Chromatofocusing : isoelectric focusing on ion-exchange columns. II. experimental verification

Theoretical considerations indicate the possibility of producing focusing effects in the ion-exchange chromatography of proteins in a pH gradient similar to those in the electrophoretic method of isoelectric focusing. A pH gradient can be produced "externally" by mixing two buffers of a different pH in a mixing chamber, or "internally" by taking advantage of the buffering action of the ion exchanger and running a buffer, initially adjusted to one pH, through a column initially adjusted to another pH. The latter method has the advantage of not subjecting the proteins to a pH more extreme than about its isoelectric pH value. Equations are derived for the width of the protein bands, taking into account axial diffusion and axial dispersion by non-uniformity of flow and for the pH at which proteins are expected to emerge from the column. These equations serve as a guide for establishing the conditions for efficient separations.