Separation of steroids using temperature-dependent inclusion chromatography

The influence of temperature on retention and separation of estrogens, progesterone derivatives and β-cyclodextrin in reversed-phase high-performance liquid chromatography has been studied. Steroids were detected using direct UV detection at 240 and 280 nm. Detection of β-cyclodextrin was achieved using a post-column indirect photometric method. Chromatographic experiments were performed using an acetonitrile–water mobile phase (30%, v/v) and a wide range of column temperatures from 0 to 80°C with 20°C steps. Linear Van’t Hoff plots were observed for steroids and β-cyclodextrin when an unmodified binary mobile phase was applied. The retention of steroids was strongly influenced by temperature when the mobile phase was modified with β-cyclodextrin at a concentration of 12 mM. Particularly, for 17β-estradiol and 20α-hydroxyprogesterone a strong deviation from the linear Van’t Hoff plots and a remarkable affinity for β-cyclodextrin was observed. Polynomial regression calculations were performed to fit the set of experimental data points. Using third-order polynomial equations, minimum separation factor values (αmin) were calculated for temperatures from −10 to +100°C with 1°C steps. The best chromatographic conditions for separation of multicomponent samples were chosen. A possible retention mechanism for solutes in the presence of macrocyclic additives is discussed. The results presented describe the role of temperature in high-performance liquid chromatography systems in which the mobile phase is modified with an inclusion agent.