CN113484438A - Method for detecting tannic acid in wine grapes and wines - Google Patents
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Abstract
The invention belongs to the technical field of substance detection, and particularly provides a method for detecting tannic acid in wine grapes and wines. The method takes methanol water solution as an extracting agent, and efficiently and quickly extracts the tannic acid from the sample to be detected under the oscillation condition; and the content of the tannic acid in the sample to be detected can be rapidly and accurately determined by combining a high performance liquid chromatography-ultraviolet detection technology, and the blank of a method for detecting and analyzing the content of the tannic acid in the wine grapes and the wine is filled. Furthermore, the pretreatment method disclosed by the invention is simple to operate, safe and reliable, low in cost and less in time consumption. The data of the examples show that: the limit of quantification of the detection method is 0.5mg/L, and the limit of detection is 0.3 mg/L; the recovery rate of tannic acid in the wine grapes is 86-99%; the recovery rate of the tannic acid in the wine is 81-92%.
Description
Technical Field
The invention relates to the technical field of substance detection, in particular to a method for detecting tannic acid in wine grapes and wine.
Background
Tannic acid is also known as tannic acid, and is also known as tannin, tannic acid, gallotannic acid, tannin, etc. It has been widely recognized for its antioxidant and astringent properties. Tannin as the skeleton of red wine can bring bitter taste and astringent taste in sense, influence the flavor and texture of red wine, stabilize the pigment of red wine, keep fresh color, make the wine body firmer and fuller and have lasting aftertaste. The wine lacking tannin is often thin and weak and has insufficient flavor.
Many quantitative detection methods of tannin are reported in research, and the classical method comprises a leather powder method, a casein method, an oxidation titration method and a spectrophotometry method; many novel measurement methods such as high performance liquid chromatography and flow injection analysis have been developed, but in practice, spectrophotometry has been the main technique.
For fruits, vegetables and wine, the currently popularized industry standard "determination of tannin content in fruits, vegetables and products thereof" (NY/T1600-2008) belongs to spectrophotometry, which mainly determines the total amount of tannin compounds mainly containing gallic acid in extracts, but not the content of tannic acid. The high performance liquid chromatography which is presented subsequently has the advantages of high separation efficiency, high analysis speed, small sample consumption and the like, and is also used for detecting the content of the tannic acid in the sample at present, the reported detection samples comprise gallnut, walnut residue, emblic leafflower fruit, tobacco leaves, plukenetia volubilis and the like, but the pretreatment process has the defects of complicated extraction steps and the like. Moreover, there is no relevant report on the determination of the content of tannic acid in wine grapes and wines at present.
Disclosure of Invention
In view of the above, the present invention provides a method for detecting tannic acid in wine grapes and wine, which has simple extraction steps and can quickly and efficiently detect tannic acid in wine grapes and wine.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for detecting tannic acid in wine grapes and wine, which comprises the following steps:
mixing a sample to be detected with an extracting agent, and extracting to obtain a solution to be detected;
performing high performance liquid chromatography-ultraviolet detection on the solution to be detected to obtain the content of the tannic acid in the sample to be detected;
the extractant is methanol water solution;
the extraction is carried out under the condition of oscillation, the speed regulation of the oscillation is 180-220 r/min, and the time is 20-60 min;
the sample to be detected comprises wine grape to be detected or wine to be detected.
Preferably, the volume ratio of methanol to water in the methanol aqueous solution is (3-9): 1.
preferably, when the sample to be detected is the wine grape to be detected, the dosage ratio of the wine grape to be detected to the extracting agent is (2.00-10.00) g: (15-50) mL.
Preferably, when the sample to be detected is wine to be detected, the volume ratio of the wine to be detected to the extracting agent is (2.00-10.00): (15-50).
Preferably, after the extraction, the method further comprises the step of performing centrifugal separation on the obtained extraction system, and taking the supernatant as a solution to be detected.
Preferably, the parameters of the high performance liquid chromatography-ultraviolet detection comprise:
a chromatographic column: UltimateAQ-C18 column, 4.6 × 250mm, 5 μm;
the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with the volume concentration of 0.1%;
a flow rate; 0.8 mL/min;
column temperature: 25 ℃;
sample introduction volume: 10 mu L of the solution;
and (3) an elution mode: gradient elution;
the procedure for the gradient elution was:
0-15 min: 5-8% of a mobile phase A;
15-20 min: 8-10% of a mobile phase A;
20-20.1 min: 10-80% of a mobile phase A;
20.1-25 min: 80-5% of a mobile phase A;
25-30 min: 5% mobile phase A;
detection wavelength: 280 nm.
Preferably, after the high performance liquid chromatography-ultraviolet detection, the method further comprises the step of substituting the obtained peak area of the tannic acid into a concentration-peak area standard curve of a tannic acid standard sample to obtain the content of the tannic acid in the sample to be detected.
The invention provides a method for detecting tannic acid in wine grapes and wine, which comprises the following steps: mixing a sample to be detected with an extracting agent, and extracting to obtain a solution to be detected; performing high performance liquid chromatography-ultraviolet detection on the solution to be detected to obtain the content of the tannic acid in the sample to be detected; the extractant is methanol water solution; the extraction is carried out under the condition of oscillation, the speed regulation of the oscillation is 180-220 r/min, and the time is 20-60 min; the sample to be detected comprises wine grape to be detected or wine to be detected. The method takes methanol water solution as an extracting agent, and efficiently and quickly extracts the tannic acid from the wine-making grape to be detected or the wine to be detected under the oscillation condition; and the content of the tannic acid in the sample to be detected can be rapidly and accurately determined by combining a high performance liquid chromatography-ultraviolet detection technology, and the blank of a method for detecting and analyzing the content of the tannic acid in the wine grapes and the wine is filled. Furthermore, the pretreatment method disclosed by the invention is simple to operate, safe and reliable, low in cost and less in time consumption, can be used for simultaneously extracting a large number of samples, and greatly improves the detection efficiency; in addition, in the mobile phase gradient elution procedure, the consumption of an organic reagent acetonitrile is greatly reduced, and the test cost is saved. The data of the examples show that: the detection method provided by the invention has the advantages of good precision, good stability and good recovery rate.
Drawings
FIG. 1 is a concentration-peak area standard curve diagram of a tannic acid standard sample (concentration is 0.5-100 mg/L);
FIG. 2 is a liquid chromatogram of a tannic acid standard sample solution at a concentration of 5.0 mg/L;
FIG. 3 is a liquid chromatogram of tannic acid from commercially available wine grapes;
FIG. 4 is a liquid chromatogram of tannic acid in a commercial wine.
Detailed Description
The invention provides a method for detecting tannic acid in wine grapes and wine, which comprises the following steps:
mixing a sample to be detected with an extracting agent, and extracting to obtain a solution to be detected;
and carrying out high performance liquid chromatography-ultraviolet detection on the solution to be detected to obtain the content of the tannic acid in the sample to be detected.
In the present invention, the starting materials used in the present invention are preferably commercially available products unless otherwise specified.
The method mixes the sample to be detected with an extracting agent, and extracts the mixture to obtain the solution to be detected.
In the invention, the sample to be detected is preferably wine grape or wine to be detected; when the sample to be detected is the wine grape to be detected, the wine grape to be detected is preferably crushed before being mixed with the extracting agent, the crushing operation is not particularly limited, and the whole wine grape can be crushed; in a specific embodiment of the present invention, when the sample to be tested is preferably a wine grape to be tested, a wall breaking machine is preferably adopted to break the wine grape to be tested.
In the present invention, the extractant is an aqueous methanol solution; the volume ratio of methanol to water in the methanol aqueous solution is preferably (3-9): 1, more preferably 9: 1. in the invention, when the sample to be detected is preferably the wine grape to be detected, the dosage ratio of the wine grape to be detected and the extracting agent is preferably (2.00-10.00) g: (15-50) mL, more preferably 3.00 g: 25.0 mL. In the invention, when the sample to be detected is preferably wine to be detected, the volume ratio of the wine to be detected to the extracting agent is preferably (2.00-10.00): (15-50), and more preferably 3.00: 25.
in the invention, the extraction is carried out under the condition of oscillation, the speed of the oscillation is 180-220 r/min, preferably 200r/min, and the oscillation time is 20-60 min, preferably 20 min.
After the extraction, the invention preferably further comprises the step of carrying out centrifugal separation on the obtained extraction system, and taking the supernatant as a solution to be detected.
The rotation speed and time of the centrifugal separation are not particularly limited, as long as solid-liquid separation can be realized to obtain a clear supernatant, and in a specific embodiment of the invention, the rotation speed of the centrifugal separation is preferably 4000r/min, and the time is preferably 3 min.
After the solution to be detected is obtained, the high performance liquid chromatography-ultraviolet detection is carried out on the solution to be detected, and the content of the tannic acid in the sample to be detected is obtained.
In the present invention, the parameters of the hplc-uv detection preferably include:
a chromatographic column: UltimateAQ-C18 column, 4.6 × 250mm, 5 μm;
the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with the volume concentration of 0.1%;
a flow rate; 0.8 mL/min;
column temperature: 25 ℃;
sample introduction volume: 10 mu L of the solution;
and (3) an elution mode: gradient elution;
the procedure for the gradient elution is shown in table 1.
TABLE 1 procedure for gradient elution
Detection wavelength: 280 nm.
After the high performance liquid chromatography-ultraviolet detection, the method preferably further comprises the step of substituting the obtained peak area of the tannic acid into a concentration-peak area standard curve of the tannic acid to obtain the content of the tannic acid in the sample to be detected.
The method for acquiring the concentration-peak area standard curve of the tannic acid is not particularly limited, and the standard curve acquisition method known to a person skilled in the art can be adopted.
The following examples are provided to illustrate the method for detecting tannins in wine grapes and wines according to the present invention in detail, but they should not be construed as limiting the scope of the present invention.
The parameters of hplc-uv detection used in the following examples include:
a chromatographic column: an UltimateAQ-C18 column (4.6X 250mm, 5 μm); the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with the volume concentration of 0.1%; the gradient elution procedure is shown in table 1; flow rate: 0.8 mL/min; column temperature: 25 ℃; detection wavelength: 280 nm; sample introduction volume: 10 μ L.
Establishment of a Standard Curve
0.01011g (to the nearest 0.0001g) of tannic acid as a solid powder was weighed into a brown vial, transferred to 10.00mL of methanol with a graduated pipette, and dissolved by vortexing to prepare a 1000mg/L stock solution.
Sequentially diluting and preparing 0.5, 2, 10, 20, 40, 60, 80 and 100mg/L tannic acid standard solution for sample injection, detecting by using the parameters of high performance liquid chromatography-ultraviolet detection, and drawing a concentration-peak area standard curve graph of the tannic acid standard sample by taking the concentration as an abscissa and taking a peak area as an ordinate, wherein the concentration-peak area standard curve of the tannic acid standard sample is shown in figure 1: 2736.9x-1931.8, R2When the concentration is 0.9997, r is 0.9998, the limit of quantitation of the detection method is 0.5mg/L, and the limit of detection is 0.3 mg/L.
FIG. 2 is a liquid chromatogram of a tannic acid standard sample solution at a concentration of 5.0 mg/L; as can be seen from fig. 2: the peak shape of tannic acid is sharp and symmetrical, and the retention time of tannic acid is about 11.91 min.
Pretreatment of a sample to be detected: weighing 3.00g of a wine grape sample (sourced from commercially available cabernet sauvignon wine grapes) and 3.00mL of a wine sample (sourced from commercially available cabernet sauvignon dry red wine) in 50mL of a centrifuge tube, adding 25.0mL of methanol aqueous solution (the volume ratio of methanol to water is 9: 1), oscillating for 20min under the condition of speed regulation of 200r/min, centrifuging for 3min at 4000r/min, sucking supernatant, passing through a membrane, and filling into a sample vial to obtain a solution to be measured; the liquid chromatogram of tannic acid in the wine grape and wine obtained by detecting with the high performance liquid chromatography-ultraviolet detection parameters is shown in FIGS. 3 and 4. As can be seen in fig. 3 and 4: and no interference of a foreign peak exists at the retention time of the target peak, which indicates that the detection method provided by the invention has good specificity.
Repeatability test
The results of measuring the recovery rates of 0.5, 5.0 and 20mg/kg tannic acid standard samples added to wine grapes are shown in Table 2.
TABLE 2 recovery of tannic acid from wine grapes (n ═ 5)
As can be seen from table 2: at the concentration addition levels of 0.5, 5.0 and 20mg/kg 3, the average recovery rate of tannic acid in the wine grapes is between 86 and 99 percent, and the relative standard deviation is between 0.7 and 6.0 percent, which all meet the requirements of the detection method.
The results of measuring the recovery rates of standard samples of tannic acid added to wine at 5.0, 20 and 40mg/kg are shown in Table 3.
TABLE 3 recovery of tannic acid from wine (n ═ 5)
As can be seen from table 3: at the concentration addition levels of 5.0, 20 and 40mg/kg 3, the average recovery rate of the tannic acid in the wine is between 81 and 92 percent, and the relative standard deviation is between 1.3 and 4.8 percent, which all meet the requirements of the detection method.
Precision test
Continuously feeding a tannic acid standard sample solution with the mass concentration of 5.0mg/L for 7 times, recording the peak area, and calculating the RSD value of the tannic acid standard sample peak area, wherein the results are shown in Table 4.
TABLE 4 results of precision test
| Number of samples taken | 1 | 2 | 3 | 4 | 5 | 6 | 7 | Average | RSD％ |
| Peak area | 12754 | 12732 | 12749 | 12727 | 12781 | 12793 | 12835 | 12767 | 0.3 |
As can be seen from table 4: and continuously injecting a tannic acid standard sample solution with the mass concentration of 5.0mg/L for 7 times to obtain a tannic acid standard sample with the peak area RSD value of 0.3 percent, so that the precision is good, and the requirement of the detection method is met.
Stability test
Taking the same wine grape sample extract, respectively standing the sample at room temperature for 0, 2, 5, 8, 12, 16, 20 and 24 hours, analyzing by a high performance liquid chromatograph, recording peak area, and calculating the peak area RSD value of tannic acid in the sample, wherein the results are shown in Table 5.
TABLE 5 stability test results
| Standing time/h | 0 | 2 | 5 | 8 | 12 | 16 | 20 | 24 | RSD％ |
| Peak area | 19553 | 19344 | 20957 | 20062 | 20299 | 20354 | 20605 | 20043 | 2.6 |
As can be seen from table 5: after the same wine grape sample extracting solution is respectively placed for 0, 2, 5, 8, 12, 16, 20 and 24 hours, the peak area RSD is 2.6, which shows that the stability of the test solution in 24 hours is good.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A method for detecting tannic acid in wine grapes and wines comprises the following steps:
mixing a sample to be detected with an extracting agent, and extracting to obtain a solution to be detected;
performing high performance liquid chromatography-ultraviolet detection on the solution to be detected to obtain the content of the tannic acid in the sample to be detected;
the extractant is methanol water solution;
the extraction is carried out under the condition of oscillation, the speed regulation of the oscillation is 180-220 r/min, and the time is 20-60 min;
the sample to be detected comprises wine grape to be detected or wine to be detected.
2. The detection method according to claim 1, wherein the volume ratio of methanol to water in the methanol aqueous solution is (3-9): 1.
3. the detection method according to claim 2, wherein when the sample to be detected is a wine grape to be detected, the usage ratio of the wine grape to be detected and the extracting agent is (2.00-10.00) g: (15-50) mL.
4. The detection method according to claim 2, wherein when the sample to be detected is wine to be detected, the volume ratio of the wine to be detected to the extracting agent is (2.00-10.00): (15-50).
5. The detection method according to claim 1, further comprising, after the extraction, centrifuging the obtained extraction system, and taking a supernatant as a solution to be detected.
6. The detection method according to claim 1, wherein the parameters of the high performance liquid chromatography-ultraviolet detection comprise:
a chromatographic column: UltimateAQ-C18 column, 4.6 × 250mm, 5 μm;
the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with the volume concentration of 0.1%;
a flow rate; 0.8 mL/min;
column temperature: 25 ℃;
sample introduction volume: 10 mu L of the solution;
and (3) an elution mode: gradient elution;
the procedure for the gradient elution was:
0-15 min: 5-8% of a mobile phase A;
15-20 min: 8-10% of a mobile phase A;
20-20.1 min: 10-80% of a mobile phase A;
20.1-25 min: 80-5% of a mobile phase A;
25-30 min: 5% mobile phase A;
detection wavelength: 280 nm.
7. The detection method according to claim 1 or 6, wherein after the HPLC-UV detection, the method further comprises substituting the obtained peak area of the tannic acid into a concentration-peak area standard curve of a tannic acid standard sample to obtain the content of the tannic acid in the sample to be detected.
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