STEROID FOCUSING BY MICELLE COLLAPSE IN MICELLAR ELECTROKINETIC CHROMATOGRAPHY

Abstract

Preliminary studies on the separation of neutral steroids through analyte focusing by micelle collapse (AFMC) are presented to investigate its efficiency, sensitivity and limit of detection (LOD). 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 (S) is prepared using sodium dodecyl sulphate (SDS) at a concentration above the critical micelle concentration (cmc) with higher conductivity than the running buffer. Normal mode–micellar electrokinetic chromatography (NM–MEKC) separation was initially performed on 100 mg/L of six neutral steroids in methanol using 20 mM SDS, 10% (v/v) methanol and sodium borate buffer (pH 9.0) with positive applied voltage of 25 kV and pressure injection of 50 mbar for 1 sec at 25°C. The same buffer condition has been applied to AFMC–MEKC, whereby the mixture of six neutral steroids was dissolved in 2 mM SDS and 250 mM sodium borate buffer which gave a conductivity ratio of 0.49. Results showed a good separation of prednisolone, prednisone, betamethasone, testosterone, 17–α–methyltestosterone, and 4–androstene–3,17–dione at 240 nm with sensitivity enhancement factors of 2.08, 1.17, 0.92, 16.9, 0.8, and 1.21 respectively. AFMC–MEKC allowed several folds improvement in sensitivity compared to NM–MEKC.