Improving selectivity in multimodal chromatography using controlled pH gradient elution

Externally generated pH gradients are employed on a multimodal cation exchange chromatographic resin to improve the selectivity for a mixture of model proteins. By combining controlled pH gradients with the unique selectivities arising from the multiple interaction types exhibited by the multimodal resin, the separation of the protein mixture is significantly improved as compared to linear salt gradient operation. Several gradient conditions are explored and a shallow gradient from pH 3.8 to 5.5 is shown to be able to resolve the proteins. This work provides proof of concept for the use of pH gradients in multimodal chromatography and sets the stage for future applications.

► Proof-of-concept study for the use of pH gradients in multimodal chromatography. ► Separation of a protein mixture using externally generated pH gradients. ► pH gradient produced improved separation of proteins as compared to salt gradients.