HIGH-PERFORMANCE MEMBRANE CHROMATOGRAPHY - HIGHLY EFFICIENT SEPARATION METHOD FOR PROTEINS IN ION-EXCHANGE, HYDROPHOBIC INTERACTION AND REVERSED-PHASE MODES

High-performance membrane chromatography (HPMC) is a very effective ch romatographic method in which all the mobile phase flows through the s eparation medium. The effects of process variables such as concentrati on of displacement agent, flow-rate and gradient slope on HPMC separat ions in the ion-exchange, hydrophobic interaction and reversed-phase m odes were studied using model protein mixtures. The basic relationship s characterizing column HPLC also apply in HPMC. Whereas the efficienc y of the HPMC membrane does not depend on flow-rate, the resolution in creases with increasing gradient volume. Separations obtained with a c ontinuous linear gradient were used for the design of a stepwise gradi ent profile which decreases the consumption of both time and mobile ph ase in separations of proteins. According to calculations, the protein diffusivity enhanced by the convective flow through the membrane is a bout four orders of magnitude higher than the ''free'' diffusivity of the protein in the stagnant mobile phase located in the pores of a sta ndard separation medium. This considerably speeds up the process and i mproves the efficiency of the separation.