High-Performance Aqueous Size-Exclusion Chromatography Using Diol-Bonded Porous Glass Packing Materials. Retention Behavior of Some Proteins

Abstract

Retention volume of proteins increased or decreased with increasing phosphate buffer or neutral electrolyte concentrations in the mobile phase. This variation suppressed or accelerated by changing pH values in the mobile phase. The behavior of proteins can be interpreted by knowing isoelectric points (pI) of proteins and pKa value of the residual silanol groups on the surface of diol-bonded porous glasses. Positively charged surface of proteins below pH 8.0 (cytochrome c, lysozyme) retarded the elution by the ion-adsorption effects and negatively charged proteins around pH 7.0 (egg albumin, bovin serum albumin) eluted earlier than expected by the ion-exclusion effects. These effects suppressed by increasing phosphate buffer and neutral electrolyte concentrations in the mobile phase. Size-exclusion separation was attained in the mobile phase over 0.1 M phosphates and 0.1 M NaCl concentrations at pH 7.0. Mcllvaine buffer and Gomori buffer showed opposite action to proteins for retention comparing with Soerensen phosphate buffer. Potassium thiocyanate showed the different action for retention of proteins comparing with other neutral electrolytes and acted like sodium dodecyl sulphonate.