PH transients in hydroxyapatite chromatography columns—Effects of operating conditions and media properties

Abstract

pH transitions occur in hydroxyapatite (HAP) columns that are subject to step changes in salt concentration, which have been shown to be controlled by proton exchange on the HAP surface. The pH temporarily decreases before gradually returning to the feed value when the salt concentration increases, potentially compromising the stability of the HAP when either the magnitude or duration of the pH drop is excessive. The opposite happens when the salt concentration decreases. In this work we address the effects of several key variables: the flow rate, the particle size, the use of salt gradients instead of steps, the use of different co-buffers, the surface area of the HAP, and the use of a slightly alkaline wash prior to increasing the salt concentration. Flow rate and particle size were found to have virtually no effect, demonstrating that the pH transitions are equilibrium rather than kinetically driven. Salt gradients resulted in smaller pH drops compared to steps since the exchanged protons are diluted over the gradient volume. MES and histidine used as co-buffers were effective at reducing the duration of the pH transitions but did not affect their magnitude. The same result was found when comparing HAP samples with different surface areas, with the lower surface area HAP yielding much shorter duration but similar pH drops and rises. Finally, washing the HAP column with a pH 7.5 buffer prior to the salt step was found to dramatically reduce the subsequent pH drop. In general, there was good agreement between these results and predictions based on our previously developed model.