Postcolumn derivatization of peptides with fluorescamine in capillary electrophoresis.
Fluorescamine is used as a postcolumn derivatization reagent for fluorescence detection detection of peptides after separation by capillary electrophoresis. The problems resulting from the use of an organic solvent have been solved by introducing LiC1O4 and 5% water into the postcolumn derivatization reagent. The reaction rate and detection sensitivity of amino acids and small peptides observed with fluorescamine and OPA were compared. Fluorescamine gives much higher sensitivity than o-phthaldialdehyde (OPA) for small peptides, with detection limits for the selected peptides and amino acids below 0.1 mumol 1-1. Under optimized experimental conditions, the method has a good reproducibility and separation efficiency for peptides. The method was applied for the analysis of the protein tryptic digests. Only submicromolar concentrations of proteins were required.
Laboratory for Analyical Chemistry, University of Amsterdam, The Netherlands.
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D000596:Amino Acids / Q000032:analysis; D019075:Electrophoresis, Capillary; D005450:Fluorescamine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D010455:Peptides / Q000737:chemistry*; D012997:Solvents; D014357:Trypsin; D009764:o-Phthalaldehyde
D000596:Amino Acids; D007202:Indicators and Reagents; D010455:Peptides; D012997:Solvents; D005450:Fluorescamine; D009764:o-Phthalaldehyde; D014357:Trypsin