Date of Award

12-2015

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Chemistry

Advisor

Marcus, Richard K

Committee Member

Anker, Jeffrey

Committee Member

Brown, Phillip

Abstract

Capillary-channeled polymer (C-CP) fibers have been studied in this laboratory as stationary phases for protein separations in high-performance liquid chromatography (HPLC). C-CP fibers are uniquely shaped so as to include eight continuous capillary-channels which interdigitate once packed into a column. The packed column resembles a monolithic structure of unobstructed flow through capillary channels which reduces backpressure and increases linear velocity, reducing separation time. Fibers are effectively nonporous with respect to macromolecules, resulting in fast mass transfer and high sample recovery. C-CP fibers made from polypropylene (PP) yield a fairly homogenous hydrophobic surface suitable for reversed phase (RP) chromatography. In a microbore C-CP column, separations can be done very quickly, in less than ten minutes, even at low flow rates, saving time as well as money in column cost, solvent cost and waste generation. Electrospray ionization mass spectrometry (ESI-MS) analysis provides more analytical information than multi-wavelength detectors. However, the low flow rates needed (<0.5 mL min-1) for optimal spectral clarity generally imply low linear velocities and slow separations if liquid chromatography (LC) in tandem with mass spectrometry (MS) is desired. To this end, decreased C-CP column diameters (0.5 mm i.d.) were employed to increase linear velocity, and therefore speed separation time, without the need to increase flow rates. Ribonuclease A, cytochrome c, myoglobin and lysozyme were loaded in phosphate buffered saline and urine. Matrix was removed using a water loading phase before applying a gradient of ACN to elute proteins according to hydrophobicity into ESI-MS. The truly interesting and research relevant proteins are often found in small concentrations in serum. Analysis of biofluids for potential biomarkers (i.e. proteins) has been of great interest in medicinal research; however, most biomarkers are typically found in minute concentrations and masked by more abundant proteins, prominently, serum albumin. A large portion of albumin hinders the collection of the proteins of interest. Modifying nylon C-CP fibers with cibacron blue dye, an albumin-specific ligand has the potential to selectively remove albumin from serum samples.

Included in

Chemistry Commons

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