CN117949579B - Method for detecting methoxy stable isotope ratio of biodegradable lignin - Google Patents
Method for detecting methoxy stable isotope ratio of biodegradable lignin Download PDFInfo
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- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 title claims abstract description 79
- 238000000034 method Methods 0.000 title claims abstract description 40
- 239000007789 gas Substances 0.000 claims abstract description 51
- 239000000126 substance Substances 0.000 claims abstract description 29
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000000463 material Substances 0.000 claims abstract description 14
- 239000001257 hydrogen Substances 0.000 claims abstract description 13
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 13
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 11
- 230000003647 oxidation Effects 0.000 claims abstract description 8
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 8
- 238000000197 pyrolysis Methods 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims description 55
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 48
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- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 9
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 claims description 9
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- WHIXFMOBEKBEQW-UHFFFAOYSA-L dichlorozinc tetrahydrate Chemical group O.O.O.O.[Cl-].[Cl-].[Zn+2] WHIXFMOBEKBEQW-UHFFFAOYSA-L 0.000 claims description 7
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention belongs to the technical field of biology, and discloses a method for detecting methoxy stable isotope ratio of biodegradable lignin. The invention utilizes physical chemistry and phytochemistry means to purify lignin substances of biodegradable matters, utilizes selective organic chemistry substitution reaction to carry out lignin methoxy substitution to obtain steady-state headspace methyl iodide gas, and respectively carries out quantitative calibration of stable carbon isotope ratio and stable hydrogen isotope ratio in high-temperature oxidation and high-temperature pyrolysis modes by a gas chromatograph-gas stable isotope ratio mass spectrometer. The relative standard deviation of the lignin methoxy stable carbon and stable hydrogen isotope ratio of the biodegradable material obtained by the invention is respectively smaller than 0.15 per mill and 0.50 per mill, and a novel technical scheme is provided for effectively utilizing the lignin methoxy stable isotope ratio to indicate the biological degradation process of the biomass in agriculture, forestry and gardens and participating in the bio-geochemical carbon and water circulation.
Description
Technical Field
The invention belongs to the technical field of biological material analysis, and discloses a method for detecting methoxy stable isotope ratio of a biodegradable lignin.
Background
The agriculture and forestry garden biomass is agriculture and forestry garden waste generated by agricultural production, gardening management and garden operation, such as crop straw, fruit and vegetable melon vine, dead branches and fallen leaves, flowers and grass branches and stem pruning and the like. The main chemical component of the agriculture, forestry and garden biomass is lignocellulose, which is a macromolecular cross-linked body composed of cellulose, hemicellulose and lignin. Naturally, lignocellulose is water insoluble and bio-resistant, and is difficult to hydrolyze or directly metabolize by microorganisms, thereby preventing rapid degradation and recycling thereof.
Biodegradation, i.e., biological enzymolysis, is the main way to promote hydrolysis of cellulose and hemicellulose, and lignin cleavage, thereby converting into usable small molecule substances. Due to the intrinsic difference of chemical structures, lignin is difficult to degrade and utilize, and can be used as a biodegradation marker for representing the degree of participation of agriculture, forestry and garden biomass in the bio-geochemical cycle.
The key to the chemical behavioural process of lignin participation in biodegradation is its monomer composition type and chemical structure specificity. Three hydrogen atoms of lignin methylation come from photosynthetic water molecules and have photo-cleavage irreversibility, namely once lignin methylation is completed, a stable isotope signal carried by methoxy groups of lignin methylation can reflect biological geographic information of lignin biosynthesis. At present, application researches on using lignin methoxy stabilized carbon and hydrogen isotope ratios (delta 13CLM and delta 2HLM) as markers are mainly focused on the index field of climatology, and most of test samples are tree-wheel logs with high lignin content. While the application research of the biomass as a biodegradation marker in the bio-geochemical circulation is considered, although the research reviews that the biomass rich in lignin is involved in the organic geochemical evolution, especially the methoxy content and delta 13CLM are involved in the evaluation of methane resources in marsh and coalbed methane, the technical system of the high-resolution and accurate quantitative evaluation method is still lacking in delta 13CLM and delta 2HLM. In addition, the quantitative determination and evaluation method for the corresponding delta 13CLM in the biomass sample rich in lignin is established by using an external standard curve method, has the advantages of simple operation steps, short time consumption, small reagent amount and the like, and still cannot effectively meet the determination requirements of samples such as peat samples with low organic matter content, lignite or long bituminous coal with high deposition evolution degree and the like in the biodegradation process. Currently, a quantitative determination method for Guan 2HLM in biomass samples with low methoxy content is still a technical blank.
As a novel green solvent, the ionic liquid is usually molten salt at room temperature, and has the physical and chemical advantages of strong polarity, difficult volatilization, easy synthesis, dissolubility and the like. Can realize dissolution and regeneration of lignocellulose. The method mainly forms intramolecular hydrogen bonds through a melt blending mode, reduces the crystallinity of cellulose, and has the effects of promoting depolymerization of lignocellulose and improving enzymolysis conversion rate. Inorganic molten salt hydrate is taken as an ionic liquid-like system, is a good medium for hydrolyzing cellulose and hemicellulose, has limited dissolving capacity for lignin, for example zinc chloride hydrate is the most commonly accepted cellulose dissolving hydrolysis medium, but cannot be directly used for lignin. However, the conventional extraction of lignin from zinc chloride hydrate requires more solvent, higher temperature and longer aging, which prevents lignin extraction from being achieved in a gentle and efficient manner. In addition, although the ionic liquid like ferrous ammonium sulfate hexahydrate is the most commonly used oxygen scavenger for lignin extraction from soil sample derivatives, the conditions of application are severe in achieving depolymerization of lignin polymers.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a method for detecting the methoxy stable isotope ratio of a biodegradable lignin. The lignin methoxy stable isotope ratio obtained by the method has stability and reliability, and can provide probes for representing carbon fixation and water retention potential of agriculture, forestry and garden biomass and participating in dynamic process of bio-geochemical circulation.
Based on the above object, the invention provides a method for detecting methoxy stable isotope ratio of biodegradable lignin, which comprises the following steps:
Pretreating a biodegradable material to obtain a fine powder sample, purifying the fine powder sample to obtain lignin substances, deriving the lignin substances through organic chemical reaction to obtain headspace methyl iodide gas (CH 3 I), and measuring the headspace methyl iodide gas to obtain the lignin methoxy stable isotope ratio.
Further, the invention provides a detection method, wherein the biodegradable material comprises a biodegradable precursor, a biodegradable intermediate and/or an end product; the biodegradable precursors comprise plant source precursors such as dead branches, fallen leaves, layer fallen matters, garden gardening production waste and the like; the biodegradable intermediate and/or end product comprises soil source morphology such as humus soil, sediment peat and the like.
Further, the detection method provided by the invention comprises the following steps: removing impurities from the biodegradable material, drying, grinding and sieving; the particle size of the fine powder sample is 80-150 mu m.
Further, the detection method provided by the invention comprises the steps of purifying by taking the biodegradable precursor as a reaction substrate or purifying by taking the biodegradable intermediate and/or the final product as the reaction substrate.
The purification by taking the biodegradable precursor as a reaction substrate comprises the following steps: taking an equal proportion mixture of ionic liquid zinc chloride hydrate and ionic liquid 1-ethyl-3-methylimidazole ferric chloride ([ Emim ] FeCl 4) as a first biphasic extraction system, mixing the first biphasic extraction system and the biodegradable precursor according to a mass ratio of 4-15:1, adjusting pH to 4-5 at 21-23 ℃, sealing at 80 ℃ for reacting for 10-20 min, -2 ℃ for quenching reaction, obtaining a first reaction product, extracting the first reaction product for 2-3 times by using ethyl acetate, and performing reduced pressure rotary evaporation to obtain lignin substances.
The purification by taking the biodegradable intermediate and/or the final product as a reaction substrate comprises the following steps: and taking an equal proportion mixture of ionic liquid ammonium ferrous sulfate hexahydrate ((NH 4)2Fe(SO4)2·6H2 O) and ionic liquid 1-ethyl-3-methylimidazole ferric chloride ([ Emim ] FeCl 4) as a second double-phase extraction system, mixing the second double-phase extraction system with the biodegradable intermediate and/or the final product according to a mass ratio of 3-15:1, adding copper oxide with the same mass as the biodegradable intermediate and/or the final product, adjusting the pH value to 8-9, sealing the microwave reaction at 60-180 ℃ for 60-80 min, quenching the reaction at-2 ℃ to obtain a second reaction product, extracting the second reaction product for 2-3 times by using ethyl acetate, and performing reduced pressure rotary evaporation to obtain lignin substances.
Further, according to the detection method provided by the invention, the ionic liquid zinc chloride hydrate comprises zinc chloride tetrahydrate, and the purity is more than or equal to 98%; the purity of the ionic liquid ferrous ammonium sulfate is more than or equal to 99 percent.
Further, the detection method provided by the invention comprises the steps that lignin substances comprise monolignol and/or a mixture derived from lignin phenol and aldehyde with meta-methoxy groups reserved; the organic chemical reaction derivatization refers to: and (3) mixing the lignin substance with hydroiodic acid (HI) according to a ratio of 8-12:3.65, performing light-shielding sealed microwave reaction at 110-130 ℃ for 20-40 min, and standing at 21-23 ℃ for 40-60 min to obtain the headspace methyl iodide gas.
Further, the determining includes determining the specie species using a chromatographic chemical isotope ratio mass spectrometer, including: performing on-line separation on the headspace CH 3 I gas by a Gas Chromatograph (GC), and respectively measuring and obtaining the lignin methoxy stable isotope ratio by a high-temperature oxidation mode and a high-temperature pyrolysis mode by using a gas stable Isotope Ratio Mass Spectrometer (IRMS); the lignin methoxy stable isotope ratio includes lignin methoxy stable carbon isotope ratio (delta 13CLM) and stable hydrogen isotope ratio (delta 2HLM).
Further, the online separation means that 60-90 mu L of steady-state headspace CH 3 I gas is extracted through a gas sensitive injector, CH 3 I online separation is carried out through a GC instrument (TRACE 1310,Thermo Fisher) provided with a TG-5MS chromatographic column, the temperature of a sample inlet is set to be 200 ℃, and the temperature rise program is as follows: the initial temperature is 40 ℃, the temperature is kept for 3.8min, the temperature is increased to 80 ℃ at 20 ℃/min, the temperature is kept for 1min, the temperature is increased to 100 ℃ at 40 ℃/min, and the temperature is kept for 3min; the sample injection split ratio is 8-12:1, the high-purity helium is used as carrier gas, and the flow rate is 0.6-0.8 mL/min.
Further, the high-temperature oxidation mode is that the reaction furnace temperature is 960-1100 ℃, high-purity carbon dioxide is used as reference gas, raw wood delta 13CLM values (-35.0 permillage and-29.7 permillage) verified by an international atomic energy organization are used as double-point correction standards, and the relative standard deviation of lignin methoxy delta 13CLM values is less than or equal to 0.15 permillage.
Further, the high-temperature cracking mode is that the temperature of a reaction furnace is 1250-1500 ℃, high-purity hydrogen is used as reference gas, the range of H 3 + value is 4.3-4.5 ppm/nA, log delta 2HLM values (-307.5 permillage and-210.1 permillage) verified by an international atomic energy organization are used as double-point correction standards, and the relative standard deviation of lignin methoxy delta 2HLM value is less than or equal to 0.50 permillage.
In the description of the present invention, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or as implicitly indicating the number or order of technical features indicated.
Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects or advantages:
According to the invention, an agricultural and forestry garden biomass biodegradation precursor, a biodegradation intermediate and/or a final product are used as a determination substrate of lignin methoxy stable isotope ratio, separation of plant source or soil source lignin substances is realized through a one-step physicochemical biphase extraction system, lignin mixture is purified through a phytochemistry solid-phase or liquid-phase extraction technology, a selective organic substitution derivatization reaction is further utilized to replace the methoxy of the biodegradation lignin, so that stable headspace methyl iodide gas is obtained, and corresponding stable carbon and hydrogen isotope ratio determination is realized through a gas chromatography-gas stable isotope ratio mass spectrometer, and corresponding relative standard deviation is respectively lower than 0.15 per mill and 0.50 per mill, so that a stable index is provided for quantifying the participation of agricultural and forestry garden biomass in a bio-geochemical circulation process, and a biomarker is also provided for carbon and water circulation research of corresponding elements.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of condition optimization for the measurement of the biodegradation lignin methoxy delta 13CLM (a) and delta 2HLM (b).
FIG. 2 is a standard curve of lignin content provided in comparative example 1.
FIG. 3 is a standard curve of the meta-methoxy content of lignin provided in comparative example 2.
FIG. 4 shows the effect of the rest time of the methoxy organic substitution reaction of the biodegradation lignin on the stability of the delta 13CLM (a) and delta 2HLM (b) values.
FIG. 5 is a graph showing monolignol content of a methoxy-rich biodegradable precursor at various derivatization rest times. A in fig. 5 is a content spectrum for standing for 20min, and b in fig. 5 is a content spectrum for standing for 30 min.
Detailed Description
The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples.
The experimental methods and the detection methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.
Example 1
The embodiment provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
Drying the withered matters of the withered branches and fallen leaves in the shade, mechanically crushing and grinding by a ball mill to obtain a fine powder sample with the particle size of 80 mu m, and drying to constant weight.
An equiproportion mixture of ionic liquid zinc chloride tetrahydrate (ZnCl.4H 2 O) with the purity of 98 percent and ionic liquid 1-ethyl-3-methylimidazole ferric chloride ([ Emim ] FeCl 4) is used as a first biphasic extraction system. Mixing the first biphasic extraction system and the fine powder sample according to the mass ratio of 4:1, slowly dripping 1.5mol/L dilute hydrochloric acid at 21 ℃ to adjust the pH value to 4, sealing the microwave reaction at 80 ℃ for 10min, and quenching the reaction at-2 ℃ to obtain a first reaction product.
The first reaction product was extracted 2 times with ethyl acetate, and the mass to volume ratio of the first reaction product to ethyl acetate was 1:3 for each extraction. The extracted organic phase is dried by anhydrous sodium sulfate and subjected to reduced pressure rotary evaporation to obtain lignin substances, and the meta-methoxy groups of lignin monomers are reserved.
8Mg of the lignin substance is weighed and added into a brown headspace liquid-phase vial (relative density 1.70) containing 0.5mL of 55% hydroiodic acid by mass fraction, and the mixture is subjected to 110 ℃ microwave reaction at 20mm in a closed state and is kept stand at 21 ℃ for 40min, so that steady-state headspace CH 3 I gas with constant isotope value is obtained.
A Thermo Fisher model TRACE 1310 GC instrument was used and equipped with a TG-5MS column (30 m. Times.0.32 mm. Times.0.25 μm) and a gas-sensitive syringe was used to withdraw 60. Mu.L of steady-state CH 3 I gas. Setting the temperature of the sample inlet to be 200 ℃, and heating the sample to be: the initial temperature is 40 ℃, the temperature is kept for 3.8min, the temperature is increased to 80 ℃ at 20 ℃/min, the temperature is kept for 1min, the temperature is increased to 100 ℃ at 40 ℃/min, and the temperature is kept for 3min; the split ratio of the sample injection is 8:1, the high-purity helium is used as carrier gas, and the flow rate is 0.6mL/min.
The CH 3 I reagent is in a liquid state in a normal state, so that the gasification and calibration of the reagent are difficult. In this example, two kinds of check logs identified by the international atomic energy agency, siberian larch (Larix gmelinii) and cedar (Cryptomeria fortunei) (see in particular Lu Q Q, Liu X H, Treydte K, et al., Altitude-specific differences in tree-ring δ2H records of wood lignin methoxy in the Qinling mountains, central China. 2023, Quaternary Science Reviews） as calibrators, corresponding lignin methoxy delta 13CLM and delta 2HLM values were-35.0%o and-29.7%o and-307.5%o and-210.1%o, respectively.
Using CH 3 I obtained by GC online separation for IRMS measurement, adopting a high-temperature oxidation mode corresponding to lignin methoxy delta 13CLM value measurement, setting the temperature of a reaction furnace to 960 ℃, using calibrated high-purity carbon dioxide as reference gas, and using calibrated log delta 13CLM values (-35.0 mill and-29.7 mill) as a double-point correction standard; the corresponding lignin methoxy delta 2HLM value is measured by adopting a high-temperature pyrolysis mode, the temperature of a reaction furnace is 1250 ℃, high-purity hydrogen is used as reference gas, the H 3 + value is 4.3ppm/nA under the self-checking state, and the checked log delta 2HLM values (-307.5%mill and-210.1%mill) are used as double-point correction standards. The delta 13CLM value and the delta 2HLM value are respectively-29.4 per mill and-254.5 per mill. 9 sets of repeatability tests were performed, yielding relative standard deviations of 0.14% and 0.49% for the delta 13CLM value and the delta 2HLM value, respectively.
Example 2
The embodiment provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
Drying the withered matters in the dry and fallen leaves layer, mechanically crushing and grinding by a ball mill to obtain a fine powder sample with the particle size of 90 mu m, and drying to constant weight.
An equiproportion mixture of ionic liquid zinc chloride tetrahydrate (ZnCl.4H 2 O) with the purity of 98 percent and ionic liquid 1-ethyl-3-methylimidazole ferric chloride ([ Emim ] FeCl 4) is used as a first biphasic extraction system. Mixing the first biphasic extraction system and the fine powder sample according to the mass ratio of 6:1, slowly dripping 1.5mol/L dilute hydrochloric acid at 22 ℃ to adjust the pH value to 4.5, sealing the microwave reaction at 80 ℃ for 15min, and quenching the reaction at 0 ℃ to obtain a first reaction product.
The first reaction product was extracted 3 times with ethyl acetate, and the mass to volume ratio of the first reaction product to ethyl acetate was 1:3 for each extraction. The extracted organic phase is dried by anhydrous sodium sulfate and subjected to reduced pressure rotary evaporation to obtain lignin substances, and the meta-methoxy groups of lignin monomers are reserved.
10Mg of the lignin substance is weighed and added into a brown headspace liquid-phase vial containing 0.5mL of 55% hydroiodic acid (with relative density of 1.70), the mixture is subjected to microwave reaction at 120 ℃ for 30min in a closed state, and is kept stand at 22 ℃ for 50min, so that steady-state headspace CH 3 I gas with constant isotope value is obtained.
A Thermo Fisher model TRACE 1310 GC instrument was used and equipped with a TG-5MS column (30 m. Times.0.32 mm. Times.0.25 μm) and a gas-sensitive syringe was used to withdraw 70. Mu.L of steady-state CH 3 I gas. Setting the temperature of the sample inlet to be 200 ℃, and heating the sample to be: the initial temperature is 40 ℃, the temperature is kept for 3.8min, the temperature is increased to 80 ℃ at 20 ℃/min, the temperature is kept for 1min, the temperature is increased to 100 ℃ at 40 ℃/min, and the temperature is kept for 3min; the split ratio of the sample injection is 10:1, the high-purity helium is used as carrier gas, and the flow rate is 0.7mL/min.
The calibrator was the same as in example 1.
Using CH 3 I obtained by GC online separation for IRMS measurement, adopting a high-temperature oxidation mode corresponding to lignin methoxy delta 13CLM value measurement, setting the temperature of a reaction furnace to 1000 ℃, using calibrated high-purity carbon dioxide as reference gas, and using calibrated log delta 13CLM values (-35.0%mill and-29.7%mill) as a double-point correction standard; the corresponding lignin methoxy delta 2HLM value is measured by adopting a high-temperature pyrolysis mode, the temperature of a reaction furnace is 1400 ℃, high-purity hydrogen is used as reference gas, the H 3 + value is 4.4ppm/nA under the self-checking state, and the checked log delta 2HLM values (-307.5%mill and-210.1%mill) are used as double-point correction standards. The delta 13CLM value and the delta 2HLM value are respectively-32.5 per mill and-213.6 per mill. 9 sets of repeatability tests were performed, yielding relative standard deviations of 0.13% and 0.48% for the delta 13CLM value and the delta 2HLM value, respectively.
Example 3
The embodiment provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
And (3) carrying out shade drying treatment on garden and gardening production waste, mechanically crushing and ball mill grinding to obtain a fine powder sample with the particle size of 100 mu m, and drying to constant weight.
An equal proportion mixture of ionic liquid zinc chloride tetrahydrate with the purity of 98 percent and ionic liquid 1-ethyl-3-methylimidazole ferric chloride ([ Emim ] FeCl 4) is used as a first biphasic extraction system. Mixing the first biphasic extraction system and the fine powder sample according to the mass ratio of 15:1, slowly dripping 1.5mol/L dilute hydrochloric acid at 23 ℃ to adjust the pH value to 5, sealing the microwave reaction at 80 ℃ for 20min, and quenching the reaction at 2 ℃ to obtain a first reaction product.
The first reaction product was extracted 3 times with ethyl acetate, and the mass to volume ratio of the first reaction product to ethyl acetate was 1:3 for each extraction. The extracted organic phase is dried by anhydrous sodium sulfate and subjected to reduced pressure rotary evaporation to obtain lignin substances, and the meta-methoxy groups of lignin monomers are reserved.
12Mg of the lignin substance is weighed and added into a brown headspace liquid-phase vial containing 0.5mL of 55% hydroiodic acid (with relative density of 1.70), and the mixture is subjected to 130 ℃ microwave reaction 40 mm in a closed state and is kept stand at 23 ℃ for 60min, so that steady-state headspace CH 3 I gas with constant isotope value is obtained.
A TRACE 1310 model GC instrument from Thermo Fisher was used and equipped with a TG-5MS column (30 m. Times.0.32 mm. Times.0.25 μm) and a gas-sensitive syringe was used to withdraw 90. Mu.L of steady-state CH 3 I gas. Setting the temperature of the sample inlet to be 200 ℃, and heating the sample to be: the initial temperature is 40 ℃, the temperature is kept for 3.8min, the temperature is increased to 80 ℃ at 20 ℃/min, the temperature is kept for 1min, the temperature is increased to 100 ℃ at 40 ℃/min, and the temperature is kept for 3min; the split ratio of the sample injection is 12:1, the high-purity helium is used as carrier gas, and the flow rate is 0.8mL/min.
The calibrator was the same as in example 1.
Using CH 3 I obtained by GC online separation for IRMS measurement, adopting a high-temperature oxidation mode corresponding to lignin methoxy delta 13CLM value measurement, setting the temperature of a reaction furnace to 1000 ℃, using calibrated high-purity carbon dioxide as reference gas, and using calibrated log delta 13CLM values (-35.0%mill and-29.7%mill) as a double-point correction standard; the corresponding lignin methoxy delta 2HLM value is measured by adopting a high-temperature pyrolysis mode, the temperature of a reaction furnace is 1400 ℃, high-purity hydrogen is used as reference gas, the H 3 + value is 4.5ppm/nA under the self-checking state, and the checked log delta 2HLM values (-307.5%mill and-210.1%mill) are used as double-point correction standards. The delta 13CLM value and the delta 2HLM value are respectively-33.8 per mill and-242.4 per mill. 9 sets of repeatability tests were performed, resulting in relative standard deviations of 0.30% and 0.12% for the delta 13CLM value and the delta 2HLM value, respectively. FIG. 1 is a graph of condition optimization for the measurement of the biodegradation lignin methoxy delta 13CLM (a) and delta 2HLM (b). Examples 1 to 3 set the Gas Chromatograph (GC) parameters measured by δ 13CLM and δ 2HLM, and the peak type is complete and has no shoulder seam and a strong timeliness of retention time as evaluation indexes. As can be seen from the results of FIG. 1, the TRACE 1310 type GC apparatus of Thermo Fisher was equipped with a TG-5MS column (30 m. Times.0.32 mm. Times.0.25 μm), the inlet temperature was set at 200℃and the temperature-raising program was: the initial temperature is 40 ℃, the temperature is kept for 3.8min, the temperature is increased to 80 ℃ at 20 ℃/min, the temperature is kept for 1min, the temperature is increased to 100 ℃ at 40 ℃/min, and the temperature is kept for 3min; the sample injection split ratio is 8-12:1, high-purity helium is used as carrier gas, and when the flow rate is 0.6-0.8 mL/min, the peak values of delta 13CLM and delta 2HLM obtained by detection are the most complete.
Example 4
The embodiment provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
Drying humus soil in the shade and removing impurities, obtaining homogenized soil through a 2mm screen, finally grinding the homogenized soil by a ball mill to obtain a fine powder sample with the particle size of 80 mu m, and drying the fine powder sample to constant weight.
Mixing the second double-phase extraction system with a fine powder sample according to the mass ratio of 3:1, adding copper oxide with the same mass as the fine powder sample, adjusting the pH value to 8, sealing the microwave reaction at 60 ℃ for 60min and quenching the reaction at-2 ℃ to obtain a second reaction product.
The first reaction product was extracted 2 times with ethyl acetate, and the mass to volume ratio of the first reaction product to ethyl acetate was 1:3 for each extraction. The extracted organic phase is dried by anhydrous sodium sulfate and is subjected to reduced pressure rotary evaporation to obtain the lignin phenol and aldehyde derivative mixture with the meta-methoxy group reserved.
Referring to the method for manufacturing lignin methoxy standard curve and the standard curve provided in comparative example 3, lignin methoxy content is 8.2%.
The method for detecting the intermediate methoxy group of the lignin phenol/aldehyde derivative mixture obtained in the above is the same as that for detecting the meta methoxy group of the lignin monomer in example 1. The delta 13CLM value and the delta 2HLM value are respectively-33.2 per mill and-227.9 per mill. 9 sets of repeatability tests were performed, yielding relative standard deviations of 0.11% and 0.34% for the delta 13CLM and delta 2HLM values, respectively.
Example 5
The embodiment provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
Drying 5cm surface soil in the shade and removing impurities, obtaining homogenized soil through a 2mm screen, finally grinding through a ball mill to obtain a fine powder sample with the particle size of 100 mu m, and drying to constant weight.
Mixing the second double-phase extraction system with a fine powder sample according to the mass ratio of 9:1, adding copper oxide with the same mass as the fine powder sample, adjusting the pH value to 8.5, sealing at 120 ℃ for 70min, and quenching at 0 ℃ to obtain a second reaction product.
The first reaction product was extracted 3 times with ethyl acetate, and the mass to volume ratio of the first reaction product to ethyl acetate was 1:3 for each extraction. The extracted organic phase is dried by anhydrous sodium sulfate and is subjected to reduced pressure rotary evaporation to obtain the lignin phenol and aldehyde derivative mixture with the meta-methoxy group reserved.
Referring to the method for manufacturing lignin methoxy standard curve and the standard curve provided in comparative example 3, lignin methoxy content is 12.4%.
The method for detecting the intermediate methoxy group of the obtained mixture of lignin phenol and aldehyde derivative is the same as that of the method for detecting the meta methoxy group of lignin monomer in example 2. The delta 13CLM value and the delta 2HLM value are respectively-33.5 per mill and-228.1 per mill. 9 sets of repeatability tests were performed, yielding relative standard deviations of 0.15% and 0.37% for the delta 13CLM and delta 2HLM values, respectively.
Example 6
The embodiment provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
Drying the soil with the section of 40cm in the shade and removing impurities, obtaining homogenized soil through a 2mm screen, finally grinding the homogenized soil through a ball mill to obtain a fine powder sample with the particle size of 150 mu m, and drying the fine powder sample to constant weight.
Mixing the second double-phase extraction system with a fine powder sample according to the mass ratio of 15:1, adding copper oxide with the same mass as the fine powder sample, adjusting the pH to 9, carrying out sealing reaction for 80min at 180 ℃, and quenching reaction at 2 ℃ to obtain a second reaction product.
The first reaction product was extracted 3 times with ethyl acetate, and the mass to volume ratio of the first reaction product to ethyl acetate was 1:3 for each extraction. The extracted organic phase is dried by anhydrous sodium sulfate and is subjected to reduced pressure rotary evaporation to obtain the lignin phenol and aldehyde derivative mixture with the meta-methoxy group reserved.
Referring to the method for manufacturing lignin methoxy standard curve and the standard curve provided in comparative example 3, lignin methoxy content is 6.8%.
The method for detecting the intermediate methoxy group of the lignin phenol/aldehyde derivative mixture obtained in the above is the same as that for detecting the meta methoxy group of the lignin monomer in example 3. The delta 13CLM value and the delta 2HLM value are respectively-33.6 per mill and-228.5 per mill. 9 sets of repeatability tests were performed, yielding relative standard deviations of 0.15% and 0.29% for the delta 13CLM and delta 2HLM values, respectively.
Comparative example 1
The embodiment provides a method for detecting the lignin content of a biodegradable material.
The pretreatment and biphasic extraction system of litters from the fallen leaves layer of the dried branches in this comparative example was the same as in example 1, except that the standard curve was prepared using the obtained lignin material.
10.0+ -0.5 Mg of lignin substance is weighed and added into an acetyl bromoacetic acid solvent containing 5mL of fresh preparation volume fraction of 25%, the reaction is carried out by heating and magnetically stirring in a water bath at 70 ℃ under a closed state for 60 mm, then the reaction system is cooled to room temperature, 5mL of 2moL/L aqueous sodium hydroxide solution, 5mL of acetic acid and 1mL of 7.5moL/L hydroxylamine are sequentially added, the volume is fixed to 20mL by acetic acid, the same operation without adding a sample is taken as a blank, and 2.5mg, 5mg, 10mg, 15mg and 20mg of alkaline lignin (purchased from Shanghai Ala Biochemical technology Co., ltd.) are taken as a control group for preparing a standard curve.
And measuring the absorbance at 280nm by using an enzyme-labeled instrument, wherein the linear fitting degree of a standard curve is R 2 =0.995, and the total lignin content in the corresponding extract is converted by using the standard curve. The standard curve is shown in fig. 2.
Comparative example 2
The comparative example provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
The pretreatment and detection method for litters of the fallen leaves of the branches are the same as in example 1, except that ionic liquids such as lithium chloride hydrate (licl.5h 2 O), zinc chloride hydrate (zncl.3h 2 O), lithium bromide hydrate (libr.3h 2 O), calcium thiocyanate hydrate (Ca (NCS) 2·3H2 O) and other inorganic molten salt hydrates are respectively mixed with the preferred ionic liquid 1-ethyl-3-methylimidazole ferric chloride ([ Emim ] FeCl 4) in equal proportion to form a biphasic extraction system of the biodegradable precursor lignin, and the mass ratio of the first biphasic extraction system to the fine powder sample is adjusted to be 4:1, 6:1, 8:1, 10:1, 12:1 and 15:1 respectively. The lignin content was obtained by passing the obtained lignin material through the standard curve provided in comparative example 1. The detection results are shown in Table 1.
TABLE 1 different ionic liquid systems and their application ratios to lignin content in biodegradable precursors
From the results in Table 1, it is clear that the ZnCl 4H 2O&[Emim]FeCl4 system composed of zinc chloride hydrate with higher solubility and 1-ethyl-3-methylimidazole ferric chloride has higher extraction efficiency on lignin in lignocellulose biomass, and both the G and S lignin monomers rich in methoxy groups are reserved. Slowly dripping 1.5mol/L dilute hydrochloric acid at normal temperature according to the mass ratio of the first biphasic extraction system to the fine powder sample of 6:1 to form an acidic medium, and performing microwave extraction at 80 ℃ for 10min to realize extraction of the lignin content of the crude extract reaching more than 85%.
Comparative example 3
The comparative example provides a method for detecting the meta-methoxy content of the biodegradable lignin.
The pretreatment and biphasic extraction system for humus soil of this comparative example was the same as in example 4, except that the standard curve was made using the resulting mixture of lignin phenol and aldehyde derivative retaining meta-methoxy groups.
The ionization color reaction of aromatic phenol, aldehyde group and ferric trichloride and copper hydroxide is used as a qualitative evaluation index for lignin purification derivative lignin phenol and aldehyde. The specific method for measuring the color reaction comprises the following steps: and adding a proper amount of lignin phenol and aldehyde derivative mixture into ferric trichloride and copper hydroxide solution respectively, oscillating and standing to obtain purple solution and red precipitate respectively, wherein the lignin phenol and aldehyde derivative mixture has a complete aromatic phenol and aldehyde structure, and can be used for further lignin methoxy selective organic substitution reaction.
Weighing 10.0+/-0.5 mg of lignin phenol and aldehyde derivative mixture, adding the mixture into a brown headspace liquid-phase vial containing 0.5mL of hydroiodic acid with the mass fraction of 55%, carrying out microwave reaction at 120 ℃ for 30min in a closed state, then standing at room temperature (22+/-1 ℃) for 40min, carrying out online separation and detection by using a headspace automatic sampler by using a gas chromatography mass spectrometer (GC-MS), and carrying out standard curve calibration conversion by taking solid vanillin as an external standard.
The conditions for GC-MS were: a TRACE 1310 GC instrument of Thermo Fisher is selected and provided with a TG-5MS chromatographic column (30 m multiplied by 0.32mm multiplied by 0.25 mu m), the steady-state headspace CH3I gas sample injection amount is 80 mu L, the temperature of a sample injection port is set to be 200 ℃, and the temperature rise program is as follows: the initial temperature is 40 ℃, the temperature is kept for 3.8min, the temperature is increased to 80 ℃ at 20 ℃/min, the temperature is kept for 1min, the temperature is increased to 100 ℃ at 40 ℃/min, and the temperature is kept for 3min; the split ratio of the sample injection is 12:1, the high-purity helium is used as carrier gas, the flow rate is 0.8mL/min, and the temperature of the detector is 250 ℃. The same procedure without adding samples was used as a blank, and 2.5mg, 5mg, 10mg, 15mg, 20mg of vanillin (purchased from Shanghai Ala Biotechnology Co., ltd.) was used as a control, and the linear fitting degree with the CH 3 I peak area standard curve was R 2 =0.996, and the lignin methoxy content in the corresponding extract was converted by the standard curve. The standard curve is shown in fig. 3.
Comparative example 4
The comparative example provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
The detection method of this comparative example was the same as that of example 2 except that the rest time in the derivation of the organic chemical reaction was set to 20min and 30min, and the detection results are shown in FIG. 4. And after the organic substitution reaction is finished, standing at room temperature for 20-30 min, wherein delta 13CLM and delta 2HLM values measured by the headspace methyl iodide gas are dispersed, and after 40min, delta 13CLM and delta 2HLM values measured by the headspace methyl iodide gas are stable, namely the headspace methyl iodide gas is considered to be in a steady state. FIG. 5 shows the lignin monomer content spectrum of the biodegradable precursor rich in methoxy groups under different derivative standing time, wherein a is 20min and b is 30min after standing, the content is obviously improved after 30min, the peak area is obviously higher than 20min, and the lignin monomer content of the biodegradable precursor tends to be stable when being combined with FIG. 4 and 40 min.
Comparative example 5
The comparative example provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
The detection method of this comparative example is the same as that of comparative example 2, except that the organic substitution reaction system was left to stand at 22℃for 20 minutes, and after the weak acid alkali salt sodium citrate was directly added to the headspace liquid vial via a syringe to neutralize the excess hydroiodic acid, the headspace methyl iodide gas was sampled and the corresponding delta 13CLM and delta 2HLM values were determined. As can be seen from the results in Table 2, after neutralization treatment, the values of delta 13CLM and delta 2HLM obtained by repeated measurement of the same batch of biodegradable organic substitution reaction system have large relative standard deviations, and the neutralization treatment may cause water vapor generation in the reaction system solution, thereby causing variation of detection peaks and free measurement values, namely, the neutralization treatment of excessive hydroiodic acid is considered to be an unnecessary operation procedure.
TABLE 2 repeatability of delta 13CLM and delta 2HLM values obtained by neutralization treatment of lignin methoxy organic substitution reaction system of biodegradable material
Comparative example 6
The comparative example provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
The detection method of the comparative example is the same as that of example 1, except that the ionic liquid zinc chloride tetrahydrate with the purity of 98% is used as an extraction system, and the lignin content in the extracted biodegradable precursor is only 65% of the content obtained by the preferable biphasic system through standard curve conversion provided by comparative example 1. The average values of delta 13CLM and delta 2HLM obtained by 9 groups of repeatability test detection are-25.2 per mill and-243.4 per mill respectively, and the corresponding relative standard deviation reaches 0.56 per mill and 1.52 per mill. This may be due to the fact that using only zinc chloride tetrahydrate as lignin extraction system, pectin-like material rich in methoxy functional groups in the withered and fallen leaves is retained in lignin extract and participates in headspace methyl iodide organic substitution reaction and methoxy stable isotope ratio determination, whereas gas chromatography-gas stable isotope ratio mass spectrometer is not capable of separating methyl iodide of non-lignin methoxy source, resulting in a large deviation of the detected delta 13CLM and delta 2HLM values.
Comparative example 7
The comparative example provides a method for detecting the methoxy stable isotope ratio of the biodegradable lignin.
The detection method of the comparative example is the same as that of example 4, except that ferrous ammonium sulfate hexahydrate with the purity of 99% is used as an extraction system, and the lignin methoxy content in the obtained biodegradable intermediate and/or final product is 1.2% through conversion of the lignin methoxy content standard curve provided in comparative example 3. The average values of delta 13CLM and delta 2HLM obtained by 9 groups of repeatability test detection are-35.6 per mill and-234.7 per mill respectively, and the corresponding relative standard deviation reaches 0.45 per mill and 1.26 per mill. The reason is that only ferrous ammonium sulfate hexahydrate is used as an extraction system, so that p-hydroxyphenylpropane lignin monomers which are easy to degrade and crack and do not contain meta-methoxy groups in humus soil samples are largely reserved, and Chuangji containing one methoxy group and syringyl lignin monomers containing two methoxy groups are difficult to extract and purify due to limited extraction efficiency, so that the content of methoxy substances which participate in headspace methyl iodide organic substitution reaction and methoxy stable isotope ratio measurement is low, and the detection limits of delta 13CLM and delta 2HLM corresponding to gas chromatography-gas stable isotope ratio mass spectrometers are difficult to capture or shoulder tailing is serious.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (7)
1. The method for detecting the methoxy stable isotope ratio of the biodegradable lignin is characterized by comprising the following steps of:
Pretreating a biodegradable material to obtain a fine powder sample, purifying the fine powder sample to obtain lignin substances, deriving the lignin substances through organic chemical reaction to obtain headspace methyl iodide gas, and measuring the headspace methyl iodide gas through a gas chromatography-gas stable isotope ratio mass spectrometer to obtain lignin methoxy stable isotope ratio;
The purification comprises the purification by taking a biodegradable precursor as a reaction substrate or the purification by taking a biodegradable intermediate and/or a final product as the reaction substrate;
the purification by taking the biodegradable precursor as a reaction substrate comprises the following steps: taking an equal proportion mixture of an ionic liquid zinc chloride hydrate and an ionic liquid 1-ethyl-3-methylimidazole ferric chloride as a first biphasic extraction system, mixing the first biphasic extraction system and the biodegradable precursor according to a mass ratio of 4-15:1, adjusting the pH value to 4-5 at 21-23 ℃, sealing the mixture at 80 ℃ for 10-20 min, quenching the reaction at-2 ℃ to obtain a first reaction product, extracting the first reaction product for 2-3 times by using ethyl acetate, and performing reduced pressure rotary evaporation to obtain lignin substances;
The purification by taking the biodegradable intermediate and/or the final product as a reaction substrate comprises the following steps: taking an equal proportion mixture of ionic liquid ferrous ammonium sulfate hexahydrate and ionic liquid 1-ethyl-3-methylimidazole ferric chloride as a second double-phase extraction system, mixing the second double-phase extraction system and the biodegradable intermediate and/or final product according to a mass ratio of 3-15:1, adding copper oxide with the same mass as the biodegradable intermediate and/or final product, adjusting pH to 8-9, sealing at 60-180 ℃ for microwave reaction for 60-80 min, -2 ℃ for quenching reaction, obtaining a second reaction product, extracting the second reaction product for 2-3 times by using ethyl acetate, and performing reduced pressure rotary evaporation to obtain lignin substances;
The biodegradable material is a biodegradable precursor, a biodegradable intermediate and/or an end product; the biodegradable precursor comprises withered branches, fallen leaves and layer fallen matters and garden gardening production waste; the biodegradable intermediate and/or end product comprises humus soil and sediment peat;
The lignin substance is lignin monomer and/or lignin phenol and aldehyde derivative mixture which retains meta-methoxy; the organic chemical reaction is derived by: mixing the lignin substance and hydroiodic acid according to the proportion of 8-12:3.65 in terms of mg/mu moL, performing light-shielding and sealing microwave reaction at 110-130 ℃ for 20-40 min, and standing at 21-23 ℃ for 40-60 min to obtain headspace methyl iodide gas;
the lignin methoxy stable isotope ratio is lignin methoxy stable carbon isotope ratio and stable hydrogen isotope ratio.
2. The method of detection according to claim 1, wherein the preprocessing comprises: drying and pulverizing the biodegradable material; the particle size of the fine powder sample is 80-150 mu m.
3. The method of detecting according to claim 1, wherein the determining comprises determining the lignin material, comprising: and (3) carrying out online separation on the headspace CH 3 I gas by a gas chromatograph, measuring and obtaining lignin methoxy stable carbon isotope ratio delta 13CLM by a high-temperature oxidation mode by combining a gas stable isotope ratio mass spectrometer, and measuring and obtaining lignin methoxy stable hydrogen isotope ratio delta 2HLM by a high-temperature pyrolysis mode.
4. The detection method according to claim 3, wherein 60-90 μl of CH 3 I gas is extracted from a steady-state headspace, CH 3 I on-line separation is performed by a gas chromatograph equipped with a TG-5MS column, the sample inlet temperature is set to 200 ℃, and the temperature-raising program is: the initial temperature is 40 ℃, the temperature is kept for 3.8min, the temperature is increased to 80 ℃ at 20 ℃/min, the temperature is kept for 1min, the temperature is increased to 100 ℃ at 40 ℃/min, and the temperature is kept for 3min; the sample injection split ratio is 8-12:1, the high-purity helium is used as carrier gas, and the flow rate is 0.6-0.8 mL/min.
5. The detection method according to claim 3, wherein the high-temperature oxidation mode adopts a reaction furnace temperature of 960-1100 ℃, and the relative standard deviation of lignin methoxy stable carbon isotope ratio delta 13CLM value is less than or equal to 0.15 per mill.
6. The detection method according to claim 3, wherein the high-temperature pyrolysis mode adopts a reaction furnace temperature of 1250-1500 ℃, the range of H 3 + values in a self-detection state is 4.3-4.5 ppm/nA, and the relative standard deviation of the lignin methoxy stable hydrogen isotope ratio delta 2HLM value obtained by the high-temperature pyrolysis mode is less than or equal to 0.50 per mill.
7. The detection method according to claim 1, wherein the ionic liquid-like zinc chloride hydrate is zinc chloride tetrahydrate, and the purity is more than or equal to 98%; the purity of the ionic liquid ferrous ammonium sulfate hexahydrate is more than or equal to 99 percent.
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