STEROID FOCUSING BY MICELLE COLLAPSE IN CAPILLARY ELECTROPHORETIC SEPARATION

Abstract

Analyte focusing by micelle collapse (AFMC) is an on-line sample preconcentration of neutral analyte for capillary electrophoresis (CE). Study on the separation of six neutral steroids by AFMC-micellar electrokinetic chromatography (MEKC) technique was presented to investigate its efficiency, sensitivity and limits of detection. The focusing mechanism of AFMC is based on the transport, release, and accumulation of molecules bound to micelle carriers that are made to collapse into a liquid phase zone. The sample solution of the neutral analytes is prepared using sodium dodecyl sulphate (SDS) at a concentration above the critical micelle concentration (cmc) with higher conductivity than the running buffer. The separation of selected steroids using MEKC was initially conducted as a comparison of enhancement factor to AFMC-MEKC. The use of a mixture of 20 mM SDS and methanol 10% (v/v) in 25 mM acetate buffer (pH 9.0) with a positive applied voltage of 25 kV and pressure injection of 40 mbar for 1 sec at 25°C was able to separate the steroids by MEKC. The same buffer condition was applied to AFMC–MEKC, whereby the selected steroids were prepared in a solution containing 7 mM SDS and 250 mM acetate buffer with conductivity ratio of 0.34. Results showed enhancement of sensitivity for each steroids with good repeatability and reproducibility. The limits of detection for the steroids ranged from 1.1-5.7 mg/L. The optimized method was successfully applied to analyse steroids in urine samples with good average recoveries ranging from 93% - 101%. AFMC-MEKC separation on selected steroids showed enhancement in sensitivity compared to MEKC.