CN111812047A - Method for determining content of total flavonoids in tobacco based on continuous flow analyzer - Google Patents
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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Abstract
The invention discloses a method for determining the content of total flavonoids in tobacco based on a continuous flow analyzer. Detecting at 510nm wavelength by using a continuous flow analyzer, taking sodium nitrite-aluminum nitrate as a color developing agent, preparing a sodium hydroxide solution, and obtaining a standard working curve equation with the y being 0.0022x +0.0017 and the correlation coefficient r2And (5) substituting the measured value of the total flavone instrument corresponding to the sample solution into a formula for calculation to obtain the total flavone content in the tobacco to be measured. The invention applies the continuous flow analyzer to the determination of the content of the total flavonoids in the tobacco for the first time. The recovery rate of the method is between 96.83 and 104.4 percent; the variation coefficients of 5 samples detected at different time and in different batches are all less than 5%; the detection limit and the quantification limit were 0.025mg/g and 0.082mg/g, respectively. The method is simple and convenient to operate, high in analysis speed, good in repeatability, stability and accuracy and good in application prospect.
Description
Technical Field
The invention belongs to the technical field of detection of physical and chemical indexes of tobacco, and particularly relates to a method for quickly and accurately determining the content of total flavonoids in tobacco based on a continuous flow analyzer.
Background
As an important economic crop in China, in recent years, along with the sign of the tobacco control framework convention in China, the tobacco development space is greatly limited, so that the production of high-quality tobacco leaves, the reduction of harmful components and the improvement of the quality of tobacco products become an important subject at the present stage. The growth and development of tobacco and the quality of tobacco leaves are influenced by various factors such as temperature, altitude, illumination, soil, moisture and the like, and at present, flavonoid compounds are proved to be latent fragrant substances which influence the quality of tobacco, and the metabolism of the flavonoid compounds is regulated and controlled by various factors. Therefore, the research on the metabolism path and the regulation mechanism of the flavonoid compounds has important significance for improving the quality of tobacco leaves, so that the accurate determination of the content of the total flavonoids in the tobacco is extremely important.
The method for measuring the content of total flavonoids has been reported to be mainly high performance liquid chromatography, but the method is difficult to popularize because the method uses expensive equipment, has high detection cost and long detection period and needs to be operated by professionals.
The continuous flow analyzer is cheap in equipment and low in maintenance cost, has the advantages of simple pretreatment, simple and convenient operation on a computer, high detection speed, accurate quantification, good reproducibility and the like, does not need to use an organic reagent in detection, and is more environment-friendly. Since the continuous flow analyzer was introduced in the tobacco industry in the last eighties, the detection of chemical components such as water-soluble sugar, total plant alkaloid, total nitrogen, total volatile acid, starch, free nicotine and the like in tobacco and tobacco products has been carried out, and the detection of chlorine, potassium, nitrate and the like in cigarette paper by using the continuous flow analyzer has also been carried out, but no report of using the continuous flow analyzer to detect the content of total flavonoids in tobacco has been found at present.
Disclosure of Invention
The invention aims to provide a method for rapidly, simply and accurately measuring the content of total flavonoids in tobacco based on a continuous flow analyzer aiming at the defects of the prior art.
The purpose of the invention is realized by the following technical scheme.
All percentages used in the present invention are mass percentages unless otherwise indicated.
A method for determining the content of total flavonoids in tobacco based on a continuous flow analyzer specifically comprises the following steps:
(1) accurately weighing 1.0g of tobacco powder, placing in a 150mL triangular flask with a plug, adding 100mL of 80% ethanol solution, shaking up, covering the plug tightly, placing on a shaker, performing oscillatory extraction at 150r/min for 30min, filtering the extract with qualitative filter paper, and collecting the filtrate as sample solution for later use;
(2) analysis with a continuous flow analyzer:
preparing a sodium nitrite reagent: weighing 5.00g of sodium nitrite, dissolving with 800mL of deionized water, and then fixing the volume to 1000 mL;
preparing an aluminum nitrate reagent: weighing 10.00g of aluminum nitrate, dissolving with 800mL of deionized water, and then fixing the volume to 1000 mL;
preparing a sodium hydroxide reagent: weighing 40.00g of sodium hydroxide, dissolving in 800mL of deionized water, and then fixing the volume to 1000 mL;
setting a flow path of the analyzer: the sample solution is pumped by a yellow/yellow pump tube at the flow rate of 1.20 mL/min; the flow rate of the black/black pump pipe for the sodium nitrite reagent is 0.32 mL/min; the aluminum nitrate reagent is a black/black pump pipe, and the flow rate is 0.32 mL/min; the sodium hydroxide reagent is pumped by a red/red pump tube at the flow rate of 0.78 mL/min; cleaning solution is pumped by a red/red pump tube at a flow rate of 0.78 mL/min; after the sodium hydroxide reagent is added, the mixture of the sample solution and the three reagents is fully mixed and developed through 80 circles of reaction mixing circles, and then the mixture enters a detector; detection wavelength: 510 nm; detecting speed: 30 samples/h; sample introduction/cleaning time ratio: 1: 1; baseline correction: opening; and (3) drift correction: opening;
(3) drawing a standard working curve: accurately weighing 0.200g of rutin, uniformly mixing in 200mL of ethanol, and then diluting to 1000mL with deionized water to obtain a standard stock solution of total flavonoids with the content of 200 mg/L; respectively transferring 10 mL, 30 mL, 50mL, 70 mL and 90mL of stock solutions by using a pipette, and respectively metering the volume to 100mL by using deionized water to obtain 20 mg/L, 60 mg/L, 100 mg/L, 140 mg/L and 180mg/L of standard working solutions; the standard working curve equation is: y is 0.0022x +0.0017 and the correlation coefficient r2＝0.9998；
(4) And (4) calculating a result:
substituting the measured value of the total flavone instrument corresponding to the tobacco powder sample solution to be measured into the formula (1) for calculation to obtain the total flavone content in the tobacco powder to be measured, and taking the average value of two parallel measurements as the final measurement result;
in the formula: p is the content (mg/g) of total flavonoids in the tobacco powder to be detected, C is the measured value (mg/L) of the total flavonoids corresponding to the sample solution by an instrument, V is the volume (mL) of the extract liquor, m is the mass (g) of the tobacco powder sample.
Compared with the prior art, the invention has the following advantages:
the invention applies the continuous flow analyzer to the determination of the content of the total flavonoids in the tobacco for the first time. The recovery rate of the method is between 96.83 and 104.4 percent; the variation coefficients of 5 samples detected at different time and in different batches are all less than 5%; the detection limit and the quantification limit of the method are respectively 0.025mg/g and 0.082 mg/g. The method is simple and convenient to operate, high in analysis speed, good in repeatability, stability and accuracy and good in application prospect.
Drawings
FIG. 1 schematic view of a flow path setup of a continuous flow analyzer
FIG. 2 is a standard operating curve;
FIG. 3 influence of extract concentration;
FIG. 4 extracts the effect of oscillation time.
Detailed Description
The present invention is further described in detail with reference to the drawings and examples, which are not intended to limit the technical scope of the present invention, and all changes and equivalents which are made based on the teachings of the present invention should fall within the protective scope of the present invention.
Example 1
1. Principles of experiment, materials and methods
1.1 principle of the experiment
The adjacent carbonyl and hydroxyl in the flavone and sodium nitrite-aluminum nitrate are subjected to metal complex reaction, the generated complex product absorbs at 510nm, and the total flavone content can be calculated by monitoring the change of absorbance at 510nm and contrasting with a standard working curve by adopting a continuous flow analyzer.
1.2 materials and instruments
Rutin (Standard substance, Shanghai Alatin Biotechnology Co., Ltd.); aluminum nitrate nonahydrate (AR, shanghai alading biochem technologies ltd.); sodium nitrite (AR, national drug group chemical agents limited); sodium hydroxide (AR, chemical agents ltd of national drug group); all 5 tobacco powder samples were from the tobacco quality supervision and detection station in Yunnan province.
METLER AE200 analytical balance (sensory: 0.0001g, METLER TOLEDO, Switzerland); an AllanceProxima continuous flow Analyzer (France); JA5002 balance (sensory 0.01g, shanghai balance instrument factory); HS501 reciprocating oscillator (shanghai chemical laboratory instruments ltd); millipore AQUELIX5 water purifier (MERCK Millipore, USA).
1.3 sample treatment and analysis
Accurately weighing 1.0g of tobacco powder, placing in a 150mL triangular flask with a plug, adding 100mL of 80% ethanol solution, shaking up, covering the plug tightly, placing on a shaker, performing oscillatory extraction at 150r/min for 30min, filtering the extract with qualitative filter paper, and collecting the filtrate as sample solution for later use;
analysis with a continuous flow analyzer:
preparing a sodium nitrite reagent R1: weighing 5.00g of sodium nitrite, dissolving with 800mL of deionized water, and then fixing the volume to 1000 mL;
preparing an aluminum nitrate reagent R2: weighing 10.00g of aluminum nitrate, dissolving with 800mL of deionized water, and then fixing the volume to 1000 mL;
preparing a sodium hydroxide reagent R3: weighing 40.00g of sodium hydroxide, dissolving in 800mL of deionized water, and then fixing the volume to 1000 mL;
the instrument circuit is shown in detail in fig. 1. The sample solution is pumped by a yellow/yellow pump tube at the flow rate of 1.20 mL/min; the flow rate of the black/black pump pipe for the sodium nitrite reagent is 0.32 mL/min; the aluminum nitrate reagent is a black/black pump pipe, and the flow rate is 0.32 mL/min; the sodium hydroxide reagent is pumped by a red/red pump tube at the flow rate of 0.78 mL/min; cleaning solution is pumped by a red/red pump tube at a flow rate of 0.78 mL/min; detection wavelength: 510 nm; detecting speed: 30 samples/h; sample introduction/cleaning time ratio: 1: 1; baseline correction: opening; and (3) drift correction: opening;
drawing a standard curve: accurately weighing 0.200g of rutin, uniformly mixing in 200mL of ethanol, and then diluting to 1000mL with deionized water to obtain a standard stock solution of total flavonoids with the content of 200 mg/L; respectively transferring 10 mL, 30 mL, 50mL, 70 mL and 90mL of stock solutions by using a pipette, and respectively metering the volume to 100mL by using deionized water to obtain 20 mg/L, 60 mg/L, 100 mg/L, 140 mg/L and 180mg/L of standard working solutions; the standard working curve equation is: y is 0.0022x +0.0017 and the correlation coefficient r20.9998 (fig. 2);
and (4) calculating a result: substituting the measured value of the total flavone instrument corresponding to the tobacco powder sample solution to be measured into the formula (1) for calculation to obtain the total flavone content in the tobacco powder sample to be measured, and taking the average value of two parallel measurements as the final measurement result; the value is reduced to 0.01mg/g according to GB/T8170. The relative mean deviation of the two replicates should be less than 5%.
In the formula: p is the content (mg/g) of total flavonoids in the tobacco powder to be detected, C is the measured value (mg/L) of the total flavonoids corresponding to the sample solution by an instrument, V is the volume (mL) of the extract liquor, m is the mass (g) of the tobacco powder sample.
2. Results and discussion
2.1 Condition optimization
2.1.1 optimization of extract concentration
The flavone is generally easy to dissolve in solvents with stronger polarity, such as ethanol and methanol, and the ethanol is selected as an extraction solvent in the experiment considering the toxicity of the methanol. In the experiment, 50%, 60%, 70%, 80% and 90% ethanol aqueous solutions were prepared as extraction solvents, and sample 1# was subjected to sample treatment and analysis according to section 1.3, and the absorbance thereof was measured. FIG. 1 shows that: the absorbance value is gradually increased along with the increase of the ethanol concentration, which indicates that the extraction efficiency of the total flavonoids is increased along with the increase of the ethanol concentration, but when the ethanol concentration reaches 80%, the absorbance value is not increased any more, which indicates that the total flavonoids in the sample are completely extracted under the condition, so the ethanol concentration of the extraction liquid is selected to be 80%.
2.1.2 Effect of extraction shaking time
Sample No. 1 was taken and subjected to sample treatment in 1.3 portions, and only the oscillation time was changed to examine the influence thereof on the absorbance value of the sample solution. As can be seen from fig. 2: with the increase of the oscillation time, the total flavonoids are gradually extracted from the sample, when the oscillation time is within 30min, the absorbance value gradually increases with the increase of the oscillation time, and the response value tends to be stable after more than 30min, which indicates that the total flavonoids in the formazan are completely extracted, and the oscillation time is determined to be 30min in consideration of the detection efficiency of the sample.
2.1.3 pipeline preparation of Primary reagents for continuous flow Analyzer
The main reagents of the method are three: the method comprises the following steps of (1) selecting a proper pipeline configuration by comparing the influences of different pipeline configurations of a sample solution, a sodium nitrite reagent and an aluminum nitrate reagent on a series of standard working solutions.
Transferring 10, 30, 50, 70 and 90mL of total flavone standard stock solution (200mg/L) by using a pipette respectively, and metering the volume to 100mL by using deionized water respectively to obtain 20, 60, 100, 140 and 180mg/L of standard working solution respectively. As shown in table 1, the peak shape, the separation degree, the highest total flavone content corresponding to linearity, the linear correlation coefficient, and the like are comprehensively considered, and the following pipeline configuration is selected: the sample solution was pumped through a yellow/yellow pump line (flow rate of 1.20mL/min), a black/black pump line for sodium nitrite (flow rate of 0.32mL/min), a black/black pump line for aluminum nitrate (flow rate of 0.32mL/min), and a red/red pump line for sodium hydroxide (flow rate of 0.78 mL/min).
TABLE 1 tubing configuration for the principal reagents of a continuous flow analyzer
2.1.4 selection of the number of turns of the chromogenic reaction mixing Ring
The detection flow path of the method has no hot bath ring, so the number of turns of the reaction mixing ring needs to be increased to prolong the reaction time. Since the color reaction occurs after the sodium hydroxide reagent is added, the color reaction time is determined by changing the influence of the total number of turns of the reaction mixing ring on the peak appearance condition after the sodium hydroxide reagent is added.
As shown in table 2, when the number of turns of the reaction mixing ring is 40 and 60, the separation degree of the peak is not good, and the linear correlation coefficient is poor, and when the number of turns of the reaction mixing ring is 80 and 90, the separation degree of the peak is good, and the difference of the linear correlation coefficient is not large, which indicates that after the sodium hydroxide reagent is added, 80 turns of the reaction mixing ring are added to sufficiently complete the color reaction, and 80 turns are selected from the viewpoints of shortening the detection time and improving the detection efficiency.
TABLE 2 selection of color reaction time for detection of pipeline in continuous flow analyzer
2.2 evaluation of method
2.2.1 detection and quantitation limits
The minimum cup standard solution is used, sample introduction is carried out for 10 times, and the standard deviation of the measurement result is 0.0082mg/g, so the detection limit of the method is 3 SD-0.025 mg/g, and the quantification limit is 10 SD-0.082 mg/g.
2.2.2 repeatability
The results of the 3 replicates of 5 tobacco dust samples, each measured according to the method of the invention by the same operator, over the same period of time are shown in Table 3. As can be seen from Table 3, the RSD values of the total flavone content measured by the method are respectively 3.29%, 1.17%, 0.88%, 0.84% and 1.06%, which are all less than 5%, and the repeatability is good.
TABLE 3 repeatability
2.2.3 stability
The results of 5 samples of tobacco dust measured by the same operator according to the method of the invention 3 times in different time periods, 2 replicates each time, were averaged, see table 4. As can be seen from Table 4, the RSD values of the total flavone content measured by the method are respectively 2.10%, 1.03%, 0.80%, 0.66% and 1.02%, and are all less than 5%, and the stability is good.
TABLE 4 stability
2.2.4 actual sample analysis and recovery Rate of spiked samples
The normalized recovery of 5 tobacco powder samples by the method of the invention was measured in parallel 3 times and found to be between 96.83% and 104.4% (Table 5). The method has the advantages of good accuracy and reliable detection result.
TABLE 5 sample determination and recovery by spiking
3. Conclusion
The method utilizes the continuous flow analyzer to measure the content of the total flavonoids in the tobacco, has simple and convenient operation, accurate result and high precision, and can provide a powerful tool for researching the metabolic pathway of the flavonoids compounds and the regulating mechanism thereof.
Claims (1)
1. A method for determining the content of total flavonoids in tobacco based on a continuous flow analyzer specifically comprises the following steps:
(1) accurately weighing 1.0g of tobacco powder, placing in a 150mL triangular flask with a plug, adding 100mL of 80% ethanol solution, shaking up, covering the plug tightly, placing on a shaker, performing oscillatory extraction at 150r/min for 30min, filtering the extract with qualitative filter paper, and collecting the filtrate as sample solution for later use;
(2) analysis with a continuous flow analyzer:
preparing a sodium nitrite reagent: weighing 5.00g of sodium nitrite, dissolving with 800mL of deionized water, and then fixing the volume to 1000 mL;
preparing an aluminum nitrate reagent: weighing 10.00g of aluminum nitrate, dissolving with 800mL of deionized water, and then fixing the volume to 1000 mL;
preparing a sodium hydroxide reagent: weighing 40.00g of sodium hydroxide, dissolving in 800mL of deionized water, and then fixing the volume to 1000 mL;
setting a flow path of the analyzer: the sample solution is pumped by a yellow/yellow pump tube at the flow rate of 1.20 mL/min; the flow rate of the black/black pump pipe for the sodium nitrite reagent is 0.32 mL/min; the aluminum nitrate reagent is a black/black pump pipe, and the flow rate is 0.32 mL/min; the sodium hydroxide reagent is pumped by a red/red pump tube at the flow rate of 0.78 mL/min; cleaning solution is pumped by a red/red pump tube at a flow rate of 0.78 mL/min; after the sodium hydroxide reagent is added, the mixture of the sample solution and the three reagents is fully mixed and developed by 80 circles of reaction mixing circles and then enters a detector; detection wavelength: 510 nm; detecting speed: 30 samples/h; sample introduction/cleaning time ratio: 1: 1; baseline correction: opening; and (3) drift correction: opening;
(3) drawing a standard working curve: accurately weighing 0.200g rutin, mixing well in 200mL ethanol, and diluting to 1000mL with deionized water to obtain total flavone standard with content of 200mg/LA quasi-stock solution; respectively transferring 10 mL, 30 mL, 50mL, 70 mL and 90mL of stock solutions by using a pipette, and respectively metering the volume to 100mL by using deionized water to obtain 20 mg/L, 60 mg/L, 100 mg/L, 140 mg/L and 180mg/L of standard working solutions; the standard working curve equation is: y is 0.0022x +0.0017 and the correlation coefficient r2＝0.9998；
(4) And (4) calculating a result:
substituting the measured value of the total flavone instrument corresponding to the tobacco powder sample solution to be measured into the formula (1) for calculation to obtain the total flavone content in the tobacco powder to be measured, and taking the average value of two parallel measurements as the final measurement result;
in the formula: p is the content (mg/g) of total flavonoids in the tobacco powder to be detected, C is the measured value (mg/L) of the total flavonoids corresponding to the sample solution by an instrument, V is the volume (mL) of the extract liquor, m is the mass (g) of the tobacco powder sample.
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