Capillary zone electrophoresis separations of enantiomers present in complex ionic matrices with on-line isotachophoretic sample pretreatment

Abstract

Analytical capabilities of capillary zone electrophoresis (CZE) with on-line coupled capillary isotachophoresis (ITP) sample pretreatment in the column-coupling capillary electrophoresis equipment to separate and determine enantiomers present in multicomponent ionic matrices were studied. Tryptophan was used as a model analyte in the ITP-capillary zone electrophoresis experiments performed in this context while a 90-component model mixture of UV-light absorbing organic anions and urine served as multicomponent sample matrices. Various working modes in which the on-line coupled capillary isotachophoresis–capillary zone electrophoresis combination in the column-coupling separation system can operate were employed in the anionic regime of the separation with direct injections of the samples. Advantages and limitations of these working modes in the separations of enantiomers present in model and urine matrices were assessed. Experiments with model mixtures of tryptophan enantiomers revealed that the two were resolved in the capillary zone electrophoresis stage with the aid of α-cyclodextrin also when their concentration ratio in the sample was 1:200 while the concentration of L(−)-tryptophan was 25 nmol/l. The limits of detection for the enantiomers were at ∼10 nmol/l (∼1.5 ng/ml) concentrations for a 220 nm detection wavelength of the UV detector employed in the capillary zone electrophoresis stage and for a 30 μl sample load. A high sample load capacity of the on-line coupled capillary isotachophoresis stage was effective in separating the samples corresponding to 3–6 μl volumes of undiluted urine. The results from the runs with urine samples showed that only the capillary isotachophoresis–capillary zone electrophoresis combination with a post-column on-line coupled capillary isotachophoresis sample clean-up (responsible for a removal of more than 99% of the sample anionic constituents migrating in the on-line coupled capillary isotachophoresis stack and detectable in the capillary zone electrophoresis stage) provided a universal alternative for the detection and quantitation of the model analyte ( L(−)-tryptophan).