Method for separating and determining content of wax compounds in tobacco leaves based on column chromatography and GC-MS (Gas Chromatography-Mass Spectrometer)

Abstract

The invention discloses a method for separating and measuring the content of wax compounds in tobacco leaves based on column chromatography and GC-MS. The method relates to the technical field of extraction, separation and analysis of the wax compounds in the tobacco leaves, and comprises the following steps: mixing tobacco leaf powder with ethanol, heating and stirring, performing reflux extraction and suction filtration, and retaining filtrate; then separating the wax compounds in the tobacco leaves by using a column chromatography stepped elution method, and quantitatively determining the content of each component of the wax compounds in a tobacco leaf system by using a GC-MS (Gas Chromatography-Mass Spectrometer) through an external standard method. According to the method, 13 compounds, namely cedrol, phytone, menthol, delta5-oat sterol, cholesterol, isosestenocycloenol, 1-heptadecanol, dehydropalmitol, 1-tetradecanol, hexadecane, 1-hexadecene, 1-tetradecene and dodecane, are separated and detected in the tobacco leaves for the first time, and the technological process is simple, convenient and easy to operate; the extraction solvent is nontoxic and harmless and can be recycled, the detection result is rapid and accurate, and the credibility is high.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction, separation and analysis of waxy compounds in tobacco leaves, and particularly relates to a method for separating and determining the content of waxy compounds in tobacco leaves based on column chromatography and GC-MS. Background Art

[0002] Tobacco wax is a hydrophobic layer composed of lipophilic compounds that coats the surface of tobacco leaves, stems, flowers, and fruit. Tobacco wax plays a significant role in tobacco growth and development, such as protecting against pests and diseases, retaining moisture and resisting drought, and protecting against ultraviolet radiation.

[0003] Modern research often uses gas chromatography-mass spectrometry (GC-MS) to identify wax components. [Severson, RF, Johnson, AM and Jackson, DM (1985) Cuticular Constituents of Tobacco: Factors Affecting Their Production and Their Role in Insect and Disease Resistance and Smoke Quality. Rec. Adv. Tob. Sci., 11, 105-173] studied the surface aliphatic hydrocarbons of different tobacco leaf types. These are alkanes with 25 to 36 carbon atoms, and their content can reach up to 25% of the extract. [Arrendale, RF, Severson, RF, Chorty, DT and Stephenson, MG (1988) Isolation and Identification of the Wax Esters from the Cuticular Waxes of Green Tobacco Leaf. Beitragezur Tabakforschung International / Contributions to Tobacco Research, 14, 67-84] A detailed study of the wax components in fresh tobacco leaves NC2326 was conducted, and 172 wax esters with carbon atoms ranging from 30 to 52 were identified.

Chen Kaibo, Xu Yingbo, Yan Xiangyang, Ge Shaolin, Yang Rui, Zhu Fengling, Liu Shaomin. Isolation and identification of non-polar and weakly polar components of tobacco leaf surface wax and their moisturizing properties[J]. Agricultural Science, 2017, 7(2): 73-81

[0004] As can be seen, existing research on the identification of tobacco leaf wax components is not comprehensive enough. Most studies have only identified one or two components within the tobacco leaf wax composition, and some studies have focused on tobacco leaf epidermal wax. No research has yet systematically and comprehensively studied all tobacco leaf waxes. Furthermore, the types of chemical components isolated and detected in tobacco leaves are relatively small, mostly known chemicals, with little exploration of new and potential chemicals.

[0005] Chinese patent application publication number CN110542736A discloses a method for the simultaneous analysis of nine alkaloids and 11 aroma components in tobacco leaves. The method involves immersing the sample in an alkaline solution to release the target alkaloids and aroma compounds from the sample matrix. Chloroform is then added and vortexed to transfer the alkaloids and aroma compounds into a chloroform phase. After centrifugation and separation, the chloroform phase is sampled for instrumental analysis. Compared to existing methods for analyzing alkaloids and aroma components in tobacco leaves, the method is simpler to use and more stable. However, the patent does not address the analysis of tobacco wax compounds and covers a relatively limited range of analysis, leaving room for further improvement. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to provide a convenient and quick method for separating and detecting multiple waxy compound components in tobacco leaves.

[0007] The present invention solves the above technical problems through the following technical means: The present invention provides a method for separating and determining the content of waxy compounds in tobacco leaves based on column chromatography and GC-MS, comprising the following steps: (1) Mix tobacco leaf powder with ethanol, heat and stir, reflux extraction, filter, and retain the filtrate; add silica gel to the filtrate and evaporate it to dryness, transfer it to a silica gel column, and elute it with an organic solvent to prepare a sample to be tested; (2) Select internal standard and standard to prepare internal standard solution and standard solution respectively, and use GC-MS technology to test according to the following chromatographic mass spectrometry conditions; use the ratio of the peak area of ​​the target compound to the internal standard as the vertical axis and the ratio of the concentration of the target compound to the internal standard as the horizontal axis to draw the standard working curve, and the linear correlation coefficient R 2 Greater than 0.995; The specific chromatographic mass spectrometry conditions are as follows: Chromatographic conditions were as follows: chromatographic column: HP-5ms (190915-433UI, 30 m × 0.25 mm × 0.25 μm); carrier gas: 99.99% pure He; flow rate: 10 mL / min; injection volume: 1 μL; injection mode: split injection, split ratio 10:1; injection port temperature: 250°C; heating program: 50°C for 2 min; heating to 120°C at a rate of 2.5°C / min and hold for 2 min, heating to 200°C at a rate of 1°C / min and hold for 2 min, heating to 250°C at a rate of 1°C / min and hold for 2 min, heating to 300°C at a rate of 2.5°C / min and hold for 2 min; The mass spectrometry conditions were as follows: ion source: electron impact (EI); electron energy: 70 eV; ion source temperature: 230°C; analyzer: quadrupole mass analyzer, quadrupole temperature: 150°C; scan mode: full scan; mass scan range: 50-550 amu; solvent delay: 4.5 min; (3) Adding an internal standard solution to the sample obtained in step (1), and detecting the content of waxy compounds in the sample using GC-MS technology according to the chromatographic mass spectrometry conditions in (2).

[0008] Preferably, in step (1), the material-liquid ratio of tobacco leaf powder to ethanol is 1 g: (5-15) mL, more preferably 1 g: 10 mL.

[0009] Preferably, in step (1), the concentration of ethanol is 85-95% (v / v), more preferably 90% (v / v).

[0010] Preferably, in step (1), the heating temperature is 50-70°C, and the reflux extraction time is 1-5 hours, more preferably 60°C and 3 hours.

[0011] Preferably, in step (1), the organic solvent comprises one or more of petroleum ether (PE), dichloromethane (DCM) and ethyl acetate (EA). Further preferably, in step (1), silica gel powder (mass ratio 20:1) is added to the extract obtained by reflux extraction and mixed well, and then added to the upper layer of a pre-prepared thin layer chromatography column after rotary evaporation to dryness; petroleum ether, dichloromethane, and ethyl acetate (mass ratio of sample to elution solvent is 1:500) are used in sequence for step elution, and petroleum ether eluate, dichloromethane eluate, and ethyl acetate eluate are obtained in sequence as the sample to be tested.

[0012] Preferably, in step (2), the internal standard substances include 5 kinds, specifically phenylethyl acetate, n-heptadecane, n-pentacosane, 1-eicosyl alcohol and 1-octacosanol.

[0013] Preferably, in step (2) and step (3), the instrument used for GC-MS testing is: Agilent 7820A gas chromatograph-Agilent 5977B mass spectrometer.

[0014] Preferably, in step (2), the standard substances include 46 kinds of alkanes, alkanes, fatty alcohols, fatty acids, sterols and the like, specifically dodecane, tridecane, tetradecane, pentadecane, hexadecane, octadecane, nonadecane, eicosane, heneicosane, docosane, tricosane, tetracosane, hexacosane, heptacosane, octacosane, triacontan, dotriacontane, tricosane, pentatriacontane, hexatriacontane, octatriacontane, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-nonadecane, 1-dodecene, 1-dodecanol, 1-tridecyl alcohol, 1-tetradecyl alcohol, 1-pentadecanol, 1-hexadecanol, 1-heptadecanol, 1-octadecyl alcohol, 1-nonadecanol, 1-docosane, 1-tetracosyl alcohol, 1-hexacosyl alcohol, palmitic acid, stearic acid, linolenic acid, cholesterol, stigmasterol, sitosterol, phytol, and scopoletin. On the one hand, the components of the wax compounds in the tobacco leaves can be further determined by comparing the retention times; on the other hand, the content of the wax compounds in the tobacco leaves can be determined by the standard working curve.

[0015] The beneficial effects of the present invention are: 1. The present invention proposes a method for separating and determining the content of waxy compounds in tobacco leaves based on column chromatography and GC-MS. By pre-treating the tobacco leaves (column chromatography) and conducting GC-MS, and optimizing the chromatography-mass spectrometry conditions, more accurate detection and qualitative analysis were performed. A total of 48 waxy compounds were identified from tobacco leaf powder. Among them, 13 compounds, including cedarwood, phyton, menthol, Δ5-avenasterol, cholesterol, isosepharophenone, 1-heptadecanol, dehydrotoluol, 1-tetradecanol, hexadecane, 1-hexadecene, 1-tetradecene, and dodecane, were isolated and detected in tobacco leaves for the first time, further clarifying the types of chemical components in tobacco leaves and having great significance.

[0016] 2. The method of the present invention is the first to separate and detect previously undetected chemical components from tobacco leaves, providing an efficient and rapid analytical method for the qualitative analysis of chemical components in tobacco leaves and their quality control, and providing data support for further elucidation of the chemical basis of tobacco leaves and further resource development and utilization.

[0017] 3. The preparation process of the present invention is simple, convenient and easy to operate; the extraction solvent is non-toxic and harmless and can be recycled; the detection results are fast, accurate and highly reliable.

[0018] 4. The method established by the present invention conducts a systematic and comprehensive qualitative and quantitative analysis of the compounds in the tobacco wax components, separates and detects a variety of compounds, and has important research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a process flow chart for the extraction, separation and quantitative detection of waxy compounds in tobacco leaves according to Example 1 of the present invention.

[0020] Figure 2 This is a chromatogram of the tobacco wax compound PE component prepared in Example 1 of the present invention.

[0021] Figure 3 This is a chromatogram of the tobacco wax compound DCM component prepared in Example 1 of the present invention.

[0022] Figure 4 This is a chromatogram of the tobacco wax compound EA component prepared in Example 1 of the present invention.

[0023] Figure 5 The chromatogram of the mixed standard working solution prepared in Example 1 of the present invention is shown.

[0024] Figure 6 This is a chromatogram of tobacco wax compounds prepared in Example 2 of the present invention.

[0025] Figure 7 This is a chromatogram of tobacco wax compounds prepared in Example 3 of the present invention.

[0026] Figure 8 This is a chromatogram of tobacco wax compounds obtained in Example 4 of the present invention.

[0027] Figure 9 This is a chromatogram of tobacco wax compounds prepared in Example 5 of the present invention.

[0028] Figure 10 This is a chromatogram of tobacco wax compounds prepared in Example 6 of the present invention.

[0029] Figure 11 This is a chromatogram of tobacco wax compounds obtained in Example 7 of the present invention.

[0030] Figure 12 This is a chromatogram of tobacco wax compounds prepared in Example 8 of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below have the same meaning as those understood by professional and technical personnel in this field.

[0032] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources or prepared by known methods.

[0033] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples were repeated three times and the results were averaged.

[0034] Example 1: A method for separating and determining the content of waxy compounds in tobacco leaves based on column chromatography and GC-MS, comprising the following steps: (1) Tobacco leaf powder from southern Anhui flue-cured tobacco was mixed with 90% (v / v) ethanol at a solid-liquid ratio of 1 g:10 mL, heated at 60°C with stirring, and refluxed for 3 h. Filtration was performed to obtain filtrate I and residue I. Silica gel powder (mass ratio 20:1) was added to filtrate I and the mixture was rotary evaporated to dryness to obtain the sample. The sample was transferred to the upper layer of a pre-prepared thin-layer chromatography column and eluted in a stepwise manner using petroleum ether, dichloromethane, and ethyl acetate (the mass ratio of sample to elution solvent was 1:500), and petroleum ether eluent, dichloromethane eluent, and ethyl acetate eluent were obtained in sequence.

[0035] (2) Select internal standard and standard to prepare internal standard solution and standard solution of appropriate concentration, and use GC-MS to test according to the following chromatographic mass spectrometry conditions. The ratio of the peak area of ​​the target compound to the internal standard is the ordinate, and the ratio of the concentration of the target compound to the internal standard is the abscissa. Draw the standard working curve, and the linear correlation coefficient R 2 Greater than 0.995.

[0036] The internal standards are phenylethyl acetate, n-heptadecane, n-pentacosane, 1-eicosanol, and 1-octacosanol, a total of 5 types, numbered ①, ②, ③, ④, and ⑤ respectively.

[0037] The standards were dodecane, tridecane, tetradecane, pentadecane, hexadecane, octadecane, nonadecane, eicosane, heneicosane, docosane, tricosane, tetracosane, hexacosane, heptacosane, octacosane, triacontan, dotriacontane, tricosane, pentatriacontane, hexatriacontane, octatriacontane, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-nonadecane, 1-dodecyl alcohol, 1-tridecyl alcohol, 1-tetradecyl alcohol, 1-pentadecanol, 1-hexadecanol, 1-heptadecanol, 1-octadecanol, 1-nonadecanol, 1-docosyl alcohol, 1-tetracosyl alcohol, 1-hexacosyl alcohol, palmitic acid, stearic acid, linolenic acid, cholesterol, stigmasterol, sitosterol, phytol, and scopoletin, for a total of 46 types. The corresponding standards for different internal standards are shown in Table 2.

[0038] The specific chromatographic mass spectrometry conditions are as follows: Chromatographic conditions were as follows: chromatographic column: HP-5ms (190915-433UI, 30 m × 0.25 mm × 0.25 μm); carrier gas: He, 99.99%; flow rate: 10 mL / min; injection volume: 1 μL; injection mode: split injection, split ratio 10:1; injection port temperature: 250°C; heating program: 50°C for 2 min; heating to 120°C at a rate of 2.5°C / min and hold for 2 min, heating to 200°C at a rate of 1°C / min and hold for 2 min, heating to 250°C at a rate of 1°C / min and hold for 2 min, heating to 300°C at a rate of 2.5°C / min and hold for 2 min; Mass spectrometry conditions were as follows: ion source: electron impact (EI); electron energy: 70 eV; ion source temperature: 230°C; analyzer: quadrupole mass analyzer, quadrupole temperature: 150°C; scan mode: full scan; mass scan range: 50–550 amu; solvent delay: 4.5 min. The gas chromatography-mass spectrometry instrument used was an Agilent 7820A gas chromatograph coupled to an Agilent 5977B mass spectrometer. (3) The petroleum ether eluate, dichloromethane eluate, and ethyl acetate eluate obtained in step (1) are sequentially used as test samples, and an appropriate amount of internal standard solution is added to the test sample. The content of the waxy compounds in the test sample is detected by GC-MS under the same chromatographic mass spectrometry conditions as in step (2). It should be noted that since the standard products of some tobacco waxy compounds are not available for purchase, a semi-quantitative method is used for quantification in the subsequent actual sample detection. In step (3), 46 standard products are selected as external standards. On the one hand, the components of the waxy compounds in the tobacco leaves can be further determined by comparing the retention times; on the other hand, the content of the waxy compounds in the tobacco leaves can be determined by the standard working curve.

[0039] This example uses GC-MS to quantitatively determine the content of each component of waxy compounds in tobacco leaves. Figures 2-4and Table 1; a summary of the mixed standard working solution chromatogram and standard working curve, as shown in Figure 5 As shown in Table 2. The test results showed that when tobacco wax components were step-wise eluted using solvents of varying polarity, a total of 48 compounds were identified. The PE fraction (Fraction I) primarily contained a large number of alkanes and alkanes. The DCM fraction (Fraction II) and the EA fraction (Fraction III) primarily contained alkanols, acids, sterols, and tobacco-characteristic compounds. Thirteen compounds, including cedarwood, phytonadione, menthol, Δ5-avenasterol, cholesterol, isosetylcyclohexanol, 1-heptadecanol, dehydrotoluol, 1-tetradecanol, hexadecane, 1-hexadecene, 1-tetradecene, and dodecane, were isolated and detected in tobacco leaves for the first time using this method.

[0040] The total content of tobacco leaf wax components was 14,514.28 μg / g. Among these components, alkanes were 494.77 μg / g, alkenes were 246.86 μg / g, alkanols were 336.91 μg / g, acids were 160.38 μg / g, aldehydes and ketones were 647.20 μg / g, and sterols were 12,624.98 μg / g. Among these, compounds with higher concentrations included stigmasterol (10,856.64 μg / g), campesterol (838.37 μg / g), cholesterol (321.64 μg / g), cephanetriadiol (181.31 μg / g), 3-methylhexadecane (148.31 μg / g), palmitic acid (145.80 μg / g), and 1-hexacosanol (131.27 μg / g). The detection method is simple, the results are accurate, the reproducibility is good, and the coverage is comprehensive.

[0041] Table 1: Summary of the total content of tobacco wax compounds obtained in this example

[0042]

[0043] Table 2: Summary of the prepared standard working curves

[0044]

[0045]

[0046]

[0047] Example 2: The difference between this embodiment and embodiment 1 is that the stepwise elution with petroleum ether, dichloromethane and ethyl acetate in step (1) is replaced by elution with petroleum ether only, and the rest is the same as in embodiment 1.

[0048] The results showed that only 16 compounds were identified, with a total content of 518.89 μg / g, mainly consisting of a large amount of alkanes and a small amount of alkanes and alkanols. Compared with Example 1, the number and content of waxy compounds were significantly reduced, and the detection results were not as ideal as those in Example 1.

[0049] Example 3: The difference between this embodiment and embodiment 1 is that the stepwise elution with petroleum ether, dichloromethane and ethyl acetate in step (1) is replaced by elution with dichloromethane only, and the rest is the same as in embodiment 1.

[0050] The results showed that only 29 compounds were identified, with a total content of 4742.59 μg / g. Compared with Example 1, the number and content of waxy compounds were greatly reduced, and the detection results were not as ideal as those in Example 1.

[0051] Example 4: The difference between this embodiment and embodiment 1 is that the stepwise elution with petroleum ether, dichloromethane and ethyl acetate in step (1) is replaced by elution with ethyl acetate only, and the rest is the same as in embodiment 1.

[0052] The results showed that only 35 compounds were identified, with a total content of 10873.83 μg / g. Compared with Example 1, the number and content of waxy compounds were greatly reduced, and the detection results were not as ideal as those in Example 1.

[0053] Example 5: The difference between this example and Example 1 is that the stepwise elution using petroleum ether (PE), dichloromethane (DCM), and ethyl acetate (EA) in step (3) is replaced by elution using a mixed solvent of petroleum ether (PE): dichloromethane (DCM): ethyl acetate (EA) = 1:1:1. The rest is the same as in Example 1.

[0054] The results showed that only 26 compounds were identified, with a total content of 925.31 μg / g. Compared with Example 1, the number and content of waxy compounds were greatly reduced, and the detection results were not as ideal as those in Example 1.

[0055] Example 6: The difference between this embodiment and embodiment 1 is that the stepwise elution using petroleum ether (PE), dichloromethane (DCM), and ethyl acetate (EA) in step (3) is replaced by elution using a mixed solvent of petroleum ether (PE): dichloromethane (DCM): ethyl acetate (EA) = 3:1:1. The rest is the same as in embodiment 1.

[0056] The results showed that only 19 compounds were identified, with a total content of 726.85 μg / g. Compared with Example 1, the number and content of waxy compounds were greatly reduced, and the detection results were not as ideal as those in Example 1.

[0057] Example 7: The difference between this embodiment and embodiment 1 is that the stepwise elution using petroleum ether (PE), dichloromethane (DCM), and ethyl acetate (EA) in step (3) is replaced by elution using a mixed solvent of petroleum ether (PE): dichloromethane (DCM): ethyl acetate (EA) = 1:3:1. The rest is the same as in embodiment 1.

[0058] The results showed that only 31 compounds were identified, with a total content of 3622.92 μg / g. Compared with Example 1, the number and content of waxy compounds were significantly reduced, and the detection results were not as ideal as those in Example 1.

[0059] Example 8: The difference between this example and Example 1 is that the stepwise elution using petroleum ether (PE), dichloromethane (DCM), and ethyl acetate (EA) in step (3) is replaced by elution using a mixed solvent of petroleum ether (PE): dichloromethane (DCM): ethyl acetate (EA) = 1:1:3. The rest is the same as in Example 1.

[0060] The results showed that only 30 compounds were identified, with a total content of 965.81 μg / g. Compared with Example 1, the number and content of waxy compounds were greatly reduced, and the detection results were not as ideal as those in Example 1.

[0061] Example 9: The difference between this embodiment and embodiment 1 is that in step (1), the material-liquid ratio of tobacco leaf powder to ethanol is 1 g:5 mL, the ethanol concentration is 95% (v / v), the heating temperature is 70°C, and the reflux extraction time is 1 h. The rest is the same as in embodiment 1.

[0062] The results of this example are similar to those of Example 1.

[0063] Example 10: The difference between this embodiment and embodiment 1 is that in step (1), the material-liquid ratio of tobacco leaf powder to ethanol is 1 g:15 mL, the ethanol concentration is 85% (v / v), the heating temperature is 50°C, and the reflux extraction time is 5 h. The rest is the same as in embodiment 1.

[0064] The results of this example are similar to those of Example 1.

[0065] Comparative Example 1: The difference between this comparative example and Example 1 is that the stepwise elution with petroleum ether, dichloromethane and ethyl acetate in step (1) is replaced by elution with acetonitrile only, and the rest is the same as Example 1.

[0066] Results: Only 12 waxy compounds were detected, and the content of each waxy compound was low. Compared with the amount of waxy compounds in Example 1, the detection results were far less ideal than those in Example 1.

[0067] Comparative Example 2: The difference between this comparative example and Example 1 is that the stepwise elution with petroleum ether, dichloromethane and ethyl acetate in step (1) is replaced by elution with methanol only, and the rest is the same as Example 1.

[0068] Results: Only 10 waxy compounds were detected, and the content of each waxy compound was low. Compared with the amount of waxy compounds in Example 1, the detection results were far less ideal than those in Example 1.

[0069] Comparative Example 3: The difference between this comparative example and Example 1 is that in step (1), the heating temperature is 85°C, and the rest is the same as Example 1.

[0070] Results: Only 8 waxy compounds were detected, and the content of each waxy compound was low. Compared with the amount of waxy compounds in Example 1, the detection results were far less ideal than those in Example 1.

[0071] Comparative Example 4: The difference between this comparative example and Example 1 is that in step (1), the heating temperature is 40°C, and the rest is the same as Example 1.

[0072] Results: Only 13 waxy compounds were detected, and the content of each waxy compound was low. Compared with the amount of waxy compounds in Example 1, the detection results were far less ideal than those in Example 1.

[0073] Comparative Example 5: The difference between this comparative example and Example 1 is that the model of the chromatographic column is changed to DB-WAX (123-7033, 30 m×0.32 mm×0.50 μm), and the rest is the same as Example 1.

[0074] Results: The number of chromatographic peaks was extremely limited, making it impossible to accurately detect waxy compounds in tobacco leaves.

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for separating and determining the content of waxy compounds in tobacco leaves based on column chromatography and GC-MS, characterized in that: The following steps are involved: (1) Mix tobacco leaf powder with ethanol, heat and stir, reflux extraction, filter, and retain the filtrate; add silica gel to the filtrate and evaporate it to dryness, transfer it to a silica gel column, and elute it with an organic solvent to prepare a sample to be tested; (2) Select internal standard and standard to prepare internal standard solution and standard solution respectively, and use GC-MS technology to test; use the ratio of target compound to internal standard peak area as the vertical axis and the ratio of target compound to internal standard concentration as the horizontal axis to draw the standard working curve, and the linear correlation coefficient R 2 Greater than 0.995; (3) Adding an internal standard solution to the sample obtained in step (1) and detecting the content of waxy compounds in the sample using GC-MS technology.

2. The method according to claim 1, characterized in that In step (1), the material-liquid ratio of tobacco leaf powder to ethanol is 1 g: 5-15 mL.

3. The method according to claim 1, characterized in that In step (1), the volume concentration of ethanol is 85-95%.

4. The method according to claim 1, wherein In step (1), the heating temperature is 50-70° C., and the reflux extraction time is 1-5 h.

5. The method according to claim 1, wherein In step (1), the organic solvent includes one or more of petroleum ether, dichloromethane and ethyl acetate.

6. The method according to claim 7, characterized in that In step (1), petroleum ether, dichloromethane and ethyl acetate are used in sequence for step elution, and petroleum ether eluate, dichloromethane eluate and ethyl acetate eluate are obtained in sequence as samples to be tested.

7. The method according to claim 1, characterized in that In step (2), the internal standard substances include phenylethyl acetate, n-heptadecane, n-pentacosane, 1-eicosyl alcohol and 1-octacosanol.

8. The method according to claim 1, characterized in that In step (2) and step (3), the instrument used for GC-MS testing is: Agilent 7820A gas chromatograph-Agilent 5977B mass spectrometer.

9. The method according to claim 1, characterized in that The specific chromatographic mass spectrometry conditions in step (2) and step (3) are as follows: Chromatographic conditions were as follows: chromatographic column: HP-5ms (190915-433UI, 30 m × 0.25 mm × 0.25 μm); carrier gas: 99.99% pure He; flow rate: 10 mL / min; injection volume: 1 μL; injection mode: split injection, split ratio 10:1; injection port temperature: 250°C; heating program: 50°C for 2 min, then increase the temperature to 120°C at a rate of 2.5°C / min and hold for 2 min, then increase the temperature to 200°C at a rate of 1°C / min and hold for 2 min, then increase the temperature to 250°C at a rate of 1°C / min and hold for 2 min, then increase the temperature to 300°C at a rate of 2.5°C / min and hold for 2 min. The mass spectrometry conditions were as follows: ion source: electron impact EI; electron energy: 70 eV; ion source temperature: 230°C; analyzer: quadrupole mass analyzer, quadrupole temperature 150°C; scan mode: full scan; mass scan range: 50-550 amu; solvent delay: 4.5 min.

10. The method according to claim 1, wherein: In step (3), 46 standards are selected as external standards; on the one hand, the components of the wax compounds in the tobacco leaves can be further determined by comparing the retention times; on the other hand, the content of the wax compounds in the tobacco leaves can be determined by the standard working curve.

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