Adsorption chromatography of proteins: Determination of optimum conditions

A simple and convenient method for predicting optimum chromatographic conditions from linear gradient elution experiments with a small column is presented and verified experimentally. The method was found to be very useful for a rapid survey of the nature of unknown proteins, for the estimation of optimum chromatographic conditions, and for scaling up the separation of proteins in ion exchange and hydrophobic interaction chromatography. Two applications were successfully carried out on the basis of the proposed method: (1) Separation of a protein mixture of ovalbumin and β‐lactoglobulins A and B with both anion and cation ion exchange columns by means of column switching technique. In this method, the sample is always subjected to the elution process until it is collected as a purified fraction through the two columns. A special valving system is devised for this method. (2) Large‐scale ion exchange chromatography separation of β‐galactosidase. Linear gradient elution of crude β‐galactosidase was carried out with a 30 L ion exchange gel column on the basis of data obtained with a small (23 mL) column.