The influence of temperature on the multiple separation of estrogenic steroids using mobile phases modified with...

The effect of temperature on the retention and multiple separation of six estrogenic steroids in reversed-phase liquid chromatography has been studied. Capacity factors (k') of estriol, 17 beta-estradiol, 17 alpha-estradiol, d-equilenin, equilin and estrone were measured using mobile phase modified with different concentrations of beta-cyclodextrin (from 0-16 mM), a fixed solvent composition (acetonitrile-water) and a wide range of column temperatures (from 5 to 80 degrees C). The plots of capacity factors vs. reciprocal of absolute temperature are nonlinear in each case when mobile phase modified with beta-cyclodextrin was used. Particularly strong nonlinearity was observed at lower temperature and at higher beta-cyclodextrin concentration. The complex chromatograms were evaluated using optimization parameters such as capacity factor of the last-eluted peak (k'max), the smallest resolution between adjacent peaks (Rs,min) and relative resolution product (r). The results presented describe precisely the role of temperature in high-performance liquid chromatography systems in which mobile phases modified with cyclodextrin were used. Moreover, the elution order of estrogenic steroids on modified and unmodified mobile phases has been discussed.