Method for analyzing amino acid by utilizing RP-HPLC (Reverse Phase High-Performance Liquid Chromatography)

Abstract

The invention relates to a method for analyzing amino acid by utilizing RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), which comprises the following steps of: weighing 50ml of sample solution, centrifuging for 10 minutes at the speed of 4000r / min, weighing 25ml of supernatant, adding 25ml of trichloroacetic acid with the mass fraction of 2 percent, placing at the temperature of 4 DEG C for settling for 2 hours, then centrifuging for 10 minutes at the speed of 4000r / min, weighing 2ml of supernatant, and sieving through a 0.2 mu m microporous filter membrane; weighing 0.5ml of treatment fluid, adding 0.5ml of DTDPA (3,3'-Dithiodipropionic Acid) solution, uniformly mixing, and placing at the temperature of 80 DEG C for water-bath heating for 1 hour to obtain a protection fluid; and setting an automatic sampler for pre-column derivation: sucking 0.5mul of protection fluid , sucking 0.5mul of OPA (O-Phthaldialdehyde), mixing, sucking 0.5mul of FMOC (Fluorenl Methoxy Carbonyl) for adding and mixing, then sucking 32mul of water for adding and mixing, sampling, and carrying out gradient elution and detection.

Description

technical field [0001] The invention belongs to the technical field of instrumental analysis, and in particular relates to a method for analyzing free amino acids in a sample by using a reversed-phase high-performance liquid chromatograph. Background technique [0002] So far, more than 180 kinds of amino acids have been found in nature, of which only more than 20 kinds of amino acids participate in protein synthesis, which are called basic amino acids. Because amino acid analysis plays an important role in the research of protein chemistry, biochemistry, food science, clinical medicine and other fields, people attach great importance to the research and improvement of amino acid analysis methods. In 1958, Spackman et al. first proposed the method of combining cation exchange chromatography with post-column ninhydrin derivatization to analyze amino acids in proteins, realizing the automation of amino acid analysis. This method is the amino acid analyzer, but the structure of...

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Problems solved by technology

[0004] Lu Yan et al disclosed the detection method of amino acids in proteins in "Reverse-Phase High Performance Liquid Chromatography Quantitative Analysis of Amino Acid Composition in Polypeptides" in the 2nd issue of "Zhejiang Agricultural Sciences" in 2006. )" "OPA pre-column derivatization method for reversed-phase high-performance liquid chromatography analysis of amino acids" in the third issue of 1991 disclosed the detection of amino acids in cytochrome C enzymolysis products. The method of Lu Yan et al. cannot analyze all amino acids, and has limitations on the types of amino acid analysis, while the method of Wu Anzhen also cannot analyze all amino acids, and its method also has limitations.

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