Effect of temperature on separation of estradiol stereoisomers and equilin by liquid chromatography using mobile phases modified with beta-cyclodextrin.

The unusual temperature effect on the retention of 17 alpha-estradiol, 17 beta-estradiol and equilin in reversed-phase liquid chromatography has been observed. Capacity factors (k') of the steroids were measured using mobile phase with different concentrations of beta-cyclodextrin (from 0 to 16 mM) in a fixed mobile phase composition (acetonitrile-water) and wide range of column temperatures (from 5 degrees to 80 degrees C). The plots of capacity factors vs reciprocal of absolute temperature are nonlinear in each case. At subambient temperatures the capacity factors decreased with temperature decrease. This effect is more evident for the natural 17 beta-estradiol than for its 17 alpha-isomer.