Amino acid analysis by post-column derivatization

The analysis of amino acids is performed for the accurate characterization of proteins and peptides. It also helps in the quantification of free amino acids present in the food samples.

Analysis of amino acid was done for the first time by Moore and Stein in 1963. This analysis was performed by using ion-exchange chromatography (IEC) and post-column derivatization (PCD) with ninhydrin reagent for absorbance detection or o-phthalaldehyde for fluorescence detection. The 20 amino acids are characterized individually and separately without mixing of the sample matrix.

A process of post-column derivatization:

The protein or peptide chain is broken to release free amino acids to determine their absolute quantity in the sample.

  1. Ion-exchange chromatography is used for the separation of amino acids in the column.
  2. Ninhydrin reagent is used for the derivatization of amino acids. This is done at an elevated temperature, i.e. the mixing of amino acids with the reagent.
  • Derivatization is actually done to gain selectivity and sensitivity in the analysis.
  • Derivatization is performed separately for each amino acid.
  • The final stage is the detection of derivatives (free amino acid) via absorption in the visible range (440 nm and 570 nm).

Advantages:

  1. It is a non-destructive method
  2. It is straight-forward and more useful than pre-column derivatization
  • It detects both primary as well as secondary amino acids
  1. It also detects the derivatives of amino acids
  2. It can be easily automated and is a simple method
  3. It has excellent quantitation performance

Disadvantages:

  1. PCD is quite difficult to use for highly-sensitive analysis
  2. It can use only a limited range of derivatizing reagent
  • Reverse-phase chromatography cannot be used rapidly for immediate analysis of samples
  1. Reagent consumption is high

PCD with other methods:

High-performance liquid chromatography (HPLC) was accomplished to be used with PCD. Especially, reverse-phase HPLC has given better results; therefore, it is widely used a chromatographic technique for amino acid analysis. It requires a minimal sample for the analysis as compared w.r.t. pre-column method. HPLC is the most effective and efficient method for this purpose since it is widely used for the health and functional foods.

Also, cation-exchange chromatography when used with PCD gave the most accurate and precise result for the determination of the exact composition of pure protein in food and feed samples.

Our laboratory is committed to providing the latest in the field of analysis so clients can be provided with the best analytical solutions. The new range of amino acid analytical methods is well suited for the food and pharmaceutical industry so food manufacturers, biological researchers, and developers of pharmaceutical formulations and food supplement manufacturers can use the amino acid analysis for quick identification of nutrition in foods,

You can call us now on +91-8588851888 and our team will be happy to share a proposal with you.

 

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