Retention Studies and Protein Separation by Potential Barrier Chromatography

Abstract

Potential barrier chromatograpy (PBC) exploits the fact that the depth of the adsorption well of the interaction potential between adsorbate and adsorbent can become moderately deep when it is controlled by opposing van der Waals attractive forces and repulsive double layer forces. The moderately deep potential well gives rise to repeated cycles of adsorption and desorption, thereby allowing isocratic elution of proteins to occur. Separation of proteins is achieved because the depth of the adsorption well, which affects the retention time, is very sensitive to the size, charge, and hydrophobicity of the adsorbate molecules. Here, a mixture of six model proteins has been separated using PBC on a commercially available strong anion-exchange column. The selection of the appropriate mobile phase conditions of pH and ionic strength led to fast separations using a two-step isocratic elution procedure. The effects of temperature and various organic additives on the resolution of protein separations have also been investigated.