pH Transients in Hydroxyapatite chromatography columns

Ceramic Hydroxyapatite (CHT), with empirical formula (Ca5(PO4)3OH)2, is a material used as ion exchange resin in chromatographic applications. The material, having both positive and negative charged sites, can be used in many different contexts encountered in the protein purification processes. Nevertheless, the resin shows an intrinsic limitation for this kind of applications: even if the material solubility in water is very low at pH values higher than 6.5, it sharply increases in more acidic environments, reducing the life of the material and increasing the operative costs of the process, making problematic the CHT application at these conditions. There is a further complication related to the CHT application: it is experimentally reported that the result of the resin interactions with salts is a pH transient occurring in the liquid phase of the chromatographic column. These pH variations are temporary but remarkable, they may influence in an important manner the material solubility, affecting the number of utilization cycle of the fixed bed and, eventually, the stability of the proteins involved in the separation process. In this work the principal aspects contributing at the selection of the operating pH and of the buffering specie are analyzed, discussing also the effect of the buffer itself on the material solubility. Then, it is reported a mathematical model useful for the description of the dynamic behavior of the column and it is proposed an interaction mechanism between the salts of the mobile phase and the stationary phase. In particular, it is emphasized the discussion regarding the different affinity shown by the stationary phase for the different counter-ions which may accompanies the buffering specie. Finally, a series of equations are developed at the scope to obtain a model useful to describe the observed pH transients.