CN107543882A - A kind of method for determining liquor flavor component synthesis molecular weight - Google Patents

Abstract

The invention discloses a method for measuring the comprehensive molecular weight of liquor flavor components, and belongs to the technical field of liquor flavor analysis. The present invention quantitatively analyzes 51 kinds of volatile skeleton component substances in liquor, calculates the proportion of the substance in the whole with the molar concentration of each substance, and then calculates the weighted molecular weight of the volatile components; Eight non-volatile sugar alcohol compounds commonly found in liquor, and the proportion of the substance in the total was calculated based on the molar concentration of each substance, and then the weighted molecular weight of the non-volatile components was calculated. The weighted molecular weight of the volatile skeleton components and the weighted molecular weight of the non-volatile components were summed to obtain the comprehensive molecule of liquor. Because the method calculates the weighted molecular weight based on the molar concentration of volatile and non-volatile skeleton components, it can fully reflect the overall molecular weight level of aroma and taste substances in liquor, and provides a basis for the molecular structure and health and efficiency of molecular liquor products.

Description

A method for determining the comprehensive molecular weight of liquor flavor components

technical field

The invention relates to a method for measuring the comprehensive molecular weight of liquor flavor components, belonging to the technical field of liquor flavor analysis.

Background technique

The diversity of Chinese liquor fermentation process forms the richness of liquor aroma. So far, the independent fragrance types recognized in the industry include light fragrance type, strong fragrance type, sauce fragrance type, rice fragrance type, mixed fragrance type, phoenix fragrance type, medicine (Dong) fragrance type, soy sauce fragrance type, and special fragrance type There are 13 kinds of fragrances, such as sesame fragrance, Laobaigan fragrance, fragrant fragrance and pottery fragrance.

From a nutritional point of view, generally speaking, substances with relatively small molecular weights are easier to digest and absorb. Then, it is of great significance to establish an accurate comprehensive molecular weight detection method for the flavor substance system of Chinese liquor to provide a theoretical reference for the study of the comfort of liquor before, during and after drinking.

At present, the molecular weight analysis methods commonly used in food include: end group analysis method, solution colligative method, osmotic pressure method, gas phase osmosis method and viscosity method, etc., mainly for long-chain polysaccharides, polypeptides, fatty acids and other macromolecular polymers After pretreatment and hydrolysis, the average length distribution of short chains was investigated to provide a basis for optimizing the nutrition and biological activity of the above foods. In food analysis, different methods are determined according to the molecular weight and structure of the polymer. The statistical significance and scope of application of the average molecular weight obtained by different methods are different. Due to the complexity of the solution and the accuracy of the method, the accuracy of the method is often only orders of magnitude. . For example: the terminal group analysis method needs to clarify the functional group of the substance and realize the quantification of the functional group. This requires the molecular properties of the short-chain substance to be as uniform as possible to achieve accurate molecular molecular analysis; the solution colligative method is based on the physical and chemical properties of the substance (heat capacity, boiling point, etc.), suitable for non-volatile and non-dissociated compounds; the osmotic pressure method measures the osmotic pressure phenomenon caused by the chemical potential difference between the solute and the solution, and the applicable range of molecular weight is 3×10 ⁴ Da-1 ×10 ⁶ Da range; gas phase osmosis method is to measure the average molecular weight of the solution by indirectly measuring the vapor pressure drop of the solution. The method has simple equipment and is easy to operate, but this method requires a large relationship between the viscosity of the solution and the molecular weight, and the measured value is only a relative value.

Liquor is a special flavor favorite product, its essence is a colloidal substance composed of various flavor active substances such as alkyd esters, the average molecular weight of the liquor is between 100Da-200Da, in view of its complex liquor characteristics, adopt Ordinary physical and chemical methods cannot achieve accurate but comprehensive molecular weight research.

At present, there is no relevant research and report in the industry on the analysis method of the comprehensive molecular weight in wine products. Only the concept of small molecules or micromolecules is used in the industry, but how to define, detect, and analyze and compare, there is no definite research conclusion.

Contents of the invention

In order to solve the above problems, the method of the present invention combines modern chromatographic analysis techniques and adopts the molar concentration weighted molecular weight analysis method to accurately analyze the comprehensive molecular weight of flavor substances in liquor, to accurately investigate the overall molecular weight of liquor, and to investigate the structure and various components of liquor body. This functional characteristic is of great significance.

The purpose of the invention is to provide a method for measuring the comprehensive molecular weight of flavor components of liquor. The main components of liquor are ethanol and water, accounting for 97-99% of the total amount; the numerous trace organic compounds such as alcohols, aldehydes, acids, esters, etc. dissolved in the wine only account for 1-3% of the total amount; The trace aroma and taste substances with a content of only 1-3% are the key to determine the aroma, taste and style of liquor; these trace components are divided into two categories: volatile components and non-volatile components. Therefore, the method of the present invention measures the comprehensive molecular weight of liquor from two aspects of volatilization and non-volatility, and presents taste with 51 kinds of volatile skeletons that are ubiquitous in liquor (these 51 kinds of substances include liquor skeleton components confirmed in the industry, that is, the content is between 20-30mg/L or more), and 8 kinds of non-volatile components of sugar alcohols commonly found in liquor (the 8 kinds of substances are the 8 kinds of non-volatile substances with the highest content in ion chromatography analysis) as the main body to carry out comprehensive calculation of liquor flavor Substance-weighted molecular weight. The amount of polyols produced is closely related to the strain, number of bacteria, raw materials, fermentation speed, and components of the fermented grains. According to the characteristics of the sample, the polyols in liquor mainly include glycerol, erythritol, xylitol, and arabinose. alcohol, sorbitol, galactitol, mannitol, and maltitol.

In the present invention, use the molar concentration of each key substance in the wine body, because what the molar concentration represents is the molecular number of this substance in the solution, it can better reflect the arrangement and ratio of this substance in the wine body, adopt the weighted (%) method , which can more accurately determine the molecular weight of flavor substances in liquor as a whole:

Liquor weighted molecular weight = Σ skeleton component molar concentration ratio (%) × skeleton component molecular weight

The method for measuring the comprehensive molecular weight of liquor flavor components of the present invention comprises:

(1) Determination of mass concentration (mg/L) of volatile skeleton components in liquor;

(2) Calculate the weighted molecular weight of volatile skeleton components in liquor; Wherein,

Weighted molecular weight of volatile skeleton components in liquor = ∑ proportion of molar concentration of volatile skeleton components (%) × molecular weight of skeleton components

Molar concentration ratio of volatile skeleton components (%) = molar concentration M of the volatile skeleton components/total molar concentration of volatile skeleton components

The molar concentration M (mol/L) of the volatile skeleton component = the mass concentration of the substance/the molecular weight of the substance

The total molar concentration of volatile skeleton components (mol/L) = the sum of the molar concentrations of more than 51 volatile skeleton components

(3) Determination of the mass concentration (mg/L) of non-volatile skeleton components in liquor;

(4) Calculate the weighted molecular weight of non-volatile skeleton components in liquor; Wherein,

Weighted molecular weight of non-volatile skeleton components in liquor = ∑ Proportion of molar concentration of non-volatile skeleton components (%) × molecular weight of skeleton components

Molar concentration ratio of non-volatile skeleton components (%)=molar concentration M of the non-volatile skeleton components/total molar concentration of non-volatile skeleton components

Molar concentration of non-volatile skeleton components M (mol/L) = mass concentration of the substance/molecular weight of the substance

The total molar concentration of non-volatile skeleton components M (mol/L) = the sum of the molar concentrations of more than 8 sugar alcohols non-volatile skeleton components

(5) Calculation of comprehensive molecular weight of liquor flavor components:

Comprehensive molecular weight of flavor components in liquor = weighted molecular weight of volatile skeleton components in liquor + weighted molecular weight of non-volatile skeleton ingredients in liquor.

In one embodiment, the volatile skeleton components include: acetaldehyde, n-propionaldehyde, ethyl formate, ethyl acetate, acetal, methanol, isovaleraldehyde, 2-pentanone, ethyl butyrate, S-butanol, n-propanol, 1,1-diethoxy-2-methylbutane, 1,1-diethoxy-3-methylbutane, isobutanol, isoamyl acetate, pentyl ethyl acetate, 2-pentanol, n-butanol, 2-methyl-1-butanol, isoamyl alcohol, ethyl hexanoate, n-pentanol, 3-hydroxy-2-butanone, ethyl heptanoate, Ethyl lactate, n-hexanol, butyl caproate, ethyl caprylate, isopentyl caproate, acetic acid, furfural, benzaldehyde, propionic acid, 2,3-butanediol (Levorotatory), isobutyric acid, 2,3 -Butanediol (meso), Propylene Glycol, Ethyl Caprate, Butyric Acid, Furfuryl Alcohol, Isovaleric Acid, Valeric Acid, Ethyl Phenylacetate, Hexanoic Acid, β-Phenylethyl Ethanol, Heptanoic Acid, Ethyl Myristate , caprylic acid, ethyl palmitate, ethyl oleate, ethyl linoleate.

The present invention also provides the determination of the volatile skeleton components, which is quantitatively analyzed by gas chromatography.

In one embodiment, the conditions of the gas chromatography are: using a capillary column, the temperature of the injection port is 210-250°C; 2-6min, 4-8°C/min to 180-210°C, keep for 15-25min; carrier gas is inert gas; flow rate is 0.5-2mL/min; sampling method is split injection.

In one embodiment, the split ratio of the split injection is 20:1-40:1.

Instrument: Agilent 7890B gas chromatograph (FID detector);

Chromatographic conditions: Agilent CP-Wax 57CB capillary column [50m×0.25mm (inner diameter)×0.2μm]; inlet temperature 230°C; initial column temperature 35°C, 2°C/min to 60°C, keep for 4min , 6°C/min to 195°C, hold for 20min; the carrier gas is nitrogen, the purity ≧99.999%; the flow rate is 1mL/min; the injection method is split injection, the split ratio is 30:1; the injection volume is 1μL.

In one embodiment, the specific conditions of the gas chromatography are:

Instrument: Agilent 7890B gas chromatograph (FID detector);

Chromatographic conditions: Agilent CP-Wax 57CB capillary column [50m×0.25mm (inner diameter)×0.2μm]; inlet temperature 230°C; initial column temperature 35°C, 2°C/min to 60°C, keep for 4min , 6°C/min to 195°C, hold for 20min; the carrier gas is nitrogen, the purity ≧99.999%; the flow rate is 1mL/min; the injection method is split injection, the split ratio is 30:1; the injection volume is 1μL.

In one embodiment, the quantitative analysis by gas chromatography is to first prepare the standard products of the same skeleton component with different concentrations, add a certain concentration of internal standard to prepare a standard working solution with gradient concentration, and then perform gas chromatography analysis , and then the standard curve of each skeleton component is obtained; the content of the sample to be tested is obtained by substituting the gas chromatographic analysis results of the sample to be tested into the standard curve.

In one embodiment, the non-volatile backbone components include: glycerin, erythritol, xylitol, arabitol, sorbitol, galactitol, mannitol, and maltitol.

The invention also provides the determination of the non-volatile skeleton components, which is quantitative analysis by ion chromatography.

In one embodiment, the conditions of the ion chromatography are: the temperature of the chromatographic column is 25-35° C.; the eluent is NaOH solution, and the flow rate is 0.2-0.6 mL/min.

In one embodiment, the conditions of the ion chromatography are: the ion chromatograph is ICS-5000+, equipped with an amperometric detector, an Au working electrode, and an Ag reference electrode; the chromatographic column is CarboPac MA1, and the column temperature is 30°C; The eluent is 480mM NaOH, the flow rate is 0.4mL/min; the injection volume is 25μL. The method is optimized through experiments and is suitable for the analysis of polyols in liquor samples.

Beneficial effects of the present invention:

1. The method of the present invention carries out quantitative analysis to the volatile skeleton component and the non-volatile skeleton component in liquor, after determining respective molar concentration, then take the molar concentration ratio of each substance as a basis, calculate the weighted molecular weight of this substance respectively , so as to determine the comprehensive molecular weight of liquor. This method calculates the weighted molecular weight based on the molar concentration of the skeleton components in liquor, which reflects the molecular weight of the product as a whole.

2. The present invention is the first comprehensive algorithm that combines the weighted molecular weight of volatile skeleton components and non-volatile skeleton components in liquor, which can more comprehensively reflect the comprehensive molecular weight of liquor products.

3. The present invention provides a calculation method for the comprehensive molecular weight of liquor for the first time, which provides a reference for understanding the overall molecular weight state and distribution of flavor substances in liquor.

Description of drawings

Fig. 1: the standard curve of ethyl hexanoate detection;

Figure 2: Example of overall chromatogram;

Figure 3: Standard curve for glycerol detection;

Figure 4: Standard sample spectra of 8 non-volatile components.

specific implementation plan

The following is a detailed description of the present invention.

Example 1

Carry out the determination of the comprehensive molecular weight of liquor flavor components according to the following method:

First divide the sample into two parts, analyze the volatile skeleton components and non-volatile skeleton components respectively, then calculate the weighted molecular weight of the volatile skeleton components in liquor and the weighted molecular weight of non-volatile skeleton components in liquor, and finally get the liquor flavor group sub-comprehensive molecular weight.

Specific steps are as follows:

(1) Determination of mass concentration of volatile skeleton components in liquor (mg/L):

Detection method:

a Instrument: Agilent 7890B gas chromatograph (FID detector)

b Reagent: tert-amyl alcohol, n-pentyl acetate, 2-ethylbutyric acid chromatographic standard substance (content not less than 99.5%, Tianjin Guangfu Fine Chemical Research Institute)

c Chromatographic conditions: Chromatographic column: Agilent CP-Wax 57CB capillary column [50m×0.25mm (inner diameter)×0.2μm], inlet temperature 230°C; carrier gas: nitrogen, purity ≧99.999%; flow rate: 1mL/min; Injection method: split injection, split ratio: 30:1; injection volume: 1 μL.

dEstablishment of the standard curve: draw 10 mL of the mixed standard substance of the skeleton components at the gradient concentration, and add 10 μL of the mixed internal standard of a certain concentration (tert-amyl alcohol, n-pentyl acetate, 2-ethylbutyric acid standard solution) to prepare the gradient concentration Standard working solution. Signals were collected under the above-mentioned chromatographic conditions, and tert-amyl alcohol, n-pentyl acetate, and 2-ethylbutyric acid were used as internal standards, and a standard curve for each component was drawn using an offline workstation.

Take ethyl hexanoate as example: standard curve is y=1.01789x (y is peak area ratio, x is content ratio), linear correlation coefficient is 0.9996 (Fig. 1), measured concentration 352.23mg/L and 2817.8mg/L two The recoveries of the points are all between 80%-120%.

Analysis of 51 volatile skeleton components in liquor: acetaldehyde, n-propionaldehyde, ethyl formate, ethyl acetate, acetal, methanol, isovaleraldehyde, 2-pentanone, ethyl butyrate, sec-butanol, n- Propanol, 1,1-diethoxy-2-methylbutane, 1,1-diethoxy-3-methylbutane, isobutanol, isoamyl acetate, ethyl valerate, 2 -Pentanol, n-butanol, 2-methyl-1-butanol, isoamyl alcohol, ethyl hexanoate, n-pentanol, 3-hydroxy-2-butanone, ethyl heptanoate, ethyl lactate, n-hexyl Alcohol, butyl caproate, ethyl caprylate, isopentyl caproate, acetic acid, furfural, benzaldehyde, propionic acid, 2,3-butanediol (Levorotatory), isobutyric acid, 2,3-butanediol ( meso), propylene glycol, ethyl caprate, butyric acid, furfuryl alcohol, isovaleric acid, valeric acid, ethyl phenylacetate, caproic acid, beta-phenylethyl alcohol, heptanoic acid, ethyl myristate, caprylic acid, palmitic acid Ethyl ester, ethyl oleate, ethyl linoleate.

An example of the overall chromatogram is shown in Figure 2. Wherein, by adopting the gas chromatograph for direct sampling and the FID detector, the linear correlation coefficients of the detection of the 51 substances are all above 0.99, and the recoveries are all between 80% and 120%.

(2) Calculation of weighted molecular weight of volatile skeleton components in liquor

Weighted molecular weight of volatile skeleton components in liquor = ∑ proportion of molar concentration of skeleton components (%) × molecular weight of skeleton components Proportion of molar concentration of volatile skeleton components (%) = molar concentration M of this component/total molar concentration of volatile skeleton components

Total molar concentration (mol/L)=the sum of 51 kinds of volatile component molar concentrations Volatile skeleton component molar concentration M (mol/L)=mass concentration/molecular weight of this substance

(3) Determination of the mass concentration of non-volatile skeleton components in liquor (mg/L):

Quantitative analysis of 8 non-volatile skeleton components represented by sugar alcohols in liquor by ion chromatography: glycerin, erythritol, xylitol, arabitol, sorbitol, galactitol, mannitol, maltose alcohol.

Detection method:

Ion chromatograph: ICS-5000+, equipped with amperometric detector, Au working electrode, Ag reference electrode;

Chromatographic column: CarboPac MA1, column temperature: 30°C;

Eluent: 480mMNaOH, flow rate: 0.4ml/min;

Injection volume: 25 μL.

Taking glycerol as an example, its standard curve is shown in Figure 3.

The standard sample spectrograms of these 8 components under the detection conditions of this embodiment are shown in Figure 4, and the coefficients of the standard curve are shown in Table 1. In addition, the inventors conducted a recovery experiment, and the results are shown in Table 2. It can be seen from Table 1-2 that the linearity of each component coefficient is above 0.9997, and the standard addition recovery rate is between 85% and 102%, indicating that the detection effect of the ion chromatography is good.

Each component standard curve coefficient of table 1:

serial number component name linear serial number name linear 1 glycerin 0.9999 5 Sorbitol 0.9999 2 Erythritol 0.9998 6 Galactitol 0.9998 3 Xylitol 0.9999 7 Mannitol 0.9999 4 Arabitol 0.9999 8 Maltitol 0.9997

The recovery rate of each component of table 2

serial number component name Spiked concentration (mg/L) Test result (mg/L) Recovery rate(%) 1 Glycerol 0.52 0.53 101.92 2 Erythritol 0.46 0.39 84.78 3 Xylitol 0.36 0.32 88.89 4 Arabitol 0.50 0.49 98.00 5 Sorbitol 0.42 0.39 92.86 6 Galactitol 0.40 0.33 82.50 7 Mannitol 0.56 0.48 85.71 8 Maltitol 0.56 0.52 92.86

Remarks: The background content has been deducted from the test results.

(4) Calculation of weighted molecular weight of non-volatile skeleton components in liquor

Weighted molecular weight of non-volatile skeleton components in liquor = ∑ proportion of molar concentration of skeleton components (%) × molecular weight of skeleton components

Molar concentration ratio of non-volatile skeleton components (%) = molar concentration M of the component/total molar concentration of non-volatile skeleton components

Molar concentration of non-volatile skeleton components M (mol/L) = mass concentration/molecular weight of the substance

Total molar concentration (mol/L) = the sum of the molar concentrations of 8 non-volatile components

(5) Calculation of the comprehensive molecular weight of liquor:

Liquor comprehensive molecular weight = weighted molecular weight of volatile skeleton components in liquor + weighted molecular weight of non-volatile skeleton components in liquor

The comprehensive molecular weight of the liquor flavor components measured by the method of the present invention comprehensively and effectively reflects the overall molecular weight level of the aroma and taste substances in the liquor, and the liquor with a small comprehensive molecular weight obtained by the measurement is indeed better than the liquor with a relatively large comprehensive molecular weight obtained by the measurement. It is easier to digest and absorb, can be used to evaluate the ease of alcohol body metabolism, and provides a theoretical reference for the study of liquor comfort before, during and after drinking, which is of great significance.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. A method for measuring comprehensive molecular weight of flavor components of white spirit is characterized by comprising the following steps:

(1) measuring the mass concentration of volatile skeleton components in the white spirit;

(2) calculating the weighted molecular weight of volatile skeleton components in the white spirit; wherein,

the weighted molecular weight of the volatile skeleton component in the white spirit is ═ Sigma molar concentration ratio (%) of the volatile skeleton component and the molecular weight of the skeleton component

A molar concentration ratio (%) of the volatile skeleton component (M mol/total mol) of the volatile skeleton component

Molar concentration M (mol/L) of volatile skeleton component (mass concentration of the substance)/molecular weight of the substance

(3) Measuring the mass concentration of non-volatile skeleton components in the white spirit;

(4) calculating the weighted molecular weight of the non-volatile framework components in the white spirit; wherein,

weight ratio (%) of mole concentration of non-volatile skeleton component ═ Σ of mole concentration of non-volatile skeleton component in white spirit and molecular weight of skeleton component

Molar concentration ratio (%) of non-volatile skeleton component to total molar concentration of non-volatile skeleton component

Molar concentration of non-volatile skeleton component M (mol/L) ═ mass concentration of the substance/molecular weight of the substance

(5) Calculating the comprehensive molecular weight of the flavor components of the white spirit:

the comprehensive molecular weight of the flavor components of the white spirit is the weighted molecular weight of volatile skeleton components in the white spirit and the weighted molecular weight of non-volatile skeleton components in the white spirit.

2. The method of claim 1, wherein the volatile matrix component comprises acetaldehyde, n-propionaldehyde, ethyl formate, ethyl acetate, acetal, methanol, isovaleraldehyde, 2-pentanone, ethyl butyrate, sec-butanol, n-propanol, 1-diethoxy-2-methylbutane, 1-diethoxy-3-methylbutane, isobutanol, isoamyl acetate, ethyl valerate, 2-pentanol, n-butanol, 2-methyl-1-butanol, isoamyl alcohol, ethyl hexanoate, n-pentanol, 3-hydroxy-2-butanone, ethyl heptanoate, ethyl lactate, n-hexanol, butyl hexanoate, ethyl octanoate, isoamyl hexanoate, acetic acid, furfural, benzaldehyde, propionic acid, 2, 3-butanediol (levo), isobutyric acid, 2, 3-butanediol (meso), propylene glycol, ethyl decanoate, butyric acid, furfuryl alcohol, isovaleric acid, valeric acid, ethyl phenylacetate, hexanoic acid, β -phenylethyl alcohol, heptanoic acid, ethyl tetradecanoate, caprylic acid, ethyl palmitate, ethyl oleate, linoleic acid, ethyl linoleate.

3. The method of claim 1, wherein the non-volatile framework components comprise: glycerol, erythritol, xylitol, arabitol, sorbitol, galactitol, mannitol, maltitol.

4. The method of claim 1, wherein the volatile matrix composition is determined quantitatively using gas chromatography.

5. The method of claim 1, wherein the non-volatile framework components are determined quantitatively by ion chromatography.

6. The method according to claim 4, wherein the quantitative analysis by gas chromatography comprises preparing standard substances of the same skeleton component with different concentrations, adding an internal standard with a certain concentration to prepare a standard working solution with a gradient concentration, and performing gas chromatography to obtain a standard curve of each skeleton component; and substituting the meteorological chromatographic analysis result of the sample to be detected into the standard curve to obtain the content of the sample to be detected.

7. The method according to claim 4, characterized in that the conditions of the gas chromatography are: adopting a capillary column, wherein the temperature of a sample inlet is 210-250 ℃; the initial column temperature is 30-40 ℃, the temperature is raised to 55-65 ℃ at 1-3 ℃/min, the temperature is kept for 2-6min, the temperature is raised to 210 ℃ at 4-8 ℃/min, and the temperature is kept for 15-25 min; the carrier gas is inert gas; the flow rate is 0.5-2 mL/min; the sample introduction mode is split-flow sample introduction.

8. The method according to claim 4, characterized in that the specific conditions of the gas chromatograph are:

the instrument comprises the following steps: agilent 7890B gas chromatograph (FID detector);

chromatographic conditions are as follows: the chromatographic column is Agilent CP-Wax 57CB capillary column [50m × 0.25mm (inner diameter) × 0.2 μm ]; the temperature of a sample inlet is 230 ℃; the carrier gas is nitrogen, and the purity is not less than 99.999 percent; the flow rate is 1 mL/min; the sample injection mode is divided sample injection, and the dividing ratio is 30: 1; the injection volume was 1. mu.L.

9. The method of claim 5, wherein the conditions of the ion chromatography are: the conditions of the ion chromatography are: the temperature of the chromatographic column is 25-35 ℃; the leacheate is NaOH solution, and the flow rate is 0.2-0.6 mL/min.

10. The method of claim 5, wherein the conditions of the ion chromatography are: the ion chromatograph is ICS-5000+, and is provided with an ampere detector, an Au working electrode and an Ag reference electrode; the chromatographic column is CarboPac MA1, and the column temperature is 30 ℃; the eluent is 480mM NaOH, and the flow rate is 0.4 mL/min; the amount of sample was 25. mu.L.