Separation method capable of overcoming plastic interference in extraction process of soil soluble organic matters

Abstract

The invention discloses a separation method capable of overcoming plastic interference in a soil soluble organic matter extraction process. The method comprises the following steps: dissolving and separating: sequentially dissolving collected soil with ultrapure water, oscillating, centrifuging and filtering, and then collecting filtrate; adsorption desalination: carrying out adsorption desalination on the obtained filtrate, eluting soluble organic matters in the filtrate, and collecting an eluent; concentrating and enriching: concentrating and enriching the obtained eluent, dissolving the concentrated sample with a methanol aqueous solution, and collecting a dissolved solution; centrifugal separation: centrifuging the obtained dissolved solution by using a high-speed centrifuge to obtain supernate; and mass spectrum detection: detecting the obtained supernatant on a high-resolution mass spectrometer, and analyzing the influence of centrifugation on the molecular level of the DOM sample. According to the method, the soluble organic matters in the soil can be efficiently extracted, the interference of plastic factors can be overcome, and different types of DOM molecules can be obtained.

Description

Technical Field

[0001] The invention belongs to soil environmental analytical chemistry, and in particular relates to a separation method for overcoming plastic interference in the extraction process of soil soluble organic matter. Background Art

[0002] Dissolved organic matter (DOM) is a heterogeneous substrate composed of carbohydrates, proteins, lipids and humic acid, which is active and complex in composition. It contains a large amount of lignin and polyphenol compounds containing oxygen and active functional groups. It is widely present in terrestrial, lake and marine environments. Generally speaking, the sources of soil DOM are divided into two aspects. On the one hand, it is the weathering and mineralization process of the soil itself, and on the other hand, it is affected by the environment, including the metabolites of microorganisms in the process of death and decay of plants and animals, animal excrement, etc. This reason makes DOM complex and active in nature. As one of the largest organic carbon pools on the earth, DOM participates in the global carbon cycle and the distribution of nutrients, has high bioavailability, affects many biogeochemical processes through its bioavailability and adsorption capacity, and has an extremely important impact on environmental ecological functions and global biogeochemical nutrient cycles. Therefore, understanding the composition of DOM at the molecular level is an essential subject frontier.

[0003] In the process of extracting DOM, it is necessary to consider the removal of inorganic salts, the removal of chloride ions, and the acquisition of as many DOM molecules as possible. There are currently a variety of DOM extraction methods, such as ultrafiltration, reverse osmosis, liquid-liquid extraction, resin adsorption separation and other treatment methods. Ultrafiltration can achieve molecular weight classification according to the different molecular weights of DOM, but the separation effect is determined by the pore size of the membrane; reverse osmosis effectively removes inorganic salts, microorganisms and other impurities in water through different pressures to improve the purity of DOM, but a high-pressure pump is required to provide pressure during the experimental operation, which consumes a lot of energy. In addition, since plastic materials themselves contain a variety of additives, such as antioxidants, plasticizers, and stabilizers, these compounds can easily leach from plastic containers and merge into DOM components during the extraction of DOM, and plastic-derived DOM and natural DOM may have similarities in molecular structure, making it difficult to distinguish between plastic-interfered DOM and natural DOM in subsequent analysis and research. It can be seen that it is difficult to avoid the influence of plastic factors on DOM components during the experiment, and the above methods do not avoid the interference of plastic factors during the experiment. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a separation method for overcoming the interference of plastics in the extraction process of soil soluble organic matter. The extraction method of the present invention can effectively solve the interference of plastic factors in the extraction process of soil DOM while ensuring that organic matter will not be lost during the extraction process, and obtain a high-concentration DOM sample. At the same time, the method is easy to operate and is applicable to different types of soluble organic matter.

[0005] Technical solution: In order to achieve the above-mentioned purpose, the separation method of the present invention for overcoming the interference of plastics in the extraction process of soil soluble organic matter comprises the following steps:

[0006] (1) Dissolution and separation: The collected soil is dissolved, shaken, centrifuged, filtered, and the filtrate is collected;

[0007] (2) adsorption desalination: subjecting the filtrate obtained in step (1) to adsorption and desalination, and then eluting the dissolved organic matter in the filtrate, and collecting the eluate;

[0008] (3) Concentration and enrichment: The eluate obtained in step (2) is concentrated and enriched, and the sample is dissolved after concentration, and the sample after extraction is the DOM sample;

[0009] (4) Centrifugal separation: The DOM sample obtained in step (3) is centrifuged at high speed to obtain a supernatant sample, which is a high-purity, salt-free DOM sample without plastic interference.

[0010] Preferably, the separation method for overcoming plastic interference in the extraction process of soil soluble organic matter comprises the following steps: (1) dissolution separation: the collected soil is sequentially dissolved in ultrapure water, shaken, centrifuged, filtered, and the filtrate is collected;

[0011] (2) adsorption desalination: the filtrate obtained in step (1) is subjected to adsorption and desalination by solid phase extraction, and then the dissolved organic matter in the filtrate is eluted, and the eluate is collected;

[0012] (3) Concentration and enrichment: The eluate obtained in step (2) is concentrated and enriched, and the concentrated sample is dissolved in a methanol-water solution to obtain an extracted sample, which is a DOM sample;

[0013] (4) Centrifugal separation: The DOM sample obtained in step (3) is centrifuged in a high-speed centrifuge to obtain a supernatant sample, which is a high-purity, salt-free DOM sample without plastic interference.

[0014] (5) Mass spectrometry detection: The DOM sample obtained in step (4) is detected on a high-resolution mass spectrometer to analyze the effect of centrifugation at the molecular level on the sample at the molecular level. In step (1), the soil is dissolved with ultrapure water, and the mass volume ratio of the soil to the ultrapure water is 1:5-15 g / mL.

[0015] Wherein, the shaking conditions of step (1) are: 100-200 rpm, 20-30° C., 10-24 h; the centrifugation conditions are: 5000-10000 rpm, 5-10 min; the filtration conditions are: polytetrafluoroethylene (PTFE) filter head, pore size of 0.45 μm or nylon filter membrane, pore size of 0.45 μm.

[0016] Wherein, the filtrate in step (1) is shaken, centrifuged and filtered, which can be repeated 1 to 10 times.

[0017] Wherein, in step (2), the filtrate obtained in step (1) is subjected to adsorption and desalination by solid phase extraction, and then the soluble organic matter in the filtrate is eluted. The solid phase extraction column of the solid phase extraction is an XAD resin, C18 or PPL polymer-based extraction column; the solid phase extraction column is activated before the solid phase extraction, and the activation process is methanol soaking activation for 2 to 4 hours, and pH = 2 to 4 hydrochloric acid washing 2 to 4 times. At present, the methods for adsorbing DOM also include ultrafiltration, dialysis, etc., but these methods have certain disadvantages. For example, although the ultrafiltration method can be classified according to the molecular weight of DOM, it is not effective for extracting low molecular weight DOM; the dialysis method can simulate the diffusion and migration process of DOM in the natural environment, but its dialysis time is long and the separation efficiency is low. The solid phase extraction used in the present invention can effectively remove impurities from the sample while improving the efficiency.

[0018] The adsorption rate in step (2) is 2-5 mL / min. -1 The eluent in the elution process is methanol or acetonitrile.

[0019] Wherein, the concentration and enrichment in step (3) is carried out by nitrogen blowing or evaporation.

[0020] Wherein, the conditions of the high-speed centrifugation in step (4) are 5000-20000 rpm, 10-20° C., and 10-20 min.

[0021] The DOM obtained in step (4) is detected on a high-resolution mass spectrometer to analyze the effect of centrifugation on the sample at the molecular level. The flow rate of the sample injected into the ion source is controlled at 100-150 μL·h -1 , the ESI needle voltage is 3-4 kV, and the lower and upper limits of the mass are m / z = 200-1000 Da.

[0022] More preferably, in the separation method:

[0023] The dissolution ratio of the soil to ultrapure water in step (1) is generally 1:10 g / mL; the shaking conditions in step (1) are: 100 rpm, 25° C., 24 h; the centrifugation conditions are: 5500 rpm, 5 min; the filtration conditions are: polytetrafluoroethylene filter head, pore size of 0.45 μm; the dissolution separation in step (1) can be repeated 5 times; the centrifuge tube material is selected as polytetrafluoroethylene centrifuge tube.

[0024] Before the solid phase extraction in step (2), the solid phase extraction column is activated. The activation process is to soak in methanol (mass spectrometry grade) for 4 hours and rinse with hydrochloric acid at pH = 2 to 4 for 3 times. The adsorption rate through the column in step (2) is 2 to 5 mL min -1 The elution liquid in the elution process is methanol. The sampling material in step (2) is a glass rubber-tipped dropper.

[0025] Wherein, the concentration and enrichment in step (3) is concentrated by a nitrogen blower. The methanol in step (3) is a methanol aqueous solution with a volume fraction of 50%.

[0026] Wherein, the centrifugation conditions described in step (4) are: 10000-15000 rpm, 20°C, 10 min.

[0027] The sample in step (5) can be stored in a refrigerator at 0°C.

[0028] Preferably, the specific detection conditions of the present invention include the following aspects:

[0029] (1) All samples were injected into the ion source using a Bruker SolariX FT-ICRMS equipped with a 9.0 T superconducting magnet and an ESI ion source. The flow rate of the sample injection into the ion source was controlled at 120 μL h -1 .

[0030] (2) The instrument parameters were set as follows: ① the negative ionization model was adopted; ② the ESI needle voltage was set to 3.8 kV; ③ the lower and upper limits of the mass were set to m / z = 200 Da and 1000 Da, respectively; ④ the ions were introduced into the cyclotron ion chamber after accumulating in the hexapole ion trap for 0.1 s; ⑤ the number of scans was set to 300 times.

[0031] The separation method for overcoming plastic interference in the process of extracting soluble organic matter in soil of the present invention extracts and separates soluble organic matter in soil.

[0032] The dissolved organic matter in the present invention can include, according to the classification of compounds: lipids (0 < O / C < 0.3, 1.5 < H / C < 2.0), aliphatics and proteins (0.3 < O / C < 0.67, 1.5 < H / C < 2.2), lignin (0.1 < O / C < 0.67, 0.7 < H / C < 1.5), carbohydrates (0.67 < O / C < 1.2, 1.5 < H / C < 2.4), unsaturated hydrocarbons (0 < O / C < 0.1, 0.7 < H / C < 1.5), aromatic structures (0 < O / C < 0.67, 0.2 < H / C < 0.7), tannins (0.67 < O / C < 1.0, 0.6 < H / C < 1.5).

[0033] With the help of a high-speed centrifuge, the present invention overcomes the interference of plastic factors during the extraction of soil dissolved organic matter. The solvent used in the present invention is ultrapure water. The solid-phase extraction technology is used to enrich the soluble organic matter in the soil and remove salts. Then, the interference of plastics during the experiment is removed by high-speed centrifugation. The centrifugal supernatant is obtained as a mass spectrometry sample for subsequent mass spectrometry analysis. During the extraction of DOM, the plastic molecules contained in the experimental equipment will affect the DOM components, and there may be similarities in the molecular structures between plastic-derived DOM and natural DOM, confusing the classification of DOM by people. In the present invention, it is proposed to add a centrifugation step after DOM extraction. Through the optimization and adjustment of the rotation speed, first, from the molecular level, it is found that after centrifugation, molecular fractionation with relatively stable molecular structures (relatively large relative intensities) can be achieved. The molecules reduced in the centrifugal supernatant are the main molecules or intermediates of plastics; in addition, from the peak shape of the mass spectrometry results, the mass spectrometry peaks of general dissolved organic matter usually show a normal distribution, but due to the large intensity of the plastic peaks, the peak shape is affected. After high-speed centrifugation, it is found that this problem can be solved. Based on the traditional extraction method, the present invention effectively avoids the influence of plastic factors on DOM molecules, is simple to operate, practical and convenient, has good practicability and repeatability, and is a reliable method for overcoming plastic interference during the extraction of soil dissolved organic matter.

[0034] Compared with the existing extraction method, the present invention adopts a high-speed centrifuge with a certain rotation speed, and realizes stratification of DOM molecules according to different selection of rotation speed, thereby simply and effectively removing the interference of plastic on the extraction of soil dissolved organic matter in the experimental process, and using water as a solvent, it can effectively extract relatively high content of dissolved organic matter. The traditional extraction method extracts DOM molecules and is affected by plastic factors, while the present invention proposes a method of first enriching DOM and then centrifuging, which overcomes the influence of plastic macromolecules on the mass spectrometry results of DOM at the molecular level while ensuring that a large number of organic molecules are not lost, thereby achieving more extraction and separation of dissolved organic matter in soil, obtaining more molecular weights and dissolved organic matter of different molecular weight ranges and different types in mass spectrometry analysis, saving time and cost.

[0035] The plastic molecules in the present invention mainly come from the equipment (such as PPL columns, filter heads, etc.) used in the experimental process. The present invention proposes for the first time to use a high-speed centrifugation method to classify molecules with stable molecular structures (such as plastic molecules), thereby avoiding the influence of plastic factors contained in experimental equipment such as centrifuge tubes, filter heads, and PPL columns used in the DOM extraction process on DOM components. In addition, although the number of molecules obtained by high-resolution mass spectrometry after high-speed centrifugation of the extracted DOM is less than the number of molecules without high-speed centrifugation, the difference is not large. At the same time, analysis shows that the molecules reduced after high-speed centrifugation are the main components of plastics: high molecular polymers. Therefore, it is shown that the lost molecules are plastic molecules and not organic matter. The present invention uses centrifugation at different speeds to extract DOM to explore a method of removing the influence of plastic factors on DOM component analysis, and achieves classification of stable structure molecules in DOM through centrifugation, and separates plastic molecules with relatively large strength, thereby obtaining high-purity DOM that is not affected by plastic factors.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) After the extracted DOM sample is processed to a certain extent, the invention centrifuges the extracted DOM sample at different speeds by adjusting and optimizing the speed. Under the premise of ensuring that organic matter is not lost, the plastic molecules in the DOM sample are removed, and the supernatant after centrifugation can be used for high-resolution mass spectrometry detection.

[0038] (2) The processing method of the present invention has better analysis results. The mass spectrum peaks of the DOM sample after centrifugation are normally distributed and are not affected by peaks with larger relative intensities.

[0039] (3) The present invention effectively improves the recognition rate of dissolved organic matter and is relatively simple to operate. Compared with traditional technologies, the present invention can analyze different types of samples and effectively avoid the loss of organic matter.

[0040] In summary, the present invention adopts solid phase extraction technology combined with high-speed centrifugation to establish a separation method for overcoming plastic interference in the extraction process of soluble organic matter in soil, obtains a wider molecular weight range and more soluble organic matter, and is an extraction and separation method that effectively overcomes plastic interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 To extract the high-resolution mass spectrum and relative abundance distribution of DOM in paddy soil without centrifugation;

[0042] Figure 2 To extract the high-resolution mass spectrometry and relative abundance distribution of DOM in paddy soil, the centrifuge speed was 5000 rpm;

[0043] Figure 3 To extract the high-resolution mass spectra and relative abundance distribution of DOM in paddy soil, the centrifuge speed was 10000 rpm;

[0044] Figure 4 To extract the high-resolution mass spectra and relative abundance distribution of DOM in paddy soil, the centrifuge speed was 15000 rpm;

[0045] Figure 5 To extract the high-resolution mass spectra and relative abundance distribution of DOM in paddy soil, the centrifuge speed was 20000rpm;

[0046] Figure 6 Extract the high-resolution mass spectrum and relative abundance distribution of DOM in forest soil without centrifugation;

[0047] Figure 7 Extract high-resolution mass spectra and relative abundance distribution of DOM in forest soil at a centrifugal speed of 5000 rpm;

[0048] Figure 8 Extract high-resolution mass spectra and relative abundance distribution of DOM in forest soil at a centrifugal speed of 10,000 rpm;

[0049] Fig. 9 Extract high-resolution mass spectra and relative abundance distribution of DOM in forest soil at a centrifugal speed of 15,000 rpm;

[0050] Fig.10 High-resolution mass spectrometry and relative abundance distribution of DOM extracted from forest soil at a centrifugal speed of 20,000 rpm. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below in conjunction with the embodiments.

[0052] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0053] Source and treatment method of soil samples in the embodiment:

[0054] The soil samples in the embodiment were obtained from Changshu ecological paddy soil of Nanjing Soil Research Institute and forest soil of Zigui Ecological Experiment Station. The soil was free of fine roots and stones, and then sieved through a 2 mm sieve and air-dried to obtain pre-treated samples.

[0055] The operating conditions of mass spectrometry detection in the embodiment are as follows: all samples were injected into the ion source at a flow rate of 120 μL·h using a Bruker SolariX FT-ICR MS equipped with a 9.0 T superconducting magnet and an ESI ion source. -1 The instrument parameters were set as follows: 1) negative ionization model was used; 2) the ESI needle voltage was set to 3.8 kV; 3) the lower and upper limits of the mass were set to m / z = 200 Da and 1000 Da, respectively; 4) the ions were introduced into the cyclotron ion chamber after accumulating in the hexapole ion trap for 0.1 s; 5) the number of scans was set to 300.

[0056] The concentration range of the samples in the implementation case was controlled at 50-100 mg L -1 According to the TOC dilution factor, the eluent volume is generally determined to be 10 mL. For example, if the injection concentration is 100 mg L -1 The control calculation formula is:

[0057]

[0058] Example 1

[0059] The DOM samples after concentration and enrichment from paddy soil were not subjected to high-speed centrifugation.

[0060] The pretreated paddy soil sample was dissolved in ultrapure water at a mass volume ratio of 1:10 g / mL, and then shaken (100 rpm, 25 °C, 24 h), centrifuged (5500 rpm, 5 min), and filtered (polytetrafluoroethylene filter with a pore size of 0.45 μm) in sequence. The shaking, centrifugation, and filtration process were repeated 5 times to collect the filtrate; 10 mL of the filtrate was diluted 5 times to determine the TOC concentration, which was 12.04 mg·L -1 , according to the mass spectrometry injection concentration of 50 mg·L -1 , the required injection volume at this concentration was calculated to be 17 mL. The solid phase extraction method was used to adsorb and elute the dissolved organic matter in the filtrate. The PPL polymer-based solid phase extraction column (Agilent Technologies, 500 mg / 6 mL) was activated by soaking in methanol for 4 h before use and rinsed with hydrochloric acid at pH = 3 three times. 30 mL of the filtrate was washed at 2 mL min -1After the sample was adsorbed through the column at a rate of 1.5, it was washed with pH=3 hydrochloric acid and ultrapure water for 3 times for desalination, and finally eluted with 10 mL of methanol, and the eluate was collected; the obtained eluate was concentrated and enriched using a nitrogen blower, and the concentrated sample was dissolved with 10 mL of methanol water (volume ratio of 50%), and the dissolved sample was preserved for high-resolution mass spectrometry analysis. The mass spectrometry analysis results are shown in the attached Figure 1 . Figure 1 The distinction is mainly based on the intensity of the impurity peaks and the normal distribution of the peaks; the vertical axis of the bar graph represents the DBE (double bond equivalent, that is, the number of double bonds) value of molecules with different nitrogen-oxygen ratios, and the horizontal axis is different because the nitrogen-oxygen ratios obtained under different centrifugal conditions for each DOM component are different.

[0061] The collected filtrate was detected by high-resolution mass spectrometry, and the C c H h O o N n The number of molecular formulas (S / N≥6) is 2484, and N x There are 1209 molecular formulas with O>2; at the same time, there are many strong peaks between 200-600Da in the mass spectrum peak, which interferes with the mass spectrum peak shape.

[0062] Example 2

[0063] Example 2 uses the same method as in Example 1, except that the dissolved sample is centrifuged at 20°C, 5000 rpm for 10 min using a high-speed centrifuge, and the supernatant is collected after centrifugation and stored for high-resolution mass spectrometry analysis. The mass spectrometry analysis results are shown in the attached Figure 2 .

[0064] The collected filtrate was detected by high-resolution mass spectrometry, and the C c H h O o N n The number of molecular formulas (S / N≥6) is 2480, and N x The number of molecular formulas with O>2 is 1207; at the same time, there are many relatively strong impurity peaks at 200-600Da that affect the peak shape.

[0065] Example 3

[0066] Example 3 uses the same method as in Example 1, except that the dissolved sample is centrifuged at 20°C, 10,000 rpm for 10 min using a high-speed centrifuge, and the supernatant is collected after centrifugation and stored for high-resolution mass spectrometry analysis. The mass spectrometry analysis results are shown in the attached Figure 3 .

[0067] The collected filtrate was detected by high-resolution mass spectrometry, and the Cc H h O o N n The number of molecular formulas (S / N≥6) is 2433, and N x The number of molecular formulas with O>2 is 1175; at the same time, the mass spectrum peaks are normally distributed.

[0068] Example 4

[0069] Example 4 uses the same method as in Example 1, except that the dissolved sample is centrifuged at 20°C, 15000 rpm for 10 min using a high-speed centrifuge, and the supernatant is collected after centrifugation and stored for high-resolution mass spectrometry analysis. The mass spectrometry analysis results are shown in the attached Figure 4 .

[0070] The collected filtrate was detected by high-resolution mass spectrometry, and the C c H h O o N n The number of molecular formulas (S / N≥6) is 2447, and N x The number of molecular formulas with O>2 is 1182. Meanwhile, the mass spectrum peaks show an obvious normal distribution.

[0071] Example 5

[0072] Example 5 uses the same method as in Example 1, except that the dissolved sample is centrifuged at 20°C, 20,000 rpm for 10 min using a high-speed centrifuge, and the supernatant is collected after centrifugation and stored for high-resolution mass spectrometry analysis. The mass spectrometry analysis results are shown in the attached Figure 5 .

[0073] The collected filtrate was detected by high-resolution mass spectrometry, and the C c H h O o N n The number of molecular formulas (S / N≥6) is 2077, and N x The number of molecular formulas with O>2 is 867; at the same time, the mass spectrum peaks no longer have normal distribution characteristics.

[0074] It can be seen from the above five examples that in Example 1, the paddy soil was extracted under the same conditions and the filtrate was not centrifuged. The mass spectrometry results showed that C c H h O o N n The number of molecular formulas is 2484, which is not significantly different from the case when the speed is 5000 rpm. Figure 1It can be seen that the peak shape is disturbed by the peak with larger relative intensity. By measuring the mass spectrum and combining data processing, the relevant molecular information about DOM is obtained. By comparing the centrifuged and uncentrifuged molecules, it is found that the reduced molecules are mainly N x O>2 molecules, N x O>2 means that in C c H h O o N n The atomic ratio of nitrogen to oxygen in the compound is greater than 2, and when N x When O>2C c H h O o N n The compound contains amine or reduced nitrogen functional groups (such as amine, imine, etc.), and the plastic contains such functional groups. Specifically, after high-speed centrifugation, especially at the speeds of 10000 and 15000 rpm mentioned in Examples 3 and 4, it was found that the number of molecules decreased by 51 and 37, respectively. The reduced molecules were mainly N x O>2 macromolecules, N x The number of molecular formulas with O>2 decreased by 34 and 27 respectively. Figure 2 , Figure 3 It can also be seen that the mass spectrum peak shape is less affected by plastic factors and is normally distributed. In addition, when the speed is selected at 20000rpm, the number of molecular formulas is significantly reduced. Figure 4 It can be seen that the number of peaks has decreased. In addition, according to the mass spectrometry results, the molecular information was obtained through data processing. At the molecular level, it was found that the reduced peaks were mainly plastic molecules. From a molecular perspective, the reduced peaks at the speed of 10000 and 15000 rpm are plastic molecules. For example, m / z is 292.0493, which may be 1-butoxyaniline, which can be used as plastic and dye; m / z is 179.2059, which is 4-hydroxybutyric acid anilide, which is often used as a stabilizer for polymer materials such as plastics; m / z is 197.1402, which is 3-nitro-4-hydroxybenzoic acid methyl ester, which is often used as a binder for plastics. The above experiment proves that the molecular weight does not change much at 10,000rpm high-speed centrifugation, which means that it does not have much impact on the organic matter of DOM molecules, but reduces plastic molecules. For example, the molecule at m / z 292.0493 belongs to plastic molecules and will be separated by centrifugation. At the same time, the effect is not obvious when the speed is too low, and some organic matter in DOM is lost when the speed is too high, such as 20,000rpm. This shows that at speeds of 10,000 and 15,000, not only can plastic molecules be removed, but also DOM organic matter can be prevented from being lost. The main elements in DOM are C, H, O, N, P, and S. The molecular compounds obtained by mass spectrometry detection in the implementation case are mainly CHO, CHON, CHOS, and CHONS compounds. The obtained samples are high-purity, salt-free DOM samples without plastic interference.

[0075] Example 6

[0076] The high-speed centrifugation conditions for the DOM sample after concentration and enrichment of paddy soil are: 5000rpm, 10min.

[0077] The pretreated paddy soil sample was dissolved in ultrapure water at a mass volume ratio of 1:15 g / mL, and then shaken (100 rpm, 25 °C, 12 h), centrifuged (5500 rpm, 5 min), and filtered (nylon filter with a pore size of 0.45 μm) in sequence. The shaking, centrifugation, and filtration process were repeated 5 times to collect the filtrate; 10 mL of the filtrate was diluted 5 times to determine the TOC concentration, which was 13.63 mg·L -1 , according to the mass spectrometry injection concentration of 50 mg·L -1 , the required injection volume at this concentration was calculated to be 15 mL. The solid phase extraction method was used to adsorb and elute the dissolved organic matter in the filtrate. The silica-based adsorbent C18 column was activated by soaking in methanol for 4 hours before use and washed three times with hydrochloric acid at pH = 3. 30 mL of filtrate was washed at 2 mL min -1 After adsorbing through the column at a rate of , it was desalted by washing with hydrochloric acid and ultrapure water for 3 times with pH = 3 respectively, and finally eluted with 10mL of methanol, and the eluate was collected; it was then concentrated and enriched with a nitrogen blower, and the concentrated sample was dissolved with 10mL of methanol water (volume ratio of 50%), and the dissolved sample was centrifuged at 20°C, 5000rpm for 10min in a high-speed centrifuge, and the supernatant was taken after centrifugation. The supernatant was preserved and can be used for high-resolution mass spectrometry analysis.

[0078] Example 7

[0079] The high-speed centrifugation conditions for the DOM sample after concentration and enrichment of paddy soil are: 10000rpm, 10min.

[0080] The pretreated paddy soil sample was dissolved in ultrapure water at a mass volume ratio of 1:10 g / mL, and then shaken (100 rpm, 25 °C, 24 h), centrifuged (5500 rpm, 5 min), and filtered (polytetrafluoroethylene filter with a pore size of 0.45 μm) in sequence. The shaking, centrifugation, and filtration process were repeated 5 times to collect the filtrate; 10 mL of the filtrate was diluted 5 times to determine the TOC concentration, which was 16.78 mg·L -1 , according to the mass spectrometry injection concentration of 50 mg·L -1 , the required injection volume at this concentration was calculated to be 12 mL. The solid phase extraction method was used to adsorb and elute the dissolved organic matter in the filtrate. The PPL polymer-based solid phase extraction column was activated by soaking in methanol for 4 hours before use and rinsed with hydrochloric acid at pH = 3 three times. 30 mL of filtrate was washed at 2 mL min -1After adsorbing through the column at a rate of , the sample was washed with hydrochloric acid of pH = 3 and ultrapure water for 3 times respectively for desalination, and finally eluted with 10 mL of methanol, and the eluate was collected; the obtained eluate was concentrated and enriched using a nitrogen blower, and the concentrated sample was dissolved with 10 mL of methanol water (volume ratio of 50%), and the dissolved sample was centrifuged at 20°C, 10000 rpm for 10 min using a high-speed centrifuge, and the supernatant was taken after centrifugation. The supernatant can be preserved and used for high-resolution mass spectrometry analysis.

[0081] Example 8

[0082] The high-speed centrifugation conditions for the DOM sample after concentration and enrichment of paddy soil are: 15000rpm, 10min.

[0083] The pretreated paddy soil sample was dissolved in ultrapure water at a mass volume ratio of 1:10 g / mL, and then shaken (100 rpm, 25 ° C, 24 h), centrifuged (5500 rpm, 5 min), and filtered (polytetrafluoroethylene filter with a pore size of 0.45 μm) in sequence. The shaking, centrifugation, and filtration process were repeated 5 times to collect the filtrate; 10 mL of the filtrate was diluted 5 times to determine the TOC concentration, which was 11.70 mg·L -1 , according to the mass spectrometry injection concentration of 50 mg·L -1 , the required injection volume at this concentration was calculated to be 17 mL. The solid phase extraction method was used to adsorb and elute the dissolved organic matter in the filtrate. The PPL polymer-based solid phase extraction column was activated by soaking in methanol for 4 hours before use and rinsed with hydrochloric acid at pH = 3 three times. 30 mL of filtrate was washed at 2 mL min -1 After adsorbing through the column at a rate of , it was rinsed three times with hydrochloric acid and ultrapure water with pH = 3 respectively for desalination, and finally eluted with 10 mL of methanol, and the eluate was collected; it was then concentrated and enriched using a nitrogen blower, and the concentrated sample was dissolved with 10 mL of methanol water (volume ratio of 50%), and the dissolved sample was centrifuged in a high-speed centrifuge at 20°C, 15000 rpm for 10 minutes, and the supernatant was taken after centrifugation. The supernatant was preserved and can be used for high-resolution mass spectrometry analysis.

[0084] Example 9

[0085] The high-speed centrifugation conditions for the DOM sample after concentration and enrichment of paddy soil are: 20000rpm, 10min.

[0086] The pretreated paddy soil sample was dissolved in ultrapure water at a mass volume ratio of 1:10 g / mL, and then shaken (100 rpm, 25 ° C, 24 h), centrifuged (5500 rpm, 5 min), and filtered (nylon filter with a pore size of 0.45 μm) in sequence. The shaking, centrifugation, and filtration process were repeated 5 times to collect the filtrate; 10 mL of the filtrate was diluted 5 times to determine the TOC concentration, which was 12.67 mg·L-1. According to the mass spectrometry injection concentration of 50 mg·L -1 , the required injection volume at this concentration was calculated to be 16 mL. The solid phase extraction method was used to adsorb and elute the dissolved organic matter in the filtrate. The silica-based adsorbent C18 column was activated by soaking in methanol for 4 h before use and washed three times with hydrochloric acid at pH = 3. 30 mL of filtrate was added at 2 mL min -1 After adsorbing through the column at a rate of , it was washed with pH = 3 hydrochloric acid and ultrapure water for 3 times respectively for desalting, and finally eluted with 10mL of methanol, and the eluate was collected; it was then concentrated and enriched with a nitrogen blower, and the concentrated sample was dissolved with 10mL of methanol water (volume ratio of 50%), and the dissolved sample was centrifuged in a high-speed centrifuge at 20°C, 20000rpm for 10min, and the supernatant was taken after centrifugation. The supernatant was preserved and can be used for high-resolution mass spectrometry analysis.

[0087] Example 10

[0088] The DOM samples after forest soil concentration and enrichment were not subjected to high-speed centrifugation.

[0089] The pretreated forest soil sample was dissolved in ultrapure water at a mass-volume ratio of 1:10 g / mL, and then shaken (100 rpm, 25 °C, 24 h), centrifuged (5500 rpm, 5 min), and filtered (polytetrafluoroethylene filter with a pore size of 0.45 μm) in sequence. The shaking, centrifugation, and filtration process were repeated 5 times to collect the filtrate. 10 mL of the filtrate was diluted 5 times to determine the TOC concentration, which was 12.77 mg·L -1 , according to the mass spectrometry injection concentration of 100 mg·L -1 , the required injection volume at this concentration was calculated to be 31 mL. The solid phase extraction method was used to adsorb and elute the dissolved organic matter in the filtrate. The PPL polymer-based solid phase extraction column was activated by soaking in methanol for 4 hours before use and rinsed with hydrochloric acid at pH = 3 three times. 30 mL of filtrate was washed at 2 mL min -1 After the column is adsorbed at a rate of 1.5, it is washed with pH=3 hydrochloric acid and ultrapure water for 3 times to remove salt, and finally eluted with 10mL methanol, and the eluent is collected; the obtained eluent is concentrated and enriched with a nitrogen blower, and the concentrated sample is dissolved with 10mL methanol water (volume ratio is 50%), and the dissolved sample is preserved for high-resolution mass spectrometry analysis. The mass spectrometry analysis results are attached Figure 6 .

[0090] The collected filtrate was detected by high-resolution mass spectrometry, and the C c H h O o N n The number of molecular formulas (S / N≥6) is 2005, and N x There are 765 molecular formulas with O>2; at the same time, there are many strong peaks between 200-600Da in the mass spectrum peak, which interferes with the mass spectrum peak shape.

[0091] Embodiment 11

[0092] Example 11 adopts the same method as in Example 10, except that: the dissolved sample is centrifuged at 20°C, 5000 rpm for 10 min using a high-speed centrifuge, and the supernatant is collected after centrifugation. The supernatant is stored and can be used for high-resolution mass spectrometry analysis. The mass spectrometry analysis results are shown in the attached Figure 7 .

[0093] The collected filtrate was detected by high-resolution mass spectrometry, and the C c H h O o N n The number of molecular formulas (S / N≥6) is 1997, and N x The number of molecular formulas with O>2 is 760; at the same time, there are many relatively strong impurity peaks at 200-600Da that affect the peak shape.

[0094] Example 12

[0095] Example 12 adopts the same method as in Example 10, except that the dissolved sample is centrifuged at 20°C, 10000 rpm for 10 min using a high-speed centrifuge, and the supernatant is collected after centrifugation and stored for high-resolution mass spectrometry analysis. The mass spectrometry analysis results are shown in the attached Figure 8 .

[0096] The collected filtrate was detected by high-resolution mass spectrometry, and the C c H h O o N n The number of molecular formulas (S / N≥6) is 1982, and N x The number of molecular formulas with O>2 is 751; meanwhile, the mass spectrum peaks tend to be normally distributed.

[0097] Embodiment 13

[0098] Example 13 adopts the same method as in Example 10, except that the dissolved sample is centrifuged at 20°C, 15000 rpm for 10 min using a high-speed centrifuge, and the supernatant is collected after centrifugation and stored for high-resolution mass spectrometry analysis. The mass spectrometry analysis results are shown in the attached Fig. 9 .

[0099] The collected filtrate was detected by high-resolution mass spectrometry, and the C c H h O o N n The number of molecular formulas (S / N≥6) is 1977, and N x The number of molecular formulas with O>2 is 746; at the same time, the mass spectrum peaks tend to be normally distributed.

[0100] Embodiment 14

[0101] Example 14 The same method as in Example 10 was used, except that the dissolved sample was centrifuged at 20°C, 20,000 rpm for 10 min using a high-speed centrifuge, and the supernatant was collected after centrifugation. The supernatant was stored and can be used for high-resolution mass spectrometry analysis. The dissolved sample was centrifuged at 10,000 rpm for 10 min using a high-speed centrifuge, and the supernatant was collected after centrifugation. The supernatant was stored and can be used for high-resolution mass spectrometry analysis. The mass spectrometry analysis results are shown in the attached Fig.10 .

[0102] The collected filtrate was detected by high-resolution mass spectrometry, and the C c H h O o N n The number of molecular formulas (S / N≥6) is 1533, and N x The number of molecular formulas with O>2 is 405; meanwhile, the mass spectrum peaks have no normal distribution characteristics.

[0103] It can be seen from the above Examples 10-14 that Example 10 is the forest soil extracted under the same conditions and the final filtrate is not centrifuged. The mass spectrometry results show that C c H h O o N n The number of molecular formulas is 2005, which is not significantly different from the case when the speed is 5000 rpm. Figure 6 It can be seen that the peak shape is disturbed by the peak with larger relative intensity. However, after high-speed centrifugation, especially the rotation speed of 10000 and 15000 rpm mentioned in Examples 12 and 13, it was found that the number of molecules decreased by 28 and 23 respectively. The reduced molecules were mainly N x O>2 macromolecules, N x The number of molecular formulas with O>2 decreased by 14 and 19 respectively. Figure 8 , Fig. 9 It can also be seen that the mass spectrum peak type is less affected by plastic factors and is normally distributed. In addition, the number of molecular formulas is significantly reduced when the rotation speed is 20000rpm. In addition, from a molecular perspective, the peaks that can be reduced at the rotation speed of 10000 and 15000rpm are plastic molecules, such as m / z 340.0674, which is difluoro-hydroxy-(isopropyldioxypropyl)-ethyl propionate, which can be used as an active agent for plastics; m / z 339.2284, which is bis(dimethylaminopropyl)amine isopropanol, which is often used as a catalyst for the preparation of plastics; m / z 636.9592, which is hexamethylenediamine, which is often used as an antioxidant for plastics.

[0104] Embodiment 15

[0105] The high-speed centrifugation conditions for the DOM samples after forest soil concentration and enrichment are: 5000rpm, 10min.

[0106] The pretreated forest soil sample was dissolved in ultrapure water at a mass-volume ratio of 1:15 g / mL, and then shaken (100 rpm, 25 °C, 12 h), centrifuged (5500 rpm, 10 min), and filtered (nylon filter with a pore size of 0.45 μm) in sequence. The shaking, centrifugation, and filtration process were repeated 5 times to collect the filtrate. 10 mL of the filtrate was diluted 5 times to determine the TOC concentration, which was 11.78 mg·L -1 , according to the mass spectrometry injection concentration of 100 mg·L -1 , the required injection volume at this concentration is calculated to be 34 mL. The solid phase extraction method is used to adsorb and elute the dissolved organic matter in the filtrate. The PPL polymer-based solid phase extraction column is activated by soaking in methanol for 4 hours before use and washed with hydrochloric acid at pH = 3 three times. 30 mL of filtrate is washed at 2 mL min -1 After adsorbing through the column at a rate of , it was washed with hydrochloric acid of pH = 3 and ultrapure water for 3 times respectively for desalting, and finally eluted with 10 mL of methanol, and the eluate was collected; the obtained eluate was concentrated and enriched by a nitrogen blower, and the concentrated sample was dissolved with 10 mL of methanol water (volume ratio of 50%), and the dissolved sample was centrifuged at 20°C, 5000 rpm for 10 minutes in a high-speed centrifuge, and the supernatant was taken after centrifugation. The supernatant can be preserved and used for high-resolution mass spectrometry analysis.

[0107] Example 16

[0108] The high-speed centrifugation conditions for the DOM samples after forest soil concentration and enrichment are: 10000rpm, 10min.

[0109] The pretreated forest soil sample was dissolved in ultrapure water at a mass volume ratio of 1:10 g / mL, and then shaken (100 rpm, 25 °C, 24 h), centrifuged (5500 rpm, 5 min), and filtered (polytetrafluoroethylene filter with a pore size of 0.45 μm) in sequence. The shaking, centrifugation, and filtration process were repeated 5 times to collect the filtrate; 10 mL of the filtrate was diluted 5 times to determine the TOC concentration, which was 14.35 mg·L -1 , according to the mass spectrometry injection concentration of 100 mg·L -1 , the required injection volume at this concentration is calculated to be 28 mL. The solid phase extraction method is used to adsorb and elute the dissolved organic matter in the filtrate. The PPL polymer-based solid phase extraction column is activated by soaking in methanol for 4 hours before use and washed with hydrochloric acid at pH = 3 three times. 30 mL of filtrate is washed at 2 mL min -1 After adsorbing through the column at a rate of , the sample was washed three times with hydrochloric acid and ultrapure water at pH = 3 respectively for desalination, and finally eluted with 10 mL of methanol, and the eluate was collected; the obtained eluate was concentrated and enriched using a nitrogen blower, and the concentrated sample was dissolved with 10 mL of methanol water (volume ratio of 50%), and the dissolved sample was centrifuged at 20°C, 10000 rpm for 10 min using a high-speed centrifuge, and the supernatant was taken after centrifugation. The supernatant can be preserved and used for high-resolution mass spectrometry analysis.

[0110] Embodiment 17

[0111] The high-speed centrifugation conditions for the DOM samples after forest soil concentration and enrichment are: 15000rpm, 10min.

[0112] The pretreated forest soil sample was dissolved in ultrapure water at a mass-volume ratio of 1:10 g / mL, and then shaken (100 rpm, 25 °C, 24 h), centrifuged (5500 rpm, 5 min), and filtered (polytetrafluoroethylene filter with a pore size of 0.45 μm) in sequence. The shaking, centrifugation, and filtration process were repeated 5 times to collect the filtrate. 10 mL of the filtrate was diluted 5 times to determine the TOC concentration, which was 12.57 mg·L -1 , according to the mass spectrometry injection concentration of 100 mg·L -1 , the required injection volume at this concentration was calculated to be 32 mL. The solid phase extraction method was used to adsorb and elute the dissolved organic matter in the filtrate. The PPL polymer-based solid phase extraction column was activated by soaking in methanol for 4 hours before use and rinsed with hydrochloric acid at pH = 3 three times. 30 mL of filtrate was washed at 2 mL min -1After adsorbing through the column at a rate of , it was washed three times with hydrochloric acid and ultrapure water with pH = 3 respectively for desalination, and finally eluted with 10 mL of methanol, and the eluate was collected; the obtained eluate was concentrated and enriched using a nitrogen blower, and the concentrated sample was dissolved with 10 mL of methanol water (volume ratio of 50%), and the dissolved sample was centrifuged at 20°C, 15000 rpm for 10 min using a high-speed centrifuge, and the supernatant was taken after centrifugation. The supernatant can be preserved and used for high-resolution mass spectrometry analysis.

[0113] Embodiment 18

[0114] The high-speed centrifugation conditions for the DOM samples after forest soil concentration and enrichment are: 20,000 rpm, 10 min.

[0115] The pretreated forest soil sample was dissolved in ultrapure water at a mass-volume ratio of 1:10 g / mL, and then shaken (100 rpm, 25°C, 24 h), centrifuged (5500 rpm, 10 min), and filtered (nylon filter with a pore size of 0.45 μm) in sequence. The shaking, centrifugation, and filtration process were repeated 5 times to collect the filtrate. 10 mL of the filtrate was diluted 5 times to determine the TOC concentration, which was 13.24 mg·L -1 , according to the mass spectrometry injection concentration of 100 mg·L -1 , calculate the required injection volume at this concentration, which is 30mL. The solid phase extraction method is used to adsorb and elute the dissolved organic matter in the filtrate. The PPL polymer-based solid phase extraction column is activated by soaking in methanol for 4 hours before use and washed with hydrochloric acid at pH = 3 three times. 30mL of filtrate is washed at 2mL·min -1 After adsorbing through the column at a rate of , the sample was washed three times with hydrochloric acid and ultrapure water at pH = 3 respectively for desalination, and finally eluted with 10 mL of methanol, and the eluate was collected; the obtained eluate was concentrated and enriched using a nitrogen blower, and the concentrated sample was dissolved with 10 mL of methanol water (volume ratio of 50%), and the dissolved sample was centrifuged at 20°C, 10000 rpm for 10 min using a high-speed centrifuge, and the supernatant was taken after centrifugation. The supernatant can be preserved and used for high-resolution mass spectrometry analysis.

[0116] From the process and results of the above embodiments, it can be seen that the present invention can directly observe the test results and has a high degree of credibility. The entire experimental process is simple and easy to operate. c H h O o N n For example, after centrifugation, the number of molecules decreases, N xThe method proposed by the present invention is applicable to samples in different states, and after high-speed centrifugation, the influence of plastic macromolecules on the results is effectively removed, and the peak shape of the mass spectrum is better modified. In summary, this method is a reliable extraction and separation method for overcoming plastic factors in the extraction process of soluble organic matter.

Claims

1. A separation method for overcoming plastic interference in the extraction process of soil soluble organic matter, characterized in that: The following steps are involved: (1) Dissolution and separation: The collected soil is dissolved, shaken, centrifuged, filtered, and the filtrate is collected; (2) adsorption desalination: subjecting the filtrate obtained in step (1) to adsorption and desalination, and then eluting the dissolved organic matter in the filtrate, and collecting the eluate; (3) Concentration and enrichment: The eluate obtained in step (2) is concentrated and enriched, and the sample is dissolved after concentration, and the sample after extraction is the DOM sample; (4) Centrifugal separation: The DOM sample obtained in step (3) is centrifuged at high speed to obtain a supernatant sample, which is a high-purity, salt-free DOM sample without plastic interference.

2. The separation method for overcoming plastic interference in the extraction process of soil soluble organic matter according to claim 1, characterized in that: The soil in step (1) is dissolved with ultrapure water, and the mass volume ratio of the soil to the ultrapure water is 1:5-15 g / mL.

3. The separation method for overcoming plastic interference in the extraction process of soluble organic matter in soil according to claim 1, characterized in that: The shaking conditions of step (1) are: 100-200 rpm, 20-30° C., 10-24 h; the centrifugation conditions are: 5000-10000 rpm, 5-10 min; the filtration conditions are: polytetrafluoroethylene (PTFE) filter head, pore size of 0.45 μm or nylon filter membrane, pore size of 0.45 μm.

4. The separation method for overcoming plastic interference in the extraction process of soil soluble organic matter according to claim 1, characterized in that: The filtrate in step (1) is shaken, centrifuged and filtered, which can be repeated 1 to 10 times.

5. The separation method for overcoming plastic interference in the extraction process of soil soluble organic matter according to claim 1, characterized in that: In step (2), the filtrate obtained in step (1) is subjected to adsorption and desalination by solid phase extraction, and then the soluble organic matter in the filtrate is eluted. The solid phase extraction column of the solid phase extraction is an XAD resin, C18 or PPL polymer-based extraction column. Before the solid phase extraction, the solid phase extraction column is activated, and the activation process is methanol soaking activation for 2 to 4 hours, and pH = 2 to 4 hydrochloric acid washing 2 to 4 times.

6. The separation method for overcoming plastic interference in the extraction process of soil soluble organic matter according to claim 1, characterized in that: The adsorption rate in step (2) is preferably 2 to 5 mL min -1 The eluent in the elution process is methanol or acetonitrile.

7. The separation method for overcoming plastic interference in the extraction process of soil soluble organic matter according to claim 1, characterized in that: The concentration and enrichment in step (3) is carried out by nitrogen blowing or evaporation.

8. The separation method for overcoming plastic interference in the extraction process of soil soluble organic matter according to claim 1, characterized in that: The conditions of the high-speed centrifugation in step (4) are 5000-20000 rpm, 10-20° C., and 10-20 min.

9. The separation method for overcoming plastic interference in the extraction process of soil soluble organic matter according to claim 1, characterized in that: The DOM obtained in step (4) was detected on a high-resolution mass spectrometer to analyze the effect of centrifugation on the sample at the molecular level. The flow rate of the sample injected into the ion source was controlled at 100-150 μL·h -1 , the ESI needle voltage is 3-4 kV, and the lower and upper limits of the mass are m / z = 200-1000 Da.

10. Dissolved organic matter in soil extracted and separated by a separation method for overcoming plastic interference in the extraction process of dissolved organic matter in soil as described in any one of claims 1 to 9.

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