Amino acid analysis method

A technology for free amino acid and quantitative analysis, which is applied in the field of analyzing free amino acids in samples by reversed-phase high performance liquid chromatography, which can solve the problems of inability to analyze amino acids, inability to analyze amino acids, etc., and achieves high efficiency, variety, and detection sensitivity. high effect

Inactive Publication Date: 2010-12-15
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 chroma...

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[0053] 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:

[0054] time (min)

0

16.0

...

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Abstract

The invention relates to an amino acid analysis method. The method comprises the following steps of: (1) centrifuging a proper amount of sample solution at a speed of 4,000 revolutions per minute for 10 to 30 minutes; adding a proper amount of trichloroacetic acid into supernatant and standing at the temperature of between 0 and 10 DEG C for 0.5 to 2 hours; and performing centrifugal treatment again, and filtering a proper amount of supernatant by using a 0.2 to 0.45-micron microfiltration membrane; (2) adding 1 to 3 parts of DTDPA solution into one part of sample treatment solution obtained by the step (1), mixing uniformly, and heating the mixture in a 60 to 100 DEG C water bath for 0.5 to 2 hours; and (3) absorbing one part of protection liquid obtained by the step (2), absorbing 1 to 3 parts of OPA and mixing; absorbing 1 to 3 parts of FMOC and mixing; absorbing 50 to 100 parts of water and mixing to obtain pre-column derivatization treatment liquid; and performing 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...

Claims

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Application Information

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IPC IPC(8): G01N30/89
Inventor 邓莉郝学财邢海鹏
Owner TIANJIN CHUNFA BIO TECH GRP
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