CN111089914A - High performance liquid detection method for metabolites in ethanol produced by microbial fermentation - Google Patents
High performance liquid detection method for metabolites in ethanol produced by microbial fermentation Download PDFInfo
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- G01N30/02—Column chromatography
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Abstract
The invention provides a high performance liquid chromatography detection method for metabolites in fuel ethanol produced by CO gas-containing microbial fermentation, which is characterized in that the content of the metabolites in fermentation liquor of the fuel ethanol produced by CO gas-containing microbial fermentation is determined through high performance liquid chromatography analysis, wherein the metabolites comprise ethanol, acetic acid, lactic acid and 2, 3-butanediol, and the chromatographic conditions are as follows: a chromatographic column: an Alltech IOA-2000Organic Acid chromatography column; mobile phase: sulfuric acid solution with the concentration of 5 mmol/L; flow rate: 0.7 mL/min; column temperature: 55 ℃; a detector: and a RID detector.
Description
Technical Field
The invention relates to the field of industrial fermentation, in particular to a high-efficiency liquid phase detection method for metabolites in fuel ethanol produced by microbial fermentation of CO-containing gas.
Background
In the process of producing ethanol by fermenting CO gas-containing microorganisms, the content of fermentation metabolites such as ethanol, acetic acid, lactic acid and 2, 3-butanediol in fermentation liquor plays an extremely important role in the control of the whole process, and the quality of a final product or the health of thalli can be adversely affected by too high or too low content.
Disclosure of Invention
In view of the above, the present invention has been developed to provide a method for detecting metabolites in ethanol produced by fermentation of microorganisms that overcomes or at least partially solves the above-mentioned problems.
The embodiment of the invention provides a high performance liquid chromatography detection method for metabolites in fuel ethanol produced by CO gas microbial fermentation, which is used for determining the content of the metabolites in fermentation liquor for producing ethanol by microbial fermentation through high performance liquid chromatography analysis, wherein the metabolites comprise ethanol, acetic acid, lactic acid and 2, 3-butanediol, and the method is characterized in that the chromatographic conditions are as follows:
a chromatographic column: an Alltech IOA-2000Organic Acid chromatography column;
mobile phase: the concentration of the sulfuric acid is 4-6mmol/L, and the solvent is ultrapure water;
flow rate: 0.5-1 mL/min;
column temperature: 50-60 ℃;
a detector: RID;
operating time: 9-11 min.
Further, the chromatographic conditions are:
a chromatographic column: an Alltech IOA-2000Organic Acid chromatography column;
mobile phase: the concentration of sulfuric acid is 5mmol/L, and the solvent is ultrapure water;
flow rate: 0.7 mL/min;
column temperature: 55 ℃;
a detector: RID;
operating time: for 10 min.
Furthermore, in the high performance liquid chromatography analysis, the injection volume is 9-11 muL.
Further, the method comprises the steps of:
s1, preparing a standard solution, wherein the standard solution is a mixed solution of ethanol, acetic acid, lactic acid and 2, 3-butanediol;
s2, respectively carrying out liquid chromatography analysis on each standard solution by using a high performance liquid chromatograph, recording peak areas obtained by analysis, and respectively drawing a standard curve regression equation of the content-peak areas of ethanol, acetic acid, lactic acid and 2, 3-butanediol by using an external standard method;
and S3, performing liquid chromatography analysis on the solution to be detected containing the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol by adopting a detection instrument and chromatographic conditions which are completely the same as those of the standard solution, recording peak areas, and calculating the contents of the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol in the solution containing the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol according to a standard curve regression equation in the step S2.
Further, the standard solution comprises a first standard solution and a second standard solution, wherein the first standard solution is obtained by mixing an ethanol solution, an acetic acid solution, a lactic acid solution and a 2, 3-butanediol solution with the mass concentrations of 30g/L, 5g/L and 10g/L respectively, and the second standard solution is obtained by mixing an ethanol solution, an acetic acid solution, a lactic acid solution and a 2, 3-butanediol solution with the mass concentrations of 15g/L, 2.5g/L and 5g/L respectively; and the solvents of the ethanol solution, the acetic acid solution, the lactic acid solution and the 2, 3-butanediol solution are ultrapure water.
Further, the solution containing ethanol, acetic acid, lactic acid and 2, 3-butanediol is prepared as follows:
adding a 5-sulfosalicylic acid solution into fermentation liquor for producing ethanol by fermentation of microorganisms to be detected, and uniformly mixing to obtain a mixed solution;
the preparation method of the 5-sulfosalicylic acid solution comprises the following steps: uniformly mixing ultrapure water, concentrated sulfuric acid and 5-sulfosalicylic acid to obtain a 5-sulfosalicylic acid solution, wherein the volume-mass ratio of the ultrapure water to the concentrated sulfuric acid to the 5-sulfosalicylic acid is 250 ml: 13.89 ml: 5g of the total weight of the mixture;
centrifuging the mixed solution to obtain a centrifuged clear solution; the centrifugal clear liquid is a solution containing ethanol, acetic acid, lactic acid and 2, 3-butanediol.
Further, the volume ratio of the fermentation liquor to the 5-sulfosalicylic acid solution is 3-5: 1.
further, the centrifugal operation rotating speed is 12000-18000 r/min.
Further, the centrifugal operation time is more than or equal to 3 min.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
1. the method has the advantages of high sensitivity, high accuracy, small error and simple operation;
2. realizing the simultaneous detection of all peaks of ethanol, acetic acid, lactic acid and 2, 3-butanediol;
3. the effective separation of ethanol, acetic acid, lactic acid and 2, 3-butanediol is realized, and the peak shape is good;
4. the detection time is shortened, and the production efficiency is greatly improved.
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Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:
FIG. 1 is a graph showing the results of detection of ethanol, acetic acid, lactic acid and 2, 3-butanediol in example 1 of the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example 1
The application provides a high performance liquid chromatography detection method for metabolites in ethanol produced by microbial fermentation, which is characterized in that the content of the metabolites in a fermentation liquid for producing ethanol by microbial fermentation is determined through high performance liquid chromatography analysis, wherein the metabolites comprise at least one of ethanol, acetic acid, lactic acid and 2, 3-butanediol, and the chromatographic conditions are as follows:
a chromatographic column: an Alltech IOA-2000Organic Acid chromatography column;
mobile phase: the concentration of sulfuric acid is 5mmol/L, and the solvent is ultrapure water;
flow rate: 0.7 mL/min;
column temperature: 55 ℃;
a detector: RID;
operating time: for 10 min.
Specifically, in the high performance liquid chromatography, the injection volume is 10 μ L.
Specifically, the method comprises the following steps:
s1, preparing a standard solution, wherein the standard solution is a mixed solution of ethanol, acetic acid, lactic acid and 2, 3-butanediol;
s2, respectively carrying out liquid chromatography analysis on each standard solution by using a high performance liquid chromatograph, recording peak areas obtained by analysis, and respectively drawing a standard curve regression equation of the content-peak areas of ethanol, acetic acid, lactic acid and 2, 3-butanediol by using an external standard method;
and S3, performing liquid chromatography analysis on the solution to be detected containing the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol by adopting a detection instrument and chromatographic conditions which are completely the same as those of the standard solution, recording peak areas, and calculating the contents of the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol in the solution containing the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol according to a standard curve regression equation in the step S2.
Specifically, the standard solution comprises a first standard solution and a second standard solution, wherein the first standard solution is obtained by mixing an ethanol solution, an acetic acid solution, a lactic acid solution and a 2, 3-butanediol solution with the mass concentrations of 30g/L, 5g/L and 10g/L respectively, and the second standard solution is obtained by mixing an ethanol solution, an acetic acid solution, a lactic acid solution and a 2, 3-butanediol solution with the mass concentrations of 15g/L, 2.5g/L and 5g/L respectively; and the solvents of the ethanol solution, the acetic acid solution, the lactic acid solution and the 2, 3-butanediol solution are ultrapure water.
Specifically, the preparation method of the solution containing ethanol, acetic acid, lactic acid and 2, 3-butanediol comprises the following steps:
putting fermentation liquor for producing ethanol by fermenting microorganisms to be detected into a centrifugal tube, adding 5-sulfosalicylic acid solution, and uniformly mixing to obtain a mixed solution;
centrifuging the mixed solution to obtain a centrifuged clear solution; the centrifugal clear liquid is a solution containing ethanol, acetic acid, lactic acid and 2, 3-butanediol.
Specifically, the volume ratio of the fermentation liquor to the 5-sulfosalicylic acid solution is 4: 1.
specifically, the centrifugal operation rotating speed is 15000 r/min.
Specifically, the centrifugal operation time is 3 min.
The detection method of the present application will be described in detail with reference to specific examples.
Example 2
A high performance liquid chromatography detection method for metabolites in fuel ethanol produced by CO gas microbial fermentation is characterized in that the content of the metabolites in fermentation liquor for producing ethanol by microbial fermentation is determined through high performance liquid chromatography analysis, wherein the metabolites comprise ethanol, acetic acid, lactic acid and 2, 3-butanediol, and the chromatographic conditions are as follows:
a chromatographic column: an Alltech IOA-2000Organic Acid chromatography column;
mobile phase: the concentration of the sulfuric acid is 4mmol/L, and the solvent is ultrapure water;
flow rate: 0.5 mL/min;
column temperature: 50 ℃;
a detector: RID;
operating time: and 9 min.
Specifically, in the high performance liquid chromatography, the injection volume is 9 μ L.
Specifically, the method comprises the following steps:
s1, preparing a standard solution, wherein the standard solution is a mixed solution of ethanol, acetic acid, lactic acid and 2, 3-butanediol;
s2, respectively carrying out liquid chromatography analysis on each standard solution by using a high performance liquid chromatograph, recording peak areas obtained by analysis, and respectively drawing a standard curve regression equation of the content-peak areas of ethanol, acetic acid, lactic acid and 2, 3-butanediol by using an external standard method;
and S3, performing liquid chromatography analysis on the solution to be detected containing the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol by adopting a detection instrument and chromatographic conditions which are completely the same as those of the standard solution, recording peak areas, and calculating the contents of the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol in the solution containing the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol according to a standard curve regression equation in the step S2.
Specifically, the standard solution comprises a first standard solution and a second standard solution, wherein the first standard solution is obtained by mixing an ethanol solution, an acetic acid solution, a lactic acid solution and a 2, 3-butanediol solution with the mass concentrations of 30g/L, 5g/L and 10g/L respectively, and the second standard solution is obtained by mixing an ethanol solution, an acetic acid solution, a lactic acid solution and a 2, 3-butanediol solution with the mass concentrations of 15g/L, 2.5g/L and 5g/L respectively; and the solvents of the ethanol solution, the acetic acid solution, the lactic acid solution and the 2, 3-butanediol solution are ultrapure water.
Specifically, the preparation method of the solution containing ethanol, acetic acid, lactic acid and 2, 3-butanediol comprises the following steps:
adding a 5-sulfosalicylic acid solution into fermentation liquor for producing ethanol by fermentation of microorganisms to be detected, and uniformly mixing to obtain a mixed solution;
the preparation method of the 5-sulfosalicylic acid solution comprises the following steps: uniformly mixing ultrapure water, concentrated sulfuric acid and 5-sulfosalicylic acid to obtain a 5-sulfosalicylic acid solution, wherein the volume-mass ratio of the ultrapure water to the concentrated sulfuric acid to the 5-sulfosalicylic acid is 250 ml: 13.89 ml: 5g of the total weight of the mixture;
centrifuging the mixed solution to obtain a centrifuged clear solution; the centrifugal clear liquid is a solution containing ethanol, acetic acid, lactic acid and 2, 3-butanediol.
Specifically, the volume ratio of the fermentation liquor to the 5-sulfosalicylic acid solution is 3: 1.
specifically, the centrifugal operation rotating speed is 12000 r/min.
Specifically, the centrifugal operation time is 4 min.
Example 3
A high performance liquid chromatography detection method for metabolites in fuel ethanol produced by CO gas microbial fermentation is characterized in that the content of the metabolites in fermentation liquor for producing ethanol by microbial fermentation is determined through high performance liquid chromatography analysis, wherein the metabolites comprise ethanol, acetic acid, lactic acid and 2, 3-butanediol, and the chromatographic conditions are as follows:
a chromatographic column: an Alltech IOA-2000Organic Acid chromatography column;
mobile phase: the concentration of sulfuric acid is 6mmol/L, and the solvent is ultrapure water;
flow rate: 1 mL/min;
column temperature: 60 ℃;
a detector: RID;
operating time: and (4) 11 min.
Specifically, in the high performance liquid chromatography, the sample injection volume is 11 μ L.
Specifically, the method comprises the following steps:
s1, preparing a standard solution, wherein the standard solution is a mixed solution of ethanol, acetic acid, lactic acid and 2, 3-butanediol;
s2, respectively carrying out liquid chromatography analysis on each standard solution by using a high performance liquid chromatograph, recording peak areas obtained by analysis, and respectively drawing a standard curve regression equation of the content-peak areas of ethanol, acetic acid, lactic acid and 2, 3-butanediol by using an external standard method;
and S3, performing liquid chromatography analysis on the solution to be detected containing the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol by adopting a detection instrument and chromatographic conditions which are completely the same as those of the standard solution, recording peak areas, and calculating the contents of the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol in the solution containing the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol according to a standard curve regression equation in the step S2.
Specifically, the standard solution comprises a first standard solution and a second standard solution, wherein the first standard solution is obtained by mixing an ethanol solution, an acetic acid solution, a lactic acid solution and a 2, 3-butanediol solution with the mass concentrations of 30g/L, 5g/L and 10g/L respectively, and the second standard solution is obtained by mixing an ethanol solution, an acetic acid solution, a lactic acid solution and a 2, 3-butanediol solution with the mass concentrations of 15g/L, 2.5g/L and 5g/L respectively; and the solvents of the ethanol solution, the acetic acid solution, the lactic acid solution and the 2, 3-butanediol solution are ultrapure water.
Specifically, the preparation method of the solution containing ethanol, acetic acid, lactic acid and 2, 3-butanediol comprises the following steps:
adding a 5-sulfosalicylic acid solution into fermentation liquor for producing ethanol by fermentation of microorganisms to be detected, and uniformly mixing to obtain a mixed solution;
the preparation method of the 5-sulfosalicylic acid solution comprises the following steps: uniformly mixing ultrapure water, concentrated sulfuric acid and 5-sulfosalicylic acid to obtain a 5-sulfosalicylic acid solution, wherein the volume-mass ratio of the ultrapure water to the concentrated sulfuric acid to the 5-sulfosalicylic acid is 250 ml: 13.89 ml: 5g of the total weight of the mixture;
centrifuging the mixed solution to obtain a centrifuged clear solution; the centrifugal clear liquid is a solution containing ethanol, acetic acid, lactic acid and 2, 3-butanediol.
Specifically, the volume ratio of the fermentation liquor to the 5-sulfosalicylic acid solution is 5: 1.
specifically, the centrifugal operation rotating speed is 18000 r/min.
Specifically, the centrifugal operation time is 5 min.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (9)
1. A high performance liquid chromatography detection method for metabolites in fuel ethanol produced by CO gas microbial fermentation is characterized in that the content of the metabolites in fermentation liquor for producing ethanol by microbial fermentation is determined through high performance liquid chromatography analysis, wherein the metabolites comprise ethanol, acetic acid, lactic acid and 2, 3-butanediol, and the chromatographic conditions are as follows:
a chromatographic column: an Alltech IOA-2000Organic Acid chromatography column;
mobile phase: the concentration of the sulfuric acid is 4-6mmol/L, and the solvent is ultrapure water;
flow rate: 0.5-1 mL/min;
column temperature: 50-60 ℃;
a detector: RID;
operating time: 9-11 min.
2. The high performance liquid chromatography detection method for the metabolites in ethanol produced by CO gas microbial fermentation according to claim 1, wherein the chromatographic conditions are as follows:
a chromatographic column: an Alltech IOA-2000Organic Acid chromatography column;
mobile phase: the concentration of sulfuric acid is 5mmol/L, and the solvent is ultrapure water;
flow rate: 0.7 mL/min;
column temperature: 55 ℃;
a detector: RID;
operating time: for 10 min.
3. The method for detecting the metabolites in the ethanol produced by the microbial fermentation according to claim 1 or 2, wherein the sample injection volume is 9-11 μ L in the HPLC analysis.
4. The method for detecting the metabolites in the ethanol produced by the microbial fermentation according to claim 1 or 2, wherein the method comprises the following steps:
s1: preparing a standard solution, wherein the standard solution is a mixed solution of ethanol, acetic acid, lactic acid and 2, 3-butanediol;
s2: respectively carrying out liquid chromatography analysis on each standard solution by adopting a high performance liquid chromatograph, recording peak areas obtained by analysis, and respectively drawing a standard curve regression equation of the content-peak area of ethanol, acetic acid, lactic acid and 2, 3-butanediol by adopting an external standard method;
s3: and (3) performing liquid chromatography analysis on the solution to be detected containing the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol by adopting a detection instrument and chromatographic conditions which are completely the same as those of the standard solution, recording peak areas, and calculating the contents of the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol in the solution containing the ethanol, the acetic acid, the lactic acid and the 2, 3-butanediol according to a standard curve regression equation in the step S2.
5. The method for detecting the metabolites in the ethanol produced by the microbial fermentation as claimed in claim 4, wherein the standard solutions comprise a first standard solution and a second standard solution, the first standard solution is obtained by mixing ethanol solutions with the mass concentrations of 30g/L, 5g/L and 10g/L, an acetic acid solution, a lactic acid solution and a 2, 3-butanediol solution, and the second standard solution is obtained by mixing ethanol solutions with the mass concentrations of 15g/L, 2.5g/L and 5g/L, an acetic acid solution, a lactic acid solution and a 2, 3-butanediol solution; and the solvents of the ethanol solution, the acetic acid solution, the lactic acid solution and the 2, 3-butanediol solution are ultrapure water.
6. The method for detecting metabolites in ethanol produced by microbial fermentation according to claim 4, wherein the solution containing ethanol, acetic acid, lactic acid and 2, 3-butanediol is prepared by the following steps:
adding a 5-sulfosalicylic acid solution into fermentation liquor for producing ethanol by fermentation of microorganisms to be detected, and uniformly mixing to obtain a mixed solution;
the preparation method of the 5-sulfosalicylic acid solution comprises the following steps: uniformly mixing ultrapure water, concentrated sulfuric acid and 5-sulfosalicylic acid to obtain a 5-sulfosalicylic acid solution, wherein the volume-mass ratio of the ultrapure water to the concentrated sulfuric acid to the 5-sulfosalicylic acid is 250ml to 13.89ml to 5 g;
centrifuging the mixed solution to obtain a centrifuged clear solution; the centrifugal clear liquid is a solution containing ethanol, acetic acid, lactic acid and 2, 3-butanediol.
7. The method for detecting the metabolites in the ethanol produced by the microbial fermentation according to claim 6, wherein the volume ratio of the fermentation liquid to the 5-sulfosalicylic acid solution is 3-5: 1.
8. The method as claimed in claim 6, wherein the rotation speed of the centrifugal operation is 12000-18000 r/min.
9. The high performance liquid chromatography detection method for metabolites in ethanol produced by microbial fermentation as claimed in claim 6, wherein the centrifugation time is not less than 3 min.
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| CN114350476A (en) * | 2022-01-12 | 2022-04-15 | 河北首朗新能源科技有限公司 | System and method for inhibiting acidification of fermented liquor after fermentation |
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Application publication date: 20200501 |