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Method for analyzing free amino acids by utilizing reversed phase high performance liquid chromatography

A reversed-phase high-efficiency liquid phase, free amino acid technology, applied in the field of instrument analysis, can solve the problems of amino acid analysis and amino acid analysis, and achieve the effect of multiple types, high efficiency, and short analysis time

Inactive Publication Date: 2010-11-24
TIANJIN CHUNFA BIO TECH GRP
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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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  • Method for analyzing free amino acids by utilizing reversed phase high performance liquid chromatography
  • Method for analyzing free amino acids by utilizing reversed phase high performance liquid chromatography

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[0052] Take 50ml of the sample solution, centrifuge at 4000r / min for 10min, take 25ml of the supernatant, add 25ml of trichloroacetic acid (mass fraction 2%), put it at 4°C for 2h, then centrifuge at 4000r / min for 10min, take the supernatant 2ml, pass through a 0.2um microporous membrane; take 0.5ml of the above treatment solution, add 0.5ml of DTDPA solution with a concentration of 0.1g / ml and a pH of 10.0, mix well, and heat in a water bath at 80°C for 1 hour; configure mobile phase A: 0.1mol / L sodium acetate: acetocyanide = 93:7, adjust the pH value to 6.5; mobile phase B: acetocyanide: water = 4:1; set the autosampler for pre-column derivatization: draw 0.5ul of sample, draw 0.5 ul of OPA with a concentration of 2.5mg / ml, mix, absorb 0.5ul of FMOC with a concentration of 10mg / ml and add to the mix, then absorb 32ul of water and add to the mix, inject the sample; carry out gradient elution according to the following table 4:

[0053] time (min)

0

16.0

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Abstract

The invention relates to a method for qualitatively and quantitatively analyzing free amino acids in a sample by utilizing reversed phase high performance liquid chromatography, which comprises the following steps of: first pre-treating the sample; then protecting the -SH of cysteine in the sample; next performing pre-column derivatization treatment on primary and secondary amino acids with derivatization agents orthophaladetyde (OPA) and fluorene methyl choroformate (FMOC) respectively; and finally performing reversed phase high performance liquid chromatographic (RP-HPLC) analysis. The method has the advantage of synchronously detecting 25 free amino acids in the sample solution, particularly performing -SH protection on the cysteine with a dithio-dipropionic acid (DTDPA) and then synchronously deriving the cysteine together with the other amino acids, along with detection diversity and high efficiency.

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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 邓莉郝学财邢海鹏
Owner TIANJIN CHUNFA BIO TECH GRP
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