CF separates proteins according to differences in isoelectric point (pI), the pH at which a protein has a zero surface charge. It is a powerful method for high resolution, analytical separations since it can resolve very small differences in pI (down to 0.02 pH units) and thus separate very similar proteins. However, the capacity of the method is low and should ideally be used for partially pure samples.
A pH gradient is generated on the column as buffer and chromatography medium interact. The medium is a weak anion exchanger, and the buffer is composed of a large number of buffering substances such as Polybuffer. Proteins with different pI values migrate at different rates down the column, along the descending pH gradient, continually binding and dissociating while being focused into narrow bands and finally eluted.
The protein with the highest pI elutes first, and the protein with the lowest pI elutes last. The upper limit of the gradient is defined by the pH of the start buffer, and the lower limit of the gradient is defined by the pH of the elution buffer. Polybuffer performs optimally over pH intervals of 3 pH units or less, and the narrowest pH intervals are likely to give the highest resolution.