Anal. Chem. 1996, 68, 2248-2250
Chiral Separation of Chloroquine Using Heparin as a Chiral Selector in High-Performance Liquid Chromatography Apryll M. Stalcup,* Kyung H. Gahm, and Michelle Baldueza
Department of Chemistry, 2545 The Mall, University of HawaiisManoa, Honolulu, Hawaii 96822
The chiral selectivity of a commercially available heparin affinity column was investigated for chloroquine. The (+) enantiomer eluted first, which is consistent with results reported previously using heparin as a chiral additive in capillary zone electrophoresis. The effects of pH, ionic strength, and organic modifier on the enantiodiscrimination were explored. A combination of electrostatic and hydrophobic interactions seems to play an important role in the enantiodiscrimination exhibited by this novel phase. Recently,1,2 we reported the application of heparin as a chiral additive (CA) in capillary zone electrophoresis (CZE). Heparin (MW 10 000-30 000) is a naturally ocurring linear glycosaminoglycan, polydisperse with regard to molecular weight as well as sites and degree of sulfation. The basic subunit of heparin may be either a di-, tetra-, or hexasaccharide composed of uronic acid and glucosamine residues. Heparin was shown to be effective as a CA for the enantioresolution of a variety of underivatized drugs, including antimalarials and antihistamines.1 The anionic character of heparin enhanced its aqueous solubility while conferring considerable intrinsinc electrophoretic mobility which opposed the electroosmotic flow, thus widening the chiral “window”. In addition, the chirality of the carbohydrate subunits and the proposed helicity of heparin suggested potential for chiral selectivity. Further, its fortuitous UV transparency permitted analyte detection when heparin was used as CA. Although heparin affinity columns have been used extensively for the purification and isolation of proteins,3-5 to the best of our knowledge, this is the first report of heparin as a chiral stationary phase (CSP) in high-performance liquid chromatography (HPLC). In this report, we explore the chiral recognition for chloroquine (Figure 1) of a commercially available heparin affinity column. Chloroquine was chosen as an analyte because it exhibited one of the highest affinities for heparin of all the analytes used in the previous CZE study.1 EXPERIMENTAL SECTION Materials. The Progel-TSK Heparin-5PW affinity column (7.5 cm × 7.5 mm i.d.) was obtained from Supelco, Inc. (Bellefonte, PA). Chloroquine was obtained from Sigma Chemical Co. (St. (1) Stalcup, A. M.; Agyei, N. M. Anal. Chem. 1994, 66, 3054-3059. (2) Agyei, N. M.; Gahm, K. H.; Stalcup, A. M. Anal. Chim. Acta 1995, 307, 185-191. (3) Karlsson, K.; Marklund, S. L. Biochem. J. 1988, 255, 223-228 (4) DiBella, E. E.; Maurer, M. C.; Scheraga, H. A. J. Biol. Chem. 1995, 270, 163-169. (5) Rosenthal, R. A.; Moses, M. A.; Shintani, Y.; Megyesi, J. F.; Langer, R.; Folkman, J. J. Cell. Biochem. 1994, 56, 97-105.
2248 Analytical Chemistry, Vol. 68, No. 13, July 1, 1996
Figure 1. Structure of chloroquine.
Louis, MO). Mobile phase components were obtained from Fisher Scientific (St. Louis, MO). Apparatus. The HPLC system used for these experiments consisted of a Shimadzu LC-600 and SPD-6A UV variable wavelength detector interfaced to a Chromatopac CR-501 data station. The isocratic separations were performed at 19 °C at a flow rate of 0.8 mL/min. Supporting evidence for chiral separation was supplied by repeating the separation at different wavelengths. In addition, individual peaks were collected and analyzed by CZE and by optical rotary dispersion. Typical mobile phases consisted of either sodium phosphate or ammonium acetate buffer (0-50 mM, pH 5-7). The phosphate buffers were prepared by mixing equimolar solutions of mono- and dibasic sodium phosphate to produce the desired pH. The ammonium acetate buffer was adjusted to the desired pH by addition of acetic acid. The instrument used for the CZE experiments was a Waters Quanta 4000 capillary zone electrophoresis system equiped with a UV detector (214 nm) interfaced to a Shimadzu Chromatopac CR-501 data station. The CZE system was operated with the cathode at the injector end of the capillary. The fused silica capillary columns were 75 m i.d., with a column length of 60 cm (52.4 cm to detector window). Further details are provided in ref 1. The optical rotary dispersion measurements were conducted using a Jasco Model DIP-370 digital polarimeter. RESULTS AND DISCUSSION The column used in this study consists of heparin, immobilized on a 10 µm polymer bead which is thought, by the vendor technical staff, to be comprised of styrene-divinylbenzene. The nature of the resin limits the flow rate (0.5-1.0 mL/min) and the amount (
