Evaluation of a Vancomycin Chiral Stationary Phase in Capillary

Anal. Chem. 2000, 72, 4394-4401

Evaluation of a Vancomycin Chiral Stationary Phase in Capillary Electrochromatography Using Polar Organic and Reversed-Phase Modes Charlotte Karlsson,† Lars Karlsson,† Daniel W. Armstrong,‡ and Paul K. Owens*,†

Analytical Development, AstraZeneca R&D Mo¨lndal, S-431 83 Mo¨lndal, Sweden, and Department of Chemistry, University of Missouri-Rolla, Rolla, Missouri 65409

A vancomycin chiral stationary phase (CSP) was fully evaluated in capillary electrochromatography (CEC) in reversed-phase and polar organic modes for a number of racemic pharmaceutical compounds. High efficiency and resolution values were obtained for a number of compound classes including thalidomide in both the polar organic mode (190 000 plates meter-1 and Rs ) 13.8) and reversed-phase mode (125 000 plates meter-1 and Rs ) 13.0). Experimental parameters, including organic modifier, organic solvent ratio, ionic strength, pH, temperature, and voltage, were examined in both the aqueous and nonaqueous modes to deduce their effect on the resultant EOF, retention times, resolution, and efficiency of chiral separations. All results were consistent with and found to be a combination of what is known from existing literature on CEC theory and experience obtained with macrocyclic antibiotic CSPs in LC. Column stability was excellent, and each column packed was found to offer repeatable separations even when switching from the aqueous to the nonaqueous mode. Capillary electrochromatography (CEC) is a relatively new separation technique that combines the selectivity of liquid chromatography (LC) with the high resolution and efficiency of capillary electrophoresis (CE).1-10 The advantages principally arise from the electroosmotic transport of mobile phase through the column which leads to reduced plate heights due to the plug-like * Corresponding author: (Tel) + 46 31 7761961; (Fax) + 46 31 7763768; (email) [email protected]. † AstraZeneca R&D Mo ¨lndal. ‡ University of Missouri. (1) Crego, A. L.; Gonzalez, A.; Marina, M. L. Crit. Rev. Anal. Chem. 1996, 26, 261-304. (2) Colo´n, L. A.; Reynolds, K. J.; Aliceamaldonado, R.; Fermier, A. M. Electrophoresis 1997, 18, 2162-2174. (3) Pesek, J. J.; Matyska, M. T. Electrophoresis 1997, 18, 2228-2238. (4) Altria, K. D.; Smith, N. W.; Turnbull, C. H. Chromatographia 1997, 46, 664-674. (5) Cikalo, M. G.; Bartle, K. D.; Robson, M. M.; Myers, P.; Euerby, M. R. Analyst (Cambridge, U.K.) 1998, 123. (6) Dermaux, A.; Sandra, P. Electrophoresis 1999, 20, 3027-3065. (7) Knox, J. H.; Grant, I. H. Chromatographia 1987, 24, 135-143. (8) Rathore, A. S.; Horvath, C. Anal. Chem. 1998, 70, 3271-3274. (9) Euerby, M. R.; Gilligan, D.; Johnson, C. M.; Roulin, S. C. P.; Myers, P.; Bartle, K. D. J. Microcolumn Sep. 1997, 9, 373-387. (10) Angus, P. D. A.; Victorino, E.; Payne, K. M.; Demarest, C. W.; Catalano, T.; Stobaugh, J. F. Electrophoresis 1998, 19, 2073-2082.

4394 Analytical Chemistry, Vol. 72, No. 18, September 15, 2000

flow and the ability to employ smaller particles and longer columns for even higher efficiency. These advantages of CEC have contributed to its rapid development, which has resulted in a diverse range of applications from small molecules to large proteins.1-10 The separation selectivity and efficiency values that are possible in CEC thus make it an attractive complimentary technique to those available for the separation of enantiomers. The majority of chiral selectors previously utilized for chiral chromatographic and electrophoretic separations have now been successfully applied for the separation of enantiomers in either open-tubular, packed, or monolithic capillary electrochromatography (CEC) columns.11-14 The earlier review by Hatajik and Brown11 has now been updated to include the application of additional chiral stationary phases (CSPs) and the evaluation of nonaqueous chiral CEC12-14 but does not include more recent reports.15-21 Traditional approaches using cyclodextrin additives and cyclodextrin, protein, π-complex, and cellulose derivative CSPs have all been reported for various racemic analytes with various degrees of success.4-6,12-14 Molecularly imprinted,22,23 weak anion-exchange,24 poly-N-acryloylL-phenylalanineethylester (Chiraspher),25 Whelk-O,26 cellulose,27,25 chiral monolithic,28 and helically chiral polydiphenyl-2-pyridylm(11) Hatajik, T. D.; Brown, P. R. J. Capillary Electrophor. 1998, 5, 143-151. (12) Wistuba, D.; Schurig, V. J. Chromatogr., A 2000, 875, 255-276. (13) Schurig, V.; Wistuba, D. Electrophoresis 1999, 20, 2313-2328. (14) Gubitz, G.; Schmid, M. G. Enantiomer 2000, 5, 5-11. (15) Wikstro¨m, H.; Svensson, L. A.; Torstensson, A.; Owens, P. K. J. Chromatogr., A 2000, 869, 395-409. (16) Tobler, E.; La¨mmerhofer, M.; Lindner, W. J. Chromatogr., A 2000, 875, 341-352. (17) Mayer, S.; Briand, X.; Francotte, E. J. Chromatogr., A 2000, 875, 331-339. (18) Karlsson, C.; Wikstro ¨m, H.; Armstrong, D. W.; Owens, P. K. J. Chromatogr., A 2000, in press. (19) Koide, T.; Ueno, K. J. High Resolut. Chromatogr. 2000, 23, 59-66. (20) Takeuchi, T.; Matsui, J. J. High Resolut. Chromatogr. 2000, 23, 44-46. (21) Wolf, C.; Spence, P. L.; Pirkle, W. H.; Cavender, D. M.; Derrico, E. M. Electrophoresis 2000, 21, 917-924. (22) Schweitz, L.; Andersson, L. I.; Nilsson, S. J. Chromatogr. 1998, 817, 5-13. (23) Owens, P. K.; Karlsson, L.; Lutz, E. S. M.; Andersson, L. I. Trends Anal. Chem. 1999, 18, 146-154. (24) La¨mmerhofer, M.; Lindner, W. J. Chromatogr. 1998, 829, 115-125. (25) Krause, K.; Girod, M.; Chankvetadze, B.; Blaschke, G. J. Chromatogr. 1999, 837, 51-63. (26) Wolf, C.; Spence, P. L.; Pirkle, W. H.; Derrico, E. M.; Cavender, D. M.; Rozing, G. P. J. Chromatogr. 1997, 782, 175-179. (27) Francotte, E.; Jung, M. Chromatographia 1996, 42, 521-527. (28) Peters, E. C.; Lewandowski, K.; Petro, M.; Svec, F.; Frechet, J. M. J. Anal. Commun. 1998, 35, 83-86. 10.1021/ac0002792 CCC: $19.00

© 2000 American Chemical Society Published on Web 08/10/2000

ethyl methacrylate29 type CSPs have also been applied successfully in chiral CEC. These applications have utilized the traditional aqueous mobile-phase compositions to obtain efficient and highresolution separations. Despite the advantages of nonaqueous electrolyte systems in LC and CE, their application in achiral30-33 and chiral,15,25,29 however, has been relatively unexplored. Although a number of macrocyclic antibiotic chiral selectors have shown an extremely broad degree of enantioselectivity in a number of techniques,34-43 there have been fewer applications reported in the CEC mode of operation. Dermaux et al., in a short communication, described a chiral separation of warfarin and hexobarbital on an in-house vancomycin CSP in the reversedphase mode.44 Wikstro¨m et al. on another in-house vancomycin CSP extended the application to a number of analytes.15 A second macrocyclic antibiotic, teicoplanin, has also been applied in the CEC mode.18,45,46 The electroosmotic flow (EOF) in CEC is used to transport the mobile phase and the analytes through the packed column when an axial electrical field is applied.7 It is generated at the interface between the charged silica surfaces within the column and the electrolytic mobile phase. Factors for consideration when developing a CEC method are thus those that directly affect the electroosmotic mobility (µeo) which can be shown by arranging the Smoluchowski equation

µeo )

x

σE ‚ η

0 r RT 2cF2

(1)

where σ is the charge density at the surface of shear, E is the electric field strength, η is the viscosity of the mobile phase, 0 is the permittivity of a vacuum, r is the dielectric constant, R is the universal gas constant, T is the absolute temperature, c is the molar salt concentration, and F is the Faraday constant. The transport of molecules in CEC, as outlined in eq 1, is governed by both chromatographic and electrophoretic principles. The chiral and achiral chromatographic interaction of each enantiomer (29) Krause, K.; Chankvetadze, B.; Okamoto, Y.; Blaschke, G. Electrophoresis 1999, 20, 2772-2778. (30) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr., A 1981, 208, 209-216. (31) Whitaker, K. W.; Sepaniak, M. J. Electrophoresis 1994, 15, 1341-1345. (32) Wright, P. B.; Lister, A. S.; Dorsey, J. G. Anal. Chem. 1997, 69, 32513259. (33) Marusˇka, A.; Pyell, U. J. Chromatogr. 1997, 782, 167-174. (34) Armstrong, D. W.; Tang, Y. B.; Chen, S. S.; Zhou, Y. W.; Bagwill, C.; Chen, J. R. Anal. Chem. 1994, 66, 1473-1484. (35) Armstrong, D. W.; Liu, Y.; Ekborg-Ott, K. H. Chirality 1995, 7, 474-497. (36) Ekborg-Ott, K. H.; Liu, Y.; Armstrong, D. W. Chirality 1998, 10, 434-483. (37) Ekborg-Ott, K. H.; Kullman, J. P.; Wang, X.; Gahm, K.; He, L.; Armstrong, D. W. Chirality 1998, 10, 627-660. (38) Armstrong, D. W.; Rundlett, K. L.; Chen, J. R. Chirality 1994, 6, 496-509. (39) Armstrong, D. W.; Gasper, M. P.; Rundlett, K. L. J. Chromatogr., A 1995, 689, 285-304. (40) Desiderio, C.; Fanali, S. J. Chromatogr. 1998, 818, 281-282. (41) Medvedovici, A.; Sandra, P.; Toribio, L.; David, F. J. Chromatogr. 1997, 785, 159-171. (42) Do ¨nnecke, J.; Svensson, L. A.; Gyllenhaal, O.; Karlsson, K. E.; Karlsson, A.; Vessman, J. J. Microcolumn Sep. 1999, 11, 521-533. (43) Svensson, L. A.; Owens, P. K. Analyst (Cambridge, U.K.) 2000, 125, 10371039. (44) Dermaux, A.; Lynen, F.; Sandra, P. J. High-Resolut. Chromatogr. 1998, 21, 575-576. (45) Carter-Finch, A. S.; Smith, N. W. J. Chromatogr. 1999, 848, 375-385. (46) Miyawa, J. H.; Alasandro, M. S. LC-GC 1998, 16, 36-41.

with a CSP will be governed similarly to that observed in LC, where stronger affinity is obtained for the enantiomer with the higher adsorption constant for that chiral selector. If the enantiomers carry an overall charge, then electrophoretic principles will also be superimposed on its transport through the column, which may be assisted or impeded by electrophoretic mobility. It is more complex when applying a CSP that contains ionizable functionalities which may potentially contribute to the EOF magnitude and, possibly, direction. This issue has already been reported and examined by other groups24,29 by assessing CSPs that contain an ionizable functionality. The magnitude and even the direction of EOF on an anion-exchange CSP was found to change at various pH values by La¨mmerhofer and Lindner.24 At high pH values, a conventional cathodic EOF was observed, but at pH values below 6.2, a strong anodic EOF was attributed to both the combination of silanol charge and positive charge from the basic functionalities of the selector which predominated. In this paper we report our results on an extensive evaluation of a vancomycin-bonded CSP using both the traditional reversedphase mode as well as the polar organic mode. A number of racemic compounds including acids, bases, and neutrals are screened for the first time on this CSP in CEC to determine its general applicability, enantioselectivity, and efficiency. A number of CEC columns were packed and prepared to assess the repeatability from column to column and their stability using this approach. The experimental conditions, including organic modifier concentration, ionic strength, pH, voltage, and temperature, were investigated for their effect on the EOF, resolution, and efficiency of enantiomeric separations. These data have thus allowed an interpretation on the presence and/or the contribution to the EOF of the ionized functionalities present on the immobilized vancomycin chiral selector. EXPERIMENTAL SECTION Chemicals. The vancomycin bonded CSP (5-µm Chirobiotic V) was a gift from Advanced Separation Technologies, Inc. (Astec, New Jersey). Acetonitrile (MeCN), methanol (MeOH), glacial acetic acid (HOAc), and sodium chloride (NaCl) were purchased from E. Merck AG (Darmstadt, Germany). Triethylamine (TEA), pindolol, atenolol, fenoterol, labetalol, sotalol, bromacil, dopa, 5-4methylphenyl-5-phenylhydantoin, 5,5-phenylhydantoin, 5-4-hydroxyphenyl-5-phenylhydantoin, phenylpropanolamine, propranolol, verapamil, terbutaline, coumachlor, and binaphthol were purchased from Sigma-Aldrich Sweden AB (Stockholm, Sweden). Metoprolol, felodipine, alprenolol, thalidomide, bupivacaine, and warfarin, were obtained from medicinal chemistry, AstraZeneca R&D Mo¨lndal (Mo¨lndal, Sweden). Ketamine was purchased from the Apotek production laboratories (Stockholm, Sweden). Acetone was purchased from Rathburn Chemicals Ltd. (Walkerburn, UK). Fused silica capillaries were obtained from Polymicro Technologies, Inc. (Phoenix, AZ). Organic solvents were of HPLC grade. Deionized water (18.2 MΩ) used throughout the study was taken from a Maxima water purification system (Elga, High Wycombe, UK). Instrumentation. The capillary packing pump (Shimadzu LC5A, Kyoto, Japan) and conditioning pump (µLC-500, ISCO, Gottingen, Germany) were both capable of operating in both constant flow and pressure mode up to 500 bar. Production of retaining frits was carried out using the Advanced Capillary Burner Analytical Chemistry, Vol. 72, No. 18, September 15, 2000

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(InnovaTech Ltd., Hertfordshire, UK). All CEC experiments were carried out using the Hewlett-Packard 3DCE system (HewlettPackard, Waldbronn, Germany) modified to allow pressure of up to 12 bar to both the inlet and the outlet mobile-phase vials. Data were collected and analyzed using the HP 3D-CE ChemStation Rev. A.05.04 (Hewlett-Packard, Waldbronn, Germany). Methods. Aqueous mobile phases were prepared by adding the desired volume of organic solvent to pH controlled buffer solutions and degassed by sonication or using helium for at least 10 minutes. Polar organic mobile phases were prepared by combining the desired ratio of MeCN and MeOH to which either HOAc and/or TEA were added. Analyte stock solutions were prepared for each analyte in MeCN at a concentration of 3.0 mg/ mL (unless stated otherwise) and stored at 4 °C. Analytical samples for reversed-phase CEC and the polar organic mode were prepared by a 50% dilution of each stock solution with MeCN (unless stated otherwise). Column Preparation. Capillaries (75 µm) were packed with the vancomycin CSP in acetone (10 mg/mL) using the slurry packing technique, very similar to that recently described by our group.15 The packing solvent, acetone, was initially delivered at 0.4 mL/min until 490 bar was reached, after which constant pressure was applied for 2 h. Prior to retaining frit production, the capillary was washed with 10 mM sodium chloride for 30 min at 450 bar since a silica with high sodium content (∼1500 ppm) is generally required before porous frits of sodium silicate can be produced at high temperatures.5 The retaining frits were prepared under pressure at 450 bar by threading it through a resistance coil and applying heat at approximately 600 °C for 10 s. The column was then conditioned with the desired mobile phase for 45 min and a detection window prepared prior to use. In these studies, the success rate at packing capillaries was nearly 90%, a much more repeatable and less fragile procedure than the production of retaining frits. Traditionally, water is used for flushing the column prior to frit preparation; however, the stationary phase must have a high sodium content (∼1500 ppm) so that porous plugs of sodium silicate may be produced at sufficiently high temperatures.5 The problem of difficult-to-fabricate retaining frits on the Chirobiotic materials has already been addressed by Carter-Finch and Smith.45 They observed that an increase in current and loss of resolution occurred, which were attributed to the quality of the retaining frit. The problem was overcome by fabricating the inlet and outlet frits from pure silica material which had to be packed and prepared prior to and after packing of the CSP, respectively. These problems were also experienced at an early stage of this evaluation but were overcome using a salt-solution flush (10 mM NaCl) as opposed to the traditional water flush prior to frit fabrication, which was carried out directly on the CSP itself. Column Evaluation. Each new column packed, frited and successfully mounted to the instrument was evaluated or examined with a system suitability test (SST) prior to further exploratory experiments. The SST included a chiral separation of the β-blocking drug metoprolol and measurement of linear velocities at various voltages for the nonretained marker, acetone. Determination of resolution and retention time repeatability (relative standard deviation (RSD) n ) 5) were also calculated for each column packed. The polar organic mode (MeCN/MeOH/TEA/ 4396 Analytical Chemistry, Vol. 72, No. 18, September 15, 2000

Figure 1. Polar organic mode chiral CEC separation of metoprolol on the vancomycin CSP using the SST conditions: MeOH/MeCN/ TEA/HOAc, (80:20:0.1:0.1, v/v/v/v), 335 mm × 75 µm i.d. (Ld 250 mm), 25 kV, 2-s injection at 10 kV, 15 °C, 10 bar, and detection at 200 nm.

HOAc, 20:80:0.1:0.1, v/v/v/v) was chosen as a suitable mobile phase for the SST since we recently found this to be a more universally selective and stable system on our in-house-prepared vancomycin CSP.15 Once the selectivity and repeatability of the column were verified, a number of racemic pharmaceutical solutes were screened in the polar organic mode. Mobile phase and electrochromatographic running conditions were subsequently examined to determine their effect on the observed EOF and on resolution and efficiency of the column. The vancomycin CSP was similarly evaluated in the reversed-phase mode by screening a number of racemic compounds and assessing the effect of each mobile phase and electrochromatographic parameter on the EOF, selectivity, and efficiency of the column. RESULTS AND DISCUSSION Column Evaluation. A chiral separation of the β-blocking drug, metoprolol, in the polar organic mode was used, in part, to test each new column packed (Figure 1). The enantiomers are separated in under 10 min with resolution and efficiency values of 1.41 and 40 000 plates meter-1, respectively. The repeatability of this separation was examined (n ) 5), resulting in RSD values for retention time (0.12%), resolution (2.1%), area (3.0%) and efficiency (2.2%). Column-to-column repeatability, using five columns packed at different intervals over a three-month period, was also determined to assess the repeatability of both the chiral SST separation and packing procedures adopted. RSD values for retention time, resolution, and efficiency were found to be 10, 13, and 23%, respectively. These data are acceptable and similar in magnitude to those recently reported in a similar study in which the Chirobiotic T CSP was packed and evaluated.45 It may therefore be concluded that columns may be prepared repeatably to obtain similar results from day to day in the same laboratory by the same analyst but greater control over the entire column preparation, including packing procedures and precautions, together with retaining frit fabrication procedures, must be attained. Polar Organic Mode. This chromatographic mode of operation, which closely resembles the normal-phase mode, was first introduced for enantioseparations on cyclodextrin47 and later for

Table 1. Racemic Analytes Examined on the 5-µm Vancomycin CSP in CEC Using the Polar Organic Mode racemate pindolol alprenolol atenolol fenoterol metoprolol sotalol propranolol bupivacaine labetalol verapamil terbutaline thalidomide ketamine phenylpropanolamine 5-(4-methylphenyl)5-phenylhydantoin 5,5-diphenylhydantoin bromacil binaphthol dopa benzoin felodipine 5-(4-hydroxyphenyl)5-phenylhydantoin

tR1 (min)

N1 (plates m-1)

N2 (plates m-1)

Rs

b b b a a a a d b b b c b a, d a, d

10.5 9.3 16.5 20.3 15.9 19.2 16.2 6.7 22.8 18.9 11.7 10.9 11.1 5.5 12.8

138 700 73 668 52 380 27 364 79 364 36 572 31 872 44 516 64252 106 428 121 496 189 804 113 492 17 252 57 908

123 724 68 336 53 152 22 056 29 256 31 692 25 704 26 308 16 428 81 264 111 456 113 776 39 616

2.74 2.21 2.11 1.11 1.22 1.66 1.63 0.92 1.4, 3.1, 2.2 1.90 2.42 13.8 0.5

a, d a, d a, d a, d a, d a, d a, d

13.2 11.4 10.9 10.9 10.9 10.6 21.5

75 244 117 636 78 344 45 812 96 444 236 652 73 308

conditions

a MeCN/MeOH/TEA/HOAc, (20:80:0.1:0.1, v/v/v/v), 10 kV, 15 °C. b MeCN/MeOH/TEA/HOAc, (20:80:0.1:0.1, v/v/v/v), 15 kV, 15 °C. c MeCN/ MeOH/TEA/HOAc, (20:80:0.1:0.1, v/v/v/v), 20 kV, 15 °C. d MeCN/MeOH/TEA/HOAc, (15:85:0.1:0.1, v/v/v/v), 25 kV, 15 °C.

the macrocyclic antibiotic CSPs.34-36 The mobile phase consists predominantly of the polar solvent MeCN, which has poor hydrogen-bonding capabilities. Traditionally, MeOH, triethylamine, and/or glacial acetic acid additives were added to regulate retention and selectivity. It has been noted that macrocyclic antibiotic-based CSPs are sometimes even more effective when MeCN is eliminated from the mobile phase, leaving methanol as the solvent with small amounts of acetic or trifluoroacetic acid in combination with triethylamine or ammonium hydride.48 The old polar organic mode of operation, however, has also been shown to be particularly selective with a number of different antibiotic CSPs when applied in both LC and SFC.36,49 Since this is a relatively new mobile phase of application in CEC,15,50 the effect of applied field strength, the predominant parameter for influencing the linear velocity of the mobile phase in CEC, was examined on this vancomycin-bonded CSP. A plot of linear velocity with applied field strength was constructed and shown to be linear, indicating the absence of both thermal and double-layer overlap effects under these polar organic conditions employed.51 A van Deemter curve using the neutral marker, acetone, was also constructed, resulting in a reduced plate height of 2.2 (∼90 000 plates meter-1) at 0.6 mm/s. Acetone was chosen since it was found to be the least retained molecule when acetone, thiourea, and dimethyl sulfoxide were simultaneously examined under identical conditions in both polar organic- and reversedphase modes. It would still be difficult, however, to know or state unequivocally that acetone is truly nonretained on this CSP. (47) Chang, S. C.; Reid, I. G.; Chen, S.; Chang, C. D.; Armstrong, D. W. Trends Anal. Chem. 1993, 12, 144-153. (48) Chirobiotic Handbook, 3rd ed.; Advanced Separation Technologies Inc. 1999. (49) Svensson, L. A. Uppsala University, Uppsala, 1998. (50) Tobler, E.; Lammerhofer, M.; Lindner, W. J. Chromatogr., A 2000, 875, 341-352. (51) Wan, Q. H. J. Chromatogr. 1997, 782, 181-189.

The polar organic cathodic EOF (6.7 × 10-5 cm2 V-1 s-1), although a little lower, is comparable to those recently reported on anion-exchange (7.5× 10-5 cm2 V-1 s-1),24 antibiotic (10 × 10-5 cm2 V-1 s-1),44 and cellulose (11 × 10-5 cm2 V-1 s-1)25 type chiral CSPs examined in reversed-phase chiral CEC. Although the EOF is relatively slow in the SST conditions given above, an increased EOF was observed, to 10.9 × 10-5 cm2 V-1 s-1, at higher MeCN concentrations in the polar organic mode. This can be attributed to the resultant phase having a higher dielectric-constant-toviscosity ratio (/η) which directly contributes to the EOF as outlined in eq 1. This higher EOF is now even more comparable to those reversed-phase EOF values obtained on other CSPs in CEC. They are a little lower, however, than those obtained in achiral nonaqueous CEC studies where values of 15.2 and approximately 19 × 10-5 cm2 V-1 s-1 were obtained on a octadecylated cellulose and an ODS Hypersil stationary phase, respectively.33,52 These higher reported values are reasonable and expected since each were obtained in pure MeCN, which will result in even higher EOF values as described above. These data indicating high electroosmotic mobility in the polar organic mode in this study therefore outline the potential of using this mobile phase as an alternative to the traditional aqueous phases applied in chiral or even achiral CEC. A number of racemic pharmaceutical molecules were subsequently screened in the polar organic mode for enantioselectivity. The majority of these were basic molecules, but both neutral and acidic molecules were also examined. High resolution separations were obtained for many analytes (Table 1) using capillaries packed with this CSP. Of greater interest are the high efficiency values obtained on this CSP, since the potential advantage of this technique has not generally been realized for chiral separations (52) Lister, A. S.; Dorsey, J. G.; Burton, D. E. J. High-Resolut. Chromatogr. 1997, 20, 523-528.

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Figure 3. Evaluation of MeCN content in the polar organic mobile phase for its effect on the (a) resolution and efficiency of terbutaline enantiomers and (b) the EOF and retention time of the terbutaline first-eluting enantiomer. All other conditions are held constant and are described in Figure 1.

Figure 2. Polar organic mode chiral CEC separations of (a) thalidomide, (b) alprenolol, and (c) terbutaline enantiomers on the vancomycin CSP. Conditions: MeOH/MeCN/TEA/HOAc, (80:20:0.1: 0.1, v/v/v/v), 335 mm × 75 µm i.d. (Ld 250 mm), (a) 20 kV, (b-c) 15 kV, 2-s injection at 10 kV, 15 °C, 10 bar, and detection at 200 nm.

to date. The combination of high resolution and efficiency is shown for thalidomide, alprenolol, and terbutaline enantiomers in Figure 2a-c, respectively. The enantiomers of the former are well separated (Rs ) 13.8) with efficiency values of 190 000 and 114 000 plates meter-1 for the first and second eluted enantiomers, respectively. This separation is surprising since the data in Table 1 indicate that this CSP is more selective for analytes containing basic functionalities in the polar organic mode and particularly for the β-blocking drugs, where the majority of molecules in this class were separated. This is also the case for this CSP in LC and is reflected accordingly in the CEC results with the addition of the high efficiency values possible using the electrodriven technique. A study was subsequently carried out to investigate effects of mobile-phase composition including solvent mix, acid/base concentration, and temperature on the EOF of the system, resolution and efficiency of terbutaline enantiomers. Terbutaline was selected 4398 Analytical Chemistry, Vol. 72, No. 18, September 15, 2000

as a probe for evaluating the mobile-phase composition since it was well separated (Rs ) 2.36) in approximately 11 min with high efficiency values (90 272 plates meter-1) under standard conditions. The effects described below for terbutaline enantiomers were also observed for other racemic solutes. The mobile phase consisted of a mixture of two organic solvents, MeOH and MeCN, and the ratio of these was examined and shown in Figure 3a and 3b. On decreasing the MeCN content (or increasing MeOH), the resolution and efficiency of terbutaline enantiomers was increased (Figure 3a). This effect of MeCN concentration is in agreement with LC data on this CSP, where generally better separations are obtained using a high MeOH concentration in combination with acid/base additives. This may in part be attributed to less nonselective hydrogen interactions at higher MeOH concentrations which results in less overall retention but a greater degree of enantioselective retention. This is supported by the observation that the retention times of terbutaline enantiomers were not significantly changed by decreasing the MeCN concentration but EOF was decreased by almost 75%, from 11.5 to 3.0 × 10-5 cm2 V-1 s-1, as shown in Figure 3b. This is an interesting observation and may also indicate the mixed nature of chromatographic and electrophoretic properties of this separation. MeOH has a lower dielectric-constant-to-viscosity ratio (/ η) and will therefore, according to equation 1, generate a lower EOF. The retention times of the terbutaline enantiomers would therefore be expected to increase proportionally, if the contribution from electrophoretic mobility remained constant, to the decrease in EOF on lowering the MeCN concentration. This is not observed, however, and is likely to be a result of faster chromato-

graphic elution of the enantiomers at higher MeCN concentrations and is not surprising since a linear relationship exists between log k′ and percent acetonitrile content in the mobile phase.9 These higher resolution and efficiency values but lower EOF (Figure 3a and 3b) at high MeOH contents may indicate a potential limitation of this CSP in the polar organic mode. If elimination of MeCN from the mobile phase is preferred, thus resulting in lower EOF values, the transport of analyte enantiomers will predominantly rely on electrophoretic mobility which may then limit the potential of separating neutral enantiomers in a reasonable time. TEA/HOAc concentration in a 1:1 ratio was also examined, at a constant solvent mix, for its effect on the EOF and resolution of terbutaline enantiomers. The resolution was not changed significantly over the range examined, but the EOF was decreased at higher concentrations of acid and base additive. Since very little has been published on the use of the polar organic mode in CEC, it is uncertain if the observed decrease in EOF at higher concentrations of acid/base can be attributed, as outlined in eq 1, to an increased double layer thickness and thus a reduced zeta potential with increased ionic strength which was derived for aqueous systems.51 This will be the focus of another polar organic mode investigation in CEC which will extend to a greater range of organic solvent mixtures each at different acid/base concentrations and ratios down to nonaqueous mixtures without added ionic species. Temperature, an influential parameter in both chiral LC separations using CSPs and in CEC in general, was also examined in order to determine its effect on the EOF, separation, and efficiency of terbutaline enantiomers. The EOF was increased as expected from 5.0 to 8.4 × 10-5 cm2 V-1 s-1 at higher temperatures since it directly affects the electroosmotic mobility (eq 1). The resolution was found to decrease from 2.5 to 1.3 at higher temperatures, and this is analogous to chiral LC where higher stability constants and thus resolution values are generally observed at lower temperatures.34,53 The observed efficiency for terbutaline enantiomers is slightly decreased from a reduced plate height of 1.6 to 2.6 over a 45 °C temperature change. Similar effects on the EOF and efficiency values have been obtained and reported for ODS material and are thus not surprising on this CSP. Since temperature has an obvious effect on all three parameters investigated, it is conceivable that it could be used as a chromatographic tool to optimize difficult chiral separations, as it is utilized in chiral LC. Reversed-Phase Mode. The use of reversed-phase mobile phases, which are traditionally applied in CEC, has also been investigated in this study. Reversed-phase chiral CEC has been reported on two in-house vancomycin CSPs but not to date on this commercial material. Wikstro¨m et al.15 and Dermaux et al.44 both utilized a triethylammonium acetate (TEAA) buffer in MeCN mobile phase, which resulted in acceptable resolution and efficiency values in each case. This mobile phase was therefore subsequently examined on this CSP in the CEC mode. Of the racemic solutes tested, enantioselectivity was only observed for five racemic compounds (Table 2) and it is shown in Figure 4 for the separation of thalidomide enantiomers. The resolution and efficiency values (Rs ) 13; 125 000 plates meter-1) are comparable in value to those obtained in the polar organic mode above, although the overall enantioselectivity appears to be much lower. (53) Pe´ter, A.; To ¨ro ¨k, G.; Armstrong, D. W.; To´th, G.; Tourwe´, D. J. Chromatogr., A 1998, 828, 177-90.

Table 2. Reversed-Phase Enantioselectivity on the 5-µm Vancomycin CSP in CEC racemate

conditions

tR1 (min)

N1 (plates m-1)

N2 (plates m-1)

Rs

warfarin coumachlor felodipine binaphthol thalidomide

a b c c d

15.5 8.5 8.8 8.5 25.9

40,716 37,224 28,144 36,272 125,832

43,984 64,428 12,948 18,892 100,592

3.4 2.2 2.4 0.6 13.0

a MeCN/0.2% TEAA, pH 5 30/70. b MeCN/0.1% TEAA, pH 5 40/ 60. c MeCN/0.1% TEAA, pH 5 30/70. d MeCN/0.1% TEAA, pH 4 30/70.

Figure 4. Reversed-phase chiral CEC separation of thalidomide on the vancomycin CSP, outlining the high efficiency (125 832 and 100 832 plates meter-1) and resolution (13.0) of this CSP in CEC. MeCN/0.1% TEAA (pH 4), 30:70, v/v, 335 mm × 75 µm i.d. (Ld 250 mm), 25 kV, 2-s injection at 10 kV, 15 °C, 10 bar, and detection at 214 nm.

It is interesting to note that no basic molecules were separated in the reversed-phase mode and that thalidomide was the only molecule to be separated in both modes of operation. A number of neutral and acidic molecules, particularly the nonsteroidal antiinflammatory drugs, were also examined, but with little success in the reversed-phase mode. This is surprising since a diverse range of solutes, including basic, neutral, and acidic molecules are readily separated on this CSP in LC.34 A similar study to that shown above for the polar organic mode was also carried out in reversed-phase to investigate the effects of MeCN content, pH, ionic strength, and temperature on the resultant EOF, resolution, and efficiency of the thalidomide enantiomers. As described above for terbutaline enantiomers, thalidomide was selected as a probe for evaluating the mobilephase composition since it was separated to the largest extent (Rs ) 13) with high efficiency values (125 000 plates meter-1) under standard conditions. In each case, the parameter under investigation was varied while others were held constant at the following set of conditions (unless stated otherwise): MeCN/0.1% TEAA, (pH 5.0), (30:70, v/v), 20 kV, 2-s injection of sample solution at 10 kV, 20 °C, 200-nm detection, and 10 bar pressurization. The effect of MeCN content on the magnitude of EOF, retention time, resolution, and efficiency of thalidomide enantiomers is shown in Figure 5a and 5b. The EOF increases, resulting in lower retention times, with increased MeCN content, as expected. These data are different than those shown above for the polar organic mode (Figure 3a) where the retention time was not shown to decrease at high MeCN concentrations which may Analytical Chemistry, Vol. 72, No. 18, September 15, 2000

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Figure 6. Evaluation of pH on the observed EOF and the efficiency and resolution of thalidomide enantiomers on the vancomycin CSP. All other conditions are held constant as described in Figure 4.

Figure 5. Evaluation of MeCN content in the aqueous triethylamine acetate buffer for its effect on the (a) EOF and retention time of the thalidomide first-eluting enantiomer and (b) the resolution and efficiency of thalidomide. All other conditions are held constant and described in Figure 4.

indicate that different chromatographic and electrophoretic mechanisms are occurring in each mode of operation. The effect of MeCN on efficiency is also different for each mode of operation where higher and lower values were obtained in the reversedphase and polar organic modes, respectively, with increasing MeCN content. The effect of MeCN on resolution, however, is analogous in each mode and shown to decrease at higher concentrations. This is also not surprising, since in both achiral CEC using ODS materials and in chiral LC using this CSP, resolution values are generally lower at higher modifier concentrations.10,34 MeCN was the only organic modifier studied since (a) this modifier has been shown to offer a greater degree of enantioselectivity and efficiency than any other on this CSP in LC34,36 and (b) it is a more attractive solvent to work with under electrophoretic conditions because it offers high electroosmotic mobility and thus lower retention times.52 The next phase of this work will need to examine the application of this CSP in greater detail, particularly examine a range of organic modifiers with different aqueous buffer systems, which may then allow a broader degree of enantioselectivity than that shown above. Macrocyclic antibiotic CSPs contain a number of ionizable groups, and their charge and conformation will change at various pH values which will affect enantioselectivity.34-36 The effect of mobile phase pH on the EOF, efficiency, and separation of thalidomide enantiomers is shown in Figure 6. In accordance with electrophoretic theory, the EOF is shown to increase as the pH of the mobile phase is increased, due to a higher charge density 4400 Analytical Chemistry, Vol. 72, No. 18, September 15, 2000

at the silica surface. The increase is almost linear, which may indicate that the predominant source of the EOF is resulting from the silica base material analogous to that observed in achiral CEC, employing ODS materials. There is of course the potential that the fused silica wall is also contributing to the EOF, but this is an ongoing debate in the literature and not the focus of this work. These data above indicate that the ionized vancomycin molecule itself is not significantly contributing to the EOF since both its acidic and basic functionalities undergo only small ionization changes over the pH range examined. This CSP has six pKa values, 2.9, 7.2, 8.6, 9.6, 10.4, and 11.7 ,48 the first three of which could possibly contribute to ionization changes on the molecule between the pH values examined.4-7 At pH 4 the carboxylic acid (pKa -2.9) and both amine functionalities (pKa 7.2 and 8.6) will be significantly and fully ionized, respectively. As the pH is raised to 7, the ionization state of the acid will only increase a little (approximately 10%), while the amine functionalities will experience a decrease in ionization which will result in the net ionization of a vancomycin molecule to become more anionic and therefore only contribute positively to the EOF. The decreased resolution values at higher pH values of the aqueous phase are also in accordance with earlier findings on these CSPs in LC where better values are generally obtained at lower pH values.34 The efficiency of the separation is also shown in Figure 6 at various pH values and found to decrease to a small extent up to pH 6, after which it significantly decreased. This decrease can predominantly be attributed to the higher EOF, and thus shorter retention times, which allows less interaction with the CSP. Another factor that influences both enantioselectivity on this CSP and results obtained in the CEC mode is the concentration of the aqueous buffer. The concentration of the TEAA buffer was examined, and the EOF was shown to gradually decrease while efficiency increased at higher ionic strengths. The former is easily understood since electroosmotic mobility will decrease at higher ionic strength aqueous mobile phases. The latter, however, although only a small effect is more difficult to understand since there is no clear theoretical explanation for higher efficiency values at higher ionic strength mobile phases other than the contributions from the EOF profile which is dependent on the ionic strength. These effects have also been observed in a more detailed achiral CEC study carried out to determine the effects of electrolyte concentration on the EOF and efficiency.51 In that study, a significant increase in efficiency was observed, a 5-µm decrease in plate height, when ionic strength was increased which

Figure 7. Effect of column temperature on the EOF, efficiency, and resolution of thalidomide enantiomers on the vancomycin CSP in reversed-phase CEC. All other conditions are held constant as described in Figure 4.

incidentally was not observed in an open-tubular CEC column. The author could not offer a firm explanation for this effect, indicating that it was more complicated than previously thought. A more simple explanation may be that higher efficiency values may be obtained in a similar way to LC when TEA is acting as a competing base, resulting in less interaction between the analytes and the base silica material. Temperature, normally a very influential parameter in both chiral LC separations and achiral separations in CEC, was examined in the reversed-phase mode to determine its effect on the EOF, separation, and efficiency of thalidomide enantiomers (Figure 7). The EOF was significantly increased from 8.5 to 16.0 × 10-5 cm2 V-1 s-1, since the temperature directly affects electroosmotic mobility. The resolution was found to decrease from 6.6 to 3.5 with an increase of 45 °C and is analogous to chiral LC and CEC studies, respectively.34,45 The observed efficiency for first-eluting thalidomide enantiomer is slightly decreased from a reduced plate height of 1.3 to 1.6 when temperature is varied over 45 °C. Similar effects on the EOF and efficiency values have been obtained and reported for ODS material and are thus not surprising on this CSP. Given the large changes in resolution and EOF with temperature, it is clear that this parameter may be used, without compromising peak efficiency, just like in LC, as a chromatographic factor for fine-tuning challenging chiral separations. The effect of applied field should exert a far greater influence on the linear velocities obtained than any other parameter examined in both the polar organic and reversed-phase modes. Plots of linear velocity with applied field strength and van Deemter curves were constructed for the first enantiomer of thalidomide. Linear plots for the former were obtained in both modes, confirming the absence of both thermal and double-layer overlap effects. Additionally, several Ohm’s plot curves were carried out to determine if Joule heating was contributing to enhanced or decreased chromatographic response values but were not considered significant since typical values for current generated when using the polar organic mode and reversed-phase mode were ∼4 and 2 µamps, respectively. Van Deemter curves were subsequently plotted for each mode and found to be similar. The profile of the

polar organic mode curve was a little flatter when higher voltages were applied compared with that of the reversed-phase mode, where efficiency drops off a little at higher voltages. Both modes of operation indicated, however, that optimum reduced plate height values could be obtained at relatively lower voltages (∼1020 kV). It is difficult to propose a theory to explain differences in the enantioselectivity in CEC in general and for the reversed-phase mode over the polar organic mode. It is known that changes in pH and organic modifier type and content will affect the conformation and thus the degree of enantioselectivity obtained in LC.34 A possible extension to this knowledge in the CEC mode may be the effect of applied field on the enantiorecognition process where electrostatic interactions are occurring, which are much stronger in the reversed-phase mode. The immobilized vancomycin molecule is predominantly ionized and will certainly undergo some changes when a field is applied. This change may simply be a tendency to change orientation by electrophoretic mobility or a direct change in the vancomycin conformation. This change would be expected to result in different enantioselective properties in a similar fashion to those described above as being obtained in LC. CONCLUSIONS It has been shown that the columns packed with vancomycin CSP (Chirobiotic V) can be used repeatably in CEC for the separation of enantiomers. In addition, a number of racemic compounds were screened to estimate the broad enantioselectivity in CEC which resulted in separations characterized by both high resolution and efficiency. Similar to LC, this CSP has shown to be multimodal in nature where CEC offered complimentary enantioselectivity in the reversed-phase and polar organic modes. A number of chromatographic and electrophoretic parameters were evaluated to determine their effect on the observed EOF, enantioselectivity, and efficiency of separations. The results were consistent with, and found to be a combination of, what is known from existing literature on CEC theory and experience obtained on this CSP in LC. EOF values were found to be influenced by voltage, pH, organic modifier content, temperature, and ionic strength of the mobile phase. Enantioselectivity was found to depend on a number of parameters but particularly temperature and organic modifier content. Interestingly, in the reversed-phase mode, it was shown that on systematically changing the pH of buffer and thus the ionization profile of the vancomycin selector itself, a linear relationship existed between EOF and pH which may indicate that the ionizable groups are not significantly contributing to the observed EOF. These data, indicating efficient and high-resolution chiral separations, show the potential of transferring and/or commercialization of this selective CSP designed for LC to CEC. Further studies are now underway to corroborate this belief, relating to an even wider screen of compounds, a continuation of mobile phases parameters, and a batch-to-batch reproducibility study.

Received for review March 9, 2000. Accepted June 22, 2000. AC0002792

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