Gradient Hydroxyapatite Chromatography with Small Sample Loads. II. Experimental Analysis and the Resolving Power of the Columns

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Abstract
Several chromatograms of biological macromolecules on hydroxyapatite columns were analyzed on the basis of a theory previously developed. The experimental data confirm the validity of the theory. The optimal chromatographic conditions were explored by introducing experimental data into the theory. In many instances it is indispensable that the slope of the molarity gradient of competing ions used in elution be extremely small to obtain high resolution; this slope generally is much too steep in published chromatograms. The differences in adsorption energies per molecule between the collagen components present in the neighboring peaks of the multipeak chromatogram obtained in an earlier paper were measured. These gave a value of about 0.5 kcal/mol for any pair of peaks. This should correspond to the energy of adsorption for a functional carboxyl group onto a C site. The same value was obtained in an earlier paper by an independent method on the basis of a micro-heterogeneous model of collagen. The agreement strongly supports the micro-heterogeneous model.