Chiral Monolithic Columns for Enantioselective Capillary

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Anal. Chem. 2000, 72, 4623-4628

Chiral Monolithic Columns for Enantioselective Capillary Electrochromatography Prepared by Copolymerization of a Monomer with Quinidine Functionality. 2. Effect of Chromatographic Conditions on the Chiral Separations Michael La 1 mmerhofer, Frantisek Svec, and Jean M. J. Fre´chet*

Department of Chemistry, University of California, Berkeley, California 94720-1460 Wolfgang Lindner

Institute of Analytical Chemistry, University of Vienna, A-1090 Vienna, Austria

The effect of chromatographic conditions on the performance of chiral monolithic poly(O-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine-co-ethylene dimethacrylate-co-2-hydroxyethyl methacrylate) columns in the capillary electrochromatography of enantiomers has been studied. The flow velocity was found to be proportional to the pore size of the monolith and both the pH and the composition of the mobile phase. The length of both open and monolithic segments of the capillary column was found to exert a substantial effect on the run times. The use of monoliths as short as 8.5 cm and the “short-end” injection technique enabled the separations to be achieved in ∼5 min despite the high retentitivity of the quinidine selector. Very high column efficiencies of close to 250 000 plates/m and good selectivities were achieved for the separations of numerous enantiomers using the chiral monolithic capillaries with the optimized chromatographic conditions. Research efforts in enantioselective capillary electrochromatography (CEC) have primarily been focused on the modification of well-established approaches known from the field of HPLC and their transfer to the capillary format. The most common technology uses capillary columns packed with conventional particulate chiral stationary phases (CSP)smostly modified silica beads. A wide variety of the enantioselective HPLC packings with typical selectors such as proteins,1,2 derivatized cyclodextrin,3-6 anitibiotics such as vancomycin and teicoplanin,7-9 quinine-derived chiral (1) Li, S.; Lloyd, D. K. Anal. Chem. 1993, 65, 3684-3690. (2) Lloyd, D. K.; Li, S.; Ryan, P. J. Chromatogr., A 1995, 694, 285-296. (3) Li, S.; Lloyd, D. K. J. Chromatogr., A 1994, 666, 321-335. (4) Lelie`vre, F.; Yan, C.; Zare, R. N.; Gareil, P. J. Chromatogr., A 1996, 723, 145-156. (5) Wistuba, D.; Czesla, H.; Roeder, M.; Schurig, V. J. Chromatogr., A 1998, 815, 183-188. (6) Wistuba, D.; Schurig, V. Electrophoresis 1999, 20, 2779-2785. (7) Dermaux, A.; Lynen, F.; Sandra, P. J. High Resolut. Chromatogr. 1998, 21, 575-576. (8) Carter-Finch, A. S.; Smith, N. W. J. Chromatogr., A 1999, 848, 375-385. 10.1021/ac000323d CCC: $19.00 Published on Web 09/02/2000

© 2000 American Chemical Society

anion exchangers,10-12 and Pirkle’s brush-type selectors 13 have been tested for CEC. Similarly, silica particles coated with polymeric chiral phases based on polyacrylamide,14 poly(diphenyl2-pyridylmethyl methacrylate),15 and polysaccharides14,16 have recently been evaluated for CEC enantioseparations. Yet another approach involves the use of irregular polymeric particles prepared by molecular imprinting and packed into capillary columns.17 Column effciencies ranging from 20 000 to 100 000 theoretical plates/m were found for the majority of the enantioseparations reported to date. Currently, the highest efficiencies of ∼200 000 plates/m were obtained for enantioselective CEC with capillaries packed with “Pirkle-type” CSPs.13 All of these data indicate that more efficient chiral separations can be achieved using CEC than is the case with conventional and µ-HPLC. The feasibility of open-tubular formats for the electrically driven separation of enantiomers has been demonstrated with fused-silica capillaries coated with cyclodextrin derivatives,18-20 cellulose derivatives,21 terguride,22 and molecularly imprinted polymers23 and with capillaries covered with adsorbed lysozyme layer24 or (9) Wikstro¨m, H.; Svensson, L. A.; Torstensson, A.; Owens, P. K. J. Chromatogr., A 2000, 869, 395-409. (10) La¨mmerhofer, M.; Lindner, W. J. Chromatogr., A 1998, 829, 115-125. (11) La¨mmerhofer, M.; Tobler, E.; Lindner, W. J. Chromatogr., A 2000, 877, 421-437. (12) Tobler, E.; La¨mmerhofer, M.; Lindner, W. J. Chromatogr., A 2000, 875, 341-352. (13) Wolf, C.; Spence, P. L.; Pirkle, W. H.; Derrico, E. M.; Cavender, D. M.; Rozing, G. P. J. Chromatogr., A 1997, 782, 175-179. (14) Krause, K.; Girod, M.; Chankvetadze, B.; Blaschke, G. J. Chromatogr., A 1999, 837, 51-63. (15) Krause, K.; Chankvetadze, B.; Okamoto, Y.; Blaschke, G. Electrophoresis 1999, 20, 2272-2778. (16) Mayer, S.; Briand, X.; Francotte, E. J. Chromatogr., A 2000, 875, 331-339. (17) Lin, J.-M.; Uchiyama, K.; Hobo, T. Chromatographia 1998, 47, 625-629. (18) Mayer, S.; Schurig, V. J. High Resolut. Chromatogr. 1992, 15, 129-131. (19) Armstrong, D. W.; Tang, Y.; Ward, T.; Nichols, M. Anal. Chem. 1993, 65, 1114-1117. (20) Schurig, V.; Wistuba, D. Electrophoresis 1999, 20, 2313-2328. (21) Francotte, E.; Jung, M. Chromatographia 1996, 42, 521-527. (22) Sinibaldi, M.; Vinci, M.; Federici, F.; Flieger, M. Biomed. Chromatogr. 1997, 11, 307-310. (23) Tan, Z. J.; Remcho, V. T. Electrophoresis 1998, 19, 2055-2060.

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modified with (R)-1-(R-naphthyl)ethylamine.25 Although efficient enantiomer separations are feasible on each of these columns, both the amount of adsorbed or bonded chiral selector and the sample loading capacity are low, thus requiring high-sensitivity detection. Monolithic stationary phases for enantioselective CEC remain less common, and only a few examples have been reported until recently. Koide and Ueno prepared charged cross-linked polyacrylamide gels covalently attached to the capillary wall with physically entrapped26 or covalently bonded β-cyclodextrin derivatives.27,28 Nilsson’s group used “superporous” molecularly imprinted monoliths prepared directly within vinylized capillaries.29-33 Capillary columns filled with irregular particles of molecularly imprinted polymer embedded in polyacrylamide gels34 or siliceous matrix35 have also been tested for enantioselective CEC. In our previous study, we prepared monolithic capillaries for chiral separations by copolymerization of a valine-based monomer with a negatively charged monomer (2-acrylamido-2-methyl-1propanesulfonic acid), ethylene dimethacrylate, and butyl or glycidyl methacrylate. These columns enabled the separation of enantiomers with efficiencies of up to ∼60 000 plates/m.36 In the previous part of this study,37 we used a quinidine carbamate-based monomer that combines the structural features required for both EOF generation (basic quinuclidine moiety) and chiral recognition.38-40 In this single molecule, the cationic quinuclidine provides ionic interaction, the carbamate group contains hydrogenbonding sites, the quinoline moiety represents a π-π-binding site, and bulky groups afford steric interactions. Monolithic columns were prepared in a single step through thermal- or UV-initiated polymerization processes involving this chiral monomer, a polar comonomer, and a cross-linker in the presence of porogens. As a result of the polyvalent nature of the quinidine derivative, only three different types of monomers, with one of them being chiral, are needed to prepare enantioselective monoliths for CEC. A series of experiments allowed us to optimize the polymerization conditions that afford chiral monolithic columns with material properties well suited for CEC. In this report, we now discuss the effects of the separation variables on EOF velocity, enantioselectivity, column efficiency, and speed of separations. (24) Liu, Z.; Zou, H. F.; Ni, J. Y.; Zhang, Y. K. Anal. Chim. Acta 1999, 378, 73-76. (25) Pesek, J. J.; Matyska, M. T.; Menezes, S. J. Chromatogr., A 1999, 853, 151158. (26) Koide, T.; Ueno, K. Anal. Sci. 1998, 14, 1021-1023. (27) Koide, T.; Ueno, K. J. High Resolut. Chromatogr. 2000, 23, 59-66. (28) Koide, T.; Ueno, K. Anal. Sci. 1999, 15, 791-794. (29) Schweitz, L.; Andersson, L. I.; Nilsson, S. Anal. Chem. 1997, 69, 11791183. (30) Schweitz, L.; Andersson, L. I.; Nilsson, S. J. Chromatogr., A 1997, 792, 401409. (31) Nilsson, S.; Schweitz, L.; Petersson, M. Electrophoresis 1997, 18, 884-890. (32) Schweitz, L.; Andersson, L. I.; Nilsson, S. J. Chromatogr., A 1998, 817, 5-13. (33) Schweitz, L.; Andersson, L. I.; Nilsson, S. Chromatographia Suppl. I 1999, 49, S-93-S-94. (34) Lin, J. M.; Nakagama, T.; Uchiyama, K.; Hobo, T. Chromatographia 1996, 43, 585. (35) Chirica, G.; Remcho, V. T. Electrophoresis 1999, 20, 50-56. (36) Peters, E. C.; Lewandowski, K.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Commun. 1998, 35, 83-86. (37) La¨mmerhofer, M.; Peters, E. C.; Yu, C.; Svec, F.; Fre´chet, J. M. J.; Lindner, W. Anal. Chem., preceding paper in this issue. (38) La¨mmerhofer, M.; Lindner, W. J. Chromatogr., A 1996, 741, 33-48. (39) Maier, N. M.; Nicoletti, L.; La¨mmerhofer, M.; Lindner, W. Chirality 1999, 11, 522-528. (40) Schefzick, S.; Lindner, W.; Lipkowitz, K. B. Chirality 2000, 12, 7-15.

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EXPERIMENTAL SECTION Materials and Methods. Suppliers and/or the preparation methods for the analytes N-3,5-dinitrobenzoyl (DNB), N-benzoyl (Bz), N-acetyl (Ac), N-9-fluorenylmethoxycarbonyl (Fmoc), N-3,5dinitrobenzyloxycarbonyl (DNZ), N-benzyloxycarbonyl (Z), and N-2,4-dinitrophenyl (DNP) derivatives of leucine (Leu), valine (Val), phenylalanine (Phe), serine (Ser), and glutamine (GlN), respectively, as well as 2-(4-chloro-2-methylphenoxy)propionic acid (Mecoprop) and 2-(2,4,5-trichlorophenoxy)propionic acid (Fenoprop) were described elsewhere.38,41 Fused-silica capillaries (100µm i.d.) with conventional polyimide coating or UV-transparent coating were obtained from Polymicro Technologies (Phoenix, AZ). A description of the preparation of the monolithic capillary columns by in situ free-radical copolymerization of O-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine (1), 2-hydroxyethyl methacrylate (HEMA), and ethylene dimethacrylate (EDMA) can be found in the first part of this study.37 CEC Experiments. CEC experiments were carried out using a HP 3DCE capillary electrophoresis instrument (Hewlett-Packard) equipped with a diode array detector and external pressurization system. An external pressure of 0.6 MPa (87 psi) was applied at both ends of the capillary column. Unless otherwise specified, the separations were performed with a typical nonaqueous standard mobile phase consisting of 0.4 mol/L acetic acid and 4 mmol/L triethylamine solution in an acetonitrile/methanol mixture (80: 20, v/v). The sample solutions (0.5 mg/mL) were injected electrokinetically using -15 kV for 5 s, and the separations were performed at a voltage of -25 kV at 50 °C. The peaks were monitored at 250 nm using ChemStation software. Acetone was used as the EOF marker. RESULTS AND DISCUSSION Flow-Through Monoliths under CEC Conditions. The quinidine moieties of the selector involve two basic sites: (i) quinoline, which is an aromatic amine, and (ii) quinuclidine with a tertiary amine group. The pKa values for both these groups of quinidine are reported in The Merck Index to be 5.4 and 10.0, respectively. As a result, the chiral monolithic columns operate best in acidic mobile phases, since the monolith surface is positively charged under these conditions. Due to the cationic nature of the quinidine functionality, the positive ζ-potential drives EOF toward the anode. Figure 1shows the effect of the pH of the mobile phase on electroosmotic mobility (µeo). Since the monolithic stationary phase is a weak anion exchanger, the ionization and consequently the electroosmotic flow vary within the studied pH range in relation to the pKa values of the charged sites. The negative values of µeo indicate that the anodic flow increases as the protonation of the weak anion-exchange functionalities increases. It should be pointed out that anodic flow is advantageous in the separation of anionic analytes, since both electroosmotic and electrophoretic transports have the same direction and the electrophoretic migration may help to accelerate the separation. According to the theory of CEC developed by Knox,42,43 the electroosmotic flow velocity in columns packed with beads is independent of the particle size. Since there are always voids (41) La¨mmerhofer, M.; Maier, N. M.; Lindner, W. Am. Lab. 1998, 30, 71-78. (42) Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 317-325. (43) Knox, J. H. J. Chromatogr., A 1994, 680, 3-13.

Figure 1. Effect of pH of the mobile phase on electroosmotic mobility in quinidine-functionalized chiral monoliths. Conditions: polymerization mixture, chiral monomer 8 wt %, 2-hydroxyethyl methacrylate 16 wt %, ethylene dimethacrylate 16 wt %, dodecanol 32 wt %, and cyclohexanol 28 wt %; UV-initiated polymerization for 16 h at room temperature; capillary column, 335 mm (250-mm active length) × 0.1 mm i.d.; EOF marker, acetone; mobile phase, 80:20 acetonitrile/ 0.1 mol/L 4-morpholinoethanesulfonic acid; apparent pH adjusted with triethylamine; voltage, -15 kV; separation temperature, 20 °C; injection at -5 kV for 5 s.

Figure 2. Variation of linear flow velocity as a function of pore size. Conditions: polymerization mixture, chiral monomer 8 wt %, 2-hydroxyethyl methacrylate 16 wt %, ethylene dimethacrylate 16 wt %, and porogenic solvent 60 wt % (consisting of 1-dodecanol and cyclohexanol in different proportions); UV-initiated polymerization 16 h at room temperature (0) and thermally initiated polymerization 20 h at 60 °C (9); capillary columns, 335 mm (250-mm active length) × 0.1 mm i.d.; EOF marker, acetone; mobile phase, 0.4 mol/L acetic acid and 4 mmol/L triethylamine in 80:20 acetonitrile/methanol; separation temperature, 50 °C; voltage, -25 kV.

between the particles of the packing material, and the size of the voids is related to that of the particles, this also implies that CEC does not depend on the size of these voids unless the electrical double layers overlap. When applied to the monolithic technology, this infers the independence of EOF on the pore size. However, we have previously found44,45 that this is not the case and that monoliths with larger pore diameters afford higher linear flow velocities. Despite the completely different chemistry used in this study, the effect of pore size on flow velocity shown in Figure 2is again quite noticeable. This dependence of flow velocity on pore size appears to be characteristic of rigid porous polymer monoliths for CEC. Although no direct evidence is currently available, the (44) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1998, 70, 2288-2295. (45) Yu, C.; Svec, F.; Fre´chet, J. M. J. Electrophoresis 2000, 21, 120-127.

Figure 3. Column efficiencies for S-enantiomer determined from DNZ-(R,S)-Leu separations on quinidine-functionalized monoliths as a function of pore diameter. Conditions: polymerization mixture, chiral monomer 8 wt %, 2-hydroxyethyl methacrylate 16 wt %, ethylene dimethacrylate 16 wt %, and porogenic solvent 60 wt % (consisting of 1-dodecanol and cyclohexanol in different proportions); UV-initiated polymerization for 16 h at room temperature (0) and thermally initiated polymerization for 20 h at 60 °C (9); capillary columns, 335 mm (250mm active length) × 0.1 mm i.d.; mobile phase, 0.4 mol/L acetic acid and 4 mmol/L triethylamine in 80:20 acetonitrile/methanol; separation temperature, 50 °C; voltage, -25 kV.

decrease in flow velocity with pore size can be related to an overall increase in resistance to flow through the tortuous channels of the monolithic column. The effects of both tortuosity of the packed structure and variations in cross-sectional area on the conductance of CEC capillaries packed with beads have recently been reported by Horva´th.46 Another possible reason for this behavior is the increase in channel length that may be expected for monolithic columns with smaller pores. This leads to a decrease in the strength of the electric field E that is defined as applied voltage per total length of the separation channel. Since the electroosmotic velocity ueo is directly proportional to E, the compounds move through the monolith slower. In addition to the slower flow velocity enacted by lower E, the more tortuous path the compounds have to traverse is longer, and therefore, the time required to traverse this distance is also greater. Chromatographic Properties of the Monoliths. Effect of Pore Size. Consistent with our previous findings using different systems,44,45,47 the column efficiency of quinidine-functionalized monolithic capillaries clearly depends on the pore size. Figure 3illustrates that this holds for monoliths prepared by either thermal or UV initiation. As previously found for the reversedphase separations of alkylbenzenes, the effect of pore size on the separation of enantiomers is also rather complex. The dependency of column efficiency on pore size again exhibits two distinct maximums, one at ∼1000 nm and the second in the range 400500 nm, with a minimum efficiency at ∼900 nm. The origin of these curves with dual maximums is currently not completely clear. We assume that the unusual shape of these curves is a reflection of changes in the overall morphology within the monoliths rather than the effect of chemical functionality since all of the monoliths are chemically identical. The mode pore sizes (46) Rathore, A. S.; Wen, E.; Horva´th, C. Anal. Chem. 1999, 71, 2633-2641. (47) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3646-3649.

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Table 1. Effect of Acetonitrile/Methanol Ratio (%, v/v) on CEC Enantioseparations of Selected Analytesa DNP-Val MeOH (%, v/v)

current (µΑ)

u (mm/s)

keff (R)

keff (S)

R

0 20 30 40 50 60 70 80 100

-6.2 -4.0 -6.1 -8.3 -9.5 -10.5 -11.7 -11.8 -8.8

1.35 0.81 0.68 0.60 0.52 0.48 0.44 0.41 0.35

16.49 15.90 10.42 7.61 6.59 5.91 5.76 6.30 10.03

20.26 19.89 13.24 9.79 8.52 7.69 7.55 8.28 13.28

1.23 1.25 1.27 1.29 1.29 1.30 1.31 1.32 1.32

Fmoc-Leu

RS

N(R) (m-1)

N(S) (m-1)

keff (S)

keff (R)

R

RS

N(S) (m-1)

N(R) (m-1)

4.41 4.02 7.88 8.70 9.59 9.36 9.39 9.70 9.07

29 874 20 430 77 970 89 841 107 063 97 830 95 359 97 470 60 859

30 889 22 478 74 833 89 030 107 796 100 596 96 348 97 519 67 393

4.94 3.99 2.87 2.08 2.00 1.67 1.63 1.87 2.22

5.41 4.53 3.25 2.38 2.32 1.96 1.93 2.23 2.67

1.10 1.13 1.13 1.15 1.16 1.17 1.19 1.19 1.20

1.19 2.21 1.97 2.35 2.81 2.94 3.37 3.85 3.71

14 396 27 741 26 770 41 167 41 981 49 256 55 804 62 667 51 804

13 815 27 626 25 222 33 922 47 141 46 622 58 000 64 422 45 926

a Polymerization mixture, chiral monomer 1 12 wt %, HEMA 20 wt %, EDMA 8 wt %, cyclohexanol 10 wt %, and 1-dodecanol 50 wt %. UV-initiated polymerization 16 h at room temperature. Capillary column 335 mm (250-mm active length) × 0.1 mm i.d., mobile phase 0.4 mol/L acetic acid and 4 mmol/L triethylamine in acetonitrile/methanol, capillary temperature 50 °C, and voltage: -25 kV.

used for the plot, determined by mercury porosimetry, indicate only the positions of the maximums (modes) of the pore size distribution curves rather than the frequency of occurrence of all of the pores. As discussed in the first part of this study, monoliths with smaller and smaller modal pore sizes exhibit considerable differences in their microporous structure as confirmed by an increase in their specific surface areas. Deviations from a pluglike flow profile were found to occur in the smaller transport channels,43,48 leading to decreased efficiencies. These effects would also be expected to be more prevalent for the monoliths with smaller pore diameters. Effect of Mobile Phase. The monolithic columns are chemically very stable. Therefore, the mobile-phase parameters such as apparent pH, electrolyte concentration, and type and content of organic solvents can be varied broadly without negatively affecting the stability of the monolithic chiral stationary phase. Optimization of these mobile-phase variables is another tool that enables further improvements in column efficiency and enantioselectivity. A high degree of ionization of the quinidine moieties, which is achieved in acidic mobile phases, is required to maintain high EOF and to allow for the primary anion-exchange retention mechanism. Variations of elution conditions within this range proved to be an effective means for optimization of both enantioselectivity and column efficiency. However, changes in the counterion concentration appear to be even more effective. Although substantial amounts of counterion must be used to offset the strong ionic interactions between the selector and the analyte, a further increase in the counterion concentration improves column efficiency without affecting the enantioselectivity and is consistent with previous findings.12 For example, a 2-fold increase in acetic acid concentration in the 80:20 acetonitrile/methanol mobile phase from 0.4 to 0.8 mol/L at a fixed acid/triethylamine ratio of 100:1, improves the column efficiency for the separation of DNZ-Leu enantiomers from 66 000 to a remarkable 95 000 theoretical plates/m. Although the higher acetic acid concentration generates a slower electroosmotic flow, retention times are shorter because of much lower adsorption of analytes on the ionexchange sites. Enantioselectivity is not effected by this change and remains almost constant with R ) 1.30 and 1.32, respectively. (48) Li, D.; Remcho, V. T. J. Microcolumn Sep. 1997, 9, 389-397.

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However, this increase should not exceed the specific upper limit of counterion concentration, which depends on the solvent, porous structure of the monolith, length of open and packed segments, applied voltage, and temperature, since the concomitant generation of Joule heat may create instability of current and poor reproducibility of results. Table 1illustrates the effect of the acetonitrile/methanol ratio at a constant electrolyte concentration on the CEC separations of racemic DNP-Val and Fmoc-Leu. In contrast to CEC columns packed with silica beads and functionalized with a similar selector for which the highest flow velocity was found for a 60:40 acetonitrile/methanol mixture,12 the flow velocity in the monolithic column decreases continuously in the whole range as the acetonitrile content decreases. Simultaneously, enantioselectivity for both selected racemates is higher in the methanol-rich mobile phases. Similar improvements in selectivity were also observed in HPLC separations using analogous CSPs.38,41 The mobile-phase composition also exerts a large effect on the column efficiency that reaches a maximum of 108 000 theoretical plates/m for the separation of DNP-Val enantiomers in 50:50 methanol/acetonitrile mixtures. However, this effect appears to be analyte specific, since the highest efficiency for the separation of Fmoc-Leu enantiomers is observed in the mobile phase containing 80% methanol. A further improvement in column efficiency is achieved in an aqueous/organic mobile phase of the type used in reversed-phase separations. Figure 4compares the separations of DNZ-Leu enantiomers using the nonaqueous polar organic phase consisting of acetonitrile/methanol (80:20) with that achieved in the reversedphase mode with acetonitrile/water mixture (80:20) containing the same electrolyte concentration. Although the aqueous mobile phase affords a lower flow velocity of 0.35 mm/s compared to 0.59 mm/s in the purely organic phase, this decrease is compensated by the higher elution strength of the more polar aqueous medium, resulting in lower effective retention factors. Due to the lower retention in the aqueous medium, the overall run times are of comparable length for both mobile phases. While enantioselectivity is equal in both mobile phases (R ) 1.36), column efficiency improves significantly by replacing the entirely organic with the aqueous/organic mobile phase. As shown in Figure 4, efficiency for the (R) and (S)-DNZ-Leu enantiomers increases from

Figure 4. Effect of mobile phase on the separation of DNZ-Leu enantiomers. Conditions: polymerization mixture, chiral monomer 8 wt %, 2-hydroxyethyl methacrylate 24 wt %; ethylene dimethacrylate 8 wt %, 1-dodecanol 45 wt %, and cyclohexanol 15 wt %; UV-initiated polymerization for 16 h at room temperature; pore diameter, 1265 nm’ capillary column, 335 mm (250-mm active length) × 0.1 mm i.d.; EOF marker, acetone; mobile phase, 0.4 mol/L acetic acid and 4 mmol/L triethylamine in 80:20 mixtures of acetonitrile/methanol (a) and acetonitrile/water (b); separation temperature, 50 °C; voltage, -15 kV. The arrow points at the unretained peak of void volume marker (acetone).

37 400 to 68 600 plates/m and from 46 400 to 158 300 plates/m, respectively. The nonaqueous polar organic mobile phase has the intrinsic advantage of generating much lower currents. This might be an important feature in CEC with beds packed with silica beads that contain in situ fabricated frits, since it helps to avoid problems with outgassing and bubble formation. However, this problem does not exist in monolithic columns.47,49 Therefore, the aqueous mobile phase can be safely used despite an increase in current from 2.9 to 13.5 µA. Effect of Temperature. Temperature considerably affects the separation of enantiomers. This is also true for the monolithic capillary columns. For example, resolution calculated from the enantioseparations of the DNZ-Leu model racemate slightly increases from 2.45 to 2.53 in the temperature range of 30-50 °C and then rapidly decreases to a value of 2.17 at 60 °C for the monolith containing 20% of the chiral monomer. The highest resolution achieved at 50 °C reflects the 2-fold increase in efficiency compared to the separation at 30 °C, although a slight decrease in enantioselectivity from 1.36 to 1.26 is observed as the temperature increases. Since higher temperature enhances the electroosmotic velocity and decreases the retention factors, both effects lead to an acceleration of the CEC separations. Therefore, we used a temperature of 50 °C in the majority of our experiments. Acceleration of Enantioseparations. Strong selector/analyte interactions are required to get suitable enanatioselectivities. In CEC, this is often accompanied by relatively long run times for columns with the standard bed lengths of 25 cm that is dictated by requirements of the common HP 3DCE instrumentation. Since the resolution of our monolithic columns is sufficiently high, the long run times can be decreased by simply reducing the length of the monolithic segment while increasing the length of the open (49) Chen, J.-R.; Dulay, M. T.; Zare, R. N.; Svec, F.; Peters, E. C. Anal. Chem. 2000, 72, 1224-1227.

segment as demonstrated in Figure 5. For example, a reduction of the monolithic bed length from 25 to 15 cm results in a decrease in run time from 40 to 16 min for the enantioseparation of DNZLeu. Although the resolution RS of 3.55 is lower than that of 6.78 for the 25-cm column, it is still sufficient for a very good separation as demonstrated in Figure 5b. A further reduction in the length of the monolithic segment to 8.5 cm affords RS ) 2.03, which is still adequate for practical application and also reduces the run time to only 7 min (Figure 5c). Unfortunately, due to the duplex nature of capillary columns consisting of a packed and an open segment and the specifics of the instrumentation, the conventional setup does not allow detection to be performed immediately after the monolithic segment, when capillaries with shorter 15- and 8.5cm monoliths are used. Another problem typical of this approach is the effect of the opposite charge on surfaces in packed and open segments. While the monolith surface is positively charged, the open segment has a negative surface charge due to the silanol groups of the fused-silica capillary. In theory, if the volumetric flow rate generated in the open segment were higher than that of the packed segment, flow reversal could occur. Obviously, this is not the case with our monoliths, since dissociation of the silanols is minimized in the acidic mobile phase and the monolith itself generates higher volumetric flow rates. A theoretical framework for flow velocities in open and packed segments of a CEC capillary column was recently discussed by Rathore and Horva´th.50 Another approach to short columns is the so-called “short-end injection” technique that has been advocated by Euerby for the HP 3DCE instrumentation and silica-based reversed-phase CEC with the typical cathodic flow.51 It uses the standard cassette; however, the 8.5-cm-long packed segment is at the outlet end and detection can occur immediately after the packed segment. Obviously, the sample must also be introduced through the outlet end and the electrochromatogram is developed with reversed polarity (in our case in the positive polarity mode). The electroosmotic flow is generated from the outlet end toward the inlet end. Using this technique, capillaries with a monolithic segment of 8.5 cm can be used without concomitant loss in resolution and efficiency and the run time for the present separation of DNZLeu enantiomers can be reduced to only 5 min (Figure 5d). Since this approach improves resolution to RS ) 2.13, the deleterious effect of the open segment between the end of the monolith and the detection window observed in the separation shown in Figure 5c is clearly confirmed. Examples of CEC Enantioseparations. The results summarized in Table 2show the separation of a wide variety of enantiomers of racemic analytes using a quinidine-functionalized monolithic column cross-linked with 10% ethylene dimethacrylate and optimized chromatographic conditions. Good enantioselectivity and excellent efficiencies were observed for a number of derivatized amino acids. For example, Figure 6a shows the separation of racemic DNP-Val for which very high efficiencies of 242 400 and 193 700 plates/m are achieved for the first- and second-eluting enantiomers, respectively. To our knowledge, this is currently the highest efficiency reported for enantioselective electrochromatography and reaches the levels heretofore reached (50) Rathore, A. S.; Horva´th, C. Anal. Chem. 1998, 70, 3069-3077. (51) Euerby, M. R.; Johnson, C. M.; Cikalo, M.; Bartle, K. D. Chromatographia 1998, 47, 135-140.

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Figure 5. Enantioseparation of DNZ-(R,S)-Leu on standard columns with 25- (a), 15- (b), 8.5-cm- (c) long monolith and 8.5-cm-long monolith using the short-end injection (d). Conditions: polymerization mixture, chiral monomer 12 wt %, 2-hydroxyethyl methacrylate 20 wt %, ethylene dimethacrylate 8 wt %, 1-dodecanol 50 wt %, and cyclohexanol 10 wt %; UV-initiated polymerization for 16 h at room temperature; pore diameter, 1317 nm; capillary columns, 335 mm × 0.1 mm i.d., active length 250 (a-c) and 85 mm (d); EOF marker, acetone; mobile phase, 0.4 mol/L acetic acid and 4 mmol/L triethylamine in 80:20 mixture of acetonitrile and methanol; separation temperature, 50 °C; voltage, -30 (a-c) and +30 kV (d); injection at - 15 (a-c) and + 15 kV (d) for 5 s. Table 2. Enantioseparation of Various Analytes on Quinidine-Functionalized Chiral Monolith by CECa analyte

keff 1

keff 2

R

RS

N1 (m-1)

N2 (m-1)

eob

DNB-Leu Bz-Leu Ac-Phe Fmoc-Leu Fmoc-Val Z-Phe DNZ-Phe DNP-Ser DNP-Gln DNP-Leu DNP-Val Mecoprop Fenoprop

1.49 1.10 1.72 0.84 0.97 1.46 1.91 4.02 5.47 2.38 2.43 1.46 2.47

3.90 1.30 1.96 0.93 1.15 1.56 2.58 4.68 6.19 2.96 2.95 1.63 2.88

2.62 1.18 1.14 1.11 1.19 1.07 1.35 1.17 1.13 1.24 1.21 1.12 1.17

15.11 2.26 1.89 1.17 2.11 0.98 5.14 3.54 2.77 6.87 6.28 1.87 4.03

103 393 70 560 54 313 63 880 54 920 64 953 75 073 84 840 75 360 213 380 242 400 84 060 153 320

45 093 60 147 53 220 57 907 69 527 56 373 58 460 89 013 75 367 194 647 193 713 73 000 123 067

S S S S S S S R R R R R R

a Polymerization mixture, chiral monomer 1 8 wt %, HEMA 28 wt %, EDMA 4 wt %, cyclohexanol 30 wt %, and 1-dodecanol 30 wt %. UVinitiated polymerization 16 h at room temperature. Capillary column 335 mm (150-mm active length, 250 mm from inlet to detection) × 0.1 mm i.d., mobile phase 0.6 mol/L acetic acid and 6 mmol/L triethylamine in acetonitrile/methanol (80:20), capillary temperature 50 °C, voltage -25 kV (-10 mA), and u ) 0.70 mm/s. b Elution order; configuration of first eluting enantiomer.

only with capillary electrophoresis.52 This chiral monolithic column also enables the separation of analytes that do not contain a strong π-acidic functionality such as N-benzoylleucine (Figure 6b) with a good selectivity. In addition, molecules such as the R-aryloxycarboxylic acid herbicides Mecoprop and Fenoprop are also well separated. Figure 6c shows as an example the separation of two Fenoprop enantiomers with efficiencies of 153 200 and 123 100 plates/m. CONCLUSION In addition to suitability for the reversed-phase separations that we demonstrated recently, monolithic columns prepared by the in situ polymerization of quinidine-functionalized chiral monomer are also well suited for the preparation of CEC columns for enantioseparations. This technique enables easy adjustment and fine-tuning of porous properties and good control over the surface chemistry through chemical composition of the porous polymer. As a result, the chromatographic properties can be readily (52) La¨mmerhofer, M.; Zarbl, E.; Lindner, W. J. Chromatogr., A, in press.

4628 Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

Figure 6. CEC separations of DNP-Val (a), Bz-Leu (b), and Fenoprop (c) enantiomers on a 150-mm-long quinidine-functionalized chiral monolith. Conditions: polymerization mixture, chiral monomer 8 wt %, 2-hydroxyethyl methacrylate 28 wt %, ethylene dimethacrylate 4 wt %, 1-dodecanol 30 wt %, and cyclohexanol 30 wt %; UV-initiated polymerization for 16 h at room temperature; pore diameter, 1097 nm; capillary column, 335 mm (250-mm active length) × 0.1 mm i.d.; EOF marker, acetone; mobile phase, 0.6 mol/L acetic acid and 6 mmol/L triethylamine in 80:20 mixture of acetonitrile and methanol; separation temperature, 50 °C; voltage, -25 kV.

optimized. Remarkably high plate numbers of close to 250 000 m-1 were easily achieved with optimized monolithic columns. This represents a substantial improvement over the current state of the art in CEC enantioseparation. This efficiency is particularly impressive when compared to the efficiencies typical of conventional packed HPLC columns. ACKNOWLEDGMENT Support of this research by a grant of the National Institute of General Medical Sciences, National Institutes of Health (GM48364) and by the Division of Materials Sciences of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 is gratefully acknowledged. M.L. is grateful to the Max Kade Foundation for granting a research scholarship.

Received for review March 17, 2000. Accepted June 28, 2000. AC000323D