Transport of amino acids and their phosphonic acid analogues through supported liquid membranes containing macrocyclic carriers. Experimental parameters

Journal of Membrane Science, 56 (1991) 167-180 Elsevier Science Publishers B.V.. Amsterdam

167

Transport of amino acids and their phosphonic acid analogues through supported liquid membranes containing macrocyclic carriers. Experimental parameters Marek Bryjak Institute of Organic and Polymer Technology, Technical University of WrocZaw,50-370 WrocZaw(Poland)

Piotr Wieczorek Institute of Chemistry, Pedagogical University of Opole, 45-052 Opole (Poland)

Pawel Kafarski*

and Barbara

Lejczak

Institute of Organic and Physical Chemistry, Technical University of Wroclaw, 50-370 WrocZaw (Poland) (Received December

21,1989; accepted in revised form August 20,199O)

Abstract Amino acid hydrochlorides are well transported through l-decanol membranes containing Kryptofix 5 or 222 and supported in a porous polyacrylonitrile hollow fiber matrix. Factors which influence the transport of phenylalanine hydrochloride were studied in some detail using this sheet- as well as hollow fibre-supported liquid membranes. These studies show that the choice of the membrane phase, the kind of polymeric support and the mode of membrane preparation are of great importance for the efficiency of the process. The most vital step in the membrane preparation appears to be its activation by soaking in a solution of phenylalanine hydrochloride in a water-ethanol or water-propanol mixture. This probably improves the contact between membrane and the aqueous phase. Keywords: Liquid membrane; facilitated transport, organic separations; riers; amino acid transport; supported liquid membrane

diffusion; membrane

car-

Introduction Liquid membrane separations, which combine the solvent extraction and stripping processes in a single step, deserve special attention because of their potential applications, which include the separation and concentration of specific chemical species, the decontamination of wastes or biological fluids and *To whom correspondence

0376-7388/91/$03.50

should be addressed.

0 1991-

Elsevier Science Publishers

B.V.

168

direct analysis for a particular compound in a mixture. Membrane separations are attractive from a preparative standpoint because they can be used in a continuous process and the cost and energy requirements are often reasonable. Such applications, however, depend on a complete analysis of the transport mechanisms. There are three general types of liquid membrane: bulk membranes, supported liquid membranes immobilized in a porous polymeric film, and liquid surfactant membranes which involve water-oil-water emulsions. Supported liquid membranes consist of an organic solvent phase which is contained within the pores of the polymeric solid, separating two aqueous phases. Chemical species may pass from one phase to the other phase through the membrane if they have some solubility in the membrane. This transfer may be accomplished by simple diffusion or by carrier facilitated transport, wherein species are ushered across the membrane by lipophilization via a co-substrate. Biological transport of amino acids is usually mediated by carrier proteins buried in biomembranes. The application of models to such transport systems has long been desired in the separation science and technology of amino acids and is an active area of research [l-16]. Previously we described preliminary studies on the crown ether-mediated permeation of amino acids through a decalin membrane supported on polyethylene film [ 161, a model system that can be controlled and is metabolically independent. The purpose of the present paper is to examine this issue more closely, with special emphasis on the influence of the kind of membrane phase, macrocyclic carrier and polymeric support as well as the method for membrane preparation on the efficiency of the transport process. Experimental Materials L-Amino acids were purchased from commercial sources and were used as received. Their phosphonic acid analogues were synthesized according to a recently described modification [ 171 of the standard amidoalkylation technique [ 181, or were kindly supplied by Dr. M. Soroka (Technical University of Wroclaw ). The macrocyclic carriers Kryptofix 5 (K5 ) , Kryptofix 22 (K22 ), Kryptofix 222 (K222), Kryptofix 22DD (K22DD), dibenzo-18-crown-6 (DB18C6) and dibenzopyridyl-18-crown-6 (DBP18C6) were a generous gift from Merck (Darmstadt, Germany). The structures and systematic names of these carriers are given in Fig. 1. All the solvents were of analytical grade purity. Preparation of thin sheet-supported liquid membranes Thin sheet polymeric films were prepared from technical fibres of modified polyacrylonitrile (Anilana, Lodz, Poland) by pouring a 17.5% (w/w) solution

169

onon

1

&$ 0

0

-

N

-

/“\I -

\

H K22

KS

K222

C12H25 KZZDD

DBl8C6 DBPlBC6

Fig. 1. Chemical structures pentaoxatridecane); K22

of the carriersused: K5 (Kryptofix 5; 1,13-bis(8-quinolyl)-1,4,7,10,13(Kryptofix 22; 1,7,10,16-tetraoxa-4,13-diazacyclooctadecane); K222

(Kryptofix 222; 4,7,13,16,21,24-hexaoxa-l,lO-diazabicyclo[8.8.8]hexacosane); K22DD (Kryptofix 22 DD; 7,16-didecyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane); DB18C6 (dibenzo-18crown-6; 1,4,7,14,17,20-hexaoxa[7.7]orthocyclophan); DPB18C6 (dibenzopyridyl-18-crown-6; 1,4,7,14,23-pentaoxa[7.2]orthocyclo[2] (2,6)pyridinophan).

of the polymer in dimethylformamide onto a flat glass surface and gelling in water at 25°C. In this manner membranes of 150-250 pm thickness, 79-82% porosity (as determined by the weighing method [ 191) and 5 nm pore diameter (as determined by the Ferry-Elford procedure [ 201) were obtained. The membranes were swollen in methanol (24 hr ) and 1-decanol(24 hr ) and then soaked in a 10W4A4 solution of the appropriate macrocyclic ligand in 1-decanol (5-6 days). The concentration of the ligands was chosen on the basis of preliminary studies, which indicated that this concentration ensures an effective amino acid transport and does not cause precipitation of the carrier. Preparation l

of hollow fibre membranes

The following ultrafiltration hollow fibre membranes were used: polysulphone asymmetric membrane (PS) for protein filtration (cut-off

170

10,000; porosity 80-85%; diameter 1.1 mm; thickness loo-150 pm) produced by W. Ekstein (Warsaw, Poland), polyacrylonitrile asymmetric membrane (PAN) for protein filtration (cutoff 10,000; porosity 80-G%; diameter 1.1 mm; thickness loo-150 pm) produced by the Institute of Synthetic Fibers (Lodz, Poland). polyethylene symmetric membrane (PE; porosity 45%; diameter 1.0 mm; thickness loo-150 pm) prepared in our laboratories. The membranes were assembled in 250 mm long glass modules in sets of six (in the case of PAN and PS) or seven (for PE) using Elamed polyurethane cement (W. Ekstein). In this way, fiber modules 210 mm in length and 43.5 cm2 in surface area for PAN and PS membranes or 46.1 cm2 for PE membranes were obtained. The modules were washed with water (72 hr ), swollen by successive washings with methanol, ethanol and 1-decanol, soaked in a 5 x low4 M solution of macrocyclic carrier in the alcohol (48 hr ) and rewashed with water. l

l

Thin sheet membrane transport experiments Membranes were conditioned by soaking them in 0.025 Methanol-water or propanol-water solutions of phenylalanine hydrochloride for 24 hr. They were then surface dried and placed between the two compartments of the Metaplax apparatus described previously [ 161. A 0.025 M solution of phenylalanine hydrochloride (source phase: pH 2.1) was placed in one compartment, and the second one was filled with deionized water or a water-ethanol mixture (receiving phase; pH 5.5-5.8; conductivity 2-4 PUS). The increase of the amino acid hydrochloride concentration in the receiving phase was monitored conductimetrically (Radelkis OK 102 1 conductimeter, Hungary). For this purpose the apparatus was calibrated using phenylalanine hydrochloride solutions of known concentrations in the receiving phase compartment, with the membrane replaced by non-permeable polyethylene film. After each series of three to five measurements, possible leakage of the membrane was checked using a lop3 M hydrochloric acid solution as the source phase. Fluxes lower by at least three orders of magnitude demonstrated a leak free membrane. Each experiment was repeated at least three times, and the results are reported as the average value from these determinations. Hollow fibre membrane experiments The system used for the transport experiments is illustrated in Fig. 2. The membrane was conditioned with a 0.025 Methanol-water (1:10v/v)solution of the appropriate amino acid hydrochloride followed by washing with water. The experiments were carried out by placing 50 ml of 0.025 M aqueous amino acid hydrochloride solution in the external loop F (source phase) and deionized water in the internal loop I (receiving phase). The increase of amino acid

171

Fig. 2. Apparatus used for the transport experiment: (I) internal cycle, receiving phase; (F) external cycle, source phase; (P) peristaltic pump (Unipan 304, Poland); (E) platinum A-9 2PBE electrode (Energopomiar, Poland); (C) conductimeter (Mera-Elwro N-572, Poland); (R) recorder (Corabid KB-5504/EII, Poland).

hydrochloride concentration in the receiving phase was monitored conductimetrically using individual calibration curves for each hydrochloride. The experiments were carried out for 4-6 hr at 25’ C with the flow of solution in each loop being 20 ml/min. Each experiment was repeated at least three times and the results are reported as the average value of these determinations. Results and discussion Studies with thin sheetpolyacrylonitrile membranes In a previous paper [ 161 we indicated that amino acid hydrochlorides are transported well through a decalin solution of Kryptofix 5 immobilized in a thin sheet porous polyethylene matrix. In expanding the scope of this investigation, we began with experiments in the permeation of amino acid hydrochlorides using the same liquid membrane system supported in porous polyacrylonitrile hollow fibers. Surprisingly, no transport was observed. Studies conducted with thin sheet membranes also indicated that replacement of a polyethylene by a polyacrylonitrile matrix resulted in the total loss of transporting ability. Intensive investigations of experimental variables led to the discovery that

172

replacement of decalin by the more polar solvent 1-decanol and the introduction of a special procedure for membrane activation gave rise to effective transport. The experimental protocol was thus designed with two goals: (a) to determine the optimal procedure for conditioning; and (b) to examine the transport efficiencies of the various macrocyclic ligands present in the membrane phase. Conditioning of the supported membrane appeared to be the most vital step in the preparation of the membrane system. It was accomplished by soaking the supported liquid membranes in a 0.025 Msolution of phenylalanine hydrochloride in aqueous ethanol, and resulted in an enormous increase in transport efficiency. Optimal conditions of this activation process involve soaking of the membrane in a 6 : 1 (v/v) water-ethanol solution of phenylalanine hydrochloride for 24 hr. It is worth noting that the use of this solution as the source phase and water-ethanol (6: 1, v/v) mixtures as the receiving phase led to a significant decrease in phenylalanine hydrochloride fluxes (Table 1). The absence of phenylalanine hydrochloride form the conditioning solutions resulted in identical rise of the observed fluxes. However, a significant lag period in the transport kinetics (up to 2 hr, with 8-12 hr duration of the whole experiment) was observed. Thus, the conditioning process was found to affect significantly the transporting ability of the membrane. Although other explanations are possible, we favour the hypothesis that it is due to two factors: (a) addition of ethanol to the activating medium results in the decrease of surface tension of the aqueous phase and enhances tight contact between the membrane and the water phases (Fig. 3 ) ; and (b ) the conditioning of the membrane changes the surface propTABLE

1

Flux of phenylalanine hydrochloride (mmol/m’-hr)” from source to receiving aqueous phases through 1-decanol membranes immobilized in a thin sheet polyacrylonitrile matrix as a function of the conditioning Carrierb

conditions

and the kind of macrocyclic Activation

conditions

carrier

(water to ethanol ratio)

Water

13:l

6:l

none

0.76

1.23

K5 K22 K222

0.75 0.59

1.25 1.67 3.42 1.55 1.32 1.32

7.8 10.9

K22DD DB18C6 DBP18C6

0.85 0.89 0.47 0.55

“For a standardized membrane thickness of 100 pm (obtained factor: 100 pm/real membrane thickness in pm). bCarrier concentration = 1 X lob4 M.

by multiplication

23.0 19.2 21.9 9.2 12.2 of the flux by the

173

Fig. 3. Conditioning of the immobilized liquid membrane: (A) non-conditioned membrane; (B) conditioning enhances the contact between the phases; (C) this property is retained during the transport experiment. 1

, 6 Percent

concentration

of

the

alcohol

in

water,

wlw

Fig. 4. The dependence of solution surface tension as a function of alcohoi concentration. The data are based on Ref. [ 2 11.

erties of the polymeric matrix, improving its wettability and, consequently, also improving the contact between the phases. If this is the case, the replacement of ethanol by propanol, a solvent of higher hydrophobicity than ethanol, would cause a stronger decrease in the surface tension of the aqueous solutions (Fig. 4)) and should lead to an increase in the transport efficiency of the membrane. This is indeed the case, since the use of water-propanol solutions for conditioning resulted in a drastic increase of phenylalanine hydrochloride flux through membranes prepared in such a way (Table 2 ) . It was also found that the water- ( 1-decanol )-water membrane sys-

174

TABLE 2 Flux of phenylalanine hydrochloride (mmol/m*-hr)” through 1-decanol membranes. Prior to the transport experiments the membranes were activated by soaking in a 0.025 M phenylalanine hydrochloride solution in water-ethanol or water-propanol (6: 1, v/v) Carrierb

Source and receiving phase solvent water-alcohol’ (6: 1, v/v)

water Water-ethanol

none K5 K22 K222 K22DD DB18C6 DBPlSC6

1.8 10.9 23.0 19.2 21.9 12.2 9.2

none K5 K22 K222 K22DD DB18C6 DBP18C6

156.6 145.5 141.3 228.1 214.0 148.0 187.7

Water-propanol

as activation solvent 5.5 7.9 9.9 6.4 10.8 9.1 6.8 as activation

solvent

31.8 36.7 25.6 33.2 39.1 25.0 35.6

“For a standardized membrane thickness of 100 ,nm. bCarrier concentration = 1 x 10e4 M. “Water-ethanol for water-ethanol activation and water-propanol for water-propanol activation.

tern was more effective than the (water-propanol)-( l-decanol)-(water-propanol) system. The hypothesis that the activation produces a change in the surface characteristics of the membranes received additional support in studies of the persistence of the conditioning effect. As seen in Fig. 5, the flux of phenylalanine hydrochloride decreased gradually for successive experiments conducted with the same membrane, although the changes were rather small. After five consecutive transport experiments the membrane properties were still satisfactory. The results presented in Tables 1 and 2 reveal that the transport efficiency is not strongly influenced by the identity of the macrocyclic ligand. All the data show that a significant portion of the transport occurs by simple diffusion rather than by macrocycle-facilitated transport. In Table 3, the percentage participation of carrier-facilitated transport in the total phenylalanine hydrochloride flux is presented for different conditions. The percentage participation of carrier-mediated transport was defined as:

175

JT-Jo @&loo=---x T JT

100

JT

where JF is the flux caused by lipophilization of the substrate by the macrocyclic ligand, and is calculated by subtraction of the diffusion-governed value found when the experiment was carried out in the absence of the carrier (Jo) from the total value (JT) observed when carrier was used. _ 220,

L

30

r

2ok---lo! 1

I

/

2

3

Number

of

I L

repetitionsa

Fig. 5. Fluxes of phenylalanine hydrochloride as a function of the number of experiment repetitions. 0 = fist experiment; 1 = first repetition without activation of the membrane, etc. Conditions: (0 ) water-ethanol (6 : 1, v/v) activation, water-ethanol (6: 1, v/v) as source and receiving phases, Kryptofix 5 as carrier; (0 ) water-ethanol (6 : 1, v/v) activation, water as source and receiving phases, Kryptofix 5 as carrier; (A ) water-ethanol (6: 1, v/v) activation, aqueous source and receiving phases, Kryptofix 22DD as carrier; (X) water-propanol (6: 1, v/v) activation, aqueous source and receiving phases, Kryptofix 5 as carrier; (0) water-propanol (6: 1, v/v) activation, aqueous source and receiving phases, Kryptofix 22DD as carrier.

176 TABLE 3 Percentage

of carrier facilitated

transport

(@) for aqueous source and receiving phases

Carrier”

@ (o/o) Activation water

none K5 K22 K222 K22DD DB18C6 DBP18C6

water-propanol 6:l

water-ethanol 13:l

6:l

10 15

0 0 26 64 21

ib ib

71

0 29 66 52 64 36 15

0 0 ib

solvent

7

0 ib ib

31 27 0 17

“Carrier concentration= 1 x 1O-4 M. bInhibition; total flux lower than that observed if no carrier was used.

The data presented in Table 3 indicate that carrier-mediated transport usually contributed to only a small percentage of the total flux of phenylalanine hydrochloride across the membrane. In only a few cases, for membranes activated with water-ethanol solutions of phenylalanine hydrochloride, the fluxes resulting from the action of macrocycles accounted for more than 50% of the total values. The efficiency of this transport component reached its maximum for certain activation conditions. Thus, in the case of membranes containing DBPlW6 and K222, the maximum appeared when the membranes were activated with a 13:1 (v/v)water-ethanol solution of phenylalanine hydrochloride, while for those containing K22 and K22DD the maximal fluxes were reached when the membranes were conditioned in 6: 1 (v/v) water-ethanol solutions. Since the selectively of the transport will depend mainly on the carrier-mediated component, the knowledge of this factor is of some practical value. Hollow fibre membrane studies In all the experiments described in this section, the membranes were conditioned in 0.025 M solutions of the appropriate amino acid hydrochloride in a water-ethanol (10 : 1, v/v) mixture prior to the measurements. The data presented in Fig. 6 reveal the influence of the type of liquid and polymeric component of the membrane on the efficiency of phenylalanine hydrochloride transport across the membranes in the absence of carrier molecules. A strong influence of these two membrane components on the flux of phenylalanine hydrochloride was found.

177

2-

c7

Number

c9

of

alcohohc

carbon

atoms

Fig. 6. Phenylalanine hydrochloride flux through liquid: heptanol (C,), octanol (CT,), nonanol (C, ) , and decanol (C 10) membranes immobilized in porous ( 0 ) polyethylene; ( 0 ) polyacrylonitrile; and (A ) polysulphone hollow fibres.

An increase of the molecular weight of the alcohol used as liquid membrane phase resulted in a decrease of the phenylalanine hydrochloride flux. Obviously, this results from two phenomena: (a) better solubility of the hydrochloride in lower alcohols; and (b) a lower viscosity of the membrane phase for the lower aliphatic alcohols. This dependence was very pronounced when polyethylene was used as the matrix, whereas for polysulphone and polyacrylonitrile the sensitivity to viscosity was much less. Although of lower porosity (45% ), the polyethylene matrix was found to be superior to the polysulphone or the polyacrylonitrile matrices (porosity 80-85% ). This probably results from the differences in the structure of the polymers used in this study. The hollow fibre matrices were symmetric polyethylene and asymmetric polysulphone and polyacrylonitrile. Thus, polyethylene was an homogeneous material, while the existence of thin (up to 1 pm) compact surface layers of porosity ca. 1% characterizes the two others. The permeation through these layers seems to limit the transport process. Finally, the transport of a wide variety of amino acid hydrochlorides through the decanol membrane supported in a porous hollow fibre polyacrylonitrile

178

matrix was studied. In this study Kryptofix 5 or Kryptofix 222 was used as carrier. In aqueous solutions amino acids exist as zwitterions, which are not transported through the membrane (data not shown). Their hydrochlorides have good solubility in the membrane phase and are easily taken up by macrocyclic ligand molecules, and thus readily transported across the membrane. For comparison, the transport of phosphonic acid analogues of several amino acid hydrochlorides was also studied. The results summarized in Table 4 clearly demonstrate that the amino acid hydrochlorides, as well as their phosphonic acid analogues, are quite well transported across the membranes containing either Kryptofix carrier. The observed fluxes are about two or three orders of magnitude higher than those reported in the literature for similar systems [ 10,11,13,14,16]. Although we have studied the transport of all protein amino acids and some of their phosphonic acid analogues, it is not possible to draw any meaningful TABLE 4 Fluxes (mmol/m’-hr) of amino acid hydrochlorides through a l-decanol membrane immobilized in a polyacrylonitrile hollow fibre matrix Hydrochloride”

Gly. HCl Ala. HCl P-Ala. HCI Val. HCI Leu. HCl Thr. HCl Pro, HCl Hyp. HCI Cys. HCl Met. HCl Phe. HCl Tyr. HCl Asn. HCl Gln. HCI Asp. HCl Glu. HCl Lys. HCl

Carrierb

Hydrochloride”

K5

K222

0.154 0.155 0.160 0.173 0.087 0.138 0.147 0.146 0.254 0.259 0.180 0.026 0.119 0.267 0.155 0.125 0.137

0.054

0.052 0.115 0.108 0.079 0.169

0.161

Carrierb K5

Lys. 2HC1 Arg. HCI Arg. 2HCl Arg. 3HCl His. HCl His. 2HCl His. 3HC1 AlaP. HCl ValP. HCl LeuP. HCl PheP. HCl TyrP. HCl P-AspP. HCl Cil (P-AlaP) Cysteic acid

0.315 0.159 0.137 0.135 0.000 0.122 0.176 0.217 0.168 0.175 0.046 0.108 0.314 0.041

K222 0.183 0.170 0.000 0.066 0.078 0.144

0.182

0.110 0.134

“The amino acid three letter IUPAC code was adopted to identify the various amino acids; their phosphonic acid analogues are abbreviated by addition of the letter P to the tree letter code of the parent amino acid; p-AspP = /3-phosphonoalanine; Cil (/3-AlaP) = ciliatine (2.aminoethanephosphonic acid). Amino acid hydrochloride concentrationz2.5 x 10e3 M bCarrier concentration = 1 X 10e4 M.

179

conclusion on the relationship between the structure of an amino acid and its ability to be transported across the membrane. There is no dependence upon the hydrophobicity of the amino acids, as shown in the case of liquid emulsion membranes [ 151, or upon the acidity of the source medium, as demonstrated in our previous study [ 161. It is also quite surprising that Kryptofix 5 mediated transport appeared to be more effective than the transport facilitated by Kryptofix 222. The opposite result was found in an analogous sheet membrane system. Conclusions

Amino acid hydrochlorides are transported well through 1-decanol membranes supported in porous polymeric matrices. This transfer is accomplished both by simple diffusion and carrier-mediated processes in a ratio which depends on the structure of the macrocyclic ligand, as well as on the mode of membrane preparation. The choice of the membrane phase, the kind of polymeric matrix and the procedure for the activation of the membrane system is of great importance for the efficiency of the transport process. The structure of the macrocyclic ligand is of less importance. Acknowledgements

This work was supported by CPBP 02.11 Research Programme. We would like to express our appreciation to Dr. Roman Gancarz for helpful discussions.

References 1 2

3 4 5

J.P. Behr and J.M. Lehn, Transport of amino acids through organic liquid membranes, J. Am. Chem. Sot., 95 (1973) 6108. M. Newcomb, J.L. Toner, R.C. Hegelson and D.J. Cram, Host-guest complexation. 20. Chiral recognition in transport as a molecular basis for catalytic resolving machine, J. Am. Chem. sot., 101 (1979) 4941. K. Maruyama, H. Tsukube and T. Araki, An artificial oligomer transport of organic substrates, J. Chem. Sot., Chem. Commun., (1980) 966. K. Maruyama, H. Tsukube and T. Araki, Active and passive transport of amino acid derivatives via metal complex carrier, Tetrahedron Lett., 22 (1981) 2001. K. Maruyama, H. Tsukube and T. Araki, Carrier-mediated transport of amino acids and simple organic anions by lipophilic metal complexes, J. Am. Chem. Sot., 104 (1982) 5197.

6

H. Tsukube, Noncyclic crown-type polyether polymers for transport of alkali cation and amino

7

acid anion, J. Polym. Sci., 20 (1982) 2989. H. Tsukube, Lipophilic macrocyclic tetraamine

8

as specific carrier of amino acids and related

anions, Tetrahedron Lett., 24 (1983) 1519. H. Tsukube, Artificial transport of amino acid, oligopeptide, and related anions by macrocyclic polyamine-transition metal complexes, J. Chem. Sot., Perkin Trans. 1, (1983) 29.

180 9

H. Tsukube, K. Takagi, T. Higahiyama,

T. Iwachido and N. Hayama, Lipophilic

polyamine

and polyamine macrocycles for membrane transport of amino acid esters and related cations, J. Chem. Sot., Perkin Trans. 2, (1985) 1541. 10

11

T. Yamaguchi, K. Nishimura, T. Shinbo and H. Sugiura, Enantiomer resolution of amino acids by a polymer-supported liquid membrane containing a chiral crown ether, Chem. Lett., (1985) 1549. T. Yamaguchi, K. Nishimura, T. Shinbo and H. Sugiura, Chiral crown ether-mediated trans-

12

port of phenylglycine through an immobilized liquid membrane, Maku, 10 (1985) 178. P. Plucinski, P. Kafarski, B. Lejczak and M. Cichocki, The permeation ofphosphonopeptides through liquid membranes, Prepr. Int. Solvent Extr. Conf., ISEC’86, 1986 Vol. III, pp. 669-

13

676. M. Yoshikawa, M. Kishida, M. Tanigaki and W. Eguchi, Novel membrane transport system for amino compounds. Amino acid transport, Maku, 12 (1987) 221.

14

T. Shinbo, T. Yamaguchi, K. Nishimura, M. Kikkawa and H. Sugiura, Enantiomer-selective membrane electrode for amino acid methyl esters, Anal. Chim. Acta, 193 (1987) 367.

15

M.P. Thien, T.A. Hatton and D.I.C. Wang, Separation and concentration of amino acids using liquid emulsion membranes, Biotechnol. Bioeng., 32 (1988) 604. M. Bryjak, P. Wieczorek, P. Kafarski and B. Lejczak, Crown-ether mediated transport of amino acids through an immobilized liquid membrane, J. Membrane Sci., 37 (1988) 287. M. Soroka, Wybrane problemy chemii kwasow aminofosfonowych (Selected problems of aminophosphonic acid chemistry), Pr. Nauk. Inst. Chem. Org. Fiz. Politech. Wroclaw., (32)

16 17

(1987). 18

19 20 21

J. Oleksyszyn, An amidoalkylation of trivalent phosphorus tions including acetic acid solutions of PC&, RPCI,, diesters phorus-111 acids, J. Prakt. Chem., 329 (1987) 19. Test manual forperm-selective membranes, Res. Dev. Progr. J.D. Ferry, Ultrafilter membranes and ultrafiltration, Chem. Handbook of Chemistry and Physics, CRC Press, Cleveland,

compounds with P(O)H funcof phosphorous acid and phosRep., U.S. Interior, No. 7, 1964. Rev., 18 (1935) 373. OH, 53rd edn., 1972.