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.