Examination of theoretical principles of gradient elution as applied to reversed-phase high-performance liquid chromatography separation of phenylthiohydantoin amino acids

More than twenty different phenylthiohydantoin (PTH)-derivatized amino acids were used in the examination of the relevance and the applicability of theoretical principles that have been postulated earlier to describe gradient elution in high-performance liquid chromatography (HPLC). Reversed-phase HPLC conditions representative of PTH-amino acid assays, i.e. chemically modified silica microspheres in conjunction with an aqueous-organic mobile phase, were utilized. As predicted by theory, inevitable compromises between resolution and retention time were observed empirically. However, an a priori assumption of equivalent linear solvent strength conditions was shown to be inappropriate for selected PTH-amino acid solutes. These findings were supported by the experimentally determined variability among the different solutes of solvent strength values, S, i.e. the slope of the plots of the logarithm of isocratic capacity factor versus the fraction of organic modifier.