Anal. Chem. 1993, 65, 3604-3690
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Direct Chiral Separations by Capillary Electrophoresis Using Capillaries Packed with an al-Acid Glycoprotein Chiral Stationary Phase Song Li and David K. Lloyd' Department of Oncology, McCill University, Mclntyre Medical Building, 3655 Drummond, Room 717, Montreal, Quebec, H3G lY6, Canada
Fused-silicacapillaries of 50-pminternal diameter were packed with an al-acid glycoprotein chiral stationaryphase. The properties of electroosmosis in these packed capillaries were studied by investigatingthe influence of field strength, pH, and solvent composition on the velocity of electroosmotic flow. Direct enantiomeric separation by capillary electrochromatographywas investigated with the use of these packed capillaries. Chiral resolution was achieved for the enantiomers of benzoin, hexobarbital, pentobarbital, ifosfamide, cyclophosphamide,disopyramide, metoprolol, oxprenolol, alprenolol,and propranolol. The effects of pH, electrolyte concentration, and concentration of organic solvents in the mobile phase on the retentionand the enantioselectivity were studied. INTRODUCTION The separation of enantiomers has long been a challenging field to analytical chemists. In recent years, there has been a rapidly increasinginterest in chiral separations. It has been found that the enantiomers of chiral bioactive molecules often differ in potency, toxicity, pharmacological actions, and metabolism.' Therefore, the ability to rapidly and accurately separate and determine the enantiomeric composition of chiral compounds is becoming increasingly important in pharmaceutical industry, food science, and agricultural chemistry, and in the last decade there has been a tremendous impetus to develop enantiomeric separation methods. Chiral separation by high-performance liquid chromatography (HPLC) using chiral stationary phases currently enjoys widespread popularity. Today, more than 50 different chiral stationary phases (CSPs) are commercially availablefor direct chiral separations. These CSPs may be subdivided into categories according to the type of chiral selector.2 One such category comprises a group of phases where immobilized proteins are used as chiral selectors. Of these, one of the most successful is that obtained by immobilizing cq-acid glycoprotein (AGP) on silica, introduced by Hermansson3 in 1983. The chiral selector,AGP, is an acidicprotein (isoelectric point 2.7) with negatively charged aspartic acid residues and terminal serine group. Its positively charged groups are present in the arginine, lysine, and histidine residues. Chiral sites appear in the peptide chain, and also in the carbohydrate units that constitute 45% of the molecular eight.^ AGP columns have been used for the direct enantiomeric sepa~~~
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(1) Wainer, I. W.; Drayer, D. E. Drug Stereochemistry: Analytical Methods and Pharmacology; Marcel Dekker: New York, 1988. (2) Wainer, I. W. Trends Anal. Chem. 1987,6,125-134. (3) Hermansaon,J. J. Chromutogr. 1983,269, 71-80. (4) Schmid,K.In ThePlasmaProteins;Putnam,F. W.,Ed.;Academic Press: New York, 1975; p 245. 0003-2700/93/0365-3684$04.0010
rations of a wide variety of chiral compounds, e.g., primary, secondary, and tertiary amines, acids, and nonproteolytic compounds.6 One observation which can be made concerning chromatographic chiral separations in general is that quite often the selectivity, a,between pairs of enantiomers is low, leading to poor resolution. For the protein-based CSPs, this problem is compounded by the fact that these columns generally exhibit relatively low separation efficiencies. In recent years, capillary electrophoresis (CE) has also been utilized for enantiomeric separations. In general, separation efficienciesare high, and baseline resolution can be achieved based on very small enantioselectivities. Chiral separations are being undertaken by the use of chiral selectors in the running buffer, including cyclodextrins,&lO chiral surfactants,11J2 bile salts,13J4 and cyclodextrin-surfactant mixtures.lsJ6 The use of cyclodextrinsimmobilized in gelmatrices for the chiral separation of dansylated amino acids has also been reported." As in HPLC, proteins may also be used for chiral separations in CE, as either buffer additives's or immobilized in gel matrices.19 Because of the wide applicability of protein-based HPLC phases, described above, it is of considerable interest to explore the use of proteins as chiral selectors in CE. The use of proteins as buffer additives in CE has limited potential for analytical purposes (although it has considerable potential for measuring the binding of chiral drugs to proteins's) because of the high background absorbance of the protein. One way to exploit proteins as chiral selectors in CE is to use capillaries packed with proteins immobilized on silica particles. To our knowledge, however, direct chiral separations by packed-capillary electrochromatography have not been reported. Enantiomer resolution by open-tubular (5) Hermamson, J. Trends Anal. Chem. 1989,8, 251-259. (6) Fanali, S. J. Chromutogr. 1991,545,437-444. (7) Jelinek, I.; Snopek, J.;Smolkova-Keulemansova,E. J.Chromutogr. 1988,439, 386-392. (8) Snopek, J.; Soini, H.; Novotny, M.; Smolkova-Keulemansova,E.; Jeliiek, I. J. Chromutogr. 1991,559, 215-222. (9) Atria, K. D.; Goodall,D. M.; Rogan,M. M. Chromatographia1992, 34, 19-24. (10) Shibukawa,A.;Lloyd,D. K.;Wainer,I. W. Chromatographia1993, 35. - - , 419-429. . -(11) Otsuka,K.;Kawahara,J.;Tatekawa,K.;Terabe,S. J.Chromatogr. 1991,559, 209-214. (12)Dobaahi, A,; Ono, T.; Hara, S.;Yamaguchi, J. J. Chromatogr. 1989,480,413-420. (13) Nishi, H.; Fukuyama,T.;Matsuo, M.; Terabe, S.J. Chromatogr. 1990,515, 233-243. (14) Cole, R. 0.;Sepaniak, M.; Hinze, W. L. J. High Resolut. Chromatogr. 1990, 13, 579-582. (15) Nishi, H.: Fukuyama, T.; Terabe, S. J. Chromatogr. 1991, 553, 503-516. (16) Terabe, S. Trends Anal. Chem. 1989,8, 129-134. (17) Guttman, A.; Paulus, A.; Cohen, A. S.;Grinberg, N.; Karger, B. L. J. Chromatogr. 1988,44.8,41-53. (18) Lloyd, D. K.; Li,S.;Ryan, P. 2nd UKInternational Symposium on Capillary Electrophoresis, York, UK, 26-28 Auguet 1992; Abstract P26. (19) Birnbaum, S.; Nilsson, S.Anal. Chem. 1992,64, 2872-2874. 0 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, lgQ3 SOW
capillary electrochromatography has been demonstrated with a cyclodextrin chiral selector coated onto a capillary surface,20 but a significant limitation of this technique is the low phase ratio unless very small capillaries are used. In packedcapillary electrochromatography (CEC),the column is packed with an HPLC packing material and the mobile phase is driven along the column by electroosmosis. The analytes are separated by normal chromatographic partitioning between the mobile and stationary phases. This was demonstrated by Pretorius et aLZ1in 1974, using 1-mm-diameter packed columns. In 1981, Jorgenson and Lukacs22 discussed the applicabilities and difficulties of working with CEC using a 64 cm X 0.17 mm i.d. capillary packed with 10-pm ODS packing. One interesting aspect of electrochromatography is that the electroosmotic flow has a flat flow profile. Thus, there is no contribution to band broadening from the parabolic flow characteristic of pressure-driven systems. Furthermore, the velocity of electroosmotic flow is independent of the geometry and the size of the channels between the packing. This means that electroosmotic flow should proceed at the same rate in a packed capillary regardless of packing irregularities down to very small packing diameters, until double-layer overlap occurs. The use of very small packing8 should lead to improved mass-transfer characteristics. Therefore, lower values of the height equivalent to a theoretical plate should be obtained with CEC systems than with equivalent pressure-driven columns. This was recently confiimed by Knox and G r a d 3 working with electrochromatographic systems using capillaries packed with 1.5-50pm-diameter silica and ODs-bonded silica gels. Reduced plate heights as low as unity were obtained for unretained solutes, and the columns (packed capillaries) driven electrically showed higher plate efficiencies than when they were driven by pressure. The possibilities and limitations of electrically driven systems in reversed-phase chromatography with packed capillarities were also discussed by Yamamoto et al.24 This paper presents the results of an investigation of the applicability of CEC using capillaries packed with an immobilized-AGP stationary phase to direct chiral separations. The properties of electroosmotic flow in the AGP-packed capillaries are described. In addition, the effect of pH, electrolyte concentration, and type and concentration of organic modifiers on the retention and enantioselectivity is detailed. EXPERIMENTAL SECTION Reagents and Materials. AGP packing material (6-pm particles)was obtained by emptying Chiral-AGPHPLC columns (Regis Chemical Co., Morton Grove, IL), which had previously been used extensively for HPLC separations. Packing material was taken from the outlet end of the column first, and the first 1-2 cm of packing at the inlet end of the column was not used. Ail measurements except those related to the effect of buffer concentration were made using packing material taken from the same column. Fused-silica capillary tubes (60-pm i.d. and 366pm 0.d.) were obtained from Polymicro Technologies (Phoenix, AZ). Benzoin was purchased from Aldrich (Milwaukee, WI). Hexobarbital and pentobarbital were from U.S.P.C. Inc. (Rockville, MD). Cyclophosphamide, metoprolol, oxprenolol, alprenolol, and disopyramide were purchased from Sigma (St. Louis, MO). Ifosfamide was obtained from Bristol-Myers (Belleville, ON, Canada). All organic solvents were purchased from Anachemia (Montreal,PQ, Canada). Analyticalgrade disodium (20) Mayer, S.;Schurig, V. J. Liq. Chromatogr. 1993, 16,915-931. (21) Pretorius, V.; Hopkine, B. J.; Schieke,J. D. J. Chromatogr. 1974, 99,23-30. (22) Jorgenson, J. W.;Lukacs, K. D. J. Chromatogr. 1981,218,209216. (23) Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 317-328. (24) Yamamoto,H.; Baumann, J.; Emi, F. J. Chromatogr. 1992,593, 313-319.
hydrogen phosphate and sodium dihydrogen phosphate were obtainedfrom BDH Inc. (Toronto,ON, Canada). Deionized water was obtained by passing distilled water through a Milli-Qm ultrapure water system (Millipore, Molshem, France). Apparatus. Electrochromatography was performed using a Model 270A CE system (Applied Biosystems, Toronto, ON, Canada). The samples were injected electrokinetically by applying a voltage of 6 kV for 1s. Based on the electroosmotic flow velocity, the length of the sample zone is ca. 0.3 mm. Oncolumn detection was carried out on UV absorbance measurementa. Temperature was set at 30 O C . A Model SP4600 integrator (Spectra-Physics,San Jose, CA) was used for recording the electropherograms. Further data manipulation was performed using Spectra-Physics Winner 386 software. A Model 1666HPLC column slurry packer (AiltechAssociates, Deerfield, IL) was used for the capillary packing. This was also used to pump liquid through the capillary when mobile phases were changed, but no other capillary washing (e.g., between run)was performed. Preparation of Packed Capillaries. Fused-silica capillaries (60-pm i.d. and 365-pm 0.d.) were used as column material. The capillaries were cut into 42-cm lengths. The first step in the preparation is to produce a frit at one end of the capillary. To do this,a small amount of silica gel (6-pmdiameter) is moistened with deionized water to form a paste. The end of the capillary is then tapped into the paste until -2 mm of the tube is packed. The silica gel is sintered at the end of the capillary by gently heating with a small flame for 10 8. The frit thus produced can retain the packing particles against packing pressure up to 7000 psi. A slurry of 6-pm AGP packing is made by mixing -60 mg of packing material with 3 mL of 1:4 acetonitrile/lOmM phosphate buffer solution in an ultrasonic bath for -6 min. The addition of a low concentration of electrolyte is useful to avoid clumping of the packing material. The rather low packing/liquid ratio also helps in reducing clumping and blockages during packing. In order to maintain the activity of immobilized proteins, we have avoided the use of balanced-densityslurries and of other pure organic solvents often used for packing micro-HPLC columns.as Although the use of pure organic solvents may not be such a severe problem with cross-linked protein phases such as AGP, it would not be suitable for other phases such as human serum albumin. The slurry is next transferred to a stainlesesteel tubing reservoir (40mm X 6 mm i.d.1. The lower end of the reeervoir is connected to the inlet of the capillary,which is retained in a reducing union with a Vespel ferrule (Chromatographic Specialties,Brockville ON, Canada). The slurry is then pumped into the capillary at a pressure of ca. 6000 psi using a HPLC column slurry packer. A 40x magnification microscope is wed to check the capillary for blockages and voids in the packing. After the desired length of capillary has been packed, the pump is switched off. A retaining frit is then made -17 cm from the end frit. The capillary is first gently heated for a few seconds at the desired site for the frit to dry the packing. Rapid heating should be avoided, since this leads to a local disruption of the packing due to violent boilingof the buffer in the capillary.Then, the retaining frit is sintered by heating in the middle of the flame for ca. 6 s. Localizationof the heating is achieved by placing the capillary behind a 4-mm-diameter hole in an aluminum plate, mounted next to a bunsen burner with a low flame. A low-pressure air jet is used to direct the flame through the hole in the plate and onto a localized region of the capillary. Before use, the packed capillary is flushed with mobile phase from both ends wing the column packer, and the polyamide coating is burned away 1-2 cm downstream of the retaining frit to make a detection window. It should be pointed out that in this case the frit is made by heating the packing material itself. This is quite effective with the AGP packing, but not with some other materials such as Pirkle-type chiralstationaryphases. In theaecases,ashortlength of capillary is packed with silica gel, after the chromatographic stationary phase, and then this is heated to make a frit. The successful production of frits with the AGP material is probably due to some reaction of the bonded phase itself.
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(25) Venele, M.;Dewaele, C. In Microbore Column Chromatography; Yang, F. J., Ed.; Marcel Dekker; New York, 1989; pp 37-66.
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ANALYTICAL CHEMISTRY, VOL. 85, NO. 24, DECEMBER 15, 1993
RESULTS AND DISCUSSION Properties of Electroosmosis. Electroosmotic flow is a fundamental process in electrochromatography, acting as the driving force for the separation. The behavior of electroosmotic flow in capillaries packed with silica gel and ODSbonded silica gel has been e ~ a m i n e d ~ ~and - ~ ~is@ broadly similar to electroosmosis in open-tubular capillaries. However, as Knox and Grant23 have pointed out, the magnitude of electroosmosis is expected to be decreased due to nonalignment of the flow channels in the packed bed with the capillary axis and to a lack of electroosmosis within microporous particles. Further modification of the electroosmotic flow may come about because of alteration of the {-potential at the packing surface because of the presence of the bonded phase. Previous articles have discussed the use of capillaries packed with silica, or silica modified with an uncharged bonded phase.22-24p26 In the work reported here, the capillary is packed with AGP-coated silica gel. As previously mentioned, AGP has an isoelectric point of 2.7. Thus it is expected that, over the normal operating pH range (3.0-7.5) of AGP stationary phase,27both capillary walls and the immobilized protein are negatively charged, although the charge density would reduce significantly at the low-pH end of this range. However, the presence of the ionic bonded phase may be expected to cause some differences in electroosmotic flow behavior in these capillaries when compared with silica- or ODS-packed capillaries. The velocity of electroosmotic flow in AGP-packed capillaries is altered by changing operating parameters which could affect {-potential, double-layer thickness, or solution viscosity. The effects of field strength, pH, and organic modifier on the velocity of electroosmotic flow were investigated in this study. The effect of field strength on electroosmotic velocity was studied by varying field strength from 120 to 600 V cm-l. Velocity measurements were made in mobile phases containing 2 5% 1-propanol, 2-propanol, and acetonitrile. These are concentrations of modifier which might typically be used on the AGP column. A low buffer concentration (2 mM phosphate) was chosen in order to avoid high currents at higher field strengths. It was noted that when applied field strengths were higher than 600 V cm-' and currents higher than 3 or 4 PA, the electroosmotic flow completely stopped, due to the formation of bubbles in the packed capillary. It has been suggested that the packing provides nucleation sites, which aids bubble formation.23It is also possible that constrictions in the current path occur, for example at the frits, leadingto locally high current densities and thus localized heating. With each mobile phase, the velocity of electroosmotic flow was found to vary linearly with electric field strength over the range 120-600 V cm-l. The maximum current was 3 pA, at the highest field strength. In open-tubular capillaries, plots of electroosmosisversus field strength may show positive deviations from linearity at high field and similar curvature has also been observed for packed c a p i l l a r i e ~ . ~ The ~ * positive ~~ curvature has been interpreted as a result of the increased mobilities due to increasedtemperatures at high currents leadingto a reduction in solution viscosity. For a 375-pm-0.d. open-tubular capillary with only convective cooling, the overall temperature rise has been estimated at 14EI (E is the field strength, V m-l, and I the current flowing in the capillary in A).23 This would suggest a maximum increase in temperature of 2.5 OC for the measurements made here. In fact, the temperature rise is likely to be considerably smaller than this value, since the packed capillary is mounted within the oven of the AB1 270A ~
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(26) Knox, J. H.Chromatographia 1988,26,329-337. (27) Schill, G.; Wainer, I. W.; Barrkan, S. A. J . Chromatogr. 1986,365, 73-88. (28) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985,57,834-841.
0.0006 Mobility (cmy/Vs) 0.0004
0.0002 I 4.5
5.5
6.5
7.5
PH
Flgure 1. Dependenceof electroosmotic mobillty on p H capillary 42 cm X 50 pm I.d., 17 cm packed with AOP statlonary phase; moblle phase 2 mM phosphate buffer/:!% 2-propanol; applled voltage 18 kV. (B) l-propanol and (A)2-propanol.
CE instrument, with forced-air temperature control. No description of temperature control is given in previous work,23.24 and we conclude that our observation of linearity in the variation of electroosmotic flow over a range of field strengths similar to those used by previous workers who observed nonlinear response^^^.^^ is due to improved heat dissipation. As in HPLC, in electrochromatography both the retention and selectivity can be controlled by adding organic modifiers in the running buffer or by changing the buffer pH. The electroosmotic flow velocity is dependent on the ratio of the mobile-phase dielectric constant to viscosity. The addition of organicsolvent will change these properties, and therefore, it is important to investigate the effect of organic modifier on the electroosmosis. Measurements of electroosmosiswere made as a function of pH, with different organic modifiers, and as a function of organic modifier concentration. The electroosmoticflowas a function of pH is open-tubular and AGP-packed capillaries was compared. The capillary used in both cases came from the same reel. Both capillaries were 42 cm long (20 cm to the detector); one contained a 17-cm length of AGP packing material. The buffers used comprised 2 mM sodium phosphate over a pH range 4.457.5, with 2% (v/v) 1-and 2-propanol. The results are shown in Figure 1. With 2-propanol as an additive in an open-tubular capillary,the electroosmoticflow reduced almost linearlywith pH over the pH range studied. Electroosmosis with l-propan01was significantly different, being higher than that with 2-propanol at all pHs. The velocity of the electroosmotic flow in the AGP-packed capillary decreases rapidly with decreasing pH with 2-propanol as additive, but varies only slightly over the pH range used here with 1-propanol as an additive. With 1-propanol, the pH dependence of electrooemosis mirrored that seen in the open-tubular capillary, with values at each pH being from 37 to 38% of the open-tubular measurements. However, for 2-propanol this is not the case. At pH 4.5 the electroosmotic flow in the packed capillary is only 12% of its open tubular value, and there is a proportional increase up to 39 5% of the open-tubular value at pH 7.5. With silica- and ODS-packed capillaries it has previously been predicted theoretically and confirmed experimentally that electroosmotic flow is in the range 4040% of open-tubular values.23 The effects seen here are probably due differences in the surface properties of the AGP caused by the two different organic modifiers. To determine the effect of the concentration of different organic modifiers, experiments were carried out with the addition of methanol, acetonitrile, 1-propanol,and 2-propanol in 2 mM phosphate buffer (pH 6.80). It was noted that the overalltrend is the same for all of these solvents, with a steady decrease in the velocity of electroosmotic flow with an
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increasing fraction of the organic solvent. For methanol, l-propanol, and 2-propanol there is a steep decrease in electroosmotic flow with increasing percentage of organic solvent, and at 30% modifier, the velocity of electroosmotic flow is reduced to '/2-l/3 of the value with no organic additive. For acetonitrile there is a smaller decrease, with the velocity reduced by only one-fourth on addition of 30% acetonitrile. The present results are similar to those obtained in opentubular capillaries.*,30 It has been observed that tpotential induced between the inner wall of a fused-silica capillary and an aqueous solution of organic solvent decreased almost linearly with increasing mole percentage of organicsolvent.m@ Schwer and Kenndler30 indicated that, with the exception of aqueous acetonitrile, aqueous solutions of protic and aprotic solvents show a steep decrease of the ratio of dielectric constant to viscosity with increasing organic solvent fraction up to -40% ;however, adding acetonitrile leads to a smaller decrease in this ratio. The magnitude of the electroosmotic velocity measured using acetonitrile, methanol, and 1-and 2-propanol varies in the same order as the ratios of the dielectric constant to the viscosity of both the pure solvent (values of 110.3,59.3,8.8, and 6.4, respectively), and also of the buffer/solvent mixtures (as shown in ref 30). The effect of electrolyte concentrationon the electroosmotic behavior in the AGP-packed capillary was studied at pH 6.8, with 2% l-propanol organic additive. The electrolyte concentration was changed from 0.5 to 10mM sodium phosphate. The electroosmoticflow mobility reduced from (2.81 A 0.03) X 10-4 cm2 V-' s-l at a concentration of 0.5 mM to (2.53 f 0.02) X 10-4 cm2 V-l s-1 with 10 mM phosphate. In opentubular capillaries, electroosmoticmobility is inversely proportional to the square root of the buffer ~oncentration;~~ however, the reduction in electroosmosis seen here does not follow quite the same behavior, with a relatively smaller effect of buffer concentration. Maintenance of a strong electroosmoticflow is important in electrochromatography, particularly if smaller diameter packing particles are used.23 For reversed-phase electrochromatographic separations, acetonitrile is the organic modifier of choice from the point of view of maintaining a strong electroosmosis, particularly when high percentages of organic modifier are needed. Also, the useful operating pH range may be limited by the need to maintain an appropriate electroosmoticflow. These two factors may limit the applications of electrically driven protein-based CSPs, since the correct choice of pH and type of organic modifier is oftan critical in achieving a chiral separation. pH limitations may be more important, since usually only relatively low (
