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

In contrast to supercritical fluid chromatography, pressure is not commonly considered an important parameter affecting solute retention in liquid chr omatography. While it is true that the bulk compressibility of polar mobile phases is minimalfor the modest pressures encountered in reversed-phase LC (<5000psi; <350bar), recent studies in our laboratory have demonstrated th at pressure-induced shifts in interaction equilibria can lead to systematic perturbations in solute retention. In this study, we address the theoretic ally predicted impact of pressure on several primary equilibria of importan ce in separations. Comparison with experimentally determined capacity facto r changes is accomplished for reversed-phase separations with and without a mobile-phase additive. Without a mobile-phase additive, capacity factors f or the nitrophenol model solutes exhibit a systematic increase of 6-8% for an average pressure increase from 65 to 280 bar. Perturbations in solute io nization are predicted to have a minor impact under these separation condit ions, and pressure-induced shifts in the partitioning equilibria are implic ated. When P-cyclodextrin is added to the mobile phase, pressure-induced ch anges in solute retention are exacerbated, leading to capacity factor shift s of up to 12%. This experimental observation is consistent with prediction s based on a Le Chatelier model of the coupled partitioning/complexation eq uilibria. These results have pragmatic implications for the practice of liq uid chromatography, especially in quality control situations where retentio n reproducibility is of key importance. Moreover, pressure-controlled liqui d chromatography is demonstrated as a fundamental measurement tool for dete rmining molar volume changes upon partitioning and complexation. (C) 1999 E lsevier Science B.V. All rights reserved.