Date of Award

12-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Mary Wirth

Committee Chair

Mary Wirth

Committee Member 1

Graham Cooks

Committee Member 2

Pete T. Kissinger

Committee Member 3

Marcy H. Towns

Abstract

Glycosylation is the most common post-translational modification affecting protein activities, including delivery to cell surface, binding affinity, and mediating different physiological effects. Separations of glycoproteins and their glycoforms are essential for drug discovery and development, identification of potential disease biomarkers and quality control. The efficiency of glycoprotein separations is limited by the bonded phases, requiring advances in chromatographic materials. Such advances would improve resolution, allowing for more species to be isolated from a mixture and studied.

Our group found that submicrometer nonporous silica stationary phases with tethered polymer chains (acrylamide) give excellent hydrophilic liquid chromatography (HILIC), a common method used in glycosylation analysis. These columns are limited by lack in reproducibility and stability. A new method to make polymer bonded phases to give more reproducible, stable, and higher efficiency HILIC columns for glycoproteins is evaluated. In this new method the polymer is grown on the silica surface bearing an initiator whilst inside the chromatographic column, rather than the conventional method of packing particles that are already modified. Compared to columns packed with previously modified particles, in-column modification showed overall higher column efficiency and stability for intact ribonuclease B and its glycoforms.

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