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Transport studies of amino acids through a liquid membrane system containing carboxylated poly (styrene) carrier
journal of MEMBRANE SCIENCE ELSEVIER
Journal of Membrane Science 104 (1995) 263-269
Transport studies of amino acids through a liquid membrane system containing carboxylated poly(styrene) carrier M. Ersoz*, U.S. Vural, A. Okdan, E. Pehlivan, S. Yildiz Department of Chemistry, Faculty of Arts and Sciences, Selcuk University.Konya 42079, Turkey Received 1 August 1994; accepted in revised form 9 February 1995
Abstract Transport mechanisms have been studied for amino acids such as p-amino benzoic acid and phenylalanine moving through a bulk organic liquid membrane system containing carboxylated poly (styrene) carder. The transport in this system, in which one side of the membrane was acidic and the other side was alkaline, was influenced significantly by the initial H ÷ ion concentration on the acidic side. The passage of amino acids through the organic bulk liquid membrane was governed by an electrochemical interaction between carboxyl groups in the membrane and transporting materials. Keywords: Aminoacids; Carboxylatedpoly(styrene) carder; Liquid membrane;Transport
1. Introduction
Amino acids are now manufactured, in most cases, by the fermentation method. Ion exchange is used for the recovery, separation and purification of amino acids from the fermentation broth. Amino acids are very important compounds because they participate in a great variety of metabolic processes; their permeation through biological membranes depends on their predominantly hydrophilic character, so that coupling with carrier systems is assumed for their transport [ 1 ]. For a better understanding of this phenomenon, it is important to study model systems capable of demonstrating various aspects of the transport mechanisms. Bulk liquid membranes are often used to investigate the complexation chemistry and transport properties of synthetic and natural ionophores. There are many reports about the transport of amino acids and their derivatives [2-13] through organic liquid membranes. 0376-7388/95/$09.50 © 1995 ElsevierScience B.V. All rights reserved SSDI0376-7388(95)00036-4
These systems are composed of the two aqueous phases (I and II) which are put on each side of an organic layer containing a carrier. The carriers, which have ionic (anionic, cationic, and zwitterionic) and nonionic properties, are selected according to the properties of samples to be transported. In recent years, liquid membranes have been widely used in studies of ion transport against a concentration gradient. The active transport of specific ions in an artificial liquid membrane system has received much attention in view of simulating biological membrane functions and developing separation science methodology. Research is in progress toward the synthesis of ionophores bearing a proton-ionizable moiety such as carboxyi, phenolic hydroxyl, and amino groups, or a donor nitrogen atom in the macrocycle which is capable of actively transporting metal ions [ 14]. Polymers with specific functional groups such as a lactone ring, amide group or a carboxyl group have been synthesised [ 15 ]. Membranes made of these pol-
264
M. Ersoz et al. / Journal of Membrane Science 104 (1995) 263-269
prepared using standard concentrated solutions. Reagent-grade chloroform was used as the membrane solvent.
2.2. The modification of carboxylated polystyrene
I COCH~
CH--
COOH m:k = (5:1)
Fig. 1. The structure of the modified carboxylated poly (styrene).
ymers are able to transport metal ions and anions down the concentration gradient between the sides and through the membrane in a diaphragm cell, one side of which is acidic, the other alkaline. The functions of active transport of these membranes were dependent on reversible and rapid opening and closing of the lactone ring, tautomerism of the N-hydroxyethyl amide group, and a dissociation-equilibrium cycle of the carboxyl group by pH changes. Active transport of amino acids through liquid membranes has been comprehensively studied so far. However, the movement of amino acids through polystyrene with a specific functional group carrier has not been investigated. Preliminary experiments show that a cation exchange membrane made of poly (styrene) is capable of transporting amino acids. In this article the transport of amino acids such asp-amino benzoic acid and phenylalanine is discussed in detail. It was the aim of this study, first to examine this issue more closely, with special emphasis on the influence of the membrane phase, carboxylated poly(styrene) carrier and also the membrane preparation on the efficiency of the transport process; second to investigate the transport of amino acids against their concentration difference between two solutions across the membrane separating these solutions under the simultaneous influence of a concentration gradient and a potential gradient within the membrane; third to explain the effect of the external solution pH on the transport process.
2. Experimental
2.1. Materials The amino acids and salt were analytical grade. All chemicals were obtained from Merck. Amino acids and salt solutions were prepared using deionized water without further purification. Standard solutions were
Chloroform was purified by distillation before use. Poly(styrene) (PS) (MW ~ 250 000) was dissolved in benzene, followed by precipitating in methanol and used after purification. BF30(C2Hs)z catalyst was purified by distillation at 123°C. Toluene was also distilled under reflux. 5.2 g (0.05 mol) poly(styrene) was dissolved in 40 ml chloroform by stirring in a three-necked flask at 20°C. Then, 0.98 g (0.01 moi) maleic anhydride was added and dissolved. 1.26 ml (0.01 mol) catalyst (BF3. OEt2) was added drop by drop to the mixture at 20°C for 30 min and the mixture stirred for 2 h. After completion of the reaction, methanol was added to precipitate the product, a modified carboxylated polystyrene. The product was dried under vacuum at 50°C for 5h. The structure of the chemically modified carboxylated poly(styrene) is given in Fig. 1. The structure of the carboxylated polystyrene was determined by spectral and chemical analysis, and the result are given in Table 1.
2.3. Transport experiments Transport experiments were performed with magnetic stirring in a conventional U-tube glass cell at room temperature (Fig. 2). Phase I is a 10 ml solution of 0.005 mol protonated amino acid in 0.075 N NaCI. The pH was adjusted with HCI in the pH range 2 to 5. The membrane phase consisted of 1 g carboxylated poly(styrene) carrier in chloroform ( 15 ml) placed at the bottom of the tube. The alkaline phase II consisted of 0.005 mol amino acid and 0.075 N NaOH, at pH 12 ( total volume 10 ml). Aliquots ( 200 p,1) of the aqueous solutions of both phases were withdrawn at appropriate Table 1 Properties of carboxylated poly (styrene) Catalyst
CO2H
m : k ratio
Molecular weight
BF3. OEh
16.6
5:1
191 000
265
M. Ersoz et al. / Journal of Membrane Science 104 (1995) 263-269
Phase 1 pH 2-5
/C%H
R ~
I
[
~- 1
]
! I
[ Phase II
pH 12 4
i
CH 2 +
" ~ NH a
3
~
/co;
R - - CH2 -
~
NH 2
Membrane phase
Fig. 2. Outline of the organic liquid membrane system: 1, phase I; 2, phase I1; 3, organic layer containing a carrier; 4, spinners; 5, magnetic stirrers.
intervals and then diluted by a factor of 25. The pH of the both sides was monitored by using an Orion model 720 pH meter. The concentration of p-amino benzoic acid and phenylalanine on both sides of the membrane were measured by a UV-visible spectrophotometer (Shimadzu W-160A) at 267 nm for p-amino benzoic acid and 254 nm for phenylalanine. Each experiment was repeated at least twice and the results were consistent within _+ 10%.
3. Results and discussion Some examples of the changes in concentration of p-aminobenzoic acid and phenylalanine on the L side (left side, phase I) and R side (right side, phase II) with time are shown in Figs. 3 and 4. Changes of the pH variation in aqueous phases are given Table 2. For all values on the L-side pH, the concentration of amino acids on the R side increased to a maximum value and then decreased, whereas on the L side it was just the opposite. Although, the pH values decreases in both phases. The changes in concentration are caused by the transport of amino acids through the membrane from the L side to the R side. Because the solution on one side of the membrane is acidic, and the solution on the other side is alkaline, the ionic forms of amino acids are different on each side. Therefore, a negatively charged form of amino acid on the alkaline side may be excluded from the membrane by repulsion, whereas a positively charged form on the acidic side can permeate the membrane and diffuse down its concentra-
tion gradient to increase the total concentration of amino acid on the alkaline side. The pH on both sides under the experimental conditions kept their respective alkaline and acidic properties over a long period. Therefore, the amino acids are charged negatively on the R side and positively on the L side for that time. Consequently the permeation of amino acids from the R side into the membrane phase is difficult because both the amino acid and the membrane are charged negatively on the R side, resulting in electrostatic repulsion between them. On the other hand, on the L side, positively charged amino acid molecules can penetrate the membrane, interact with carboxylate anions in a boundary region in which the membrane swells in alkali and contracts in acid, be released by, the O H - ion, and consequently be transferred to the R side. It is generally known that in an acidic medium amino acids are protonated according to the following reaction H3N + - R - C O O - + H + ~, H3N+-R-COOH In a basic medium the following reaction occurs; H3N + - R - C O O - + O H - ~ H2N-R-COO -- + H20 In the system studied here, the transport mechanism between the membrane carrier and amino acids can be indicated as follows; R-, . CO0H~N + - R - C O O H tcarner-ammo acid) In any geometry, transport of a substrate from one aqueous phase to another must occur via a substrate/ carrier complex in the organic phase. The transport
266
M. Ersoz et al. / Journal of Membrane Science 104 (1995) 263-269
system this diffusion occurs across the unstirred boundary layer (Nernst layer) adjacent to the interface. (ii) Dissociation of the carboxylate groups in the membrane phase in the alkaline side.
"o x T --}.
E
10
Y o <,)
%
Initial L side: pH 4.5
i
[
[
i
i
i
t
0
1
2
3
4
5
30
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40
T --4. E v
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50
J
Time (h} o
Indiar L side: pH 5.0 -II
i
r
i
i
~
i
0
1
2
3
4
5
~
,
,
2O
30
40
i
5O
Time (h)
6
lO
,,,,
4 x
o
o
o
~o
Initial L side: pH 3.2
o
E ,
,
,
,
O
1
2
3
,
II
4
,
,
,
,
20
30
40
50
T i m e (h)
¢p
o
Initial L side: pH 3.2
o 0
0
1
i
i
,
,
2
3
4
5
II
~
,
,
,
20
30
40
50
Time (h)
Fig. 4, Changes in the concentration of p h e n y ~ i n e trough an organic liquid membrance; (e) phenylalanine on the L side, (o) phenylanaline on the R side.
5
Table 2 pH Variation in aqueous phases of amino acids"
o
c)
Initial L side: pH 2.0
Time p-Amino benzoic acid (pH) ,
,
~
,
O
1
2
3
~
II
4
~
,
Ii
30
40
50
Phenylanaline ( pH )
() LS
RS
LS
RS
LS
RS
LS
RS
KS
RS
2.0 2.0 2.0 1.98 1.97 1.98 1.96
12.20 12.10 12.05 11.89 11.60 11.48 11.45
3.0 2.95 2.83 2.72 2.60 2.58 2.55
12.20 12.15 12.12 12.03 11.97 11.78 11.52
4.0 3.97 3.92 3.85 3.80 3.40 2.80
12.20 12.17 12.12 1205 11.99 II.93 11.45
3.0 2.95 2.90 2.85 2.80 2,70 2,60
12.20 12.10 12.00 11.80 11.75 11.60 11.55
5,00 3.80 3.16 2.96 2.88 2.78 2.75
12.20 12.12 12.05 11.90 11.80 11.55 11.50
T i m e (h)
Fig. 3. Changes in the concentration ofp-aminobenzoic acid through an organic liquid membrane; ( e ) p-aminobenzoic acid on the L side; (o) p-aminobenzoic acid on the R side.
process can be assumed to occur according to the following sequence: (i) Diffusion of the substrate from the bulk aqueous phase to the organic aqueous interface. In a stirred
0 1 2 6 10 25 45
"LS, left side (phase I) and RS, fight side (phase II)
267
M. Ersoz et al. / Journal of Membrane Science 104 (1995 ~263-269
(iii) Complexation at the aqueous and organic phase interface resulting in the formation of a carrier-substrate complex. (iv) The movement of the carrier-substrate complex from phase I to phase II in the organic phase. (v) The releasing of the substrate at the organic/ aqueous interface (phase II). (vi) The diffusion of the carrier in the reverse direction (phase I). Thus, the pH gradient between the two aqueous phases promotes the transport of amino acid against its concentration gradient. Consequently, the acid medium of phase I allows the formation of complex-carrier which passes through the membrane. The basic medium of the phase I! facilitates the dissociation of the complex and thus the amino acids passes from the membrane interface into phase II. The concentration of moving species in the equilibrium as a function of pH was calculated using the pK values of the amino acids by the following steps: ( 1 ) The equilibrium of amino acids in the Phase I: Ci
AT 10(PH-pKa)
i _
C AI =
(for phase I, pH 2-5)
15 15
IE
10 0~
Z o c~
c m
i
i
i
i
2.0
3.0
4.0
5.0
pH
Fig. 5. Effectof the initialpH on the acidic sideon the meantransport rate of amino acids trough the organic liquid membrane; (t) paminobenzoicacid, (A) phenylalanine. static interactions with the negative charges of the carboxylated poly (styrene). Effect of the initial concentration in the L side of the membrane on a mean transport rate (mol/min) and transport fraction (%) of amino acids, can be defined [16] by Eqs. (1) and (2): Mean transport rate
i
i _
C All =
C AT 10(pOH--pKb)
(for phase II, pH > 10)
where CAI and CAH are the protonated amino acid concentration in phases I and II, respectively; CAT, total amino acid concentration (protonated and ionized) in phase I or II, In the equilibrium: C iO H C Ai l l
K~.q- ~c i H C
AI
Partitioning of amino acids between the organic membrane phase and phase I or II: C ~ l = m k C iAm
or
i _- - m jAC iAm C AII
where mA, ' partition coefficient and CiAm, amino acid concentration in phase I or II at the membrane interface. Changes in the concentration of amino acids on both sides through the organic liquid membrane with time is shown in Figs. 3 and 4 In this case the pH values of phase II were kept above pH 10. Amino acids could be transported at pH 2-5 in phase I. Below pH 5, the amino acids have a positive charge sufficient to cause electro-
l amino acid ] m~, - [ amino acid ] o -
~1)
/max Transport fraction [amino acid]m~,- [amino acid]o -
[ amino acid] o
(2)
where [Amino acid]max is the concentration in the R side of the membrane at time t maximum and [Amino acid]o is the initial concentration in the L side of the membrane. In Figs. 5 and 6 we show the effect of the initial L-side pH on the mean transport rate and transport fraction of amino acids from the L side to the R side of the membrane. In all systems, the concentration of amino acids in the R side is increased with time. The concentration changes on the two sides were necessarily opposite. These results could suggest that amino acids were actively transported across the membrane against the concentration gradient between both sides. The mean transport rate and the transport fraction had maximum values above pH 3 in the L side. These results are because under these conditions the R side stayed alkaline and the L side acidic or nearly neutral,
268
M. Ersoz et al. / Journal of Membrane Science 104 (1995) 263-269
ylated poly(styrene) as a carrier in the liquid membrane system. The carrier system may find applications in designing different functional groups to anchor to the p o l y ( s t y r e n e ) capable of transporting amino acids, metal ions and organic ions, or in examining fundamental transport behaviours o f various transport combinations.
80
~ _ . _ _ - - - - ~ 60 zr-""
g 40 o_ c
~t
f
J f
20
Acknowledgement i
i
2.0
3.0
i
i
4.0
5.0
pH
The authors wish to thank Selcuk University for research facilities.
Fig. 6. Effect of the initial pH on the acidic side on the transport fraction of amino acids trough the organic liquid membrane; (o) paminobenzoic acid, (A) phenylalanine. References which is assigned to the fixed C O O H groups o f the polymer. The mean transport rate and the transport fraction o f amino acids from the L side to the R side increased with an increase of pH. A decrease of transport fraction over the m a x i m u m value is dependent on a reverse diffusion of amino acids from the L side to the R side due to the acidic pH in the L side. In this transport system, H ÷ ions play the role o f the driving force in the transport o f the amino acids. It is considered an isothermal-isobaric system in which two electrolyte solutions are separated by a organic liquid membrane. The interest here was in the transport of amino acids against its concentration difference in the outside solutions. The concentration gradient o f amino acids on the L side of membrane - the driving force of the transport of this amino acid - is generated by a concentration difference and pH difference of a second electrolyte between the both sides. This is in good agreement with a simple mechanism for this transport, in which the driving force of the process is the concentration gradient of the amino acids. The amino acids are transported against their concentration difference between the outside solutions and pH gradient, but they move in the direction of their electrochemical potential difference.
4. Conclusions The transport of amino acids could be achieved in a wide range o f the receiving phase pH by use of carbox-
[ 1] K. Ring, Some aspects of the active transport of amino acids, Angew. Chem. Int. Ed., 9 (1970) 345. [2] S. Takeshima and S. Hirose, Selective transport of histamine from a mixture of histidine and histamine by the use of organic liquid membranesystems, Sep. Sci. Teclmol.,25 (1990) 1201, [3] C.V. Uglea and C.V. Zanoaga, Transport of amino acids through organic liquid membranes I. p-Aminobenzoic acid, J. Membrane Sci., 47 (1989) 285. [4] C.V. Uglea and C.V. Zanoaga, Transport of amino acids through organic liquid membranes 11. Aspects of the mechanism, J. Membrane Sci., 65 (1992) 47. [5] M. Bryjak, P. Wieczorek, P. Kafarski and B. Lejczak, Crownether mediated transport of amino acids through an immobilized liquid membrane, J. Membrane Sci., 37 (1988) 287. [6] J.P. Behr and J.M. Lehn, Transport of amino acids through organic liquid membranes, J. Am. Chem. Soc., 95 (1973) 6108. [7] K. Marayoma, H. Tsukuhe and T. Araki, Carrier-mediated transport of amino acid derivatives via metal complex carrier, Tetrahedron Lett., 22 ( 1981 ) 2001. [8] T. Yamaguchi, K. Nishimura, T. Shinbo and M. Sugiura, Enantiomer resolution of amino acids by a polymer-supported liquid membrane containing a chiral crown ether, Chem. Lett., (1985) 1549. [9] K. Marayoma, H. Tsukuhe and T. Araki, Carrier-mediated transport of amino acids and simple organic anions by lipophilic metal complexes, J. Am. Chem. Soc., 104 (1982) 5197. [1o] M. Bryjak, P. Wieczorek,P. Kafarskiand B. Lejczak, Transport of amino acids and their phosphonic acid analogues through supported liquid membranes containing macrocyclic carders. Experimental parameters, J. Membrane Sci., 56 ( 1991 ) 167. [11] T. Shinbo, T. Yamaguchi, H. Yanagishita, K. Sakaki, D. Kitamoto and M. Sugiura, Supported liquid membranes of amino acid mediatedby chiral crown ether-effect of membrane
M. Ersoz et al. / Journal of Membrane Science 104 (1995) 263-269
solvent on transport rate and membrane stability, J. Membrane Sci., 84 (1993) 241. 12] H. Tsukube, K. Takagi, T. Higahiyama, T. lwachido and N. Hayama, Lipophilic polyamine and polyamine macrocycles for membrane transport of amino acid esters and related cations, J. Chem. Soc., Perkin Trans. 2, (1985) 1541. 131 T. Yamaguchi, K. Nishimura, T. Shinbo and M, Sugiura, Amino acid transport through supported liquid membranes: mechanism and its application to enantiomeric resolution, Bioelectrochem. Bioenerg., 20 (1988) 109. 141 S. Yoshida and T. Watanabe, Cooperative effect of lipophilic amine and neutral crown ether on potassium ion active transport through a chloroform membrane, Bull. Chem. Soc. Jpn., 63 (1990) 3508.
269
[15] T. Uragami, S. Watanabe and M. Sugihara, Studies on syntheses and permeabilities of special polymer membranes. 48. Transport of organic ions through crosslinked poly (isobutylene-alternative co-maleic anhydride) membranes. J. Polym. Sci., Polym. Chem. Ed., 20 (1982) 1193. i 16] T. Uragami, R. Nakamura and M. Sugihara, Studies on syntheses and permeabilities of special polymer membranes: 50. Transport of metal ions against their concentration gradient through water-insoluble poly(styrene sulphonic acid) membrane. Polymer, 24 (1983) 559.
Journal of Membrane Science 104 (1995) 263-269
Transport studies of amino acids through a liquid membrane system containing carboxylated poly(styrene) carrier M. Ersoz*, U.S. Vural, A. Okdan, E. Pehlivan, S. Yildiz Department of Chemistry, Faculty of Arts and Sciences, Selcuk University.Konya 42079, Turkey Received 1 August 1994; accepted in revised form 9 February 1995
Abstract Transport mechanisms have been studied for amino acids such as p-amino benzoic acid and phenylalanine moving through a bulk organic liquid membrane system containing carboxylated poly (styrene) carder. The transport in this system, in which one side of the membrane was acidic and the other side was alkaline, was influenced significantly by the initial H ÷ ion concentration on the acidic side. The passage of amino acids through the organic bulk liquid membrane was governed by an electrochemical interaction between carboxyl groups in the membrane and transporting materials. Keywords: Aminoacids; Carboxylatedpoly(styrene) carder; Liquid membrane;Transport
1. Introduction
Amino acids are now manufactured, in most cases, by the fermentation method. Ion exchange is used for the recovery, separation and purification of amino acids from the fermentation broth. Amino acids are very important compounds because they participate in a great variety of metabolic processes; their permeation through biological membranes depends on their predominantly hydrophilic character, so that coupling with carrier systems is assumed for their transport [ 1 ]. For a better understanding of this phenomenon, it is important to study model systems capable of demonstrating various aspects of the transport mechanisms. Bulk liquid membranes are often used to investigate the complexation chemistry and transport properties of synthetic and natural ionophores. There are many reports about the transport of amino acids and their derivatives [2-13] through organic liquid membranes. 0376-7388/95/$09.50 © 1995 ElsevierScience B.V. All rights reserved SSDI0376-7388(95)00036-4
These systems are composed of the two aqueous phases (I and II) which are put on each side of an organic layer containing a carrier. The carriers, which have ionic (anionic, cationic, and zwitterionic) and nonionic properties, are selected according to the properties of samples to be transported. In recent years, liquid membranes have been widely used in studies of ion transport against a concentration gradient. The active transport of specific ions in an artificial liquid membrane system has received much attention in view of simulating biological membrane functions and developing separation science methodology. Research is in progress toward the synthesis of ionophores bearing a proton-ionizable moiety such as carboxyi, phenolic hydroxyl, and amino groups, or a donor nitrogen atom in the macrocycle which is capable of actively transporting metal ions [ 14]. Polymers with specific functional groups such as a lactone ring, amide group or a carboxyl group have been synthesised [ 15 ]. Membranes made of these pol-
264
M. Ersoz et al. / Journal of Membrane Science 104 (1995) 263-269
prepared using standard concentrated solutions. Reagent-grade chloroform was used as the membrane solvent.
2.2. The modification of carboxylated polystyrene
I COCH~
CH--
COOH m:k = (5:1)
Fig. 1. The structure of the modified carboxylated poly (styrene).
ymers are able to transport metal ions and anions down the concentration gradient between the sides and through the membrane in a diaphragm cell, one side of which is acidic, the other alkaline. The functions of active transport of these membranes were dependent on reversible and rapid opening and closing of the lactone ring, tautomerism of the N-hydroxyethyl amide group, and a dissociation-equilibrium cycle of the carboxyl group by pH changes. Active transport of amino acids through liquid membranes has been comprehensively studied so far. However, the movement of amino acids through polystyrene with a specific functional group carrier has not been investigated. Preliminary experiments show that a cation exchange membrane made of poly (styrene) is capable of transporting amino acids. In this article the transport of amino acids such asp-amino benzoic acid and phenylalanine is discussed in detail. It was the aim of this study, first to examine this issue more closely, with special emphasis on the influence of the membrane phase, carboxylated poly(styrene) carrier and also the membrane preparation on the efficiency of the transport process; second to investigate the transport of amino acids against their concentration difference between two solutions across the membrane separating these solutions under the simultaneous influence of a concentration gradient and a potential gradient within the membrane; third to explain the effect of the external solution pH on the transport process.
2. Experimental
2.1. Materials The amino acids and salt were analytical grade. All chemicals were obtained from Merck. Amino acids and salt solutions were prepared using deionized water without further purification. Standard solutions were
Chloroform was purified by distillation before use. Poly(styrene) (PS) (MW ~ 250 000) was dissolved in benzene, followed by precipitating in methanol and used after purification. BF30(C2Hs)z catalyst was purified by distillation at 123°C. Toluene was also distilled under reflux. 5.2 g (0.05 mol) poly(styrene) was dissolved in 40 ml chloroform by stirring in a three-necked flask at 20°C. Then, 0.98 g (0.01 moi) maleic anhydride was added and dissolved. 1.26 ml (0.01 mol) catalyst (BF3. OEt2) was added drop by drop to the mixture at 20°C for 30 min and the mixture stirred for 2 h. After completion of the reaction, methanol was added to precipitate the product, a modified carboxylated polystyrene. The product was dried under vacuum at 50°C for 5h. The structure of the chemically modified carboxylated poly(styrene) is given in Fig. 1. The structure of the carboxylated polystyrene was determined by spectral and chemical analysis, and the result are given in Table 1.
2.3. Transport experiments Transport experiments were performed with magnetic stirring in a conventional U-tube glass cell at room temperature (Fig. 2). Phase I is a 10 ml solution of 0.005 mol protonated amino acid in 0.075 N NaCI. The pH was adjusted with HCI in the pH range 2 to 5. The membrane phase consisted of 1 g carboxylated poly(styrene) carrier in chloroform ( 15 ml) placed at the bottom of the tube. The alkaline phase II consisted of 0.005 mol amino acid and 0.075 N NaOH, at pH 12 ( total volume 10 ml). Aliquots ( 200 p,1) of the aqueous solutions of both phases were withdrawn at appropriate Table 1 Properties of carboxylated poly (styrene) Catalyst
CO2H
m : k ratio
Molecular weight
BF3. OEh
16.6
5:1
191 000
265
M. Ersoz et al. / Journal of Membrane Science 104 (1995) 263-269
Phase 1 pH 2-5
/C%H
R ~
I
[
~- 1
]
! I
[ Phase II
pH 12 4
i
CH 2 +
" ~ NH a
3
~
/co;
R - - CH2 -
~
NH 2
Membrane phase
Fig. 2. Outline of the organic liquid membrane system: 1, phase I; 2, phase I1; 3, organic layer containing a carrier; 4, spinners; 5, magnetic stirrers.
intervals and then diluted by a factor of 25. The pH of the both sides was monitored by using an Orion model 720 pH meter. The concentration of p-amino benzoic acid and phenylalanine on both sides of the membrane were measured by a UV-visible spectrophotometer (Shimadzu W-160A) at 267 nm for p-amino benzoic acid and 254 nm for phenylalanine. Each experiment was repeated at least twice and the results were consistent within _+ 10%.
3. Results and discussion Some examples of the changes in concentration of p-aminobenzoic acid and phenylalanine on the L side (left side, phase I) and R side (right side, phase II) with time are shown in Figs. 3 and 4. Changes of the pH variation in aqueous phases are given Table 2. For all values on the L-side pH, the concentration of amino acids on the R side increased to a maximum value and then decreased, whereas on the L side it was just the opposite. Although, the pH values decreases in both phases. The changes in concentration are caused by the transport of amino acids through the membrane from the L side to the R side. Because the solution on one side of the membrane is acidic, and the solution on the other side is alkaline, the ionic forms of amino acids are different on each side. Therefore, a negatively charged form of amino acid on the alkaline side may be excluded from the membrane by repulsion, whereas a positively charged form on the acidic side can permeate the membrane and diffuse down its concentra-
tion gradient to increase the total concentration of amino acid on the alkaline side. The pH on both sides under the experimental conditions kept their respective alkaline and acidic properties over a long period. Therefore, the amino acids are charged negatively on the R side and positively on the L side for that time. Consequently the permeation of amino acids from the R side into the membrane phase is difficult because both the amino acid and the membrane are charged negatively on the R side, resulting in electrostatic repulsion between them. On the other hand, on the L side, positively charged amino acid molecules can penetrate the membrane, interact with carboxylate anions in a boundary region in which the membrane swells in alkali and contracts in acid, be released by, the O H - ion, and consequently be transferred to the R side. It is generally known that in an acidic medium amino acids are protonated according to the following reaction H3N + - R - C O O - + H + ~, H3N+-R-COOH In a basic medium the following reaction occurs; H3N + - R - C O O - + O H - ~ H2N-R-COO -- + H20 In the system studied here, the transport mechanism between the membrane carrier and amino acids can be indicated as follows; R-, . CO0H~N + - R - C O O H tcarner-ammo acid) In any geometry, transport of a substrate from one aqueous phase to another must occur via a substrate/ carrier complex in the organic phase. The transport
266
M. Ersoz et al. / Journal of Membrane Science 104 (1995) 263-269
system this diffusion occurs across the unstirred boundary layer (Nernst layer) adjacent to the interface. (ii) Dissociation of the carboxylate groups in the membrane phase in the alkaline side.
"o x T --}.
E
10
Y o <,)
%
Initial L side: pH 4.5
i
[
[
i
i
i
t
0
1
2
3
4
5
30
J
40
T --4. E v
i
50
J
Time (h} o
Indiar L side: pH 5.0 -II
i
r
i
i
~
i
0
1
2
3
4
5
~
,
,
2O
30
40
i
5O
Time (h)
6
lO
,,,,
4 x
o
o
o
~o
Initial L side: pH 3.2
o
E ,
,
,
,
O
1
2
3
,
II
4
,
,
,
,
20
30
40
50
T i m e (h)
¢p
o
Initial L side: pH 3.2
o 0
0
1
i
i
,
,
2
3
4
5
II
~
,
,
,
20
30
40
50
Time (h)
Fig. 4, Changes in the concentration of p h e n y ~ i n e trough an organic liquid membrance; (e) phenylalanine on the L side, (o) phenylanaline on the R side.
5
Table 2 pH Variation in aqueous phases of amino acids"
o
c)
Initial L side: pH 2.0
Time p-Amino benzoic acid (pH) ,
,
~
,
O
1
2
3
~
II
4
~
,
Ii
30
40
50
Phenylanaline ( pH )
() LS
RS
LS
RS
LS
RS
LS
RS
KS
RS
2.0 2.0 2.0 1.98 1.97 1.98 1.96
12.20 12.10 12.05 11.89 11.60 11.48 11.45
3.0 2.95 2.83 2.72 2.60 2.58 2.55
12.20 12.15 12.12 12.03 11.97 11.78 11.52
4.0 3.97 3.92 3.85 3.80 3.40 2.80
12.20 12.17 12.12 1205 11.99 II.93 11.45
3.0 2.95 2.90 2.85 2.80 2,70 2,60
12.20 12.10 12.00 11.80 11.75 11.60 11.55
5,00 3.80 3.16 2.96 2.88 2.78 2.75
12.20 12.12 12.05 11.90 11.80 11.55 11.50
T i m e (h)
Fig. 3. Changes in the concentration ofp-aminobenzoic acid through an organic liquid membrane; ( e ) p-aminobenzoic acid on the L side; (o) p-aminobenzoic acid on the R side.
process can be assumed to occur according to the following sequence: (i) Diffusion of the substrate from the bulk aqueous phase to the organic aqueous interface. In a stirred
0 1 2 6 10 25 45
"LS, left side (phase I) and RS, fight side (phase II)
267
M. Ersoz et al. / Journal of Membrane Science 104 (1995 ~263-269
(iii) Complexation at the aqueous and organic phase interface resulting in the formation of a carrier-substrate complex. (iv) The movement of the carrier-substrate complex from phase I to phase II in the organic phase. (v) The releasing of the substrate at the organic/ aqueous interface (phase II). (vi) The diffusion of the carrier in the reverse direction (phase I). Thus, the pH gradient between the two aqueous phases promotes the transport of amino acid against its concentration gradient. Consequently, the acid medium of phase I allows the formation of complex-carrier which passes through the membrane. The basic medium of the phase I! facilitates the dissociation of the complex and thus the amino acids passes from the membrane interface into phase II. The concentration of moving species in the equilibrium as a function of pH was calculated using the pK values of the amino acids by the following steps: ( 1 ) The equilibrium of amino acids in the Phase I: Ci
AT 10(PH-pKa)
i _
C AI =
(for phase I, pH 2-5)
15 15
IE
10 0~
Z o c~
c m
i
i
i
i
2.0
3.0
4.0
5.0
pH
Fig. 5. Effectof the initialpH on the acidic sideon the meantransport rate of amino acids trough the organic liquid membrane; (t) paminobenzoicacid, (A) phenylalanine. static interactions with the negative charges of the carboxylated poly (styrene). Effect of the initial concentration in the L side of the membrane on a mean transport rate (mol/min) and transport fraction (%) of amino acids, can be defined [16] by Eqs. (1) and (2): Mean transport rate
i
i _
C All =
C AT 10(pOH--pKb)
(for phase II, pH > 10)
where CAI and CAH are the protonated amino acid concentration in phases I and II, respectively; CAT, total amino acid concentration (protonated and ionized) in phase I or II, In the equilibrium: C iO H C Ai l l
K~.q- ~c i H C
AI
Partitioning of amino acids between the organic membrane phase and phase I or II: C ~ l = m k C iAm
or
i _- - m jAC iAm C AII
where mA, ' partition coefficient and CiAm, amino acid concentration in phase I or II at the membrane interface. Changes in the concentration of amino acids on both sides through the organic liquid membrane with time is shown in Figs. 3 and 4 In this case the pH values of phase II were kept above pH 10. Amino acids could be transported at pH 2-5 in phase I. Below pH 5, the amino acids have a positive charge sufficient to cause electro-
l amino acid ] m~, - [ amino acid ] o -
~1)
/max Transport fraction [amino acid]m~,- [amino acid]o -
[ amino acid] o
(2)
where [Amino acid]max is the concentration in the R side of the membrane at time t maximum and [Amino acid]o is the initial concentration in the L side of the membrane. In Figs. 5 and 6 we show the effect of the initial L-side pH on the mean transport rate and transport fraction of amino acids from the L side to the R side of the membrane. In all systems, the concentration of amino acids in the R side is increased with time. The concentration changes on the two sides were necessarily opposite. These results could suggest that amino acids were actively transported across the membrane against the concentration gradient between both sides. The mean transport rate and the transport fraction had maximum values above pH 3 in the L side. These results are because under these conditions the R side stayed alkaline and the L side acidic or nearly neutral,
268
M. Ersoz et al. / Journal of Membrane Science 104 (1995) 263-269
ylated poly(styrene) as a carrier in the liquid membrane system. The carrier system may find applications in designing different functional groups to anchor to the p o l y ( s t y r e n e ) capable of transporting amino acids, metal ions and organic ions, or in examining fundamental transport behaviours o f various transport combinations.
80
~ _ . _ _ - - - - ~ 60 zr-""
g 40 o_ c
~t
f
J f
20
Acknowledgement i
i
2.0
3.0
i
i
4.0
5.0
pH
The authors wish to thank Selcuk University for research facilities.
Fig. 6. Effect of the initial pH on the acidic side on the transport fraction of amino acids trough the organic liquid membrane; (o) paminobenzoic acid, (A) phenylalanine. References which is assigned to the fixed C O O H groups o f the polymer. The mean transport rate and the transport fraction o f amino acids from the L side to the R side increased with an increase of pH. A decrease of transport fraction over the m a x i m u m value is dependent on a reverse diffusion of amino acids from the L side to the R side due to the acidic pH in the L side. In this transport system, H ÷ ions play the role o f the driving force in the transport o f the amino acids. It is considered an isothermal-isobaric system in which two electrolyte solutions are separated by a organic liquid membrane. The interest here was in the transport of amino acids against its concentration difference in the outside solutions. The concentration gradient o f amino acids on the L side of membrane - the driving force of the transport of this amino acid - is generated by a concentration difference and pH difference of a second electrolyte between the both sides. This is in good agreement with a simple mechanism for this transport, in which the driving force of the process is the concentration gradient of the amino acids. The amino acids are transported against their concentration difference between the outside solutions and pH gradient, but they move in the direction of their electrochemical potential difference.
4. Conclusions The transport of amino acids could be achieved in a wide range o f the receiving phase pH by use of carbox-
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