Stationary phase contributions to retention in high-performance anion-exchange protein chromatography: ligand density and mixed mode effects.
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 added. Data from a non-ionic hemoglobin-binding assay correlated reasonably well with anticipated matrix hydrophobicities. Possible explanations and applications of the observed phenomena are discussed.
D000588:Amines / Q000032:analysis; D001798:Blood Proteins / Q000032:analysis*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002852:Chromatography, Ion Exchange / Q000379:methods; D003432:Cross-Linking Reagents; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D010851:Picrates / Q000032:analysis; D011485:Protein Binding
D000588:Amines; D001798:Blood Proteins; D003432:Cross-Linking Reagents; D006454:Hemoglobins; D008024:Ligands; D010851:Picrates; C005858:picric acid