pH-gradient ion-exchange chromatography: an analytical tool for design and optimization of protein separations

This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (pI<6) and basic (pI>8) proteins elute roughly at their pI, whereas neutral proteins (pI 6-8) elute at pH 8-9 regardless their pI values. Because of the flat nature of protein titration curves from pH approximately 6 to approximately 9, neutral proteins indeed exhibit nearly zero net charge at pH approximately 9. The elution-pH in pH-gradient IEC or the titration curve, but not the pI, was identified as the key parameter for pH optimization of preparative IEC in a fast and rational way. The pH-gradient IEC was also applied and found to be an excellent analytical tool for the fractionation of crude protein mixtures.