Method for determining plant leaf BVOCs by headspace solid-phase microextraction method

Abstract

The invention belongs to the technical field of analysis and detection methods for volatile organic compounds released by plant leaves, and discloses a method for determining BVOCs (Blue Volatile Organic Compounds) of plant leaves by using a headspace solid-phase microextraction method, which comprises the following steps: cleaning collected plant leaves, and wiping water to obtain clean plant leaves; taking a plurality of circular leaf samples with the same size from a non-main vein area of the cleaned plant leaf; extracting BVOCs of the leaf sample with a preset weight by using a headspace solid-phase microextraction method; and carrying out analysis and quantitative analysis on the extracted BVOCs of the leaf sample. According to the method, a plurality of circular leaf samples with the same area are drilled in a non-vein area of a leaf by using a fixed-aperture drilling device, so that the uniform distribution of notches is ensured, and the influence of inconsistent damage degrees on volatile matter release can be reduced to the greatest extent. By utilizing the method, qualitative, relatively quantitative and relative emission intensity analysis and determination of BVOCs emitted by leaves of different broad-leaved plants can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection methods for volatile organic compounds released from plant leaves, and specifically relates to a method for measuring BVOCs of plant leaves by using a headspace solid phase microextraction method. Background Art

[0002] Volatile organic compounds (VOCs) refer to volatile hydrocarbons with a boiling point between 50 and 260°C and a saturated vapor pressure of more than 133.32 Pa at room temperature. They can be divided into anthropogenic and biogenic sources according to their sources. Biogenic sources are mainly emitted by vegetation. In addition to releasing CO2 and O2 through metabolic activities such as photosynthesis and respiration, green plants also release a large number of low-boiling-point, volatile, and highly reducing secondary metabolites, namely plant-derived volatile organic compounds (BVOCs), mainly through their leaves. Globally, BVOCs emissions are significantly higher than those from anthropogenic sources, accounting for 75% to 90% of the world's total VOCs emissions, and are the main source of VOCs in ecosystems.

[0003] As a product of plant physiological processes, BVOCs have important physiological and ecological significance for plants. They can regulate plant growth, development and reproduction, promote information exchange between neighboring plants, resist various environmental stresses (high temperature, strong light, oxidative damage), resist the invasion of bacteria and pathogens, and the feeding of insects and animals, and resist natural enemies. BVOCs have an important impact on both the atmospheric environment and human health. The beneficial components released by some plants have positive effects such as bactericidal and antibacterial, air purification, fatigue relief, and mood regulation, which are beneficial to human physiological and mental health. In recent years, the concepts of "forest bathing place" and "forest hospital" introduced by many countries such as Europe, the United States, and Japan, as well as the "aromatherapy" and "floral fragrance therapy" that have emerged in health care are based on the components of BVOCs released by plants that are beneficial to human health. The BVOCs components of some plants will not only directly pollute the environment, but also undergo photochemical reactions when entering the atmosphere to produce secondary pollutants, thereby exacerbating environmental pollution.

[0004] Different plants emit different BVOCs, and their chemical components, relative contents and emission intensities vary significantly. From the perspective of chemical ecology, scientific evaluation of the human health effects and ecological and environmental benefits of different plants can provide a theoretical basis for the rational selection of urban gardening tree species and the optimization of plant configuration. To achieve the above goals, the key is to analyze and determine the components of plant volatiles to explore their components, contents and emission intensity. Plant volatile organic compounds have the characteristics of low content, volatility, high activity and complex composition. The detection limit of general instruments is difficult to reach its concentration level. Therefore, establishing an efficient and sensitive determination method is the key to accurate qualitative and quantitative analysis of plant volatile organic compounds. Solid phase microextraction (SPME) is a popular solvent-free sample pretreatment technology in recent years. It is an analytical technology that integrates extraction, enrichment and injection. It can be used in conjunction with gas chromatography and mass spectrometry to achieve rapid separation and analytical identification of volatile components emitted by isolated plant leaf samples. However, different extraction time and extraction temperature will also affect the extraction results. It is difficult to quantitatively analyze the components of plant volatiles and lacks a unified standard. Summary of the invention

[0005] In order to improve the shortcomings of headspace solid phase microextraction technology for quantitative analysis of volatile organic compounds in leaves of different plants, the present invention aims to provide a method for determining BVOCs in plant leaves using headspace solid phase microextraction. The present invention can realize qualitative, relatively quantitative and relative emission intensity analysis and determination of BVOCs emitted from leaves of different broad-leaved plants.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme: A method for determining BVOCs in plant leaves using headspace solid phase microextraction method, comprising the following process: The collected plant leaves are cleaned and dried to obtain clean plant leaves; Taking a plurality of circular leaf samples of the same size from the non-main vein area of ​​the cleaned plant leaves; Using a headspace solid phase microextraction method, extracting BVOCs of a preset weight of the leaf sample; The BVOCs of the extracted leaf samples were analyzed and quantified.

[0007] Preferably, when collecting the plant leaves, broad leaves from healthy and intact plants are selected.

[0008] Preferably, when taking a plurality of circular leaf samples of the same size from the non-main vein region of the cleaned plant leaves, a fixed aperture drill is used to drill a plurality of circular leaf samples of the same size from the non-main vein region of the cleaned plant leaves.

[0009] Preferably, the collected plant leaves are rinsed with distilled water to remove pollutants on the surface of the plant leaves, and then the water is absorbed with filter paper.

[0010] Preferably, the above method of the present invention also includes a process of optimizing the extraction conditions to determine the extraction temperature and extraction time when extracting BVOCs of a preset weight of the leaf sample using the headspace solid phase microextraction method.

[0011] Preferably, the extraction temperature is 40-70°C and the extraction time is 30-60 min.

[0012] Preferably, when the BVOCs of the extracted leaf samples are analyzed and quantitatively analyzed, the extractor needle is inserted into the injection port of the GC-MS instrument for analysis, and after the analysis is completed, the GC-MS instrument is started for injection analysis.

[0013] Preferably, when the extractor needle is inserted into the injection port of the GC-MS coupling instrument for analysis, the desorption temperature is 230±1° C. and the desorption time is 3 min.

[0014] Preferred: The working parameters of headspace solid phase microextraction are as follows: the injection head selects SPME Arrow 1 fiber head; the fiber head aging temperature is 250℃, the aging time is 3 min; the extraction temperature is set to 40-70℃; the extraction time is 30-60 min); the sample desorption temperature is 230℃, and the desorption time is 3 min; The working parameters of the gas chromatography were as follows: the injection port temperature was 280°C, the carrier gas was helium, the carrier gas flow rate was 24 mL / min, and the injection was non-split; the chromatographic column was a 30 m×0.32 mm×0.25 μm HP-5MSUI column; the programmed temperature was: the initial temperature was 40°C, and after 3 min, it was increased to 160°C at a rate of 10°C / min, maintained for 1 min, and then increased to 250°C at a rate of 10°C / min, and maintained for 1 min. The constant flow rate of the chromatographic column was 1 mL / min; The working parameters of the mass spectrometer are as follows: the ionization source is EI source, the electron energy is 70 eV; the ion source temperature is 230℃; the solvent delay is 0.5 min; the interface temperature is 250℃; the scan is full scan qualitative, the mass range is 29~350 m / z, and the scan interval is 0.2s.

[0015] Preferably, the plant leaves include leaves of Magnolia grandiflora, Ligustrum lucidum, Osmanthus fragrans, Pittosporum tobira, Ligustrum lucidum and Sophora japonica.

[0016] The present invention has the following beneficial effects: Since mechanical damage to leaves can significantly increase the release of BVOCs, this method takes multiple circular leaf samples of the same size in the non-main vein area of ​​the plant leaves to ensure that the incisions are evenly distributed, which can minimize the impact of inconsistent damage on the release of volatiles. After that, the present invention uses the headspace solid phase microextraction method to extract the BVOCs of the leaf samples of a preset weight; finally, the BVOCs of the extracted leaf samples are parsed and quantitatively analyzed to obtain the type and content of the BVOCs of the leaf samples. The present invention can perform qualitative and relative quantitative analysis on the volatile components emitted by the leaves, as well as analyze the relative release intensity of volatile substances from different plants based on the total peak area of ​​their response on the chromatogram. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is an operational flow chart of the method for determining BVOCs in plant leaves using headspace solid phase microextraction method of the present invention.

[0018] Figure 2 The figure is a graph showing the number of peaks and total peak area of ​​volatile components of Ginkgo biloba leaves measured at different extraction temperatures using headspace solid phase microextraction technology in an embodiment of the present invention.

[0019] Figure 3 This is a graph showing the number of peaks and total peak area of ​​volatile components of Ginkgo biloba leaves measured at different extraction times using headspace solid phase microextraction technology in an embodiment of the present invention.

[0020] Figure 4 This is a chromatogram of volatile components released from Ginkgo leaves under the conditions of extraction temperature of 70° C. and extraction time of 50 min in an embodiment of the present invention.

[0021] Figure 5 The method provided by the invention is used to determine the chromatograms of volatile organic compounds in leaves of six plant species, namely, Magnolia grandiflora, Ligustrum lucidum, Osmanthus fragrans, Pittosporum tobira, Ligustrum lucidum and Sophora japonica.

[0022] Figure 6 The total peak area graphs of volatile components of leaves of six plant species, namely Magnolia grandiflora, Ligustrum lucidum, Osmanthus fragrans, Pittosporum tobira, Ligustrum lucidum and Sophora japonica, were determined for the present invention.

[0023] In the figure, 1-headspace bottle, 2-sample matrix, 3-volatile component, 4-extraction head, 5-injection port, 6-blade. DETAILED DESCRIPTION

[0024] In order to make the experimental operation, technical method and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the invention.

[0025] Reference Figure 1The method of the present invention for determining BVOCs in plant leaves using headspace solid phase microextraction is carried out according to the following steps: (1) Select test plants that are growing well and collect healthy leaves from them. Test plants should be broad-leaved species with large leaves.

[0026] (2) Rinse the collected leaves with distilled water and then dry them with filter paper. Rinse the collected leaves with distilled water to remove dust, debris and other pollutants attached to the leaves to prevent them from affecting instrument analysis. Use a fixed-aperture drill to drill multiple circular leaves of the same area (i.e., leaf samples) in the non-vein area of ​​the leaves. The radius of the leaf samples is 0.3 mm. Weigh 0.5 g of the leaf samples and place them in a 20 mL headspace injection bottle. The cap of the headspace bottle is equipped with a PTFE septum to seal it.

[0027] (3) Optimizing extraction conditions: Determine the optimal extraction temperature and extraction time for different types of plant leaves through experimental measurement. The extraction temperature (40°C, 50°C, 60°C, 70°C) and extraction time (30 min, 40 min, 50 min, 60 min) recommended by the present invention is to experiment with different extraction temperatures and extraction times for the leaf samples of the same plant so that the total peak area and number of volatile components shown in the chromatogram reach the peak value, thereby determining the optimal instrumental analysis conditions for detecting different plant leaves.

[0028] (4) Set the parameters of solid phase microextraction (SPME), gas chromatography (GC), and mass spectrometry (MS) in sequence. After editing and running the sequence, the autosampler will take samples in sequence. Insert the aged extraction head into the headspace bottle and place it above the headspace gas. Extract for a period of time so that the organic matter volatilized from the sample will be adsorbed by the extraction head. Then pull out the extraction needle and insert it into the injection port of the GC-MS instrument for desorption at 230°C for 3 min. The organic matter adsorbed by the extraction head will be desorbed at high temperature, and then the GC-MS will be started for injection analysis. Among them, in the above scheme of the present invention, the instruments used in this method are Agilent PAL3 three-in-one autosampler and Agilent gas chromatograph-mass spectrometer (GC-MS, GC8890A-MS7000E, Agilent Technologies, USA).

[0029] SPME-GC-MS instrument operating parameters are as follows: (1) SPME: The injection head was the SPME Arrow 1 fiber head; the fiber head aging temperature was 250°C and the aging time was 3 min; the extraction temperature was (40°C, 50°C, 60°C, 70°C); the extraction time was (30 min, 40 min, 50 min, 60 min); the sample desorption temperature was 230°C and the desorption time was 3 min.

[0030] (2) GC: injection port temperature was 280°C, carrier gas was helium, carrier gas flow rate was 24 mL / min, and splitless injection was used; the chromatographic column was a 30 m×0.32 mm×0.25 μm HP-5MSUI column; programmed temperature: the initial temperature was 40°C, which was increased to 160°C at a rate of 10°C / min after 3 min and maintained for 1 min, then increased to 250°C at a rate of 10°C / min and maintained for 1 min. The column constant flow rate was 1 mL / min.

[0031] (3) MS: The ionization source was EI source, the electron energy was 70 eV; the ion source temperature was 230 °C; the solvent delay was 0.5 min; the interface temperature was 250 °C; the scan was full scan qualitative, the mass range was 29~350 m / z, and the scan interval was 0.2 s.

[0032] The qualitative analysis process in this method is as follows: after solid phase microextraction pretreatment, the volatiles extracted from the sample are passed through a GC-MS instrument to obtain the total ion current (TIC) of plant BVOCs and the mass spectrum of each component. The computer automatically retrieves the database standard spectrum (NIST20 spectral library) and chromatographic retention time (RT) to comprehensively identify the compounds and determine the specific chemical components of BVOCs emitted by each tree species.

[0033] The quantitative analysis of this method includes two types of analysis: relative quantification and relative concentration of volatiles: 1. According to the ratio of the integrated area of ​​different components in the chromatogram to the total area of ​​TIC, the peak area is normalized to obtain the relative percentage content of each component of BVOCs emitted by each plant.

[0034] 2. Since the size of the chromatogram peak area obtained by GC-MS analysis is proportional to the actual release concentration of volatiles, and the leaf biomass of the samples is consistent and the sample cuts made from the leaves are evenly distributed, the total peak area of ​​the response of different types of plant samples on the chromatogram can reflect the relative emission intensity of volatiles from different plants. This method can be used to evaluate the emission capacity of BVOCs from different plants.

[0035] This embodiment Figure 2 , Figure 3In this paper, ginkgo was used as an example to optimize the conditions of extraction time and extraction temperature. The effects of extraction temperature (40℃, 50℃, 60℃, 70℃) and extraction time (30 min, 40 min, 50 min, 60 min) on the volatile components of ginkgo leaves were explored. From the results in the figure, 70℃ was selected as the optimal extraction temperature for ginkgo. Under this temperature condition, the total peak area and number of volatile components emitted by ginkgo leaves reached the maximum value; it can be seen from the figure that with the extension of extraction time, the total peak area and number of components also increased significantly, reaching a peak value at 50 min, and then, as time continued to extend, the total peak area and number of volatile components decreased. This may be because when the extraction time is too long, the adsorbed components may desorb, resulting in a decrease in peaks, so 50 min was selected as the optimal extraction time.

[0036] Since mechanical damage to leaves can significantly increase the release of BVOCs, this method uses a fixed-aperture drill to drill multiple circular leaf samples of the same area in the non-vein area of ​​the leaf. This ensures that the incisions are evenly distributed and can minimize the impact of inconsistent damage on volatile release. Because leaf biomass largely determines the amount of volatile release, and leaf biomass is proportional to the amount of volatile release, for leaves of different plants, during the experiment, the circular leaf samples are weighed to the same weight and placed in a headspace bottle to ensure that the extracted leaf biomass is consistent. In summary, in addition to being able to perform qualitative and relative quantitative analysis on volatile components emitted by leaves, this method can also analyze the relative release intensity of volatile substances from different plants based on the total peak area of ​​their response on the chromatogram.

[0037] In this embodiment, Figure 4 In the study, the above method was used to optimize the extraction time and temperature of Ginkgo biloba leaves. At the optimal extraction temperature of 70°C and extraction time of 50 min, 104 peaks were detected in Ginkgo biloba leaves by GC-MS injection analysis, and 89 components were identified after deducting background impurities. The relative percentage content of each component can be obtained by normalizing the peak area based on the ratio of the integrated area of ​​each component to the total peak area. The total peak area is 1.63×10 10 .

[0038] In this embodiment, Figure 5 As shown in the above method, the volatile organic compounds emitted from the leaves of six plants, namely Magnolia grandiflora, Ligustrum lucidum, Osmanthus fragrans, Pittosporum tobira, Ligustrum lucidum and Sophora japonica, were measured in turn. The qualitative and relative quantitative analysis of the volatile organic components released from the leaves of different plants was achieved, and the relative emission intensity of volatile organic compounds from the leaves of different types of plants was explored based on the size of the total peak area of ​​the response on the chromatogram. Figure 6 As shown in the figure, the emission capacity of BVOCs of the six plants is as follows: Pittosporum tobira (14.3×1010 )>Magnolia grandiflora (9.33×10 10 )> Sophora japonica (2.87×10 10 )>Ligustrum lucidum (1.35×10 10 )>Ligustrum lucidum(9.89×10 9 )>Osmanthus fragrans(4.13×10 9 ).

[0039] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for determining BVOCs in plant leaves using headspace solid phase microextraction, characterized in that: The process includes the following: The collected plant leaves are cleaned and dried to obtain clean plant leaves; Taking a plurality of circular leaf samples of the same size from the non-main vein area of ​​the cleaned plant leaves; Using a headspace solid phase microextraction method, extracting BVOCs of a preset weight of the leaf sample; The BVOCs of the extracted leaf samples were analyzed and quantified.

2. The method for determining BVOCs in plant leaves using headspace solid phase microextraction according to claim 2, characterized in that: When collecting the plant leaves, choose broad leaves from healthy and intact plants.

3. The method for determining BVOCs in plant leaves using headspace solid phase microextraction according to claim 1, characterized in that: When taking a plurality of circular leaf samples of the same size from the non-main vein region of the cleaned plant leaves, a fixed aperture drill is used to drill a plurality of circular leaf samples of the same size from the non-main vein region of the cleaned plant leaves.

4. The method for determining BVOCs in plant leaves using headspace solid phase microextraction according to claim 1, characterized in that: The collected plant leaves were rinsed with distilled water to remove pollutants on the surface of the plant leaves, and then the water was absorbed with filter paper.

5. The method for determining BVOCs in plant leaves using headspace solid phase microextraction according to claim 1, characterized in that: The method also includes a process for optimizing the extraction conditions to determine the extraction temperature and extraction time when extracting the BVOCs of a preset weight of the leaf sample using a headspace solid phase microextraction method.

6. The method for determining BVOCs in plant leaves using headspace solid phase microextraction according to claim 1, characterized in that: The extraction temperature is 40-70℃ and the extraction time is 30-60 min.

7. The method for determining BVOCs in plant leaves using headspace solid phase microextraction according to claim 1, characterized in that: When analyzing and quantitatively analyzing the BVOCs of the extracted leaf samples, the extractor needle is inserted into the injection port of the GC-MS instrument for analysis. After the analysis is completed, the GC-MS instrument is started for injection analysis.

8. The method for determining BVOCs in plant leaves using headspace solid phase microextraction according to claim 7, characterized in that: When the extractor needle is inserted into the injection port of the GC-MS instrument for analysis, the desorption temperature is 230±1℃ and the desorption time is 3 min.

9. The method for determining BVOCs in plant leaves using headspace solid phase microextraction according to claim 7, characterized in that: The working parameters of headspace solid phase microextraction are as follows: the injection head selects SPME Arrow 1 fiber head; the fiber head aging temperature is 250℃, the aging time is 3 min; the extraction temperature is set to 40-70℃; the extraction time is 30-60 min); the sample desorption temperature is 230℃, and the desorption time is 3 min; The working parameters of the gas chromatography were as follows: the injection port temperature was 280°C, the carrier gas was helium, the carrier gas flow rate was 24 mL / min, and the injection was non-split; the chromatographic column was a 30 m×0.32 mm×0.25 μm HP-5MSUI column; the programmed temperature was: the initial temperature was 40°C, and after 3 min, it was increased to 160°C at a rate of 10°C / min, maintained for 1 min, and then increased to 250°C at a rate of 10°C / min, and maintained for 1 min. The constant flow rate of the chromatographic column was 1 mL / min; The operating parameters of the mass spectrometer are as follows: the ionization source is EI source, the electron energy is 70 eV; the ion source temperature is 230℃; the solvent delay is 0.5 min; the interface temperature is 250℃; the scan is full scan qualitative, the mass range is 29~350 m / z, and the scan interval is 0.2 s.

10. The method for determining BVOCs in plant leaves using headspace solid phase microextraction according to claim 1, characterized in that: Plant leaves include leaves of Magnolia grandiflora, Ligustrum lucidum, Osmanthus fragrans, Pittosporum tobira, Ligustrum lucidum and Sophora japonica.

Images