High-efficiency enantioseparations of N-derivatized amino acids by packed capillary electrochromatography using ODS silica and a quinine-derived chiral selector as ion-pair agent

Abstract

The enantiomers of N-derivatized amino acids, e.g., 3,5-dinitrobenzoyl, 3,5-dinitrobenzyloxycarbonyl, 2,4-dinitrophenyl and 9-fluorenylmethoxycarbonyl amino acids, have been separated by enantioselective ion-pair formation and packed capillary electrochromatography (CEC) using RP18 silica particles. Thus, a CEC-Hypersil ODS-3 μm packed capillary column, 335 mm (effective length 250 mm)×0.1 mm I.D., was used in combination with a quinine carbamate type chiral ion-pair agent (selector, SO) which was added to aqueous and non-aqueous buffered mobile phases, respectively. The negatively charged analyte enantiomers interact with the positively charged chiral SO by multiple intermolecular interactions to form a pair of transient diastereomeric ion-pairs which may differentially adsorb to the ODS-stationary phase. Enantioseparation is achieved due to different observed mobilities of the analyte enantiomers originating from different ion-pair formation rates of the enantiomers and/or differential adsorption of the diastereomeric ion-pairs to the ODS-stationary phase. Countercurrent-like electrophoretic migration of oppositely charged ion-pair agent and solute enantiomers may give rise to enhanced enantioselectivity. At high electrolyte concentrations (>10 m M), e.g. if the chiral ion-pair agent is added to the mobile phase that contains, compared to typical RP-CEC conditions (<10 m M), relatively high amounts of background electrolyte, electrophoretic transport of the analytes is dominating (negative polarity mode); this method is characterized by high efficiency with theoretical plate numbers up to 170 000 per meter, e.g. for DNP-Val, but with moderate enantioselectivity. In contrast, at low electrolyte concentrations (<10 m M), e.g. if the chiral ion-pair agent itself is used as electrolyte without any other background electrolyte, the analytes are driven through the capillary column with the electroosmotic flow (positive polarity mode); this operation mode is characterized by relatively poor efficiency, but high enantioselectivity. The influence of several mobile phase parameters (aqueous versus non-aqueous, selector concentration, type of background electrolyte) on observed mobility, enantioselectivity and efficiency was evaluated.