Method for analyzing stability of organic carbon in soil based on < 13 > C-NMR and FT-ICR-MS

Abstract

The invention provides a method for analyzing soil organic carbon stability based on < 13 > C-NMR and FT-ICR-MS, and belongs to the technical field of agricultural informatization. In the soil organic carbon stability research, the two technologies are combined, and the composition, structure and change of the soil organic carbon can be comprehensively understood from the microscopic level. NMR provides detailed information about the chemical environment, while FT-ICR-MS provides high resolution molecular component analysis. The complementarity of the two technologies enables researchers to deeply and accurately discuss the dynamic stability of the soil organic carbon. The combination of the two technologies is helpful for better understanding the effect of the soil organic carbon in the soil ecosystem, and further provides a scientific basis for soil management and environmental protection.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural information technology and specifically relates to a 13 C-NMR and FT-ICR-MS methods for analyzing soil organic carbon stability. Background Art

[0002] With the intensification of global climate change, land use change and environmental pollution problems, it is particularly important to study the stability of soil organic carbon. Soil organic carbon not only affects soil health and agricultural production, but also plays a key role in the global carbon cycle and greenhouse gas emissions. The stability of soil organic carbon generally refers to the persistence and resistance to decomposition of different components in soil organic matter in the soil environment, among which the changes in the composition and aromaticity of aromatic organic carbon (such as phenolic carbon: phenol-C and aromatic carbon: aromatic-C) are mainly considered. Understanding the stability of soil organic carbon is of great significance for assessing the long-term storage capacity of soil carbon and the risk of carbon release.

[0003] Existing technologies generally use elemental analyzers, Fourier transform infrared spectroscopy (FTIR) or X-ray photoelectron spectroscopy (XPS) alone to analyze the stability of soil organic carbon.

[0004] However, the above methods require complex sample preparation and may introduce interference from external factors, leading to inaccurate measurement results. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a 13 The present invention combines 13C-NMR with FT-ICR-MS to measure the stability of organic carbon in soil, which can obtain more comprehensive analysis results, effectively eliminate interference, clearly identify different organic components, accurately measure results, and simplify sample preparation.

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

[0007] The present invention provides a 13 C-NMR and FT-ICR-MS methods for analyzing soil organic carbon stability, including 13 C-NMR analysis method and FT-ICR-MS analysis method;

[0008] Said 13 The C-NMR analysis method includes the following steps:

[0009] Prepare nuclear magnetic resonance samples of soil to be tested and blank soil; the soil to be tested includes soil with organic fertilizer added; the blank soil includes soil without organic fertilizer added;

[0010] The NMR samples of the soil to be tested and the blank soil were respectively 13 C-NMR determination, respectively, to obtain the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum;

[0011] Compare the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum, if the content of phenolic carbon and aromatic carbon in the tested soil is lower than that of the blank soil, the organic carbon stability of the tested soil is reduced, otherwise the organic carbon stability of the tested soil is good;

[0012] The FT-ICR-MS analysis method comprises the following steps:

[0013] Preparing FT-ICR-MS samples of soil to be tested and blank soil; the soil to be tested includes soil to which organic fertilizer is added; the blank soil includes soil to which organic fertilizer is not added;

[0014] The FT-ICR-MS samples of the test soil and the blank soil are subjected to FT-ICR-MS measurement respectively, to obtain the FT-ICR-MS spectrum of the test soil and the FT-ICR-MS spectrum of the blank soil respectively;

[0015] By comparing the FT-ICR-MS spectrum of the soil to be tested with the FT-ICR-MS spectrum of the blank soil, if the lignin and tannin content in the soil to be tested is lower than that in the blank soil, the organic carbon stability of the soil to be tested is reduced, otherwise the organic carbon stability of the soil to be tested is good.

[0016] Preferably, the 13 The conditions for C-NMR measurement include: the nucleus is 13 C, the number of scans is 2400 to 8000, the single test time is 0.02 to 1 s, the relaxation time is 1 to 5 s, the resonance frequency is 104.3 to 150 mHZ, and the pulse width is 3.13 to 5 μs.

[0017] Preferably, the 13 The testing method of C-NMR measurement is cross-polarization.

[0018] Preferably, the magnetic field strength of the FT-ICR-MS measurement is 7.0-15.0 T, the ion source is an electrospray ion source, and the negative ion mode.

[0019] Preferably, the injection mode of the FT-ICR-MS determination is continuous injection, the injection speed is 100 to 120 μL / h, the capillary entrance voltage is -3.8 to -2 kV, and the ion accumulation time is 0.06 to 2 s.

[0020] Preferably, the acquisition mass range of the FT-ICR-MS determination is 100 to 1600 Da, the number of sampling points is 4M 32-bit data, and the number of time domain signal superpositions is 100 to 300 times.

[0021] Preferably, the preparation of the nuclear magnetic resonance sample of the soil to be tested comprises the following steps:

[0022] The soil sample to be tested is air-dried, ground, sieved, the sieve material is mixed with hydrofluoric acid for digestion, and then shaken and centrifuged in sequence to obtain a precipitate;

[0023] The precipitate is washed, dried, ground and sieved in sequence to obtain a nuclear magnetic resonance sample of the soil to be tested.

[0024] Preferably, the shaking and centrifugation are repeated 8 times; the centrifugation speed is 3000-3500 rmin -1 , time is 10 to 15 minutes.

[0025] Preferably, the preparation of the FT-ICR-MS sample of the soil to be tested comprises the following steps:

[0026] The soil sample to be tested is mixed with water, and the mixture is shaken and centrifuged in sequence, and then the obtained supernatant is filtered to obtain a soil sample solution;

[0027] The soil sample solution is subjected to SPE solid phase extraction to obtain an FT-ICR-MS sample of the soil to be tested.

[0028] The method according to claim 9 is characterized in that the shaking time is 8 hours, the centrifugal speed is 4000-4200 rpm, and the time is 15-20 minutes.

[0029] The present invention provides a 13 C-NMR and FT-ICR-MS methods for analyzing soil organic carbon stability, including 13 C-NMR analysis method and FT-ICR-MS analysis method; 13 The C-NMR analysis method comprises the following steps: preparing nuclear magnetic samples of the soil to be tested and blank soil; the soil to be tested comprises soil with organic fertilizer added; the blank soil comprises soil without organic fertilizer added; the nuclear magnetic samples of the soil to be tested and the blank soil are respectively subjected to 13 C-NMR determination, respectively, to obtain the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum; compare the soil to be tested 13 C-NMR spectrum and blank soil 13C-NMR spectrum, if the content of phenolic carbon and aromatic carbon in the tested soil is reduced relative to that of blank soil, the organic carbon stability of the tested soil is reduced, otherwise the organic carbon stability of the tested soil is good; the FT-ICR-MS analysis method comprises the following steps: preparing FT-ICR-MS samples of the tested soil and blank soil; the tested soil comprises soil with added organic fertilizer; the blank soil comprises soil without added organic fertilizer; performing FT-ICR-MS measurement on the FT-ICR-MS samples of the tested soil and blank soil respectively, and obtaining the FT-ICR-MS spectrum of the tested soil and the FT-ICR-MS spectrum of the blank soil respectively; comparing the FT-ICR-MS spectrum of the tested soil and the FT-ICR-MS spectrum of the blank soil, if the content of lignin and tannin in the tested soil is reduced relative to that of the blank soil, the organic carbon stability of the tested soil is reduced, otherwise the organic carbon stability of the tested soil is good.

[0030] Nuclear magnetic resonance carbon spectroscopy (NMR) is rich in structural information: NMR can provide information on molecular structure, including the chemical environment of different types of carbon atoms (such as alkyl carbon, aromatic carbon, carboxyl carbon, etc.), and can identify and quantify the relative abundance of different compounds in soil organic carbon. Non-destructive: NMR is a non-destructive method that can measure samples multiple times without loss. This is very important in studying the changes and dynamics of soil organic carbon. Quantitative analysis: Through the peak area in different chemical environments, NMR can achieve quantitative analysis, helping to evaluate the relative stability of different types of carbon and their changes over time. Applicability to aqueous and non-aqueous phases: NMR can be analyzed in aqueous or non-aqueous phases, which increases its flexibility of application and is suitable for studying soil organic carbon under different conditions.

[0031] Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has high resolution and high sensitivity: FT-ICR-MS has extremely high mass resolution and can separate and identify compounds with very close molecular weights, so detailed information on a variety of organic carbon molecules in complex soil organic matter can be obtained. Multistage mass spectrometry analysis: This technology supports multistage mass spectrometry analysis (MSn), which can provide a deeper structural analysis of the target compound, reveal its fragmentation pattern, and thus infer its chemical structure and stability. Direct analysis of complex mixtures: FT-ICR-MS is capable of analyzing complex organic mixtures and is suitable for the diverse organic carbon components in the soil, while requiring less sample pretreatment. Qualitative analysis of chemical components: The qualitative analysis of different organic carbon components is achieved through signal intensity, so that the relative stability of different components can be explored in depth. Accurate identification of molecular components: FT-ICR-MS can effectively analyze polar molecules (such as organic acids, amino acids, etc.) in soil organic carbon to evaluate its stability in the soil.

[0032] In the study of soil organic carbon stability, the combination of the above two techniques can provide a comprehensive understanding of the composition, structure and changes of soil organic carbon at the microscopic level. NMR provides detailed information about the chemical environment, while FT-ICR-MS provides high-resolution molecular component analysis. The complementarity of the above two techniques enables researchers to explore the dynamic stability of soil organic carbon more deeply and accurately. The combination of these two techniques helps to better understand the role of soil organic carbon in the soil ecosystem, and thus provide a scientific basis for soil management and environmental protection.

[0033] Soil is a highly complex system composed of organic matter from various sources. 13 C-NMR and FT-ICR-MS are used to analyze the stability of soil organic carbon, which can effectively eliminate interference, clearly identify different organic components, have accurate measurement results, and simple sample preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0035] Figure 1 For forest soil, grassland soil and wetland soil after adding cow dung 13 C-NMR spectroscopy;

[0036] Figure 2 The wetland soil (i.e. wetland control) and the wetland soil after adding cow dung (i.e. wetland + cow dung) 13 C-NMR spectroscopy;

[0037] Figure 3 The soil samples were collected from grassland (i.e., grassland control) and grassland soil after adding cow dung (i.e., grassland + cow dung). 13 C-NMR spectroscopy;

[0038] Figure 4 The forest soil (i.e. forest control) and the forest soil after adding cow dung (i.e. forest + cow dung) 13 C-NMR spectroscopy;

[0039] Figure 5 Fourier transform ion cyclotron resonance (FT-ICR / MS) Van Krevelen plots, (a) wetland, (b) wetland + cow dung, (c) grassland, (d) grassland + cow dung, (e) forest, (f) forest + cow dung;

[0040] Figure 6Nominal oxidation state of carbon (NOSC) DBE of Fourier transform ion cyclotron resonance (FT-ICR / MS): double bond equivalent, (a) wetland, (b) wetland + cow dung, (c) grassland, (d) grassland + cow dung, (e) forest, (f) forest + cow dung. DETAILED DESCRIPTION

[0041] The present invention provides a 13 C-NMR and FT-ICR-MS methods for analyzing soil organic carbon stability, including 13 C-NMR analysis method and FT-ICR-MS analysis method;

[0042] Prepare nuclear magnetic resonance samples of soil to be tested and blank soil; the soil to be tested includes soil with organic fertilizer added; the blank soil includes soil without organic fertilizer added;

[0043] The NMR samples of the soil to be tested and the blank soil were respectively 13 C-NMR determination, respectively, to obtain the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum;

[0044] Compare the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum, if the content of phenolic carbon and aromatic carbon in the tested soil is lower than that of the blank soil, the organic carbon stability of the tested soil is reduced, otherwise the organic carbon stability of the tested soil is good;

[0045] The FT-ICR-MS analysis method comprises the following steps:

[0046] Preparing FT-ICR-MS samples of soil to be tested and blank soil; the soil to be tested includes soil to which organic fertilizer is added; the blank soil includes soil to which organic fertilizer is not added;

[0047] The FT-ICR-MS samples of the test soil and the blank soil are subjected to FT-ICR-MS measurement respectively, to obtain the FT-ICR-MS spectrum of the test soil and the FT-ICR-MS spectrum of the blank soil respectively;

[0048] By comparing the FT-ICR-MS spectrum of the soil to be tested with the FT-ICR-MS spectrum of the blank soil, if the lignin and tannin content in the soil to be tested is lower than that in the blank soil, the organic carbon stability of the soil to be tested is reduced, otherwise the organic carbon stability of the soil to be tested is good.

[0049] Unless otherwise specified, the raw materials and equipment used in the present invention are commercially available products.

[0050] In the present invention, the13 The C-NMR analysis method includes the following steps:

[0051] Prepare nuclear magnetic resonance samples of soil to be tested and blank soil; the soil to be tested includes soil with organic fertilizer added; the blank soil includes soil without organic fertilizer added;

[0052] The NMR samples of the soil to be tested and the blank soil were respectively 13 C-NMR determination, respectively, to obtain the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum;

[0053] Compare the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum: if the content of phenolic carbon and aromatic carbon in the tested soil is lower than that in the blank soil, the organic carbon stability of the tested soil is reduced; otherwise, the organic carbon stability of the tested soil is good.

[0054] The present invention first prepares nuclear magnetic resonance samples of the soil to be tested and blank soil. In the present invention, the soil to be tested preferably includes: peat wetland soil collected from the Zoige Plateau of the Qinghai-Tibet Plateau, grassland soil of the Qinghai-Tibet Plateau, or forest soil of the Qinghai-Tibet Plateau with the addition of organic fertilizer; the organic fertilizer is preferably cow dung slurry; the amount of organic fertilizer added is preferably 0.5 mL / mg. In the present invention, the blank soil preferably includes peat wetland soil collected from the Zoige Plateau of the Qinghai-Tibet Plateau, grassland soil of the Qinghai-Tibet Plateau, or forest soil of the Qinghai-Tibet Plateau without the addition of organic fertilizer. The present invention uses blank soil as a control.

[0055] In the present invention, the preparation of the nuclear magnetic resonance sample of the soil to be tested preferably comprises the following steps:

[0056] The soil sample to be tested is air-dried, ground, and sieved (referred to as the first sieving), the sieve material is mixed with hydrofluoric acid for digestion, and then shaken and centrifuged in sequence to obtain a precipitate;

[0057] The precipitate is washed, dried, ground and sieved (referred to as the second sieving) in sequence to obtain a nuclear magnetic resonance sample of the soil to be tested.

[0058] In the present invention, the mass concentration of the hydrofluoric acid is preferably 10%; the mass ratio of the volume of the hydrofluoric acid to the material under the sieve is preferably 10 mL: 1 g. In the present invention, the shaking and centrifugation are preferably repeated 8 times. In an embodiment of the present invention, the shaking can be repeated 4 times for 1 hour, repeated 3 times for 12 hours, and repeated once for 24 hours. Centrifugation is performed after each shaking to remove the supernatant and obtain a precipitate. The present invention can remove water-soluble and polar organic matter in the soil sample to be tested by adding hydrofluoric acid for digestion. In the present invention, the rotation speed of the centrifuge is preferably 3000-3500 rmin. -1 In the embodiment of the present invention, the specific speed may be 3000 rmin -1 、3100rmin -1 、3200rmin -1 、3300rmin -1 、3400rmin -1 or 3500rmin -1 The centrifugation time is preferably 10 to 15 minutes. In an embodiment of the present invention, it can be 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes.

[0059] After obtaining the precipitate, the present invention sequentially washes, dries, grinds and sieves the precipitate to obtain a nuclear magnetic resonance sample of the soil to be tested.

[0060] In the present invention, the washing is preferably performed with water for 5 to 6 times; the drying temperature is preferably 45° C. In the present invention, the first sieving and the second sieving are preferably performed through a 100 mesh sieve.

[0061] In the present invention, the preparation of the NMR sample of the blank soil is the same as described above and will not be repeated here.

[0062] After obtaining the nuclear magnetic resonance sample of the soil to be tested, the present invention performs nuclear magnetic resonance on the nuclear magnetic resonance samples of the soil to be tested and blank soil respectively. 13 C-NMR determination, respectively, to obtain the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum.

[0063] In the present invention, the 13 The conditions for C-NMR measurement preferably include: the nucleus is 13C, the number of scans is 2400-8000, and in the embodiment of the present invention, it can be 3400, 3000, 4000, 5000, 6000, 7000 or 8000; the single test time is 0.02-1s, and in the embodiment of the present invention, it can be 0.02s, 0.03s, 0.04s, 0.05s, 0.08s or 1s; the relaxation time is 1-5s, and in the embodiment of the present invention, it can be 1s , 2s, 3s, 4s or 5s; the resonance frequency is 104.3-150MHZ, and in the embodiment of the present invention, it can be 104.3MHZ, 110MHZ, 120MHZ, 130MHZ, 140MHZ or 150MHZ; the pulse width is 3.13-5μs, and in the embodiment of the present invention, it can be 3.13μs, 3.2μs, 3.5μs, 4μs, 4.2μs, 4.5μs or 5μs. In the present invention, the 13 The testing method of C-NMR measurement is cross-polarization.

[0064] Get the soil to be tested 13 C-NMR spectrum and blank soil 13 After the C-NMR spectrum, the present invention compares the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum: if the content of phenolic carbon and aromatic carbon in the tested soil is lower than that in the blank soil, the organic carbon stability of the tested soil is reduced; otherwise, the organic carbon stability of the tested soil is good.

[0065] Nuclear magnetic resonance carbon spectroscopy (NMR) is rich in structural information: NMR can provide information on molecular structure, including the chemical environment of different types of carbon atoms (such as alkyl carbon, aromatic carbon, carboxyl carbon, etc.), and can identify and quantify the relative abundance of different compounds in soil organic carbon. Non-destructive: NMR is a non-destructive method that can measure samples multiple times without loss. This is very important in studying the changes and dynamics of soil organic carbon. Quantitative analysis: Through the peak area in different chemical environments, NMR can achieve quantitative analysis, helping to evaluate the relative stability of different types of carbon and their changes over time. Applicability to aqueous and non-aqueous phases: NMR can be analyzed in aqueous or non-aqueous phases, which increases its flexibility of application and is suitable for studying soil organic carbon under different conditions.

[0066] In the present invention, the FT-ICR-MS analysis method comprises the following steps:

[0067] Preparing FT-ICR-MS samples of soil to be tested and blank soil; the soil to be tested includes soil to which organic fertilizer is added; the blank soil includes soil to which organic fertilizer is not added;

[0068] The FT-ICR-MS samples of the test soil and the blank soil are subjected to FT-ICR-MS measurement respectively, to obtain the FT-ICR-MS spectrum of the test soil and the FT-ICR-MS spectrum of the blank soil respectively;

[0069] By comparing the FT-ICR-MS spectrum of the soil to be tested with the FT-ICR-MS spectrum of the blank soil, if the lignin and tannin content in the soil to be tested is lower than that in the blank soil, the organic carbon stability of the soil to be tested is reduced, otherwise the organic carbon stability of the soil to be tested is good.

[0070] The present invention first prepares FT-ICR-MS samples of soil to be tested and blank soil; the soil to be tested includes soil with organic fertilizer added; the blank soil includes soil without organic fertilizer added. In the present invention, the source of the soil sample to be tested is the same as described above and will not be repeated here.

[0071] In the present invention, the preparation of the FT-ICR-MS sample of the soil to be tested preferably comprises the following steps:

[0072] The soil sample to be tested is mixed with water, and the mixture is shaken and centrifuged in sequence, and then the obtained supernatant is filtered to obtain a soil sample solution;

[0073] The soil sample solution is subjected to SPE solid phase extraction to obtain an FT-ICR-MS sample of the soil to be tested.

[0074] In the present invention, the mass ratio of the soil sample to be tested to the volume of water is preferably 1g:4mL; the shaking time is preferably 8h, and the temperature is preferably 24-26°C; the centrifugal speed is preferably 4000-4200rpm, and in an embodiment of the present invention, it can be 4000rpm, 4050rpm, 4100rpm, 4150rpm or 4200rpm; the centrifugal time is preferably 15-20min, and in an embodiment of the present invention, it can be 15min, 16min, 17min, 18min, 19min or 20min; the filtration preferably uses a 0.22μm filter membrane.

[0075] The present invention has no special requirements for the SPE solid phase extraction steps, and the steps well known in the art can be used. In the embodiment of the present invention, specifically, the pH value of the soil sample solution is adjusted to 2.0 with hydrochloric acid, and then the solid phase extraction column is activated and loaded with the sample. After the sample flows out, hydrochloric acid is added to elute the column to remove the salt; the column is dried with nitrogen, and then eluted with methanol, and the eluate is collected; the eluate is blown with nitrogen until the methanol is completely volatilized, and the sample is stored in a -20°C refrigerator for testing.

[0076] In the present invention, the magnetic field strength measured by the FT-ICR-MS is preferably 7.0-15.0 T. In the embodiments of the present invention, it can be 7.0 T, 8.0 T, 9.0 T, 10.0 T, 12.0 T, 14.0 T or 15.0 T. The ion source is preferably an electrospray ion source in negative ion mode. In the present invention, the injection mode of the FT-ICR-MS determination is preferably continuous injection, and the injection speed is preferably 100-120 μL / h. In the embodiment of the present invention, it can be 100 μL / h, 105 μL / h, 110 μL / h, 115 μL / h or 120 μL / h; the capillary entrance voltage is preferably -3.8-2 kV. In the embodiment of the present invention, it can be -3.8 kV, -3 kV or -2 kV; the ion accumulation time is preferably 0.06-2 s. In the embodiment of the present invention, it can be 0.06 s, 0.1 s, 0.2 s, 0.4 s, 0.5 s, 1.0 s, 1.5 s or 2 s; the collection mass range of the FT-ICR-MS determination is preferably 100-1600 Da, and the number of sampling points is preferably 4 M For 32-bit data, the number of time domain signal superpositions is preferably 100 to 300 times. In an embodiment of the present invention, it can be 100 times, 120 times, 140 times, 160 times, 200 times, 250 times, 280 times or 300 times.

[0077] Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has high resolution and high sensitivity: FT-ICR-MS has extremely high mass resolution and can separate and identify compounds with very close molecular weights, so detailed information on a variety of organic carbon molecules in complex soil organic matter can be obtained. Multistage mass spectrometry analysis: This technology supports multistage mass spectrometry analysis (MSn), which can provide a deeper structural analysis of the target compound, reveal its fragmentation pattern, and thus infer its chemical structure and stability. Direct analysis of complex mixtures: FT-ICR-MS is capable of analyzing complex organic mixtures and is suitable for the diverse organic carbon components in the soil, while requiring less sample pretreatment. Qualitative analysis of chemical components: The qualitative analysis of different organic carbon components is achieved through signal intensity, so that the relative stability of different components can be explored in depth. Accurate identification of molecular components: FT-ICR-MS can effectively analyze polar molecules (such as organic acids, amino acids, etc.) in soil organic carbon to evaluate its stability in the soil.

[0078] In the study of soil organic carbon stability, the combination of the above two techniques can provide a comprehensive understanding of the composition, structure and changes of soil organic carbon at the microscopic level. NMR provides detailed information about the chemical environment, while FT-ICR-MS provides high-resolution molecular component analysis. The complementarity of the above two techniques enables researchers to explore the dynamic stability of soil organic carbon more deeply and accurately. The combination of these two techniques helps to better understand the role of soil organic carbon in the soil ecosystem, and thus provide a scientific basis for soil management and environmental protection.

[0079] Soil is a highly complex system composed of organic matter from various sources. 13 C-NMR and FT-ICR-MS are used to analyze the stability of soil organic carbon, which can effectively eliminate interference, clearly identify different organic components, have accurate measurement results, and simple sample preparation.

[0080] In order to further illustrate the present invention, the following is a method based on the present invention provided by combining the drawings and embodiments. 13 The methods of analyzing soil organic carbon stability by C-NMR and FT-ICR-MS are described in detail, but they should not be construed as limiting the scope of protection of the present invention.

[0081] Example 1

[0082] 13 C-NMR: Peat wetland soil was collected from the Zoige Plateau of the Qinghai-Tibet Plateau, air-dried, ground, and passed through a 100-mesh sieve. 5 g of the sieve was weighed into a centrifuge tube, and 50 mL of a 10% HF aqueous solution was added. The tube was shaken for 1 h and then centrifuged at 3000 rmin. -1 After centrifugation for 10 min, the supernatant was removed and the soil sample precipitated at the bottom was treated with the above HF solution for a total of 8 times, with the shaking time being 4 times × 1 h, 3 times × 12 h, and 1 time × 24 h (the concentration and amount of HF added remained unchanged, only the shaking time changed, 1 h was repeated 4 times, 12 h was repeated 3 times, and 24 h was repeated once). After each shaking, centrifugation was performed and the supernatant was removed. Finally, the soil sample precipitated at the bottom of the centrifuge tube was obtained, and then washed with 20 mL of distilled water until neutral, washed 5 to 6 times, and then dried at 45 ° C, ground with an agate mortar, and passed through a 100-mesh sieve to obtain the NMR sample of the wetland soil.

[0083] Take an appropriate amount of wetland soil NMR sample and place it in the test rotor (4 mm in diameter). Use the sample loading rod to gently compact the sample. Use the capping device to tightly combine the cap with the rotor. 13 C, scanning times 2400, single test time 0.02s, relaxation time 1s, resonance frequency 104.3mHZ, pulse width 3.13μs, test mode is cross polarization, the analysis results are Figure 2 (Wetland comparison).

[0084] Among them, the peak positions are: carboxyl / amide (160~190ppm), phenol-C (145~160ppm), aromatic-C (110~145ppm), O2-alkyl (95~110ppm), O-alkyl (65~95ppm), N-alkyl / methoxy-C (45~65ppm), C-alkyl (0~45ppm).

[0085] FT-ICR-MS: 5 g of peat wetland soil samples were collected from the Zoige Plateau on the Qinghai-Tibet Plateau. The samples were mixed with 20 mL of ultrapure water, oscillated at a constant temperature of 25 ± 1 °C for 8 h, and then centrifuged at 4200 rpm for 20 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain a soil sample solution.

[0086] The soil sample solution is subjected to SPE solid phase extraction:

[0087] Specifically, the pH value of the soil sample solution is adjusted to 2.0 with 1 mol / L hydrochloric acid;

[0088] Install the PPL column on the solid phase extraction device, and use 12 mL of methanol (chromatographic grade) and 12 mL of 0.01 mol / L hydrochloric acid to elute and activate the column in sequence;

[0089] Then slowly add the soil sample solution to the PPL column, let the sample flow down naturally (what flows down is other solvents except the target component, and the target component remains on the column), and the flow rate is maintained at about 2mL / min;

[0090] After the sample flows through, add 18 mL of 0.01 mol / L hydrochloric acid to elute the column to remove salt;

[0091] The column was dried with nitrogen, then eluted with 12 mL of methanol and the eluate was collected;

[0092] The eluent was purged with nitrogen until the methanol was completely volatilized, and the FT-ICR-MS sample of the wetland soil was obtained and stored in a −20 °C refrigerator until testing;

[0093] Redissolve with 1 mL of 1:1 methanol-water solution (or methanol);

[0094] Take 200 μL and inject directly for mass spectrometry analysis, the magnetic field strength is 15.0T, the ion source is electrospray ion source (ESI), negative ion mode. The main detection parameters are: injection mode is continuous injection, injection speed is 120 μL / h, capillary entrance voltage is -3.8 kV, ion accumulation time is 0.06 s, collection mass range is 100-1600 Da, sampling points are 4M 32-bit data, and time domain signal is superimposed 300 times to improve the signal-to-noise ratio.

[0095] Before the sample test, the instrument was calibrated with a 10 mmol / L sodium formate aqueous solution. After the sample test, the internal standard calibration was performed with soluble organic matter (using 200 to 800 molecular weight, 20 to 30 soluble organic matter with known molecular formulas were selected for calibration, which is a method known in the art). After calibration, the mass error of the detection was less than 1 ppm, which further illustrates that the FT-ICR-MS detection method of the present invention is accurate. The analysis results are obtained. Figure 5 Middle (a), Figure 6 Middle (a).

[0096] 13 C-NMR: 2.5 mL of cow dung slurry was added to the wetland soil sample, and other conditions were the same as above. The analysis results were obtained Figure 1 (a) and Figure 2 (Wetland + cow dung).

[0097] FT-ICR-MS: 2.5 mL of cow dung slurry was added to the wetland soil sample, and other conditions were the same as above. Figure 5 Middle (b), Figure 6 Middle (b).

[0098] Example 2

[0099] 13 C-NMR: The soil sample was replaced with soil from the Qinghai-Tibet Plateau grassland, and other conditions were the same as in Example 1. The analysis results were obtained Figure 3 (Grassland control).

[0100] FT-ICR-MS: The soil sample was replaced with soil from the Qinghai-Tibet Plateau grassland, and other conditions were the same as in Example 1. The analysis results were obtained Figure 5 Middle (c), Figure 6 Middle (c).

[0101] 13 C-NMR: 2.5 mL of cow dung slurry was added to the grassland soil sample, and other conditions were the same as in Example 1. The analysis results were obtained Figure 1 (b) and Figure 3 Medium (grassland + cow dung).

[0102] FT-ICR-MS: 2.5 ml of cow dung slurry was added to the grassland soil sample, and other conditions were the same as in Example 1. The analysis results were obtained Figure 5 Middle (d), Figure 6 Middle (d).

[0103] Example 3

[0104] 13C-NMR: The soil sample was replaced with the Qinghai-Tibet Plateau forest soil. Other conditions were the same as in Example 1. The analysis results were obtained. Figure 4 (Forest control).

[0105] FT-ICR-MS: The soil sample was replaced with the forest soil from the Qinghai-Tibet Plateau. Other conditions were the same as in Example 1. The analysis results were Figure 5 Middle (e), Figure 6 Chinese (e).

[0106] 13 C-NMR: 2.5 mL of cow dung slurry was added to the forest soil sample, and other conditions were the same as in Example 1. The analysis results were obtained Figure 1 (c) and Figure 4 (Forest + cow dung).

[0107] FT-ICR-MS: 2.5 ml of cow dung slurry was added to the forest soil sample, and other conditions were the same as in Example 1. The analysis results were obtained Figure 5 Middle (f), Figure 6 Middle (f).

[0108] Examples 1 to 3 Soil samples 13 C-NMR measurement results: The contents of various forms of C are shown in Table 1.

[0109] Table 1 Soil samples of Examples 1 to 3 and Comparative Examples 1 to 3 13 C-NMR measurement results

[0110]

[0111] Note: AR / AL = (aromatic-C + phenol-C) / (C-alkyl + N-alkyl / methyl-C + O-alkyl + O2-alkyl + carboxyl / amide); fa% = aromatic-C + phenol-C.

[0112] From Table 1 and Figures 1 to 4 It can be seen that the aromatic content (fa%) and the ratio of aromatic to aliphatic (AR / AL) of the three soils are in the order of grassland > forest > wetland. After the influence of cow dung, the aromatic content (fa%) and the ratio of aromatic to aliphatic (AR / AL) of grassland and forest soils decreased significantly, while there was no change in wetland soil. Specifically, the percentage of aromatic carbon (110-145 ppm) in forest and grassland soils decreased with the application of manure, while it remained basically unchanged in wetland soil.

[0113] Figure 5Fourier transform ion cyclotron resonance (FT-ICR / MS) Van Krevelen diagram, (a) wetland, (b) wetland + cow dung, (c) grassland, (d) grassland + cow dung, (e) forest, (f) forest + cow dung. Figure 5 It can be seen that compared with the control (e), the variables of condensed aromatic hydrocarbons in the forest soil (f) with cow dung added were greatly reduced; unsaturated hydrocarbons with low oxidation state (O / C: 0-0.1) also decreased in grassland (d) and forest soil (f), especially CHON and CHONS, which decreased more, indicating that the stability of the soil with cow dung added was reduced. Lignin and tannin are the two major polyphenolic substances that constitute phenolic carbon. The content of grassland (d) and forest soil (f) with cow dung added also decreased, indicating that the stability of the soil with cow dung added was reduced.

[0114] Figure 6 The nominal oxidation state of carbon (NOSC) DBE of Fourier transform ion cyclotron resonance (FT-ICR / MS): double bond equivalent, (a) wetland, (b) wetland + cow dung, (c) grassland, (d) grassland + cow dung, (e) forest, (f) forest + cow dung. Figure 6 It can be seen that with the decrease of aromatic carbon and unsaturated carbon, the overall NOSC of the forest soil (f) with the addition of cow dung increased significantly, the total percentage of NOSC>0 increased from 23% to 31%, and the variable proportion of NOSC<0 decreased from 71% to 61%, indicating that the addition of cow dung has improved the decomposition potential of organic carbon; and the increase in NOSC indicates a higher degree of oxidation. An increase in NOSC usually means that organic matter is more easily metabolized and decomposed by microorganisms, which may lead to a decrease in the stability of soil organic carbon.

[0115] Combination Figures 1 to 6 As shown in Table 1, in grasslands and forests, the addition of manure changed the composition of soil organic carbon, reduced aromaticity (aromatic carbon and phenolic carbon), and increased the nominal oxidation state of carbon (NOSC), while little effect was observed in wetlands. The stability of organic carbon mainly depends on the content of phenolic carbon and aromatic carbon. On the one hand, phenolic carbon can reduce the activity of hydrolases and protect the soil from decomposition. On the other hand, due to its ring structure and high bond energy, aromatic carbon is relatively difficult to decompose and is considered to be a stable organic carbon in the soil. The reduction in aromaticity may indicate that peat soils have lower stability. The differences in soil responses to the addition of manure in different land types are related to water content, plant input, dominant bacteria, and SOC chemical composition. Compared with grasslands and forests, organic carbon in wetlands basically shows a stronger resistance to degradation, so soil organic carbon in wetlands is more stable.

[0116] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method based on 13 The method for analyzing soil organic carbon stability by C-NMR and FT-ICR-MS is characterized in that: include 13 C-NMR analysis method and FT-ICR-MS analysis method; Said 13 The C-NMR analysis method includes the following steps: Prepare nuclear magnetic resonance samples of soil to be tested and blank soil; the soil to be tested includes soil with organic fertilizer added; the blank soil includes soil without organic fertilizer added; The NMR samples of the soil to be tested and the blank soil were respectively 13 C-NMR determination, respectively, to obtain the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum; Compare the soil to be tested 13 C-NMR spectrum and blank soil 13 C-NMR spectrum, if the content of phenolic carbon and aromatic carbon in the tested soil is lower than that of the blank soil, the organic carbon stability of the tested soil is reduced, otherwise the organic carbon stability of the tested soil is good; The FT-ICR-MS analysis method comprises the following steps: Preparing FT-ICR-MS samples of soil to be tested and blank soil; the soil to be tested includes soil to which organic fertilizer is added; the blank soil includes soil to which organic fertilizer is not added; The FT-ICR-MS samples of the soil to be tested and the blank soil are subjected to FT-ICR-MS measurement respectively, to obtain the FT-ICR-MS spectrum of the soil to be tested and the FT-ICR-MS spectrum of the blank soil respectively; By comparing the FT-ICR-MS spectrum of the soil to be tested with the FT-ICR-MS spectrum of the blank soil, if the lignin and tannin content in the soil to be tested is lower than that in the blank soil, the organic carbon stability of the soil to be tested is reduced, otherwise the organic carbon stability of the soil to be tested is good.

2. The method according to claim 1, characterized in that Said 13 The conditions for C-NMR measurement include: the nucleus is 13 C, the number of scans is 2400 to 8000, the single test time is 0.02 to 1 s, the relaxation time is 1 to 5 s, the resonance frequency is 104.3 to 150 mHZ, and the pulse width is 3.13 to 5 μs.

3. The method according to claim 1 or 2, characterized in that: Said 13 The testing method of C-NMR measurement is cross-polarization.

4. The method according to claim 1, characterized in that: The magnetic field strength of the FT-ICR-MS measurement is 7.0-15.0 T, the ion source is an electrospray ion source, and the negative ion mode.

5. The method according to claim 1 or 4, characterized in that: The injection mode of the FT-ICR-MS determination is continuous injection, the injection speed is 100 to 120 μL / h, the capillary entrance voltage is -3.8 to -2 kV, and the ion accumulation time is 0.06 to 2 s.

6. The method according to claim 1 or 4, characterized in that: The acquisition mass range of the FT-ICR-MS determination is 100 to 1600 Da, the number of sampling points is 4M 32-bit data, and the number of time domain signal superpositions is 100 to 300 times.

7. The method according to claim 1 or 2, characterized in that: The preparation of the nuclear magnetic resonance sample of the soil to be tested comprises the following steps: The soil sample to be tested is air-dried, ground, sieved, the sieve material is mixed with hydrofluoric acid for digestion, and then shaken and centrifuged in sequence to obtain a precipitate; The precipitate is washed, dried, ground and sieved in sequence to obtain a nuclear magnetic resonance sample of the soil to be tested.

8. The method according to claim 7, characterized in that The shaking and centrifugation were repeated 8 times; the centrifugal speed was 3000-3500 rmin -1 , time is 10 to 15 minutes.

9. The method according to claim 1 or 4, characterized in that: The preparation of the FT-ICR-MS sample of the soil to be tested comprises the following steps: The soil sample to be tested is mixed with water, and the mixture is shaken and centrifuged in sequence, and then the obtained supernatant is filtered to obtain a soil sample solution; The soil sample solution is subjected to SPE solid phase extraction to obtain an FT-ICR-MS sample of the soil to be tested.

10. The method according to claim 9, characterized in that The shaking time is 8 hours, the centrifugal speed is 4000-4200 rpm, and the time is 15-20 minutes.

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