Peptide-protein analysis and characterization by multidimensional liquid chromatography and capillary electrophoresis
Abstract
This research was conducted to study the type of multidimensional liquid chromatography utilizing partitioning based chromatography in the first dimension. The “drifting peak” phenomenon was studied from the fundamental theory and practical viewpoint and various solutions were proposed and validated. Various types of two-dimensional HPLC which combine ion-exchange and reversed-phase chromatography or two different reversed-phase chromatography columns were explored to separation of peptides. Effects of different ion-pairing agents on selectivity and resolution of peptides were studied as well. A group of new cationic ion-pairing agents was developed to study the deamidation of recombinant human growth hormone (rhGH). Two-dimensional HPLC targeted component analysis system demonstrated its power in resolving deamidated from native peptides. A stable, hydrophilic polymer coating was developed by adsorbing polyethyleneimine to the surface of fused silica capillaries through electrostatic interaction and then cross-linked with ethyleneglycol diglycidyl ether. These columns were used to carry out electrophoretic separations and immunological assays. Excellent reproducibility and resolution of peptides was achieved in the electrophoresis mode. This coating was also used in combination with avidinbiotin chemistry for quantitative analysis of antibodies by flow-through ELISA. These open tubular columns show great promise in process monitoring by flow-through ELISA.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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