Anal. Chem. 1997, 69, 958-964
Studies of Polymerized Sodium N-Undecylenyl-L-valinate in Chiral Micellar Electrokinetic Capillary Chromatography of Neutral, Acidic, and Basic Compounds Kimberly A. Agnew-Heard, Montserrat Sa´nchez Pen˜a, Shahab A. Shamsi, and Isiah M. Warner*
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803
The polymerized surfactant poly(sodium N-undecylenyl amino L-valinate) [poly(L-SUV)] has been used in micellar electrokinetic capillary chromatography for the chiral separation of various acidic and basic drugs, as well as neutral compounds. Under the conditions studied, poly(L-SUV) was shown to be a very versatile anionic chiral selector in the pH range of 5.6-11. The micelle was used for the enantioseparation of coumarinic anticoagulant drugs with various buffers under moderately acidic conditions. Neutral and alkaline buffer conditions were used to successfully separate the neutral atropisomers (()-1,1′bi-2-naphthol, (()-1,1′-binaphthyl-2,2′-diamine, and Tro1ger’s base. Chiral separation of the cationic paveroline drugs, laudanosine, norlaudanosoline, and laudanosoline, was influenced by pH and the use of coated capillaries. The acquired data focused on optimizing the migration times, capacity and separation factors, and electrophoretic mobilities of the various racemic mixtures. The enantioselectivity of chiral compounds is important to the environmental and biological fields, as well as to synthetic chemists and the pharmaceutical industry. Our studies here focus on the pharmaceutical industry, where many chiral drugs are generated and sold as racemates. Since the emergence of strict Food and Drug Administration (FDA) policies, separating individual enantiomers has become necessary to the drug industry.1 These FDA regulations specify that the stereoisomeric composition of a drug with a chiral center should be studied for its biological activities and toxicological effects.2 One reason for this mandate is that each enantiomer of a drug may exhibit stereoselectivity in its pharmacological function. As a result, one optical antipode may produce the desired physiological response, while the other may be toxic or inactive.3 It is also necessary to determine whether each antipode is critical to the desired pharmacokinetic activity. Many drug companies now seek to market chiral drugs as single enantiomers because of this heightened concern with consumer safety and federal regulations. In the past, gas chromatography (GC) and high-performance liquid chromatography (HPLC) were the two most prominent techniques used for enantiomeric separations. By combining the high efficiency of GC with the selectivity of HPLC, capillary electrophoresis (CE) has emerged as a technique that provides a (1) Ward, T. J. Anal. Chem. 1994, 66, 632A-640A. (2) U.S. Food and Drug Administration. Chirality 1992, 4, 338-340. (3) Jamali, F.; Mehvar, R.; Pasutto, F. M. J. Pharm. Sci. 1989, 78, 695-715.
958 Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
larger application range for enantioseparation.4 Other advantages of CE include higher separation efficiency and resolution, faster analysis times, and smaller sample volumes.5 One of the most valuable advantages of CE, over direct chiral HPLC separation, is the small consumption of chiral selectors in the mobile phase. Regarding the latter advantage, only a few milliliters of buffer is needed for enantioseparation with most CE instruments. Variations of the basic mode of CE, e.g., capillary zone electrophoresis (CZE), are often used to achieve chiral separations. Direct chiral separation in CE is frequently achieved either through using an immobilized chiral phase, i.e., capillary gel electrophoresis,6 or the addition of chiral selectors as mobile phase additives. The most common chiral mobile phase selectors include host-guest additives, such as cyclodextrins (CDs)7 and crown ethers.8 Another example is the N-acylcalix[4]arene amino acid derivatives recently synthesized by our research group.9 Other background electrolyte (BGE) additives used for enantioseparation include chiral ligand exchange reagents10,11 and the newly introduced macrocyclic antibiotics,12 heparin,13-15 and dextran sulfates.15 In recent years, increased interest has been shown in the use of chiral surfactants for the micellar electrokinetic capillary chromatography (MECC) approach to enantioseparation. This technique involves the addition of a chiral surfactant into the BGE at a concentration above the critical micelle concentration (cmc). Enantioselectivity is achieved by differential interaction of each enantiomer with the chiral micelle. The utility of the chiral MECC approach has been demonstrated using sodium N-dodecanoyl-Lvalinate (SDVal),16-18 bile salts,19,20 digitonin,16 saponins,21 and (4) Krstulovic, A. M., Ed. Chiral Separations by HPLC: Applications to Pharmaceutical Compounds; John Wiley & Sons: New York, 1989. (5) Li, S. F. Y. Capillary Electrophoresis: Principles, Practice and Applications; Elsevier Science: New York, 1992. (6) Cruzado, I. D.; Vigh, G. J. Chromatogr., A 1992, 608, 421-425. (7) Fanali, S. J. Chromatogr., A 1989, 474, 441-446. (8) Kuhn, R.; Stoecklin, F.; Erni, F. Chromatographia 1992, 33, 32-36. (9) Sa´nchez Pen ˜a, M.; Zhang, Y.; Thibodeaux, S.; McLaughlin, M. L.; de la Pen ˜a, A. M.; Warner, I. M. Tetrahedron Lett. 1996, 37, 5841-5844. (10) Gassmann, E.; Kuo, J. E.; Zare, R. N. Science 1985, 230, 813-814. (11) Gozel, P.; Gassmann, E.; Michelsen, H.; Zare, R. N. Anal. Chem. 1987, 59, 44-49. (12) Armstrong, D. W.; Rundlett, K.; Reid, G. L. Anal. Chem. 1994, 66, 16901695. (13) Stalcup, A. M.; Agyei, N. M. Anal. Chem. 1994, 66, 3054-3059. (14) Nishi, H.; Nakamura, K.; Nakai, H.; Sato, T. Anal. Chem. 1995, 67, 23342341. (15) Agyei, N. M.; Gahm, K. H.; Stalcup, A. M. Anal. Chim. Acta 1995, 307, 185-191. (16) Otsuka, K.; Terabe, S. J. Chromatogr., A 1990, 515, 221-226. S0003-2700(96)00778-0 CCC: $14.00
© 1997 American Chemical Society
glucopyranoside-based phosphate and sulfate surfactants.22 In all of these studies, monomeric chiral surfactants were added to the BGE above their cmc. One problem with MECC is that chiral separation may be compromised by the dynamic equilibrium that exists between the surfactant monomer and micelle.23 In addition, micelles are species with equilibrium mixtures of monomers and species containing monomers well above the average aggregation number. It is well established that such polydispersity is detrimental to chromatographic separations. To eliminate these problems, polymerized surfactants were introduced for chiral separation. As first reported by Wang and Warner, poly(sodium N-undecylenyl-L-valinate) [poly(L-SUV)] may be used for the optical resolution of (()-1,1′-bi-2-naphthol and D,L-laudanosine with and without γ-CD.23,24 Lower concentrations than the cmc of this polymerized surfactant were added to the BGE since the surfactants are covalently linked and the surfactant monomers have been removed. Thus, the polymer is more stable and can withstand high concentrations of organic solvents. The same polymer, poly(sodium 10-undecenoyl-L-valinate), was employed for the enantioseparation of 3,5-dinitrobenzoyl amino acid isopropyl esters.25 Similar to polymerized surfactants, the high molecular mass surfactant butyl acrylate-butyl methacrylate-methacrylate acid copolymer sodium salt (BBMA) was used to separate chiral26 and achiral27-29 compounds with and without CD-modified MECC. The cmc of this surfactant was determined to be effectively zero; therefore, BBMA could be used at lower concentrations as a pseudostationary phase with MECC.27 Ozaki et al. reported the on-line coupling of MECC with electrospray ionization interfacedmass spectrometry (ESI-MS) detection. This coupling is advantageous over UV absorbance detection because structural information can be acquired and the high molecular mass of BBMA (40 000 Da) is beyond the hardware mass range. In this paper, we demonstrate the wider applicability of poly(L-SUV) for the enantioseparation of acidic, cationic, and neutral pharmaceuticals, as well as other chiral molecules. Chiral separation was achieved for Tro¨ger’s base and binaphthyl, paveroline, and coumarin derivatives. Many factors, e.g., selectivity and capacity factors, mobility, and resolution, were investigated as a function of various pH and buffer conditions. EXPERIMENTAL SECTION Chemicals and Reagents. The monomeric carboxylic acid form of N-undecylenyl-L-valine (L-UV) was synthesized according to the procedure reported by Lapidot et al.30 The carboxylic acid form was then converted to the sodium salt form, L-SUV, by adding (17) Otsuka, K.; Terabe, S. Electrophoresis 1990, 11, 982-984. (18) Otsuka, K.; Kawahara, J.; Tatekawa, K.; Terabe, S. J. Chromatogr., A 1991, 559, 209-214. (19) Terabe, S.; Shibata, M.; Miyashita, Y. J. Chromatogr., A 1989, 480, 403411. (20) Cole, R. O.; Sepaniak, M. J.; Hinze, W. L. J. High Resolut. Chromatogr. 1990, 13, 579-582. (21) Ishihama, Y.; Terabe, S. J. Liq. Chromatogr. 1993, 16, 933-944. (22) Tickle, D. C.; Okafo, G. N.; Camilleri, P.; Jones, R. F. D.; Kirby, A. J. Anal. Chem. 1994, 66, 4121-4126. (23) Wang, J.; Warner, I. M. Anal. Chem. 1994, 66, 3773-3776. (24) Wang, J.; Warner, I. M. J. Chromatrogr., A 1995, 711, 297-304. (25) Dobashi, A.; Hamada, M.; Dobashi, Y. Anal. Chem. 1995, 67, 3011-3017. (26) Ozaki, H.; Ichihara, A.; Terabe, S. J. Chromatrogr., A 1995, 709, 3-10. (27) Ozaki, H.; Terabe, S.; Ichihara, A. J. Chromatrogr., A 1994, 680, 117-123. (28) Ozaki, H.; Itou, N.; Terabe, S.; Takada, T.; Sakairi, M.; Koizumi, H. J. Chromatrogr., A 1995, 716, 69-79. (29) Takada, Y.; Sakairi, M.; Koizumi, H. Rapid Commun. Mass Spectrom. 1995, 9, 488-490. (30) Lapidot, Y.; Rappoport, S.; Wolman, Y. J. Lipid Res. 1967, 8, 142-145.
an equal molar solution of sodium bicarbonate. This procedure for the polymerization and characterization of the surfactant and polymer was previously reported by our group.23,31 The analytes (()-1,1′-bi-2-naphthol (BINOL) (99%), (R)-(+)-1,1′-bi-2-naphthol [(R)-BINOL] (99%), (S)-(-)-1,1′-bi-2-naphthol [(S)-BINOL] (99%), (R)-(+)-1,1′-binaphthyl-2,2′-diamine [(R)-DABN] (99%), (S)-(-)1,1′-binaphthyl-2,2′-diamine [(S)-DABN] (99%), (()-laudanosine (99%), (()-laudanosoline hydrogen bromide trihydrate (98%), (()tetrahydropaveroline hydrogen bromide (norlaudanosoline) (98%), (()-warfarin (98%), D,L-3-(R-acetonyl-4-chlorobenzyl)-4-hydroxycoumarin (coumachlor) (98%), and Tro¨ger’s base (98%) were all purchased from Aldrich (Milwaukee, WI). The (+) and (-) Tro¨ger’s bases (99.5%) were purchased from Fluka (Ronkonkoma, NY). All compounds were used as received. The structures of these chiral analytes are provided in Figure 1. Capillary Electrophoresis. A Biofocus 3000 automated CE system (Bio-Rad Laboratories, Hercules, CA) with a multiwavelength UV absorbance detector was used for our MECC experiments. Separations were performed with uncoated fused-silica capillaries of 50 µm i.d. with a column length of 55 or 60 cm (45.5 or 55.5 cm to detector window, respectively) purchased from Polymicro Technologies (Phoenix, AZ). The paveroline experiment used a poly(vinyl alcohol) (PVA)-coated capillary that was 50 µm × 55 cm (45.5 cm effective length) and purchased from Hewlett-Packard (Wilmington, DE). The capillaries were thermostated at 25 °C with an aqueous coolant. Separations were accomplished by applying a constant voltage of +20 kV. An output wavelength of 280 nm was used for absorbance detection. Electrolyte and Standard Preparation. For all experiments, except the coumarin derivatives, the BGE consisted of 25 mM dibasic sodium phosphate. Before adjusting the pH, 0.25% (w/v) poly(L-SUV) was added to the BGE. The pH values of 7 and 8 were achieved by adding hydrochloric acid to the BGE. Sodium hydroxide was used to adjust to higher pH values (10 and 11), while no acid or base was added to the pH 9 buffer solution. In the acidic studies with coumarin derivatives, 0.5% (w/v) poly(LSUV) was added to 25 mM phosphate or acetate buffers and the pH was respectively adjusted with phosphoric acid or acetic acid. After the pH was adjusted, the running buffer was filtered through a 0.45 µm nylon filter (Nalgene, Rochester, NY) and then degassed by use of sonication prior to use. To ensure reproducibility, the capillary was purged with 0.1 N sodium hydroxide, followed by water, and then BGE for 2 min before each run. However, purging with sodium hydroxide was not performed at pH values lower than 7 to prevent pH hysteresis. Samples were prepared in methanol at concentrations between 0.1 and 0.5 mg/mL and were introduced into the anodic end of the capillary by applying 2 psi‚s pressure injections. RESULTS AND DISCUSSION Chiral Separation with Poly(L-SUV). Racemates elute simultaneously in CZE since they have the same charge-to-mass ratio, i.e., unless a chiral medium is introduced for chiral recognition. Chiral MECC using polymerized surfactants is based on the partitioning of an enantiomer between the surfactant polymer and the aqueous phase. Consequently, enantiomers will be chromatographically resolved by differential solubilization (interaction) between the micelle and BGE, as well as differences (31) Larrabee, C. E.; Sprague, E. D. J. Polym. Sci., Polym. Lett. Ed. 1979, 17, 749-751.
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
959
Figure 1. Chemical structures of chiral drugs and compounds.
in electrophoretic mobilities of the two phases.32 According to Wren and Rowe’s theory for chiral separation,33 the strength of interaction between each racemate and chiral surfactant depends on optimizing the concentration of the chiral selector. In our model, the amino acid on the polymerized surfactant, L-valinate, is the active site for chiral recognition. Valinate forms a diastereomeric complex with each of the optical antipodes of a racemic analyte. The formation constant for each complex depends on the following interaction:34 D,L-Rac
+ L-Sel f D,L-Prod + L,L-Prod
where Rac is the racemic analyte, L-Sel is poly(L-SUV), and the diastereomer product is denoted by Prod. Each enantiomer possesses a different capacity factor, k′, when complexed with a chiral micelle, according to an equation given by Terabe et al.,35 i.e.,
k′ )
tR - t0 t0(1 - tR/tmc)
(1)
where t0, tR, and tmc are the migration times of the unretained species, the enantiomer, and the micelle, respectively. However, due to the limited elution range inherent to MECC, the term (1 - tR/tmc) is negligible as tmc approaches infinity.36 Equation 1 will then be reduced to a fundamental equation of chromatography, i.e. (32) Terabe, S. Trends Anal. Chem. 1989, 8, 129-134. (33) Wren, S. A. C.; Rowe, R. C. J. Chromatrogr., A 1992, 603, 235-241. (34) Snopek, J.; Jelı´nek, I.; Smolkova´-Keulemansova´, E. J. Chromatogr., A 1992, 609, 1-17. (35) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-113. (36) Camilleri, P., Ed. Capillary Electrophoresis: Theory and Practice; CRC: Boca Raton, FL, 1993.
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k′ ) (tR - t0)/t0
(2)
where t0 can be determined by injecting the hydrophilic solute, methanol, which moves at the electroosmotic flow (EOF) rate. Enantioseparation of Atropisomeric Racemic Mixtures. Atropisomerism occurs in compounds possessing a chiral plane with restricted rotation around a central bond because of molecular rigidity and steric hindrance.37 Unlike conventional chiral compounds, the atropisomeric compounds, such as BINOL, DABN, and Tro¨ger’s base, have chirality about an asymmetrical plane instead of an asymmetrical carbon center. As an optically active host, BINOL has been used for efficient optical resolution of guest compounds by complex formation.38 Chiral recognition of the guest molecules in solution was detected by 1H nuclear magnetic resonance (NMR) spectroscopy. This atropisomer was also used as a chiral shift reagent for determining the enantiomeric purity of a wide variety of organic compounds.39 Tro¨ger’s base, a chiral solvating agent, is a chiral heterocyclic amine whose chirality is due to the presence of two stereogenic nitrogen atoms.40 Molecular rigidity and C2 symmetry are characteristics necessary for its incorporation into biomimic systems and inclusion formation for molecular recognition.41 As previously reported,23 the weight fraction of poly(L-SUV) required for baseline separation of BINOL fell in the range of 0.20.5% (w/v). In recent studies, we could separate racemic mixtures of BINOL with DABN and Tro¨ger’s base using an optimized concentration of 0.25% polymer as illustrated in Figure 2. Under neutral and alkaline conditions, these compounds are electrically neutral, except BINOL, which is partially anionic at pH 10 and 11. It is interesting to note the elution order obtained over this (37) Allenmark, S. Chromatographic Enantioseparation: Methods and Applications, 2nd ed.; Ellis Horwood: New York, 1991. (38) Toda, F. Top. Curr. Chem. 1987, 140, 43-69. (39) Toda, F.; Mori, K.; Okada, J.; Node, M.; Itoh, A.; Oomine, K.; Fuji, K. Chem. Lett. 1988, 131-134. (40) Wilen, S. H.; Qi, J. Z., Williard, P. G. J. Org. Chem. 1991, 56, 485-487. (41) Katz, H. E. J. Chem. Soc., Chem. Commun. 1990, 126-127.
Figure 2. Chiral separation of (1) (-)-Tro¨ ger’s base, (2) (+)-Tro¨ ger’s base, (3) (S)-DABN, (4) (R)-DABN, (5) (S)-BINOL, and (6) (R)-BINOL at (a) and (b) pH 9, (c) pH 10, and (d) pH 11. CE conditions: buffer, 0.25% poly(L-SUV) with 25 mM dibasic phosphate; capillary, 50 µm × 60 cm (55.5 cm effective length); applied voltage, +20 kV; detection, 280 nm.
Figure 3. Effect of pH on resolution of the enantiomeric mixtures of (b) BINOL, ([) DABN, and (9) Tro¨ ger’s base. CE conditions: same as Figure 2.
Figure 4. Effect of pH on selectivity and effective mobility (inset) of the enantiomeric mixtures of (b) BINOL, ([) DABN, and (9) Tro¨ ger’s base. CE conditions: same as Figure 2.
pH range where BINOL shows a gradual decrease in retention time relative to DABN. Its increasing anionic character causes a decrease in binding with the anionic polymerized surfactant. This ionic repulsion results in the earlier elution of BINOL at pH 11. However, under all pH conditions, the (S)-(-) enantiomers eluted faster than its corresponding (R)-(+) form. Thus, the migration times and order of enantiomer elution are a direct indication of the analyte/micelle association.
The resolution (Rs), selectivity factors (R), and effective mobilities of BINOL, DABN, and Tro¨ger’s base in the pH range from 8 to 11 are depicted in Figures 3 and 4. Baseline resolution (Rs > 1.5) of BINOL and DABN was achieved at pH values as low as 6. Relative to Tro¨ger’s base at pH 9, the more hydrophobic binaphthyl compounds were retained longer; hence, chiral recognition was enhanced. Also, the hydroxyl groups on BINOL probably could hydrogen bond more strongly to the micelle than Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
961
the neutral amine groups on DABN and Tro¨ger’s base. The maximum resolutions were obtained at pH 10 for all three atropisomeric compounds. Thus, the selectivity factors slightly decreased until a plateau was reached over the entire pH range. It seems that the larger the mobility, shown in the inset of Figure 4, the less interactions of the racemate with the micelle. Neutral compounds separated with anionic micelles will have negative effective mobility values because the analytes migrate after the EOF. Initially, as the pH was increased from 8 to 10, complexation between poly(L-SUV) and the two atropisomers (DABN, Tro¨ger’s base) caused a slight decrease in electrophoretic mobility resulting in enhanced resolution. However, Tro¨ger’s base had higher mobilities than BINOL and DABN under all pH conditions. The racemic mixture of BINOL had a lower mobility than DABN except at pH 11 where it migrated faster. The sharp increase in mobility of BINOL might be due to ionization of the hydroxyl groups, which should decrease binding with the anionic polymerized surfactant because of charge repulsion. These data suggest that the ionization of this analyte inhibits binding with the anionic micelle but does not necessarily decrease enantiomeric resolution. In fact, interactions of the racemate with the core of the polymerized surfactant appear to be the driving force for this chiral recognition. Furthermore, because of the increase in the EOF at pH values above 10, the electrophoretic mobilities for all three enantiomers increased. Effect of Anionic Coumarin Drugs at Low pH. Warfarin is a coumarinic anticoagulant drug frequently used in the treatment of thromboembolic diseases. Although sold as a racemic mixture, it is well-known that the (S)-(-) enantiomer is more pharmacologically active than its corresponding (R)-(+) form.42 This drug displays a stereoselective metabolism and pharmacokinetics where each enantiomer follows different metabolic pathways.43 Coumachlor, an analog of warfarin, has been used in HPLC as an internal standard. Qualitative and quantitative experiments using both drugs have been documented by use of HPLC and GC.44,45 Warfarin and coumachlor are structurally related acidic drugs. They are both electronegative due to their keto-enol groups. The phenolic group on warfarin has a pKa of 5.1.46 Theoretically, both drugs were not expected to complex strongly to the anionic micelle under neutral and basic pH conditions. Ideal conditions for our model would be very acidic buffer solutions in order to increase the elution window. Acidic conditions will also increase the positive charge on the amide group on the micelle and decrease the negative charges on the valinate groups. However, at pH values below 5.5, the micelle tended to precipitate out of solution due to a decrease in the ionization of the carboxylate functionality of poly(L-SUV). In an attempt to optimize the chiral resolution of coumachlor and warfarin, four different buffers in the acidic pH range from 5.5 to 6.5 were tested with poly(L-SUV). The electrophoretic data using the four buffers are summarized in Table 1. The optimum weight fraction of poly(L-SUV) required to achieve the highest resolution of these two drugs was determined to be 0.5% (w/v, (42) D’Hulst, A.; Verbeke, N. Chirality 1994, 6, 225-229. (43) Lewis, R. J.; Trager, W. F.; Chan, K. K.; Breckenridge, A.; Orme, M.; Roland, M.; Schary, W. J. Clin. Invest. 1974, 53, 1607-1617. (44) Armstrong, D. W.; Tang, Y.; Ward, T.; Nichols, M. Anal. Chem. 1993, 65, 1114-1117. (45) DeVries, J. X.; Schmitz-Kummer, E. J. Chromatogr., A 1993, 644, 315320. (46) Hiskey, C. F.; Bulloch, E.; Whitman, C. J. Pharm. Sci. 1962, 51, 43-46.
962 Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
Table 1. Migration Times, Capacity and Selectivity Factors, and Resolution of Racemic Mixtures of Warfarin and Coumachlor with Different Buffersa warfarin b
b
coumachlor b
buffer (pH)
tR1 tR2
k1 k2
R Rs
tR1 tR2b
k1 k2
R Rs
NaH2PO4 (5.92)
21.50 21.98
1.54 1.59
1.03 1.10
29.69 30.28
2.52 2.59
1.03 0.99
acetate (5.56)
16.57 16.95
1.30 1.35
1.04 0.73
35.62 37.06
4.05 4.26
1.05 1.10
Na2HPO4 (5.80)
19.50 19.78
1.77 1.85
1.02 0.55
25.81 26.26
3.00 3.10
1.02 0.96
NaH2PO4 (6.50)
19.70 19.82
1.30 1.32
1.01 0.27
21.77 22.04
1.55 1.58
1.02 0.57
a CE conditions: 25 mM buffer, 0.5% poly(L-SUV); capillary, 50 µm × 60 cm, 55.5 cm effective length; applied voltage, +20 kV; detection, 280 nm. b tR in minutes.
Figure 5. Chiral separation of enantiomeric mixtures of warfarin (peaks 1) and coumachlor (peaks 2) at (a) pH 5.6 and (b) pH 6.5. CE conditions: same as Figure 2 except buffer, 0.5% poly(L-SUV) with 25 mM monobasic phosphate; capillary, 50 µm × 55 cm (50.5 cm effective length).
data not shown). The deterioration in separation as the pH was increased from 5.6 to 6.5 is shown in Figure 5. Enantioseparation above pH 6.5 was not observed. Although the acetate buffer had the lowest current and shortest retention times, separation was poorer than with the monobasic phosphate buffer. In all cases, baseline resolution was not obtained with any of the buffers at pH values greater than 5.5. The more polar coumachlor eluted after warfarin because it seemed to interact more strongly with the micelle. Overall, higher resolutions were achieved with coumachlor, except with the monobasic phosphate buffer at pH 5.92, where warfarin was better resolved. Coumachlor appears to have a lower electrophoretic mobility toward the cathode and a larger electronegative charge because of the chlorine group. The better enantioseparation of coumachlor is probably due to the importance of the chiral center being located between the aromatic moiety and the negative charge on the racemate.47 However, under alkaline conditions, the electrostatic repulsions between both enantiomers and the micelle are likely too great to allow chiral recognition. Otsuka et al.18 were able to separate warfarin under basic conditions, pH 9.0, only with the use of the mobile phase additives (47) Kowblansky, M. Macromolecules 1985, 18, 1776-1779.
Table 2. Migration Times, Capacity and Selectivity Factors, and Resolution of Racemic Mixtures of Laudanosoline, Norlaudanosoline, and Laudanosinea laudanosoline pH 5.6c 5.9d 7d 9e 11e
tR1b tR2b
k1′ k2′
15.39 16.12 14.04 14.79 16.61 17.57 6.23 6.50 8.27 9.59
0.45 0.52 1.17 1.28 1.68 1.84 0.34 0.40 0.66 0.93
norlaudanosoline
R Rs
tR1b tR2b
k1′ k2′
R Rs
1.16 0.78 1.10 1.09 1.06 0.64 1.04 0.95 1.16 1.07
17.12 19.68 15.51 16.14 18.12 20.58 6.31 6.45 7.98 9.95
0.62 0.86 1.39 1.49 1.91 2.31 0.37 0.40 0.63 1.03
1.39 1.78 1.07 0.97 1.14 0.83 1.02 0.49 1.25 0.95
laudanosine tR1b tR2b
k1′ k2′
23.51
1.22
17.07 18.33 19.40
1.63 1.83 2.20
7.73
0.67
7.17 7.31
0.49 0.52
R Rs
1.12 0.97
1.02 0.49
a CE conditions: buffer, 25 mM monobasic phosphate with 0.25% poly(L-SUV); applied voltage, +25 kV; detection, 280 nm. b tR in minutes. c PVA-coated capillary, 50 µm × 55 cm (50.5 cm effective length). d Uncoated capillary, 50 µm × 55 cm (50.5 cm effective length). e Uncoated capillary, 50 µm × 60 cm (55.5 cm effective length).
sodium dodecyl sulfate (SDS), methanol, and urea with SDVal. The SDS was necessary in enantioseparation because it seemed to enhance warfarin’s selectivity toward the micelle. The addition of urea increased the solubility of warfarin while methanol was needed to further increase selectivity and improve resolution. Experiments are currently being investigated to determine whether poly(L-SUV) will separate warfarin and coumachlor in alkaline conditions in the presence of nonchiral additives. If enantioseparation does occur under these conditions, then electrostatic repulsion between the acidic racemates and anionic micelles is not a major factor in enantioseparation. Thus, chiral recognition is not totally dependent on the strong ionic complexations with the micelle. Enantioseparation of Cationic Paveroline Drugs. Laudanosine, a cationic biosynthetic precursor of morphine, and its derivatives, laudanosoline and norlaudanosoline, were separated with our polymerized surfactant. The electrophoretic results are summarized in Table 2. Under neutral and alkaline conditions, laudanosine was only separated at pH 11. Better resolution was obtained (Rs ) 1.2) with 0.5% poly(L-SUV) at pH 11;23 however, it is at pH 11 where the analyte will be neutral. Laudanosoline and norlaudanosoline were partially resolved at higher pH values. The elution order was laudanosoline > norlaudanosoline > laudanosine. The amine on the paveroline derivatives appears to be important in interactions with the micelle. It was expected that laudanosine would migrate faster than its derivatives since it is a larger cationic species; however, the opposite occurred, probably because it was the most hydrophobic molecule. This higher hydrophobicity allows laudanosine to interact more with the inner core of the micelle. For this analyte, the longer interaction with the micelle did not result in better resolution or selectivity. The best data were attained with laudanosoline at higher pH values. Norlaudanosoline elutes after laudanosoline because of the electrostatic attraction and hydrogen bonding between the secondary amine on the analyte and the carboxylate group on the micelle. Although all of the paveroline derivatives studied are cationic, they do not elute before the EOF. This is indicative of the cationic species binding to the anionic micelle and migrating toward the anode. Overall, the effective mobility of laudanosoline and norlaudanosoline, depicted in Figure 6, increased at pH 9 and had
Figure 6. Effect of pH on effective mobility of (b) laudanosoline, ([) norlaudanosoline, and (9) laudanosine. CE conditions: same as Figure 2 except capillary, 50 µm × 55 cm (50.5 cm effective length) at pH 7.
Figure 7. Enantioseparation of paveroline derivatives using (a) uncoated silica capillary at pH 6 and (b) PVA-coated capillary at pH 5.6. Peaks: (1) laudanosoline, (2) norlaudanosoline, and (3) laudanosine. CE conditions: same as Figure 2 except capillary, 50 µm × 55 cm (50.5 cm effective length).
the lowest values at pH 11. The change in mobility was on the order of 1 × 10-4 cm2 V-1 s-1 for laudanosoline and norlaudanosoline. This decrease in mobility signifies that the enantiomers are interacting less with the micelle at higher pH values. The opposite effect was observed with laudanosine, which shows a trend of increasing mobility with an increase in pH. We found that the retention times were not always reproducible with poly(L-SUV) at pH values below 7. Similar problems were observed by Otsuka and Terabe16 using SDVal. Four reasons may contribute to these instabilities in separation, the first being the tighter conformation of the polymerized surfactant in acidic media. This could occur if the carboxylic groups on the polymer become protonated. Second, pH hysteresis may exist with the silanol groups within the capillary walls, causing an unpredictable EOF. The next possibility may be electrophoretic dispersion among the polymerized surfactants. Last, the hydrophilic head groups on the micelle may interact with the capillary via hydrogen bonding leading to polymer adsorption on the capillary. Once bound to the capillary walls, the micelle will not strongly complex with the analytes. A PVA-coated capillary was used to decrease micelle adsorption and diminish capillary wall interaction with the polymerized surfactant. Figure 7 depicts a comparison of the paveroline Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
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derivative mixture at pH 5.6 and 6 with a coated and uncoated capillary, respectively. The selectivity and resolution of norlaudanosoline, listed in Table 2, were enhanced with the coated capillary. Enantioseparation could not be achieved with laudanosine. Experiments are being conducted to further elucidate this anomalous behavior of laudanosine on coated capillaries. CONCLUSION MECC has proven to be an important tool to the pharmaceutical industry for chiral separation of drugs. The polymerized anionic surfactant poly(L-SUV) has demonstrated great versatility in enantioseparation with a variety of cationic, anionic, and neutral compounds under moderately acidic, neutral, and basic BGE conditions. Separation was found to be dependent on the racemates having bulky ring structures at or around their chiral centers. All of the enantiomers separated also had polar groups that seemed to be necessary for hydrogen bonding with L-valinate
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on the micelle. Negative electrophoretic mobilities confirmed enantiomer association with the anionic micelle. In all cases, hydrophobic and electrostatic interactions as well as hydrogen bonding appear to be important for chiral recognition. ACKNOWLEDGMENT This work was supported through a grant from the National Institutes of Health (GM39844). I.M.W. also acknowledges the Philip W. West endowment for partial support of this research. K.A.A.-H acknowledges the Louisiana Board of Reagents for a fellowship award in support of this research. Received for review August 1, 1996. Accepted December 16, 1996.X AC960778W X
Abstract published in Advance ACS Abstracts, February 1, 1997.
