Preparative Separation of Proteins by Gradient- And Stepwise-Elution Chromatography: Zone-Sharpening Effect

A method is developed for predicting the zone-sharpening effect in linear gradient- and stepwise-elution chromatography of proteins. Numerical calculations have shown that the elution curve by different models are almost the same when the values of zone spreading parameters in the model are chosen so that the values of the number of the theoretical plates are equal. A good correlationship curve is established on the basis of the numerical calculations, from which the degree of the zone-sharpening can be easily predicted with a single dimensionless parameter. When the modulator concentration is very high (the desorption is complete), the stepwise-elution chromatography can be regarded as the gradient elution with steep-slope of the gradient. The experimental stepwise elution cation-exchange chromatography of basic proteins from egg white has shown that the purity, the recovery and the concentration factor of the recovered fraction are very high.