Effect of pressure-induced ionization, partitioning, and complexation on solute retention in reversed-phase liquid chromatography

Abstract

In contrast to supercritical fluid chromatography, pressure is not commonly considered an important parameter affecting solute retention in liquid chromatography. While it is true that the bulk compressibility of polar mobile phases is minimal for the modest pressures encountered in reversed-phase LC (<5000 psi; <350 bar), recent studies in our laboratory have demonstrated that pressure-induced shifts in interaction equilibria can lead to systematic perturbations in solute retention. In this study, we address the theoretically predicted impact of pressure on several primary equilibria of importance in separations. Comparison with experimentally determined capacity factor changes is accomplished for reversed-phase separations with and without a mobile-phase additive. Without a mobile-phase additive, capacity factors for the nitrophenol model solutes exhibit a systematic increase of 6–8% for an average pressure increase from 65 to 280 bar. Perturbations in solute ionization are predicted to have a minor impact under these separation conditions, and pressure-induced shifts in the partitioning equilibria are implicated. When β-cyclodextrin is added to the mobile phase, pressure-induced changes in solute retention are exacerbated, leading to capacity factor shifts of up to 12%. This experimental observation is consistent with predictions based on a Le Chatelier model of the coupled partitioning/complexation equilibria. These results have pragmatic implications for the practice of liquid chromatography, especially in quality control situations where retention reproducibility is of key importance. Moreover, pressure-controlled liquid chromatography is demonstrated as a fundamental measurement tool for determining molar volume changes upon partitioning and complexation.