Mobile phase selection for the high-performance ion-exchange chromatography of proteins

Proper mobile phase selection significantly improved high-performance ion-exchange fractionations of proteins. The pH and salt content of the eluant affected chromatographic behavior on both strong and weak ion-exchange columns. The retention and resolution of a number of proteins was examined on strong and weak ion-exchange supports with regard to these mobile phase variables. The strong ion-exchange columns were found to be superior for the protein separations performed in this study. The selectivity of both weak and strong ion-exchange columns was pH dependent, however, strong ion-exchange columns were operable over a broader range of pH. Examination of the effect of salts demonstrated that their "displacing activity" could be divided into three categories: weak, intermediate, and strong with regard to the protein mixture utilized. Intermediate salts provided best resolution along with good recoveries. Strong displacing salts were useful for eluting strongly retained proteins. The selection of a mobile phase with respect to protein retention is discussed.