Date of Award

12-2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Chemistry

Advisor

MARCUS, RICHARD K

Abstract

Capillary-channeled polymer (C-CP) fibers are being developed and characterized as high-performance liquid chromatography (HPLC) stationary phases for reversed-phase separations of proteins. Conventional porous phases are not well suited for separating large macromolecules due to the slow diffusivity of the molecules, large diffusional distances, and stagnant mobile phase zones within the column. Stationary phases are continually being developed to address the challenges associated with HPLC of proteins and other macromolecules in general. C-CP fiber stationary phases are an alternative to conventional bead technology and offer a variety of chemical and physical advantages. The fibers can be selected for their chemical functionality (e.g., polypropylene, polyester, nylon) and are stable over a wide range of pH. They are non-porous, which addresses the mass transfer limitations encountered in macromolecular separations performed on porous media. Fibers within the columns interdigitate to form continuous rod-like structures that are similar to monoliths. Flow rates up to 9 mL/min can be used on conventional LC systems with minimal system backpressures, which is indicative of efficient mass transport through the column bed with minimal obstructions to fluid flow. When compared to conventional columns of the same dimensions, C-CP fiber columns exhibit 70 % lower system backpressures over a range of flow rates. Novel reversed-phase (RP) chromatographic methods using polypropylene (PP) and poly (ethylene-terephthalate) PET fiber columns have been developed, optimized, and compared to a conventional packed-bed column used in macromolecule separations. The reproducibility of the packing procedure and column efficiency were determined by chromatographic characterization, i.e., retention times, selectivity, elution order, resolution, peak shapes, peak areas, and peak widths. Overall, the PET fiber phase was best suited for protein separations when compared to the PP fiber type. Protein adsorption on surfaces is an affinity related process (kinetic process), so breakthrough analysis was used to evaluate the adsorption kinetics of the adsorbate-adsorbent system (protein-C-CP fiber). The breakthrough curve data were analyzed to determine the applicability and limitations of this technology for preparative and rapid protein separations. The steep, uniform frontal profiles of the breakthrough curves are indicative of systems with favorable mass transfer kinetics. The protein adsorption characteristics of C-CP fibers evaluated in these studies were similar to what has been reported for other stationary phases being used in rapid separations of proteins. These fundamental studies and the corresponding results presented here support the use of C-CP fibers as HPLC stationary phases for macromolecule separations and adsorption studies.

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