Journal of Membrane Science 234 (2004) 101–109

Study on the transport selectivity and kinetics of amino acids throughdi(2-ethylhexyl) phosphoric acid-kerosene bulk liquid membrane

Ming Maa,b, Bo Chenb, Xubiao Luob, Hao Tanb, Dingsheng Heb,Qingji Xie b, Shouzhuo Yaoa,b,∗

a State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR Chinab College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China

Received 5 September 2003; received in revised form 7 September 2003; accepted 21 January 2004

Abstract

Transport selectivity and kinetics of amino acids through di(2-ethylhexyl) phosphoric acid-kerosene bulk liquid membrane have been studied.The liquid-membrane system exhibited excellent transport selectivity for amino acids containing a side chain of high lipophilicity constant, likel-phenylalanine,l-tryptophan,l-leucine, andl-isoleucine. Main factors affecting the amino acids transport have been discussed. As a targetamino acid with bulky aromatic side-chain ofl-phenylalanine, the influence of its concentration and the concentration of di(2-ethylhexyl)phosphoric acid on the interfacial tension, and the effect of temperature on its transport through the bulk liquid membrane were investigated.The transport kinetics ofl-phenylalanine could be analyzed in the formalism of a reversible pseudo-first-order reaction followed by anirreversible pseudo-first-order reaction. The pseudo-first-order apparent rate constants of the interfacial transport ofl-phenylalanine for theextraction reaction, extraction back reaction, and stripping reaction were estimated.© 2004 Elsevier B.V. All rights reserved.

Keywords:Bulk liquid membrane; Amino acids;l-Phenylalanine; Di(2-ethylhexyl) phosphoric acid

1. Introduction

In recent years, liquid membrane (LM) has widely beenused to study the ion transport against a concentration gradi-ent. The ion transport through an LM plays an important rolein simulating biological membrane functions and separationtechnologies because of high transport efficiency, excellentselectivity, and economic advantages of LM. A number ofsuccessful researches involving the transport of metal ions[1–4], rare earth elements[5,6], drugs[7,8], phenols[9],fructose[10], and the treatment of seawater and waste wa-ter [11–13] through the liquid membrane have been carriedout. The transport kinetics of alkali metal ions[14], copperion [15], cadmium ion[16], andl-isoleucine (Ile)[17] hasalso been studied.

Amino acids (AAs) are important bioactive substances.Study on the transport selectivity and kinetics of AAsthrough the liquid membrane is helpful for separation and

∗ Corresponding author. Fax:+86-731-8865515.E-mail addresses:mamingm@hotmail.com, mming@public.cs.hn.cn

(M. Ma), dr-chenpo@vip.sina.com (B. Chen), szyao@hnu.net.cn (S. Yao).

concentration of AAs and for understanding the transportprocess of AAs through the cell membrane. Several pa-pers have been addressed the separation and transport ofAAs with liquid membranes. Wieczorek et al.[18] in-vestigated the transport efficiency of five kinds of AAs,namely, arginine (Arg), glutamic acid (Glu), phenylalanine(Phe), tyrosine (Tyr), and tryptophan (Trp), through thedi-2-ethylhexyl phosphoric acid (D2EHPA)-tri-2-ethylhexylphosphate supported liquid membrane (SLM), and a 56.2%of extraction efficiency for Trp was obtained. Teramotoet al.[19], Itoh et al.[20], and Hong et al.[21,22] exploredthe extraction and concentration of Phe and Trp by emul-sion liquid membrane (ELM) containing D2EHPA as a car-rier, respectively. Calzado et al.[23] studied the facilitatedtransport and separation of three kinds of aromatic aminoacids through activated composite membranes (ACM) withD2EHPA as a carrier, and found that the AA-transportselectivity through these ACM followed a sequence ofTrp > Phe> Tyr, regardless of a different AA–D2EHPAaffinity sequence, Phe> Trp > Tyr. Deblay et al.[24] de-veloped a carrier-mediated countertransport process for thesuccessful separation ofl-valine (Val) from fermentation

0376-7388/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.memsci.2004.01.014

102 M. Ma et al. / Journal of Membrane Science 234 (2004) 101–109

broths using a SLM with Aliquat 336 as a carrier. In thesestudies, the effects of experimental parameters on the ex-traction efficiency of AAs have been investigated. However,the transport selectivity of multi-AAs (more than five kindsof AAs) through the LM and the mechanism of strippingat the membrane–stripping solution interface have seldombeen mentioned.

In our previous research[17], the transport kinetics of Ile(a target compound with a bulky alkyl side-chain) throughthe D2EHPA-bulk liquid membrane (BLM) was studied,and high transport efficiency was obtained. In this paper,Phe was selected as a target amino acid with bulky aro-matic side-chain to explore the kinetic transport mechanismthrough the D2EHPA-BLM. The results indicate that thetransport kinetics of Phe could be analyzed in the formal-ism of a reversible pseudo-first-order reaction followed byan irreversible pseudo-first-order reaction. The present BLMsystem exhibited high transport efficiency for Phe. The influ-ences of the extraction time, D2EHPA concentration in theorganic phase, and pH in the feed solution on the extractionefficiency, the effect of the stripping solution compositionand concentration on the stripping efficiency, and the effectof temperature on the BLM transport of Phe were also inves-tigated. The transport selectivity through D2EHPA-keroseneBLM for 15 kinds of AAs, namely, Phe, Trp, Arg, Tyr, Glu,Val, Ile, aspartic acid (Asp), histidine (His), glycine (Gly),threonine (Thr), alanine (Ala), methionine (Met), leucine(Leu), and lysine (Lys), was studied in detail. Furthermore,the proposed transport method was used to transport AAsfrom a practical protein hydrolysate.

2. Experimental

2.1. Apparatus

The HPLC analysis was performed with a Waters model246 high performance liquid chromatograph (Waters Assoc.,USA) consisting of a model 590 solvent delivery pump, amodel 510 solvent delivery pump, a Waters 420 fluorescencedetector, a U6K manual injector, and a Baseline-810 chro-matographic workstation. Chromatograms were recordedand the peak areas were measured by the 810 workstation.

JJ-1 motor driven stirrer (Henan Gongyi Dufu InstrumentFactory), PHS-3E digital pH meter (Jiangsu Electroanalyt-ical Instrument Factory), 755B model UV-Vis spectropho-tometer (Precision Science Instrument Limited Company ofShanghai), and SZG-441C hand-hold digital rotation-speedindicator (Shanghai Automatic Instruments Corporation)were used.

2.2. Reagents

D2EHPA was a product of the Chemical Reagent Factoryof Shanghai and prepurified by recrystallization of its cop-per salt[25]. Its monobasic acid content was 99%. The dis-

tillate of sulfonated kerosene at 185–220◦C was used as themembrane solvent.l-Amino acids were purchased from theChinese National Institute for the Control of Pharmaceuticaland Biological Products (Beijing, PR China). The AAs sam-ple was the silkworm chrysalis protein hydrolysate providedby Xing Linchun Biological Medicine Health Product Fac-tory (Changsha, PR China).o-Phthaldialdehyde (OPA) and2-mercaptoethanol (2-MCE) were purchased from Sigma(St. Louis, MO). Acetonitrile and methanol were of HPLCgrade. Other chemical reagents used (Hunan Chemical Re-gent Company) were of analytical grade. Deionized watervia an ultra-water system from Millipore (Milford, MA) wasused throughout.

2.3. Analysis of AAs

A pre-column derivative RP-HPLC method was usedto investigate the transport behavior of AAs in the mixedAAs solution. A Waters 246 HPLC was used comprising abinary solvent delivery system and a 420 AC fluorescencedetector set at 338 nm excitation wavelength and 425 nmemission wavelength. For the separation of AA deriva-tives, a 250 mm× 4.6 mm ID column was used filled withspherigel ODS 5�m stationary phase (Johnsson, Dalian, PRChina). The mobile phases were as follows: (A) a mixture ofCH3OH, tetrahydrofuran (THF), and aqueous solution con-taining 50 mM sodium acetate+50 mM Na2HPO4 (pH 7.5)(2/2/96, v/v/v), (B) a mixture of CH3OH and H2O (65/35,v/v). The gradient elution profile is listed inTable 1. Forthe derivatization of AAs, the derivative reagent (the mix-ture of OPA together with 2-MCE in sodium borate buffer(pH 10.4)) was used. An amount of 1�l AAs solution and1�l of derivative reagent were mixed for 10 s and then in-jected. The column temperature was 45± 1◦C. An externalstandard mixture solution was prepared in our laboratory tocalibrate and quantify the AAs. Phe content was determinedwith the 755B spectrophotometer using triketohydrindenehydrate (λmax = 565 nm) in the single AA experiments.

2.4. Procedures

Extraction and stripping experiments were carried out at30±0.5◦C. The desired concentration of D2EHPA solution

Table 1The gradient elution profile of HPLC for determining AAs

Time (min) Flow (ml/min) A (%) B (%) Curve

0 0 100 0 –2.0 0.1 100 0 12.5 1.5 100 0 6

14.0 1.5 50 50 723.0 1.5 0 100 728.0 1.5 100 0 1135.0 0 100 0 11

A: a mixture of CH3OH, THF, and aqueous solution containing 50 mMsodium acetate+50 mM Na2HPO4 (pH 7.5) (2/2/96, v/v/v); B: a mixtureof CH3OH and H2O (65/35, v/v).

M. Ma et al. / Journal of Membrane Science 234 (2004) 101–109 103

(in the dimer of D2EHPA, H2R2) was prepared by dissolvinga calculated amount of D2EHPA in kerosene. Aqueous feedsolution (containing 1.21× 10−3 M Phe+ 0.2 M Na2SO4)was adjusted to the desired initial pH value with 2 M H2SO4.An amount of 10 ml organic solution and 10 ml of the aque-ous feed solution were added into a centrifugal tube andmixed on a vibrator for 30 min. In the stripping experiments,150 ml of feed solution was extracted with 150 ml of organicphase containing 0.5 M D2EHPA. After phase separation,10 ml of the same organic phase was stripped with 10 ml ofdifferent stripping solution. The concentration of Phe com-plex compound in the organic phase was calculated frommaterial balance.

Bulk liquid membrane (BLM): the transport experimentswere carried out at 30± 0.5◦C [17]. The aqueous feed so-lutions (40 ml) consisted of 1.21 × 10−3 M Phe+ 0.2 MNa2SO4 (adjusted to pH 3.1 with 2 M H2SO4 solution). Thestripping solution was 40 ml of 1.0 M HCl solution and themembrane phase was 20 ml of 0.5 M D2EHPA-kerosene so-lution. The AAs standard solution (containing 15 kinds ofAAs + 0.2 M Na2SO4) and the AAs sample (containing avariety of AAs+ 0.2 M Na2SO4) were adjusted the initialpH to 3.1 by carefully adding 2 M H2SO4 solution in thefeed solution whose pH was continuously monitored witha pH-meter. In view of the weaker buffer capacity of thefeed solution (ca. 12 ofcSO4

2−/cHSO4− value at pH 3.1) and

the effect of H+ (released to the feed solution among thetransport experiments) on the pH value of the feed solution,lower concentration of Phe (6.05×10−4 M) was selected inthe mixed AAs experiments, and the coexistent AAs con-centrations were in a range of 10−4 to 10−3 M, except thatof Asp in the AAs sample. The single AA feed solutionwas displaced with the AAs standard solution or the AAssample in the selectivity experiment. The feed solution, themembrane phase, and the stripping solution were stirred at62.8 rad/s (600 rpm), and the interfacial areas were approx-imately 7.2 cm2.

The interfacial tensions were measured with drop volumemethod[26].

3. Theoretical

Extraction of AAs is complicated due to their zwitteri-onic properties. For example, there are acidic carboxylic andbasic amino groups in Phe molecule. The dissociation equi-librium of protonated Phe in aqueous solution is as follows[19]:

H2A+Ka1�H+ + HA± (1)

HA±Ka2�H+ + A− (2)

where the dissociation constants pKa1 and pKa2 are 1.83 and9.13, respectively. Two ways had been proposed by Liu et al.[27] for Phe extraction with D2EHPA as the extractant. The

2

.2H AR 3HR

.

22H A

+

+

H

H2A

+High [H ]

+Low [H ]

+

+

H2A

H

2H R2

2H AR 3HR

22H R+H

+

feed solution membrane stripping solution

(and HA )

(and HA )

+−

Fig. 1. Schematic diagram of the transport processes.

first way was the ion-exchange reaction of H2A+ with H2R2at the feed solution–organic phase interface. The second waywas the proton-transfer reaction between H2R2 and HA± atthe feed solution–organic phase interface. In a number ofstudies[18–23], the first way has been used to explain theirresults. However, in this paper, over 70% of the extraction ef-ficiency at higher pH values of 5–6 (shown inSection 4.1.2)implies that the zwitterionic Phe molecule, whose fractionis close to 1 at these pH values, may also be extracted, al-though the extraction efficiency is lower than that at pH 3.1.The result suggests that both of the above extraction wayscarry out simultaneously, but the ion-exchange reaction isthought to be more crucial than the proton-transfer reaction(Section 4.3). The transport processes of Phe through theLM (shown in Fig. 1) are as follows: At the feed (d, Pheaqueous solution)-membrane (m, H2R2 in kerosene) inter-face, H2R2 reacts with H2A+ (or HA±) in the feed solutionto form Phe–D2EHPA ion-pairing compound H2AR · 3HR.The ion-pairing compound diffuses in the membrane, andreaches the membrane–stripping (a, HCl aqueous solution)interface under effective stirring. At the membrane–strippinginterface, the ion-pairing compound reacts with H+ in thestripping solution to liberate H2R2 and H2A+. H2R2 dif-fuses back to the membrane phase and H2A+ is released inthe stripping solution. In view of the ion-exchange reactionand the diffusion of the ion-pairing compound in the mem-brane phase, we suggest that the concentration of H2A+ andthe lipophilicity of AAs side-chain are the two main factorsaffecting the AAs transport from the feed solution to the or-ganic phase, and then affecting the transport efficiency of theAAs through the BLM. This idea has been verified by exper-iment (Section 4.3). According to this transport mechanism,

104 M. Ma et al. / Journal of Membrane Science 234 (2004) 101–109

the concentration gradient of H+ is the most used drivingforce. Under the same condition with the bulk liquid mem-brane transport, extraction experiments reveal that about89% of Phe in the feed solution could be extracted in the or-ganic phase and more than 98% of Phe in the organic phasecould be stripped in the stripping solution. Based on the factthat the concentration of D2EHPA in the membrane phaseis much higher than the concentration of Phe in the feedsolution, and the concentration of H+ in the stripping solu-tion is much higher than the concentration of Phe–D2EHPAion-pairing compound in the membrane phase, we assumethat the Phe transport may obey the kinetic mechanism ofa reversible pseudo-first-order reaction followed by an irre-versible pseudo-first-order reaction. The mechanism and thekinetic transport scheme of Phe through a liquid membraneare schematically described inFig. 1 and byEq. (3)

l-Phe(d)

k1�k−1

l-Phe(m)k2−→l-Phe(a) (3)

The concentrations of Phe were directly measured in bothfeed solution (donor phase,cd) and stripping solution (ac-ceptor phase,ca) in the experiments. The correspondingmolar quantity of Phe in the membrane (nm) was estab-lished from the material balance. For practical reasons, thereduced dimensionless molar fractions are used:

Rd = nd

nd0, Rm = nm

nd0, Ra = na

nd0(4)

wherend0 is the initial molar quantity of Phe in the feedsolution att = 0, nd, nm, andna are molar quantities of Phein d, m, and a phases, respectively.Rd, Rm, andRa representthe molar fractions of Phe in the feed solution, membranephase, and stripping solution, respectively. Obviously, wehaveRd + Rm + Ra = 1.

Considering the heterogeneous mass-transfer/reaction ki-netics and a spatial uniformity of the bulk concentration ofPhe in each phase through effective stirring, the above ki-netic scheme may be described by the following equations[17]:

dRd

dt= −k1Rd + k−1Rm ≡ Jd (5)

dRm

dt= k1Rd − k2Rm − k−1Rm (6)

dRa

dt= k2Rm ≡ Ja (7)

where J stands for the flux, andk1, k−1, and k2 are thepseudo-first-order apparent rate constants of feed–membraneinterfacial and membrane–stripping interfacial transport ofPhe. IntegratingEqs. (5)–(7)gives

Rd = k1(λ2 − k2)

λ2(λ2 − λ3)exp(−λ2t) + k1(k2 − λ3)

λ3(λ2 − λ3)exp(−λ3t)

(8)

Rm = − k1

λ2 − λ3exp(−λ2t) + k1

λ2 − λ3exp(−λ3t) (9)

Ra = k1k2

λ2λ3+ k1k2

λ2(λ2 − λ3)exp(−λ2t)

− k1k2

λ3(λ2 − λ3)exp(−λ3t) (10)

where

λ2 = 12(k1 + k−1 + k2 + [(k1 + k−1 + k2)

2 − 4k1k2]1/2),

λ3 = 12(k1 + k−1 + k2 − [(k1 + k−1 + k2)

2 − 4k1k2]1/2)

It is obvious that a plot ofRd versust yields a monotonicdecreasing curve, and the time variation ofRa is a mono-tonic increasing curve, whereas the time dependence ofRmpresents a maximum. The actual numerical analysis was car-ried out by non-linear curve fitting.Eqs. (8)–(10)are alsovalid in the case of unequal volumes of d, m, and a phases.

According to Eq. (9), Rm has a maximum (whendRm/dt = 0) at t = tmax:

tmax = ln(λ2/λ3)

λ2 − λ3(11)

Rmaxm = k1

λ2 − λ3

[(λ2

λ3

)−λ3/(λ2−λ3)

−(

λ2

λ3

)−λ2/(λ2−λ3)]

(12)

and att = tmax, the penetration (Jmaxd ) and exit (Jmax

a ) fluxesare equal but of opposite sign as follows:

dRd

dt

∣∣∣∣max

= − k1

λ2 − λ3

[(k2 − λ3)

(λ2

λ3

)−λ3/(λ2−λ3)

+(λ2 − k2)

(λ2

λ3

)−λ2/(λ2−λ3)]

≡ Jmaxd (13)

dRa

dt

∣∣∣∣max

= k1k2

λ2 − λ3

[(λ2

λ3

)−λ3/(λ2−λ3)

−(

λ2

λ3

)−λ2/(λ2−λ3)]

≡ Jmaxa (14)

−dRd

dt

∣∣∣∣max

= + dRa

dt

∣∣∣∣max

(15)

4. Results and discussion

4.1. Selection of extraction conditions

4.1.1. Effect of the extraction time on the extractionefficiency

In the extraction experiment, the extraction efficiency,Eis expressed as

E = no

nd0= nd0 − nd

nd0(16)

M. Ma et al. / Journal of Membrane Science 234 (2004) 101–109 105

t (min)

0 10 20 30 40 50

E

0.000

.200

.400

.600

.800

1.000

Fig. 2. Effect of extraction time on the extraction efficiency. D2EHPAconcentrations: (�) 0.1 M; (�) 0.2 M; (�) 0.5 M.

where no is the molar quantity of the analyte in the or-ganic phase after extraction. The effect of the extractiontime onE of Phe has been investigated.Fig. 2 shows thatthe extraction equilibrium was established in ca. 2, 5, and15 minutes for 0.5, 0.2, and 0.1 M D2EHPA solution, re-spectively. The result indicates that the time achieved ex-traction equilibrium increases with a decrease in D2EHPAconcentration. For insuring the establishment of the extrac-tion equilibrium in all of the extraction processes, an ex-traction time of 30 min is used for the subsequent extractionexperiments.

4.1.2. Effect of D2EHPA concentration in the organicphase and pH in the feed solution on the extractionefficiency of Phe

The extraction efficiency of Phe is affected obviously bythe concentration of D2EHPA in the organic phase and theacidity of the feed solution. As shown inFig. 3, the ex-traction efficiency of Phe increases with the increase of thepH value (at low pH value), and then decreases with thefurther increase of the pH value (at high pH value). Thismay be explained as follows: The strong acidity is favor-able to form H2A+. However, the strong acidity is unfavor-able to the ion-exchange reaction between H2A+ and H2R2(H2A+

(d) +2H2R2(m) ⇔ H2AR ·3HR(m) +H+(d)) because the

equilibrium is shifted to the left side of the above extractionreaction with an increase in H+ concentration. The final re-sults are decided by the competition of these two factors.Fig. 3also shows that the extraction efficiency increases withan increase in D2EHPA concentration in the organic phasefrom 0.1 to 0.5 M. However, a slightly decrease in extrac-tion efficiency has been observed for high concentration ofD2EHPA (0.7 M) with the feed solution pH value 3.1 or 4.3.

pH

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

E

0.000

.200

.400

.600

.800

1.000

Fig. 3. Effect of D2EHPA concentration in the organic phase and pH inthe feed solution on the extraction efficiency. D2EHPA concentrations:(�) 0.1 M; (�) 0.2 M; (�) 0.3 M; (�) 0.5 M; (�) 0.7 M.

This may be due to the interfacial adsorption of D2EHPA atthe feed solution–organic phase interface.

The interfacial tension for various concentrations ofD2EHPA and Phe had been determined at 30± 0.1◦C.Fig. 4 indicates that the change of interfacial tensions is notobvious as the Phe concentration increase from 1× 10−7 to

logC

-8.0 -7.0 -6.0 -5.0 -4.0 -3.0 -2.0 -1.0

γ/ m

Nm

-1

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

1

2

Fig. 4. Influence of D2EHPA and Phe concentrations on the interfacialtension. Curve 1: dependence of the interfacial tension on the logarithmof Phe concentration. The feed aqueous solution: Phe (pH= 3.1), 0.2 MNa2SO4; the organic phase: kerosene. Curve 2: dependence of the in-terfacial tension on the logarithm of D2EHPA concentration. The feedaqueous solution: 1.21 × 10−3 M Phe (pH= 3.1), 0.2 M Na2SO4; theorganic phase: D2EHPA-kerosene solutions.

106 M. Ma et al. / Journal of Membrane Science 234 (2004) 101–109

Table 2Influence of the stripping solution composition on the stripping efficiency

HCl solution as thestripping solution

H2SO4 solution asthe stripping solution

Concentration(M)

Strippingefficiency (%)

Concentration(M)

Strippingefficiency (%)

0.20 44.3 0.10 36.20.50 65.3 0.25 37.30.80 83.4 0.40 56.81.0 98.4 0.50 89.72.0 97.9 1.0 90.3

1 × 10−2 M, without D2EHPA in the organic phase (curve1). However, the interfacial tension decreases rapidly withan increase in D2EHPA concentration in the organic phasefrom 1 × 10−7 to 1 × 10−2 M (curve 2) (the feed solu-tion composition: 1.21 × 10−3 M Phe, 0.2 M Na2SO4, pH3.1). The results suggest that D2EHPA molecules adsorbat the interface and cause the interfacial tension change.More D2EHPA molecules adsorbed at the interface at highD2EHPA concentration will result in higher extraction effi-ciency. However, more D2EHPA molecules adsorbed at theinterface will hamper the transport of the ion-pairing com-pound H2AR · 3HR to enter in the organic phase, and thencause lower extraction efficiency. The result demonstratesthat pH 3.1 in the feed solution and 0.5 M D2EHPA in theorganic phase is the optimal conditions.

4.1.3. Effect of the stripping solution composition on thestripping efficiency

Different type acid solutions have been reported as strip-ping solution for liquid membrane process with D2EHPA ascarrier. Sulfuric acid solutions[17,21,22]and hydrochloricacid solutions[18,20,24]were used for extracting AAs usingELMs and SLMs. In this work, the effects of five concen-trations of HCl stripping solution (0.20, 0.50, 0.80, 1.0, and2.0 M) and H2SO4 stripping solution (0.10, 0.25, 0.40, 0.50,and 1.0 M) on the stripping efficiency (the ratio value of themolar quantity of Phe in the stripping solution to that in theinitial organic phase) are investigated. The results (shown inTable 2) indicate that the stripping efficiency increases withan increase in concentration of the stripping agents except aslightly decrease with 2.0 M HCl as the stripping solution,and HCl solution is a better stripping agent than H2SO4solution. According to the two main factors affecting thestripping efficiency, i.e., the concentrations of H+ and thecoexistent anion (Cl− or SO4

2−) in the stripping solution,

Table 3Effect of temperature on kinetic parameters for Phe transport through bulk liquid membrane

T (K) k1 × 102 (h−1) k−1 × 102 (h−1) k2 × 102 (h−1) Jmaxa × 102 (h−1) Jmax

d × 102 (h−1)

287 17.0± 1.5 4.49± 0.5 10.1± 0.6 4.24 −4.24293 21.0± 1.8 8.67± 0.8 11.1± 0.7 4.58 −4.58303 31.2± 2.5 20.5± 1.9 12.7± 0.9 5.16 −5.16313 43.5± 4.1 33.2± 3.0 13.6± 1.0 5.65 −5.65

k1, k−1, andk2 were calculated fromEqs. (8) and (10), respectively.

t(h)

0.0 4.0 8.0 12.0 16.0

R

0.000

.200

.400

.600

.800

1.000

Rd

Ra

Rm

Fig. 5. Variation ofRd (�), Rm (�) and Ra (�) with time at 303 K.Theoretical curves were obtained by usingEqs. (8)–(10).

this phenomenon may be explained as follows: High con-centration of H+ is favorable to the stripping of Phe (Fig. 1).However, high concentration of coexistent anion may beunfavorable for the stripping of Phe, and the influence ofSO4

2− is more obvious. According to the results shown inTable 2, 1.0 M HCl is selected as the suitable stripping agent.

4.2. Study on transport kinetics

In order to investigate the transport kinetics of Phethrough the bulk liquid membrane, the influence of tem-perature on Phe transport through the BLM containingD2EHPA in kerosene was examined at 287, 293, 303, and313 K. The experimental results are shown inFig. 5 andTable 3. FromFig. 5, Rd decreases monotonically with time,andRa follows an increasing sigmoid-type curve, while thetime dependence ofRm presents a maximum.Fig. 5 showsthat the experimental points are in good agreement with thetheoretical curves.

The results of the extraction and the transport experimentsthrough the BLM verify that Phe transport obeys the kineticmechanism of a reversible pseudo-first-order reaction fol-lowed by an irreversible pseudo-first-order reaction.Table 3indicates that the pseudo-first-order apparent rate constants

M. Ma et al. / Journal of Membrane Science 234 (2004) 101–109 107

of the interfacial transport of Phe for the extraction reaction,extraction back reaction, and stripping reaction,k1, k−1, andk2 increase with the increase of temperature. The maximumof penetration and exit fluxes,Jmax

d andJmaxa , also increase

with the increase of temperature although the apparent rateconstants of the extraction back reaction also increase withthe increase of temperature. The result indicates that the in-crease of temperature in a certain range is favorable to thetransport of Phe through the BLM.

4.3. Selectivity of AAs transport through D2EHPA-BLM

A number of studies have reported the use of D2EHPA-LMtechnique to extract and separate AAs[17–23]. The highertransport selectivity of the D2EHPA-SLM system to Arg,Glu, Phe, Tyr, and Trp[18], and the D2EHPA-ACM systemto Trp, Phe, and Tyr[23] has been revealed. In this work, theAA standard solution containing 15 kinds of AAs was usedto investigate the transport selectivity of D2EHPA-BLM,and the silkworm chrysalis protein hydrolysate was used tocarry out a practical application of the proposed AAs BLMtransport method. Based on the OPA derivation couplingRP-HPLC method, the transport information could be moni-tored effectively. The transport efficiency of the AA throughthe BLM (Ra, at 15 h of transport time), the lipophilicityconstant of amino acid side-chain (πR) [28], the fraction ofthe protonated AA (αp) at pH 3.1, the dissociation constants[29] of the AA, and the ratio of the molar quantity of thecoexistent AA to Phe (Rr) are listed inTable 4.

Table 4 clearly illustrates that the AAs with relativelybulky non-polar side-chain such as Phe, Ile, Leu, Trp, Val,and Met exhibit higher transport efficiencies (>37%) thanthose AAs with polar side-chain, such as Tyr, Thr, Gly, Glu,Asp, Lys, Arg, and His. However, a deviant low transportefficiency (∼11%) of Ala with the non-polar side-chainmay be due to the relatively short side-chain of –CH3.

Table 4The values of the transport efficiency (Ra), the dissociation constants (Ka), the lipophilicity constant of amino acid side-chain (πR), the fraction of theprotonated AA (αp), and the ratio of the molar quantity of the coexistent AA to Phe (Rr)

No. AA πR pKa αp (pH 3.1) AA standard solution AA sample

pKa1 pKa2 pKa3 Rr Ra (%) Rr Ra (%)

1 Trp 1.85 2.38 9.39 0.160 0.9 71.5 – –2 Phe 1.56 1.83 9.13 0.051 – 62.3 – 66.83 Leu 1.28 2.36 9.60 0.154 1.2 58.8 1.7 62.84 Ile 1.20 2.36 9.68 0.154 1.2 57.1 0.5 59.05 Met 0.90 2.28 9.21 0.132 1.0 45.9 0.8 45.26 Val 0.71 2.32 9.62 0.142 1.6 37.3 1.3 38.17 Lys −0.77 2.18 8.95 10.53 ∼1.0 1.3 23.4 1.8 24.68 Arg −0.79 2.17 9.04 12.48 ∼1.0 1.1 23.4 4.8 21.89 His 0.15 1.82 6.00 9.17 0.999 1.1 18.4 1.8 17.9

10 Tyr 0.89 2.20 9.11 10.07 0.112 0.7 15.4 0.8 13.611 Ala 0.23 2.34 9.69 0.148 3.7 11.0 12.0 10.412 Thr 0.17 2.11 9.62 0.093 2.1 7.1 1.7 6.413 Gly 0.00 2.34 9.60 0.148 1.0 6.5 5.9 5.014 Glu −0.51 2.19 4.25 9.67 0.103 0.8 2.2 4.0 1.915 Asp −0.61 1.88 3.65 9.60 0.045 0.3 1.6 34.6 1.5

The result demonstrates that the transport efficiency of AAthrough the LM depends on the polar behavior, i.e., thelipophilicity of AA side-chain. The lipophilicity of AAside-chain is usually expressed by the lipophilicity constantof AA side-chain (πR). The lipophilicity constant of AAside-chain, the fraction of the protonated AA in the feedsolution, and the transport efficiency of AA through theBLM are shown inFig. 6. Fig. 6 indicates thatRa is relativeto πR andαp. With lower fraction values of the protonatedAAs (0.045–0.160), the change ofRa mainly depends onthe change ofπR. Ra decreases with the decrease ofπR forthe investigated AAs (Trp, Phe, Leu, Ile, Met, Val, Tyr, Ala,Thr, Gly, Glu, and Asp). For the six kinds of AAs with therelatively bulky non-polar side-chain, i.e., Trp, Phe, Leu,Ile, Met, and Val, a rough linear relationship betweenRaandπR is obtained and shown inFig. 7. The correspondingequation is given as follows:

Ra = 19.8 + 28.5πR, n = 6, r = 0.961 (17)

Lys, Arg, and His are AAs with polar side-chain in theirmolecules, and theirπR values are lower than that of Tyr,Ala, Thr, etc. However, the deviant higher transport effi-ciency of Lys, Arg, and His (23.4, 23.4 and 18.4%) havebeen observed. The higher transport efficiencies of Lys,Arg, and His may be contributed by the high fractions(∼1) of protonated Lys, Arg, and His in the feed solutionshown inFig. 6(A) and (B). The result, i.e.,Ra of Lys andArg (with lower πR and higherαp) is higher than that ofTyr, Thr, Gly, Glu, and Asp (with higherπR and lowerαp), indicates that the ion-exchange reaction is more cru-cial between the two reaction ways of AAs with D2EHPAat the feed solution–membrane phase interface. The resultverifies that the lipophilicity constant of AA side-chain andthe fraction of the protonated AA in the feed solution arethe two main factors forRa. According to the results fromabove study, the lipophilicity constant of AA side-chain

108 M. Ma et al. / Journal of Membrane Science 234 (2004) 101–109π R

-1.00

-.50

0.00

.50

1.00

1.50

2.00

α p

0.00

.20

.40

.60

.80

1.00

Number of the AA

0 2 4 6 8 10 12 14 16

Ra

0.0

20.0

40.0

60.0

80.0

(A)

(B)

(C)

Fig. 6. The lipophilicity constant of AA side-chain (πR, A), the fractionof the protonated AA (αp, B) in the feed solution, and the transportefficiency (Ra, C) of various AAs in the AA standard solution transportexperiment. Number of AA axis: 1, Trp; 2, Phe; 3, Leu; 4, Ile; 5, Met;6, Val; 7, Lys; 8, Arg; 9, His; 10, Tyr; 11, Ala; 12, Thr; 13, Gly; 14,Glu; 15, Asp.

and the fraction of the protonated AA could be used to esti-mate the transport efficiency of AAs through the LM in theseparation industry. The results obtained from the AAssample of protein hydrolysate are in agreement with that

πR

.80 1.20 1.60 2.00

Ra

30.0

40.0

50.0

60.0

70.0

80.0

Trp

PheLeu

Ile

Met

Val

Fig. 7. The relationship betweenRa andπR.

from the AA standard solution.Table 4also indicates thatthe present LM system exhibit a better selectivity to theAAs with relatively bulky non-polar side-chain such as Phe,Ile, Leu, and Trp, and higher transport efficiencies of Pheare also obtained with the mixed AAs solutions as the feedsolution (62.3 and 66.8% of transport efficiencies in the AAstandard solution and the AA sample transport experiments).

5. Conclusion

The D2EHPA-BLM system exhibits high transport se-lectivity to different amino acids. The transport behavioris mainly affected by the lipophilicity constant of AAside-chain and the fraction of the protonated AA in the feedsolution. For the six kinds of bulky non-polar side-chainAAs, Trp, Phe, Leu, Ile, Met, and Val, with low fractions ofprotonated AAs, a rough linear relationship betweenRa andπR has been obtained. As a target amino acid with relativelybulky non-polar aromatic side-chain, the pseudo-first-orderapparent rate constants of interfacial transport of Phe forthe extraction reaction, extraction back reaction, and strip-ping reaction were calculated. The results indicated that thisliquid membrane system has good selectivity for AAs withhigh lipophilicity constant of AA side-chain like Phe Ile,Leu, and Trp. This work is significant for separation andconcentration of AAs in industry and for understanding thetransport process of AAs through the cell membrane.

Acknowledgements

This work was supported by the Natural Science Founda-tion of China (20335020), the Key Technologies Researchand Development Program of the Tenth Five-year Plan ofthe Ministry of Science and Technology of the PR Chinaand Hunan Province (2001BA746C), and the Natural Sci-ence Foundation of Hunan Province (03JJY1002).

Nomenclature

a stripping solutionca concentration of Phe in the stripping

solutioncd concentration of Phe in the feed solutiond aqueous feed solutionHA± zwitterionic molecule of amino acidH2A+ protonated amino acidH2AR · 3HR Phe–D2EHPA ion-pairing compoundH2R2 dimer of D2EHPAJ fluxJmax

a maximum exit fluxJmax

d maximum penetration fluxk1 pseudo-first-order apparent rate constants

for extraction reaction

M. Ma et al. / Journal of Membrane Science 234 (2004) 101–109 109

k−1 pseudo-first-order apparent rate constantsfor extraction back reaction

k2 pseudo-first-order apparent rate constantsfor stripping reaction

Ka dissociation constantm membrane phasena molar quantity of Phe in the stripping

solutionnd molar quantity of Phe in the feed

solutionnd0 initial molar quantity of Phe in the

feed solutionnm molar quantity of Phe in the

membrane phaseRa molar fraction of Phe in the

stripping solutionRd molar fraction of Phe in the

feed solutionRm molar fraction of Phe in the

membrane phaseRmax

m maximum ofRmRr ratio of the molar quantity of the

coexistent AAs to Phetmax time whenRm reaches the maximum

Greek lettersαp fraction of the protonated AAπR lipophilicity constant of amino

acid side-chain

References

[1] D. Nanda, M.S. Oak, M.P. Kumar, B. Maiti, P.K. Dutta, Facili-tated transport of Th(IV) across bulk liquid membrane by di(2-ethylhexyl)phosphoric acid, Sep. Sci. Technol. 36 (2001) 2489–2497.

[2] M.B. Gholivand, S. Khorsandipoor, Selective and efficient up-hill transport of Cu(II) through bulk liquid membrane usingN-ethyl-2-aminocyclopentene-1-dithiocarboxylie acid as carrier, J.Membr. Sci. 180 (2000) 115–120.

[3] F. Guyon, N. Parthasarathy, J. Buffle, Mechanism and kinetics ofcopper(II) transport through diaza-crown ether-fatty acid supportedliquid membrane, Anal. Chem. 71 (1999) 819–826.

[4] P.S. Kulkarni, S. Mukhopadhyay, M.P. Bellary, S.K. Ghosh, Studieson membrane stability and recovery of uranium(VI) from aqueoussolutions using a liquid emulsion membrane process, Hydrometal-lurgy 64 (2002) 49–58.

[5] M. Ma, D.S. He, Q.Y. Wang, Q.J. Xie, Kinetics of europium(III)transport through a liquid membrane containing HEH(EHP) inkerosene, Talanta 55 (2001) 1109–1117.

[6] J. Dolezal, C. Morenol, A. Hrdlicka, M. Valiente, Selective trans-port of lanthanides through supported liquid membranes containingnon-selective extractant, di-(2-ethylhexyl)phosphoric acid, as a car-rier, J. Membr. Sci. 168 (2000) 175–181.

[7] R.S. Juang, S.H. Lee, R.C. Shiau, Carrier-facilitated liquid membraneextraction of penicillin G from aqueous streams, J. Membr. Sci. 146(1998) 95–104.

[8] S.C. Lee, Continuous extraction of penicillin G by emulsion liquidmembranes with optimal surfactant compositions, Chem. Eng. J. 79(2000) 61–67.

[9] P.F.M.M. Correia, J.M.R. de Carvalho, Recovery of 2-chlorophenolfrom aqueous solutions by emulsion liquid membranes: batch exper-imental studies and modelling, J. Membr. Sci. 179 (2002) 175–183.

[10] M.D. Luccio, B.D. Smith, T. Kida, C.P. Borges, T.L.M. Alves,Separation of fructose from a mixture of sugars using supportedliquid membranes, J. Membr. Sci. 174 (2000) 217–224.

[11] A.A. Ensafi, H. Eskandari, Selective extraction of bromide withliquid organic membrane, Sep. Sci. Technol. 36 (2001) 81–89.

[12] D.S. He, M. Ma, Z.H. Zhao, Transport of cadmium ions through aliquid membrane containing amine extractants as carriers, J. Membr.Sci. 169 (2000) 53–59.

[13] J.C. Aguilar, M. Sánchez-Castellanos, E. Rodrı́guez de San Miguel,J. de Gyves, Cd(II) and Pb(II) extraction and transport modeling inSLM and PIM systems using Kelex 100 as carrier, J. Membr. Sci.190 (2001) 107–118.

[14] Z.L. Wang, M.C. Shen, H.Q. Luo, Studies on crown coordinationcompounds, Acta Chim. Sinica (Ch) 50 (1992) 1000–1004.

[15] M. Szpakowska, O.B. Nagy, Non-steady state vs. steady state ki-netic analysis of the coupled ion transport through binary liquidmembranes, J. Membr. Sci. 76 (1993) 27–38.

[16] D.S. He, M. Ma, Y. Wang, Q.J. Xie, Study on extraction of Cd(II)ions with N235-xylene-CH3COONH4 bulk liquid membrane, Chin.J. Inorg. Chem. (Ch) 16 (2000) 893–898.

[17] M. Ma, D.S. He, S.H. Liao, Y. Zeng, Q.J. Xie, S.Z. Yao, Kineticstudy ofl-isoleucine transport through a liquid membrane containingdi(2-ethylhexyl) phosphoric acid in kerosene, Anal. Chim. Acta 456(2002) 157–165.

[18] P. Wieczorek, J.Å. Jönsson, L. Mathiasson, Concentration of aminoacids using supported liquid membranes with di-2-ethylhexyl phos-phoric acid as a carrier, Anal. Chim. Acta 346 (1997) 191–197.

[19] M. Teramoto, T. Yamashiro, A. Inoue, A. Yamamoto, H. Matsuyama,Y. Miyake, Extraction of amino acids by emulsion liquid membranescontaining di(2-ethylhexyl) phosphoric acid as a carrier biotechnol-ogy; coupled, facilitated transport; diffusion, J. Membr. Sci. 58 (1991)11–32.

[20] H. Itoh, M.P. Thien, T.A. Hatton, D.I.C. Wang, A liquid emulsionmembrane process for the separation of amino acids, Biotech. Bioeng.35 (1990) 853–860.

[21] S.A. Hong, H.J. Choi, S.W. Nam, Concentration of amino acids bya liquid emulsion membrane with a cationic extractant, J. Membr.Sci. 70 (1992) 225–235.

[22] S.A. Hong, J.W. Yang, Process development of amino acid concen-tration by a liquid emulsion membrane technique, J. Membr. Sci. 86(1994) 181–192.

[23] J.A. Calzado, C. Palet, M. Valiente, Facilitated transport and separa-tion of aromatic amino acids through activated composite membrane,Anal. Chim. Acta 431 (2001) 59–67.

[24] P. Deblay, M. Minior, H. Renon, Separation ofl-valine from fermen-tation broths using a supported liquid membrane, Biotech. Bioeng.35 (1990) 123–131.

[25] J.A. Partridge, R.C. Jensen, Purification of di-(2-ethylhexyl)phos-phoric acid by precipitation of copper(II) di(2-ethylhexyl) phosphate,J. Inorg. Nucl. Chem. 31 (1969) 2587–2589.

[26] G.X. Sun, Y.H. Yang, S.X. Sun, J.L. Shen, Studies on interfacialproperties of HDEHP inn-octane, Chem. J. Chin. Univ. (Ch) 16(1995) 1677–1679.

[27] Y.S. Liu, Q.Y. Dai, J.D. Wang, Distribution behavior ofl-phenylalanine by extraction with di(2-ethylhexyl) phosphoric acid,Sep. Sci. Technol. 34 (1999) 2165–2176.

[28] N.E. Tayar, R.S. Tsai, P.A. Carrupt, B. Testa, Octan-1-ol-waterpartition coefficients of zwitterionic�-amino acid, determinationby centrifugal partition chromatography and factorization intosteric/hydrophobic and polar components, J. Chem. Soc., Perkin.Trans. 2 (1992) 79–84.

[29] X.C. Wang, Biological Chemistry (Ch), Press of Tsinghua University,Beijing, 2001, p. 11.