Electrophoresis zyxwvutsrqponmlk 1995, zyxwvutsrqponm 16, 1505-1509 Enantiomeric CE separation zyxwv of a-hydroxy acids 1505 Salvatore Fanali Zeineb Aturki Istituto die Cromatografia del C.N.R., Area della Ricerca di Roma, Monterotondo Scalo (Roma) Further study on the use of uncharged P-cyclodextrin polymer in capillary electrophoresis: Enantiomeric separation of some a-hydroxy acids Uncharged fi-cyclodextrin polymer was used as chiral selector for the enantio- meric separation of some a-hydroxy acids by capillary electrophoresis. Compl- exation and enantiomeric resolution of mandelic acid, m-hydroxy and p-hydro- xymandelic acid, 3,4-dihydroxymandelic acid, as well as 2- and 3-phenyllactic acid were studied, changing the concentration of the B-cyclodextrin polymer added to the background electrolyte at different pH in the range of 4.5-7. Fur- thermore, the effects of the concentration of the background electrolyte, column temperature, and applied voltage on chiral resolution were also exa- mined. The best enantiomeric separations were obtained using a background electrolyte at pH 6 containing 100 mg/mL of fbcyclodextrin polymer. 1 Introduction Capillary electrophoresis (CE) has become a useful ana- lytical technique for the separation and quantification of several classes of compounds, e.g., proteins, peptides, nucleic acids, inorganic and organic ions, enantiomers, etc. One promising area of interest is the field of enantio- meric separation, especially for pharmaceutical and clin- ical analyses where rapid and efficient separation methods are required, e.g., for chiral purity control and/ or drug monitoring. Since two enantiomers of the same compound possess similar physicochemical properties they cannot be separated by CE because they move with similar velocity unless a chiral environment is used. The chiral selector allows the formation of either stable dias- tereomers (indirect separation method) or labile diaste- reomeric complexes (direct separation method) if used before and during the electrophoretic run, respectively [I]. In the direct separation method a wide number of chiral selectors were applied in CE for enantiomeric sep- arations, either by addition to the background electrolyte (BGE), binding to the capillary wall, or inclusion to a gel matrix [2-41. Bile salts, metal complexes with chiral ligands, modified crown ether, proteins, and cyclodextrins are widely used in CE for chiral separations [2,5-81. Among them, native cyclodextrins such as a, zyxwvuts B and zyxwvuts y as well as their deriva- tives (methylated, hydroxypropylated, carboxymethyl- ated, methylamino, sulfobutylated, cyclodextrin poly- mers, etc.), were used in order to selectively modify the effective mobility of the enantiomers [I, 2, 9-13]. Only few papers deal with the use of polymeric matrix for enantiomeric separation employing cyclodextrins as chiral selectors [4, 13, 141. In our laboratory we studied the effect of charged and uncharged B-cyclodextrin poly- Correspondence: Dr. Salvatore Fanali, C.N.R. Istituto di Cromatografia, Area della Ricerca di Roma, P.O.Box 10-00016 Monterotondo Scalo (Roma) Italy (Tel: +39-6-9062-5328; Fax: +39-6-9062-5849) Nonstandard abbreviations: BGE, background electrolyte; CD, cyclo- dextrin; MA, mandelic acid; m-OH-MA, m-hydroxyrnandelic acid; p-OH-MA, p-hydroxymandelic acid; poly-p-CD, uncharged p-cyclo- dextrin polymer; 3,4-di-OH-MA, 3,4-dihydroxymandelic acid; 2-PhL, 2-phenyllactic acid; 3-PhL, 3-phenyllactic acid zyxwvuts Keywords: Enantiomers / Capillary electrophoresis / Cyclodextrin polymer / a-Hydroxy acids mers on the enantiomeric separation of several basic compounds [15, 161. In this paper we further investigated the effect of the concentration of neutral P-cyclodextrin polymer (poly-fl-CD) on the complexation and enantio- meric separation of acidic compounds by CE. The effect of the pH and the concentration of BGE, column tem- perature, and applied voltage on the enantiomeric resolu- tion was also studied. 2 Materials and methods 2.1 Instrumentation The experiments were performed with a Biofocus 3000 Automated Capillary Electrophoresis System, Bio-Rad Laboratories (Hercules, CA, USA), equipped with a UV multiwavelength detector. Detection was at 206 nm. Analyses were carried out in a fused silica capillary tube 35 cm X 0.05 mm ID (effective length 31.5 cm) of Poly- micro Technologies (Phoenix, AZ, USA), positioned into a user cartridge thermostated with circulating liquid. Injection was by pressure at 10 psi*s (1 psi = 6894.76 PA). The capillary wa3 coated with linear polyacrylamide using the coating procedure described elsewhere [ 171. The samples and the capillary were thermostated at 25 "C. The applied voltage (constant) was 15 kV at pH 4.5 (22 PA), 6 (35 PA) and 7 (44 PA). 2.2 Chemicals Acetic acid, L-mandelic acid (L-MA), D-mandelic acid (D-MA), sodium hydroxide, and phosphoric acid were purchased from Carlo Erba (Milan, Italy). ~~-3-Phenyl- lactic acid (3-PhL), L-3-phenyllacticacid ( L - ~ - P ~ L ) , DL-m- hydroxymandelic acid (m-OH-MA), DL-p-hydroxyman- delic acid (p-OH-MA), ~~-3,4-dihydroxymandelic acid (3,4-di-OH-MA) were from Sigma (St. Louis, MO, USA). ~~-2-phenyllactic acid (2-PhL) was from Fluka (Buchs, Switzerland). Uncharged B-cyclodextrin polymer (poly-B- CD) was purchased from Cyclolab (Budapest, Hungary). Bidistilled water (Menichelli, Roma, Italy) was used to prepare all solutions. The concentration of the injected samples was M prepared by diluting stock solutions M in methanol) with running buffer (10 mM without poly-8-CD. The BGE used were: 0.1 M acetic acid, titrated with Tris at pH 4.5, and 0.05 M disodium 0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1995 0173-0835/95/0808-1505 $5.00+.25/0