Method for separating leaf wax ester compound family components

Abstract

The invention provides a leaf wax ester compound group component separation method, and belongs to the technical field of organic geochemistry. The first-step column chromatography is carried out before saponification to separate out the alkane compounds, so that the loss of the alkane compounds, especially short-chain alkanes, in the saponification heating process in a traditional method is avoided. Besides, neutral components such as alkanes, alcohols and the like in a traditional method are usually extracted in the first-step extraction process, and acidic compounds are easily brought out, so that the acidic compounds are mixed in the alcohol compounds obtained after column chromatography. However, according to the method, the alcohol and acidic components are jointly extracted after saponification, and pure alcohol and acidic compounds are obtained through subsequent column chromatography separation. According to the method provided by the invention, the loss of the n-alkanes in the saponification heating process is effectively avoided, and the recovery rate of the alkane compounds is remarkably improved. Meanwhile, the alcohols and acidic compounds separated by the method are high in purity and few in impurities.

Description

Technical Field

[0001] The present invention relates to the technical field of organic geochemistry, and particularly relates to a method for separating the group components of leaf wax lipid compounds. Background Art

[0002] Leaf wax is a layer of wax covering the surface of the plant cuticle, and its main components are various lipid compounds, including alkanes, fatty alcohols, fatty acids, aldehydes, wax esters, etc. The formation of the wax on the plant epidermis and the changes in its components are usually the results of the plant's adaptation to the external environment, including responses to extreme environmental conditions such as abnormal temperature, drought, high salinity, etc. Leaf wax lipid compounds have the characteristics of stable structure and wide distribution in nature. Therefore, leaf wax lipid compounds are regarded as important biomarkers indicating climate and environmental changes, and are widely used in the differentiation of source information, paleoclimate reconstruction, and paleoenvironment research.

[0003] Among them, the n-alkanes in the alkane compounds are usually used to analyze and characterize the source of organic matter by using the distribution characteristics of n-alkanes with different carbon chain lengths in sediments, so as to reveal the changing trends of climate and environment due to the specificity and representativeness of their sources; the compositional changes of long-chain alkenones in ketone compounds can be used to indicate the changes in the surface water temperature; the main peak carbon and distribution characteristics of fatty acid compounds are of great significance in indicating the source of organic matter. In addition, the ratio of unsaturated fatty acids to saturated fatty acids can also reflect the relative change of temperature; the differences in the molecular structures of sterols in fatty alcohol compounds reflect different biological sources and can be used to indicate the input of organic matter related to marine and terrestrial organisms. Therefore, scientifically and effectively separating various lipid biomarkers in plant or sediment leaf wax is of great significance for more accurately differentiating source information and reconstructing the paleoenvironment.

[0004] Currently, in the field of organic geochemistry, the lipid compounds in leaf wax are usually separated by the methods of saponification, extraction, and column chromatography separation (as shown in Figure 1 ). However, the existing methods have certain limitations in the actual operation process: (1) Loss of short-chain alkanes: During the saponification heating process, short-chain alkane compounds are easily lost due to their high volatility, resulting in deviations in their subsequent quantitative analysis results, thereby affecting the accuracy of the experiment; (2) Component cross-interference: During the extraction process, fatty acid compounds are easily mixed with other components and enter the target component, so that the subsequent separated fatty alcohol component contains fatty acid residues, resulting in a decrease in the purity of the compound; (3) Insufficient separation efficiency: In traditional silica gel column chromatography, the polarity differences of different components are relatively large, which may lead to incomplete separation of component elution and it is difficult to obtain high-purity target compounds. Therefore, traditional separation methods are difficult to obtain relatively pure group components when separating multiple types of biomarker compounds such as alkanes, fatty acids, ketones, and fatty alcohols, and the loss of alkane compounds is relatively large. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a method for separating the components of the cutin wax lipid compound family, so as to obtain high-purity lipid compounds of each family, and significantly reduce the loss of alkane components, thereby improving the accuracy and efficiency of separation.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for separating the components of the cutin wax lipid compound family, comprising the following steps:

[0008] Step 1. After the sample is pretreated, the total lipid is extracted with an organic solvent to obtain a total lipid (TLES) extract;

[0009] Step 2. The total lipid extract is separated by chromatography silica gel, and different organic solvents are used for elution to obtain an alkane (Hdy) component and a polar component 1 respectively;

[0010] Step 3. The polar component 1 is saponified to obtain a saponification reaction solution;

[0011] Step 4. Saturated sodium chloride solution is added to the saponification reaction solution, and then acidified to pH < 3;

[0012] Step 5. n-Hexane is added to the sample acidified in Step 4 for liquid-liquid extraction, and the supernatant is collected;

[0013] Step 6. The supernatant is separated by aminopropyl silica gel, and different organic solvents are used for elution to obtain a neutral (Ne) component, an acidic (Fa) component and a polar component 2 respectively;

[0014] Step 7. The neutral component is separated by chromatography silica gel, and different organic solvents are used for elution to obtain ketoaldehyde compounds, alcohol compounds and a polar component 3 respectively.

[0015] In some embodiments of the present invention, the sample is a plant leaf or a plant leaf deposit; the pretreatment is: the sample is dried, ground and sieved in sequence.

[0016] The present invention does not make special limitations on the conditions of drying, grinding and sieving, and conventional technical means of those skilled in the art can be selected, for example: freeze-drying, grinding, and sieving through a 100-mesh sieve.

[0017] In some embodiments of the present invention, the method for extracting the total lipid with an organic solvent is an extraction method or a Soxhlet extraction method, and the organic solvents used are dichloromethane and methanol.

[0018] The present invention does not make special requirements on the specific parameters and steps of the extraction method or the Soxhlet extraction method, and they are all conventional technical means of those skilled in the art.

[0019] In some embodiments of the present invention, in step 2, when the organic solvent selected is n-hexane, an alkane component is eluted; when the organic solvent selected is a mixed solvent of dichloromethane and methanol with a volume ratio of 1:3, a polar component 1 is eluted.

[0020] In the present invention, after the eluted alkane component is concentrated, it is transferred to a sample bottle and can be directly subjected to gas chromatography analysis.

[0021] Based on the polarity difference, the present invention first uses n-hexane for elution to elute alkane compounds, and then uses a mixed solvent of dichloromethane and methanol with a volume ratio of 1:3 for elution to elute polar components.

[0022] In some embodiments of the present invention, the steps of saponification treatment are as follows: add a KOH solution and water to the polar component 1, and heat and saponify at 80 °C for 2 hours.

[0023] In some embodiments of the present invention, the concentration of the KOH solution is 1 mol / L.

[0024] The present invention does not make special limitations on the volume ratio of the polar component 1, the KOH solution and water, and the dosage can enable the polar component 1 to fully carry out the saponification reaction.

[0025] In some embodiments of the present invention, the acid solution used for acidification to pH < 3 is a 4 mol / L HCl solution.

[0026] The present invention does not make special limitations on the dosage of the saturated sodium chloride solution, and the dosage commonly used in the art that can promote the separation of the organic phase and the aqueous phase, reduce the emulsification phenomenon, and promote the extraction of fatty acids can be selected.

[0027] The present invention does not make special limitations on the number of times of liquid-liquid extraction in step 5, and multiple liquid-liquid extractions can be carried out until the last two n-hexane extraction solutions are colorless.

[0028] In some embodiments of the present invention, in step 6, when the organic solvent selected is a mixed solvent of dichloromethane and isopropanol with a volume ratio of 3:1, a neutral component is eluted; when the organic solvent selected is a 4% v / v acetic acid ether solution, an acidic component is eluted; when the organic solvent selected is methanol, a polar component 2 is eluted.

[0029] In the present invention, by taking advantage of the different polarities of the eluents, a mixed solvent of dichloromethane and isopropanol with a volume ratio of 3:1 is first used to elute the neutral components, then an acetic acid ethyl ether solution with a concentration of 4% v / v is used to elute the acidic components, and finally methanol is used to elute the polar components. Based on the principle that the polarity of the compounds gradually increases, from non-polar to polar, different polar compounds are eluted step by step, and such an order can ensure the effective separation of different polar compounds.

[0030] In the present invention, after eluting the acidic components, the acidic components are concentrated to near dryness, and then a hydrochloric acid solution of dichloromethane and methanol is added for derivatization. After derivatization, n-hexane is added to extract the supernatant, and the extraction step of n-hexane is repeated until the last two n-hexane rinsing solutions are colorless. The supernatant is collected in a new sample bottle for gas chromatography-mass spectrometry analysis.

[0031] In some embodiments of the present invention, in the hydrochloric acid solution of methanol, the volume ratio of hydrochloric acid to methanol is 5:95; the volume ratio of the hydrochloric acid solution of dichloromethane and methanol is 1:1; the reaction temperature of the derivatization is 80 °C, and the reaction time is 2 h.

[0032] The present invention does not make special limitations on the dosages of dichloromethane and the hydrochloric acid solution of methanol. The dosages commonly used by those skilled in the art that can enable the derivatization of the acidic components can be selected, for example: 100 μl.

[0033] In some embodiments of the present invention, in step 7, when the organic solvent selected is a mixed solvent of n-hexane and dichloromethane with a volume ratio of 1:1, ketoaldehyde compounds are eluted; when the organic solvent selected is a mixed solvent of n-hexane and ethyl acetate with a volume ratio of 4:1, alcohol compounds are eluted; when the organic solvent is methanol, polar component 3 is eluted.

[0034] In the present invention, by taking advantage of the different polarities of the eluents, a mixed solvent of n-hexane and dichloromethane with a volume ratio of 1:1 is first used to elute the ketoaldehyde compounds, then a mixed solvent of n-hexane and ethyl acetate with a volume ratio of 4:1 is used to elute the alcohol compounds, and finally methanol is used to elute the polar component 3. Based on the principle that the polarity of the compounds gradually increases, from non-polar to polar, different polar compounds are eluted step by step, and such an order can ensure the effective separation of different polar compounds.

[0035] After the elution is completed, in the present invention, the ketoaldehyde compounds are concentrated and transferred to a sample bottle for direct gas chromatography-mass spectrometry analysis; after the alcohol compounds are concentrated to near dryness, 100 μl of dichloromethane and 100 μl of bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization reagent are added, and derivatization is carried out at a temperature of 70 °C for 1 hour. After the derivatization is completed, the volume is fixed for gas chromatography-mass spectrometry analysis.

[0036] The present invention does not make any special limitation on the above concentration method, and conventional technical means of those skilled in the art can be selected, for example: rotary evaporator or nitrogen purging.

[0037] In the above elution process of the present invention, the amount of the eluent is not specially limited, and the amount of the eluent commonly used for compound elution in the art can be selected.

[0038] The methods and parameters for the above gas chromatography - mass spectrometry analysis in the present invention are all conventional technical means in the art and are not the technical solutions protected by the present invention.

[0039] The present invention discloses the following technical effects:

[0040] The method provided by the present invention effectively avoids the loss of n - alkanes during the saponification heating process, and significantly improves the recovery rate of alkane compounds. At the same time, the alcohols and acidic compounds separated by this method have relatively high purity and few impurities. Through the optimization of the existing technical solutions, the present invention can scientifically and efficiently separate alcohols and acidic compounds, and ensure the stability and integrity of alkane compounds during the separation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0042] Figure 1 Schematic flow chart of separating the leaf wax lipid compound family components by the traditional method of Comparative Example 1;

[0043] Figure 2 Schematic flow chart of separating the leaf wax lipid compound family components of the present invention;

[0044] Figure 3 Distribution diagram of n - alkane compounds obtained in Example 1 and Comparative Example 1, where A is Example 1 and B is Comparative Example 1;

[0045] Figure 4 Total ion chromatogram of acidic compounds obtained in Example 1 and Comparative Example 1, where A is Example 1 and B is Comparative Example 1;

[0046] Figure 5 Total ion chromatogram of alcohol compounds obtained in Example 1 and Comparative Example 1, where A is Example 1 and B is Comparative Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] The various exemplary embodiments of the present invention will be described in detail below. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation manners of the present invention.

[0048] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0050] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of the present invention are merely exemplary.

[0051] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0052] There are problems in the prior art such as short-chain alkane loss, component cross-interference, and insufficient separation efficiency in the "saponification-column chromatography" process. In particular, the loss of short-chain alkanes and component cross-interference limit the accurate separation and quantitative analysis of leaf wax lipid compounds. To overcome these deficiencies, the present invention proposes an innovative "three-step" separation method. By adjusting the process sequence (separating saturated hydrocarbons first and then performing saponification treatment) and combining the characteristics of two fillers, aminopropyl silica gel and silica gel, the recovery rate of saturated hydrocarbons and the separation efficiency of fatty acids and alcohols are significantly improved, providing a more accurate and reliable sample basis for subsequent carbon and hydrogen isotope analysis and meeting the requirements for high-precision isotope data in research such as ecological environment and paleoclimate reconstruction.

[0053] The three-step column chromatography separation method of the present invention is as follows: the first column chromatography is carried out before saponification to separate alkane compounds, thus avoiding the loss of alkane compounds, especially short-chain alkanes, during the saponification heating process in the traditional method. In addition, in the traditional method, neutral components such as alkanes and alcohols are usually extracted in the first extraction process, and acidic compounds are easily carried out, resulting in acidic compounds being mixed in the alcohol compounds obtained after column chromatography. In the present invention, the combined extraction of alcohols and acidic components is carried out after saponification, and pure alcohols and acidic compounds are obtained through subsequent column chromatography separation.

[0054] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been made public.

[0055] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments, but the content of the present invention is not limited to the following embodiments.

[0056] The chromatography silica gel used in the examples was pre-baked at a temperature of 150 °C for 4 h to remove moisture.

[0057] Example 1

[0058] Referring to Figure 2 , the separation method of various lipid compounds in leaf wax provided by the present invention is as follows:

[0059] (1) Sample treatment: The mangrove sediment sample was freeze-dried and ground into a powder, and passed through a 100-mesh sieve.

[0060] (2) Total lipid extraction: Weigh 10 g of the mangrove sediment sample ground in step (1), and extract it with dichloromethane / methanol (V 二氯甲烷 :V 甲醇 = 93:7), and concentrate by rotary evaporation to obtain the total lipid (TLES) extract.

[0061] (3) First column chromatography separation: Select a chromatography column with a specification of 10×240 mm, weigh 4 g of chromatography silica gel with a particle size of 80-100 mesh and add it to the chromatography column. Add the total lipid extract obtained in step (2) to the chromatography column, and then first use 20 ml of n-hexane to elute to obtain the alkane (Hdy) component, and then use 20 ml of dichloromethane / methanol (V 二氯甲烷 :V 甲醇 = 1:3) mixed solvent to elute to obtain polar component 1. The eluted alkane component was concentrated, transferred to a sample bottle, and can be directly subjected to gas chromatography analysis.

[0062] (4) Saponification treatment of polar component 1: Add 3 ml of 1 mol / L KOH solution and 2 ml of deionized water to the polar component 1 obtained in step (3), heat and saponify at 80 °C for 2 hours. After saponification, add 1 ml of saturated NaCl solution and 2 ml of 4 mol / L HCl solution to acidify the sample to pH = 2.

[0063] (5) Liquid-liquid extraction: Add 2 ml of n-hexane to the acidified sample in the above step (4), mix and extract the supernatant for liquid-liquid extraction. Repeat the steps of liquid-liquid extraction until the last two n-hexane extracts are colorless, and collect the supernatant in a pear-shaped flask.

[0064] (6) Second column chromatography separation: Pour the n-hexane extract collected in step (5) into an aminopropyl solid-phase extraction column. Then, first use 10 mL of dichloromethane / isopropanol (V 二氯甲烷 :V 异丙醇 = 3:1) mixed solvent for elution to obtain the neutral (Ne) component. Then, use 8 mL of 4% v / v acetic acid ethyl ether solution for elution to obtain the acidic (Fa) component. Finally, use 6 mL of methanol for elution to obtain polar component 2.

[0065] (7) Derivatization of acidic component: Concentrate the acidic component obtained in step (6) to nearly dryness, add 100 μl of dichloromethane and 100 μl of hydrochloric acid / methanol (V 盐酸 :V 甲醇 = 5:95) mixed solution, and derivatize at 80 °C for 2 hours. After derivatization, add n-hexane to extract the supernatant. Repeat the steps of n-hexane extraction until the last two n-hexane rinses are colorless, and collect the supernatant in a new sample bottle for gas chromatography-mass spectrometry analysis.

[0066] (8) Third column chromatography separation: Use a chromatography column with a specification of 6×150 mm, weigh 1 g of chromatography silica gel and add it to the chromatography column. Pour the neutral component obtained in step (6) into the chromatography column. Then, first use 8 ml of n-hexane / dichloromethane (V 正己烷 :V 二氯甲烷 = 1:1) mixed solvent for elution to obtain ketoaldehyde compounds (Ald). Then, use 10 ml of n-hexane / ethyl acetate (V 正己烷 :V 乙酸乙酯 = 4:1) mixed solvent for elution to obtain alcohol compounds (Alc). Finally, use 4 ml of methanol for elution to obtain the remaining polar component 3.

[0067] (9) Gas chromatography - mass spectrometry analysis: Concentrate the keto - aldehyde compounds obtained in step (8) and transfer them to a sample vial for direct use in gas chromatography - mass spectrometry analysis; after concentrating the alcohol compounds to nearly dryness, add 100 μl of dichloromethane and 100 μl of BSTFA derivatization reagent, and derivatize at 70 °C for 1 hour. After the derivatization is completed, make up the volume and wait for gas chromatography - mass spectrometry analysis.

[0068] Comparative Example 1

[0069] Refer to Figure 1 , a method for separating various lipid compounds in leaf wax, the steps are as follows:

[0070] (1) The same as step 1 of Example 1.

[0071] (2) The same as step 2 of Example 1.

[0072] (3) Saponification reaction: Add 3 ml of 1 mol / L KOH solution and 2 ml of deionized water to the total lipid (TLES) extract obtained in step (2), and heat and saponify at 80 °C for 2 hours.

[0073] (4) Liquid - liquid extraction: After the saponification is completed, wait for the sample temperature to return to room temperature, then add 2 ml of n - hexane to the saponified sample, mix and extract the supernatant for liquid - liquid extraction. Repeat the liquid - liquid extraction steps until the last two n - hexane extracts are colorless, collect the supernatant in a pear - shaped flask to obtain the n - hexane extract for the next column chromatography separation. After obtaining the n - hexane extract, add 1 ml of saturated NaCl solution and 2 ml of 4 mol / L HCl solution to the original saponified sample to acidify the sample to pH = 2. Add 2 ml of n - hexane to the acidified sample, mix and extract the supernatant for liquid - liquid extraction, and collect the supernatant in a pear - shaped flask to obtain the acidic component.

[0074] (5) Column chromatography separation: Use a chromatography column with a specification of 6×150 mm, weigh 1 g of chromatography silica gel and add it to the chromatography column. Pour the n - hexane extract obtained in step (4) into the chromatography column. First, use 10 ml of n - hexane to elute to obtain the alkane (Hdy) component, then use 8 ml of n - hexane / dichloromethane (V 正己烷 :V 二氯甲烷 = 1:1) mixed solvent to elute to obtain the keto - aldehyde compounds (Ald), then use 10 ml of n - hexane / ethyl acetate (V 正己烷 :V 乙酸乙酯 = 4:1) mixed solvent to elute to obtain the alcohol compounds (Alc), and finally use 4 ml of methanol to elute the remaining polar components.

[0075] (6) Gas chromatography - mass spectrometry analysis: The alkane (Hdy) component and keto - aldehyde compounds obtained in step (5) are concentrated and transferred to sample bottles respectively, and directly used for gas chromatography - mass spectrometry analysis; after the alcohol compounds are concentrated to nearly dry, 100 μl of dichloromethane and 100 μl of BSTFA derivatization reagent are added, and derivatization is carried out at 70 °C for 1 hour. After the derivatization is completed, the volume is fixed and waiting for gas chromatography - mass spectrometry analysis. The derivatization treatment of the acidic component is the same as that in step (7) of Example 1.

[0076] To verify the scientificity and effectiveness of the method of the present invention, the leaf wax lipid compounds in the surface sediment samples of mangroves were separated by using the method of the present invention (Example 1) and the traditional method (Comparative Example 1) respectively, and the contents and compositions of the n - alkane compounds, alcohols and acidic compounds separated by the two methods were compared.

[0077] For the n - alkane compounds, the Σn - C 23-35 content separated by the method of the present invention is 7.14 μg / g, while that by the traditional method is only 1.76 μg / g. From the Figure 3 distribution of the n - alkane composition, the n - alkane content separated by using the method of the present invention is significantly higher than that of the traditional method. This is mainly because the traditional method causes the loss of alkane compounds during saponification heating.

[0078] Secondly, for acidic compounds and alcohol compounds, in general experiments, acidic compounds and alcohol compounds are difficult to separate due to their large polarity, and the separated alcohol compounds often also carry acidic compounds. Through gas chromatography - mass spectrometry analysis ( Figure 4 、 Figure 5 ), it can be seen that the acidic and alcohol compounds separated by the method of the present invention are relatively pure and have less impurities; while the acidic components separated by the traditional method contain more other interfering substances, and the alcohol compounds often also carry acidic components.

[0079] The above - mentioned is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for separating the components of a family of leaf wax lipid compounds, characterized in that, It includes the following steps: Step 1. After pre-treating the sample, extract the total lipids using an organic solvent to obtain a total lipid extract; Step 2. Separate the total lipid extract using chromatography silica gel, and by eluting with different organic solvents, obtain an alkane fraction and a polar fraction 1 respectively; Step 3. Saponify the polar fraction 1 to obtain a saponification reaction solution; Step 4. Add saturated sodium chloride solution to the saponification reaction solution, and then acidify it to pH < 3; Step 5. Add n-hexane to the sample acidified in Step 4 for liquid-liquid extraction, and collect the supernatant; Step 6. Separate the supernatant using aminopropyl silica gel, and by eluting with different organic solvents, obtain a neutral fraction, an acidic fraction and a polar fraction 2 respectively; Step 7. Separate the neutral fraction using chromatography silica gel, and by eluting with different organic solvents, obtain keto-aldehyde compounds, alcohol compounds and a polar fraction 3 respectively.

2. The method for separating the cutin wax compound group components according to claim 1, characterized in that, The sample is plant leaves or plant leaf deposits; the pre-treatment is: sequentially dry, grind and sieve the sample.

3. The method for separating the leaf wax lipid compound family components according to claim 1, wherein, In Step 2, when the selected organic solvent is n-hexane, the alkane fraction is eluted, and when the selected organic solvent is a mixed solvent of dichloromethane and methanol with a volume ratio of 1:3, the polar fraction 1 is eluted.

4. The method for separating the leaf wax lipid compound group components according to claim 1, characterized in that, The steps of the saponification treatment are: add KOH solution and water to the polar fraction 1, and heat and saponify at 80 °C for 2 hours.

5. The method for separating the leaf wax lipid compound group components according to claim 1, characterized in that, In Step 6, when the selected organic solvent is a mixed solvent of dichloromethane and isopropanol with a volume ratio of 3:1, the neutral fraction is eluted, when the selected organic solvent is a 4% v / v acetic acid ethyl ether solution, the acidic fraction is eluted, and when the selected organic solvent is methanol, the polar fraction 2 is eluted.

6. The method for separating the leaf wax lipid compound family components according to claim 1, characterized in that, In Step 7, when the selected organic solvent is a mixed solvent of n-hexane and dichloromethane with a volume ratio of 1:1, the keto-aldehyde compounds are eluted, when the selected organic solvent is a mixed solvent of n-hexane and ethyl acetate with a volume ratio of 4:1, the alcohol compounds are eluted, and when the selected organic solvent is methanol, the polar fraction 3 is eluted.

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