Resolution and Retention of Proteins near Isoelectric Points in Ion-Exchange Chromatography. Molecular Recognition in Electrostatic Interaction Chromatography

Abstract

Both molecular recognition and transport phenomena play important roles in very high resolution ion-exchange (electrostatic interaction) chromatography (IEC) of proteins. A fast and simple method for obtaining important information on molecular recognition, peak retention (and the number of binding sites), and peak resolution of proteins from linear gradient elution experiments (salt concentration is increased linearly at a fixed mobile phase pH) is described. The proposed method was verified with β-lactoglobulin A and B (LgA, LgB) as model proteins. The gradient elution experimental data were obtained over a wide range of mobile phase pH with various types of IEC media. The number of binding sites involved in the retention (adsorption) decreased as the mobile phase pH approached the isoelectric points pI (= 5.1–5.2). However, even at pH 5.2 both LgA and LgB were retained on anion- and cation-exchange chromatography columns. The separation (resolution) of LgA and LgB became better when the pH approached the pI in anion-exchange chromatography columns where the number of adsorption site values are small(ca. 2–3). The two proteins were not separated even with efficient cation-exchange chromatography columns. The resolution and the retention near the pI (pH 5.2) did not significantly depend on sample loading compared with those at pH 6.0.