THE INFLUENCE OF TEMPERATURE ON THE MULTIPLE SEPARATION OF ESTROGENICSTEROIDS USING MOBILE PHASES MODIFIED WITH BETA-CYCLODEXTRIN IN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The effect of temperature on the retention and multiple separation of six estrogenic steroids in reversed-phase liquid chromatography has be en studied. Capacity factors (k') of estriol, 17 beta-estradiol, 17 al pha-estradiol, d-equilenin, equilin and estrone were measured using mo bile 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 plot s of capacity factors vs. reciprocal of absolute temperature are nonli near in each case when mobile phase modified with beta-cyclodextrin wa s used. Particularly strong nonlinearity was observed at lower tempera ture and at higher beta-cyclodextrin concentration. The complex chroma tograms were evaluated using optimization parameters such as capacity factor of the last-eluted peak (k(max)'), the smallest resolution betw een adjacent peaks (R-s,R-min) and relative resolution product (r). Th e results presented describe precisely the role of temperature in high -performance liquid chromatography systems in which mobile phases modi fied with cyclodextrin were used. Moreover, the elution order of estro genic steroids on modified and unmodified mobile phases has been discu ssed. (C) 1997 Elsevier Science B.V.