Control of the Interaction Potential for Improved Resolution in Potential Barrier Chromatography

Potential barrier chromatography (PBC) is a recently demonstrated protein separation technique which uses an isocratic elution procedure and exploits the differences in the interaction potentials between the proteins and the adsorbent. The interaction potential is determined by the van der Waals attraction, double-layer, Born, and hydration repulsions between the adsorbent and adsorbates and is very sensitive to the properties of the molecules, such as charge and size. A separation is feasible without any change in the composition of the mobile phase when the interaction potentials have surmountable potential barriers to adsorption and moderately deep adsorption energy wells. To ensure short analysis time and useful resolution, the total interaction potential must be controlled by suitably modifying the van der Waals attraction and the double-layer repulsion. The van der Waals attraction can be controlled by the introduction of small amounts of organic solvents in the aqueous mobile phase. The double-layer repulsion can be modified by changes in pH, ionic strength, or chemical nature of the ions of the mobile phase. Additionally, changes in temperature may be used to improve resolution. Here an updated high performance liquid chromatography version of PBC is reported. Using an isocratic elution procedure and an inexpensive ion-exchange column, the effect of changes in the pH, ionic strength, chemical nature of the ions, and organic solvent content of the mobile phase on the retention times and resolution of two model proteins (ovalbumin and bovine serum albumin) are demonstrated. Improved separations with high resolutions are achieved.