Stationary phase contributions to retention in high-performance anion-exchange protein chromatography: ligand density and mixed mode effects

Abstract

Several anion-exchange stationary phases (based on polyethyleneimine-coated silica) were synthesized so as to vary in ligand density and hydrophobicity. These materials were first examined for hemoglobin-binding capacity and then evaluated chromatographically. Protein binding, retention and resolution increased concomitantly with ligand density. Ferritin (molecular weight 440,000) could not be eluted from the more highly-charged surfaces, but was desorbed from a low ligand density support. The above parameters also varied with the hydrophobic character of the stationary phase. Retention and resolution increased as more hydrophobic moieties were addd. Data from a non-ionic hemoglobic-binding assay correlated reasonably well with anticipated matrix hydrophobicities. Possible explanations and applications of the observed phenomena are discussed.