1995).

45 Lee HS and Hong J, Electrokinetic separation of lysine and asparticacid using polypyrrole-coated stacked membrane system. J MembrSci 169:277–285 (2000).


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

Documents

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