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Separation of a multicomponent mixture of proteins by potential barrier chromatography

Separation of a multicomponent mixture of proteins by potential barrier chromatography

Separation Science and Technology 21(2): 111-138

Potential barrier chromatography (PBC) is a high performance liquid chromatographic method which uses an isocratic elution procedure and exploits the high sensitivity of the interaction potentials (between the proteins and the packing) to small differences in their physicochemical properties (isoelectric point, hydrophobicity, molecular size, etc.). The interaction potential is a result of the van der Waals attraction and double-layer, Born, and hydration repulsions. While a sufficiently strong van der Waals attraction has the tendency to generate a deep adsorption energy well, the double-layer repulsion prevents the occurrence of such a deep well; however, the latter repulsion together with the van der Waals attraction also generates a potential barrier to adsorption. Separation is feasible when the above-mentioned interaction potentials between adsorbent and adsorbates produce surmountable energy barriers to adsorption as well as moderately deep adsorption energy wells. The interaction potentials can be controlled by changing the pH and ionic strength of the mobile phase, thus affecting the double-layer repulsions, and by adding a small amount of organic solvent to the mobile phase, thus altering the van der Waals attraction. A separation can be achieved by properly tuning the mobile phase conditions, such as the pH and the ionic strength and/or the organic content of the mobile phase, to produce sufficiently different, but moderate, depths of adsorption energy wells for the individual adsorbates. In the present work an anion-exchange adsorbent and four model proteins (.gamma.-globulin, albumin, bovine serum albumin, and ovalbumin) are employed. The optimum conditions for the best resolution of the four model proteins are identified as pH = 4.35 and no additional electrolytes. As a result of double-layer repulsion, the adsorption energy well is not too deep and hence, the processing time is short. Second, the effects of the amount of the protein mixture injected in a single pulse and that of a succession of pulses at short time intervals on the resolution of separation are investigated.

Accession: 006391242

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