Anal. Chem. 1997, 69, 4226-4233
A Family of Single-Isomer Chiral Resolving Agents for Capillary Electrophoresis. 1. Heptakis(2,3-diacetyl-6-sulfato)-β-cyclodextrin J. Bryan Vincent, Alex D. Sokolowski, Thanh V. Nguyen, and Gyula Vigh*
Department of Chemistry, Texas A&M University, College Station, Texas 77845-3255
A new, moderately hydrophobic, single-isomer charged cyclodextrin, the sodium salt of heptakis(2,3-diacetyl-6sulfato)-β-cyclodextrin, has been synthesized and used to separate a variety of neutral, weak acid, strong base, weak base, and zwitterionic racemic enantiomers in low-pH and high-pH background electrolytes. Separation selectivity for the netural analytes rapidly decreases as the concentration of heptakis(2,3-diacetyl-6-sulfato)-β-cyclodextrin increases. For charged analytes, selectivity can increase, decrease, or pass a maximum, depending on the numeric values of the respective complexation constants and ionic mobilities. In addition to separation selectivity, the extent of peak resolution that can be realized strongly depends on the magnitude of the dimensionless electroosmotic flow. Heptakis(2,3-diacetyl-6-sulfato)-β-cyclodextrin proved to be a broadly applicable chiral resolving agent. In this decade, capillary electrophoresis (CE) became firmly established as a powerful method for the separation of enantiomers,1 thanks, in great part, to the versatility of various cyclodextrins, the most frequently used chiral resolving agents in both acidic and alkaline background electrolytes (BEs). Though spectacular separations have been achieved with native as well as derivatized neutral cyclodextrins, as recently reviewed in ref 2, the analysis of noncharged enantiomers only became possible when charged cyclodextrins entered the stage.3 The charged cyclodextrins are either weak electrolytes,3-5 which can only be used in a limited pH range (where they possess at least a fractional charge), or strong electrolytes,6-13 which can be used in BEs of any pH. Most of the charged cyclodextrins used so far are complex mixtures which contain a large number of isomers differing both in their degree of substitution (the number of charges per cyclodextrin molecule) and the loci of substitution. The use of resolving agent mixtures is fraught with at least four (1) St. Claire, R. L. Anal. Chem. 1996, 58, 569R. (2) Fanali, S. J. Chromatogr. A 1996, 735, 77. (3) Terabe, S. Trends Anal. Chem. 1989, 8, 129. (4) Fanali, S. J. Chromatogr. 1989, 474, 441. (5) Schmitt, T.; Engelhardt, H. Chromatographia 1993, 37, 475. (6) Tait, R. J.; Skanchy, D. J.; Thompson, D. O.; Chetwyn, N. C.; Dunshee, D. A.; Rajewsky, R. A.; Stella, V. J.; Stobaugh, J. F. J. Pharm. Biomed. Anal. 1992, 10, 615. (7) Tait, R. J.; Thompson, D. O.; Stobaugh, J. F. Anal. Chem. 1994, 66, 4013. (8) Dette, C.; Ebel, S.; Terabe, S. Electrophoresis 1994, 15, 799. (9) Wu, W. H.; Stalcup, A. M. J. Liq. Chromatogr. 1995, 18, 1289. (10) Desiderio, C.; Fanali, S. J. Chromatogr. A 1996, 716, 183. (11) Chakvetadze, B.; Endresz, G.; Blaschke, G. Electrophoresis 1994, 15, 804. (12) Chakvetadze, B.; Endresz, G.; Blaschke, G. J. Chromatogr. A 1995, 704, 234. (13) Stalcup, A. M.; Gahm, K. H. Anal. Chem. 1996, 68, 1360.
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distinct problems which are of varying importance in any given separation. The first problem is that the number and loci of substituents on the cyclodextrin can have a great effect on the chiral selectivity of the system, and, at our current level of understanding, the direction and magnitude of these changes cannot be predicted a priori.14 Therefore, when mixtures of different isomers of substituted cyclodextrins are used, the overall separation selectivity of the system might be reduced (due to parasitic complexation), eliminated (due to matched, opposite binding selectivities for the two enantiomers), or, in a few favorable cases, enhanced.15 The second problem is that kinetic band broadening can occur when the finite complexation rates of the different cyclodextrin isomers are slightly different; this results in an unavoidable decrease of the separation efficiency. The third drawback is that fundamental molecular level studies (e.g., NMR,16 crystallographic or molecular modeling17), aimed at improving the level of understanding of the chiral recognition process and, perhaps, leading to a rational selection of the best chiral resolving agent for a particular analyte, are impossible with mixtures of resolving agent isomers. Fourth, but not least, when a critical separation is developed with a particular mixture of resolving agent isomers (commercial or otherwise), there is always the danger that the composition of the mixture will be slightly different in the next batch, and the reproducibility of a difficult separation will be compromised. Therefore, a project was undertaken in our laboratory to develop a family of pure, well-characterized, single-isomer charged cyclodextrins which (i) are strong electrolytes and can be used at any pH required for selectivity optimization,18 (ii) offer the same intermolecular interactions (functional groups) on the 2- and 3-positions of the glucose moieties as the well-established neutral cyclodextrins do,2 and (iii) carry the maximum number of charged functional groups on the 6-position of the glucose moieties to provide for the best possible peak resolution.18-20 This paper describes the synthesis, characterization, and use of the first member of this new, single-isomer charged cyclodextrin family, heptakis(2,3-diacetyl-6-sulfato)-β-cyclodextrin. (14) Weseloh, G.; Bartsch, H.; Konig, W. A. J. Microcolumn Sep. 1995, 7, 355. (15) Stalcup, A. M.; Gahm, K. H. Anal. Chem. 1995, 67, 19. (16) Endresz, G.; Chakvetadze, B.; Bergenthal, D.; Blaschke, G. J. Chromatogr. A 1996, 732, 132. (17) Lipkowitz, K.; Pearl, G.; Coner, B.; Peterson, M. J. Am. Chem. Soc. 1992, 114, 1554. (18) Williams, B. A.; Vigh, Gy. J. Chromatogr. 1997, 776, 295. (19) Friedl, W.; Kenndler, E. Anal. Chem. 1993, 65, 2003. (20) Rawjee, Y. Y.; Vigh, Gy. Anal. Chem. 1994, 66, 428. S0003-2700(97)00252-7 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Synthesis scheme for heptakis(2,3-diacetyl-6-sulfato)-βcyclodextrin.
EXPERIMENTAL SECTION Synthesis of Heptakis(2,3-diacetyl-6-sulfato)-β-cyclodextrin. All chemicals used in this work were obtained from Aldrich Chemical Co. (Milwaukee, WI), except β-cyclodextrin, which was a generous gift from CeraStar (Hammond, IN). The schematic of the synthetic procedure is shown in Figure 1. Briefly, heptakis(2,3-diacetyl-6-sulfato)-β-cyclodextrin is produced by first reacting β-cyclodextrin with dimethyl-tert-butylchlorosilane as described in ref 21, purifying the intermediate by gradient elution preparative column chromatography22 on silica gel using n-hexane/ethyl acetate/ethanol as eluent.21 (The 50 mm i.d., 300 mm long preparative HPLC column packed with 30 nm pore size, 10 µm irregular silica (Merck, Darmstadt, Germany) was generously loaned to us by Dr. Y. Y. Rawjee of SmithKline Beecham, King of Prussia, PA.) The purified intermediate was then peracetylated with acetic anhydride as described in ref 21, and the product was once again purified by gradient elution preparative column chromatography on silica gel using ethyl acetate/ethanol as eluent.21 The purified heptakis(2,3-diacetyl-6-dimethyl-tert-butylsilyl)-β-cyclodextrin was then reacted with boron trifluoride etherate as described in ref 21 to remove the dimethyl-tertbutylsilyl protecting group, and the product was repurified by gradient elution preparative column chromatography on silica gel using ethyl acetate/ethanol as eluent.21 Next, the pure heptakis(2,3-diacetyl)-β-cyclodextrin was reacted with SO3‚pyridine in DMF as described in ref 23 to completely sulfate the primary hydroxyl groups of the cyclodextrin. Progress of the reaction was monitored by indirect UV detection capillary electrophoresis24 using 20 mM p-toluenesulfonic acid background electrolyte, whose pH was adjusted to 8 with tris(hydroxymethyl)aminomethane. Once the reaction was complete, the reaction mixture was poured into acetone, and the semisolid material was filtered out and redissolved in water. The aqueous solution was neutralized with NaOH, and the material was reprecipitated with ethanol. The solid material was filtered out and redissolved in water, and the remaining sodium sulfate was removed by repeated addition of ethanol. Finally, the end product was dried in a vacuum oven at 80 °C to obtain pure heptakis(2,3-diacetyl-6-sulfato)-β-cyclodextrin. The purity of the material was checked by indirect UV detection (21) Takeo, K.; Hisayoshi, M.; (22) Vigh, Gy.; Quintero, G.; (23) Bernstein, S.; Joseph, (24) Nardi, A.; Fanalli, S.;
Uemura, K. Carbohydr. Res. 1989, 187, 203. Farkas, Gy. J. Chromatogr. 1989, 484, 237. J.; Nair, U. U.S. Patent 4,020,160, 1977. Foret, F. Electrophoresis 1990, 11, 774.
Figure 2. Indirect UV detection electropherogram of a typical heptakis(2,3-diacetyl-6-sulfato)-β-cyclodextrin sample. BE: 20 mM p-toluenesulfonic acid, pH 8, adjusted with TRIS. Wavelength: 214 nm. Applied potential: 25 kV. Thermostat temperature: 25 °C. Capillary: 25 µm i.d., 39/45 cm effective/total length, uncoated fused silica.
Figure 3. 1H NMR spectrum of a typical heptakis(2,3-diacetyl-6sulfato)-β-cyclodextrin sample. Solvent: D2O.
capillary electrophoresis24 as above. The electropherogram of a typical heptakis(2,3-diacetyl-6-sulfato)-β-cyclodextrin sample is shown in Figure 2. The 200 MHz 1H NMR spectrum of the final product is shown in Figure 3. Electrophoretic Separations Using Heptakis(2,3-diacetyl6-sulfato)-β-cyclodextrin. All separations were carried out on a UV detector-equipped P/ACE 2100 CE unit (Beckman Instruments, Fullerton, CA). The detection wavelength was set at 214 nm, and the cartridge coolant was thermostated at 20 °C. The separations were carried out in 25 µm i.d. untreated fused silica capillaries (Polymicro Technologies, Phoenix, AZ) with a 45 cm total length and a 39 cm injector-to-detector length. The injection pressure was set at 5 psi and the injection time was 1 s. The applied potential was varied between 12 and 20 kV in order to maintain a power dissipation of 500-700 mW/m. Two buffer stock solutions were prepared as required by the charged resolving agent migration model (CHARM model) of CE enantiomer separations:18 a low-pH buffer (pH 2.5) and a highpH buffer (pH 9.5). The low-pH buffer was prepared by adding 0.0250 mol of concentrated (85% w/w) phosphoric acid to enough deionized water (Milli-Q, Millipore, Milford, MA) to obtain a solution of about 0.95 L. This solution was titrated to pH 2.5 with a saturated aqueous solution of LiOH using a combination glass electrode and a precision pH meter (both of them from Corning Analytical Chemistry, Vol. 69, No. 20, October 15, 1997
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Table 1. Effective Mobilities of the Less Mobile Enantiomer [µ, in 10-5 cm2/(V‚s)], Separation Selectivities (r), Measured Peak Resolution Values (Rs), Dimensionless EO Flow Values (β), and the Injector-to-Detector Potential Drop (U, in kV) in Low-pH (pH 2.5) HDAS-βCD BEs 10 mM structure
µ
R
15 mM
Rs
β
H
µ
R
Rs
β
Marker -27.34 ((0.07)
-28.45 ((0.07)
SO3–
U
30 mM U
µ
R
Rs
-25.93 ((0.05)
50 mM β
U
µ
Rs
R
β
U
-23.71 ((0.09)
Neutrals 1.40 0.7 -27.1 17.2 -2.7
1.19 1.7 -2.5 2.1a -3.0
1.19
3.0
-2.6 12.8
17.2 -3.6
1.08
