Chiral separation of amino acids in ultrafiltration through DNA-immobilized cellulose membranes

Journal of Molecular Structure 739 (2005) 145–152 www.elsevier.com/locate/molstruc

Chiral separation of amino acids in ultrafiltration through DNA-immobilized cellulose membranes Akon Higuchia,*, Akiyuki Hayashia, Naoki Kandaa, Kohei Sanuib, Hanako Kitamuraa a

Department of Applied Chemistry, Seikei University, Kichijoji Kitamachi, Musashino, Tokyo, Japan b Department of Chemistry, Sophia University, Kioi-cho, Chiyoda-ku, Tokyo, Japan Received 4 March 2004; revised 16 April 2004; accepted 10 May 2004 Available online 25 November 2004

Abstract Ultrafiltration experiments for the chiral separation of racemic tryptophan, phenylglycine and phenylalanine were investigated through immobilized DNA membranes having various pore sizes. L-tryptophan preferentially permeated through immobilized DNA membranes with a pore size!2.0 nm (molecular weight cut-off (MWCO)!5000) while D-tryptophan preferentially permeated through immobilized DNA membranes with a pore sizeO2.0 nm (MWCOO5000). These results are completely opposite tendency in the ultrafiltration of racemic phenylalanine through the immobilized DNA membranes. This may be originated from the different interaction between DNA and tryptophan compared to that between DNA and phenylalanine. However, in both cases the pore size of the immobilized DNA membranes regulated preferential permeation of the enantiomer through the membranes. The immobilized DNA membranes are categorized as channel type membranes and not as affinity membranes. Chiral separation models were proposed from using the chiral separation results of racemic amino acids, preferential adsorption of amino acid enantiomers and EPMA results. q 2004 Elsevier B.V. All rights reserved. Keywords: Chiral separation; Membrane; DNA; Ultrafiltration

1. Introduction Terrestrial life utilizes only the L enantiomers of amino acids; that is known as the homochirality of life [1]. The optical resolution of one specific enantiomer from others is potentially in demand for the production of pharmaceuticals and food products. Currently, separation of racemic mixtures [2,3] is typically performed by column chromatography [4–6], preferential crystallization [7] or stereoselective transformation [8]. Membrane-based enantiomeric separation [9–33] has the advantage that it can be scaled up and that it saves energy. Especially, ultrafiltration using chiral porous membranes in channel type permeation [17–27] as well as affinity-based chiral ultrafiltration [24,26–33] can facilitate industrial scale chiral separation. The mechanism of chiral separation in the affinity-based chiral ultrafiltration is based * Corresponding author. Tel.: C81 422 37 3748; fax: C81 422 37 3748. E-mail address: [email protected] (A. Higuchi). 0022-2860/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2004.05.040

on the adsorption of one specific enantiomer on the membranes that gives higher binding affinity than opposite enantiomer, while the mechanism of the channel type permeation is based on higher permeation of one specific enantiomer compared to opposite enantiomer through the membranes. In our previous studies, ultrafiltration experiments for the chiral separation of racemic amino acids were performed in a solution system (i.e. affinity ultrafiltration) [24,26–29] and membrane systems [24,26–28] using albumin or DNA as chiral recognition sites. DNA has been discovered to have several novel functions such as the carrier of electron transfer and not only the carrier of genetic information [34]. Our previous study showed that DNA has binding ability for L-phenylalanine (S-phenylalanine) and D-phenylalanine (R-phenylalanine), and that the binding constant for L-phenylalanine is higher than for D-phenylalanine. Ultrafiltration of a racemic phenylalanine solution using DNA solution system resulted in preferential concentration of

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D-phenylalanine in the permeate [28]. We also utilized immobilized DNA membranes prepared from cellulose dialysis membranes with different molecular weight cut-off (MWCO) [25,28]. D-Phenylalanine preferentially permeated through immobilized DNA membranes with a pore size!2.0 nm (molecular weight cut-off (MWCO)!5000) while L-phenylalanine preferentially permeated through immobilized DNA membranes with a pore sizeO2.0 nm (MWCOO5000) [25]. The pore size of the immobilized DNA membranes regulated preferential permeation of the enantiomer through the membranes. In contrast to albumincoated membranes, these membranes appeared to separate on the basis of channel formation rather than affinity interaction [25]. Here we report ultrafiltration experiments for the chiral separation of other racemic amino acids of phenylalanine (i.e. tryptophan and phenylglycine) using immobilized DNA membranes with various pore sizes prepared from cellulose dialysis membranes. We specifically focused on whether the pore size of the immobilized DNA membranes regulates preferential permeation of enantiomer through the membranes.

2. Experimental 2.1. Materials Base membranes used for the chemical modification were commercially available cellulose dialysis membranes (Spectra/Pro 7, MWCO (molecular weight cut-off)Z1000, 2000, 3500, 10,000, 25,000 and 50,000, Spectrum Medical Industries, Inc.). DNA (from salmon testes, D-1626) was purchased from Sigma Chemical Co. Other chemicals,

purchased from Tokyo Chemical Co., were reagent grade and were used without further purification. Ultrapure water was used throughout the experiments. 2.2. Preparation of immobilized DNA membranes 30 mg/mL of aqueous CNBr solution was adjusted to a pH of 10.5 by adding 1 mol/L NaOH solution. The cellulose dialysis membranes having diameter of 6 cm were immersed in the CNBr solution at 25 8C for 1 h (see Scheme 1). After the CNBr reaction, the membranes were sequentially washed with 0.1 mol/L NaHCO3 solution five times, immersed in ethanolamine solution adjusted to a pH of 9.1 by adding 2 mol/L HCl solution for 30 min and immersed in 5 wt% of FeCl3 solution for 30 min [25,28,35]. The CNBr activated cellulose membranes were washed with 0.5 mol/L NaCl solution four times and then immersed in aqueous hydrazine solution (50 mL hydrazine monohydrate C150 mL H2O) for 10 h at 50 8C. After the amino group was introduced into the cellulose membranes from the above procedures (see Scheme 1), the membranes were immersed in 0.2 mol/L of aqueous K2[PtCl4] solution for 8 h at 25 8C [25,28,36]. Pt-introduced cellulose membranes (i.e. immobilized Pt membranes) were immersed in 1000 ppm DNA buffer (5 mmol/L tris(hydroxymethyl)aminomethane HCl/0.5 mmol EDTA buffer) solution for 8 h at pH 8.0, and DNA-immobilized cellulose membranes (i.e. immobilized DNA membranes) were finally prepared [25,28]. 2.3. Characterization of the membranes Atomic analysis of the surface of immobilized Pt membranes and immobilized DNA membranes was

Scheme 1.

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147

Fig. 1. Schematic representation of ultrafiltration apparatus for chiral separation.

performed using XPS (ESCA-3400, Kratos Analytical Ltd). EPMA (WDX) measurements of the cross-section of immobilized DNA membranes were performed with EPMA-1610 (Shimazu Corporation). 2.4. Chiral separation through immobilized DNA membranes 25 ml of racemic phenylalanine solution (0.006 mmol/L) was ultrafiltered for 24 h through immobilized DNA membranes using a batch-type ultrafiltration apparatus (UHP-25 K, Advantec MFS, Inc.) under transmembrane pressure (Dp) of 0.05–0.3 MPa at 25 8C. Fig. 1 shows schematic representation of ultrafiltration apparatus for chiral separation used in this study. The permeate solution was sampled every 1 g and the flux was calculated. The concentration of L-enantiomer and D-enantiomer was measured using HPLC (JASCO Co.) with a Crownpak column (CR(C), Daicel Chemical Co. Ltd). The separation factor, a, was defined to be [JD/JL]/[Cfeed(D)/Cfeed(L)], where Cfeed(L) and Cfeed (D) were the concentrations of L-enantiomer and D-enantiomer in the feed solution, respectively. JD and JL were the fluxes of D-enantiomer and L-enantiomer through the membranes, respectively. Since the flux of solute was directly related to the concentration in the permeate [i.e. JD/JLZCp(D)/Cp(L)] where Cp(L) and Cp(D) were the concentrations of L-enantiomer or D-enantiomer in the permeate, respectively, a was reduced to be aZ[Cp(D)/ Cp(L)]/[Cfeed(D)/Cfeed(L)]. The separation factor in the concentrate solution, ac was also defined as the concentration ratio of D-enantiomer [Ccon(D)] to L-enantiomer [Ccon(L)] in the concentrate solution after the ultrafiltration of racemic amino acid solution.

3. Results and discussion 3.1. Characterization of the DNA membranes Immobilized DNA membranes were prepared from the surface modified reaction of the membranes. Atomic analysis on the surface of the membranes prepared using cellulose dialysis membranes having MWCOZ1000 (i.e. Pt-1000 and DNA-1000 membranes) was performed using XPS, and the results are shown in Table 1. Pt was detected on both immobilized Pt and immobilized DNA membranes. The amount of phosphate atom, which was originated from phosphate group of DNA increased on the immobilized DNA membranes compared to immobilized Pt membranes, while the amount of chloride decreased on the immobilized DNA membranes compared to immobilized Pt membranes. These results are exactly the same tendency reported previously [25]. The surface analysis by XPS surely suggested the surface reaction occurred on the membranes as described in Scheme 1. The concentration of phosphate on the cross-section of DNA-1000 and DNA-10,000 membranes, which was originated from phosphate group of DNA was investigated from EPMA (WDX) measurements where DNA-1000 and DNA-10,000 were the membranes prepared using cellulose dialysis membranes having MWCOZ1000 and 10,000, respectively. Fig. 2 shows EPMA spectra of the crosssection of DNA-1000 and DNA-10,000 membranes. High concentration of phosphate was found in the cross-section of Table 1 Atomic analysis on the surface of Pt-immobilized (Pt-CE) and DNAimmobilized (DNA-CE) membranes measured by XPS (WDX) Membranes

Cl/C

N/C

O/C

Pt/C

P/C

Pt-CE DNA-CE

0.0185 0.0029

0.0353 0.0789

0.8452 0.8333

0.0057 0.0079

0.0059 0.0151

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Fig. 2. Scanning electron microscope and EPMA spectra of the cross-section of DNA-1000 and DNA-10,000 membranes.

DNA-10,000 membranes, while only dilute concentration of phosphate was found in the cross-section of DNA-1000 membranes. Therefore, in DNA-1000 membranes with a smaller pore size than the pore size of DNA-10,000 membranes, it was estimated that DNA was immobilized only the surface of the membranes and not inside the pores, while in DNA-10,000 membranes with a bigger pore size than that of DNA-1000 membranes, DNA was immobilized inside the pores as well as the surface of the membranes. 3.2. Chiral separation in immobilized DNA membranes

the pores of the immobilized DNA (DNA-1000) membranes and permeates through the membranes due to the interaction between DNA and L-tryptophan because no optical resolution was found in the cellulose membranes having no bound DNA as found in Fig. 3. The immobilized DNA membranes could be repeatedly used on the same experimental conditions. It was found that the immobilized DNA membranes showed exactly the same separation factor and similar tendency on each experiments within the experimental errors. Based on these results, the immobilized DNA membranes are categorized

A racemic mixture of tryptophan was separated by ultrafiltration using the DNA-1000 membranes at DpZ 0.1 MPa, pH 7.0 and 25 8C. Fig. 3 shows the time dependence of the separation factor in the permeate solution at CfeedZ0.006 mmol/L. The separation factor in the permeate solution was less than a unity. This indicates that L-tryptophan preferentially existed in the permeate, which contrasts with the effects of ultrafiltration of racemic phenylalanine solution through the same DNA-1000 membranes as reported in our previous study [25]. This may be originated from the different interaction between DNA and tryptophan compared to that between DNA and phenylalanine. The concentration ratio of D-amino acid to L-amino acid in the concentrate solution (the ratio of concentrations in the concentrated feed solution after the ultrafiltration of racemic tryptophan) was also plotted as ac in Fig. 3. D-tryptophan was concentrated in the concentrate with an acO1, whereas L-tryptophan preferentially existed in the permeate solution with an a!1. This indicates that L-tryptophan preferentially enters into

Fig. 3. Dependence of separation factors in the permeate (B,,) and the concentrate (C) solutions on permeate amount during the ultrafiltration of 6 mmol/L racemic tryptophan solution using the immobilized DNA (DNA1000) membranes (B,C) and the cellulose membranes (,) at DpZ 0.1 MPa, pH 7.0 and 25 8C. The meansGS.D. of three independent measurements are shown.

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as channel type membranes and not as affinity membranes as reported for immobilized albumin membranes [28–30]. 3.3. Pressure dependence of separation factor and flux Pressure dependence of the flux and separation factor in the ultrafiltration of racemic tryptophan solution through the DNA-1000 membranes was investigated, and is shown in Fig. 4. Flux increased with the increase of pressure in the ultrafiltration as was expected from Hagen–Poiseuille’s law. Separation factor in the permeate decreased and that in the concentrate increased with the increase of the supplied pressure (Dp). This indicates that the separation factors in both permeate and concentrate were improved at lower pressure in the ultrafiltration of racemic tryptophan through the immobilized DNA membranes, although the flux decreased at lower pressure in the ultrafiltration. Therefore, the following ultrafiltration experiments were performed at DpZ0.1 MPa to obtain relatively high separation factor in the ultrafiltration of racemic amino acids through the immobilized DNA membranes.

Fig. 4. Pressure dependence of the flux (a) and separation factor (b) in the ultrafiltration of racemic tryptophan solution through the DNA-1000 membranes at pH 7.0 and 25 8C. The meansGS.D. of three independent measurements are shown.

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3.4. Dependence of separation factor on the pore size of the membranes We further investigated whether the pore size of the immobilized DNA membranes regulated preferential permeation of enantiomer through the membranes. Therefore, immobilized DNA membranes were prepared from membranes having different pore sizes (MWCOZ1000, 2000, 3500, 10,000, 25,000 and 50,000). Fig. 5 shows the dependence of the separation factor in the permeate and the concentrate solutions on the MWCO of the base membranes and on the pore size of the immobilized DNA membranes in the ultrafiltration of racemic tryptophan solution. The pore size of the DNA membranes was estimated from water flux using Hagen–Poiseuille’s law and assumption of cylindrical pores in the membranes: r Z ð8hJL=ðXDpÞÞ1=2

(1)

where r is the equivalent pore size, h is the viscosity of solution, J is flux and X is the porosity of the membranes. When the pore size of the immobilized DNA membranes was less than 2.0 nm (MWCO!5000), the separation factor in the permeate solution was !1, while the separation factor was more than 1 when DNA membranes having pore size O2.0 nm (MWCOO5000) were used. The separation factor in the concentrate solution showed exactly the opposite tendency as the separation factor in the permeate solution. Therefore, it appears that L-tryptophan preferentially permeates through the immobilized DNA membranes having pore size ! 2.0 nm, while D-tryptophan preferentially permeates through the immobilized DNA membranes with a pore size O2.0 nm. These results are completely opposite tendency in the ultrafiltration of racemic phenylalanine through the immobilized DNA membranes reported

Fig. 5. Dependence of the separation factor in the permeate (B) and the concentrate (C) solutions through the immobilized DNA membranes on the MWCO of the base membranes and on the pore size of the immobilized DNA membranes at pH 7.0 and 25 8C. The feed solution is racemic 6 mmol/L tryptophan solution. The meansGS.D. of three independent measurements are shown.

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previously [25]. In our previous study [23], D-Phenylalanine preferentially permeated through immobilized DNA membranes with a pore size !2.0 nm (molecular weight cut-off (MWCO)!5000) while L-phenylalanine preferentially permeated through immobilized DNA membranes with a pore size O2.0 nm (MWCOO5000). Again, this is originated from the different interaction between DNA and tryptophan compared to that between DNA and phenylalanine. However, in both cases the pore size of the immobilized DNA membranes regulated preferential permeation of the enantiomer through the membranes. 3.5. Chiral separation of several racemic amino acids Chiral separation of several amino acids other than tryptophan (i.e. phenylalanine and phenylglycine) was performed in the ultrafiltration through the DNA-1000 and DNA-10,000 membranes. Fig. 6 shows separation factor in the permeate and concentrate in the ultrafiltration of racemic phenylalanine, tryptophan and phenylglycine solutions through DNA-1000 and DNA-10,000 membranes. The separation factor in the permeate in the ultrafiltration of tryptophan and phenylglycine through DNA-1000 membranes was found to be !1, while that in the concentrate was O1. These results are opposite tendency to the separation factor in the ultrafiltration of phenylalanine solution. The separation factor in the permeate in the ultrafiltration of tryptophan and phenylglycine solutions through DNA10,000 membranes, which had larger pore size of DNA1000 membranes was found to be O1, while that in the concentrate was !1. These results are opposite tendency to the separation factor in the ultrafiltration of the same corresponding amino acid solutions through DNA-1000 membranes. These results indicate that the pore size of the immobilized DNA membranes regulated preferential permeation of the specific enantiomer through the membranes. 3.6. Model of chiral separation To establish chiral separation model in immobilized DNA membranes, the amount of amino acids adsorbed on the immobilized DNA (DNA-1000 and DNA-10,000) membranes (Qm(L) for L-amino acid and Qm(D) for D-amino acid) was calculated from the mass balance of the amount of amino acid enantiomer in the initial feed solution (Qf(L) for L-amino acid and Qf(D) for D-amino acid), permeate solution (Qp(L) for L-amino acid and Qp(D) for D-amino acid) and concentrate solution (Qc(L) for L-amino acid and Qc(D) for D-amino acid): Qm ðLÞ Z Qf ðLÞ K Qc ðLÞ K Qp ðLÞ

(2)

Qm ðDÞ Z Qf ðDÞ K Qc ðDÞ K Qp ðDÞ

(3)

The results are summarized in Table 2. In all cases, the immobilized DNA membranes adsorbed L-amino acids

Fig. 6. Separation factor in the permeate and concentrate in the ultrafiltration of racemic phenylalanine, tryptophan and phenylglycine solutions through DNA-1000 (a) and DNA-10,000 (b) membranes at DpZ 0.1 MPa, pH 7.0 and 25 8C. The meansGS.D. of three independent measurements are shown.

(i.e. L-phenylalanine, L-tryptophan and L-phenylglycine) preferentially independent of the pore size. Chiral separation models were examined using the separation results in previous [25] and this studies, preferential adsorption results in Table 2 and EPMA results shown in Fig. 2. In our previous study [25], ultrafiltration of a racemic phenylalanine solution through immobilized DNA membranes was investigated. It was reported that D-phenylalanine preferentially permeated through immobilized DNA membranes with a pore size!2.0 nm (molecular weight cut-off (MWCO)!5000), while L-phenylalanine

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Table 2 Adsorption of L- and D- amino acids on the immobilized DNA membranes after ultrafiltration of 0.006 mM of racemic amino acids at 25 8C, pH 7.0 and DpZ0.3 MPa. Adsorbed amount indicates Qm(L)!100/Qf(L) for L-isomer and Qm(D)!100/Qf(D) for D-isomer Membranes

Amino acid

DNA-1000 DNA-10,000 DNA-1000 DNA-10,000 DNA-1000 DNA-10,000

Phenylalanine Phenylalanine Tryptophan Tryptophan Phenylglycine Phenylglycine

Adsorbed amount (%) L-isomer

D-isomer

9.0 5.8 13. 5.0 3.4 7.6

5.3 2.7 6.0 4.4 0.9 6.3

preferentially permeated through immobilized DNA membranes with a pore sizeO2.0 nm (MWCOO5000). These results are quite opposite tendency to the separation results using racemic tryptophan solution investigated in this study. Both previous [25] and present studies suggested that the pore size of the immobilized DNA membranes regulated preferential permeation of the enantiomer through the membranes. The chiral separation model of racemic phenylalanine solution through the immobilized DNA membranes was shown in Fig. 7. When the pore size of the immobilized DNA membranes is less than 2 nm (DNA-1000, -2000 and -3500), L-phenylalanine binds to DNA on the top surface of the immobilized DNA membranes. Therefore, free D-phenylalanine permeates through the pores in the immobilized DNA membranes (Fig. 7(a)). On the other hand, when the pore size of the immobilized DNA membranes is more than 2 nm (DNA8000, 10,000 and 25,000), L-phenylalanine binds to DNA inside the pores, as EPMA results (see Fig. 2) suggest that DNA was immobilized inside the pores. Therefore L -phenylalanine inside the pores permeates through

Fig. 8. Chiral separation model in the ultrafiltration of racemic tryptophan solution through the immobilized DNA membranes.

the pores in the immobilized DNA membranes by surface diffusion of L-phenylalanine (Fig. 7(b)). In the ultrafiltration of a racemic tryptophan solution through the immobilized DNA membranes, it was found that L-tryptophan preferentially permeated through the immobilized DNA membranes with a pore size!2.0 nm (molecular weight cut-off (MWCO)!5000), while D-tryptophan preferentially permeated through the immobilized DNA membranes with a pore sizeO2.0 nm (MWCOO5000). The chiral separation model of racemic tryptophan solution through the immobilized DNA membranes was shown in Fig. 8. When the pore size of the immobilized DNA membranes is less than 2 nm (DNA-1000, -2000 and -3500), L-tryptophan binds to DNA on the top surface of the immobilized DNA membranes. Subsequently, L-tryptophan permeates through the pores in the immobilized DNA membranes (Fig. 8(a)). On the other hand, when the pore size of the immobilized DNA membranes is more than 2 nm (DNA-8000, 10,000 and 25,000), L-tryptophan binds to DNA inside the pores. D-tryptophan may interact with L-tryptophan inside the pores. Therefore D-tryptophan inside the pores permeates through the pores by surface diffusion of D-tryptophan (Fig. 8(b)).

Acknowledgements

Fig. 7. Chiral separation model in the ultrafiltration of racemic phenylalanine solution through the immobilized DNA membranes.

The authors thank to Mr T. Maki and Mr Y. Yoshida of Kratos Analytical Ltd for XPS measurements. The authors also appreciate Mr H. Hayashi of Shimazu Corporation and Mr T. Saka of Abbe Science Company, Ltd for EPMA measurements. We gratefully acknowledge financial support through a Grant-in-Aid for Scientific Research on Priority Areas (B, ‘Novel Smart Membranes Containing

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Controlled Molecular Cavity’, No. 13133202, 2001-2004) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. This research was also supported by Salt Science Foundation (0320, 0131).

References [1] [2] [3] [4] [5] [6] [7] [8]

[9] [10] [11] [12] [13] [14] [15] [16]

G.L.A. Rikken, E. Raupach, Nature 405 (2000) 932. H. Yamanaka, Y. Tashiro, Kikan Kagaku Sosetsu 6 (1989) 2. A. Higuchi, Bio Ind. 20 (6) (2003) 5. K. Brandt, Nachr. Chem. Tech. Lab. 39 (1991) M1. G. Hesse, R. Hagel, Chromatographia 6 (1973) 277. Y. Okamoto, M. Kawashima, K. Yamamoto, K. Hatada, Chem. Lett. 1984; 739. H. Nohira, K. Watanabe, M. Kurokawa, Chem. Lett. 1976; 299. J. Jacques, A. Collet, S. Wilen, Enantiomers Racemates, and Resolutions, in: J. Jacques (Ed.),, Wiley/Interscience, New York, 1981, p. 5. A. Maruyama, N. Adachi, T. Takehisa, M. Torii, K. Sanui, N. Ogata, Macromolecules 23 (1990) 2748. C. Thoelen, M. De bruyn, E. Theunissen, Y. Kondo, I.G.J. Vankelecom, P. Grobet, M. Yoshikawa, P.A. Jacobs, J. Membr. Sci. 186 (2001) 153. M. Yoshikawa, T. Ooi, J. Izumi, J. Appl. Polym. Sci. 72 (1999) 493. K. Inoue, A. Miyahara, T. Itaya, J. Am. Chem. Soc. 119 (1997) 6191. Y. Aoki, M. Ohshima, K. Shinohara, T. Kaneko, E. Oikawa, Polymer 38 (1997) 235. B.B. Lakshmi, C.R. Martin, Nature 388 (1997) 758. M. Newcomb, J.L. Toner, R.C. Helgeson, D.J. Cram, J. Am. Chem. Soc. 101 (1979) 4941. L.J. Keurentjes, W.M. Nabuurs, E.A. Vegter, J. Membr. Sci. 113 (1996) 351.

[17] J.H. Kim, J.H. Kim, J. Jegal, K.-H. Lee, J. Membr. Sci. 213 (2003) 273. [18] S. Tone, T. Masawaki, T. Hamada, J. Membr. Sci. 103 (1995) 57. [19] T. Aoki, S. Tomizawa, E. Oikawa, J. Membr. Sci. 99 (1995) 117. [20] S. Kiyahara, M. Nakamura, K. Saito, K. Sugita, T. Sugo, J. Membr. Sci. 152 (1999) 143. [21] F. Garnier, J. Randon, J.L. Rocca, Separation Purif. Tech. 16 (1999) 243. [22] Y. Osada, F. Ohta, A. Mizumoto, M. Takase, Y. Kurimura, Nippon Kagakukaishi 1986; 866. [23] T. Kobayashi, H.Y. Wang, N. Fujii, Chem. Lett. 1995; 927. [24] A. Higuchi, Y. Ishida, T. Nakagawa, Desalination 90 (1993) 127. [25] A. Higuchi, Y. Higuchi, K. Furuta, B.O. Yoon, M. Hara, S. Maniwa, M. Saitoh, K. Sanui, J. Membr. Sci. 221 (2003) 207. [26] A. Higuchi, M. Hara, T. Horiuchi, T. Nakagawa, J. Membr. Sci. 93 (1994) 157. [27] A. Higuchi, P. Wang, T.-M. Tak, T. Nohmi, T. Hashimoto, Membrane formation and modification in: I. Pinnau, B.D. Freeman (Eds.),, ACS Symposium Series 744, American Chemical Society, Washington, DC, 1999, p. 228. [28] A. Higuchi, H. Yomogita, B.O. Yoon, T. Kojima, M. Hara, S. Maniwa, M. Saitoh, J. Membr. Sci. 205 (2002) 203. [29] A. Higuchi, T. Hashimoto, M. Yonehara, N. Kubota, K. Watanabe, S. Uemiya, T. Kojima, M. Hara, J. Membr. Sci. 130 (1997) 31. [30] B. Mattiason, M. Ramstorp, Ann. N.Y. Acad. Sci. 413 (1983) 307. [31] S. Poncet, J. Randon, J. Rocca, Sep. Sci. Technol. 32 (1997) 2029. [32] J. Romero, A.L. Zydney, Biotechnol. Bioeng. 88 (2002) 256. [33] J. Romero, A.L. Zydney, J. Membr. Sci. 209 (2002) 107. [34] H.-A. Wagenknecht, E.D.A. Stemp, J.K. Barton, J. Am. Chem. Soc. 122 (2000) 1. [35] A. Denizli, J. Appl. Polym. Sci. 74 (1999) 655. [36] A. Higuchi, S. Adachi, T. Imizu, B.O. Yoon, T. Tsubomura, M. Hara, K. Sakai, J. Phys. Chem. 104 (2000) 9864.