(199c) Electrostatic Interaction Chromatography for Separation of Very Similar Proteins – Effects of Stationary and Mobile Phase Properties

It is well known that very difficult separation of protein variants or isoforms can be achieved by electrostatic interaction chromatography (ion-exchange chromatography, IEC) although the mechanism has not yet been fully clarified. Separation behaviour of several protein variant systems was investigated. The number of binding sites B values were determined from linear (salt) gradient elution (LGE) experiments as a function of pH, The B-pH curves provided important information on the biorecognition mechanism of proteins. We also examined how mobile phase properties affect the separation behaviour by using different types of column materials such as membrane-based IEC, monolithic IEC disks as well as conventional porous packing IEC. In most cases proteins are retained near or at the isoelectric points (pI), and the resolution was better near the pI where the number of binding site B values are small, ca.2-3. We also examined which parts of proteins are responsible for the separation (or retention) based on protein-structure-charge distribution information.