CN113030333A - Method for measuring plastid pigment content in tobacco shreds - Google Patents
Method for measuring plastid pigment content in tobacco shreds Download PDFInfo
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Abstract
The invention discloses a method for measuring the plastid pigment content in cut tobacco, which comprises the following steps: (1) and (3) preparing a standard curve: preparing a series of standard working solutions of the plastid pigment to be tested, carrying out high performance liquid chromatography testing, and making a linear regression equation of the plastid pigment to be tested according to a testing result of the high performance liquid chromatography; (2) and (3) measuring the plastid pigment content in the tobacco shreds: and (2) testing under a determined chromatographic condition, carrying out high performance liquid chromatography testing on the tobacco shred extracting solution according to the chromatographic condition in the step (1), bringing a testing result into a corresponding linear regression equation, and calculating to obtain the content of the plastid pigment in the tobacco shreds. In the determination method, the time for determining the tobacco plastid pigment is short, the detection efficiency is high, and the adopted high performance liquid chromatography has the advantages of rapidness, high efficiency and high analysis sensitivity.
Description
Technical Field
The invention relates to the technical field of tobacco inspection, in particular to a method for measuring the content of a plastid pigment in tobacco shreds.
Background
The tobacco leaf plastid pigment mainly comprises chlorophyll a, chlorophyll b, beta-carotene, lutein, neoxanthin, violaxanthin and the like, and most of the pigments are precursors of tobacco leaf flavor components and directly or indirectly influence the inherent quality of tobacco leaves. Whether the pigment is degraded or not in the tobacco leaf preparation process can directly influence the fragrance and color of the tobacco leaves. Research shows that the degradation product content of carotenoid accounts for about 8% -12% of the total aroma, and the carotenoid is an important aroma component in smoke. For example, megastigmatrienone, a degradation product of carotenoid, has cocoa flavor and can improve the aroma of smoke; the dihydroactinidiolide and geranylacetone can increase the fragrance of the flue-cured tobacco and reduce the irritation of the flue-cured tobacco, and the like. Therefore, the accurate determination of plastid pigments in tobacco leaves is of great significance to the understanding of the quality and usability of tobacco leaves.
The traditional assay method is to measure chlorophyll and carotenoids separately. The high performance liquid chromatography realizes the simultaneous determination of chlorophyll a, chlorophyll b, beta-carotene, lutein, neoxanthin and violaxanthin.
The measurement of the 6 kinds of plastid pigments was carried out by reversed-phase high performance liquid chromatography, but the sample high performance liquid chromatography measurement time was 40 minutes or more. And (3) separating and measuring the 6 plastid pigments by using a reversed-phase high performance liquid chromatography, wherein the high performance liquid chromatography measuring time of the sample is more than 55 minutes. Wufang Pinna and the like adopt microcolumn high performance liquid chromatography to measure 6 plastid pigments in tobacco, but the sample pretreatment is complicated and comprises the following steps: pigment extraction, solid phase extraction column activation, solid phase extraction, elution and the like, and has high detection cost and detection limit of 20-40 mug/mL.
From the research of Tianhaiying and the like and our experiments, it can be known that the cut tobacco of cigarettes only contains lutein and beta-carotene. The expanded tobacco shreds are used as the components of cigarette formula, and can provide the cigarette with thick feeling, increase air permeability, improve combustion performance, and reduce tar and nicotine. The expansion process of the expanded tobacco shreds is accompanied by the change of temperature values and humidity values, and the degradation of plastid pigments can occur in the expansion process, so that the flavor and the color of the tobacco shreds are endowed. Therefore, the change of the plastid pigment in the cut tobacco before and after expansion is monitored, and the process and the quality control of the expanded cut tobacco are facilitated. However, the measurement of the content of the plastid pigment in the tobacco shreds in the prior art has the technical problems of long test time and high cost.
In conclusion, it is necessary to establish a method for rapidly, accurately and inexpensively measuring the content of plastid pigments in tobacco shreds.
Disclosure of Invention
The invention aims to overcome the defects of the prior art based on the prior art, and provides a method for measuring the content of the plastid pigment in the tobacco shreds, so that the plastid pigment in the tobacco shreds can be quickly, accurately and cheaply measured.
In order to solve the technical problem, the embodiment of the invention discloses a method for measuring the plastid pigment content in tobacco shreds, which comprises the following steps:
(1) preparation of Standard Curve
Preparing a series of standard working solutions of the plastid pigment to be tested, and carrying out high performance liquid chromatography test:
mobile phases including phase a, phase B and phase C:
phase A is isopropanol;
phase B is ultrapure water;
the phase C is a mixed solution consisting of triethylamine and acetonitrile with the volume percentage of 0.18-0.22%;
the elution mode is gradient elution, and the gradient elution procedure is as follows:
0-10 min: the volume fraction of the mobile phase A is 0, the volume fraction of the mobile phase B is 20%, and the volume fraction of the mobile phase C is 80%;
10-15 min: the volume fraction of the mobile phase A is 40%, the volume fraction of the mobile phase B is 0, and the volume fraction of the mobile phase C is 60%;
15-18 min: the volume fraction of the mobile phase A is 0, the volume fraction of the mobile phase B is 20%, and the volume fraction of the mobile phase C is 80%;
and (4) according to the test result of the high performance liquid chromatography, making a linear regression equation of the plastid pigment to be tested.
(2) Determination of plastid pigment content in tobacco shreds
And (2) carrying out high performance liquid chromatography test on the tobacco shred extracting solution according to the chromatographic conditions in the step (1), bringing the test result into a corresponding linear regression equation, and calculating to obtain the content of the plastid pigment in the tobacco shreds.
By adopting the technical scheme, the time for extracting the cut tobacco plastid pigment is short, the detection efficiency is high, and the accuracy is high; by changing the mobile phase composition and gradient elution procedure, the separation and determination time of each plastid pigment in the cut tobacco is shortened to be within 22 min; meanwhile, the mobile phase has simple composition and low cost.
The embodiment of the invention discloses a method for measuring the content of plastid pigment in cut tobacco, wherein the plastid pigment is at least one of the following plastid pigments: lutein, beta-carotene.
The embodiment of the invention discloses a method for measuring the plastid pigment content in cut tobacco, which comprises the following steps (2): preparing tobacco shreds into tobacco powder samples, dissolving the tobacco powder samples in acetone water solution, performing ultrasonic extraction, filtering with qualitative filter paper, and filtering with organic phase filter membrane.
The embodiment of the invention discloses a method for measuring the plastid pigment content in cut tobacco, wherein the step of preparing a tobacco powder sample from the cut tobacco comprises the following steps: and (3) putting the tobacco shreds into an oven with the temperature of not higher than 40 ℃ for drying for 2 hours, taking out and grinding the tobacco shreds, and sieving the tobacco shreds with a 40-mesh sieve to obtain a tobacco powder sample.
The embodiment of the invention discloses a method for measuring the plastid pigment content in cut tobacco, wherein an acetone water solution is prepared from acetone and water according to the volume fraction of 9:1, the volume of acetone aqueous solution is 25mL, the ultrasonic extraction time is 20min, and the membrane pores of the organic phase filter membrane are 0.45 μm.
The embodiment of the invention discloses a method for measuring the plastid pigment content in cut tobacco, wherein in the step (1), the model of a high performance liquid chromatography instrument tested by the high performance liquid chromatography is Waters NOVA-Park, a chromatographic column is a C18 high performance liquid chromatography column, the specification of the high performance liquid chromatography column is 3.9mm multiplied by 150mm, and the diameter of a filler is 4.0 mu m.
The embodiment of the invention discloses a method for measuring the content of plastid pigment in cut tobacco, wherein the column temperature of a high performance liquid chromatography column is 30 ℃, the sample injection volume is 10 mu L, and the flow rate of a mobile phase is 1 mL/min.
The embodiment of the invention discloses a method for measuring the content of plastid pigment in cut tobacco, wherein in the step (1), the test result of high performance liquid chromatography is the peak area corresponding to each concentration of a series of standard working solutions of plastid pigment to be measured; the linear regression equation for preparing the plastid pigment to be tested comprises the following steps: and respectively taking the peak area of each plastid pigment as a vertical coordinate and the concentration of each plastid pigment as a horizontal coordinate to prepare a linear regression equation of each plastid pigment.
The embodiment of the invention discloses a method for measuring the content of plastid pigment in cut tobacco, wherein the measuring time of the content of plastid pigment in the cut tobacco is not more than 18 min.
The invention determines the chromatographic conditions for measuring the plastid pigment in the tobacco shreds by the high performance liquid chromatograph, can completely separate the plastid pigment in the tobacco shreds within 18min, and is favorable for identifying the plastid pigment in the sample.
Drawings
FIG. 1 shows chromatograms of lutein and beta-carotene of a standard working liquid of an embodiment of the present invention;
figure 2 shows a chromatogram of a cut tobacco sample of an embodiment of the invention.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure. While the invention will be described in conjunction with the preferred embodiments, it is not intended that features of the invention be limited to these embodiments. On the contrary, the invention is described in connection with the embodiments for the purpose of covering alternatives or modifications that may be extended based on the claims of the present invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be practiced without these particulars. Moreover, some of the specific details have been left out of the description in order to avoid obscuring or obscuring the focus of the present invention. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
The experimental procedures described in the following detailed description are conventional unless otherwise specified, and the reagents and materials, unless otherwise specified, are commercially available.
The reagents used in the present invention are shown in Table 1.
TABLE 1 reagents used in the experiments
The invention provides a method for measuring the plastid pigment content in cut tobacco, which comprises the following steps:
(1) preparation of Standard Curve
(a) Preparation of a standard stock solution:
standard stock solution 1: weighing 1mg of lutein, transferring the lutein into a 10mL brown volumetric flask, and fixing the volume of 90% acetone aqueous solution to a scale to prepare a single standard stock solution 1 with the lutein concentration of 100 mug/mL;
standard stock solution 2: 2.5mg of beta-carotene is weighed and placed in a 50mL brown volumetric flask, 90% acetone aqueous solution is added to the volume to be calibrated, and a single standard stock solution 3 with the volume of 50 mug/mL is prepared.
Wherein, the 90% acetone water solution is prepared by mixing acetone and water according to the volume ratio of 9:1, and (2) preparing a solution.
(b) Preparing a series of standard working solutions:
respectively transferring 1mL of standard stock solution 1 and 2mL of standard stock solution 2 into a 10mL volumetric flask, fixing the volume to the scale by 90% acetone aqueous solution, preparing first-grade mixed standard stock solution of 10 mu g/mL, and then gradually diluting to respectively obtain standard working solutions of 10 mu g/mL, 5 mu g/mL, 2.5 mu g/mL, 1.0 mu g/mL, 0.50 mu g/mL and 0.10 mu g/mL.
(c) Liquid chromatography analysis: detecting and analyzing the series of standard working solutions by using a high performance liquid chromatograph, wherein the chromatographic analysis conditions are as follows: the chromatographic column adopts a Waters NOVA-Park C18 column, the specification of the chromatographic column is 3.9mm multiplied by 150mm, the diameter of a filler is 4.0 mu m, the column temperature is 30 ℃, the sample injection volume is 10 mu L, the flow rate of a mobile phase is 1mL/min, and the detection wavelength of a photodiode array detector (PDA) is 448 nm; mobile phase: the phase A is isopropanol, the phase B is ultrapure water, and the phase C is a mixed solution consisting of 0.18-0.22% of triethylamine and acetonitrile; a gradient elution procedure was used: 0-10 min: the volume fraction of the mobile phase A is 0, the volume fraction of the mobile phase B is 20%, and the volume fraction of the mobile phase C is 80%; 10-15 min: the volume fraction of the mobile phase A is 40%, the volume fraction of the mobile phase B is 0, and the volume fraction of the mobile phase C is 60%; 15-18 min: the volume fraction of mobile phase A was 0, the volume fraction of mobile phase B was 20%, the volume fraction of mobile phase C was 80%, and the total elution time was 18 min.
According to tobacco industry standard YC/T382-2010 (high performance liquid chromatography for measuring plastid pigment) sample removal, 40min is needed to remove beta-carotene, which wastes time and mobile phase solution, and beta-carotene stays in a chromatographic column with certain temperature for a long time and is isomerized, so that the selection of a flow gradient is crucial. The carotenoid is a terpenoid, the molecular formula contains a plurality of unsaturated bonds, wherein two ends of the lutein are provided with hydroxyl, the beta-carotene completely belongs to a multi-double-bond compound only containing two elements of carbon and hydrogen, and the nonpolar is stronger than the lutein, so that the beta-carotene needs a nonpolar strong solvent to accelerate the elution of the beta-carotene; meanwhile, when the standard working solution is used by a method standard in the tobacco industry, the beta-carotene has a trailing phenomenon, and the invention replaces the interaction between the beta-carotene and the chromatographic column by the interaction between triethylamine and the chromatographic column in the mobile phase organic solvent, thereby reducing the trailing of the beta-carotene. Through repeated tests, the mobile phase composition is optimized as described above, namely mobile phases A + B + C are adopted, the mobile phase A is isopropanol, the mobile phase B is ultrapure water, and the mobile phase C is a mixed solution of 0.18-0.22% of triethylamine and acetonitrile, so that the elution capacity of beta-carotene is improved.
(d) And (3) preparing a standard curve: recording the peak area of each concentration of the lutein series standard working solution, taking the peak area of lutein as a vertical coordinate and the concentration of lutein as a horizontal coordinate, and making a linear regression equation of lutein; recording the peak area of each concentration of the beta-carotene series standard working solution, taking the peak area of the beta-carotene as a vertical coordinate and the concentration of the beta-carotene as a horizontal coordinate, and making a linear regression equation of the beta-carotene; the linear range, linear regression equation, correlation coefficient, detection limit, and quantification limit of each obtained plastid pigment are shown in table 2.
TABLE 2 Linear regression equation, correlation coefficient and detection limits for plastid pigments
Note: the detection limit was calculated as 3 times the signal-to-noise ratio (S/N-3).
The high performance liquid chromatography test method adopts a specific mobile phase composition and a gradient elution program, the correlation coefficients of working curves of the lutein and the beta-carotene measured in a linear range of 0.096-9.6 mu g/mL are both larger than 0.999, the linear relation is good, the detection limit (S/N ═ 3) of the lutein and the beta-carotene is between 0.0250 and 0.0320 mu g/mL, and the quantification limit is between 0.0833 and 0.1067 mu g/mL.
(2) Determination of plastid pigment content in tobacco shreds
(a) Crushing tobacco shreds to prepare tobacco powder: taking and processing tobacco shred samples according to tobacco industry standard YC/T31-1996, drying the tobacco shred samples in an oven with the temperature not higher than 40 ℃ for about 2 hours until the tobacco shred samples can be kneaded by fingers, taking out the tobacco shred samples and immediately grinding the tobacco shred samples into tobacco powder, sieving the tobacco powder with a 40-mesh sieve, uniformly mixing the tobacco powder and placing the tobacco powder in a sealed bag for later use.
(b) Pigment extraction: 2.00g of tobacco powder was accurately weighed, placed in a 100mL Erlenmeyer flask, and 25mL of 90% aqueous acetone solution (V) was addedAcetone (II)：VWater (W)9:1), ultrasonic extracting for 20min, taking out and shaking upThen filtered by qualitative filter paper, 3mL of filtrate is filtered by 0.45 μm organic phase filter membrane in 1.5mL sample bottle, and then directly injected for analysis.
(c) And (3) determination of pigment content: and (2) measuring the content of the plastid pigments in the sample to be measured according to the chromatographic conditions in the step (1), respectively recording the peak areas of the plastid pigments, respectively substituting the peak areas of the plastid pigments into a linear regression equation of the plastid pigments, quantifying by using an external standard method, calculating the content of the plastid pigments in a sample volume of 10 mu L, and converting into the content of the plastid pigments in a tobacco powder sample of 2g to obtain the content of the plastid pigments in the tobacco shred sample.
(3) Methodology validation
(a) Accurately weighing 13 parts of 2g of the same tobacco powder sample in 13 triangular flasks with 100 mu g/mL of lutein standard solution 1.15mL to form a low-concentration lutein labeling level (4.6 mu g/mL) in 5 parts of the sample, and simultaneously adding 0.588mL of 50 mu g/mL of beta-carotene standard solution to form a low-concentration beta-carotene labeling level (1.175 mu g/mL); wherein 1.635mL of a 100. mu.g/mL standard solution of lutein was added to 5 samples to form a normalized concentration level in lutein (6.54. mu.g/mL), and 1.09mL of a 50. mu.g/mL standard solution of β -carotene was added to form a normalized concentration level in β -carotene (2.18. mu.g/mL), as shown in Table 4, followed by HPLC analysis using the chromatographic conditions of step (1) with 3 samples without the mixed standard solution, calculating the recovery from the measurement values before and after the addition of each plastid pigment and the addition amount, and calculating the Relative Standard Deviation (RSD) from the parallel measurement values of 5 samples with the addition of the standard. Referring to tables 3 and 4, the results show that the cut tobacco contains two kinds of plastid pigments of lutein and beta-carotene, the Relative Standard Deviation (RSD) of the lutein and the beta-carotene is 4.66% -4.94%, is less than 10%, the repeatability is good, the recovery rate is 86.65-109.02%, and the chromatographic conditions of the high performance liquid chromatography can be suitable for analyzing the two kinds of pigments in the cut tobacco.
Table 3 repeatability tests (n ═ 5)
TABLE 4 recovery test with addition of standard
(4) Determination of lutein and beta-carotene content in actual tobacco shred sample
The method comprises the steps of taking tobacco shred samples before and after microwave expansion in copper-core cigarette factories in Guizhou, carrying out different expansion technologies on tobacco shreds of different grades, measuring the contents of lutein and beta-carotene in the tobacco shreds by adopting the high performance liquid chromatography, carrying out parallel measurement twice on each group of samples, and expressing the average value of the measurement results of the samples as shown in Table 5. The results show that: the contents of lutein and beta-carotene in X2Fys are higher than those in expanded cut tobaccos X2F31 and X2F32 expanded by SP31 and SP32 processes, which shows that the two pigments of lutein and beta-carotene are degraded in the SP31 and SP32 expansion processes. The change of lutein and beta-carotene in the mixed tobacco before and after the GYys expansion is also a descending rule.
After the flue-cured tobacco is modulated, redried and aged, a large amount of plastid pigment is degraded and disappears, and lutein and beta-carotene mainly remain; compared with the tobacco shreds before microwave expansion, the contents of lutein and beta-carotene in the tobacco shreds after expansion are reduced to different degrees, and the maximum reduction range is 9.54 percent and 14.38 percent; the color and the fragrance of the tobacco shreds after microwave expansion are changed, and have a certain relation with the pigment. Research shows that the content of the degradation products of the carotenoid accounts for about 8% -12% of the total aroma, and the degradation products of the carotenoid are important aroma components in smoke, such as megastigmatrienone, dihydroactinidiolide and geranyl acetone, which can increase the aroma of flue-cured tobacco and reduce irritation.
TABLE 5 content of lutein and beta-carotene (mg/g) in the actual tobacco samples
Note: x2Fys is X2Protofilaments of F-grade tobacco leaves, X2F31 being X2Expanded tobacco shreds obtained by subjecting F grade tobacco protofilaments to SP31 process, wherein X2F32 is X2Expanded tobacco shred X2FCO expanded by SP32 process from F grade tobacco leaf protofilament2Is X2Subjecting the protofilament of F-grade tobacco leaf to CO2Expanded cut tobacco of the process expansion; gyys is protofilament of GY-grade tobacco leaf, GY31 is expanded tobacco shred of GY-grade tobacco leaf expanded by SP31 process, GY32 is expanded tobacco shred of GY-grade tobacco leaf expanded by SP32 process, GYCO2The protofilament of GY-grade tobacco leaves is subjected to CO2Expanded cut tobacco by the process.
Example 1
(1) Tobacco shred crushing and tobacco powder making
Sampling and treating according to tobacco industry standard YC/T31-1996, drying the tobacco sample in an oven at a temperature of not higher than 40 ℃ for 2h, taking out and immediately grinding, sieving with a 40-mesh sieve, uniformly mixing the tobacco powder and placing in a sealed bag for later use.
(2) Extracting lutein and beta-carotene from tobacco shred
2.00g of tobacco powder was accurately weighed, placed in a 100mL Erlenmeyer flask, and 25mL of 90% aqueous acetone solution (V) was addedAcetone (II)：VWater (W)And (9: 1), performing ultrasonic extraction for 20min, taking out, shaking up, filtering by qualitative filter paper, filtering 3mL of filtrate by a 0.45-micron organic phase filter membrane in a 1.5mL sample bottle, and directly injecting and analyzing.
(3) High performance liquid chromatography detection
Detecting chromatographic conditions by high performance liquid chromatography:
a chromatographic column: waters NOVA-Park C18 column (3.9X 150mm, 4 μm, Waters corporation); the sample volume is 10 mu L, and the flow rate is 1 mL/min; the column temperature was 30 ℃. Mobile phase composition: the mobile phase A is isopropanol, the mobile phase B is ultrapure water, and the mobile phase C is a mixed solution of 0.18-0.22% of triethylamine and acetonitrile. The gradient elution procedure was: 0-10 min: the volume fraction of the mobile phase A is 0, the volume fraction of the mobile phase B is 20%, and the volume fraction of the mobile phase C is 80%; 10-15 min: the volume fraction of the mobile phase A is 40%, the volume fraction of the mobile phase B is 0, and the volume fraction of the mobile phase C is 60%; 15-18 min: the volume fraction of mobile phase a was 0, the volume fraction of mobile phase B was 20%, and the volume fraction of mobile phase C was 80%.
The chromatogram of the tobacco sample measured by using a photodiode array detector (PDA) with a detection wavelength of 448nm is shown in FIG. 2.
As can be seen from the chromatogram of FIG. 2, the separation of lutein and beta-carotene in tobacco shreds is good, and the peak-off time of beta-carotene is only 11.633min, so that the usage amount of the organic mobile phase is saved, and the detection time is also saved. There is a small drum peak beside the main peak of beta-carotene, and the chromatogram measured by the standard working solution also has the same small drum peak beside the main peak of beta-carotene in FIG. 1, which is the isomer peak of beta-carotene. The extraction method of the lutein and the beta-carotene is ultrasonic extraction, the ultrasonic extraction time is 20min, and the damage of plant cell walls is accelerated according to the cavitation, the thermal effect and the mechanical effect of ultrasonic waves, so that a solvent enters cells to dissolve a target object to achieve the extraction effect of the lutein and the beta-carotene. The ultrasonic extraction is a heat release process, and because the beta-carotene is sensitive to light, heat and acid solvents and the ultrasonic time is long, the cut tobacco itself shows weak acidity, which is the reason of beta-carotene isomerization, in the process of measuring the content of the beta-carotene in the cut tobacco, the small drum peak beside the main peak of the beta-carotene is also incorporated into a linear regression equation of the beta-carotene for quantitative analysis.
According to the invention, the triethylamine is added into the mobile phase, and a certain gradient elution program is matched, so that the beta-carotene can be separated from impurities, the peak pattern of the lutein is good, the separation and determination time of two plastid pigments of the lutein and the beta-carotene in the tobacco shreds is shortened to 18min, the analysis and determination time is improved, and meanwhile, the mobile phase is simple in composition and low in cost. The invention improves the determination method of the lutein and the beta-carotene in the cut tobacco by improving the mobile phase composition and the gradient elution procedure.
While the invention has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing is a more detailed description of the invention, taken in conjunction with the specific embodiments thereof, and that no limitation of the invention is intended thereby. Various changes in form and detail, including simple deductions or substitutions, may be made by those skilled in the art without departing from the spirit and scope of the invention.
Claims (9)
1. A method for measuring the plastid pigment content in tobacco shreds is characterized by comprising the following steps:
(1) preparation of Standard Curve
Preparing a series of standard working solutions of the plastid pigment to be tested, and carrying out high performance liquid chromatography test:
mobile phases including phase a, phase B and phase C:
phase A is isopropanol;
phase B is ultrapure water;
the phase C is a mixed solution consisting of triethylamine and acetonitrile with the volume percentage of 0.18-0.22%;
the elution mode is gradient elution, and the gradient elution program is as follows:
0-10 min: the volume fraction of the mobile phase A is 0, the volume fraction of the mobile phase B is 20%, and the volume fraction of the mobile phase C is 80%;
10-15 min: the volume fraction of the mobile phase A is 40%, the volume fraction of the mobile phase B is 0, and the volume fraction of the mobile phase C is 60%;
15-18 min: the volume fraction of the mobile phase A is 0, the volume fraction of the mobile phase B is 20%, and the volume fraction of the mobile phase C is 80%;
and according to the test result of the high performance liquid chromatography, making a linear regression equation of the plastid pigment to be tested.
(2) Determination of plastid pigment content in tobacco shreds
And (2) carrying out high performance liquid chromatography test on the tobacco shred extracting solution according to the chromatographic conditions in the step (1), bringing the test result into a corresponding linear regression equation, and calculating to obtain the content of the plastid pigment in the tobacco shreds.
2. The method for measuring the plastid pigment content in the tobacco shreds according to claim 1, wherein the plastid pigment is at least one of the following plastid pigments: lutein, beta-carotene.
3. The method for measuring the plastid pigment content in the tobacco shreds according to claim 1, wherein the step (2) further comprises the following steps: preparing tobacco shreds into tobacco powder samples, dissolving the tobacco powder samples in acetone aqueous solution, performing ultrasonic extraction, filtering by qualitative filter paper, and filtering by an organic phase filter membrane.
4. The method for determining the plastid pigment content in the tobacco shred according to claim 3, wherein the step of preparing the tobacco shred into the tobacco powder sample comprises the following steps: and (3) drying the tobacco shreds in an oven at the temperature of not higher than 40 ℃ for 2 hours, taking out and grinding the tobacco shreds, and sieving the tobacco shreds with a 40-mesh sieve to obtain the tobacco powder sample.
5. The method for determining the plastid pigment content in the tobacco shreds according to claim 3, wherein the acetone aqueous solution is an acetone and water according to the volume fraction of 9:1, the volume of the acetone aqueous solution is 25mL, the ultrasonic extraction time is 20min, and the membrane pores of the organic phase filter membrane are 0.45 mu m.
6. The method for determining the plastid pigment content in the tobacco shreds according to claim 1, wherein in the step (1), the instrument model of the high performance liquid chromatography test is Waters NOVA-Park, the chromatographic column is a C18 high performance liquid chromatography column, the specification of the high performance liquid chromatography column is 3.9mm x 150mm, and the diameter of the filler is 4.0 μm.
7. The method for determining the plastid pigment content in the tobacco shreds according to claim 6, wherein the column temperature of the high performance liquid chromatography column is 30 ℃, the sample injection volume is 10 μ L, and the flow rate of the mobile phase is 1 mL/min.
8. The method for determining the plastid pigment content in the tobacco shreds according to claim 1, wherein in the step (1), the test result of the high performance liquid chromatography is a peak area corresponding to each concentration of a series of standard working solutions of the plastid pigment to be tested; the linear regression equation for manufacturing the plastid pigment to be tested comprises the following steps: and respectively taking the peak area of each plastid pigment as a vertical coordinate and the concentration of each plastid pigment as a horizontal coordinate to prepare a linear regression equation of each plastid pigment.
9. The method for measuring the contents of lutein and β -carotene in tobacco shreds according to any one of claims 1 to 8, wherein the measuring time of the content of plastid pigment in tobacco shreds is not more than 18 min.
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