(325e) The Effect of Buffer Salt Species and Concentration on the Internal pH of a Strong Anion Exchange Resin

Ion exchange chromatography is commonly used in the purification of proteins and other ionic species. In porous materials, the majority of the active sites will be inside the resin phase and thus intraparticle conditions will determine the environment in which uptake occurs. Understanding the relationship between the intraparticle conditions and those of the surrounding buffer is crucial for predicting the optimum operating conditions for ion exchange. This is especially true of the intraparticle pH since pH has a significant impact on the properties of proteins and other zwitterionic species, as well as the extent of dissociation of weak electrolytes and buffer species. Despite the importance of this key parameter, only a handful published models make an effort to calculate the resin phase pH, and no direct evidence of this parameter has been presented to date. The internal pH of Q Sepharose Fast Flow anion exchange resin in equilibrium with a bistris-acetate buffer solution is investigated as a function of buffer salt concentration. Direct evidence of a resin phase pH shift is presented. At low buffer salt concentrations of 20mM NaCl the resin phase pH is found to be as much as 1.1 pH units greater than that of the buffer phase, approaching to within 0.1 units of the buffer phase at salt concentrations greater than 250 mM. An equilibrium model based on electrostatic interaction theory is shown to provide excellent agreement with experimental results and provides framework for deepening our understanding the behavior of small buffer ions. This model can be extended to protein behavior by the incorporation of the molecule's physical size and zwitterionic nature.