Overload Quasi-Static Linear Gradient Chromatography: Theory Versus Hydroxyapatite High-Performance Liquid Chromatography

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Abstract
The earlier theory of quasi-static linear gradient chromatography under overload conditions is improved. The quasi-crystalline phase model for the molecules adsorbed on the adsorbent surfaces in the column is specified; this model was introduced earlier, and it is used in the present theory. A close experimental verification of the theory is carried out for the case of hydroxyapatite high-performance liquid chromatography using both turkey and chicken egg white lysozymes as probes. It can be deduced that a quasi-crystalline phase of lysozyme molecules, in fact, is realized on the hydroxyapatite surfaces in the column. The mutual interaction energy per molecule of lysozyme occurring provided the maximum possible contact with other molecules is made on the hydroxyapatite surface can be estimated to be larger than 0.2–0.4 kcal/mol.

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