High-pressure liquid chromatography and ultraviolet spectrometry of ketonic C27 sterols

A method for the separation of ketonic C27 sterols was devised, based on high-pressure liquid chromatography (HPLC) and ultraviolet absorption (UV). The adsorption column contained silica gel, particle size 10 μm, and the eluents were dichloromethane/n-hexane/ethyl acetate (94∶5∶1) and dichloromethane/ethyl actate (99∶1) followed by dichloromethane/ethyl acetate (3∶1). The 5β-sterols were eluted before the 5α-analogs, sterols with isolated double bonds before conjugated carbonyl compounds, and ketones before hydroxy ketones. The effect of carbonyl groups on polarity depends on the position in the molecule and decreases in the order C-3>C-6>C-7. The ultraviolet absorption spectra of eleven sterols were determined, and their absorbance at 254 nm and at 280 nm was used for analyzing the column effluent with a dual detector system.