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Home / Applications / Analysis of Dabsyl Amino Acids using HPLC

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  • Technique

Analysis of Dabsyl Amino Acids using HPLC

By Kenichi Akao

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January 5, 2024

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

Keywords

430022H

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.
This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.

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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.

JASCO Application Note

Analysis of Dabsyl Amino Acids using HPLC

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.

Keywords

430022H

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.
28600 Mary’s Court, Easton, MD 21601 USA • (800) 333-5272 • Fax: (410) 822-7526 • jascoinc.com/applications

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