Prostaglandins are biological lipid mediators that control a variety of physiological and pathophysiological processes. They function as locally acting hormones and are continuously synthesized and inactivated in target tissues by respective biosynthetic and metabolic enzymes. The separation of prostaglandins and their metabolites is crucial for study of their roles in biological systems. In this article, we describe systematic studies of the retention and separation of prostaglandin E 2 (PGE 2 ) from its inactive metabolites 15-keto-PGE 2 and 13,14-dihydro-15-keto-PGE 2 . We also describe the separation of a second prostaglandin species, PGF 2α , from its metabolites 15-keto-PGF 2α and 13,14-dihydro-15-keto-PGF 2α . Chromatography was performed on RP-18W high-performance thin-layer chromatographic plates with binary mobile phases comprising water and an organic modifier (methanol, ethanol, acetonitrile, acetone, or tetrahydrofuran). The experimental data revealed that at constant temperature, plots of th...