Anal. Chem. 1994,66, 3113-3776
This Research Contribution is in Commemoration of the Life and Science of I. M. Kolthoff (1894- 1993).
Chiral Separations Using Micellar Electrokinetic Capillary Chromatography and a Polymerized Chiral Micelle Jian Wang and Isiah M. Warner’ Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803
A chiral micelle polymer, poly( sodium N-undecylenyl-Lvalinate), has been synthesized and used for chiral separation in micellar electrokineticcapillarychromatography. The chiral separationsof (&)-l,l’-bi-2-naphthol and D,L-laudanosinewere achieved through the addition of this chiral micelle polymer to buffer solutions. A comparison study of separations was conducted on the micelle polymer and the corresponding nonpolymerized surfactant under the same separation conditions. Several advantages of using the micelle polymer are noted. In addition, a model of micelle-solute interaction is proposed to rationalize the improved chiral separation ability of micelle polymers. The resolution of enantiomeric mixtures into individual optical isomers is one of the most challenging problems in separations and, more generally, in analytical chemistry. Such separations are very important to many industries, particularly the pharmaceutical industry. For example, it is now an acknowledged fact that the pharmacokinetic characteristics of individual enantiomers of a given chiral drug may be quite different. Such differences may produce serious physiological problems. Thus, a variety of chromatographic approaches for chiral separation and purification of such molecules, especially by use of HPLC, have been developed.” Recently, chiral separation by use of capillary electrophoresis (CE) has shown great promise as a potential method for the measurement of the optical purity of drugs and other racemic compound^.^.^ In CE, chiral separation is often achieved either through the use of immobilized chiral phases or through the addition of chiral selectors as mobile phase additives. In the latter case, three types of chiral selectors are often employed: (1) c y c l ~ d e x t r i n s (2) , ~ ~chiral ~ metal comp l e ~ e sand , ~ (3) chiral surfactants.lOJ1 The separation using the latter is often called micellar electrokinetic capillary chromatography (MECC). In MECC, a chiral surfactant is added to the buffered solution at appropriate concentrations (1) Jamali, F.; Mehvar, R.; Pasutto, F. M. J . Pharm. Sei. 1989, 78, 695. (2) Pirkle, W. H.; Burke, J. A., 111. Chirality 1989, 1, 57. (3) Armstrong, D. W. Anal. Chem. 1987, 59, 84A. (4) Ahuja, S., Ed. Chiral Separations in Liquid Chromatography; American Chemical Society: Washington, DC, 1991; Vol. 471. (5) Snopek, J.; Jclinek, I.; Smolkova-Keulemansova, E. J . Chromatogr. 1992, 609, 1. ( 6 ) Kuhn, R.; Hoffstetter-Kuhn, S. Chromatographia 1992, 34(9/10),505. (7) Nishi, H.; Kokusenya, Y.; Miyamoto, T.; Sato, T. J. Chromatogr. 1994,659, 449. ( 8 ) Fanli, S . J . Chromarogr. 1989, 474, 441. (9) Gassmann, E.; Kuo, J. E.; Zare, R. N. Science 1985, 230, 813. (IO) Doshi, A.; Ono, T.; Hara, S . ; Yamaguchi, J. Anal. Chem. 1989, 61, 1684. (11) Terabe, S. S.;Shibata, M.; Miyashita, Y. J . Chromatogr. 1989, 480, 403.
0003-2700/94/0366-3773$04,50/0
@ 1994 American Chemical Society
to form optically active micelles. The required enantiorecognition occurs through chiral centers which have been incorporated into the micelle. It is well established that, in MECC, such micelles form a pseudostationary phase with which solutes can interact.129’3 In addition to the necessary interactions between a micelle and the solute, it is also important to recognize the dynamic equilibrium that exists between the surfactants and the micelle since normal micelles are dynamic aggregates of the surfactant monomers. In chiral separations, the difference in interaction between the micelle and the individual enantiomers is often very small. Such dynamic equilibria do not always allow adequate discrimination for many racemic mixtures. In addition, high concentrations of surfactant must often be used in order for some surfactants to be effective in MECC. Thus, excessive heat may be generated when ionic surfactants are used. This could cause problems in CE separations. The problems identified above suggest that control of dynamic equilibrium in chiral micelles may yield improved discrimination for racemic mixtures. In the late 1970s, polymerized micellar systems were introduced as alternatives to normal micelles.14 Such polymerized micelles were demonstrated to produce some distinct advantages over normal micelles, e.g., enhanced stabilities and rigidities and controllable size, because the covalent bonds between these surfactant aggregates eliminate the normal dynamic equilibrium which occurs between the surfactant monomers and the micelle. Thus, the process of complexation between the micelle and the solute is simplified and enhanced. Numerous publications have reported on the characterization and utilization of polymerized micelles.1”18 However, few papers have reported the use of polymerized micelles and polyions in capillary electrophoresis.l*J9 To our knowledge, the use of polymerized chiral micelles in MECC has not been reported. This is surprising, considering the obvious advantages of such systems. In this manuscript, a new typeof polymerized chiral micelle, poly(sodium N-undecylenyl-L-valinate)(poly(L-SUV)), is synthesized and characterized by use of various spectroscopic (12) Terabc, S.;Otsuka, K.; Ando, T. Anal. Chem. 1985,57, 834.
(13) Sepaniak, M. J.; Cole, R. 0. Anal. Chem. 1987, 59, 472. (14) Larrabee, C. E.; Sprague, E. D. J. Polym. Sci.. Polym. Lcrr. Ed. 979,17,149. (15) Nagai, K.; Elias, H.-GMakromol. Chem. 1987, 188, 1095. (16) Fendler, J. H.;Tundo, P. Acc. Chem. Res. 1984, 17, 3 . (17) Tabor, D. G.; Underwood, A. L. J. Chromafogr. 1989, 463, 13. (18) Palmer, C. P., Khaled, M. Y.; McNair, H. M. J. High Resolur. Chromarogr. 1992, 15, 156. (19) Terabe, S.; Isemura, T. Anal. Chem. 1990, 62, 650.
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techniques. This micellar system is then used in MECC for chiral separation of (&)-binaphthol and D,L-laudanosine. In addition, the utility of poly(L-SUV) and the nonpolymerized L-SUV is compared under similar conditions in MECC chiral separation. The advantages of using poly(L-SUV) in MECC are demonstrated.
EXPER I MENTAL SECTION Synthesis of Poly(sodium N-undecylenyl-L-valinate). The monomer acid N-undecylenyl-L-valine (L-UV) was prepared from the undecylenic acid N-hydroxysuccinimide ester by treatment with L-valine according to a procedure reported by Lapidot et a1.20 (mp 91 ‘C, [.]25D = -2.9’ (c = 1.00, methanol)). This carboxylic acid was then converted to the corresponding sodium salt, L-SUV, by the addition of an equal molar solution of sodium hydroxide in an ethanol-water (1:4 by volume) mixture. The CMC of the synthesized nonpolymerized surfactant was determined to be 2.1 X M by use of surface tension measurements. The polymerization of L-SUV was achieved by 6oCo y-irradiation of a 0.05 M surfactant solution. The detailed procedure of this polymerization is as previously reported by Larrabee et al.I4 After irradiation, the polymer solution was purified by lyophilization and treatment with hot ethanol to extract unreacted monomer. The product was then dried under vacuum. Proton NMR of the purified product did not show the multiplets of double bond proton signals present in the monomers and thus verified polymerization. More importantly, NMR and FT-IR data indicate that key functional groups associated with the surfactant monomers remained intact in the polymerized micelles and that the chirality of the micelles has not been destroyed ([CX]25D = -8.19O (c = 1.00 in water)). The polymer was found to be 99% pure as calculated from elemental analysis. Further characterization of this system is underway in our laboratory. Materials. The (&)-l,lt-bi-2-naphthol (99%), (I?)-(+)1,I’-bi-Znaphthol(99%), and S-(-)-l ,lt-bi-2-naphthol(99%) were purchased from Aldrich (Milwaukee, WI). D,LLaudanosine (95%), L-valine (>99%), and undecylenic acid (>99%) were obtained from Sigma (St. Louis, MO). These items were used as received. Capillary Electrophoresis Procedure. Micellar electrokinetic capillary chromatography experiments were conducted by use of a CES I capillary electrophoresis system (Dionex Co., Sunnyvale, CA). Data were collected by use of an AI450 chromatography workstation. An untreated fused silica capillary (effective length 60 cm, 75 mm i.d.) was purchased from Polymicro Technologies (Phoenix, AZ) and used as a separation column. The solution was buffered with 25 mM borate (pH = 9.0 or pH = 10.0 as noted in experiments). The micelles were added directly to the buffer system. The buffer solutions were filtered through a 0.45 mm membrane filter prior to use. Separations were performed at 12 kV with UV detection at 290 or 254 nm. Samples were prepared in methanol-water mixture, and the concentrations were typically about 0.1 mg/mL. RESULTS AND DISCUSSION The base line separation of (f)-1 ,l’-bi-2-naphthol using 25 mM sodium borate buffer (pH = 9.0) containing 0.5% (20) Lapidot, Y.;R a p p p r t , S.; Wolman, Y . J . Lipid Res. 1967, 8, 142
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(a)
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’,20 , ,
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Flgure 1. Comparison between polymerized micelle and nonpoty,l‘-bi-Pnaphthol. (a) 0.5% polymerizedmicelle for separation of (f)-l (L-SUV);(b) 0.5% L-SUV; (c) 1 % L-SUV. Buffer, 25 mM borate buffer (pH = 9.0); applied voltage, 12 kV; current, (a) 39, (b) 40, and (c) 51 mA: UV detection at 290 nm.
(w/v) poly(L-SUV) is shown in Figure la and demonstrates the chiral recognition ability of the polymerized micelle. The (S)-(-)-l,l’-bi-a-naphthol enantiomer eluted faster than the corresponding (R)form. This suggests that (R)-(+)-1 ,l’bi-2-naphthol has a higher affinity with the ( S ) (L) form of the chiral polymer. In addition, as expected, our preliminary study showed a reversal of elution order of (*)-1,l’-bi-2naphthol in the presence of the (R)(D) form of the micelle polymer (poly(D-SUV)). Figures l b and IC show electropherograms of (A)-1,l’-bi-Znaphthol under the same conditions as in Figure la. However, the surfactant used in these figures is the nonpolymerized surfactant at concentrations of 0.5% (w/v) and 1% (w/v), respectively. No separation is noted at a surfactant concentration of 0.5% since this concentration is just below the normal CMC of the nonpolymerized surfactant. Chiral separation is noted only when the surfactant concentration is increased to 1%. Thus, the polymerized micelle allows for better discrimination of the enantiomers than the nonpolymerized micelle. In a normal (nonpolymerized) micelle system, the dynamic equilibrium that exists between the monomers and micelle aggregates, as well as the interaction of solutes with the aggregates (Figure 2a), is a disadvantage, as noted previously. Moreover, the difference in affinities between the micelle and a pair of enantiomers is usually very small, and the dynamic equilibrium between the monomers and the micelle may reduce the enantiorecognition ability of the micelle. In contrast, polymerized micelles are void of such problem since the covalent bonds formed between monomers eliminate dynamic equilibrium (Figure 2b). Thus, a stable pseudostationary phase can be formed in the buffer, and the interaction between solutes and micelle is simplified and enhanced. Another distinct advantage of polymerized micelles is that there is no CMC of the polymerized micelles. Thus, the micelle polymer is not concentration dependent and can be used at any concentration. However, for nonpolymerized micelles, the concentration of the surfactant has to be higher than the CMC in order to be effective, because concentrations below the CMC will break up the micelle. This is demonstrated in Figures l b and IC. It should also be noted that if the CMC
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Figure 2. Schematic dlagram of the prlnciple of surfactant, micelle, and solute Interactions. (a) Normal (nonpolymerized) mlcelle. (b) Poiymerlzed micelle. S,solute; asterisk denotes chlral center. (a)
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Figure 3. Effects of poly(~-SUV)concentration on chiral separation of (f)-l,l’-bi-2-naphthol. (a) 0.02% poly(~-SUV);(b) 0.05% poly(^SUV); (c) 0.1 % poiy(~-SUV);(d) 0.5 % poly(~-SUV).Other condltlons are as in Figure 1.
of a charged surfactant is very high, the high concentration of the surfactant used in MECC will generate considerable heat, which is deleterious to separations by use of CE. Another advantage of the polymerized micelle systems in MECC is also shown in Figures l a and IC. It is noted that the peaks are more broad in Figure IC than those in Figure la. In terms of numbers of theoretical plates, the value of N i s 102 240 in Figure l a and 28 073 in Figure IC. An inherent disadvantage of micellar liquid chromatography and also of MECC is peak broadening associated with slow mass transfer of the solute between the micelle and the bulk solvent. These observations are consistent with the spectroscopic data reported by Paleos et alaz1 In that study, polymerized micelles were noted to have more compact structures than normal micelles. Thus, the solute does not penetrate as deeply into the polymerized micelle as into normal micelles, and an increase in the mass transfer rate of the solute should be noted. Figure 3 shows the effect of changing the concentration of the poly(L-SUV) in the chiral separation of (i)-l,l’-bi-2naphthol. In this figure, the concentration of poly(~-SUV) varies from 0.02%to 0.5%. It is clear that there is an optimum concentration of poly(L-SUV) to achieve highest resolution (21) Paleos, C. M.; Stasslnopoulou, C. I.; Mallaris, A. J. Phys. Chem. 1983, 87, 251.
Figure 4. Influence of poly(~-SUV)concentrationon the resolutlon of (f)-1 ,l‘-bi-2-naphthol.
of (f)-l,l’-bi-2-naphthol. It is noted that the resolution of (f)- 1,l’-bi-Znaphthol increased while the concentration of poly@-SUV) was initially increased. At some optimum concentration (0.27&0.5%), further increases in the concentration of the poly(L-SUV) did not improve the resolution and actually led to a slight decrease in resolution (Figure 4). This observation can be explained by using the chiral separation model proposed by Wren.22 The two enantiomers of (i)l,l’-bi-2-naphthol have the same electrophoretic mobility in a nonchiral buffer. When they interact with the chiral selector poly(L-SUV), which is dissolved in the buffer, they form complexes with the polymer as diagrammed in Figure 2b. One assumes that the complexes of the individual enantiomers have the same electrophoretic mobility. However, if the two enantiomers have different binding constants with poly(LSUV), then chiral resolution is achievable since the electrophoretic mobilities of the free and complexed binaphthols are different. The optimum concentration of poly(L-SUV) is the concentration that maximizes the apparent mobility difference of the enantiomers. This set of data is another demonstration of the concentration advantage of the polymerized micelles, because the concentrations of the polymer used in our studies were well below the normal CMC of the monomer. We have also used poly(L-SUV) to resolve another pair of enantiomers, D,L-laudanosine. In this separation, the pH of the buffer plays a very important role. As shown in Figure 5, only slight resolution of the enantiomers is achieved at pH = 9.0. However, adjustment to pH = 10.0 produced a near base line separation (R, = 1.2). The pH effects on polymerized micelles have been reported by Chu and Thomas.23 At lower pH, negatively charged polymerized micelles have a compact conformation, while at higher pH, the highly negatively charged polymerized micelles may have a looser conformation because of the electrostatic repulsion. Our data are consistent with these observations. The looser conformation of the micelle at higher pH must provide better interactions with the laudanosine enantiomers. A detailed study of the pH effect of the interaction between this polymer and the solute is currently underway in this laboratory. CONCLUSIONS A novel polymerized chiral micelle was synthesized and used for chiral separations in MECC. Several advantages (22) Wren, S. A. C.; Rowe, R. C. J. Chromutogr. 1992, 603, 235. (23) Chu, D. Y.;Thomas, T.K. Mucromolecules 1991, 24, 2212.
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are noted for polymerized micelles over normal micelles in chiral separations. First, the elimination of dynamic equilibrium between monomer and micelle can enhance chiral recognitionof the racemic mixture. Thus, better enantiomeric
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resolution is expected with use of this approach. Second, the lack of a CMC in polymerized micelles makes them more practical for use in MECC separation, Le., the micelle polymers operate over all concentration ranges. Finally, the rigidity of the polymerized micelle improves the mass transfer rate and thus reduces peak broadening. We believe that polymerized chiral micelles represent an exciting new approach to chiral separations in capillary electrophoresis.
ACKNOWLEDGMENT The authors thank Ms. Michelle Butterfield for technical assistance and Dr. Arthur Underwood of Emory University for useful discussions regarding this work. This work was supported in part by a grant from the National Science Foundation (CHE 9224177). Scientific Parentage of the Authors. J. Wang, Ph.D. candidate under I. M. Warner, Ph.D. under G. D. Christian, Ph.D. under W. Purdy, Ph.D. under D. Hume, Ph.D. under I. M. Kolthoff. Received for review May 18, 1994. Accepted July 27, 1994.'
* Abstract published in Advance ACS Abstracts, September 15, 1994.
