Date of Award
Doctor of Philosophy
Silica supports containing "spacer arms" with terminal carboxylic acid groups were prepared by refluxing diol-bonded silica in a solution of a cyclic anhydride. Surface densities ranging from a few percent to complete monolayer coverage were obtained by varying the reaction time, temperature, and anhydride concentration. These supports were directly used for cation-exchange chromatography of proteins. In addition, the carboxylate silica was further derivatized to form reversed-phase and affinity chromatographic supports;In the ion-exchange chromatography, the Z numbers (defined as numbers of interaction sites on a protein surface when it comes into contact with the stationary phase) of cytochrome C and lysozyme were determined on silica cation exchange supports as a function of stationary phase ligand density. It was found that the elution order reversal, or selectivity change, for cytochrome C and lysozyme with ligand density was due to the heterogeneous binding energy of different interaction sites on a protein surface. This led to variations in the binding strength difference between these proteins as the ligand density was changed. The elution order reversal was accompanied by nearly constant values of the Z number difference (Z of lysozyme - Z of cytochrome C) and the ratio Z(lysozyme) / Z(cytochrome C), indicating that the degree of protein-resin interaction is not necessarily dependent upon the Z number in ion exchange chromatography;In the reversed-phase chromatography, C-bonded phases were prepared by coupling octylamine to the carboxyl silica using a water-soluble carbodiimide. Chromatography of proteins on the lower ligand density phases resulted in higher mass recovery and less denaturation of the proteins, while the resolution was insensitive to ligand density;In the affinity chromatography, the activation of carboxylated silica gels using N-hydroxysuccinimide and the coupling of protein to the activated supports were examined in terms of pore size, condensing reagent, and pH. It was found that the specific activities of several immobilized proteins were increased as the ligand density was decreased.
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Wu, Danlin, "Effects of stationary phase ligand density in high-performance liquid chromatography of proteins " (1987). Retrospective Theses and Dissertations. 8600.