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HPLC method for simultaneously detecting L-homoserine and free amino acid in fermentation broth

A technology of free amino acid and homoserine, applied in the field of HPLC for simultaneous detection of L-homoserine and free amino acid in fermentation broth, to achieve the effects of stable analysis results, short analysis time and good repeatability

Active Publication Date: 2020-05-12
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the HPLC method for the simultaneous detection of non-protein amino acids and protein amino acids.

Method used

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  • HPLC method for simultaneously detecting L-homoserine and free amino acid in fermentation broth
  • HPLC method for simultaneously detecting L-homoserine and free amino acid in fermentation broth
  • HPLC method for simultaneously detecting L-homoserine and free amino acid in fermentation broth

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The high-performance liquid chromatography detection of L-homoserine hydrochloride standard substance comprises the following steps:

[0034] (1) Weigh 2.75mg L-homoserine hydrochloride standard substance, add 1mL ultrapure water to dissolve, make 2.75g / L L-homoserine hydrochloride solution, and dilute to 1.375g / L, 0.55g / L, 0.275g / L L-homoserine hydrochloride solution;

[0035] (2) Take 200 μL of the above-mentioned L-homoserine hydrochloride solution, add 350 μL of boric acid buffer solution and 150 μL of 0.5% DEEMM methanol solution, and then derivatize at 70° C. for 2 h;

[0036] The boric acid buffer solution was prepared as follows: weigh 6.183g of boric acid, add 70mL of ultrapure water to dissolve, adjust the pH to 9.0 with 1mol / L NaOH solution, add ultrapure water to 100mL.

[0037] The DEEMM methanol solution was prepared as follows: draw 0.5mL DEEMM into a 100mL volumetric flask, and dilute to 100mL with pure methanol.

[0038] (3) The above-mentioned deriv...

Embodiment 2

[0047] The high-performance liquid chromatography detection of 11 kinds of amino acid mixed standard liquid chromatography, comprises the following steps:

[0048] (1) Draw 200 μL of the amino acid mixed standard solution, add 350 μL of boric acid buffer and 150 μL of 0.5% DEEMM methanol solution, and then derivatize at 70°C for 2 hours;

[0049] (2) The above-mentioned derivatized solution is detected by high performance liquid chromatography, and the detection method is:

[0050] Detection instrument: Agilent Technologies 1260 Infinity II high performance liquid chromatography system;

[0051] Chromatographic column: J&K Scientific HPLCC-18Column (4.6×250mm, 5μm);

[0052] Mobile phase: mobile phase A is pure methanol; mobile phase B is 25mmol / L ammonium acetate solution, A:B=40%:60%, flow rate 0.6mL / min;

[0053] Column temperature: 25°C;

[0054] Detection wavelength: 250nm.

[0055] 11 kinds of amino acid mixed standard liquid chromatogram see image 3 , image 3 The ...

Embodiment 3

[0058] The chromatographic detection of L-homoserine and free amino acids in the fermentation broth comprises the following steps:

[0059] (1) Take 1 mL of fermentation broth sample in a centrifuge tube, centrifuge at 12000r for 2min, and collect the supernatant for derivatization.

[0060] (2) Take 100 μL of the supernatant and 100 μL of the amino acid mixed standard solution, add 350 μL of 1 mol / L boric acid buffer solution, and add 150 μL of 0.5% DEEMM (diethyl ethoxymethylene malonate) methanol solution , after derivatization at 70°C for 2h, filter with a 0.22μm organic filter.

[0061] (3) The above-mentioned derivatized solution is detected by high performance liquid chromatography, and the detection method is:

[0062] Detection instrument: Agilent Technologies 1260 Infinity II high performance liquid chromatography system;

[0063] Chromatographic column: J&K Scientific HPLCC-18Column (4.6×250mm, 5μm);

[0064] Mobile phase: mobile phase A is pure methanol; mobile ...

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Abstract

The invention relates to an HPLC method for simultaneously detecting L-homoserine and free amino acid in fermentation broth. The method comprises the following steps of: pretreating a fermentation broth sample, carrying out derivatization treatment on the fermentation broth sample by using a DEEMM derivatization reagent, and separating and determining L-homoserine and other free amino acids in thefermentation broth by using high performance liquid chromatography and a universal C18 chromatographic column. The used configuration is the standard liquid phase configuration of the high performance liquid chromatography system, such that the method is suitable for the amino acid analysis, can be used for the analysis work of other substances, has advantages of strong universality, high detection sensitivity, simple sample treatment, short analysis time, stable analysis result, good repeatability and the like, and is suitable for the conventional laboratory.

Description

[0001] (1) Technical field [0002] The invention belongs to the technical field of amino acid detection, in particular to an HPLC method for simultaneously detecting L-homoserine and free amino acids in a fermentation broth. [0003] (2) Background technology [0004] Amino acids are the most basic substances that constitute biological proteins, and are also important components of active peptides, enzymes, and other biologically active molecules in organisms, and are closely related to life activities. So far, more than 180 kinds of amino acids have been found in nature, among which more than 20 kinds of amino acids participate in protein synthesis, which are called basic amino acids. L-homoserine, a naturally occurring non-protein amino acid, is a versatile platform compound for the synthesis of L-methionine, γ-butyrolactone, and other high value-added chemicals. Among these chemicals, pharmaceutical grade L-methionine is used in the treatment of liver disease and other rel...

Claims

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Application Information

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IPC IPC(8): G01N30/06G01N30/02
CPCG01N30/06G01N30/02G01N2030/067
Inventor 柳志强刘吉颂张博郑裕国
Owner ZHEJIANG UNIV OF TECH
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