Pressure-Dependent Retention and Selectivity in Reversed-Phase Liquid Chromatographic Separations Using β-Cyclodextrin Stationary Phases

Abstract

The influence of pressure on solute retention in liquid chromatography is commonly ignored due to the small compressibility of polar mobile phases. However, the equilibrium processes driving solute retention may be significantly affected by pressure, even under the modest conditions commonly encountered in HPLC (<350 bar). In this paper, we examine the role of pressure in separations where the primary mechanism for solute retention is inclusion complexation. Using the positional isomers of nitrophenol as model solutes, pressure-induced decreases in solute capacity factor ranging from −2.1% to −35.1% are observed experimentally for pressures from 40 to 340 bar. Individual contributions of pressure-induced solute ionization and complexation to this pressure-dependent solute retention are isolated by controlling mobile-phase pH. Pressure-induced dissociation of the cyclodextrin−solute complex appears to play the primary role in determining the pressure dependence of solute retention. Experimentally observed selective perturbation in solute retention with pressure is shown to have a direct impact on chromatographic resolution. In addition, the magnitude of the pressure-induced decrease in solute retention is demonstrated to be a function of the mobile-phase solvent strength. This previously underappreciated pressure effect has clear implications for the practical application of cyclodextrin stationary phases, as well as for the fundamental interpretation of those thermodynamic parameters central to the separation process.