Rational Design Calculation Method for Stepwise Elution Chromatography of Proteins

Although linear gradient elution ion-exchange chromatography is best suited for very fine separation of proteins, it is not commonly employed for the actual protein purification process. Instead, stepwise elution is usually used. However, it is difficult to design an efficient stepwise elution process as small changes in the buffer salt concentration often affect the retention volume and the peak width significantly. In this paper, a design calculation method for stepwise elution proposed previously was modified and extended. This method uses the data obtained by linear gradient elution experiments. The efficiency of the process was examined on the basis of the peak width and the separation time. The advantages using convection-aided chromatography media (monolithic columns) were investigated.