Stability and selectivity of a cholesterol-coated C18 stationary phase

This paper details the use of cholesterol as a mobile phase additive and stationary phase complexing agent in reversed-phase liquid chromatography. Cholesterol loading onto a typical C18 stationary phase is examined. It is found that, when using a standard 150 mm × 4.6 mm column, between 5.0 and 50.0 mg of cholesterol can be loaded by choosing appropriate values of mobile phase composition and cholesterol concentration. Adding cholesterol to the stationary phase is shown to have an effect on shape and phenyl selectivities, but not on methylene selectivity. Most notably, selectivity of triphenylene and o-terphenyl is increased from 1.08 to 1.49 by adding cholesterol to the chromatographic system. Phenyl-group selectivity shows a more modest but real increase in selectivity as well. Cholesterol-loaded stationary phases are demonstrated to be stable after cholesterol is removed from the mobile phase, for at least 250 column volumes, when mobile phases of 70% methanol or less are used.