Studies of Polymerized SodiumN-Undecylenyl-L-valinate in Chiral MicellarElectrokinetic Capillary Chromatography ofNeutral, Acidic, and Basic CompoundsKimberly A. Agnew-Heard, Montserrat Sanchez Pena, Shahab A. Shamsi, and Isiah M. Warner*

Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803

The polymerized surfactant poly(sodium N-undecylenylamino L-valinate) [poly(L-SUV)] has been used in micellarelectrokinetic capillary chromatography for the chiralseparation of various acidic and basic drugs, as well asneutral compounds. Under the conditions studied, poly-(L-SUV) was shown to be a very versatile anionic chiralselector in the pH range of 5.6-11. The micelle was usedfor the enantioseparation of coumarinic anticoagulantdrugs with various buffers under moderately acidic condi-tions. Neutral and alkaline buffer conditions were usedto successfully separate the neutral atropisomers (()-1,1′-bi-2-naphthol, (()-1,1′-binaphthyl-2,2′-diamine, and Tro1g-er’s base. Chiral separation of the cationic paverolinedrugs, laudanosine, norlaudanosoline, and laudanosoline,was influenced by pH and the use of coated capillaries.The acquired data focused on optimizing the migrationtimes, capacity and separation factors, and electrophoreticmobilities of the various racemic mixtures.

The enantioselectivity of chiral compounds is important to theenvironmental and biological fields, as well as to syntheticchemists and the pharmaceutical industry. Our studies here focuson the pharmaceutical industry, where many chiral drugs aregenerated and sold as racemates. Since the emergence of strictFood and Drug Administration (FDA) policies, separating indi-vidual enantiomers has become necessary to the drug industry.1

These FDA regulations specify that the stereoisomeric composi-tion of a drug with a chiral center should be studied for itsbiological activities and toxicological effects.2 One reason for thismandate is that each enantiomer of a drug may exhibit stereose-lectivity in its pharmacological function. As a result, one opticalantipode may produce the desired physiological response, whilethe other may be toxic or inactive.3 It is also necessary todetermine whether each antipode is critical to the desiredpharmacokinetic activity. Many drug companies now seek tomarket chiral drugs as single enantiomers because of thisheightened concern with consumer safety and federal regulations.

In the past, gas chromatography (GC) and high-performanceliquid chromatography (HPLC) were the two most prominenttechniques used for enantiomeric separations. By combining thehigh efficiency of GC with the selectivity of HPLC, capillaryelectrophoresis (CE) has emerged as a technique that provides a

larger application range for enantioseparation.4 Other advantagesof CE include higher separation efficiency and resolution, fasteranalysis times, and smaller sample volumes.5 One of the mostvaluable 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 isneeded for enantioseparation with most CE instruments.

Variations of the basic mode of CE, e.g., capillary zoneelectrophoresis (CZE), are often used to achieve chiral separa-tions. Direct chiral separation in CE is frequently achieved eitherthrough using an immobilized chiral phase, i.e., capillary gelelectrophoresis,6 or the addition of chiral selectors as mobile phaseadditives. The most common chiral mobile phase selectorsinclude host-guest additives, such as cyclodextrins (CDs)7 andcrown ethers.8 Another example is the N-acylcalix[4]arene aminoacid derivatives recently synthesized by our research group.9

Other background electrolyte (BGE) additives used for enanti-oseparation include chiral ligand exchange reagents10,11 and thenewly introduced macrocyclic antibiotics,12 heparin,13-15 anddextran sulfates.15

In recent years, increased interest has been shown in the useof chiral surfactants for the micellar electrokinetic capillarychromatography (MECC) approach to enantioseparation. Thistechnique involves the addition of a chiral surfactant into the BGEat a concentration above the critical micelle concentration (cmc).Enantioselectivity is achieved by differential interaction of eachenantiomer with the chiral micelle. The utility of the chiral MECCapproach has been demonstrated using sodium N-dodecanoyl-L-valinate (SDVal),16-18 bile salts,19,20 digitonin,16 saponins,21 and

(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.

(4) Krstulovic, A. M., Ed. Chiral Separations by HPLC: Applications to Pharma-ceutical 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) Sanchez Pena, M.; Zhang, Y.; Thibodeaux, S.; McLaughlin, M. L.; de la Pena,

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, 1690-

1695.(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, 2334-

2341.(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.

Anal. Chem. 1997, 69, 958-964

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glucopyranoside-based phosphate and sulfate surfactants.22 In allof these studies, monomeric chiral surfactants were added to theBGE above their cmc. One problem with MECC is that chiralseparation may be compromised by the dynamic equilibrium thatexists between the surfactant monomer and micelle.23 In addition,micelles are species with equilibrium mixtures of monomers andspecies containing monomers well above the average aggregationnumber. It is well established that such polydispersity isdetrimental to chromatographic separations. To eliminate theseproblems, polymerized surfactants were introduced for chiralseparation. As first reported by Wang and Warner, poly(sodiumN-undecylenyl-L-valinate) [poly(L-SUV)] may be used for theoptical resolution of (()-1,1′-bi-2-naphthol and D,L-laudanosine withand without γ-CD.23,24 Lower concentrations than the cmc of thispolymerized surfactant were added to the BGE since the surfac-tants are covalently linked and the surfactant monomers have beenremoved. Thus, the polymer is more stable and can withstandhigh concentrations of organic solvents. The same polymer, poly-(sodium 10-undecenoyl-L-valinate), was employed for the enanti-oseparation of 3,5-dinitrobenzoyl amino acid isopropyl esters.25

Similar to polymerized surfactants, the high molecular masssurfactant butyl acrylate-butyl methacrylate-methacrylate acidcopolymer sodium salt (BBMA) was used to separate chiral26 andachiral27-29 compounds with and without CD-modified MECC. Thecmc of this surfactant was determined to be effectively zero;therefore, BBMA could be used at lower concentrations as apseudostationary phase with MECC.27 Ozaki et al. reported theon-line coupling of MECC with electrospray ionization interfaced-mass spectrometry (ESI-MS) detection. This coupling is advanta-geous over UV absorbance detection because structural informa-tion 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 neutralpharmaceuticals, as well as other chiral molecules. Chiral separa-tion was achieved for Troger’s base and binaphthyl, paveroline,and coumarin derivatives. Many factors, e.g., selectivity andcapacity factors, mobility, and resolution, were investigated as afunction of various pH and buffer conditions.

EXPERIMENTAL SECTIONChemicals and Reagents. The monomeric carboxylic acid

form of N-undecylenyl-L-valine (L-UV) was synthesized accordingto the procedure reported by Lapidot et al.30 The carboxylic acidform was then converted to the sodium salt form, L-SUV, by adding

an equal molar solution of sodium bicarbonate. This procedurefor the polymerization and characterization of the surfactant andpolymer 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-hydroxy-coumarin (coumachlor) (98%), and Troger’s base (98%) were allpurchased from Aldrich (Milwaukee, WI). The (+) and (-)Troger’s bases (99.5%) were purchased from Fluka (Ronkonkoma,NY). All compounds were used as received. The structures ofthese chiral analytes are provided in Figure 1.

Capillary Electrophoresis. A Biofocus 3000 automated CEsystem (Bio-Rad Laboratories, Hercules, CA) with a multiwave-length UV absorbance detector was used for our MECC experi-ments. Separations were performed with uncoated fused-silicacapillaries of 50 µm i.d. with a column length of 55 or 60 cm (45.5or 55.5 cm to detector window, respectively) purchased fromPolymicro Technologies (Phoenix, AZ). The paveroline experi-ment used a poly(vinyl alcohol) (PVA)-coated capillary that was50 µm × 55 cm (45.5 cm effective length) and purchased fromHewlett-Packard (Wilmington, DE). The capillaries were ther-mostated at 25 °C with an aqueous coolant. Separations wereaccomplished by applying a constant voltage of +20 kV. An outputwavelength of 280 nm was used for absorbance detection.

Electrolyte and Standard Preparation. For all experiments,except the coumarin derivatives, the BGE consisted of 25 mMdibasic sodium phosphate. Before adjusting the pH, 0.25% (w/v)poly(L-SUV) was added to the BGE. The pH values of 7 and 8were achieved by adding hydrochloric acid to the BGE. Sodiumhydroxide was used to adjust to higher pH values (10 and 11),while no acid or base was added to the pH 9 buffer solution. Inthe acidic studies with coumarin derivatives, 0.5% (w/v) poly(L-SUV) was added to 25 mM phosphate or acetate buffers and thepH was respectively adjusted with phosphoric acid or acetic acid.After the pH was adjusted, the running buffer was filtered througha 0.45 µm nylon filter (Nalgene, Rochester, NY) and then degassedby use of sonication prior to use. To ensure reproducibility, thecapillary was purged with 0.1 N sodium hydroxide, followed bywater, and then BGE for 2 min before each run. However, purgingwith sodium hydroxide was not performed at pH values lowerthan 7 to prevent pH hysteresis. Samples were prepared inmethanol at concentrations between 0.1 and 0.5 mg/mL and wereintroduced into the anodic end of the capillary by applying 2 psi‚spressure injections.

RESULTS AND DISCUSSIONChiral Separation with Poly(L-SUV). Racemates elute

simultaneously in CZE since they have the same charge-to-massratio, i.e., unless a chiral medium is introduced for chiralrecognition. Chiral MECC using polymerized surfactants is basedon the partitioning of an enantiomer between the surfactantpolymer and the aqueous phase. Consequently, enantiomers willbe chromatographically resolved by differential solubilization(interaction) between the micelle and BGE, as well as differences

(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, 403-

411.(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.

(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

in electrophoretic mobilities of the two phases.32 According toWren and Rowe’s theory for chiral separation,33 the strength ofinteraction between each racemate and chiral surfactant dependson optimizing the concentration of the chiral selector. In ourmodel, the amino acid on the polymerized surfactant, L-valinate,is the active site for chiral recognition. Valinate forms a diaster-eomeric complex with each of the optical antipodes of a racemicanalyte. The formation constant for each complex depends onthe following interaction:34

where Rac is the racemic analyte, L-Sel is poly(L-SUV), and thediastereomer product is denoted by Prod.

Each enantiomer possesses a different capacity factor, k′, whencomplexed with a chiral micelle, according to an equation givenby Terabe et al.,35 i.e.,

where t0, tR, and tmc are the migration times of the unretainedspecies, 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 willthen be reduced to a fundamental equation of chromatography,i.e.

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 planewith restricted rotation around a central bond because of molec-ular rigidity and steric hindrance.37 Unlike conventional chiralcompounds, the atropisomeric compounds, such as BINOL,DABN, and Troger’s base, have chirality about an asymmetricalplane instead of an asymmetrical carbon center. As an opticallyactive host, BINOL has been used for efficient optical resolutionof guest compounds by complex formation.38 Chiral recognitionof the guest molecules in solution was detected by 1H nuclearmagnetic resonance (NMR) spectroscopy. This atropisomer wasalso used as a chiral shift reagent for determining the enantiomericpurity of a wide variety of organic compounds.39 Troger’s base,a chiral solvating agent, is a chiral heterocyclic amine whosechirality is due to the presence of two stereogenic nitrogenatoms.40 Molecular rigidity and C2 symmetry are characteristicsnecessary for its incorporation into biomimic systems and inclu-sion 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 0.2-0.5% (w/v). In recent studies, we could separate racemic mixturesof BINOL with DABN and Troger’s base using an optimizedconcentration of 0.25% polymer as illustrated in Figure 2. Underneutral and alkaline conditions, these compounds are electricallyneutral, except BINOL, which is partially anionic at pH 10 and11. It is interesting to note the elution order obtained over this

(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.; Jelınek, 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.

(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 1. Chemical structures of chiral drugs and compounds.

D,L-Rac + L-Sel f D,L-Prod + L,L-Prod

k′ )tR - t0

t0(1 - tR/tmc)(1)

k′ ) (tR - t0)/t0 (2)

960 Analytical Chemistry, Vol. 69, No. 5, March 1, 1997

pH range where BINOL shows a gradual decrease in retentiontime relative to DABN. Its increasing anionic character causes adecrease in binding with the anionic polymerized surfactant. Thisionic repulsion results in the earlier elution of BINOL at pH 11.However, under all pH conditions, the (S)-(-) enantiomers elutedfaster than its corresponding (R)-(+) form. Thus, the migrationtimes and order of enantiomer elution are a direct indication ofthe analyte/micelle association.

The resolution (Rs), selectivity factors (R), and effectivemobilities of BINOL, DABN, and Troger’s base in the pH rangefrom 8 to 11 are depicted in Figures 3 and 4. Baseline resolution(Rs > 1.5) of BINOL and DABN was achieved at pH values aslow as 6. Relative to Troger’s base at pH 9, the more hydrophobicbinaphthyl compounds were retained longer; hence, chiral rec-ognition was enhanced. Also, the hydroxyl groups on BINOLprobably could hydrogen bond more strongly to the micelle than

Figure 2. Chiral separation of (1) (-)-Troger’s base, (2) (+)-Troger’s base, (3) (S)-DABN, (4) (R)-DABN, (5) (S)-BINOL, and (6) (R)-BINOLat (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 × 60cm (55.5 cm effective length); applied voltage, +20 kV; detection, 280 nm.

Figure 3. Effect of pH on resolution of the enantiomeric mixturesof (b) BINOL, ([) DABN, and (9) Troger’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)Troger’s base. CE conditions: same as Figure 2.

Analytical Chemistry, Vol. 69, No. 5, March 1, 1997 961

the neutral amine groups on DABN and Troger’s base. Themaximum resolutions were obtained at pH 10 for all threeatropisomeric compounds. Thus, the selectivity factors slightlydecreased until a plateau was reached over the entire pH range.

It seems that the larger the mobility, shown in the inset ofFigure 4, the less interactions of the racemate with the micelle.Neutral compounds separated with anionic micelles will havenegative effective mobility values because the analytes migrateafter the EOF. Initially, as the pH was increased from 8 to 10,complexation between poly(L-SUV) and the two atropisomers(DABN, Troger’s base) caused a slight decrease in electrophoreticmobility resulting in enhanced resolution. However, Troger’s basehad higher mobilities than BINOL and DABN under all pHconditions. The racemic mixture of BINOL had a lower mobilitythan DABN except at pH 11 where it migrated faster. The sharpincrease in mobility of BINOL might be due to ionization of thehydroxyl groups, which should decrease binding with the anionicpolymerized surfactant because of charge repulsion. These datasuggest that the ionization of this analyte inhibits binding withthe anionic micelle but does not necessarily decrease enantiomericresolution. In fact, interactions of the racemate with the core ofthe polymerized surfactant appear to be the driving force for thischiral recognition. Furthermore, because of the increase in theEOF at pH values above 10, the electrophoretic mobilities for allthree enantiomers increased.

Effect of Anionic Coumarin Drugs at Low pH. Warfarin isa coumarinic anticoagulant drug frequently used in the treatmentof thromboembolic diseases. Although sold as a racemic mixture,it is well-known that the (S)-(-) enantiomer is more pharmaco-logically active than its corresponding (R)-(+) form.42 This drugdisplays a stereoselective metabolism and pharmacokinetics whereeach enantiomer follows different metabolic pathways.43 Cou-machlor, an analog of warfarin, has been used in HPLC as aninternal standard. Qualitative and quantitative experiments usingboth 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. Thephenolic group on warfarin has a pKa of 5.1.46 Theoretically, bothdrugs were not expected to complex strongly to the anionicmicelle under neutral and basic pH conditions. Ideal conditionsfor our model would be very acidic buffer solutions in order toincrease the elution window. Acidic conditions will also increasethe positive charge on the amide group on the micelle anddecrease the negative charges on the valinate groups. However,at pH values below 5.5, the micelle tended to precipitate out ofsolution due to a decrease in the ionization of the carboxylatefunctionality of poly(L-SUV).

In an attempt to optimize the chiral resolution of coumachlorand warfarin, four different buffers in the acidic pH range from5.5 to 6.5 were tested with poly(L-SUV). The electrophoretic datausing the four buffers are summarized in Table 1. The optimumweight fraction of poly(L-SUV) required to achieve the highestresolution of these two drugs was determined to be 0.5% (w/v,

data not shown). The deterioration in separation as the pH wasincreased from 5.6 to 6.5 is shown in Figure 5. Enantioseparationabove pH 6.5 was not observed. Although the acetate buffer hadthe lowest current and shortest retention times, separation waspoorer than with the monobasic phosphate buffer. In all cases,baseline resolution was not obtained with any of the buffers atpH values greater than 5.5. The more polar coumachlor elutedafter warfarin because it seemed to interact more strongly withthe micelle. Overall, higher resolutions were achieved withcoumachlor, except with the monobasic phosphate buffer at pH5.92, where warfarin was better resolved. Coumachlor appearsto have a lower electrophoretic mobility toward the cathode anda larger electronegative charge because of the chlorine group.The better enantioseparation of coumachlor is probably due tothe importance of the chiral center being located between thearomatic moiety and the negative charge on the racemate.47

However, under alkaline conditions, the electrostatic repulsionsbetween both enantiomers and the micelle are likely too great toallow chiral recognition.

Otsuka et al.18 were able to separate warfarin under basicconditions, pH 9.0, only with the use of the mobile phase additives

(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, 315-

320.(46) Hiskey, C. F.; Bulloch, E.; Whitman, C. J. Pharm. Sci. 1962, 51, 43-46. (47) Kowblansky, M. Macromolecules 1985, 18, 1776-1779.

Table 1. Migration Times, Capacity and SelectivityFactors, and Resolution of Racemic Mixtures ofWarfarin and Coumachlor with Different Buffersa

warfarin coumachlor

buffer (pH) tR1b tR2

b k1 k2 R Rs tR1b tR2

b k1 k2 R Rs

NaH2PO4 21.50 1.54 1.03 29.69 2.52 1.03(5.92) 21.98 1.59 1.10 30.28 2.59 0.99

acetate 16.57 1.30 1.04 35.62 4.05 1.05(5.56) 16.95 1.35 0.73 37.06 4.26 1.10

Na2HPO4 19.50 1.77 1.02 25.81 3.00 1.02(5.80) 19.78 1.85 0.55 26.26 3.10 0.96

NaH2PO4 19.70 1.30 1.01 21.77 1.55 1.02(6.50) 19.82 1.32 0.27 22.04 1.58 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 (50.5cm effective length).

962 Analytical Chemistry, Vol. 69, No. 5, March 1, 1997

sodium dodecyl sulfate (SDS), methanol, and urea with SDVal.The SDS was necessary in enantioseparation because it seemedto enhance warfarin’s selectivity toward the micelle. The additionof urea increased the solubility of warfarin while methanol wasneeded to further increase selectivity and improve resolution.Experiments are currently being investigated to determine whetherpoly(L-SUV) will separate warfarin and coumachlor in alkalineconditions in the presence of nonchiral additives. If enantiosepa-ration does occur under these conditions, then electrostaticrepulsion between the acidic racemates and anionic micelles isnot a major factor in enantioseparation. Thus, chiral recognitionis not totally dependent on the strong ionic complexations withthe micelle.

Enantioseparation of Cationic Paveroline Drugs.Laudanosine, a cationic biosynthetic precursor of morphine, andits derivatives, laudanosoline and norlaudanosoline, were separatedwith our polymerized surfactant. The electrophoretic results aresummarized in Table 2. Under neutral and alkaline conditions,laudanosine was only separated at pH 11. Better resolution wasobtained (Rs ) 1.2) with 0.5% poly(L-SUV) at pH 11;23 however, itis at pH 11 where the analyte will be neutral. Laudanosoline andnorlaudanosoline were partially resolved at higher pH values. Theelution order was laudanosoline > norlaudanosoline > laudanosine.The amine on the paveroline derivatives appears to be importantin interactions with the micelle. It was expected that laudanosinewould migrate faster than its derivatives since it is a larger cationicspecies; however, the opposite occurred, probably because it wasthe most hydrophobic molecule. This higher hydrophobicityallows laudanosine to interact more with the inner core of themicelle. For this analyte, the longer interaction with the micelledid not result in better resolution or selectivity. The best datawere attained with laudanosoline at higher pH values. Nor-laudanosoline elutes after laudanosoline because of the electro-static attraction and hydrogen bonding between the secondaryamine on the analyte and the carboxylate group on the micelle.Although all of the paveroline derivatives studied are cationic, theydo not elute before the EOF. This is indicative of the cationicspecies binding to the anionic micelle and migrating toward theanode. Overall, the effective mobility of laudanosoline andnorlaudanosoline, depicted in Figure 6, increased at pH 9 and had

the lowest values at pH 11. The change in mobility was on theorder of 1 × 10-4 cm2 V-1 s-1 for laudanosoline and norlaudano-soline. This decrease in mobility signifies that the enantiomersare interacting less with the micelle at higher pH values. Theopposite effect was observed with laudanosine, which shows atrend of increasing mobility with an increase in pH.

We found that the retention times were not always reproduciblewith poly(L-SUV) at pH values below 7. Similar problems wereobserved by Otsuka and Terabe16 using SDVal. Four reasons maycontribute to these instabilities in separation, the first being thetighter conformation of the polymerized surfactant in acidic media.This could occur if the carboxylic groups on the polymer becomeprotonated. Second, pH hysteresis may exist with the silanolgroups within the capillary walls, causing an unpredictable EOF.The next possibility may be electrophoretic dispersion among thepolymerized surfactants. Last, the hydrophilic head groups onthe micelle may interact with the capillary via hydrogen bondingleading to polymer adsorption on the capillary. Once bound tothe capillary walls, the micelle will not strongly complex with theanalytes. A PVA-coated capillary was used to decrease micelleadsorption and diminish capillary wall interaction with the polym-erized surfactant. Figure 7 depicts a comparison of the paveroline

Table 2. Migration Times, Capacity and SelectivityFactors, and Resolution of Racemic Mixtures ofLaudanosoline, Norlaudanosoline, and Laudanosinea

laudanosoline norlaudanosoline laudanosine

pHtR1

b

tR2b

k1′k2′

RRs

tR1b

tR2b

k1′k2′

RRs

tR1b

tR2b

k1′k2′

RRs

5.6c 15.39 0.45 1.16 17.12 0.62 1.39 23.51 1.2216.12 0.52 0.78 19.68 0.86 1.78

5.9d 14.04 1.17 1.10 15.51 1.39 1.07 17.07 1.63 1.1214.79 1.28 1.09 16.14 1.49 0.97 18.33 1.83 0.97

7d 16.61 1.68 1.06 18.12 1.91 1.14 19.40 2.2017.57 1.84 0.64 20.58 2.31 0.83

9e 6.23 0.34 1.04 6.31 0.37 1.02 7.73 0.676.50 0.40 0.95 6.45 0.40 0.49

11e 8.27 0.66 1.16 7.98 0.63 1.25 7.17 0.49 1.029.59 0.93 1.07 9.95 1.03 0.95 7.31 0.52 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).

Figure 6. Effect of pH on effective mobility of (b) laudanosoline,([) norlaudanosoline, and (9) laudanosine. CE conditions: sameas 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 pH5.6. Peaks: (1) laudanosoline, (2) norlaudanosoline, and (3)laudanosine. CE conditions: same as Figure 2 except capillary, 50µm × 55 cm (50.5 cm effective length).

Analytical Chemistry, Vol. 69, No. 5, March 1, 1997 963

derivative mixture at pH 5.6 and 6 with a coated and uncoatedcapillary, respectively. The selectivity and resolution of nor-laudanosoline, listed in Table 2, were enhanced with the coatedcapillary. Enantioseparation could not be achieved with laudanosine.Experiments are being conducted to further elucidate thisanomalous behavior of laudanosine on coated capillaries.

CONCLUSIONMECC has proven to be an important tool to the pharmaceuti-

cal industry for chiral separation of drugs. The polymerizedanionic surfactant poly(L-SUV) has demonstrated great versatilityin enantioseparation with a variety of cationic, anionic, and neutralcompounds under moderately acidic, neutral, and basic BGEconditions. Separation was found to be dependent on theracemates having bulky ring structures at or around their chiralcenters. All of the enantiomers separated also had polar groupsthat seemed to be necessary for hydrogen bonding with L-valinate

on the micelle. Negative electrophoretic mobilities confirmedenantiomer association with the anionic micelle. In all cases,hydrophobic and electrostatic interactions as well as hydrogenbonding appear to be important for chiral recognition.

ACKNOWLEDGMENTThis work was supported through a grant from the National

Institutes of Health (GM39844). I.M.W. also acknowledges thePhilip W. West endowment for partial support of this research.K.A.A.-H acknowledges the Louisiana Board of Reagents for afellowship award in support of this research.

Received for review August 1, 1996. Accepted December16, 1996.X

AC960778W

X Abstract published in Advance ACS Abstracts, February 1, 1997.

964 Analytical Chemistry, Vol. 69, No. 5, March 1, 1997