Anal. Chem. 1997, 69, 1179-1183
Capillary Electrochromatography with Predetermined Selectivity Obtained through Molecular Imprinting Leif Schweitz,† Lars I. Andersson,‡ and Staffan Nilsson*,†
Division of Technical Analytical Chemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden, and Department of Bioanalytical Chemistry, Astra Pain Control, S-151 85 So¨ derta¨ lje, Sweden
This article presents a novel approach to capillary electrochromatography by which predetermined selectivity is achieved. A simple and quick method for the in situ preparation of monolithic molecularly imprinted flowthrough polymers inside fused silica capillaries is described. The superporous structure of the polymers permits rapid solvent and electrolyte exchange, as well as easy regeneration of the capillaries by hydrodynamic pumping. Chiral stationary phases were prepared by molecular imprinting of the β-adrenergic antagonists propranolol and metoprolol. The separation systems were operational within 3 h of the start of capillary preparation. Chiral separations with baseline resolution could be carried out in less than 120 s. One of the great challenges facing the pharmaceutical industry today is that of satisfying the need for new and improved means for the enantioselective preparation, purification, and analysis of optically active compounds. The primary motivation for scientific and technological efforts here is to be found in the frequently differing pharmacological and pharmacokinetic profiles of the enantiomers of chiral drugs. Enantiomer-dependent differences in biological activity are well documented, the horrific consequences of enantiomeric contamination of thalidomide during the 1960s being particularly dramatic.1 The β-adrenergic antagonist propranolol was launched as a racemate, as most new drugs were until recent years, despite the S-form being approximately 100 times as potent as the R-form.2 Capillary electrophoresis (CE) and capillary electrochromatography (CEC)3,4 are important analytical tools in a number of application areas, including chiral separation. CE and CEC provide a high degree of separation efficiency, short separation times, and a minimal consumption of materials and chemicals. This leads to cost-effective and environmentally safe operations, high throughput, and the ability to analyze samples available in only limited amounts. Several chiral selectors, such as cyclodextrins, crown ethers, chiral micelles, and proteins, have been used as mobile-phase additives and/or stationary phases in CE and CEC.5-7 In capillary affinity gel electrophoresis (CAGE), such †
Lund University. Astra Pain Control. (1) De Camp, W. H. Chirality 1989, 1, 2-6. (2) Barrett, A. M.; Cullum, V. C. Br. J. Pharmacol. 1968, 34, 43-55. (3) Behnke, B.; Bayer, E. Anal. Chem. 1995, 67, 3656-3658. (4) Lelie`vre, F.; Yan, C.; Zare, R. N.; Gareil, P. J. Chromatogr. 1996, 723, 145156. (5) Ward, T. J. Anal. Chem. 1994, 66, 633-640. (6) Terabe, S.; Otsuka, K.; Nishi, H. J. Chromatogr. 1994, 666, 295-319. ‡
S0003-2700(96)00792-5 CCC: $14.00
© 1997 American Chemical Society
chiral selectors as antibodies and proteins are immobilized in the capillary.8,9 Cyclodextrins are currently the most popular chiral selectors because of their applicability and ease of use. It is, however, impossible to predetermine the selectivity as in this study. The use of antibody- and protein-derived capillaries often suffers from preparation being complicated and stability poor, due to the tendency of air bubbles being trapped in the antibody or protein gel during preparation or electrochromatography.8,9 This causes conductivity cut off, which prevents further use of the column. The use of molecular imprinting as a general method for preparing chiral sorbents for CE and CEC would, we reasoned, overcome these difficulties. Molecular imprinting10-13 entails polymerization around the imprint species, employing monomers selected for their ability to form specific and definable interactions with the imprint molecule. The polymerization reaction fixes the functional monomers in the polymer network, their spatial position being determined by their interaction with the imprint species (Figure 1). The subsequent removal of the imprint species yields a material filled with cavities, the shape, size, and chemical functionality of which are complementary to those of the template. These cavities give rise to a specific affinity for the compound that was present during the preparation of the molecularly imprinted polymer (MIP). Thus, imprinted chiral stationary phases can be made with separative properties that are predetermined for a particular application and with a predictable elution order of the enantiomers, the imprinted enantiomer always being the one most strongly retained. The enantiomeric liquid chromatographic (LC) separation on imprinted columns of a wide variety of compounds, including drug compounds,14,15 amino acid derivatives,16,17 sugars, and sugar derivatives,18,19 has been reported. We describe here the in situ preparation of imprinted polymers, in which the MIPs are covalently linked to the inner wall of fused silica capillaries. Molecular imprinting of enantiomeric forms of (7) Novotny, M.; Soini, H.; Stefansson, M. Anal. Chem. 1994, 66, 646-655. (8) Birnbaum, S.; Nilsson, S. Anal. Chem. 1992, 64, 2872-2874 (9) Ljungberg, H.; Nilsson, S. J. Liq. Chromatogr. 1995, 18, 3685-3698. (10) Mosbach, K. Trends Biochem. Sci. 1994, 19, 9-14. (11) Wulff, G. Trends Biotechnol. 1993, 11, 85-87. (12) Shea, K. J. Trends Polym. Sci. 1994, 2, 166-173. (13) Mallik, S.; Plunkett, S. D.; Dhal, P. K.; Johnson, R. D.; Pack, D.; Shnek, D.; Arnold, F. H. New J. Chem. 1994, 18, 299-304. (14) Fischer, L.; Mu ¨ ller, R.; Ekberg, B.; Mosbach, K. J. Am. Chem. Soc. 1991, 113, 9358-9360. (15) Kempe, M.; Mosbach, K. J. Chromatogr. 1994, 664, 276-279. (16) Andersson, L. I.; Mosbach, K. J. Chromatogr. 1990, 516, 313-322. (17) Kempe, M.; Mosbach, K. J. Chromatogr. A 1995, 694, 3-13. (18) Wulff, G.; Minarik, M. J. Liq. Chromatogr. 1991, 13, 2987-3000. (19) Mayes, A.; Andersson, L. I.; Mosbach, K. Anal. Biochem. 1994, 222, 483488.
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Figure 2. Schematic representation of the preparation procedure for capillary columns with molecularly imprinted polymer. (1) A mixture of imprint molecule, functional and cross-linking monomer, radical initiator, and a solvent is prepared, and plastic tubing is placed on both ends of a capillary derivatized with [(methacryloxy)propyl]trimethoxysilane. (2) The capillary is filled with the mixture using a syringe. (3) The capillary is sealed by placing clips on the plastic tubings. The polymerization is performed under a UV source (350 nm) at -20 °C, for 80 min. (4) The polymerization reaction is interrupted by flushing remaining monomer, radical initiator, and imprint molecule out of the capillary. (5) The capillary column is then ready for CEC.
Figure 1. Schematic representation of imprint formation. A mixture of functional monomer, methacrylic acid (MAA, 1), cross-linking monomer, trimethylolpropane trimethacrylate (TRIM, 2), and the imprint molecule, in this instance (R)-propranolol (3), is prepared. As a result of noncovalent interactions between complementary chemical functionalities, the functional monomers come to be arranged around the imprint molecule. After polymerization, the imprint molecule is removed by extraction, exposing recognition sites complementary in size, shape, and chemical functionality to the imprint molecule.
the β-adrenergic antagonists (β-blockers) propranolol and metoprolol was undertaken, and the resulting polymer-filled capillaries were used for chiral separation in the CEC mode. EXPERIMENTAL SECTION Materials and Methods. Trimethylolpropane trimethacrylate (TRIM) was purchased from Aldrich. 2,2′-Azobis(isobutyronitrile) (AIBN) and racemic propranolol hydrochloride were obtained from Sigma (St. Louis, MO). R- and S-enantiomers of propranolol 1180 Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
were from ICN Biomedicals Inc. (Aurora, OH). (S)- and racmetoprolol were kind gifts from Astra Ha¨ssle AB (Mo¨lndal, Sweden). All other chemicals were purchased from Merck (Darmstadt, Germany). Fused silica capillary (TSU075, 75 µm i.d., 375 µm o.d.; Polymicro Technologies, Phoenix, AZ) was derivatized with [(methacryloxy)propyl]trimethoxysilane, according to the basic procedure developed by Hjerte´n,20 by flushing the capillary with 1 M NaOH followed by water for at least 30 min each. The capillary was filled with a solution of 4 µL of [(methacryloxy)propyl]trimethoxysilane in 1 mL of 6 mM acetic acid, and the solution was kept in the capillary for at least 1 h. The capillary was then flushed with water for several minutes and finally emptied and dried with a flow of nitrogen. The polymers were characterized by scanning electron microscopy of cross sections of a polymer-filled capillary. Preparation of Capillaries Containing Molecularly Imprinted Polymers. Imprint species (0.030 mol/L) in their free (20) Hjerte`n, S. J. Chromatogr. 1985, 347, 191-195.
Figure 3. Scanning electron micrographs of a polymer-filled capillary column. (A) Micrometer-sized globular units of macroporous molecularly imprinted polymer surrounded by 1-20 µm wide interconnected superpores. (B) A superpore, about 7 µm wide, magnified from the square present in (A). (C) The covalent attachments of the polymer to the capillary wall can be seen.
base form, initiator (AIBN, 3.6 g/L), methacrylic acid (MAA, 0.24 mol/L), and TRIM (0.24 mol/L) were dissolved in sodium-dried toluene. This mixture was degassed for 10 min by ultrasonication. A capillary (length 35 cm, i.d. 75 µm, o.d. 375 µm), with a detection window prepared by removing a part of the protecting polymer layer 8.5 cm from one end and with plastic tubing placed at both ends, was filled with the mixture using a syringe. The ends were sealed by placing clips on the plastic tubing. Polymerization was carried out at -20 °C in a freezer for 80 min under a UV source (350 nm). The capillary was then removed from the UV source and was immediately flushed by several column volumes of acetonitrile and electrolyte (acetonitrile/acetate pH 3.0 (80/20 v/v), prepared as described in the following section) (Figure 2). A reference capillary without any imprint molecule was prepared in the same way. The capillaries were stored at room temperature until use. Capillary Electrochromatography. Electrochromatographic experiments were performed with a Hewlett Packard HP 3D capillary electrophoresis system. The electrolyte was flushed through the capillary for several minutes with pressure (5-8 bar) applied. The capillary was then equilibrated until a stable current and baseline were achieved. The electrolytes were prepared by titrating 4 or 2 M acetic acid with 4 or 2 M ammonium acetate, respectively, to the desired pH value. These buffers were then mixed with acetonitrile in the proportions desired and degassed by sonication before use. Samples were dissolved in Milli-Q water to a concentration of 10 mM and were diluted in the electrolyte to the desired concentration. Prior to injection, the samples were sonicated for 10 min. Samples were injected electrokinetically. The resolution was calculated using either f/g21,22 or Rs, f being the distance from a line connecting the peaks of the eluting bands to the valley between the bands and g being the corresponding distance to the baseline. Rs, was calculated according to Rs ) (tr(b) - tr(a))/0.5(W(b) + W(a)), where tr is the retention time and W is the width at the baseline between tangents drawn to the inflection points for the peak.
RESULTS AND DISCUSSION The use of MIP-derived capillaries for electrochromatography requires that the polymer-filled capillaries possess good flowthrough properties, since before these are used the organic solvent employed for polymerization must be replaced by an electrolyte. This necessitates hydrodynamic pumping. The polymerization can be interrupted at specified time intervals by simply removing the UV source and flushing the capillary, so as to remove remaining portions of the initiator and unreacted monomers (Figure 2). By varying the duration and temperature of polymerization, polymers having the desired flow-through properties can be obtained. The preparation of MIP-filled capillaries was judged successful when the capillary was filled with a rigid, monolithic polymer (determined visually in a microscope) and flushing of the capillary column at low pressures using a syringe was possible. Careful optimization of the extent of polymerization is required, since too long a polymerization time results in a dense polymer, through which hydrodynamic pumping is not possible. Reducing the polymerization time, however, reduces the number of imprints due to there being less material in the capillary, which in turn leads to poor separation abilities of that capillary column. Once the conditions for the preparation of a particular type of MIPfilled capillary were established, the success rate was 100%. In contrast to methods for preparing continuous imprinted polymers that have been suggested previously,23-25 the method presented here does not interfere with the formation of well-defined imprints of the template molecule (see below). Electron micrographs showed there to be aggregates of micrometer-sized globular particles extending throughout the capillary (Figure 3). The aggregates were surrounded by 1-20 µm wide interconnected superpores which permitted bulk flow (21) Mayer, V. R. Chromatographia 1987, 24, 639-645. (22) Kaiser, R. E. Gaschromatographie; Geest and Portig: Leipzig, 1960. (23) Matsui, K.; Kato, T.; Takeuchi, T.; Suzuki, M.; Yokoyama, K.; Tamiya, E.; Karube, I. Anal. Chem. 1993, 65, 2223-2224. (24) Sellergren, B. J. Chromatogr. A 1994, 673, 133-141. (25) Nilsson, K.; Lindell, J.; Norrlo ¨w, O.; Sellergren, B. J. Chromatogr. A 1994, 680, 57-61.
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to occur throughout the entire capillary (the inner diameter of the capillary was 75 µm). The small size of the globular units, ∼0.5-1 µm, provides good diffusion properties and thus rapid access of the sample to the imprinted sites in the globules. This is facilitated further by the macroporous (∼200 Å) structure of the polymer.26 The covalent attachment points of the polymer to the capillary wall can be seen in Figure 3C. These are formed during polymerization when the growing polymer chains react with the [(methacryloxy)propyl]trimethoxysilane units with which the inner wall of the capillary is coated. Without such anchoring elements, the polymer was eluted from the capillary column during both hydrodynamic pumping and electrochromatography. The stability of these covalent attachments was shown by the fact that the polymer-filled capillaries were stable toward hydrodynamic pumping using pressures up to at least 600 bar. Due to the good flow characteristics of the capillary columns, electrolyte exchange could be easily carried out by hydrodynamic pumping, after which a stable current and stable UV absorption baselines could usually be obtained within 0.5-1 h. Air bubbles formed in the capillary, although representing a notorious problem in packed CEC and in CAGE,8,9 could easily be removed by hydrodynamic pumping. The enantiomers of propranolol and metoprolol were well resolved in their respective capillaries (Figures 4-6). The (R)propranolol MIP could achieve a separation of the enantiomers of propranolol with a retention time ratio of the enantiomer peaks, tr(R)/tr(S), of 1.12, resolution, f/g, of the enantiomers of propranolol of 0.98, and Rs ) 1.26. By definition, the f/g value ranges from 0 to 1, where 1 represents complete resolution of the eluting compounds. Nonracemic mixtures of propranolol (R:S of 9:1 and 99:1) were also separated (Figure 5). The enantiomers of metoprolol were resolved at tr(S)/tr(R) ) 1.08, f/g ) 0.99, and Rs ) 1.17 on the (S)-metoprolol MIP. A nonimprinted reference capillary, showed no enantiomeric separation ability. Propranolol and metoprolol were transported through the capillary by both electrophoresis and electroosmosis. The degree of electroosmotic flow was shown by mesityl oxide, a neutral marker, whose mobility was calculated to be 7.79 × 10-9 m2/V‚s under the conditions described in Figure 5. In CAGE in which cellobiohydrolase I was utilized as the chiral selector,9 the separation of rac-propranolol showed separation efficiencies similar to those observed here. The use of MIP capillaries in the present study, however, allowed the separation time to be reduced 10-fold, due mainly to the possibility of applying a higher separation voltage without destroying the capillary column. This demonstrates the advantage of superior stability, which the use of MIP capillaries has. During several weeks, the MIP capillaries were used continuously, and the separation system was able to repeat the analyses in a reproducible manner. The capillaries can be stored at ambient temperatures for at least 12 weeks without any observable changes in selectivity or in electrochromatographic performance. A partially or completely dried capillary is easily regenerated through fresh electrolyte being pumped hydrodynamically into the capillary. Previously, a general procedure had been described for the in situ preparation of continuous rods of macroporous polymer (26) Andersson, L. I. Anal. Chem. 1996, 68, 111-117. (27) Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1992, 64, 820-822. (28) Vlatakis, G.; Andersson, L. I.; Mu ¨ ller, R.; Mosbach, K. Nature 1993, 361, 645-647. (29) Andersson, L. I.; Mu ¨ ller, R.; Vlatakis, G.; Mosbach, K. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4788-4792.
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Figure 4. Separation using a capillary column containing imprints of (R)-propranolol. The electrochromatograms show the separation of 100 µM rac-propranolol (A), 50 µM (S)-propranolol (B), and 50 µM (R)-propranolol (C). The samples were injected electrokinetically (5 kV, 3 s) and were separated at a constant voltage of 30 kV (857 V/cm). Acetonitrile/4 M acetate pH 3.0 (80/20 v/v) served as the electrolyte. UV detection was carried out at 214 nm (bw 16 nm; reference 450 nm, bw 100 nm). The capillary was thermostated to 60 °C, and an overpressure of 7 bar was applied.
for LC separation.27 This method has been employed in three studies in the preparation of imprinted polymer rods for use in LC23,24 and in CE.25 Efficient bulk flow through the rods was obtained in the LC and CE modes. The enantiomeric separations recorded in the LC mode were, however, much less efficient than those obtained using traditional packed imprinted columns, and such separations were not achieved at all in the CE mode.25 This observation has been confirmed in our laboratory, where it has been found that MIP-filled capillaries prepared by that method25 fail to resolve the enantiomers of propranolol. We consider this to be due to the polymerization method necessitating the use of porogenic agents such as cyclohexanol and 1-dodecanol. These are added as pore formers so as to make the polymer rods sufficiently porous. Protic solvents, however, interfere with the ionic and hydrogen bonds, giving rise to complexation between the imprint molecule and the functional monomers. Hence, the formation of well-defined imprints with such a method is less
Figure 5. Separation of nonracemic mixtures of propranolol on a capillary column containing imprints of (R)-propranolol. The electrochromatograms show a 9:1 mixture of (R)- and (S)-propranolol (A) and a 99:1 mixture of (R)- and (S)-propranolol (B). The samples were injected electrokinetically (5 kV, 5 s) and were separated at a constant voltage of 15 kV (428 V/cm). Acetonitrile/4 M acetate pH 3.0 (80/20 v/v) served as the electrolyte. UV detection was carried out at 214 nm (bw 16 nm; reference 450 nm, bw 100 nm). The capillary was thermostated to 60 °C.
Figure 6. Separation using a capillary containing imprints of (S)metoprolol. The electrochromatograms show the separation of 100 µM rac-metoprolol (A) and 50 µM (S)-metoprolol (B). The samples were injected electrokinetically (3 kV, 3 s). A separation voltage of 5 kV (143 V/cm) was used, the capillary being thermostated to 60 °C. Acetonitrile/2 M acetate pH 3.0 (80/20 v/v) served as the electrolyte. UV detection was carried out at 195 nm (bw 10 nm; reference 450 nm, bw 100 nm).
efficient. As a rule, for noncovalent imprinting the polymerization should be performed using a porogen that is as apolar as possible.26,28,29 In the present study, we used toluene, which has (30) Kempe, M.; Mosbach, K. Tetrahedron Lett. 1995, 36, 3563-3566. (31) Kempe, M. Anal. Chem. 1996, 68, 1948-1953. (32) O’Shannessy, D. J.; Ekberg, B.; Mosbach, K. Anal. Biochem. 1989, 177, 144-149.
previously been reported to be an excellent polymerization solvent for preparation of (S)-propranolol MIPs.26 Due to its proven versatility in the molecular imprinting of (S)-propranolol,26 methacrylic acid, which has been employed widely in molecular imprinting studies,10,15-17,28,29 was employed as the functional monomer. Trimethylolpropane trimethacrylate (TRIM) has been shown to be superior, in terms of improved load capacity, selectivity, and resolving capability, to alternative cross-linking monomers in the preparation of MIPs for liquid chromatographic applications.30,31 The same observation was made for capillary electrochromatography, and TRIM was used throughout these studies. Regarding the polymerization temperature, it has been reported that a low temperature is favorable for molecular imprinting.32 In the present study, the polymerization reaction was carried out at -20 °C, which improved the column efficiency compared to that of columns prepared at room temperature. We believe that the complexes of monomers and imprint molecule present in the prepolymerization mixture are more stable at lower temperatures, due to a more favorable entropy term,32 leading to more well-defined imprints in the resultant polymer. An even lower temperature may further improve the column efficiency. To conclude, easy and rapid access to monolithic MIP-filled capillaries of predetermined specificity for CE and CEC has been demonstrated here. Preparation is simple and can be performed in any reasonably equipped laboratory environment. We believe the method is applicable to the preparation of MIP-derived capillaries generally, since it does not involve the use of any additives or any steps that may interfere with the ability of the polymerization system to form well-defined imprints. Molecular imprinting technology represents a widely applicable strategy for producing recognition systems for a predetermined ligand, and its utility has been shown repeatedly in the imprinting of a range of functionally distinct compounds.14-19,28,29 Since the total volume of the capillary is approximately 1.5 µL, the consumption of chemicals is minimal. The amount of the imprint species required per capillary column is only 45 nmol, which for propranolol and metoprolol is approximately 10 µg. This is advantageous in situations in which the analyte (the imprint species) is expensive or is otherwise difficult to obtain in large quantities. The whole process from the start of capillary preparation to having the separation system running, polymerization, exchange of solvent to electrolyte, and equilibration included, can be completed within 3 h. The chemical resistance of the polymer also permits the use of organic solvents, strong acidic and basic buffers, and high voltages, which may lead to separation times less than 120 s. ACKNOWLEDGMENT We thank Mr. Peter Alberius-Henning for the scanning electron micrographs. Financial support from the Carl Tryggers Foundation, the Crafoord Foundation, the Royal Physiographical Society of Lund, the Swedish Natural Science Research Council, the Swedish Research Council for Engineering Sciences, and Astra Ha¨ssle AB is acknowledged. This study has, in part, been presented at the Analysdagarna, June 10-13, 1996, Stockholm, Sweden, and at the 20th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 96), June 16-21, 1996, San Francisco, CA. Received for review August 6, 1996. Accepted January 2, 1997.X AC9607929 X
Abstract published in Advance ACS Abstracts, February 1, 1997.
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