Gradient Hydroxyapatite Chromatography with Small Sample Loads. I. Fundamental Theory

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Abstract
On the basis of experimental data, it can be deduced that, on a hydroxyapatite column, the effect of thermodynamic longitudinal diffusion of molecules is “hidden” within diffusion occurring due to the heterogeneity in the flow rate of the solution. This can be assumed to occur caused by the heterogeneity in interspaces among hydroxyapatite crystals packed in the column. The chromatographic process is virtually a quasi-static process. By taking into account the longitudinal diffusion in the column, a theory of hydroxyapatite chromatography was developed for small sample loads for the linear gradient elution. The chromatographic mechanisms are fundamentally different between gradient and stepwise chromatographies. No theories that have been developed over many years for stepwise chromatography are applicable to gradient chromatography. Relations of the present theory to both classical “equilibrium” and recent “rate” theories are discussed.