EFFECTS OF STATIONARY PHASE LIGAND DENSITY ON HIGH-PERFORMANCE ION-EXCHANGE CHROMATOGRAPHY OF PROTEINS

Cation-exchange matrices with ligand densities from 10 to 500-mu-mol/g were prepared by reaction of diglycolic anhydride with diol-bonded silica. Lysozyme and cytochrome c were isocratically eluted from these columns under various conditions. The data was used to examine a retention model of proteins in which the slope (Z number) of a plot of log k' vs. log (1/sodium ion activity) was assumed to represent the number of points of binding between the protein and the matrix. As expected from the model, the Z number increased as the ligand density of the matrix increased. However, many qualitative and quantitative deviations from the model were also found, some of which may have been due to heterogeneity of the distribution of the ion-exchange sites on the matrix. An interesting observation was a change in elution order of lysozyme and cytochrome c as the ligand density changed; however, this may not have much practical benefit dual to the large band-broadening observed at low ligand densities.