The Separation of Dansyl Amino Acids by Reversed-Phase High Performance Liquid Chromatography

Abstract

The dansyl amino acids have been separated by reversed phase HPLC on a single column in about 30 min, using a linear gradient formed from acetonitrile and sodium phosphate buffers of approximately neutral pH. The effect on retention times of the pH and ionic strength of the eluting buffer has been investigated and, by an appropriate choice of these variables, a separation of most of the derivatives of the 20 amino acids commonly found in proteins may be made on a column of either μBondapak® C₁₈ or Spherisorb 5ODS. The greatest difference between the two columns was found to be in the retention of the basic dansyl derivatives, particularly α-Dns-His and Dns-Arg. Owing to the quenching of the fluorescence of the dansyl amino acids in aqueous solutions, the UV absorbance at 250 nm has been used for detection giving a sensitivity of about 100 pmol for a single component. It is suggested that this system may be useful for the analysis of peptides at high sensitivity and for the quantitation of N- and C-terminal amino acids.