The Development of Reversed-Phase Capillary Electrochromatography for the Separation of Steroids

Abstract

This paper describes the preparation and optimization of packed capillary columns for reversed-phase separation of steroids with CEC. The fabrication of on-column frits is considered to be the most important step for obtaining a reproducible packed column for CEC separation. Porous silicate frits were generated in a fused-silica capillary by heating the silica gel/sodium hydroxide solutions electrically. The optimized conditions involve silica gel (10.8%), sodium hydroxide (5.8%), and heating time (5 sec) with heating voltage (5V) for obtaining a 100-μ end-frit that can withstand pressure over 6000 psi. A HPLC pump was utilized to pack the 5-μm ODS particle slurry into the capillary column. The ODS packed capillaries were then utilized for the separation of four anabolic cholesterols with a capillary electrophoresis system without pressurization of the column. The reproducibility of the packed columns was evaluated by measuring the relative standard deviations of four steroids. The relative standard deviations of migration time for column-to-column, day-to-day, and run-to-run are less than 7%, 2%, and 1% for four steroids, respectively.