Gradient chromatofocusing (GCF) utilizing linear pH gradients provide unique capabilities in pI based protein separation when compared with the conventional ion-exchange and conventional chromatofocusing chromatography. By exploiting the advantages of GCF technique, the present work demonstrates the comparison of chromatograms of proteins chromatographed on a DEAE weak anion-exchange column employing the same descending linear pH- gradient with two buffer systems which differ in number of buffer components. These linear pH-gradients over 6.5-3.5 pH range generated by the mixing of high pH application buffer with low pH acidic elution buffer via a gradient HPLC pump system. Also reported is a new way of generating linear pH-gradients that eliminates non-linear drops in pH that are problematic in current gradient HPLC techniques by introducing the bridging buffer components between application and elution buffers. Experiments compared chromatography of five standard proteins for pH gradient generated by buffer system I (less buffer components with spacing of buffer pKa of approximately 1 pH unit) with employing the exact same pH gradient using buffer system II (more buffer components with spacing of buffer pKa of approximately 0.5 pH units).Results showed that proteins were focused into narrow bands and improved resolution with more component buffer system II compared to buffer system I.

Effect of the number of buffer components on separation of protein in high performance gradient chromatofocusing ion-exchange using linear pH gradients

246th National Meeting (2013)

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