Enantioseparation of tetrahydropalmatine and Tröger's base by molecularly imprinted monolith in capillary electrochromatography

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1 J. Biochem. Biophys. Methods 70 (2007) Enantioseparation of tetrahydropalmatine and Tröger's base by molecularly imprinted monolith in capillary electrochromatography Junjie Ou, Jing Dong, Tuijun Tian, Jiwei Hu, Mingliang Ye, Hanfa Zou National Chromatographic R and A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian , China Received 15 February 2006; received in revised form 13 June 2006; accepted 25 July 2006 Abstract Two chiral compounds, Tröger's base and tetrahydropalmatine, were enantioseparated on the (5S, 11S)-(-)-Tröger's base and l-tetrahydropalmatine imprinted monolithic capillary columns with CEC, respectively. The monoliths were prepared by in situ thermal-initiated copolymerization of methacrylic acid (MAA) and ethylene dimethacrylate (EDMA). After optimizing the ratio of porogens (toluene and dodecanol), the obtained monolithic capillary columns show good flow-through property and enantioselectivity. The influences of CEC parameters such as ph of the buffer, organic solvent and salt concentration on the electroosmotic flow (EOF) and recognition selectivity were systematically investigated. Under the optimal conditions, baseline resolutions of two chiral compounds were achieved. In addition, the fast separation was obtained within 4 min by applying higher voltage and assisting pressure of 6 bar Elsevier B.V. All rights reserved. Keywords: Tetrahydropalmatine; Tröger's base; Molecular imprinting polymer; Capillary monolithic column; Chiral separation; Capillary electrochromatography 1. Introduction Corresponding author. Tel.: ; fax: address: hanfazou@dicp.ac.cn (H. Zou). In many aspects enantiomers have to be dealt with as different molecular entities. This applies in particular to the enantiomers of biologically active compounds like drugs and food additives, which may have different activity and transformation profiles. Thus the enantioseparation of these chiral compounds are very important in life science and other fields. Generally, the resolution of enantiomers is performed on chiral stationary phases (CSPs) by high performance liquid chromatography (HPLC) [1 4]. Capillary electrochromatography (CEC) as a hybrid technique of HPLC and capillary electrophoresis (CE) may offer significant advantages for enantioseparation, such as high separation efficiency, short separation time and small consumption of packing materials, solvents and samples. Many CSPs used in HPLC have been adapted to the CEC mode [5 10] for enantioseparation. Molecular imprinting technique has attracted attention as a method to prepare high-selective host polymer [11 14]. The polymers with specific binding sites are prepared by the copolymerization of functional monomers and cross-linkers in the presence of the template molecules. The template molecules are subsequently removed from the polymer, leaving recognition sites complementary to the template molecules in shape and in the position of functional groups. The resultant polymers can recognize the imprinted molecules and show substrate and/or enantio-selectivity. Thus the molecularly imprinted polymer (MIP) can be classified as a specific type of CSP as novel stationary phase for the separation of racemates. To date, enantioseparations of a number of chiral compounds have been successfully performed by MIP-CEC [15 19]. Tröger's base (TB) is a concave molecule, the chirality of which results from the blocked configuration of two stereogenic nitrogen atoms. Tetrahydropalmatine (THP) containing one chiral nitrogen atom is one of the active ingredients isolated from Corydalis yanhusuo, a traditional Chinese herbal medicine [20]. Xu et al. [21] have reported that two enantiomers of dl-thp act on different targets in the central nervous system. Enantioseparation of these two basic compounds has been performed on CSPs by HPLC and CEC [22,23]. Adobo and coworkers have prepared the (5R, 11R)-(+)-Tröger's base (R-TB) imprinted polymers by bulk polymerization [24,25] for chiral separation of racemic TB with HPLC. The preparation method of bulk polymerization is time X/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi: /j.jbbm

2 72 J. Ou et al. / J. Biochem. Biophys. Methods 70 (2007) consuming and not cost-efficient. Recently, the l-thp imprinted monolith was synthesized in a stainless-steel column tube of 50 mm 4 mm I.D. by in situ polymerization in our lab [26].Inthe present works, (5S, 11S)-(-)-Tröger's base (S-TB) and l-thp were used as the template molecules, respectively, to prepare MIPs in fused-silica capillary. The obtained monoliths were evaluated by CEC and exhibited good enantioselectivity for these two basic racemates. 2. Materials and methods 2.1. Materials Racemic dl-thp and enantiomer l-thp (98%) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products of China (Beijing, China). S-TB and R-TB were obtained from Fluka (Buchs, Switzerland). Their structures are shown in Fig. 1. Ethylene dimethacrylate (EDMA) from Sigma (St Louis, MO, USA) was extracted with 10% aqueous sodium hydroxide and water, and dried over anhydrous magnesium sulfate. Methacrylic acid (MAA) from Acros (Geel, Belgium) was distilled under vacuum. 2,2 -Azobis-isobutyronitrile (AIBN) was obtained from Shanghai Chemical Plant (Shanghai, China) and recrystallized in ethanol before used. γ- Methacryloxypropyltrimethoxysilane (γ-maps) was purchased from Sigma (St Louis, MO, USA). Thiourea from Tianjin Chemical Reagent (Tianjin, China) was used as EOF marker. Acetic acid and sodium acetate were purchased from Shanghai Chemical Reagent (Shanghai, China). Fused-silica capillary with 75 μm I.D. 375 μm O.D. was purchased from the Yongnian Optic Fiber Plant (Hebei, China). Water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore Inc., Milford, MA, USA). HPLC-grade acetonitrile (ACN) was used for preparation of running buffer Preparation of monolithic capillaries for CEC The capillary was prepared according to the general procedure described elsewhere [27]. First the capillary was derivatized with γ-maps to provide anchoring sites for the polymer. After subsequent flushing with methanol for 10 min, it was dried by passage of nitrogen gas in oven. γ-maps dissolved in methanol (1:1,v/v) was injected into the capillary with a syringe. It was then kept at 40 C overnight with both ends sealed with Fig. 1. Structures of (a) Tröger's base and (b) tetrahydropalmatine. Table 1 Effect of porogen ratio on the enantiomers separation of dl-thp on the l-thp imprinted monolithic capillary column Polymers Percent of toluene in porogen (%) t d /min t l /min R s P1 4 P P P P5 20 CEC conditions: column, effect length 24.5 cm (total length 33 cm) 75 μm I.D. 375 μm O.D.; mobile phase, 10 mm acetate buffer containing 85% acetonitrile, ph 6.0; applied voltage, 10 kv; injection, 5 kv for 3 s; temperature, 25 C; detection wavelength, 214 nm. rubber. Finally, the capillary was rinsed with methanol and water successively to flush out the residual reagents and dried again. The template (l-thp), functional monomer (MAA), crosslinker (EDMA) and initiator (AIBN) were dissolved in porogenic solvents (toluene and dodecanol) to form homogenous solution. The compositions are indicated in Table 1. The solution was sonicated for 10 min and purged with dry nitrogen for 15 min to remove oxygen. The next step was to fill the capillary with the polymerization mixtures. Then both ends of the capillary were plugged with silicon rubber and the capillary was immersed in a water bath at 55 C for 10 h. The prepared MIP monolithic capillary column was washed with ACN/acetic acid (9/1,v/v) by an HPLC pump to remove the unreacted monomers, porogenic solvents and template molecules. Finally, the capillary with MIP stationary phase was equilibrated with the running buffer before separation. Similarly, the S-TB imprinted monolithic capillary was prepared according to the procedures described above Electrochromatographic experiments Capillary electrochromatography was performed on an Agilent CE system (Hewlett Packard, Waldbronn, Germany) equipped with a UV detector. Data were acquired and processed with the ChemStation software. The electrolytes composed of acetonitrile with varying concentrations of acetate buffer were filtered before use. A detection window was created by burning out a 2 3 mm segment of the polyamide outer coating at 8.5 cm from the outlet end of the monolithic capillary (total length, 33 cm). The obtained capillary column was placed in the instrument and equilibrated by applying a voltage of 10 kv until the baseline signal was stabilized. A pressure of 3 bar was applied to the both inlet and outlet vials simultaneously if not otherwise stated. The samples of 3 mg/ml were degassed by sonication, and injected electrokinetically by applying a voltage of 5 kv for 3 s in CEC. The temperature was kept at 25 C, and the detection wavelength was set at 214 nm. All data were obtained based on three runs. The retention factor (k ) can be defined as (t r t 0 )/t 0, where t r is the migration time of a solute, and t 0 is the migration time of a neutral and chromatographic nonretained solute [28]. The resolution is calculated from the equation R s =2(t 2 t 1 )/(w 1 + w 2 ), where t 1 and t 2 are the retention times of the first and second eluted enantiomers,

3 J. Ou et al. / J. Biochem. Biophys. Methods 70 (2007) Fig. 2. Effect of ph of buffer solution on the EOF( ) and retention factor of (a) d-thp( ), l-thp( ); (b) R-TB ( ), S-TB ( ) on the l-thp and S-TB imprinted monolithic capillary column, respectively. CEC conditions were the same as the Table 1 except the running buffer containing 80% acetonitrile with different ph. respectively, and w 1 and w 2 are the baseline peak widths of the first and second eluted enantiomers, respectively. 3. Results and discussion 3.1. Preparation of capillary MIP monolith For the basic template molecules, TB and THP in our cases, MAA was generally selected as the functional monomer to synthesize the MIPs, which also generates the electroosmotic flow (EOF) in CEC. The molar ratio of l-thp/maa/edma and S-TB/MAA/EDMA in polymerization solution was 1/2/13 and 1/3/20, respectively, which were the optimal composition for the preparation of imprinted column in HPLC reported by us. As the flow-through property of capillary monolithic column is very important in CEC, the optimization of volume ratio of porogenic solvents is needed [29]. Toluene and dodecanol have been used in the preparation of traditional monolithic MIP columns in HPLC, which was reported previously in our lab [30]. We also selected toluene and dodecanol as the binary porogens for the preparation of capillary monolithic columns, while the volume ratio of them had to be optimized. A series of columns were prepared, as can be seen in Table 1. When the proportion of toluene was lower than 4% in porogens, the polymer had a soft gel-like appearance. With the increase of the content of toluene, the monolithic polymer showed good flowthrough property and enantioselectivity, however, too much toluene made the polymer so dense that the electrolyte could not flow through the column. The best selectivity for THP was obtained when 12% of toluene was used for the preparation of monolith. Similarly, the optimal composition for the preparation of S-TB imprinted polymer was also acquired when the 16% of toluene was used. The relative standard deviation (RSD) values for EOF was less than 5% for three consecutive runs, which indicated the good stability of the obtained monolith. The column-to-column reproducibility was also evaluated, and the RSD for EOF was less than 8% Effect of mobile phase composition on the chiral resolution Effect of ph value of buffer The influence of ph of running buffer ranged from 4.0 to 7.0 on the resolution was studied with the mobile phase of ACNacetate buffer (80/20, v/v). Fig. 2a indicated the EOF generated by the l-thp imprinted monolithic column was increased from 7.51 to 9.11 cm 2 kv 1 min 1 with the increase of ph value. The retention factor of both d-thp and l-thp decreased with the increase of ph value, but the retention factor of l-thp decreased remarkably. Thus the resolution of racemic THP was decreased. These results may be due to both an increase of EOF and a decrease of interaction between the enantiomers of dl-thp and functional groups on the surface of polymers. The EOF on the prepared MIP monolithic column is mainly generated by the dissociation of carboxyl groups incorporated in the polymer. It is known that the pk a of carboxyl groups on the surface of polymer is approximately equal to the pk a of acetic acid, 4.75, which lead to ph-dependant EOF. With the increase of ph ranged from 4.0 to 7.0, the number of ionized carboxyl Fig. 3. Effect of content of acetonitrile on the EOF( ) and retention factor of (a) d-thp( ), l-thp( ); (b) R-TB ( ), S-TB ( ) on the l-thp and S-TB imprinted monolithic capillary column, respectively. CEC conditions were the same as the Table 1.

4 74 J. Ou et al. / J. Biochem. Biophys. Methods 70 (2007) Fig. 4. Effect of buffer concentration on the EOF( ) and retention factor of (a) d-thp( ), l-thp( ); (b) R-TB ( ), S-TB ( ) on the l-thp and S-TB imprinted monolithic capillary column, respectively. CEC conditions were the same as the Table 1. groups is increased and as a result, the EOF increased [31]. Additionally, as THP is a weak basic compound (pk a N9.25), a lot of THP molecules are protonated at ph of when the acetonitrile content was as high as 80% in the electrolyte. Although the content of protonated THP is decreased with the increase of ph, the retention factor of d-thp was less than zero at the ph of 7.0, which indicates that the chromatographic interaction between the d-thp and the polymers was so weak that the electrophoretic mobility of d-thp controlled the migration behavior at higher ph value. However, the chromatographic retention based on ion interaction played an important role in the enantioseparation of dl-thp [32]. The similar results could be seen in Fig. 2b. With the increase of ph, the EOF became bigger, while the retention factor of R- TB and S-TB decreased, and the resolution of racemic TB decreased too. Considering both run time and resolution of dl- THP and R,S-TB, the ph 6.0 of buffer was selected for the separation of them in the following experiments Effect of acetonitrile content in the electrolyte The effect of ACN content in running buffer on the EOF and the retention factors of THP and TB on the l-thp and S-TB imprinted monoliths was investigated, respectively, in the range of 75 95% (v/v) by keeping the ph value and acetate concentration of the electrolyte. The results were shown in Fig. 3. It can be seen that the increasing content of ACN resulted in the increase of EOF generated by both MIP columns. Xu et al. [33] and Quaglia et al. [34] have also observed the same phenomenon. The retention factors of both dl-thp and R,S-TB decreased with the increase of ACN content, but the migration time of S-TB decreased sharply, which led to the decrease of enatioseparation of R,S-TB. According to the resolution and migration time, 85% and 80%(v/v) ACN content was selected as the optimal condition for the enantioseparation of dl-thp and R, S-TB, respectively, in the following experiments. Additionally, Fig. 3a indicated the migration of d-thp was faster than that of thiourea when the ACN content in the electrolytes was higher than 90% (v/v). The similar result can be found in Fig. 3b for the migration of R-TB. These phenomena can be explained by the protonation of the THP and TB in the higher ACN content of mobile phase. Since the basic THP and TB molecules are almost fully protonated in the mobile phase of ACN/acetate buffer (90/10, v/v, ph 6.0), the electrophoretic migration of them was more significant when the chromatographic interaction became weak. It can be deduced that the hydrogen-bond/ion interaction between the analytes and the polymers determines the molecular recognition and chiral separation, and the electrophoretic mobility only partly affects the retention Effect of ionic strength Fig. 4a illustrated the effect of concentration of acetate buffer on the EOF and the retention factor of dl-thp on the l-thp imprinted monolith, which was obtained by using different ionic Fig. 5. Enantioseparation of racemic THP (a) and TB (b) on l-thp and S-TB imprinted monolithic capillary column, respectively. CEC conditions were the same as the Table 1 except that mobile phase were (a) ACN/10 mm HOAc buffer (85/15, v/v, ph 6.0) and (b) ACN/5 mm HOAc buffer (80/20, v/v, ph 6.0).

5 J. Ou et al. / J. Biochem. Biophys. Methods 70 (2007) Fig. 6. Fast enantioseparation of racemic THP (a) and TB (b) on l-thp and S-TB imprinted monolithic capillary column, respectively. CEC conditions were the same as the Fig. 5 except that only the inlet vial was applied pressure of 6 bar. strength of acetate buffer from 2 mm to 20 mm in the electrolytes containing 85% (v/v) ACN. It can be seen that EOF decreased with an increase of ionic strength. The retention of both d-thp and l-thp became weaker as the acetate concentration increased from 2 mm to 10 mm, while the retention of them became stronger when the acetate concentration was further increased from 10 mm to 20 mm. However, the retention factor obtained at 15 mm of acetate buffer was higher than that obtained at 20 mm of acetate buffer. These results may be due to the increase of the elution ability of mobile phases with an increase of ionic strength from 2 mm to 20 mm. It is well known that acetic acid was generally used as additive for the mobile phases in HPLC, which can weaken the binding of template molecule to the MIP. When the concentration of acetate buffer in the electrolyte was only 2 mm the EOF was the highest, but the elution ability of the buffer was the weakest. As a result, although the EOF decreased with the increase of acetate buffer concentration, it could be deduced that the elution ability of the buffer increased dramatically, which led to the THP molecules eluted quickly from the MIP. When the concentration of acetate buffer was further increased to 15 mm the EOF was further decreased, as a result, the retention factor of THP was increased. The effect of concentration of acetate on the EOF and retention of TB on the S-TB imprinted monolith was also investigated by using different ionic strength of the buffer/acn, and the results were indicated in Fig. 4b. It can be seen that the EOF also decreased with an increase of concentration of acetate, and the strongest retention of S-TB on the MIP was exhibited as the concentration of acetate was 15 mm. It may be due to the both a decrease of EOF and an increase of elution ability. These results further indicated that the ion interaction between the analytes and the polymers plays an important role in the molecular recognition and chiral separation Chiral separations Under the optimal conditions mentioned above, the chiral separations of racemic THP and TB were successfully obtained. It can be seen from Fig. 5 that the THP and TB were completely separated on the l-thp and S-TB imprinted monolithic capillary column, respectively. The resolution reached to 1.43 for THP and 1.56 for TB. However, the running time was too long and the second eluted peak was seriously tailing and asymmetry. It is well known that the EOF velocity increases approximately linearly with the increase of the voltage. Thus the separation time will be shortened when the applied voltage is increased. As can be seen from Table 2, although both enantioseparation and resolution of THP decreased with the increase of the applied voltage, the running time was shortened. In addition, fast chiral separation can be obtained by applying gas pressure, which was only applied on the inlet of the vial. It can be seen from Fig. 6 that the chiral separation of these two compounds could be accomplished in 4 min. 4. Simplified description of the method and its application Molecularly imprinted polymers have attracted considerable attention as one of CSPs in CEC, as the polymer characterizes the high enantioselectivity and predetermined elution order. The MIP stationary phases were directly synthesized in the capillary by use of an in situ thermal-initiated polymerization reaction with (5S, 11S)-(-)-TB and l-thp as the template, respectively, which was dissolved in the solution of MAA, EDMA, toluene and dodecanol before polymerization. The enantioseparations of these two chiral drugs, Tröger's base and tetrahydropalmatine, were achieved in CEC. The process of column preparation is very simple, which can be applied for other molecular imprinting. Table 2 Effect of voltage applied on the resolution of dl-thp on the l-thp imprinted monolithic capillary column Voltage/kV t d /min t l /min R s CEC conditions were the same as the Table 1 except with different applied voltage.

6 76 J. Ou et al. / J. Biochem. Biophys. Methods 70 (2007) Acknowledgements Financial supports from the China State High-Tech Program Grants (2003AA233061) and the Knowledge Innovation Program of DICP to Dr. Hanfa Zou are gratefully acknowledged. References [1] Millot MC. Separation of drug enantiomers by liquid chromatography and capillary electrophoresis, using immobilized proteins as chiral selectors. J Chromatogr, B, Biomed Sci Appl 2003;797: [2] Srinivas NR. Evaluation of experimental strategies for the development of chiral chromatographic methods based on diastereomer formation. Biomed Chromatogr 2004;18: [3] Yashima E. Polysaccharide-based chiral stationary phases for highperformance liquid chromatographic enantioseparation. J Chromatogr, A 2001;906: [4] Szymura-Oleksiak J, Bojarski J, Aboul-Enein HY. Recent applications of stereoselective chromatography. Chirality 2002;14: [5] Lammerhofer M. Chiral separations by capillary electromigration techniques in nonaqueous media II. Enantioselective nonaqueous capillary electrochromatography. J Chromatogr, A 2005;1068: [6] Gübitz G, Schmid MG. Recent advances in chiral separation principles in capillary electrophoresis and capillary electrochromatography. Electrophoresis 2004;25: [7] Kang J, Wistuba D, Schurig V. Recent progress in enantiomeric separation by capillary electrochromatography. Electrophoresis 2002;23: [8] Mangelings D, Maftouh M, Heyden YV. Capillary electrochromatographic chiral separations with potential for pharmaceutical analysis. J Sep Sci 2005;28: [9] Fanali S, Catarcini P, Blaschke G, Chankvetadze B. Enantioseparations by capillary electrochromatography. Electrophoresis 2001;22: [10] Maier NM, Franco P, Lindner W. Separation of enantiomers: needs, challenges, perspectives. J Chromatogr, A 2001;906:3 33. [11] Ulbricht M. Membrane separations using molecularly imprinted polymers. J Chromatogr, B, Biomed Sci Appl 2004;804: [12] Blanco-Lopez MC, Lobo-Castanon MJ, Miranda-Ordieres AJ, Tunon- Blanco P. Electrochemical sensors based on molecularly imprinted polymers. Trends Anal Chem 2004;23: [13] Ansell RJ. Molecularly imprinted polymers in pseudoimmunoassay. J Chromatogr, B Biomed Sci Appl 2004;804: [14] Alvarez-Lorenzo C, Concheiro A. Molecularly imprinted polymers for drug delivery. J Chromatogr, B Biomed Sci Appl 2004;804: [15] Turiel E, Martin-Esteban A. Molecular imprinting technology in capillary electrochromatography. J Sep Sci 2005;28: [16] Schweitz L, Spegel P, Nilsson S. Approaches to molecular imprinting based selectivity in capillary electrochromatography. Electrophoresis 2001;22: [17] Wistuba D, Schurig V. Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography. J Chromatogr, A 2000;875: [18] Nilsson J, Spegel P, Nilsson S. Molecularly imprinted polymer formats for capillary electrochromatography. J Chromatogr B Biomed Sci Appl 2004;804:3 12. [19] Quaglia M, Sellergren B, Lorenzi ED. Approaches to imprinted stationary phases for affinity capillary electrochromatography. J Chromatogr, A 2004;1044: [20] Chang CK, Lin MT. DL-tetrahydropalmatine may act through inhibition of amygdaloid release of dopamine to inhibit an epileptic attack in rats. Neurosci Lett 2001;307: [21] Xu SX, Jin GZ, Yu LP, Liu GX, Liu WW, Fang SD. Brain dopamine depleted by d-tetrahydropalmatine. Acta Pharm Sin 1987;8: [22] Cass QB, Bassi AL, Calafatti SA, Matlin SA, Tiritan ME, Campos LM. Carbohydrate carbamate coated Onto microporous silica: application to chiral analysis of commercial pharmaceutical drugs. Chirality 1996;8: [23] Qin F, Xie CH, Feng S, Ou JJ, Kong L, Ye ML, et al. Monolithic silica capillary column with coated cellulose tris(3,5-dimethylphenylcarbamate) for capillary electrochromatographic separation of enantiomers. Electrophoresis 2006;27: [24] Adobo K, Nichols IA. Enantioselective solid-phase extraction using Tröger's base molecularly imprinted polymers. Anal Chim Acta 2001;435: [25] Adobo K, Andersson HS, Ankarloo J, Karlsson JG, Norell MC, Olofsson L, et al. Enantioselective Tröger's base synthetic receptors. Bioorgan Chem 1999;27: [26] Ou JJ, Kong L, Pan CS, Su XY, Lei XY, Zou HF. Determination of dltetrahydropalmatine in Corydalis yanhusuo by l-tetrahydropalmatine imprinted monolithic column coupling with reversed-phase high performance liquid chromatography. J Chromatogr, A 2006;1117: [27] Dong J, Xie C, Tian R, Wu R, Hu J, Zou H. Capillary electrochromatography with neutral monolithic column for classification of analytes and determination of basic drugs in human serum. Electrophoresis 2005;26: [28] Fu HJ, Jin WH, Xiao H, Huang HW, Zou HF. Peptide separation in hydrophilic interaction capillary electrochromatography. Electrophoresis 2003;24: [29] Schweitz L, Andersson LI, Nilsson S. Capillary electrochromatography with molecular imprint-based selectivity for enantiomer separation of local anaesthetics. J Chromatogr, A 1997;792: [30] Huang X, Zou H, Chen X, Luo Q, Kong L. Molecularly imprinted monolithic stationary phases for liquid chromatographic separation of enantiomers and diastereomers. J Chromatogr, A 2003;984: [31] Quaglia M, Lorenzi ED, Massolini G, Sulitzky C, Sellergren B. Surface initiated molecularly imprinted polymer films: a new approach in chiral capillary electrochromatography. Analyst 2001;126: [32] Yan W, Gao R, Zhang Z, Wang Q, Jiang CV, Yan C. Capillary electrochromatographic separation of ionizable compounds with a molecular imprinted monolithic cationic exchange column. J Sep Sci 2003;26: [33] Xu Y, Liu Z, Wang H, Yan C, Gao R. Chiral recognition ability of an (S)- naproxen-imprinted monolith by capillary electrochromatography. Electrophoresis 2005;26: [34] Quaglia M, Lorenzi ED, Sulitzky C, Caccialanza G, Sellergren B. Molecularly imprinted polymer films grafted from porous or nonporous silica: Novel affinity stationary phases in capillary electrochromatography. Electrophoresis 2003;24:952 7.