Deriviatization, Hydrolysis, and HPLC Analysis of Amino Acids on SUPELCOSIL™ and ASCENTIS® Columns

Introduction

In many cases, an exact knowledge of protein quantities is required for protein chemistry applications. Amino acid analysis enables precise determination of protein quantities and provides detailed information regarding the relative amino acid composition and free amino acids. The relative amino acid composition gives a characteristic profile for proteins, which is often sufficient for protein identification, and is often used as a deciding factor for choosing proteases for protein fragmentation. 

The procedure includes the following steps:

  • Hydrolysis
  • Separation
  • Detection
  • Quantification

Hydrolysis

Hydrolysis is typically achieved by using acidic conditions. A standard procedure is hydrolysis with 6 M hydrochloric acid (24 hours, 110°C). The standard procedure is a compromise between time requirement and temperature. Sensitive amino acids (especially tryptophan and cysteine) will be partially destroyed. Gas-phase hydrolysis and addition of other acids (e.g., propionic acid, TFA, methansulfonic acid) can be used to shorten the hydrolysis time and improve the yield of sensitive amino acids.

Separation, detection and quantification

Hydrolysed samples (amino acids) are derivatized for sensitive detection,separated by HPLC. Post-column derivatization was typical; however, pre-column derivatization has gained importance and can be achieved by a broader range of derivatization reagents. The use of internal and external standards is crucial.

Column choice is a critical factor for experimental design. Supelco’s SUPELCOSIL™ and ASCENTIS® columns are an excellent choice to meet your amino acid analysis needs.

Materials

     
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