Analysis of Dabsyl Amino Acids using HPLC

Download PDF August 25, 2017

Introduction

The pre-column derivatization using HPLC for amino acids analysis has prevailed as a simultaneous separation method of multiple components using a selective, high-sensitivity detector and a reversed phase column.

Many results of amino acids analysis using dabsyl chloride (4-(Dimethylamino)azobenzene-4’-sulfonyl chloride:DABS-Cl) as a reagent for pre-column derivatization have been reported because the amino acids derivatized by dabsyl chloride are more stable than those by other derivatization agents. Also a simply a UV/VIS detector can be used with high sensitivity. Here the standard mixture of amino acids was measured by the system using dabsyl chloride.

Jasco PU-2080, DG-2080-53, LG-2080-02, AS-2055,CO-2060, UV-2070

Experimental

Results

Figure 1 shows the chromatogram of standard mixture of amino acids. 17 kinds of amino acids were separated in 45 minutes.

Figure 1. Chromatogram of standard mixture of amino acids. Aspartic acid, Glutamic acid, Serine, Threonine, Arginine, Glycine, Alanine, Proline, Valine, Methionine, Isoleucine, Leucine, Phenylalanine, Cystine, Lysine, Histidine, Tyrosine

Advantages of Dabsylation

  • By using the kit (DAB Label), the procedure of intricate preparation of derivatization can be avoided.
  • Amino acid composition of 0.5 µg of proteins or peptides can analyzed with good accuracy and reproducibility.
  • 17 kinds of the derivatized amino acids can be separated within 45 minutes.
  • The derivatized amino acids are very stable. (for one month at room temperature)
  • The derivatized amino acids can be detected by visible light at 465 nm, which allows high-sensitivity analysis without the interference from other components having UV absorption.

DABS-Cl reacts with α-amino groups, ε-amino groups, phenolic hydroxyl groups and imidazole groups.

Figure 2. Reaction formula with α-amino group.

About the Author

Mr. Akao, is the Applications Group Manager at JASCO Corporation, with many years at the company working on applications in electronic and vibrational spectroscopy, and is an expert in IR and Raman spectroscopy.