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Historically, separation of a protein mixture after adsorption to a cation-exchange column is effected by alteration in ionic strength.An alternative separation method using pH induced gradient in the range of 4¨C7.5was studied.A cation-exchange column with large particle beads containing excessive carboxyls was employed.A pH-gradient across the columnwas generated by a step change at the column entrance using a non-retained buffer system. Consistency and accuracy of pH values in timed intervalswere demonstrated in three different batches. In development of the application, we found a correlation coefficient of >0.9 between the elution pH values of six acidic proteins and their isoelectric points.One case study showed the resolution between a monoclonal antibody and non-retained protein species from a protein A column. Another case study showed the feasibility of separating polyethylene glycol conjugated protein from native protein.

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