Chemically l-Phenylalaninamide-Modified ... - ACS Publications

Jun 19, 2001 - Zilin Chen , Katsuyoshi Hayashi , Yuzuru Iwasaki , Ryoji Kurita , Osamu Niwa , Kenji Sunaawa. Electroanalysis .... Chuzo FUJIMOTO. Anal...
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Anal. Chem. 2001, 73, 3348-3357

Chemically L-Phenylalaninamide-Modified Monolithic Silica Column Prepared by a Sol-Gel Process for Enantioseparation of Dansyl Amino Acids by Ligand Exchange-Capillary Electrochromatography Zilin Chen* and Toshiyuki Hobo

Department of Applied Chemistry, Graduate School of Engineering, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachioji, Tokyo 192-0397, Japan

A new type of chiral monolithic column was successfully developed for the enantioseparation of dansyl amino acids by ligand exchange-capillary electrochromatography (LECEC) in this work. The monolithic column matrix was prepared by a sol-gel process and then chemically modified with the spacer (3-glycidoxypropyl)trimethoxysilane and the chiral selector L-phenylalaninamide. After being conditioned with Cu(II) aqueous solution, the ligand exchange-chiral stationary phase (LE-CSP) possesses positive charges. When the external electric field was applied in CEC, electroosmotic flow (EOF) was generated on the surface of LE-CSP in the direction from the cathode to the anode. The EOF was found to be dependent on the applied electric field strength and the composition of the mobile phase. With the increase of pH of the mobile phase, the EOF showed a tendency to decrease. Scanning electron microscopy showed that the chiral monolithic column has a continuous skeleton and large through-pore structure. The separation efficiency (theoretic plate numbers) for the separation of Dns-DL-Leu reached up to 9.0 × 104 plates m-1 for the D-enantiomer and 6.6 × 104 plates m-1 for the L-enantiomer, by using pH 5.5, acetonitrile/0.50 mM Cu(Ac)2-50 mM NH4Ac (7:3) as mobile phase. The reproducibility and lifetime were satisfactory. CEC was carried out with conventional capillary electrophoresis equipment without pressurizing the ends of the capillary. No bubble was formed during the operation, after degassing the mobile phase and conditioning the column. Capillary electrochromatography (CEC), a separation mode integrating both features of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), has been received great interest during the 1990s, despite having origins in the 1970s.1 Like HPLC, the chromatographic resolution in CEC is based on the interaction between the analytes and stationary * Corresponding author: (tel) 0426-77-2825; (fax) 0426-77-2821; (e-mail) [email protected]. (1) Cikalo, M. G.; Bartle, K. D.; Robson, M. M.; Myers, P.; Euerby, M. R. Analyst 1998, 123, 87R-102R.

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phases. However, in CEC, the analytes move through the separation bed of the column, driven by the electroosmotic flow (EOF), which drives the mobile phase, as well as the self-electrophoretic mobility when charged analytes are separated. Because of the flat pluglike profile of the EOF, CEC offers greatly enhanced separation efficiency compared to HPLC. The column plays a key role in CEC, because it serves not only as the separation bed but also as the pumping device of the mobile phase. Therefore, further development of CEC needs a significant advancement in column technology. The development of monolithic columns has become a fascinating research field in the chromatographic sciences. The reason could be that monolithic columns offer many advantages over conventional packed capillary columns. For example, monolithic columns could eliminate the tasks of particle synthesis, the difficulty of packing columns with discrete particles, and moreover the need of end frits to maintain the stationary phase. Depending on the monolithic material, the monolithic column can be classified into two categories: (i) organic polymer-based2-4 and (ii) bonded silica-based.5-9 In the first type, the fabrication of monolithic columns is accomplished through a single-step polymerization reaction of organic monomeric precursors. One critical drawback associated with this type of monolithic capillary is its tendency to swell/shrink during exposure to various solvents in the mobile phases. In the latter, the monolithic column is often bonded silica stationary phase through the use of a sol-gel process. Some work, however, still cannot escape from column packing, although a monolithic column bed can be made well by sol-gel methods. Therefore, the functionally modified sol-gel monolith column may be a promising direction for the development of monolithic column (2) Chirica, G.; Remcho, V. Anal. Chem. 2000, 72, 3605-3610. (3) Gusev, I.; Huang, X.; Horvath, C. J. Chromatogr., A 1999, 855, 273-290. (4) Peters, E. C.; Petro, M.; Svec, F.; Frechet, J. M. Anal. Chem. 1998, 70, 2296-2303. (5) Tang, Q.; Lee, M. L. J. High Resolut. Chromatogr. 2000, 23 (1), 73-80. (6) Hayes, J. D.; Malik, A. Anal. Chem. 2000, 72, 4090-4099. (7) Tanaka, N.; Nagayama, H.; Kobayashi, H.; Ikegami, T.; Hosoya, K.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Cabrera, K.; Lubda D. J. High Resolut. Chromatogr. 2000, 23, 111-116. (8) Fujimoto, C. J. High Resolut. Chromatogr. 2000, 23, 89-92. (9) Dulay, M. T.; Kulkarni, R. P.; Zare, R. N. Anal. Chem. 1998, 70, 51035107. 10.1021/ac010243p CCC: $20.00

© 2001 American Chemical Society Published on Web 06/19/2001

technology. Moreover, although these works reported the monolithic column, they are limited in the achiral separation. Chiral separation is an important area in the chromatographic sciences. Chromatographic scientists have been paying great attention to the development of chiral separation technology, since it has been recognized that the chirality of a molecule would considerably affect its physiological activity. Although CEC has been widely used as a chromatographic technique, quite a few papers were published on chiral CEC. For example, Wistuba and Schurig reported enantiomer separation by CEC on a cyclodextrinmodified monolith.10 Koide and Ueno reported enantioseparation by CEC with β-cyclodextrin-bonded polyacrylamide gels.11,12 Schweitz et al.13 prepared chiral imprinted polymers using (R)propranolol or (S)-ropivacaine as a print molecule. Hjerte´n et al.14 resolved the enantiomers of kynurenine by CEC on an acrylatebased continuous bed with immobilized human serum albumin (HAS). Lammerhofer et al.15,16 prepared chiral monolithic columns by copolymerization of monomers with a quinidine functionality for chiral CEC. Schmid et al.17 recently developed a continuous bed column for separation of amino acid enantiomers based on ligand exchange by a copolymeriztion procedure using methacrylamide, piperazine diacrylamide, vinylsulfonic acid, and N-(2-hydroxy-3-allyloxypropyl)-L-4-hydroxyproline. However, these papers were almost restricted in polymer-based chiral monolithic columns. It has been known that polymer-based monolithic columns usually suffer from solvent swelling phenomena in mobile phases containing organic modifiers. From this point of view, silica-based monolithic columns could be full of promise. Up to now, unfortunately, no any publications reported the use of a silica-based chiral monolithic column. Ligand exchange, introduced in liquid chromatography by Davankov and Rogozhin18 in the 1970s, is based on a chiral discrimination principle with high enantioselectivity for the chiral resolution of chelate complex-forming compounds such as amino acids, hydroxy acids, and peptides.19 It has widely been used in the chromatographic separations in the modes of ligand exchangeliquid chromatography (LE-LC),19,20 ligand exchange-capillary electrophoresis (LE-CE),21-23 and ligand exchange-micellar electrokinetic chromatography (LE-MEKC).24-28 Advances in chiral separations based on ligand exchange were reviewed in our recent (10) Wistuba, D.; Schurig, V. Electrophoresis 2000, 21, 3152-3159. (11) Koide, T.; Ueno, K. J. High Resolut. Chromatogr. 2000, 23 (1), 59-66. (12) Koide, T.; Ueno, K. J. Chromatogr., A 2000, 893, 177-187. (13) Schweitz, L.; Andersson, L. I.; Nilsson, S. J. Chromatogr. A 1998, 817, 5-13. (14) Hjerte´n, S.; Vegvari, A.; Srichaiyo, T.; Zhang, H.-X.; Ericson, C.; Eaker, D. J. Capillary Electrophor. 1998, 5, 13-26. (15) Lammerhofer, M.; Peters, E. C.; Yu, C.; Svec, F.; Frechet, J. M. Anal. Chem. 2000, 72, 4614-4622. (16) Lammerhofer, M.; Svec, F.; Frechet, J. M.; Lindner, W. Anal. Chem. 2000, 72, 4623-4628. (17) Schmid, M. G.; Grobuschek, N.; Tuscher, C.; Gu ¨ bitz, G.; Vegvarl, A.; Maruska, E.; Maruska, A.; Hjerten, S. Electrophoresis 2000, 21, 3141-3144. (18) Davankov, V. A.; Rogozhin, S. V. J. Chromatogr. 1971, 60, 280-283. (19) Davankov, V. A. J. Chromatogr., A 1994, 666, 55-76. (20) Gu ¨ bitz, G.; Mihellyes, S.; Kobinger, G.; Wutte, A. J. Chromatogr., A 1994, 666, 91-97. (21) Gassmann, E.; Kuo, J. E.; Zare, R. N. Science 1985, 230, 813-814. (22) Gozel, P.; Gassmann, E.; Michelsen, H.; Zare, R. N. Anal. Chem. 1987, 59, 44-49. (23) Schmid, M. G.; Gu ¨ bitz, G. Enantiomer 1996, 1, 23-27. (24) Chen, Z.; Lin, J.; Uchiyama, K.; Hobo, T. J. Chromatogr., A 1998, 813, 369378. (25) Chen, Z.; Lin, J.; Uchiyama, K.; Hobo, T. Chromatographia 1999, 49, 436443.

paper.29 Although ligand exchange has been used in chiral separations for 30 years, no paper using ligand exchange in CEC, except for the recent report,17 was published. Therefore, ligand exchange-capillary electrochromatography (LE-CEC) was expected to be developed by chromatographic scientists. We focused on the development of silica-based chiral monolithic column technology and its application to CEC for the chiral separations. The chiral monolithic column was prepared by a solgel process and chemical modifications with the chiral selectors on the monolithic silica matrix. Galli et al.30 reported the enantioseparation of dansyl and dabsyl amino acids by conventional LC with (R)- or (S)-phenylalaninamide-modified silica gel. Our recent work31 reported a L-prolinamide-modified monolithic silica column for the enantioseparation of hydroxy acids and dansyl amino acids. This work reported on the development of a chemically Lphenylalaninamide-modified monolithic silica column and its applications to the enantioseparation of dansyl amino acids by LECEC. The characterization of EOF and the chromatographic performance of ligand exchange-chiral stationary phase (LE-CSP) were investigated. EXPERIMENTAL SECTION Apparatus. CEC was carried out on an instrumental setup, involving an HCZE-30PNO25-LD high-voltage power supply (Matsusada Precision Devices, Tokyo, Japan), a CE-1570 intelligent UV/visible detector (Jasco, Tokyo, Japan) and a C-R7A plus Chromatopac integrator (Shimatzu, Tokyo, Japan). Scanning electron micrograph (SEM) was carried out on JSM-6100 scanning electron microscope (JEOL, Tokyo, Japan). Programmed temperature heating was performed within a GC-17A oven (Shimadzu). Fused-silica capillary (0.375-mm o.d., 0.10-mm i.d.) was obtained from GL Sciences. Reagents. Tetramethoxysilane (TMOS) and (3-glycidoxypropyl)trimethoxysilane were obtained from Shin-Etsu Chemical (Tokyo, Japan). Poly(ethylene glycol) (PEG; Mw 10 000), Lphenylalaninamide, alaninamide, and dansyl amino acids were obtained from Sigma (St. Louis, MO). Dehydrated toluene, dehydrated N,N-dimethylformamide (DMF), acetonitrile for HPLC, copper(II) acetate monohydrate, and ammonium acetate were obtained from Kanto Chemical (Tokyo Japan). Pretreatment of Capillary. To clean and activate the inner surface of capillaries, the capillaries were washed by following procedures: water for 30 min, 1 M sodium hydroxide overnight at 40 °C in an LC oven, water for 30 min, 0.1 M HCl overnight at 40 °C, and then water, acetone, and diethyl ether for 30 min, respectively. Finally, the washed capillary was purged with helium at 180 °C for 2 h prior to use. Preparation of the Silica-Based Monolithic Column Matrix by a Sol-Gel Process. The monolithic silica matrix was prepared by referring to the procedures of refs 7 and 8 with modifications. (26) Chen, Z.; Lin, J.; Uchiyama, K.; Hobo, T. J. Microcolumn Sep. 1999, 11, 534-540. (27) Chen, Z.; Lin, J.; Uchiyama, K.; Hobo, T. Anal. Sci. 2000, 16, 131-137. (28) Chen, Z.; Lin, J.; Uchiyama, K.; Hobo, T. Anal. Chim. Acta 2000, 403, 173178. (29) Chen, Z.; Hobo, T. Recent Research Developments in Chemical & Pharmaceutical Sciences; Transworld Research Network: Trivandrum, India, 2001. (30) Galli, B.; Gasparrini, F.; Misiti, D.; Villani, C.; Corradini, R.; Dossena, A.; Marchelli, R. J. Chromatogr., A 1994, 666, 77-89. (31) Chen, Z.; Hobo, T. Electrophoresis, in press.

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Briefly, 0.5 mL of 0.01 M acetic acid, 54 mg of PEG and 0.2 mL of TMOS were mixed in a 1.5-mL vial bottle. The solution was stirred for ∼30 min at ice bath. When a transparent solution was observed, it was then introduced into an appropriate length of pretreated capillary with a syringe. The ends of capillary were connected with a Teflon tube to form a circle and then placed in a 40 °C LC oven for 24 h. The silica gel made within the capillary was washed with water and subsequently treated with 0.2 M ammonium hydroxide solution. The columns were placed into the 40 °C oven for another 24 h. The ends of column were opened and placed into a GC oven for heating by a programmed temperature. While the temperature was increasing from 60 to 300 °C at a rate of 1.0 °C/min, it was kept for 4 h at 80, 120, 180, and 300 °C, respectively. Finally, after being purged with helium at 180 °C for 1 h, the column was cooled to room temperature at the rate of -1.0 °C/min. This column was used for the chemical modifications in the next steps. Chemical Modifications of L-Phenylalaninamide on the Surface of the Monolithic Silica Column. Before use, a LC pump was purged by dehydrated toluene. (3-Glycidoxypropyl)trimethoxysilane (12.6%) in dehydrated toluene was pumped through the monolithic silica column immersed in a 110 °C oil bath for 6 h with the LC pump at the constant pressure of 1960 kPa. To have the whole monolithic column bed immersed in the oil bath and to introduce the modifier solution into a waster reservoir outside the oil bath, a 15-cm capillary was connected with the monolithic column by a Teflon tube sleeve during the pumping. Then, the column was connected with a Teflon tube to form a circle and then placed inside a 110 °C GC oven for another 5 h. The column was washed with toluene, methanol, and acetone and then purged with helium for 2 h at 50 °C. L-Phenylalaninamide (10%) in dehydrated DMF was introduced through the column with a water pump. The pumping was continues for 30 min after the liquid drops was seen at the end of column. The column was kept at room temperature for one week. The column was washed with methanol and conditioned with 16 mM CuSO4 aqueous solution for 30 min and mobile phase. The detection window (5 mm) was made right after the CSP bed by burning out the polyimide coating on the surface by a lighter. Separation Conditions of CEC. CEC was carried out on a home-built CE instrument without the support of pressure. The monolith column sizes were 0.375-mm-o.d., 100-µm-i.d., 26.5-cm effective length (EL) of LE-CSP and 37-cm total length (TL). Sample solutions were injected by electrokinetic method for 3-5 s at the same electric field strength applied for the separation. The EOF was determined by using acetone as a marker. UV detection wavelength was kept at 254 nm. Mobile phases were prepared by mixing acetonitrile and the electrolyte solution containing ammonium acetate and copper acetate and then adjusting the pH with ammonia or acetic acid. Before use, all solutions were filtered through a 0.45-µm membrane (Nihon Millipore Ltd.) and degassed by vacuum and ultrasonication. Water was purified by distillation apparatus (Advantec, Tokyo, Japan). Sample solutions were dissolved in the mobile phase at the range of 5.0-10 × 10-4 M. The calculations of resolution (Rs), separation factor (R), and EOF are the same as previous work.24,25 To avoid bubble formation, it is important to electrokinetically condition the column at low electric field strength before high 3350

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electric field strength is applied to a new column. Sometimes, minute bubbles were observed at the interface of the monolithic column bed and the mobile phase; electromagnetic stirring was very usefully used to prevent bubble formation at the interface. Without pressurizing the ends of column, CEC was carried out by conventional CE equipment in this work. RESULTS AND DISCUSSION Sol-Gel Process and Chemical Modification in the Preparation of Monolithic LE-CSP. Based on the sol-gel chemistry, a monolithic silica network matrix was prepared inside the capillary. The chemical reactions of the sol-gel process include three steps:32 (i) the hydrolysis of TMOS, (ii) the condensation of the hydrated silica tetrahedra forming tSisOsSit bonds, and (iii) the polycondensation of linkage of additional tSisOH tetrahedra eventually resulting in a SiO2 network having silanol groups on the surface. The silanol groups on the skeleton surface of the monolithic silica matrix prepared by the sol-gel process were used for chemical modifications, including two steps: (i) the modification of a spacer, (3-glycidoxypropyl)trimethoxysilane, and (ii) the modification of a chiral selector, L-phenylalaninamide. After conditioning with CuSO4 aqueous solution, the Cu(II) was grafted on the surface of the L-phenylalaninamide-modified CSP, as shown in Scheme 1. This LE-CSP can be used for the enantiomeric separation by LE-CEC. Principle of Retention in Chiral CEC. Two components for the retention of solutes are associated with the separated process in chiral CEC. One involves solute retention based on the interaction with CSP, as in HPLC. Another component involves differential migrations, characterized by the electrophoretic mobility (µep) of solutes, which is similar to CE. Although enantiomers have the same electrophoretic mobility, the enantioseparation is based on the chiral interaction between the enantiomers and CSP. The retention of a solute can be expressed as the following equations.33

k* ) k + k(µep/µeof) + (µep/µeof)

(1)

k ) (uep + ueof)/us - 1

(2)

where k*, k, µep, µeof, us, ueof, and uep are electrochromatographic retention factor, the actual retention factor caused by chromatography alone in CEC, electrophoretic mobility of solute, electroosmotic mobility, migration velocity of solute, electroosmotic velocity, and electrophoretic velocity of solute in the absence of the chromatographic interaction, respectively. The experiments showed that the peaks of enantiomers always appeared in the front of the acetone peak (the electrochromatograms are not shown here). It is suggested that analytes possess negative charges under the experimental conditions and move through the column bed not only by EOF driving the mobile phase but also by the selfelectrophoretic mobility of negatively charged analytes at the experimental conditions. On the basis of these results, it is easy to understand the retention mechanism of CEC as the concurrence of chromatography and electrophoresis. In other words, CEC is a hybrid of HPLC and CE. (32) Hench, L. L.; West, J. K. Chem. Rev. 1990, 90, 33-72. (33) Colon, L. A.; Guo Y.; Fermier, A. Anal. Chem. 1997, 461A-467A.

Scheme 1. Proposed Chiral Discrimination on the Ligand Exchange-Chiral Stationary Phase

As can be seen in eqs 1 and 2, the exact calculation of retention factor (k*) in CEC is very complicated, because many factors must be determined. Therefore, the separation factors (R) were simply calculated as R ) t2/t1 by the use of retention times, although the t0 can be easily determined. Chiral Discrimination in LE-CEC. Chiral discrimination is based on the principle of ligand exchange, which is attributed to the exchange of one ligand in the Cu(II) complex on the stationary phase by an analyte ligand, forming ternary mixed copper complexes with different stabilities. As shown in Scheme 1, DnsDL-amino acids (Dns-DL-AA) interact with LE-CSP by exchanging the ligands, forming two complexes: (L-phenylalaninamide)Cu(II) (Dns-D-AA) and (L-phenylalaninamide)Cu(II) (Dns-L-AA).

Because the stabilities of these two complexes are different, resulting from the different stereostructures, the retention times of Dns-D-AA and Dns-L-AA are different. Since the phenyl group of the L-phenylalaninamide and the R-group of the Dns-L-AA locate at the different sides of the complex plane of Dns-L-AA, and at the same side of the complex of Dns-D-AA, complex Dns-L-AA is more stable than Dns-D-AA, resulting in the L-analytes having a longer retention time than D-analytes. Morphology, Permeability, and Mechanical Strength of the Chiral Monolithic Columns. SEM photographs in Figure 1 show that the monolithic column has the morphology of a continuous skeleton and large through-pores. Panels A and B of Figure 1 are at magnifications of 800× and 4300×, respectively. Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

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Figure 1. SEM photographs of sol-gel monolithic column modified with L-phenylalaninamide: 20 kV, WD 39, and magnification (A) 800×, (B) 4300×.

As can be seen in the photographs, the silanol groups at the inner surface of the capillary took part in the sol-gel reactions so that the monolithic bed was bonded to capillary wall. The permeability of the column was examined by the pressure drop. When a mobile phase of acetonitrile/0.1 M NH4Ac-0.25 mM Cu(Ac)2 (7:3 v/v) (pH 7.6) was used at a flow rate of 1 µL/min, the pressure drop of the column (i.d., 100 µm; EL, 26.5 cm; TL, 37 cm) was 294 kPa. It shows the monolithic column with a good permeability. To obtain a macroporous structure, it is important to control the composition of sol solution, the temperature, the pH, and the velocity of gelation during the sol-gel process.34 Since the monolithic column has a silica-based matrix, it shows good mechanical strength and lifetime of column. After the frequent use during the chromatographic evaluation and separation of samples for several months, no obvious decline of column efficiency was observed. It also indicated that the chemically bonded CSP was very stable under exposure to the external electric field and the mobile phases. After strictly degassing mobile phases and conditioning the column, the bubble formation during the CEC operation can be prevented. Characterization of EOF in the Monolithic LE-CSP. EOF is a very important factor in CEC, because analytes are moved through the separation bed of the column by the mobile phase driven by the EOF, of course, as well as the self-electrophoretic (34) Nakanishi, K. J. Porous Mater. 1997, 4, 67-112.

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mobility of analytes when charged analytes are separated. Therefore, EOF affects the retention time, resolution, and separation efficiency. Knowing the characteristics of the EOF will be helpful to the understanding of separation behavior and the mechanism in CEC. As shown in Scheme 1, the LE-CSP possesses positive charges, forming the electrical double layer on the surface of CSP. When the external electric field was applied, the EOF was generated with the direction from the cathode to the anode. Therefore, it is necessary to apply a negative voltage at the inlet end of the monolithic column to achieve the separations during operation. The effect of pH on EOF is shown in Figure 2 A. With the increase of pH, EOF shows a tendency to decrease at the pH range of 4.5-6.5. The EOF at higher pHs such as 7.5 and 8.5 was examined; it was so slow that the acetone peak was not observed for 2 h. It can be explained by the influence of unmodified silanol groups residing on the surface of the monolithic matrix and the inner wall of capillary, as shown in Scheme 1. In general, the EOF generated by silanol groups on silica matrix, from anode to cathode, increases with the increase of pH. In this work, the chemically modified LE-CSP with positive charges on the surface formed a reversed EOF with the direction from cathode to anode. Therefore, the observed EOF was contributed by the LE-CSP and residing silanol groups. Since the directions of EOF formed by the LE-CSP and residing silanol groups are opposite, the net EOF shows a tendency to decrease with the increase of pH. The effect of applied electric field strength on EOF was investigated, and the result is presented in Figure 2B. It was shown that the EOF linear velocity was proportional to the applied electric field strength at the range of -150 to -500 V/cm. At pH 5.5, acetonitrile/0.25 mM Cu(Ac)2-50 mM NH4Ac (7:3) was used as the mobile phase and acetone as an EOF marker. This result agrees with the theoretical eq 3, derived from an open tube in the absence of thermal and double-layer overlap effects.1

veof ) (ξ0ξrζ/η)E

(3)

where νeof, o, r, ζ, η, and E are the EOF velocity, the permittivity of the vacuum, the dielectric constant of the mobile phase, the zeta potential, the viscosity of the mobile phase, and the external electric field strength, respectively. It is suggested that both the thermal and double-layer overlap effects on electroosmotic velocity are insignificant under the conditions employed and that the EOF of the monolith column has similar behavior in the open tube. The effects of mobile-phase compositions, such as the content of acetonitrile and the concentration of Cu(II) ion in the mobile phase, on the EOF were investigated. The effects of acetonitrile content in the range of 50-80% (v/v) and the Cu(II) concentration in the range of 0.1-1.0 mM on the EOF are shown in Figure 2C and D, respectively. With the increase of acetonitrile content in the mobile phase, the EOF mobility decreases; however, the EOF mobility increases with the increase of Cu(II) concentration. The electroosmotic mobility, µeof, can be expressed as eq 4, where µeof,

µeof )

veof δσ σ ) ) 7 E η (3 × 10 |Z|xC)η

(4)

Figure 2. Effects of pH (A), electric field strength (B), acetonitrile concentration (C), and Cu(II) concentration (D) on the EOF. Monolithic column: 100-µm i.d., 375-µm o.d., TL 35 cm, EL of CSP 26.5 cm. Applied electric field strength: -300 V/cm. EOF marker was acetone. Mobile phases: (A) acetonitrile/0.25 mM Cu(Ac)2-50 mM NH4Ac (7:3) at different pHs; (B) pH 5.5, acetonitrile/0.25 mM Cu(Ac)2-50 mM NH4Ac (7:3); (C) pH 5.5, acetonitrile/0.25 mM Cu(Ac)2-50 mM NH4Ac at different ratios; and (D) pH 5.5, acetonitrile/50 mM NH4Ac-0.10∼1.0 mM Cu(Ac)2 (7:3).

δ, σ, Z, and C are the electroosmotic mobility, the electrical doublelayer thickness, the amount of charge per unit surface area in the stern plane, the number of valence electrons, and the electrolyte concentration, respectively.35 On the basis of this equation, it is easy to understand the effects of acetonitrile and Cu(II) concentrations. Equation 4 shows that the EOF mobility is proportional to the amount of charge per unit surface area in the stern plane, σ. With the increase of acetonitrile content, the Cu(II) concentration in the mobile phase became diluted, resulting in the decrease of the amount of charge per unit surface area in the stern plane, σ, and consequently, the decrease of EOF mobility. On the other hand, eq 4 also shows that Z, C, and η affect the EOF mobility. With the increase of acetonitrile content, the electrolyte concentration was diluted. It seems to cause the increase of EOF mobility. Results in Figure 2C show that EOF mobility decreases with the increase of acetonitrile content; it suggests that the influence of acetonitrile content on the amount of charge per unit surface area in the stern plane is significant. This suggestion can be confirmed by the results in Figure 2D. The increase of Cu(II) concentration causes the increase of the (35) Tsuda, T.; Nomura, K.; Nakagawa, G. J. Chromatogr. 1982, 248, 241-247.

amount of charge per unit surface areas in the stern layer, resulting in the increase of EOF mobility. However, the EOF slowly increases when Cu(II) concentration is higher than 0.5 mM. It suggests that the loading of the Cu(II) cation on the surface of CSP gradually approaches saturation. Chromatographic Characterization of Monolithic LE-CSP. Van Deemter plots, as depicted in Figure 3 A, were constructed through variations in the operating voltages (-7.0 to -17.5 kV). The plate height of neutrally unretained acetone versus the linear velocity of the EOF was examined by using acetonitrile/0.25 mM Cu(Ac)2-50 mM NH4Ac (7:3), pH 5.5, as mobile phase. The relatively flat right portion of the H-u curves indicates an efficient mass-transfer process between the mobile phase and the monolithic separation bed. In other words, the resistance to mass transfer can be negligible at the experimental conditions. Because the electroosmotic velocity is relatively low, the A and B terms of van Deemter’s equation predominate. When H was plotted with 1/u, as shown in Figure 3B, the linear regression is obtained as

H ) 11.427 + 4.9627/u (R2 ) 0.9211) Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

(5) 3353

Figure 4. Effect of pH on the enantioseparation of dansyl amino acids. Other conditions are the same as Figure 2A.

Figure 3. Effect of EOF linear velocities (u) on the plate height (H) of unretained neutral acetone (A). Plot of H vs 1/u (B). Other conditions are the same as Figure 2B.

Compared with van Deemter’s equation, H ) A + B/u + Cu, it indicates that column efficiency (plate height) is dominated by the molecular diffusion of the solutes (second term of van Deemter equation) and the geometric property of the monolithic separation bed, such as the nonuniformity and nonalignment of through-pore (channels) and skeleton (the first term). On the other hand, the 3354 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

H-u plot is very similar to the plot of open tube with low inner diameter (10 µm).33 It suggests that the monolithic column with large through-pore structure has some features in common with the open tube. As seen in Figure 3A, a relatively low plate height (average 30 µm) for neutrally unretained solute (acetone) can be obtained when the EOF linear velocity reaches 0.25 mm/s, which corresponds to the electric field strength of -400 V/cm. When the electrophoretic mobility of negatively charged analytes is taken into account, analytes will have faster migration velocity than the neutral marker, resulting in higher separation efficiency. The highest theoretical plate number (∼90 000 plates m-1 for Dns-DLeu) was obtained by the resolution of Dns-DL-Leu. pH plays an important role in CEC separation. It usually affects the magnitude of EOF, the charges of both the analytes and stationary phases, and the interactions between the analytes and CSPs, resulting in different enantioselectivity. The effect of pH on the EOF has been discussed in the above section. Here we discuss the effect of pH on the separation factors of some representative samples of Dns-DL-AAs. As can be seen in Figure 4, at pH 6.5, these analytes show relatively high separation factors than at other pH values. On the other hand, although the higher separation factor can also be obtained at a high pH value such as 8.5, the migration times of analytes are very long, because of the low velocity of EOF caused by the unmodified silanol groups residing on the CSPs. Based on these considerations, the pH range between 5.5 and 7.5 would be recommended. The effect of acetonitrile content on the separation factor of Dns-DL-Thr and the migration time of D-enantiomers is shown in Figure 5. As can be seen, the highest separation factor was obtained when the ratio of acetonitrile to electrolyte solution containing 0.25 mM Cu(Ac)2-50 mM NH4 was kept at 6:4. When the ratio was increased to 7:3, the separation factor was reduced. With further increase of the ratio to 8:2, the separation factor increased again. The migration times increased with the increase of the acetonitrile content. To explain this behavior, the changes of EOF and the nonpolarity of the mobile phase resulting from the change of acetonitrile content could be considered as the main reasons. With increase of the acetonitrile content, the EOF decreases (Figure 2C), and the nonpolarity of the mobile phase increases. These two factors determine the separation selectivity

Figure 5. Effect of acetonitrile concentration on the separation factor of Dns-DL-Thr and the migration time of Dns-D-Thr. Other conditions are the same as Figure 2C.

and the migration time. At the high content of 80% acetonitrile, the EOF is very low; the analytes move through the separation bed very slowly. It results in longer interaction time with the CSPs; consequently, a high separation factor could be obtained. It suggests that the effect of EOF is predominant at acetonitrile content higher than 80%. The effect of buffer compositions such as the concentrations of Cu(II) and NH4Ac was examined. It was shown that when an NH4Ac concentration higher than 100 mM was used, the electric current became high and resulted in the generation of bubbles during the separation due to the high Joule heat. When a low concentration of NH4Ac was used, the conductivity and the pH buffering capacity were not satisfactory. In this work, NH4Ac was used in the concentration range of 25 and 50 mM. The effect of Cu(II) concentration on separation was investigated. As shown in Figure 6, the highest separation factor was obtained at 0.25 mM Cu(II). With further increase of Cu(II) concentration, the separation factor showed a reducing tendency. The probable reason may also be the influence of Cu(II) concentration on the EOF. With the increase of Cu(II) concentration, the velocity of EOF increases (Figure 2D), resulting in a decrease of the time of interaction between CSP and the analytes, eventually decrease of the separation factor. Effect of the Chemical Structures of Chiral Selectors on the Enantioselectivity. To investigate the mechanism of chiral discrimination, the effect of the chemical structures of the chiral selectors on enantioselectivity was compared. Chiral monolithic columns chemically modified with L-alaninamide or L-prolinamide were also prepared by the same procedures. It was shown that only Dns-DL-Ser (R ) 1.10), Dns-DL-Thr (R ) 1.03), and Dns-DLAsp (R ) 1.06) were resolved by using a L-alaninamide-modified

Figure 6. Effect of Cu(II) concentrations on the separation factor of Dns-DL-Thr. Other conditions are the same as Figure 2D.

monolithic column (EL, 32 cm; TL, 44 cm) and using pH 5.5 acetonitrile/0.50 mM Cu(Ac)2-50 mM NH4Ac (7:3) as the mobile phase at the electric field strength of -300 V/cm. It implied that the phenyl group in the phenylalaninamide plays an important role in the interaction between the LE-CSPs and the analytes. An L-Prolinamide-modified monolithic column has different enantioselectivity compared to the L-phenylalaninamide-modified monolithic column; it can be used for the enantioseparation of not only dansyl amino acids but also hydroxy acids.31 Further, the enantiomers of free amino acids were not resolved by use of these columns; it suggests that the dansyl groups also play important roles in chiral recognition. Reproducibility and Lifetime of Column. The reproducibility of two columns, which were prepared in two different batches and made the same lengths (EL, 21 cm; TL, 30 cm), was investigated by employing Dns-DL-Thr and Dns-DL-Leu as the test samples. The statistical results are listed in Table 1. As can be seen, both reproducibility of run-to-run and column-to-column were satisfactory. It should be mentioned that the CEC instrument was not thermostated and the experiments were carried out at room temperature (roughly 25-26 °C). Good reproducibility suggests that chiral monolithic columns can be prepared and used for reproducible routine analysis. Besides, it should also be mentioned that an obvious decline of column efficiency was not observed after having been used for hundreds of operations with each column. It suggests that the columns have a good lifetime. Because the monolithic column has a silica matrix and a chiral selector chemically bonded, it is stable under exposure to the external electric field and the mobile phase containing the organic solvents.

Table 1. Reproducibility of Two Columns Prepared by Different Batchesa column

samples

tD (min)

tL (min)

R

Rs

ND (plates m-1)

NL (plates m-1)

1

Dns-DL-Thr Dns-DL-Leu Dns-DL-Thr Dns-DL-Leu

6.50 ( 0.37 6.20 ( 0.35 4.47 ( 0.06 4.88 ( 0.02

8.86 ( 0.60 6.92 ( 0.06 5.79 ( 0.23 5.45 ( 0.06

1.36 ( 0.017 1.12 ( 0.007 1.30 ( 0.045 1.12 ( 0.007

2.39 ( 0.24 1.13 ( 0.07 1.69 ( 0.20 0.87 ( 0.01

4294 ( 258 9605 ( 2035 5477 ( 1180 7242 ( 63

4893 ( 923 7556 ( 2716 2396 ( 94 3400 ( 798

2

a Mobile phase: pH 6.5, 0.50 mM Cu(Ac) /50 mM NH Ac (7:3); applied voltage, -12.0 kV. Column sizes: EL 21cm, TL 30 cm, i.d. 100 µm, o.d. 2 4 375 µm. x ) x ( SD (n ) 3).

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Figure 7. Electrochromatograms of racemates of representative dansyl amino acids. Mobile phase: pH 7.6, acetonitrile/0.1 M NH4Ac-0.25 mM Cu(Ac)2 (7:3). Monolithic column: 100-µm i.d., 375-µm o.d., TL 37 cm, EL of CSP 28.5 cm. Applied electric field strength -315 V/cm. All D-enantiomers were eluted as the first peaks.

Table 2. Enantioseparation of Dansyl Amino Acids by LE-CEC Using Monolithic Columna samples

tD

R (tL/tD)

Rs

Dsn-DL-Asp Dsn-DL-Glu Dsn-DL-Leu Dsn-DL-Met Dsn-DL-NorLeu Dsn-DL-NorVal Dsn-DL-Phe Dsn-DL-Ser Dsn-DL-Thr Dsn-DL-Trp Dsn-DL-Val Dsn-DL-R-amino-nbutyric acid

10.72 12.76 12.00 11.34 11.28 10.25 13.98 9.70 9.92 17.89 8.62 9.68

1.28 1.31 1.40 1.51 1.27 1.27 1.23 1.67 1.76 1.18 1.13 1.20

1.52 2.80 4.52 3.78 2.31 2.63 1.61 4.80 5.41 2.02 2.03 2.04

a Separation conditions: mobile phase, pH 7.6, acetonitrile/0.1M NH4Ac-0.25 mM Cu(Ac)2 (7:3); monolithic column, 100-µm i.d., 375µm o.d., TL 37 cm, EL of LE-CSP 28.5 cm; applied electric field strength -315 V/cm.

Sample Separations. Twelve commercially available dansyl amino acids have successfully been resolved by LE-CEC. The results are listed in Table 2. As for the enantiomer migration order (EMO), all the D-enantiomers were eluted as the first peaks., Since L-enantiomers can form more stable ternary complexes than D-enantiomers, as discussed in the section on chiral discrimination of LE-CEC, L-enantiomers show a stronger retention on the LECSP. Therefore, the EMO shows D-enantiomers are faster than L-ones. Figures 7 and 8 show the electochromatograms of the single pair of racemates of representative Dns-DL-AAs and the simultaneous separation of six enantiomers of three pairs of DnsDL-AAs, respectively. It has been demonstrated that the monolithic 3356 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

Figure 8. Electrochromatogram of six enantiomers of three pairs of dansyl amino acids. Peak identification: 1, Dns-D-Thr; 2, Dns-DSer; 3, Dns-L-Thr; 4, Dns-D-Leu; 5, Dns-L-Leu; 6, Dns-L-Ser. Column: L-phenylalaninamide-modified monolithic column (TL 35 cm, EL 26.5 cm; i.d. 100 µm, o.d. 375 µm). Mobile phase: pH 5.5 acetonitrile/0.50 mM Cu(Ac)2-50 mM NH4Ac (7:3). Applied electric field strength -300 V/cm; UV detection 254 nm; electrokinetic injection 3-5 s.

LE-CSP can successfully be used in the enantioseparations of dansyl amino acids with high selectivity and efficiency. The chiral monolithic column was also used for the separation of dansyl amino acids by HPLC; the results are not shown here. It indicated that both separation selectivity and column efficiency by HPLC are poorer than by CEC. Although the single pair of the enantiomers of dansyl amino acids can be resolved in HPLC, the mixtures of dansyl amino acids cannot be resolved well. It demonstrated that LE-CEC is the most promising technique for the chiral separations.

selectivity and separation efficiency for the separation of dansyl amino acids. Because of the silica-based matrix with large throughpore structure, it showed good mechanical strength and permeability. Compared to the results in the publications in the past 30 years, this work seems to have made use of chiral ligand exchange for the enantioseparation at the top of the mountains of separation selectivity and efficiency. In addition, this work demonstrated that the chemically modified monolithic silica columns prepared by sol-gel offer significant promise to further development of chiral CEC column technology.

CONCLUSION A new type of L-phenylalaninamide-modified monolithic silica column, prepared by the sol-gel process and chemical modifications, has been developed and successfully used for the enantiomeric separation of dansyl amino acids by LE-CEC. It integrates the advantages of high enantioselectivity in ligand exchange and the high efficiency in CEC, as well as the merits of a silica-based monolithic column. EOF with direction from cathode to anode was generated on the LE-CSP. The LE-CSP offered high enantio-

ACKNOWLEDGMENT Z.C. thanks the Japan Society for the Promotion of Science (JSPS) for providing his postdoctoral fellowship and Grant-in-Aid (99301) for scientific research from the Ministry of Education, Science, Sports and Culture of Japan. Received for review February 27, 2001. Accepted May 9, 2001. AC010243P

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