pH transients in hydroxyapatite chromatography columns-Effects of operating conditions and media properties.

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.

pH Transients in hydroxyapatite chromatography columns-experimental evidence and phenomenological modeling.

Hydroxyapatite (HAP) columns, widely used for chromatographic separation of proteins and other biomolecules because of their unique selectivity and ability to resolve complex mixtures, exhibit limited stability at acidic conditions requiring careful control of pH. Even with buffered solutions, however, unintended pH transients can occur when the salt concentration varies. For example, the pH temporarily decreases below the feed value when the salt concentration increases and increases above the feed value when the salt concentration is decreased. The intensity and duration of these transients depend on the particular buffer used and the magnitude of the salt concentration step, but in extreme cases the pH can drop by as much as 1.5 pH units creating conditions where the HAP stability is potentially compromised. This work examines the mechanisms leading to pH transients in HAP columns generated by salt steps. The pH excursions are similar to those observed for weak cation exchange columns, but are accompanied by a transient evolution of phosphate which temporarily decreases below the feed value when the salt concentration is increased and increases sharply when the salt concentration is reduced before returning to the feed value. A phenomenological model is developed to describe this behavior by considering the reversible uptake of sodium ions by the P-sites and binding of phosphate ions by the C-sites. The interplay of these two adsorption mechanisms results in complex pH patterns that are consistent with those observed experimentally. In addition to helping understand the underlying mechanisms, the model also provides a useful tool to predict the effects of different buffers and salt concentration and develop corrective measures that can reduce the intensity and duration of the pH transients such as the addition of unretained co-buffers.