Date of Award

12-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Chemistry

Advisor

Marcys, R. Kenneth

Committee Member

Christensen , Kenneth A

Committee Member

Anker , Jeffrey N

Committee Member

Brown , Phili J

Abstract

High performance liquid chromatography (HPLC) is a fundamental methodology for the characterization and purification of macromolecules. Since its development in the 1970's, HPLC has significantly advanced its instrumentation and column technology to become a powerful analytical technique. A considerable amount of research focuses on advancements to the stationary phase, or solid support, on which molecules interact and separate. To overcome limitations of other phases that lead to poor mass transfer, slow speeds, and low efficiency, nonporous and superficially porous phases have been developed. Specifically, fiber based polymer stationary phases show great promise as stationary phases for protein separations. The Marcus laboratory has investigated capillary-channeled polymer (C-CP) fibers as an HPLC stationary phase for the past decade. These novel shaped fibers have advantageous attributes including increased surface area over cylindrical cross section fibers, low cost, rapid mass transfer, and the ability to operate at high linear velocities without high pressure. The fibers are available in several base which allow for a wide variety of chemical interactions. What's more, surface modifications can allow for a broader range of chemical specificity. Therefore, the focus of this work is on adosprtion-based surface modification of C-CP fibers. First, modification with recombinant protein A ligand allowed for the capture and recovery of immunoglobulin G (IgG) antibody. This small scale study evaluated maximum protein A ligand density, the stability of the modification, and demonstrated the selective capture of IgG from a mixture with myoglobin (as a surrogate host cell protein) with minimal non-specific binding through the use of a sodium citrate (pH 4) wash buffer. IgG was recovered with high yield with a 0.1 M acetic acid elution buffer. The second modification involves the adsorption of head-group functionalized poly(ethylene glycol) lipids (PEG-lipids), where the lipid tail strongly adsorbs to the polypropylene surface and the hydrophilic PEG group extends away from the surface allowing for the functional ligand to interact with the analyte of interest. This modification was first evaluated as an initial proof-of-concept study, where biotin-PEG-lipid modified PP C-CP fibers were able to selectively capture streptavidin from a complex mixture that also contained a green fluorescent protein. Non-specific binding was minimized through the use of a 0.1 % PBS-Tween buffer. Next, a more in-depth study of surface loading characteristics determined maximum binding capacity when using FITC-PEG-lipid to modify the fibers. This modified surface was also exposed to several test solvents to reveal a highly robust surface modification. Finally, the mechanism of interaction between the lipid and the polypropylene was determined through modification with an environmentally sensitive probe, NBD. Fibers were modified with lipids containing an NBD group attached to either the head group or the fatty acid tail. Fluorescence imaging revealed that the lipid tail intercalates into the PP structure to yield an efficient, robust surface modification. Overall, modifications of the polypropylene fiber surface show promise in increasing the efficiency of affinity separations.

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