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Enantioselective Membranes

Membrane Applications

  1. Masakazu Yoshikawa1,
  2. Akon Higuchi2,3

Published Online: 19 APR 2013

DOI: 10.1002/9781118522318.emst131

Encyclopedia of Membrane Science and Technology

Encyclopedia of Membrane Science and Technology

How to Cite

Yoshikawa, M. and Higuchi, A. 2013. Enantioselective Membranes. Encyclopedia of Membrane Science and Technology. 1–22.

Author Information

  1. 1

    Kyoto Institute of Technology, Matsugasaki, Kyoto, Japan

  2. 2

    National Central University, Jhongli, Taoyuan, Taiwan

  3. 3

    National Research Institute for Child Health and Development, Setagaya-ku, Tokyo, Japan

Publication History

  1. Published Online: 19 APR 2013

1 Background

  1. Top of page
  2. Background
  3. Permeation and Selective Theory for Enantioseparation
  4. Resolution Mechanism Through the Membranes
  5. Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers
  6. Enantioseparation Membranes Prepared from Polymers with a Chiral Branch
  7. Enantioseparation Membranes with Immobilized Stereoselective Ligands
  8. Enantioseparation by Nanofibers
  9. Enantioseparation by Nonchiral Membranes
  10. Future Perspectives
  11. References
  12. Further Reading

Many drugs, pharmaceuticals, and flavoring compounds are racemic mixtures consisting of chiral isomers that have nearly identical physical and chemical properties (1-4). However, the enantiomers of chiral therapeutic chemicals often exhibit significant differences in toxicity. An example of this phenomenon is the tragedy that occurred from the use of thalidomide in the early 1960s, where it was discovered that only the (R)-enantiomer of thalidomide had a pain-relieving effect, whereas the (S)-enantiomer caused serious deformities in unborn children (1, 5).

The (S, S)-diastereomer of ethambutol is effective in the treatment of tuberculosis, but the (R, R)-diastereomer may cause blurred vision and eye pain and might result in complete blindness (3, 6). The FDA and the Committee for Proprietary Medicinal Products (CPMP) now require pharmaceutical companies to produce only a single enantiomer as the therapeutic agent or to clearly demonstrate the advantages of using a racemic mixture (3, 5, 7). This requirement has resulted in considerable demand for separation techniques appropriate for the large-scale resolution (purification) of chiral molecules. Worldwide, the market for chiral fine chemicals sold as single enantiomers was $6.63 billion in 2000; the market is expected to grow at a rate of 13.2% annually, reaching $16.0 billion in 2007 (3, 8).

There is a growing need for separation techniques appropriate for the large-scale resolution of chiral molecules, although many single-enantiomer drugs are produced by stereoselective synthesis. However, relatively low cost pharmaceuticals cannot be produced by stereoselective synthesis. The most widely used methods for the separation of racemic mixtures are diastereomeric salt crystallization, column chromatography, and stereoselective enzyme catalysis (3). Liquid membranes with immobilized chiral ligands have also been used for enantioseparation (chiral separation), although these techniques could be difficult to apply in commercial systems because of the instability of the liquid membranes (3). An alternative approach is to use an affinity ultrafiltration system in which a large stereoselective ligand is added to the bulk solution to selectively bind and thus retain one of the stereoisomers (5). Enantioseparation of pharmaceuticals through polymeric membranes with an immobilized chiral selector could be very promising for commercial systems in the future. Membrane-based enantioseparation techniques are advantageous because they avoid the issues of low throughout and poor flow distribution, which plague current chromatographic separation techniques. Membrane-based enantioseparation techniques are also energy saving and more economical than many other separation technologies. Examples of such techniques used to facilitate industrial scale enantioseparations include ultrafiltration using chiral porous membranes with a channel-type permeation and affinity-based chiral ultrafiltration (1). Enantioseparation in the affinity-based ultrafiltration method is achieved by the adsorption of one specific enantiomer that possesses a higher binding affinity than its opposite enantiomer onto a membrane, whereas separation using channel-type permeation occurs via the differential permeation of the enantiomers through the membranes (1). This article summarizes materials used for the enantioseparation of pharmaceuticals and discusses the future trend of membrane materials for enantioseparation (3).

2 Permeation and Selective Theory for Enantioseparation

  1. Top of page
  2. Background
  3. Permeation and Selective Theory for Enantioseparation
  4. Resolution Mechanism Through the Membranes
  5. Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers
  6. Enantioseparation Membranes Prepared from Polymers with a Chiral Branch
  7. Enantioseparation Membranes with Immobilized Stereoselective Ligands
  8. Enantioseparation by Nanofibers
  9. Enantioseparation by Nonchiral Membranes
  10. Future Perspectives
  11. References
  12. Further Reading

Enantioseparation (chiral separation) membranes preferentially allow a specific enantiomer to adsorb to or diffuse on to the membrane. This specificity is generated by chiral recognition sites in the membranes such as chiral side chains, chiral backbones, or immobilized chiral selectors in polymeric enantioseparation membranes. These enantioselective membranes act as selective barriers in the resolution process by preferentially allowing only one enantiomer to diffuse across the membrane because of the stereospecific interaction between the enantiomer and chiral recognition sites (3, 9).

The transport process of enantiomers through the membranes can be categorized as filtration, dialysis, electrodialysis, and pervaporation, depending on the main driving force of the permeation of enantiomers through the membranes (pressure gradient, concentration difference, electric field difference, or vapor difference, respectively) (3).

A solution-diffusion mechanism determines the permeation of enantiomers through the homogeneous dense membranes and is described as follows:

  • mathml alt image(1)

where P, D, and S refer to the permeability coefficient, diffusion coefficient, and sorption coefficient (solubility), respectively (3).

The diffusion coefficient, D, is a kinetically determined coefficient influenced by the characteristics and interaction(s) between the membrane and the enantiomer. The sorption coefficient is a thermodynamically determined parameter defined as the ratio of the concentration in the membrane (Cm) to that in the solution (Co), as shown in Equation 2 (3).

  • mathml alt image(2)

The separation factor α is evaluated from the concentration of the upstream side and downstream side and is defined as follows:

  • mathml alt image(3)

or

  • mathml alt image(4)

where Cf(R) and Cf(S) are the concentrations of the R-enantiomer and S-enantiomer, respectively, in the feed solution (the solution at the upstream side) (3). Cp(R) and Cp(S) are the concentrations of the R-enantiomer and S-enantiomer, respectively, in the permeate solution (solution at the downstream side). The concentrations in the upstream side, Cf(R) and Cf(S), are the same in some cases. In this case, α reduces to

  • mathml alt image(5)

The enantioselectivity of transport through the membrane can be divided into two factors, solubility selectivity and diffusion selectivity (3).

  • mathml alt image(6)

or

  • mathml alt image(7)

where D(R) and D(S) are the diffusion coefficients of the R-enantiomer and S-enantiomer, respectively. S(R) and S(S) are the solubility coefficients of the R-enantiomer and S-enantiomer, respectively.

The chiral selectivity of transport through membranes is also evaluated in terms of the enantiomeric excess (ee) of permeates (9). The ee value is defined as the ratio of the concentration difference to the total concentration of both enantiomers in the permeate (3).

  • mathml alt image(8)

or

  • mathml alt image(9)

When the concentrations in the feed side, Cf(S) and Cf(R), are the same, the separation factor can be calculated from ee using the following Equation (3):

  • mathml alt image(10)

3 Resolution Mechanism Through the Membranes

  1. Top of page
  2. Background
  3. Permeation and Selective Theory for Enantioseparation
  4. Resolution Mechanism Through the Membranes
  5. Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers
  6. Enantioseparation Membranes Prepared from Polymers with a Chiral Branch
  7. Enantioseparation Membranes with Immobilized Stereoselective Ligands
  8. Enantioseparation by Nanofibers
  9. Enantioseparation by Nonchiral Membranes
  10. Future Perspectives
  11. References
  12. Further Reading

The mechanism of enantioseparation (chiral separation) on polymeric membranes can be categorized as diffusion-selective membranes and sorption-selective membranes. Diffusion-selective membranes are usually made of an intrinsically chiral polymer without specific foreign chiral selectors (e.g., albumin or other proteins, chiral polysaccharide chains or segments, DNA, crown ether derivatives, and oligopeptides). Sorption-selective membranes can be made by embedding or immobilizing chiral selectors in polymer membranes or on the membrane surfaces; these membranes have less selective diffusion but show highly selective sorption. Examples of chiral selectors include crown ether derivatives, CD, albumin and other proteins, and DNA (3).

In most cases of enantioseparation through polymeric membranes, there is a trade-off between diffusion selectivity and solution selectivity; the membranes showing diffusion selectivity for one chiral isomer have sorption selectivity for the opposite chiral isomer. Therefore, the permeation selectivity is determined by whichever selectivity is higher, either sorption selectivity or diffusion selectivity (3). In general, enantioseparation membranes with sorption selectivity should be designed with no diffusion selectivity, and vice versa. One method that can be used to reduce the diffusion selectivity of sorption-selective concentration-driven permeation in enantioseparation membranes (dialysis membranes) is to apply an electrical potential that makes the concentration-driven permeation of chiral pharmaceuticals or chemicals a potential-driven permeation. Potential-driven permeation generally results in the same diffusion coefficient for each isomer (3).

The driving force responsible for the permeation and separation of chiral pharmaceuticals and chemicals for the dialysis method is the concentration difference and a pressure-driven force for ultrafiltration and nanofiltration. Most studies of enantioseparation membranes have been performed using dialysis membranes. The main disadvantages of the dialysis method are that the concentration of the final product is more dilute than that of the feed solution and that permeation is extremely slow. Owing to these disadvantages, enantioseparation in industrial applications may instead require ultrafiltration or nanofiltration through enantioseparation membranes (3).

In addition to dialysis and filtration, pervaporation via membranes is also useful for the enantioseparation process (here, the driving force of the permeation is a vapor pressure difference). Shinohara et al. (10) reported the enantioseparation of racemates of 1,3-butanediol, 2-butanol, and their derivatives by pervaporation through a (+)-poly{1-[dimethyl(10-pinanyl)silyl]-1-propyne} membrane. They reported a permeation rate of 1.19 × 10−3 g m/h and 41.7% ee for racemic 1,3-butanediol. Enantioselective vapor permeation is also effective for enantioseparation if the racemic compounds are more or less volatile. Only a few examples have been reported of enantioseparation of pharmaceuticals and racemates using the pervaporation method (10), although it is expected that this technology will be used more often in the future (3).

4 Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers

  1. Top of page
  2. Background
  3. Permeation and Selective Theory for Enantioseparation
  4. Resolution Mechanism Through the Membranes
  5. Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers
  6. Enantioseparation Membranes Prepared from Polymers with a Chiral Branch
  7. Enantioseparation Membranes with Immobilized Stereoselective Ligands
  8. Enantioseparation by Nanofibers
  9. Enantioseparation by Nonchiral Membranes
  10. Future Perspectives
  11. References
  12. Further Reading

Several enantioseparation (chiral separation) membranes were prepared from chiral polymers where enantioselectivity was generated from chiral carbons in the main chain. Poly(γ-methyl-l-glutamate), alginate, chitosan, cellulose, and their derivatives (Fig. 1) are typically used as chiral polymers for the preparation of enantioseparation membranes (3).

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Figure 1. Chemical scheme of (a) PMLG, (b) sodium alginate, (c) cellulose, and (d) chitosan used as chiral polymers for the preparation of enantioseparation membranes. Source: Reproduced from Reference 3 with permission. Copyright 2012 Taylor & Francis Group, LLC.

Kim et al. (11) prepared cross-linked sodium alginate and chitosan membranes with glutaraldehyde for enantioseparation of racemic tryptophan and tyrosine by ultrafiltration. Both cross-linked sodium alginate and chitosan membranes were found to be applicable for the enantioseparation of racemic tryptophan and tyrosine by a pressure-driven process. When a cross-linked chitosan membrane with a 70% swelling index was used for the enantioseparation of a racemic tryptophan mixture (0.49 mmol/l aqueous solution), over 98% ee and 6.4 mg/(m2 h) of flux were obtained (11). The presence of five chiral carbons located on the ring structure of sodium alginate and chitosan seemed to induce a chiral environment in the membrane, similar to the function of cellulose and its derivatives, making the membrane enantioselective. As the degree of cross-linking increased, the membrane showed higher enantioselectivity by increasing the interaction between the chiral environment of the membrane and penetrating chiral isomers (11). Other factors that could decrease the intermolecular interaction between the membrane and solutes, such as an increase in operating pressure and an increase in the concentration of the feed solution, acted against the enantioselectivity (3, 11).

Ultrathin polymeric membranes were prepared by Rmaile and Schlenoff (12) through a multilayer stacking approach using chiral polyelectrolytes. The chiral polyelectrolyte multilayer membranes composed of poly(d-lysine) and poly(d-glutamic acid) showed preferential permeation of d-ascorbic acid compared to l-ascorbic acid (α = 1.3). The membranes composed of poly(l-lysine) and poly(l-glutamic acid) exhibited preferential permeation of l-ascorbic acid (α = 1.3). The combination of the l-form of one polyelectrolyte and the d-form of its oppositely charged partner effectively neutralized the chiral selectivity (no selectivity). The permeation selectivity was reported by Rmaile and Schlenoff (12) to be generated by diffusion selectivity, based on permeation and sorption experiments of l- and d-ascorbic acids. The polyelectrolyte multilayer membranes offered high flux due to the thinness of the membranes, but a high separation factor (α > 2) has not yet been achieved.

5 Enantioseparation Membranes Prepared from Polymers with a Chiral Branch

  1. Top of page
  2. Background
  3. Permeation and Selective Theory for Enantioseparation
  4. Resolution Mechanism Through the Membranes
  5. Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers
  6. Enantioseparation Membranes Prepared from Polymers with a Chiral Branch
  7. Enantioseparation Membranes with Immobilized Stereoselective Ligands
  8. Enantioseparation by Nanofibers
  9. Enantioseparation by Nonchiral Membranes
  10. Future Perspectives
  11. References
  12. Further Reading

It is rather difficult to generate excellent enantioseparation (chiral separation) membranes from chiral polymers alone because racemic penetrants mainly encounter the flexible side chains of the membrane polymers. Therefore, enantioseparation membranes were prepared using polymers with a chiral branch (3).

Lee and Frank (13) prepared polypeptide-modified poly(vinylidene fluoride) (PVDF) membranes for the separation of chiral molecules via ultrafiltration. Poly(γ-benzyl-l-glutamate) (PBLG) was vapor deposited on the PVDF membranes, and the PBLG on PVDF membranes were modified through either debenzylation or an ester exchange reaction to produce poly(l-glutamic acid) (PLGA) and polyglutamate with triethylene glycol monomethyl ether side chains (PLTEG). The enantioselectivities for chiral α-amino acids (tryptophan, phenylalanine, and tyrosine) and chiral drugs (propranolol, atenolol, and ibuprofen) were measured by concentration-driven experiments. The separation factors were reported to range from 1.04 to 1.47 (13). The selectivity increased with the helical content of PLGA immobilized on the PVDF membranes. The enantioselectivity was observed to be higher for chemically grafted polypeptide-modified PVDF membranes compared to polypeptide-physisorbed PVDF membranes. This difference is attributed to the higher molecular weight and density of the polypeptide chains, which enhances the interaction between the chiral compounds and the surface-bound polypeptides (3, 13).

Enantioseparation of racemic amino acids through polymeric membranes with saccharide side chains has been reported. Satoh (14) prepared enantioseparation membranes composed of polyacrylonitrile-graft-(1[RIGHTWARDS ARROW]6)-2,5-anhydro-3,4-di-O-methyl-d-glucitol. The permeation rates of the amino acids increased in the order of phenylglycine < phenylalnine < tryptophan, in accordance with the molecular size of the permeating solutes. The permeation rate of the d-isomer was higher than that of the l-isomer for the amino acids, although the l-isomer was more highly adsorbed than the d-isomer. In this case, the enantioseparation by these membranes was caused by diffusion selectivity (3, 14).

6 Enantioseparation Membranes with Immobilized Stereoselective Ligands

  1. Top of page
  2. Background
  3. Permeation and Selective Theory for Enantioseparation
  4. Resolution Mechanism Through the Membranes
  5. Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers
  6. Enantioseparation Membranes Prepared from Polymers with a Chiral Branch
  7. Enantioseparation Membranes with Immobilized Stereoselective Ligands
  8. Enantioseparation by Nanofibers
  9. Enantioseparation by Nonchiral Membranes
  10. Future Perspectives
  11. References
  12. Further Reading

Specific interactions between large biological molecules and specific pharmaceuticals or drugs were reported to be used for enantioseparation (chiral separation). Crown ether derivatives and CDs are typically used as stereoselective ligands in enantioseparation membranes (Fig. 2). The idea of attaining affinity binding of specific enantiomers with large biological macromolecules such as proteins and DNA in ultrafiltration dates back to the early work of Higuchi et al. (3, 15) on the purification of an amino acid isomer by exploiting its binding to albumin on the membrane.

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Figure 2. Chemical scheme of (a) α-cyclodextrin, (b) β-cyclodextrin, (c) γ-cyclodextrin, (d) 18-crown-6, (e) dibenzo-18-crown-6, and (f) diaza-18-crown-6 used as stereoselective ligands in enantioseparation membranes. Source: Reproduced from Reference 3 with permission. Copyright 2012 Taylor & Francis Group, LLC.

Three mechanisms are considered for enantioseparation using membranes with immobilized large molecules as chiral selectors: (i) affinity membranes, (ii) selective sorption membranes, and (iii) selective diffusion membranes. The enantioseparation mechanism of affinity membranes is based on the selective adsorption of specific isomers among other isomers (3). These membranes cannot obtain steady-state enantioseparation, and enantioseparation using affinity membranes is not focused on in this article.

6.1 Immobilized Cyclodextrin Membranes

Native CD are cyclic oligosaccharides consisting of six to eight d-(+)-glucopyranose units that provide three-point interactions for the chiral recognition of various organic molecules by hydrophobic interaction with the CD cavity and two hydrogen bonds with the hydroxyl groups at the opening of the CD (16).

CD-functionalized membranes have a lower cost and might have wider applicability and higher tolerance to various environments compared to other chiral-selector-immobilized membranes. However, enantioseparation using immobilized CD membranes has the disadvantage of low selectivity because native cyclodextrins have limited chiral recognition ability and limited flexibility, both of which are important to enable interaction with the enantiomers. Chemical modification of CD and the preparation of CD derivatives may be an interesting topic of research; enantioseparation can be performed using the immobilized CD-derivative membranes (3).

Acetylated-β-cyclodextrin-immobilized cellulose dialysis membranes for enantioseparation of racemic tryptophan were reported by Xiao et al. (17). The acetylated CD-immobilized membranes expressed enantioselectivity in the range 1.26–1.33 depending on the acetylation time, whereas native CD-immobilized membranes exhibited an enantioselectivity of only 1.11. The enhancement in enantioselectivity after acetylation was mainly attributed to the increased discrimination ability of acetylated CD and the decreased membrane pore size (17). From molecular modeling simulations, the acetylation of hydroxyl groups was suggested to result in a CD conformation with toroidal distortions that generate a greater steric hindrance for phenylalanine interaction. The acetylated CD has less effective binding but better discrimination of enantiomers compared to the original CD (3, 17).

6.2 Immobilized Albumin Membranes

Serum albumin is reported to have a high affinity binding site for l-tryptophan. Its binding constant was reported to be 4.4 × 104 M−1 by Kragh-Hansen (18), and weakly bound d-tryptophan has been reported to displace l-tryptophan (19). Therefore, Higuchi et al. (15, 19) developed a method for enantioseparation of racemic amino acids using ultrafiltration of solutions of various racemic amino acids through immobilized serum albumin membranes, making use of the binding site of bovine serum albumin (BSA) to the l-isomer. The immobilized BSA membranes demonstrated efficient enantioseparation of racemic tryptophan, as well as leucine and phenylalanine (Fig. 3) (19). The target molecules for enantioseparation using the immobilized albumin membranes should be small organic chemicals with aromatic or hydrophobic side groups because of the specific binding site characteristics of albumin. The enantioseparation of low molecular weight pharmaceuticals (such as ibuprofen) using the immobilized BSA membranes should be an interesting topic of research in the future (3).

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Figure 3. Enantioseparation of racemic amino acids (phenylalanine, leucine, or tryptophan) by ultrafiltration through immobilized albumin membranes (black bar) and a solution containing albumin (white bar) at pH 7.0 and 25 °C. Source: Reprinted from Reference 19 with permission. Copyright 2012 Elsevier Inc.

6.3 Immobilized Apoenzyme Membranes

Enzymes are well known to have high substrate selectivity, explained by the lock and key model. Enzymes could be a good selection for use in the recognition of chiral molecules in enantioseparation membranes. However, enzymes not only recognize the specific enantiomer but also catalyze a chemical reaction of the molecule. It is known that an apoenzyme requires a cofactor to perform the enzymatic reaction; thus, unwanted chemical conversion of the substrate molecule in enzyme-based separation can be circumvented when using this molecular recognition agent (3). Lakshmi and Martin (20) investigated enantioseparation using apoenzymes immobilized in porous polymeric membranes (Fig. 4). The membranes selectively transported the specific substrate molecule without the unwanted chemical conversion of the molecule in their system. When d-amino acid oxidase apoenzyme was loaded in the membranes, facilitated transport of d-phenylalanine relative to l-phenylalanine was reported; the maximum d- versus l-phenylalanine selectivity coefficient was found to range from 3.3 to 4.9 (3, 20).

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Figure 4. Schematic cross-section of the polypyrrole/polycarbonate/polypyrrole sandwich membrane with the apoenzyme entrapped in the pores for enantioseparation. Source: Modified from Reference 20 with permission. Copyright 2012 Macmillan Publishers Ltd.

6.4 Immobilized Antibody Membranes

Antibodies specifically bind to a variety of targets (antigens) depending on the type of antibody. The specificity between the antibody and antigen is extremely high; this allows antibodies to identify and bind to only their unique antigen from among millions of different molecules, using an induced fit binding (21). The specific recognition of an antigen by an antibody could be used for the recognition of chiral molecules in enantioseparation membranes. However, the binding constants for the antibody are generally too large to bind with the specific antigen reversibly; this is undesirable because the enantioseparation membrane must ultimately release the target molecules to be collected in the permeate solution. This problem can be solved by tuning the binding constant of the antibody with the addition of dimethyl sulfoxide (DMSO) to the racemic feed solution and the permeate solution (21).

Lee et al. (21) prepared enantioseparation membranes with immobilized antibodies for 4-[3-(4-fluorophenyl)-2-hydroxy-1-[1,2,4]triazol-1-yl-propyl]-benzonitrile, a drug serving as an inhibitor of aromatase enzyme activity. This molecule has two chiral centers and thus four stereoisomers: RR, SS, SR, and RS. The RS-enantiomer is preferentially selected by the antibody relative to the SR-enantiomer, and the membranes based on the Fab fragment of this antibody were used to separate this enantiomeric pair. Nanopore alumina membranes (20 and 35 nm of pore diameter) were used as host membranes for immobilization of the anti-RS molecules (21).

A separation factor of approximately 2.0 was reported to be obtained for the membranes prepared from the alumina membranes with a pore diameter of 35 nm when both the RS- and the SR-enantiomers were dissolved in 10% DMSO–phosphate buffer saline (PBS) at pH 8.5 as a feed solution. A Langmuirian-shaped flux curve was observed for the permeation of the RS-enantiomer, but not for that of SR-enantiomer, for the effect of the concentration of the enantiomers in the racemic feed solution on the flux. This result is the evidence of facilitated transport of the RS-enantiomer because of the binding site of the enzyme (21).

Porous polymeric membranes with immobilized antibodies or different types of apoenzymes may be candidates for enantioseparation membranes in future research.

6.5 Immobilized DNA Membranes

DNA has been discovered to have several novel functions aside from carrying genetic information, such as electron transfer and DNA enzymatic activity (22). DNA can also intercalate some enantiomers with a binding constant that depends on the stereoenantiomer (3).

DNA is contained in several common protein preparations as an impurity on the order of parts per billion (23). Therefore, the effect of the DNA in the albumin solution was investigated in the enantioseparation of amino acids using ultrafiltration by Higuchi et al. (24). These results brought about the idea that DNA has a binding site for enantiomers, with the binding constant depending on the stereoenantiomer. They established that DNA was used as a biomacromolecular chiral selector in enantioseparation technology (23-25). When affinity ultrafiltration experiments were performed for the enantioseparation of racemic phenylalanine using DNA as chiral selectors in the feed solution with a DNA concentration of less than 0.5 ppm, d-(R)-phenylalanine was preferentially present in the collected permeate solution, but the separation factor fluctuated from 0.5 to 20 depending on the permeation time. This fluctuation was attributed to the fact that DNA sometimes releases phenylalanine in a dilute DNA solution (i.e., 0.01–0.5 ppm) because of the conformational change of DNA over time (25).

The immobilized DNA membranes were prepared from cellulose dialysis membranes with different pore sizes, and the effect of the pore size on enantioseparation was investigated using the immobilized DNA membranes (24-27). d-Phenylalanine preferentially permeated through the immobilized DNA membranes with pore sizes of less than 2.0 nm [molecular weight cut off (MWCO) < 5000], whereas l(S)-phenylalanine preferentially permeated through the immobilized DNA membranes with a pore size of greater than 2.0 nm (MWCO of the base membranes >5000) (Fig. 5) (26). The pore size of the immobilized DNA membranes regulated preferential permeation of the stereoenantiomer through the membranes. It was found that the immobilized DNA membranes adsorbed l-phenylalanine preferentially, independent of the pore size (25). Figure 6 shows the model of enantioseparation by the immobilized DNA membranes (27).

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Figure 5. Dependence of the separation factor in the permeate (open circle) and the concentrate (closed circle) solutions through the immobilized DNA membranes on the molecular weight cut-off (MWCO) of the base membranes and on the pore size of the immobilized DNA membranes at pH 7.0 and 25 °C. Source: Reprinted from Reference 26 with permission. Copyright 2012 Elsevier Inc.

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Figure 6. Enantioseparation model of the ultrafiltration of a racemic phenylalanine solution through immobilized DNA membranes. Source: Reprinted from Reference 27 with permission. Copyright 2012 Elsevier Inc.

It was determined that the immobilized DNA membranes are potentially useful for enantioseparation. Enantioseparation by these immobilized DNA membranes is based on the interaction between DNA and a specific stereoenantiomer, as determined by the absence of enantioseparation observed in the cellulose membranes without bound DNA. The immobilized DNA membranes were categorized as channel-type membranes and not as affinity membranes (such as those used for affinity ultrafiltration using albumin) (15, 19). The membrane pore size and the binding affinity of the specific stereoenantiomer are the most important factors for the preparation of channel-type membranes. The immobilized albumin membranes reported in the literature (15, 19) worked as affinity membranes and not as channel-type membranes, even though the membranes have a similar pore size (e.g., MWCO = 13, 000) to the membranes used in DNA immobilized membranes. The strong binding affinity of l-amino acids to albumin interferes with the generation of the channel-type permeation mechanism. The specific enantiomer cannot permeate through the membrane but may adsorb to the membrane when the binding affinity of the specific stereoenantiomer to the membranes is too strong. The weaker binding affinity of DNA to l-amino acids compared to that of albumin causes the immobilized DNA membranes to work as channel-type membranes (26).

7 Enantioseparation by Nanofibers

  1. Top of page
  2. Background
  3. Permeation and Selective Theory for Enantioseparation
  4. Resolution Mechanism Through the Membranes
  5. Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers
  6. Enantioseparation Membranes Prepared from Polymers with a Chiral Branch
  7. Enantioseparation Membranes with Immobilized Stereoselective Ligands
  8. Enantioseparation by Nanofibers
  9. Enantioseparation by Nonchiral Membranes
  10. Future Perspectives
  11. References
  12. Further Reading

First, we roughly calculated the surface areas for a usual cast membrane and a nanofiber membrane, both of which are hypothetically fabricated from a given membrane material with a density of 1.0 g/cm3. The surface area for the nanofiber membrane was calculated to be approximately 40 m2/g, whereas the usual cast membrane had a surface area of approximately 0.2 m2/g. The surface area of the nanofiber membrane is 200 times greater than that of the usual membrane sheet. Figure 7 shows the relationship between the surface area of the nanofiber membrane and its diameter, assuming that the density of the candidate polymer is 1.0 g/cm3. In the case that the membrane material shows a specific interaction toward one target enantiomer and not to the corresponding antipode, the target enantiomer would be incorporated into the membrane preferentially over the antipode. If the target molecule is in contact with two types of membrane, such as a usual cast membrane and a nanofiber membrane, the amount of the target molecule incorporated into the nanofiber membrane should be greater than that incorporated into the usual membrane. Assuming that the amount of substrate incorporated into a membrane is proportional to its surface area, the amount incorporated into a nanofiber membrane would be 200 times higher than that for a usual membrane. From this, it follows that the solubility coefficient of the target enantiomer for a nanofiber membrane would be higher than that for a usual membrane.

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Figure 7. Relationship between the surface area of nanofiber membrane and its diameter (calculations were carried out assuming the density of the polymer to be 1.0 g/cm3).

It is necessary to think about the porosity of those two types of membrane. In general, the porosity of a nanofiber membrane is reported to be approximately 80%, whereas a usual cast membrane possesses low porosity (ca. 5–10%) (28). Membranes with a high porosity give a high diffusion coefficient for the permeant.

As described in Section 2, membrane transport ability is determined by solubility and diffusivity. As described above, a nanofiber membrane has high solubility and high diffusivity, which leads to high membrane transport ability. Precisely speaking, in the case of membrane transport with a nanofiber membrane, the term partition is more suitable than solution. In membrane separation, both the flux and permselectivity are important. These two factors often exhibit a trade-off relationship (in other words, an enhancement of flux leads to a concurrent reduction in permselectivity and vice versa). Membranologists have perceived that the simultaneous enhancement of both flux and permselectivity has been an unsolved and/or unsolvable problem in membrane separation. The membrane form of nanofiber mats (membrane), fabricated by electrospray deposition, is expected to solve this trade-off relationship problem of membrane separation. Nanofiber membranes have higher surface area and higher porosity, as mentioned above. This is expected to lead to a higher permselectivity and an enhancement of flux. The enhancement of both the flux and permselectivity was first proved by enantioseparation of racemic mixtures with molecularly imprinted nanofiber membranes (4, 29, 30). The possibility of a breakthrough in membrane separation is further discussed in Section 8.1. Next, enantioseparation with nanofiber membranes made from natural chitin is discussed.

Chitin, an abundant green polymer and a main component of the exoskeletons of arthropods such as crustaceans and insects, is one promising green polymer for use as a membrane material. Chitin is synthesized at a rate of 1010−1011 tons/year (31). Chitin is a polymer of N-acetyl glucosamine and contains a chiral environment that is expected to discriminate between absolute configurations of a given mixture of enantiomers. Ifuku and his coworkers isolated chitin nanofibers from the exoskeletons of crabs and shrimps. The obtained chitin nanofibers had a highly uniform structure of 10–20 nm in diameter. On the basis of their characteristic nanoform and other excellent physical properties, chitin nanofibers are strong candidates for an enantioseparation membrane. To this end, chitin nanofiber membranes were fabricated from crab shells and their enantioseparation ability was studied (32). Racemic mixtures of amino acids such as glutamic acid (Glu), phenylalanine (Phe), and lysine (Lys) were adopted as model racemates. In the enantioseparation of Phe, the l-isomer was preferentially incorporated into the chitin nanofiber membrane from a racemic mixture of Phe. The adsorption selectivity toward l-Glu was determined to be 2.33. The interaction between l-Phe and chitin was relatively strong. Here, as described in Section 3, the trade-off relationship between solubility selectivity and diffusivity selectivity was applicable to membrane transport, and consequently, the diffusion of the l-isomer in the membrane was retarded. As a result, the permselectivity toward l-Phe was decreased to 0.86. In other words, d-Phe was selectively transported, even though the l-Phe was preferentially incorporated into the chitin nanofiber membrane. For other amino acids, such as Gly and Lys, the d-isomers were selectively transported through the chitin nanofiber membrane, although the permselectivities for those enantiomers were not so high.

8 Enantioseparation by Nonchiral Membranes

  1. Top of page
  2. Background
  3. Permeation and Selective Theory for Enantioseparation
  4. Resolution Mechanism Through the Membranes
  5. Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers
  6. Enantioseparation Membranes Prepared from Polymers with a Chiral Branch
  7. Enantioseparation Membranes with Immobilized Stereoselective Ligands
  8. Enantioseparation by Nanofibers
  9. Enantioseparation by Nonchiral Membranes
  10. Future Perspectives
  11. References
  12. Further Reading

The central dogma that chirality can only be generated from chiral molecules may be extended to predict that membranes without any chiral molecules cannot contribute to enantioseparation (chiral separation). Therefore, most researchers have developed enantioseparation membranes prepared from natural chiral polymers, synthetic chiral polymers from asymmetric synthesis, polymers with chiral branches, or immobilized chiral molecules, proteins, polysaccharides, and DNA. However, enantioseparation membranes can be prepared from nonchiral polymers with “chiral memory,” accomplished with molecularly imprinted membranes or membranes with a chiral memory of the polymer helicity. An original landmark work on enantioseparation by molecularly imprinted membranes composed of an achiral polymer was first reported by Yoshikawa et al. (33) in 1995. Those enantioseparation membranes are described in detail in the following sections.

8.1 Molecularly Imprinted Membranes

As described in Section 2, membrane separation consists of two mechanisms: the incorporation of the permeant into a membrane and the diffusion of the permeant within the membrane from the feed side to the permeate side. Diffusion of permeant within the membrane is predominantly governed by its dimensions and/or shape. From this, the control of permselectivity with diffusion should be intrinsically limited. Contrary to this idea, the incorporation of permeant into a membrane is expected to range from naught to infinity. The introduction of molecular recognition sites that rigorously discriminate the target molecule from others into the membrane will lead to an enhancement of its permselectivity.

The introduction of molecular recognition sites into polymeric membranes is usually time consuming, costly, and annoying. However, molecular imprinting could introduce molecular recognition sites into polymeric membranes without requiring these drawbacks (34-37). Molecular imprinting might be the most facile method to give polymeric membranes substrate specificity (38-41).

Among molecular imprinting methods, alternative molecular imprinting is facile and easily introduces molecular recognition sites into polymeric membranes. As opposed to the pioneering and conventional molecular imprinting methods, molecular recognition sites are constructed at the same time as the molecularly imprinted materials are prepared from a polymer solution or polymer melt. In other words, any polymeric material (such as synthetic polymers (42), oligopeptide derivatives (43-45), derivatives of natural polymer (46), and natural polymers (47)) can be directly converted into molecular recognition materials by applying the alternative molecular imprinting method and without any laborious laboratory work.

A similar approach was proposed by Michaels and his colleagues (48) in 1962. Polyethylene membranes conditioned by p-xylene, which worked as a print molecule, transported p-isomers preferentially over the corresponding o- and m-xylenes by pervaporation. This study is not just the first report on alternative molecular imprinting, but it is also the first application of molecularly imprinted polymeric membranes to membrane separation. Michaels' paper (48) is a commendable paper in the fields of molecular imprinting and membrane separation.

Membrane transport using a molecularly imprinted polymeric membrane prepared by conventional molecular imprinting (noncovalent molecular imprinting) was first reported in 1990 (49). Since then, various molecularly imprinted membranes have been studied by adopting noncovalent molecular imprinting. Enantioseparation membranes were prepared from methacrylic acid (functional monomer) and 1,1,1-tris(hydroxymethyl)propane trimethacrylate (cross-linker) in the presence of N-α-benzyloxycarbonyl-l-tyrosine (Z-l-Tyr) as a print molecule (50). Membrane transport experiments of Z-d-Tyr and Z-l-Tyr were performed separately. The transport rate of Z-l-Tyr through the Z-l-Tyr-imprinted membrane was higher than that of Z-d-Tyr, whereas both enantiomers gave the same flux through the nonimprinted control membrane. As described later, the transport of an enantiomer preferentially incorporated into a membrane is often retarded by a relatively strong interaction between the enantiomer and the membrane. As a result, the antipode is selectively transported from among a racemic mixture of enantiomers. If a racemic mixture of Z-Tyr is transported through the Z-l-Tyr-imprinted membrane, which enantiomer will be selectively transported? In most cases, Z-d-Tyr would be preferentially transported; at worst, enantioseparation would hardly be observed.

Enantioseparation using molecularly imprinted membranes has been intensively studied by using alternative molecular imprinting, a technique that can convert any polymeric material into an enantioseparation membrane. By applying alternative molecular imprinting, polymeric materials such as entirely achiral synthetic polymers (42, 51), oligopeptide derivatives (43-45), and derivatives of natural polymers (46) have been converted into enantioseparation membranes. In the application of alternative molecular imprinting to achiral synthetic polymers, both d- and l-isomers have worked well as print molecules; in other words, as summarized in Figure 8, the d-isomer recognition membranes were converted by adopting the d-isomer and vice versa. The oligopeptide tweezers (52) and derivative of natural polymers, cellulose acetate (CA) (46), were classified into the same category, even though they have a chiral environment.

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Figure 8. Construction of molecular recognition sites in achiral synthetic polymers [polysulfone (PSf) and SELAR], derivatives of natural polymers (CA), and oligopeptide tweezers by an alternative molecular imprinting.

Contrary to this, the chiral recognition ability of molecularly imprinted membranes from oligopeptide derivatives showed different dependencies based on the absolute configuration of the print molecule adopted. As shown in Figure 9, the membrane containing an oligopeptide residue from a d-amino acid and imprinted by a d-amino acid derivative recognized the d-isomer in preference to the corresponding l-isomer and vice versa. This led to the conclusion that it is not necessary to adopt an optically pure d- or l-isomer as a print molecule to generate a chiral recognition site. Subsequently, enantioselective membranes were prepared using racemic print molecules instead of an optically pure print molecule (45) The membranes imprinted with a given amino acid print molecule recognized not only the print molecule analogue but also other racemic α-amino acids whose absolute configuration was the same as that of the print molecule (43). The control membranes, which were prepared without the print molecule, hardly showed any adsorption selectivity.

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Figure 9. Construction of molecular recognition sites in oligopeptide derivatives by an alternative molecular imprinting.

As described above, by adopting CA (with an acetyl content of 40%) and print molecules (such as Z-d-Glu and Z-l-Glu), two types of molecularly imprinted membrane were obtained (46). The membrane imprinted by Z-d-Glu selectively transported d-Glu with the optimal applied potential acting as a driving force for membrane transport and vice versa. When using two types of molecularly imprinted membrane at the same time, as shown in Figure 10, d-Glu was selectively transported through the d-Glu preferentially incorporated membrane and vice versa. The dual direction enantioselective membrane transport is an interesting membrane separation system. Molecularly imprinted membranes from carboxylated polysulfone also separated racemic mixtures of Glu by dual direction electrodialysis (42).

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Figure 10. Enantioseparation of a racemic mixture of Glu using two types of molecularly imprinted CA membrane and dual direction electrodialysis. Source: Reprinted from Reference 46 with permission. Copyright 2012 John Wiley and Sons, Inc.

As mentioned, by applying molecular imprinting such as conventional molecular imprinting or alternative molecular imprinting, molecular or chiral recognition sites are easily introduced into polymeric membranes. From this, it is easy to enhance the permselectivity of a given membrane by applying those molecular imprinting methods. However, neither the flux nor the permselectivity of those membranes were enough for practical application. The enhancement of flux is especially indispensable to allow for molecularly imprinted membranes to be able to be utilized on an industrial scale. Molecularly imprinted membranes with higher surface area and higher porosity are expected to give higher flux and higher permselectivity. Molecularly imprinted membranes with a membrane form of a nanofiber mat are a suitable (or may be the best) membrane morphology for attaining high flux and high permselectivity. Electrospray deposition is expected to give such a membrane.

The enhancement of flux without a concurrent reduction in permselectivity was proven with molecularly imprinted nanofiber membranes made from polysulfones bearing a carboxylic acid (29) or aldehyde moiety (4) and CA (Fig. 11) (30). Membrane performances for molecularly imprinted nanofiber membranes and usual molecularly imprinted membranes prepared from polysulfone with an aldehyde moiety are summarized in Table 1. The molar mobility, u (mol·cm/cm2/J/h), of Glu for each membrane is a suitable parameter to compare the membranes' performance. The molar mobility u was determined by the following equation, derived from the Nernst–Planck equation (53).

  • mathml alt image(11)

where J is the sum of the flux of d-Glu and l-Glu, c is the concentration of each Glu in the upstream side, and dμ/dx is the potential gradient at that point. The molar mobility u is defined as the mobility and is simply the flux per unit membrane area, per unit membrane thickness, per unit concentration, and per unit driving force. In Table 1, relative mobility is also shown for convenience. The upper part of Table 1, which shows membrane performances for the membranes from PSf-CHO-10, gives the relative molar mobility relative to MIPM-10-L. The lower part of Table 1 gives the relative molar mobility relative to MIPM-05-L. The flux values for the molecularly imprinted nanofiber membranes were one to two orders of magnitude higher than those for the usual molecularly imprinted membranes.

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Figure 11. Chemical structures of a polysulfone with an aldehyde group (PSf-CHO) and a print molecule (Z-d-Glu or Z-l-Glu). Source: Reprinted from Reference 4 with permission. Copyright 2012 Elsevier Inc.

Table 1. Results of Chiral Separation with Molecularly Imprinted Nanofiber (MINFM's) and Molecularly Imprinted (MIPM's) Membranes
MembraneZ-d-Glu-Imprinted MembraneZ-l-Glu-imprinted membrane
 αL/DuaαD/Lua
  1. a

    u = (−J/C)/(dμ/dx) {[(mol cm/cm2/h)/(mol/cm3)]/(J/mol/cm) =mol cm3/J/h}.

  2. b

    Figures in parentheses are the relative values; the u value for MIPM-10 imprinted by Z-l-Glu being set as unity.

  3. c

    Figures in parentheses are the relative values; the u value for MIPM-05 imprinted by Z-l-Glu being set as unity.

  4. Source: Reprinted from Reference 4 with permission. Copyright 2012 Elsevier Inc.

MINFM-10b1.241.15 × 10−9 (28)1.201.67 × 10−9 (41)
MIPM-10b1.204.20 × 10−11 (∼1)1.204.10 × 10−11 (1)
MINFM-05c1.127.00 × 10−9 (231)1.202.20 × 10−9 (72)
MIPM-05c1.256.64 × 10−11 (2.2)1.163.05 × 10−11 (1)

The permselectivity for these molecularly imprinted nanofiber membranes was not very high compared to previously reported membranes (3, 9). The permselectivity is thought to be greatly dependent on adsorption selectivity; therefore, the enhancement of adsorption selectivity is indispensable for increasing the permselectivity of a given molecularly imprinted nanofiber membrane.

In membrane separation, flux and the corresponding permselectivity often show a trade-off relationship. However, as shown in Table 1 and in previous studies (4, 29, 30), the enhancement of flux without a concurrent reduction in permselectivity has been achieved. In other words, molecularly imprinted nanofiber membranes have the potential to simultaneously enhance both throughput (flux) and permselectivity.

8.2 Membranes Composed of an Achiral Polymer with a One-Handed Helical Conformation

The preparation of one-handed helical polymers has usually been achieved by one of the following methods: (i) polymerization of optically active monomers, (ii) polymerization of prochiral monomers using optically active catalysts or initiators, (iii) induction of chirality by interaction between achiral polymers and chiral additives, and (iv) induction of chirality in achiral polymers with a chiral functional group followed by removal of the chiral functional group. Method (iv) enables the construction of chiral membranes from achiral polymers. This “chiral memory” phenomenon of polymer helicity was first discovered by Yashima's group, but they did not apply this technique to enantioseparation technology (2, 54).

Teraguchi and Masuda (55) verified enantioselective permeation using helical polymeric membranes composed of achiral poly(diphenylacetylene). The chiral memory of the membranes was created using poly(diphenylacetylenes) with a high content of pinanylsilyl groups, thus generating a chiral polymer, followed by depinanylsilylation after preparation of self-stable membranes. Depinanylsilylation of the membranes was carried out by exposure of the membranes to a mixture of hexane/trifluoroacetic acid at room temperature (55). The chiral selectivity of racemic tryptophan solutions through the membranes was found to be as high as 58.6% ee, while that of the chiral poly(diphenylacetylenes) retaining the pinanylsilyl group was 80.5% ee. These studies verified that membranes composed of achiral polymers with chiral helical memory can contribute to the enantioseparation of chemicals and drugs (3, 55).

9 Future Perspectives

  1. Top of page
  2. Background
  3. Permeation and Selective Theory for Enantioseparation
  4. Resolution Mechanism Through the Membranes
  5. Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers
  6. Enantioseparation Membranes Prepared from Polymers with a Chiral Branch
  7. Enantioseparation Membranes with Immobilized Stereoselective Ligands
  8. Enantioseparation by Nanofibers
  9. Enantioseparation by Nonchiral Membranes
  10. Future Perspectives
  11. References
  12. Further Reading

More than 100 articles have been published on the development of polymeric membranes for enantioseparation (chiral separation). Most enantioseparation membranes have relatively low separation factors, except for affinity membranes. There are four reasonable ways to obtain superior enantioseparation membranes in the near future. One of the methods is the utilization of natural materials as chiral environments or chiral selectors; these materials may possess greater enantioseparation ability than existing materials. As described in Section 6, natural materials have already been used as components of enantioseparation membranes or enantioseparation membrane systems; however, their enantioseparation abilities were not very high. To attain higher membrane performance, some modification of those natural materials will be required.

The second possible method is the application of a multistage enantioseparation process, an idea that has been rationalized by Higuchi et al. (26). If such a process is applied, existing enantioseparation membranes will continue to play an active part in future enantioseparation. Application of multistage process(es) might lead to a reduction in throughput, an important factor for membrane performance.

In connection with the second method, as the third one, a membrane form of nanofiber fabric may be able to reasonably enhance both throughput (flux) and permselectivity, as described in Section 8.1.

The fourth possible method is the use of a membrane form of polymeric pseudo-liquid membrane (56). Construction of a liquid membrane is very easy; a liquid membrane is constructed by one easy operation, the dissolution of transporter into solvent. A liquid membrane with a transporter for a target molecule will directly reflect the affinity between the transporter and the target substrate. However, the drawback of liquid membranes is their long-term stability; problems include the evaporation of the membrane solution and “washout” of the transporter and/or the transporter/target substrate complex during the process. The polymeric pseudo-liquid membrane, which consists of a transporter and a rubbery polymer, will work as a stable liquid membrane for enantioseparation.

There is a growing demand in industries involved with products such as pharmaceuticals, agrochemicals, fragrances, and food additives for the production of enantiomerically pure compounds. Membranologists struggle to provide the ultimate enantioseparation membranes, which is expected.

References

  1. Top of page
  2. Background
  3. Permeation and Selective Theory for Enantioseparation
  4. Resolution Mechanism Through the Membranes
  5. Enantioseparation Membranes Prepared from the Chiral Main Chain of Polymers
  6. Enantioseparation Membranes Prepared from Polymers with a Chiral Branch
  7. Enantioseparation Membranes with Immobilized Stereoselective Ligands
  8. Enantioseparation by Nanofibers
  9. Enantioseparation by Nonchiral Membranes
  10. Future Perspectives
  11. References
  12. Further Reading