Separation as an analytical tool for peptide and protein analysis
Abstract
The objective of this research was to examine various analytical systems including high performance liquid chromatography (HPLC), capillary electrophoresis (CE), capillary electrocgromatography (CEC) and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS) in the analysis of peptides and proteins. HPLC was used to explore the nature of interactions between protein analytes and immobilized proteins. Organic additive was added to the mobile phase to optimize and overcome selectivity changes during the separation of analyte proteins. Chromatographic retention was found to be determined by a relatively small number of amino acids located in a chromatographic contact region on the surface of polypeptides. Methods were developed to study the location of the polypeptide contact region involved in the ion exchange separation by HPLC. Packed capillary electrochromatography (CEC) has advantages of being able to separate mixtures with both high resolution and selectivity. The disadvantage is that the packing and frits cause air bubble formation when high voltage is applied across the column. Capillary columns with low phase ratio were examined as an alternative approach to CEC and found to eliminate both the air bubble and frits problems. Capillary columns were coated with polymer to stabilize electroosmotic flow and prevent adsorption of proteins during isocratic separations. The problem in capillary electrophoresis (CE) separation is that the separation is limited in a narrow window of analytes and adsorption of analytes on the column. A method was developed to separate protein mixtures which range in pI from 9.0 to 5.1 by coating capillaries with the polymer to prevent adsorption.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry|Organic chemistry|Biochemistry
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