PECULIARITIES OF ZONE MIGRATION AND BAND BROADENING IN GRADIENT REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF PROTEINS WITH RESPECT TO MEMBRANE CHROMATOGRAPHY

The peculiarities of zone migration and band broadening in the reverse d-phase gradient HPLC of proteins were investigated. In the isocratic mode a critical composition of the mobile phase was found at which all proteins regardless of their molecular mass migrate with equal veloci ty and have a capacity factor equal to the phase ratio (V(P)/V0) the s ame capacity factor as a marker of total accessible volume would have in steric exclusion chromatography. It is shown that steric exclusion conditions are never achieved in gradient HPLC. In the first (adsorpti on stage) of gradient elution where the separation takes place the vel ocity of a protein increases until it becomes equal to the velocity of the desorbing solvent front at a critical distance X0 from column ent rance. Strong broadening is characteristic of this stage. In the secon d (critical) stage the protein travels the remaining distance (L - X0) with the velocity of the solvent. A definition of X0 is given allowin g one very simple calculation of the minimum permissible column length as a function of gradient steepness, mobile phase velocity and protei n adsorption parameter. When x = X0 the protein zone has the smallest dispersion. Making L < X0 is especially disadvantageous. as it leads t o anomalous bandspreading. The theory of gradient HPLC was refined on this basis and the usefulness of this approach in high-performance mem brane chromatography is demonstrated.