High-performance liquid chromatography of amino acids, peptides and proteins. LXXXVI. The influence of different displacer salts on the retention and bandwidth properties of proteins separated by isocratic anion-exchange chromatography.
The influence of different displacer salts on the retention behaviour of seven globular proteins ranging in molecular weight from 12,000 to 69,000 was investigated using the Mono Q anion-exchange resin as the stationary phase. Isocratic retention data were collected using several different alkali metal halides as the displacing salt, thereby systematically varying the anion and cation species in the series F-, Cl- and Br- and Li+, Na+ and K+. The different anions were found to reduce protein retention in order of their decreasing hydrated ionic radii. Protein Zc values were found to be lower for fluoride and bromide than for chloride. It was demonstrated that the cationic co-ions also influence solute retention properties with this anion-exchange resin through, inter alia, preferential interactions with the protein solute. Protein band-broadening was found to systematically vary with the choice of displacer salt. These changes were related to known Hofmeister effects on protein aggregation kinetics and solubility and the degree of ion penetration at the double layer of the stationary phase-mobile phase interface. These studies now provide a rapid comparative basis for evaluating the mechanism of co- and counter-ion interactions with proteins in high-performance ion-exchange chromatographic systems.