A Practical Approach to Improve the Resolution of Dansyl-Amino Acids by High-Performance Liquid Chromatography

Abstract

We describe an HPLC procedure for the separation of dansyl (Dns)-derivatives of all common amino acids present in polypeptide hydrolyzates. A linear gradient of solvent B [methanol: water, 70:30, (v/v)] in solvent A [30 mM sodium phosphate buffer, pH 7.4, containing 5 mL methanol and 6.5 mL tetrahydrofuran (THF)], from 0 to 100% B in 30 min, at 1.0 mL/min and 25°C, was used to elute the Dns-amino acids from a 4 μm NovaPak C₁₈ column with good resolution. The Dns-amino acids were detected fluorometrically at 338 nm wavelength excitation and 455 nm emission.

The concentration of sodium phosphate buffer was critical for the resolution of the dansyl-derivatives of: Arg from Ser and Thr, NH₃ from Val and Met and of cysteic acid from Asp and Glu. THF mainly improved the resolution of the dansyl-derivatives of: Arg from Ser and Thr, NH₃ from Val, and Leu from Ile and Trp. The effects of sodium phosphate buffer and THF on the column capacity factor provided a convenient and reproducible manner to adjust elution conditions for Dns-amino acid separation when using new columns.

Using this procedure, we showed that Asx is the N-terminal amino acid residue of human pancreatic secretory trypsin inhibitor, in agreement with sequence data.