High-performance liquid chromatography of amino acids, peptides and proteins. LXXXIX. The influence of different displacer salts on the retention properties of proteins separated by gradient anion-exchange chromatography

The influence of eight different displacer salts on the retention properties of four globular proteins, ranging in molecular weight from 14 000 to 43 000, was investigated by using the Mono-Q strong-anion-exchange resin as the stationary phase. Proteins were eluted under gradient conditions with a range of alkali metal halides to vary systematically the anion and cation species in the series F^-, Cl^-, and Br^- and Li⁺, Na⁺ and K⁺. Protein Z_c values (i.e. slopes of the ion-exchange retention plots, as derived from the dependency of the logarithmic capacity factor log k′ on the concentration of the ionic displacer) generally increased when both the anion and cation were either chaotropic, e.g. KBr, or kosmotropic, e.g. NaF, in nature. Conversely, Z_c values decreased when the displacer salt contained an anion-cation combination of a chaotropic and a kosmotropic ion, e.g. KF. These results indicate that the lyotropic properties of salts are additive in their effect on the interactive properties of proteins in anion-exchange chromatography. The Z_c values were also found to depend on the manner in which the ionic strength was manipulated to affect elution, i.e. isocratic or gradient change in concentration of the displacing salt. Thus, isocratic experiments and gradient experiments with varied gradient time or varied flow-rate were observed to result in log k′ versus log 1/c dependencies with non-coincident Z_c values. The relationship between protein Z_c values, the electrostatic contact area or ionotope, A_c, and the electrostatic potential of the protein surface, ψ_s, is discussed.