Performance of pH elution in high-performance affinity chromatography of proteins using non-porous silica

Abstract

Non-porous silica with an average diameter of 1.4 μm was synthesized and derivatized for the high-performance affinity chromatography of proteins. The elution behaviour of proteins on a non-porous silica-based adsorbent was investigated both theoretically and experimentally using human immunoglobulin G (IgG) and immobilized protein A as the affinity pair. An equation used to predict the elution peak profiles was obtained. A comparison was also made between predicted and experimental elution peaks from chromatography of IgG on immobilized protein A. Experimental data indicated that samples with IgG contents up to 360 μg could be analysed chromatographically using a column packed with the non-porous silica-based affinity adsorbent. Alternation of the mobile phase pH is one of the conventional techniques employed to perform the elution of bound proteins from the column. Using this technique, the desorption rate constant and equilibrium association constant under elution conditions were found to have substantial effects on the elution time and the shape of the elution peak. The influence of sample load on the peak height was also examined.