Analysis of Peptides and Amino Acids by Pre-column Derivatization High Performance Liquid Chromatography with Fluorescence Detection

Abstract

Abstract: Anew activated ester fluorescence derivatization reagent, N-hydroxysucci-nimidyl-3-indolylacetate(SIIA) is used for analysis of small peptides and their hydrolysates by HPLCand fluorescence detection. In a one-step procedure, SIIAreacts with peptides and amino acids to form stable derivatives. Studies on derivatization conditions demonstrate ex-cellent derivative properties of the reagent. Maximal yields are observed with a molar reagent excess of approximately twenty five or greater and heated at 60℃ for 10 min in pH 8.0 buffer. SIIAderivatives of glutathionide, diglycine, glutamic acid, cystine and glycine are separated on C₁₈, reverse-column with the mobile phase of CH₃OH/H₂O (20/80, V/V)containing 10mmol/LpH 3.6 citric acid-Na₂HPO₄ buffer. They are detected at λ_cx/λ_cm＝278 nm/355 nm with the detection limits ranged from 0. 2 pmol to 2. 5 pmol.

Key words: Activated ester reagent, Fluorescence derivatization, High performance liquid chromatography, Peptides, Amlno acids

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LIU Xun, XU Guo-Liang, ZHANG Hua-Shan, CHENG Jie-Ke. Analysis of Peptides and Amino Acids by Pre-column Derivatization High Performance Liquid Chromatography with Fluorescence Detection[J]. Chem. J. Chinese Universities, 1997, 18(6): 873.

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Analysis of Peptides and Amino Acids by Pre-column Derivatization High Performance Liquid Chromatography with Fluorescence Detection

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