Peculiarities of gradient ion-exchange high-performance liquid chromatography of proteins

Since the influence of column length on protein resolution in high-performance liquid chromatography (HPLC) is not clear, different viewpoints presented in the literature are analysed in detail. The influence of gradient steepness on the length of the working column part ( X 0) or the part of a column in which the quasi-steady state is attained was studied. The equation for estimating the X 0 value was obtained for the general case of the retention model. It was shown that at steep gradients only a short part of the column is used as the working part on which all separation processes develop. The other part of a column is a ballast where the protein zone migrates in a regime of parallel transfer. These results form a theoretical basis for high-performance membrane chromatography. As was shown experimentally, this method makes it possible to perform protein separation at low gradient times with appropriate resolution, comparable with that of HPLC.