Amino acid analysis in High Performance Liquid Chromatography (HPLC)

Principle: Protein is hydrolyzed to constituent amino acids by 6N hydrochloric acid. The amino acids are separated in a HPLC equipped with an ion exchange column.

Preparation of Sample: About 100 mg of homogenized fish mince was weighed in to a test tube. The tube was then sealed after filling with nitrogen and digested by keeping at 120 º C for 24 hrs in an oven. The test tube was then cooled and the content was filtered using Whatman No 1 filter paper. The tube was rinsed with distilled water and filtered. The filtrate was then evaporated in a vacuum flash evaporator. The contents were made acid free by repeated washing with distilled water and subsequent evaporation.

HPLC analysis: 20 µL of the hydrolyzed sample was injected in HPLC (1525, Waters) equipped with a C18 reverse phase (RP) column and a fluorescence detector (2475, Waters). The amino acids were identified and quantified by comparing the retention times and peak areas with that of amino acid standard (WAT088122, Waters).

Estimation of Tryptophan

Principle: The 5-hydroxy furfural resulting from sucrose under acidic conditions of reaction forms pale green colored condensation product with thioglycolic acid which reacts with tryptophan in the hydrolyzed protein giving a pink colored complex. The color intensity of the solution is measured at 500 nm.

Procedure

Sample preparation: 200mg of finely homogenized fish mince was taken in a test tube and 10 ml 5% NaOH was added to it. The tube was then sealed after filling with nitrogen and digested by keeping at 120 º C for 24 hrs in an oven. The contents after hydrolysis was neutralized to pH 7.0 using 6N HCl. The total volume was made to 100 ml and filtered through Whatman No. 1 filter paper.

Estimation: 0.1ml of 2.5% sucrose and 0.1 ml of 0.6% thioglycolic acid was added successively in a test tube containing 4 ml of 50% H2SO4. The test tubes were then kept in a water bath at 45-50 º C for 10 mins. Sample aliquots (0.1-0.8ml) was added to the test tubes and mixed. The volume was made 5 ml with 0.1 N HCl and kept aside for 5 mins. The colored intensity was measured at 500 nm.

Reference

• 1. Ishida Y, Fujita T and Arai K (1981) New detection and separation methods for amino acid by high performance liquid chromatography.Journal of Chromatography 204: 143-148.
  

• 2. Sastry CSP and Tammuru MK (1985) Spectrophotometric determination of tryptophan in protein.Journal of Food Science and Technology 22: 146-147.