Open tubular capillary electrochromatography-laser induced fluorescence for the separation and detection of proteins and amino acids.

摘要

With its high efficiency and reproducibility, open tubular capillary electrochromatography (OTCEC) is beginning to prove itself as a promising method for the separation of proteins, peptides and pharmaceuticals. OTCEC, when coupled to an absorbance-based detector, suffers from poor sensitivity and high concentration limits of detection. However, laser induced fluorescence coupled to CEC has shown picomolar limits of detections for some compounds. This thesis describes the coupling of OTCEC to a laser induced fluorescence (LIF) detector to optimize both the separation and detection aspects of protein and amino acid analysis. OTCEC capillaries were etched and silanized, and the 4,4' cyanopentoxy biphenyl phase was attached through a radical-initiated process. Such biphenyl phases have shown unique dependence on temperature and mobile phase composition and demonstrated higher selectivity for certain compounds3 than bare silica capillaries. Proteins and amino acids were modified using pre-column derivatization formats with two different fluorescein-based dyes (fluorescein isothiocyanate, FITC) and Atto-tag FQ (3-(2-furoyl)quinoline-2-carboxaldehyde). These conditions were varied to attempt consistent labeling conditions with varying protein size. Optimizations of the analyses were achieved by modifying conditions such as background electrolyte, pH, voltage, and percent organic modifiers. These modified conditions were used to achieve the highest resolution on protein and amino acid derivatives. Results determined that high sensitivity and efficiency are achieved when LIF detection is coupled to cyano pentoxy biphenyl coated capillaries using optimal conditions. These conditions include (1) the use of Atto-tag FQ derivatization and (2) an 80:20 acetonitrile: 50 mM phosphate buffer (pH 2.00) background electrolyte with a positive electrical current applied.