Superheated water as eluent in high-temperature high-performance liquid chromatographic separations of steroids on a polymer-coated zirconia column

Abstract High-temperature liquid chromatography (HTLC), with a superheated water mobile phase, has been shown to be a feasible replacement for medium-polarity acetonitrile–water mixtures as an eluent in reversed-phase HPLC. Instrumental parameters of flow-rate, injection volume and mobile phase preheating were shown to have significant effects on the quality of the chromatographic peaks. The selectivity and retention patterns of testosterone and several related compounds were investigated on a porous zirconia, polybutadiene-coated column at temperatures up to 200°C and compared with that of a porous silica, octadecylsilane-coated column and the zirconia column under traditional reversed-phase conditions of an acetonitrile–water mobile phase at 40°C. The selectivity differences observed for testosterone and related compounds show that the separation mechanisms are complementary and unique selectivity is obtained with the zirconia column under HTLC conditions.