Characterization method of lignin in environmental sample

Abstract

The invention discloses a characterization method of lignin in an environmental sample, and belongs to the technical field of lignin detection.The characterization method comprises the steps that a CuO / NaOH system is adopted for oxidizing the sample, supernate is acidified and extracted with diethyl ether, diethyl ether extract liquor is further purified with a pretreated silica gel column, and the lignin in the environmental sample is obtained; the method comprises the following steps: carrying out methylation reaction on a sample by using tetrabutyl ammonium hydroxide / methyl iodide in dichloromethane, neutralizing a methylation product mixture, diluting by using diethyl ether, and purifying by using a silica gel column reaction mixture. Most impurities in a sample are removed by combining diethyl ether extraction and silica gel column chromatography, and a solvent can be rapidly removed by reducing pressure, so that the operation complexity is greatly reduced. And the time consumed by gas chromatography maintenance is greatly reduced. The main operation is carried out under the condition of no protective gas, so that the operation complexity is greatly reduced. The methylated lignin phenol is stable to air and water and does not change obviously after being stored in a freezing chamber for one week. The method does not limit the quantity of analysis which can be performed at the same time, and is suitable for conventional lignin characterization of geochemical samples with high lignin content.

Description

Technical Field

[0001] The invention belongs to the technical field of lignin detection, and particularly relates to a method for characterizing lignin in an environmental sample. Background Art

[0002] Lignin is an aromatic macromolecule that comes only from vascular plants and is one of the main organic components in environmental samples. Plant litter is the main source of lignin in soil, natural water bodies and sediments. Lignin is mainly composed of three subunits: pinoresinol, abietol and p-coumarol, which are linked together by hydrolysis-resistant carbon-carbon bonds and carbon-oxygen bonds. Therefore, lignin in soil changes slowly under the influence of biological and environmental parameters (such as pH, humidity, temperature) and contributes to the global carbon cycle.

[0003] Copper oxide oxidation of lignin produces 11 lignin phenol monomers. They are divided into four types: vanillylphenol (V type), eugenol (S type), p-hydroxyphenol (H type) and cinnamylphenol (C type). Each of the V type, S type and H type contains an aldehyde, a ketone and an acid; usually, there are more aldehyde phenols than ketophenols and acid phenols in the same type. The relative content of each type of phenol in a sample is determined by the plant source (Fate of lignins in soils: A review). For example, gymnosperm wood contains about 80% V type phenol and a small amount of C type phenol; in contrast, angiosperm wood mainly produces approximately equal amounts of V type and S type phenol. Therefore, the relative contribution of the three types of phenols (such as S / V and C / V ratios) is often used to characterize the lignin in a sample. On the other hand, as lignin changes (such as biodegradation), the composition of phenols in a sample also changes. Compared with phenols containing aldehyde groups in V type and S type phenols, the increase of phenols containing carboxylic acid groups leads to an increase in the acid / aldehyde ratio in these two types of phenols. During lignin degradation, syringyl and cinnamyl units (S- and C-types) are lost faster than guaiacyl units (V-type), resulting in a decrease in the S / V and C / V ratios. Since H-type phenols are of non-lignin origin, they are rarely used as geochemical tracers. Therefore, accurate characterization of lignin provides important information on the properties of geochemical samples. CuO / NaOH aqueous solutions are traditionally used as oxidizing agents to depolymerize lignin into phenols, such as Figure 1As shown. The resulting lignin phenolic mixture needs to be purified and derivatized into a form suitable for gas chromatography (GC) analysis. Hedges et al. purified the mixture by ether extraction, and for the first time used a mixture of N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) and 1% trimethylchlorosilane (TMCS) to derivatize phenol into trimethylsilyl derivatives, and analyzed its composition by capillary GC equipped with a flame ionization detector (FID) (Characterization of lignin by gascapillary chromatography of cupric oxide oxidation products). and Bochter (Characterization of lignin in forest humus layers by high-performance liquid chromatography of cupric oxide oxidation products) improved the method by extracting phenols with C18 solid phase extraction (SPE) instead of ether. Many research groups now use C18 columns to extract phenols because this method produces simpler chromatograms than those obtained by solvent extraction.

[0004] However, the recovery efficiency of the C18 column has been questioned because Kogel et al. only reported the recovery of vanillin, while the recovery of other monomers remains unknown. Kaiser et al. used a polymer-based SPE (copolymer of N-vinylpyrrolidone and divinylbenzene) instead of a C18 column to extract monomeric lignin phenols. The recovery of all lignin phenols was very good, ranging from 87% to 90%. Ting et al. compared the recovery of lignin monomers using C18 SPE columns and polymer-based SPE columns and found that the C18 column had a lower recovery of carboxyl-containing monomers (such as syringic acid and ferulic acid). Perhaps due to concerns about the low recovery of the C18 column, some researchers still use ethyl acetate or ether to purify the mixture. However, the C18 column is still widely used to purify lignin. In contrast, Kaiser's polymer-based SPE method is rarely used.

[0005] Extraction with organic solvents results in the concentration of other soluble compounds along with ligninphenol. BSTFA / TMCS is a highly active derivatization reagent that reacts not only with the phenolic hydroxyl and carboxyl groups of ligninphenol, but also with the alcoholic hydroxyl groups of impurities, resulting in complex volatile mixtures and complex chromatograms. Complex chromatograms pose challenges to the identification and quantification of products; complex components can contaminate the inlet, capillary column, and FID detector of the gas chromatograph, as well as the vacuum chamber and ion source of the mass spectrometer. In addition, the trimethylsilane (TMS) derivative of ligninphenol is sensitive to moisture and undergoes isomerization and decomposition at room temperature. Typically, samples should be analyzed by GC or GC-MS immediately after derivatization. The immediacy of analysis limits the analytical throughput of samples, as leaving samples on the rack of the autosampler overnight may result in decomposition.

[0006] Thermally assisted hydrolysis and methylation (THM) is another widely studied method for characterizing lignin in environmental samples. Lignin is depolymerized in the presence of tetramethylammonium hydroxide (TMAH) and the resulting lignin monomers are methylated at 250–770 °C. Due to different depolymerization mechanisms, the TMH process produces 18 methylated lignin monomer phenols, some of which are different from the phenols produced by CuO oxidation. For most of these methylated monomers, there are no commercially available standards, which makes the determination of retention times and response factors difficult. High temperatures also produce complex chromatograms (Characterisation of wood by pyrolysisderivatisation-gas chromatography / mass spectrometry), and only MS detectors can be used to characterize lignin. In addition, many factors in the THM process, including TMAH concentration, solvent, and temperature, affect the distribution of products (Influence of parameters on pyrolysis-GC / MS of lignin in the presence oftetramethylammonium hydroxide). Comparative studies have shown that the CuO oxidation process provides more accurate lignin information than the THM process (Comparison of two thermochemolytic methods for the analysis of lignin in decomposing gymnosperm wood: the CuO oxidation method and the methodof thermochemolysis with tetramethylammonium hydroxide (TMAH)). Those skilled in the art are in urgent need of developing a method for characterizing lignin in environmental samples with high sample throughput and low maintenance costs. Summary of the invention

[0007] In view of this, the present invention provides a method for characterizing lignin in environmental samples. The present invention discloses for the first time a new derivatization method for converting lignin monomers obtained from CuO oxidation. These monomers are quantitatively methylated with tetrabutylammonium hydroxide / methyl iodide in dichloromethane. The stable methylated product can be purified by silica gel column chromatography, making the gas chromatogram of the sample simpler, making GC-FID or GC-MS analysis possible and reliable, and using toluene as a solvent to dissolve the sample and setting appropriate programmed temperature conditions can significantly reduce the tailing phenomenon of the chromatographic peak and reduce the burden of instrument maintenance. In addition, the stable product can be analyzed at any time, thereby improving the sample throughput.

[0008] Copper oxide oxidation of lignin produces 11 lignin phenol monomers. They are divided into four types: vanillylphenol (V type), eugenol (S type), p-hydroxyphenol (H type) and cinnamylphenol (C type), as shown in the attached Figure 1 shown.

[0009] A method for characterizing lignin in an environmental sample comprises the step of performing an alkylation reaction on the environmental sample after a purification step using tetraalkylammonium hydroxide / alkylating agent in an organic solvent, and further comprising the following steps: (1) oxidizing the environmental sample using a CuO / NaOH reaction system; (2) purifying: extracting an acidified aqueous solution with ether, and further purifying the ether extract with a pretreated silica gel column to obtain a purified environmental sample; (3) diluting an alkylation product mixture with ether, and then performing subsequent purification on the reaction mixture with a silica gel column to prepare a sample solution; (4) injecting the sample solution into a gas chromatograph or a gas chromatograph-mass spectrometer for analysis and detection; wherein the alkylation reaction is between step (2) and step (3).

[0010] The organic solvent is selected from dichloromethane or 1,2-dichloroethane; the alkylating agent is selected from one of methyl iodide, ethyl iodide, ethyl bromide, dimethyl sulfate and diethyl sulfate; these alkylating agents can all undergo quantitative alkylation reaction with ligninphenol, but methyl iodide has the fastest alkylation reaction rate and relatively low toxicity, so methyl iodide is preferred;

[0011] The tetraalkylammonium hydroxide is selected from one of tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, tetrapentylammonium hydroxide and tetrahexylammonium hydroxide; these reagents can play the same role of phase transfer catalyst in the alkylation reaction to achieve the alkylation reaction of ligninphenol, but tetrabutylammonium hydroxide has the lowest price, so tetrabutylammonium hydroxide is preferred.

[0012] As an example, the organic solvent is dichloromethane; the alkylating agent is selected from iodomethane; and the tetraalkylammonium hydroxide is tetrabutylammonium hydroxide.

[0013] As a preferred embodiment of the present invention, a method for characterizing lignin in an environmental sample comprises the steps of methylating the purified environmental sample with tetrabutylammonium hydroxide / methyl iodide in dichloromethane, and further comprising the following steps:

[0014] (1) The environmental sample is oxidized using a CuO / NaOH reaction system; (2) Purification: The acidified aqueous solution is extracted with ether, and the evaporated sample is further purified using a pretreated silica gel column to obtain a purified environmental sample; (3) The methylation product mixture is extracted and purified with ether, and the concentrated sample is subsequently purified using a silica gel column to prepare a sample solution; (4) The sample solution is injected into a gas chromatograph for analysis and detection.

[0015] Further, the environmental sample was methylated with tetrabutylammonium hydroxide / methyl iodide in dichloromethane. The methylation of lignin phenol was carried out in a 5 mL Hungate vial, which was dried under vacuum and filled with nitrogen. 250-500 μL of a 40% aqueous solution of tetrabutylammonium hydroxide, 0.5 mL of dichloromethane and 100 μL of methyl iodide were added to the vial in sequence, and the reaction was carried out at 28-30° C. for 90 min-12 h. The pressure generated in the vial was released by inserting a syringe through the diaphragm. After neutralization with 50 μL of 1 mol / L hydrochloric acid, the mixture was evaporated and concentrated on a rotary evaporator, 1 mL of water was added to the vial, and the mixture was extracted with 1 mL of ether. The extract was passed through an anhydrous magnesium sulfate column, and the extraction process was repeated 4 times. Rinse the desiccator with an additional 5 mL of ether, receive the ether solution in a 25 mL flask, concentrate the ether solution to dryness under reduced pressure on a rotary evaporator at room temperature, and purify the residue in the flask by passing it through a 1 g silica gel column. The silica gel column has been pre-rinsed with water, methanol, ether, and n-hexane. First, rinse the flask with 1 mL of n-hexane and pass the n-hexane solution through the column. Repeat this process seven times, discard the eluent, then wash the flask with 1 mL of ether, pass the ether solution through the column, repeat the ether wash four times, and then wash the silica gel column with an additional 6 mL of ether. Receive the eluent in a 25 mL flask and concentrate it to about 0.5 mL on a rotary evaporator. Transfer the residue to a 5 mL vial with ether and concentrate it again to dryness. Dissolve the residue in the vial with 200 microliters of internal standard solution and mix well.

[0016] As another preferred embodiment, the environmental sample is methylated with tetrabutylammonium hydroxide / methyl iodide in dichloromethane, and the methylation of ligninphenol is carried out in a 5 mL Hungate vial, which is filled with nitrogen after drying under vacuum, and 100-500 μL of a 40% mass fraction tetrabutylammonium hydroxide aqueous solution, 0.3-1.5 mL of dichloromethane and 50-100 μL of methyl iodide are added to the vial in sequence, and the reaction is carried out at 26-30°C for 90 min-12 h. After neutralization with 50 μL of 1 mol / L hydrochloric acid, add 1.5 mL of ether, stir for 30 seconds, and then transfer the slurry to the upper part of the purification column. The filler of the purification column consists of a desiccant located at the upper part of the column and silica gel located at the lower part of the column. Use a brown glass bottle to receive the effluent; wash the reaction bottle with 2 mL of ether twice, and add the washing liquid to the upper part of the purification column, and apply pressure to make the solution flow through the column; wash the purification column with 4.5 mL of ether; and concentrate the solution in the receiving bottle to dryness using a rotary evaporator. Add a certain volume of toluene solution of ethyl 2-iodobenzoate to the bottle and mix well. The sample can be analyzed by GC-MS or GC-FID. If the sample impurities interfere with the GC-FID chromatographic peak, the concentrated sample can be further purified according to the following method and then added toluene solution of ethyl 2-iodobenzoate, and then analyzed by GC-FID: prepare a column with 2g of silica gel, wash the concentrated sample with 4mL of n-hexane 4 times, pass the washing liquid through the silica gel column, then wash the silica gel column with 4mL of n-hexane, and discard all the effluent. Wash the concentrate with 1.5mL of ether 3 times, pass the washing liquid through the silica gel column, and finally wash the silica gel column with 4mL of ether, and collect all the ether solution in a dark brown glass bottle; concentrate the ether solution to dryness under reduced pressure; add toluene solution of ethyl 2-iodobenzoate, and then analyze by GC-FID.

[0017] Furthermore, the environmental sample in step (1) is one of plants, water, soil, sediment, sewage, and sludge.

[0018] Optionally, when the environmental sample is a soil sample, the specific steps of the characterization method are as follows:

[0019] 1. Alkaline copper oxide oxidation

[0020] Add 100 mg Fe(NH4)2(SO4)2·6H2O, 1.000 g CuO powder, 0.500 g soil sample, and a stirrer into a stainless steel reactor. If the organic carbon content is less than 5 mg, add 10-20 mg glucose.

[0021] Place the reactor and lid in a nitrogen glove box, and use a pipette to add 7-20 mL of 2M NaOH solution to the reactor. Take the reactor out of the glove box and place it on a stirrer. Heat the temperature to 150-170°C over 30 minutes while stirring. Keep the temperature constant for 120-180 minutes after reaching the set temperature. Cool the reactor with running tap water for 5 minutes after reaching the set time, or remove the heating device and cool it naturally and stir overnight (operation time 40 minutes).

[0022] 2. Purification

[0023] Open the lid of a reactor, quickly add 250μL (48ng / μL) of 2M NaOH solution to recover the internal standard (2-bromo-4-methoxybenzoic acid), and cover tightly. Centrifuge the reactor (7000rpm, 1min), and quickly pour the supernatant into a centrifuge tube filled with concentrated hydrochloric acid. Immerse 1 / 3 of the centrifuge tube in an ice water bath. Add 2mL of 0.5-2M NaOH solution or water to the inner wall of the reactor, tighten the lid, and stir vigorously for 1-5min. Centrifuge and pour the supernatant into a centrifuge tube. Repeat the above washing twice (a total of 3 times).

[0024] The aqueous solution in the centrifuge tube was acidified to pH 1-1.5 with 6M HCl.

[0025] Extract the aqueous solution with 11 mL of ether. If the interface layer is emulsified, centrifuge to accelerate the separation. Add the ether extract to the purification column. Repeat the ether extraction 2 times (3 times in total).

[0026] The purification column consists of two columns connected in series, the upper column is filled with 3-5g of anhydrous sodium sulfate, the lower column is filled with 0.5-1g of silica gel or diatomaceous earth, and the surface of the silica gel or diatomaceous earth is covered with glass wool. A brown eggplant-shaped bottle is used to receive the outflow solution of the purification column.

[0027] Wash the desiccant with 6 mL of ether. Remove the sodium sulfate drying column on the top. Wash the silica gel column with 3 mL of ether / methanol (9:1, v / v) solution. Combine all the effluent with the ether extract.

[0028] The ether solution was concentrated to dryness using a rotary evaporator.

[0029] The concentrated residue was transferred into a reaction tube with ether and concentrated again to dryness.

[0030] Add a stirrer and about 20 mg of ferrous ammonium sulfate into the reaction tube and dry under vacuum at 26-30°C for 5-20 min.

[0031] (Operation time 3h).

[0032] 3. Methylation Derivatization

[0033] The reaction bottle was transferred to a nitrogen glove box, and 100-500 μL of tetramethylammonium hydroxide (TBAOH,

[0034] 40 wt% aqueous solution), 0.3-1.5 mL dichloromethane and 100 μL MeI. Stir at 26-30°C for 90-120 min

[0035] (rotation speed 600rpm).

[0036] 4. Separation of methylation products

[0037] Add 50 μL 1M HCl to the reaction bottle and stir for 10 seconds. Add 1.5 mL ether and stir for 1 minute. Add the resulting slurry to the top of the purification column. Collect the effluent in a brown glass bottle.

[0038] The lower part of the purification column is 0.5g of silica gel, and the upper part is a desiccant, which can be magnesium sulfate, sodium sulfate or calcium chloride. Wash the reaction bottle twice with 2mL of ether, and the washing liquid also passes through the purification column. Wash the purification column with 4-5mL of ether. Combine all the outflows.

[0039] The ether solution was concentrated to dryness under reduced pressure (operation time 30 min).

[0040] Furthermore, the conditions for the gas chromatograph analysis in step (5) are as follows: the temperature of the injection port and the detector is 300° C., the chromatographic column is a capillary chromatographic column, the capillary chromatographic column is an SE-54 capillary column, with a length of 50 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 mm, the stationary phase is 5% phenyl and 95% methylpolysiloxane, the detector is a hydrogen flame ionization detector, the carrier gas is nitrogen with a purity of >99.999%, the carrier gas flow rate is 1 mL / min, the injection volume is 1.0 to 2.0 μL, and the split ratio is 2:1.

[0041] Furthermore, the gas chromatograph used in step (5) is a Shimadzu GC-2014C gas chromatograph.

[0042] Furthermore, the heating program of the gas chromatograph in step (5) is: starting temperature 50°C, heating to 170°C at a heating rate of 4°C / min and maintaining for 20 min, then heating to 270°C at a heating rate of 10°C / min and maintaining for 20 min.

[0043] Furthermore, the oxidation reaction in step (1) is carried out in a cylindrical stainless steel reaction container with a total capacity of 10 mL, the reaction container is equipped with a polytetrafluoroethylene magnetic stirrer, 500 mg of copper oxide, 200 mg of ammonium ferrous sulfate hexahydrate and 500 mg of an environmental sample, the reaction container is transferred to a plastic bag, connected to a vacuum nitrogen pipeline and the plastic bag is sealed, evacuated and backfilled with nitrogen four times, 7 mL of a 2 mol / L NaOH solution is added to the reaction container, the mixture in the reaction container is stirred at 1800 rpm, the temperature is maintained at 40° C. for 30 min, increased to 170° C. at 5° C. / min, and maintained for 180 min, and then the reaction container is cooled with tap water for 10 min.

[0044] Furthermore, in step (2), the reaction vessel is opened, and 200 μL of an aqueous solution of ethyl vanillin in 2 mol / L NaOH is immediately injected therein, the reaction vessel is closed and magnetically stirred at 1800 rpm for 30 s, each reaction vessel is centrifuged at 9000 rpm for 60 s, the supernatant is quickly poured into a 50 mL plastic centrifuge tube, 1 mL of concentrated hydrochloric acid and a stirrer are pre-added to the centrifuge tube, and the bottom 1 / 3 of the centrifuge tube is immersed in ice water, 2 mL of a 0.5 mol / L NaOH solution is injected along the inner wall of each reaction vessel, the lid is tightened by hand and rotated horizontally for 30 s, the mixture in the container is stirred at 1800 rpm for 60 s, and then each container is centrifuged again, and the supernatant is added to the centrifuge tube, the washing process is repeated twice, and the solution in the centrifuge tube is acidified to a pH of 6. 2. Extract the acidified solution with 8 mL of ether and centrifuge at 4500 rpm for 60 s. Transfer the ether layer to a 50 mL conical flask with a glass pipette. Repeat the extraction process three times. Dry the ether solution with anhydrous sodium sulfate for 10 min. Filter the ether extract through a layer of glass wool into a 50 mL evaporating flask. Wash the conical flask with 2 mL of ether three times and concentrate to dryness on a rotary evaporator at room temperature. During evaporation, the flask can be immersed in a water bath below 25°C. Purify the residue in the flask with a 0.5 g silica gel column. Wash the residue in the flask with ether and add the solution to the top of the silica gel column. Repeat the washing three more times. Wash the column with an additional 7 mL of ether. Collect the eluate in a 25 mL flask and concentrate it on a rotary evaporator at room temperature. Transfer the concentrated residue to a 5 mL Hungate reaction vial with ether. Concentrate the solution and further dry it under vacuum for 20 min after adding a stir bar.

[0045] As another preferred embodiment, in step (2), the reaction vessel is opened, 200 μL of a solution containing 2 mol / L NaOH and 50 ng / μL 2-bromo-4-hydroxybenzoic acid is immediately injected therein, the reaction vessel is closed and magnetically stirred at 7000 rpm for 1 min, 1.5 mL of concentrated hydrochloric acid and a stirrer are pre-added in a centrifuge tube, and the bottom 1 / 3 of the centrifuge tube is immersed in ice water, the supernatant is added to the centrifuge tube, 2 mL of a 1 mol / L NaOH solution is injected along the inner wall of the reaction vessel, the lid is tightened by hand and magnetically stirred for 2 min, centrifugation is performed again, and the supernatant is added to the centrifuge tube, the washing process is repeated twice, and the solution in the centrifuge tube is acidified to pH 5 with 6 mol / L hydrochloric acid. 1-1.5, extract the acidified solution with 11 mL of ether, stir for 2 minutes and let stand, transfer the ether layer to the upper part of the purification column with a glass pipette after complete stratification, pressurize the ether to make it flow through the purification column, and use a brown flask to receive the effluent, repeat the extraction process twice, wash the purification column with 6 mL of ether, remove the drying column, and wash the silica gel column with 3 mL of ether / methanol solution (9:1, v / v); concentrate the ether solution with a rotary evaporator, and flush with nitrogen at the end; transfer the residue in the flask with ether to a 5 mL reaction bottle, and concentrate it to dryness again; and further dry it under vacuum for 5-20 minutes after adding a stirring bar.

[0046] Furthermore, the methylation reaction of lignin phenol was carried out in a 5 mL Hungate vial. After drying under vacuum, nitrogen was filled into the vial, 250 μL of a 40% aqueous solution of tetrabutylammonium hydroxide by mass, 0.5 mL of dichloromethane and 100 μL of iodomethane were added to the vial in sequence, and the reaction was carried out at 28° C. for 12 h. The pressure generated in the vial was released by inserting a syringe through the diaphragm, and 50 μL of 1 mol / L After neutralization with HCl, the mixture was concentrated by evaporation under reduced pressure, 1 mL of water was added to the vial, the mixture was extracted with 1 mL of ether, the extract was passed through an anhydrous magnesium sulfate column, the extraction operation was repeated four times, the desiccant was washed with an additional 5 mL of ether, the eluted solution was received in a 25 mL flask, and the ether solution was concentrated to dryness using a rotary evaporator at room temperature, the residue in the flask was purified by passing through a 1 g silica gel column, the silica gel column was previously washed with water, methanol, ether and n-hexane, the flask was first washed with 1 mL of n-hexane, and the n-hexane solution was passed through the column, the process was repeated seven times, the eluate was discarded, and the flask was then washed with 1 mL of ether, and the ether solution was passed through the column, the ether wash was repeated four times, the column was eluted with an additional 6 mL of ether, the eluate was received in a 25 mL flask and concentrated using a rotary evaporator, the residue was transferred to a 5 mL vial with ether and concentrated again, the residue in the vial was dissolved and mixed with 200 μL of internal standard solution.

[0047] Beneficial effects of the present invention:

[0048] (1) The characterization method of the present invention has good repeatability and reliability: the relative deviations of 7 repeated characterizations, except for CAD and FAD (10%), are all less than 5%.

[0049] (2) The purification method has a good recovery rate: the recovery rate of all lignin phenols is between 70-92%.

[0050] (3) The derivatization reaction is mild and fast: it takes only 90-120 minutes to react at room temperature.

[0051] (4) High yield of derivatization reaction: Sample impurities have little effect on the yield of derivatization reaction.

[0052] (5) Suitable for high-throughput analysis: This method can use equipment similar to the SPE high-throughput integrated tube device, and one device can complete the analysis of 20 samples in one day.

[0053] (6) High time efficiency: The two stages of sample preparation only take about 3.5 hours.

[0054] (7) Low cost of standard reagents: The compounds used to determine the relative response factor are stable, widely available, and inexpensive (~30 yuan / 25g). The price of each lignin phenol standard used to determine the relative response factor by the traditional method is 1440 yuan / 10 mg (Sigma-Aldrich).

[0055] (8) The price of derivatization reagents is low: the price of tetrabutylammonium hydroxide and methyl iodide is much lower than that of the silylation reagent bis(trimethylsilyl)trifluoroacetamide (25 mL, price ~1500 yuan).

[0056] (9) Very weak chromatographic peak tailing: The analytical interference and instrument maintenance problems caused by the contamination of the sample matrix at the gas chromatography inlet are difficult problems that traditional lignin characterization methods often need to face and solve. However, the mass chromatogram obtained by the characterization method based on lignin phenol methyl derivatives is less affected by matrix contamination.

[0057] The results of the comparative study with the traditional lignin characterization method showed that the lignin content measured by the new method was higher than that measured by the traditional method, up to 6 times the value measured by the C18-SPE purification method and 3 times the value measured by the liquid-liquid extraction purification method. Therefore, the lignin content measured by the traditional lignin characterization method is unreliable.

[0058] In particular, the best results were obtained with tetrabutylammonium hydroxide / methyl iodide in dichloromethane. Specifically, the samples were first oxidized with CuO / NaOH. A specially designed stainless steel reaction vessel was used, and the reaction was conveniently carried out with magnetic stirring. The second step was the purification of the CuO oxidation product. The acidified aqueous solution was extracted with ether, and the evaporated sample was further purified with a pretreated silica gel column. Too much silica gel could not be used due to concerns about the partial loss of carboxyl-containing lignin phenol due to tailing effects. The third step was the methylation of the sample with tetrabutylammonium hydroxide / methyl iodide in dichloromethane. Although we found that trace amounts of ethyl acetate could hinder the conversion of lignin phenol to the methylated form, the impurities remaining after the purification of the tested soil samples did not interfere with the methylation reaction. In contrast, the derivatization of lignin phenol using traditional BSTFA / TMCS is sensitive to compounds containing active protons, such as water and alcohols. Finally, the methylation product mixture was purified by extraction with ether, and the concentrated sample was subsequently purified with a silica gel column. The use of diethyl ether is crucial to shorten the overall operation time, as it can be removed quickly and safely by rotary evaporation.

[0059] Ether extraction and silica gel column purification removed most of the impurities in the sample and gave a simplified chromatogram. Therefore, the time consumed by GC maintenance was significantly reduced. Except for the CuO oxidation reaction and the methylation reaction, all other operations were carried out without the protection of argon or nitrogen. Therefore, the complexity of the operation was greatly reduced. Methylated lignin phenol is stable to air and water, and we did not observe the decomposition of the toluene solution of methylated lignin phenol after storage in a freezer (-20°C) for one week; in contrast, the TMS derivative of lignin phenol decomposes due to the gradual introduction of water during storage, so it is necessary to analyze it within 24 hours. These new features of this method make it suitable for routine lignin characterization of geochemical samples with high lignin content. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The present invention is described in detail below in conjunction with the accompanying drawings:

[0061] Figure 1 are 11 lignin phenols obtained from CuO oxidation of geochemical samples;

[0062] Figure 2 is the chromatogram of the methylated phenol standard mixture shown in Table 1;

[0063] Figure 3 is a GC graph of a sample obtained after CuO oxidation of a forest soil sample, wherein a mixture of an acidified aqueous solution of lignin phenol and a methylated product is extracted and purified only with ether, and the peaks marked with vertical short lines are the peaks of the internal standard and lignin phenol;

[0064] Figure 4 This is a chromatogram of a forest soil sample that was oxidized with CuO / NaOH, the acidified aqueous solution was extracted with ether, and cleaned up with a silica gel column. The methylation product mixture was also extracted with ether and cleaned up with a silica gel column. DETAILED DESCRIPTION

[0065] Example 1

[0066] (1) Gas chromatography-flame ionization detector analysis:

[0067] A Shimadzu GC-2014C gas chromatograph equipped with a flame ionization detector was used. The sample was diluted with 200 μL of internal standard (1,4-diisopropylbenzene) toluene solution and mixed thoroughly. For samples with concentrations >0.1 mg / mL and <0.1 mg / mL, the injection volumes were 1 μL and 2 μL, respectively, and the split ratio for all samples was 2:1. The temperature of the injector and detector was set to 300 °C. The diaphragm purification flow rate was 3 mL / min. A straight quartz liner filled with undeactivated glass wool was used. A SE-54 capillary column (50 m long, 0.25 mm inner diameter, 0.25 mm film thickness) was used for separation, and the stationary phase was 5% phenyl and 95% methylpolysiloxane. Nitrogen with a purity of >99.999% was used as the carrier gas (flow rate of 1 mL / min). The temperature program of the gas chromatograph is: 50°C to 170°C, 4°C / min (hold for 20 min), to 270°C, 10°C / min (hold for 20 min). The identification of the product is carried out by comparing its retention time in the standard mixture solution. Quantification is calculated based on the GC peak area and response factor of the component relative to the internal standard. The response factor R for component x is f (x) is calculated according to the following equation:

[0068]

[0069] Among them C STD and C x Represent the concentrations of internal standard and component x, A STD and A x are the peak areas of the internal standard and component x, respectively. Therefore, the concentration of component x in the sample is C x It is calculated by the following equation:

[0070]

[0071] where the parameters have the same meanings as in the previous equation. f (x) Usually determined by injection of a standard mixture at the beginning of a GC analysis.

[0072] (2) Copper oxide oxidation:

[0073] The oxidation reaction used a specially designed cylindrical stainless steel reaction vessel. The lid of the vessel contained a polytetrafluoroethylene gasket (20 mm in diameter and 2 mm in thickness). The total capacity of the vessel was 10 mL. The reaction vessel was equipped with a magnetic stirring bar (14 × 7 mm) covered with polytetrafluoroethylene. 500 mg of copper oxide, 200 mg of Fe(NH4)2(SO4)2·6H2O, and 500 mg of soil sample were added to the vessel. The vessel was then transferred to a plastic bag (30 × 40 mm) and connected to a vacuum nitrogen line. The bag was sealed and then evacuated and backfilled with nitrogen four times. 7 mL of 2 mol / L NaOH solution was injected into the vessel, the lid was closed, and the vessel was removed from the bag. The mixture in the vessel was stirred at 1800 rpm. The temperature was maintained at 40 °C for 30 min, increased to 170 °C at 5 °C / min, and maintained for 180 min. The reaction vessel was cooled with tap water for 10 min. After each oxidation, the polytetrafluoroethylene gasket of the reaction vessel was replaced with a new one.

[0074] (3) Extraction with ether and purification with silica gel column

[0075] The reaction vessel was opened and immediately injected with 200 μL of an aqueous solution of ethyl vanillin in NaOH with a concentration of 2 mol / L. The vessel was closed and magnetically stirred at 1800 rpm for 30 s. The vessel was centrifuged at 9000 rpm for 60 s. The supernatant was quickly poured into a 50 mL plastic centrifuge tube containing 1 mL of concentrated hydrochloric acid and a stir bar (7×14 mm). The bottom 1 / 3 of the centrifuge tube was immersed in ice water. 2 mL of 0.5 M NaOH solution was injected along the inner wall of the container. The lid was tightened by hand and rotated horizontally for 30 s. The mixture in the container was stirred at 1800 rpm for 60 s. Each container was then centrifuged again and the supernatant was added to the centrifuge tube. The washing process was repeated twice. The solution in the centrifuge tube was acidified to pH 2 with 6 M hydrochloric acid. The acidified solution was extracted with 8 mL of ether and centrifuged at 4500 rpm for 60 s. The ether layer was transferred to a 50 mL Erlenmeyer flask using a glass pipette. The extraction process was repeated three times. Dry the ether solution over anhydrous sodium sulfate for 10 min. Filter the ether extract through a layer of glass wool into a 50 mL evaporating flask and wash the Erlenmeyer flask three times with 2 mL of ether. Concentrate the ether solution to dryness on a rotary evaporator at room temperature. During evaporation, the flask can be immersed in a water bath below 25°C.

[0076] The residue in the flask was purified with a 0.5 g silica gel column. The column was washed with water (2 mL x 2), methanol (2 mL x 3) and ether (2 mL x 3) in succession. The residue in the flask was washed with 0.7 mL ether and the solution was added to the top of the silica gel column. The flask was washed three times repeatedly. The column was then eluted with an additional 7 mL ether. The eluate was received in a 25 mL flask and concentrated with a rotary evaporator at room temperature. The residue was transferred to a 5 mL Hungate reaction vial with ether. The solution was concentrated and further dried under vacuum (100 Pa) for 20 min after adding a stirrer (5 x 10 mm) to remove any volatile components that may interfere with the subsequent methylation reaction.

[0077] (4) Methylation with tetrabutylammonium hydroxide / methyl iodide and purification with silica gel column:

[0078] The methylation of ligninphenol was carried out in a 5 mL Hungate reaction vial. After drying under vacuum, the vial was filled with nitrogen. 250 μL of tetrabutylammonium hydroxide (40% w / w aqueous solution), 0.5 mL of dichloromethane and 100 μL of iodomethane were added to the reaction vial in sequence. The reaction was allowed to proceed at 28 °C for 12 h. The pressure generated in the vial was released by inserting a syringe through the septum. After neutralization with 50 μL of 1 M HCl, the mixture was concentrated using a rotary evaporator. 1 mL of water was added to the vial and the mixture was extracted 5 times with 1 mL of ether. Each extract was dried over a 0.6 g anhydrous magnesium sulfate column. The desiccant was rinsed with an additional 5 mL of ether. The ether solution was received in a 25 mL flask and concentrated to dryness using a rotary evaporator at room temperature. The residue in the flask was purified by passing through a 1 g silica gel column. The silica gel column was previously washed with water (3 mL × 2), methanol (3 mL × 3), ether (3 mL × 3) and n-hexane (3 mL × 3). First, wash the flask with 1 mL of n-hexane and pass the n-hexane solution through the silica gel column. Repeat the n-hexane washing process seven times. Discard the eluent. Then wash the flask with 1 mL of ether four times, and pass the ether solution through the silica gel column each time. Elute the column with an additional 6 mL of ether. Use a 25 mL flask to receive the ether eluent and concentrate the ether solution on a rotary evaporator at room temperature. Transfer the residue to a 5 mL vial with ether and concentrate again. Dissolve the residue in the vial with 200 μL of the internal standard in toluene and mix well.

[0079] Example 2

[0080] Experimental Results

[0081] 1. Methylation of lignin phenol standard mixture

[0082] Although we have previously reported that individual lignin monomers can be quantitatively methylated using iodomethane tetrabutylammonium hydroxide / iodomethane in dichloromethane at 28 °C (58), the lignin phenol standard mixture (see Figure 1 ) remains unknown. We prepared a methanol solution of a mixture of 14 compounds, including 3-hydroxybenzoic acid and 3,5-dihydroxybenzoic acid, which are frequently found in CuO oxidation products in environmental samples, and 3-ethoxy-4-hydroxybenzaldehyde, a commonly used recovery standard, at a concentration of ∼0.2 mg / mL for each compound. 200 μL of the solution was injected into a 5 mL reaction vial equipped with a septum and a screw cap using a syringe. The solvent was removed using a rotary evaporator and further dried under vacuum (∼100 Pa) for 5 min after adding a stir bar. After nitrogen was injected into the vial, 100 μL of tetrabutylammonium hydroxide, 300 μL of dichloromethane, and 100 μL of iodomethane were added in sequence. The reaction was carried out at 28 °C for 10 h. 50 μL of hydrochloric acid (1.0 M) was added, and dichloromethane was removed using a rotary evaporator. After adding 1.0 mL of water, the mixture was extracted with ether (1.0 mL×4). The extract was passed through a 0.6 g magnesium sulfate column and a 1.0 g silica gel column in succession, and the ether solution was dried and purified. The column system was washed with 6.0 mL of ether. The eluate was concentrated and the residue was transferred to a 5 mL vial. After adding 200 μL of a toluene solution of the internal standard (1,4-diisopropylbenzene), the methylated product was analyzed by GC-FID equipped with a SE-54 capillary column (50 m×0.25 mm×0.25 μm). The symbols and conversion rates of the compounds are listed in Table 1. The chromatogram of the standard (~0.07 mg / mL) of the methylated product is shown in FIG. Figure 2 shown.

[0083] Except for 3,5-dihydroxybenzoic acid (DiOHBA), the average conversion values ​​of other monomers (three tests) ranged from 91.6% to 105.2% (Table 1). The relatively low conversions of PAL and PON may be due to the mass loss caused by their lower boiling points (the boiling points of methylated PAL and PON are 249°C and 264°C, respectively) during the reduced pressure evaporation process. The two hydroxyl groups in DiOHBA, or the corresponding methoxy groups in the intermediates, have a strong induced electron withdrawal effect on the carboxylate ion. Therefore, the stability of the ester formed by DiOHBA is reduced. We also studied the conversion of the ethyl acetate solution of the lignin phenol standard mixture. Despite the removal of ethyl acetate by evaporation and further drying under vacuum, the conversion of the four aldehyde-containing lignin phenols (PAL, VAL, EVAL and SAL) ranged from 0 to 11%. This result shows that even the presence of trace amounts of some components may have a huge impact on the conversion. Among them, the compound symbols and the conversion and recovery of their methylation reactions are shown in Table 1. The standard mixture of 13 compounds (about 40 μg of each compound) was used to determine the conversion. The methylation reaction was carried out at 28 °C for 10 h using 0.3 mL of dichloromethane, 100 μL of tetrabutylammonium hydroxide and iodomethane. The average deviation was calculated as:

[0084] Table 1 Compound symbols and conversion and recovery rates of methylation reactions

[0085]

[0086] 2. Silica gel column purification and effect verification

[0087] The composition of CuO oxidation products of typical geochemical samples is usually quite complex and may contain impurities that affect the methylation reaction. In addition, non-volatile components also lead to the deterioration of capillary column performance. Silica gel column chromatography is a widely used technique for the purification of organic chemicals. By silica gel column chromatography, impurities with polarity less than or higher than ligninphenol or methylated ligninphenol can be partially removed by eluting the mixture in the column with different solvents. However, to the best of our knowledge, silica gel column chromatography has not been applied to the purification of ligninphenol obtained from CuO oxidation in geochemical samples. Therefore, we tested the feasibility of silica gel column chromatography for the purification of phenols and methylated ligninphenol.

[0088] We first analyzed the recovery of a standard mixture of lignin phenols (containing ~0.1 mg of each compound). 200 μL of the mixture in ether was loaded onto a 0.5 g silica gel column and eluted with 10 mL of ether. The eluate was concentrated and the residue was transferred to a reaction vial. The residue was further dried under vacuum for 20 min and methylated under previously described conditions. The reaction mixture was neutralized with 1 M HCl and the dichloromethane was removed by rotary evaporation. 1 mL of water was added to the residue and the methylated product was extracted with ether (1 mL×5). Each extract was passed through a 0.6 g anhydrous magnesium sulfate column and a 1 g silica gel column in succession. We then washed the column system with an additional 5 mL of ether. The received ether solution was concentrated using a rotary evaporator and the residue was transferred to a 5 mL vial with ether. After removing the ether by rotary evaporation, a toluene solution of the internal standard was added and the product was analyzed by GC-FID. The fourth column of Table 1 lists the analytical recoveries of each phenol.

[0089] The average recoveries of the two tests for lignin phenols ranged from 87.9% for SAD to 105.5% for VAL. The relatively low recovery of SAD may be due to its partial loss during the first silica gel column chromatography. SAD is the most polar of the 11 lignin phenols. The silica gel column chromatography of SAD alone showed that the silica gel column chromatography has a tail effect, which may be the reason for its low recovery. The standard mixture of methylated compounds in Table 1 (each compound <unk>1 mg) of toluene solution was loaded onto a 1 g silica gel column, and we found that 5 mL of ether could completely elute all compounds from the column. These results indicate that silica gel column chromatography is suitable for the purification of ligninphenol and methylated ligninphenol.

[0090] 3. CuO Oxidation of Forest Soil Samples

[0091] We first selected forest soil samples (organic carbon content 13.6 g / kg) to study the new method for CuO oxidation and methylation. We used the reaction conditions reported by Hedge (2). CuO, Fe(NH4)2(SO4)2·6H2O, a stirrer and 0.5 g of soil were added to a reaction vessel. The atmosphere was easily exchanged by placing the reaction vessel in a plastic bag, which was then sealed, evacuated and filled with nitrogen four times. It was first kept at 40°C for 30 minutes to remove trace amounts of molecular oxygen by reaction with Fe(NH4)2(SO4)2·6H2O. The reaction was then continued at 170°C for 3 hours.

[0092] 4. Purification by ether extraction only

[0093] In order to clarify the purification effect of silica gel column chromatography, we did not purify the ether extract with a silica gel column. The ether extract was concentrated and the residue was transferred to a reaction vial by washing the flask 5 times with 1 mL of ether. The ether solution in the vial was concentrated again to obtain about 3 mg of residue. After methylation with tetrabutylammonium hydroxide / methyl iodide at room temperature for 10 h, the reaction mixture was neutralized, concentrated and extracted with ether. The extract was only dried over magnesium sulfate, but was not purified with a silica gel column. After adding 200 μL of a toluene solution of the internal standard, the sample solution was analyzed by GC-FID, and the chromatogram is shown in the following figure. Figure 3 In addition to the peaks of the 13 analyzed compounds (marked by blue vertical bars), a large number of impurity peaks illustrate the compositional complexity of typical soil samples. The content of aldehyde phenols in vanillyl and syringyl lignin phenols is higher than that of the corresponding ketone and carboxylic acid phenols ( Figure 2 ) is a typical characteristic of CuO oxidation products in soil samples (2). In addition, the baseline is wavy and cannot return to a smooth level even after 150 minutes. This situation raises great concerns about GC maintenance. Kaiser et al. reported that even if the sample is purified by a polymer SPE column, the packing in the injection liner should be replaced frequently (every 24 samples) and the front end of the capillary column should be trimmed after about 300 samples to avoid the peak tail effect (4). The quantitative results of 11 lignin phenols and DiOHBA are shown in the second column of Table 2. The concentrations of lignin phenols obtained by oxidizing forest soil samples are shown in Table 2, where the error is the standard deviation.

[0094] Table 2 Concentrations of lignin phenol obtained by oxidation of forest soil samples

[0095]

[0096] 5. Ether Extraction and Double Silica Column Purification

[0097] The above results show that purification of CuO oxidation products by ether extraction alone results in a rather complex chromatogram ( Figure 3 ). The successful validation of using a silica gel column to purify a standard mixture of ligninphenols and a subsequent mixture of methylated products encouraged us to investigate the effects of purifying a mixture of CuO oxidation products and methylated products. The ether extract obtained from the CuO oxidation products of a forest soil sample was concentrated by rotary evaporation. The residue was purified using a pretreated 0.5 g silica gel column. The silica gel in the used column had a light orange color, indicating that some components were retained in the column. The methylated reaction mixture was also purified using a 1.0 g silica gel column (see Materials and Methods for details). The chromatograms measured are shown in Figure 2. Figure 4 The contents of various lignin phenols were analyzed and listed in the fourth column of Table 2.

[0098] and Figure 3 Compared with the above, the chromatogram is significantly simplified, indicating that after lignin phenol and methylated lignin phenol were purified by silica gel column, most of the volatile by-products in the sample were removed, and all volatile components were eluted from the capillary column within 75 minutes, and the baseline was stable within 90 minutes. After 50 injections, the filler in the injection port liner was gray to a certain extent, but no tailing was observed, and no non-volatile components were deposited on the liner wall. Comparing the data of the same lignin phenol in columns 2 and 4 of Table 2, it was found that within the standard deviation range, the yields of PAL, PAD, VAL and SAL obtained by ether extraction and silica gel column purification were consistent with those obtained by simple ether extraction, and the yields of PON, CAD and FAD in column 2 were 30, 133 and 132% higher than those in column 4, respectively. This may be due to the co-elution of by-products and lignin phenol. On the other hand, the yields of VON, DiOHBA, SON and SAD in column 2 were lower than those obtained by ether extraction and silica gel column purification. We inferred that the reduction in yield was due to some impurities in the mixture that hindered the conversion rate in the methylation reaction of these ligninphenols. These results indicate that the use of ether extraction and silica gel column chromatography is essential to achieve a simplified chromatogram and enable the quantitative conversion of ligninphenols to their methylated forms. To further verify the efficiency of the new analytical method, we determined the recoveries of 11 ligninphenols, DiOHBA, CiAD, and EVAL.

[0099] 6. Recovery of lignin phenol by ether extraction and silica gel column purification

[0100] The recoveries of EVAL, CiAD and all ligninphenols were determined by adding 200 μL of a standard mixture of these compounds in 2 M NaOH (the concentration of each compound was 0.1 mg / mL) to the reaction mixture immediately after the CuO oxidation of the forest soil sample was stopped and cooled to room temperature. The recovery is a comprehensive indicator of the efficiency of multiple sample transfers, ether extraction, silica gel column purification and methylation reactions. The recovery was calculated by subtracting the value shown in column 4 of Table 2 from the corresponding analytical value of each ligninphenol, dividing it by the spiked value of the ligninphenol, and multiplying it by 100%. The recoveries of these compounds are shown in column 5 of Table 2. The recoveries of all compounds (except DiOHBA) were in the range of 81 to 103%. These results indicate that the two silica gel column purifications and methylation reactions did not result in significant losses of ligninphenols. The low recovery of DiOHBA was due to its 50% conversion in the methylation reaction.

[0101] 7. Method Performance

[0102] We evaluated the performance of the method using precision and reproducibility. The relative standard deviations (% Deviations) of individual ligninphenols in forest soil samples ranged from 3.2 to 16% (n = 5) (Table 2, column 5). There was an inverse correlation between the ligninphenol content and the % Deviation: the higher the ligninphenol content, the lower the % Deviation, and vice versa. The higher % Deviation when the ligninphenol content was low may be caused by two factors: first, interference from side components eluting before or after the target peak in the sample, and second, the lower sensitivity of the FID detector. The higher % Deviations of PAL, PON, and PAD may also be related to their higher volatility, especially when they are present in low amounts. Therefore, overdrying of methylated ligninphenols should be avoided. The % Deviations of the individual ligninphenol recoveries ranged from 4 to 10% (Table 2, column 7). The substantial improvement in % Deviation was due to the addition of the standard mixture, which increased the content of each individual ligninphenol.

[0103] In order to further study the precision and reproducibility of the new analytical method, as well as the interference of impurity components in the soil on the conversion rate of ligninphenol in the methylation reaction, we characterized the lignin in three different soils on different dates: tomato soil, grassland soil and freshwater sediment. Due to the different sources of the samples, the impurities in these samples should be very different. The results of the quantitative analysis are shown in Table 3. It was found that the average % deviation (n=2~4) of these three samples showed a similar variation pattern: the average % deviation of ligninphenol>10μg / g was 2~8%, and the average % deviation of ligninphenol<10μg / g was 12~19%. The chromatograms of these samples showed a chromatogram similar to that of the forest soil samples. The concentrations of CuO oxidation products in grassland soil, tomato soil and freshwater sediments are shown in Table 3. These products are obtained by Figure 4 and purified as described in the text.

[0104] Table 3 Concentrations of CuO oxidation products in grassland soil, tomato soil and freshwater sediments

[0105]

[0106]

[0107] Example 2: Characterization of lignin in forest soil samples

[0108] S1. Add a magnetic stirrer to a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 1.00 g of CuO, and 0.5000 g of soil. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and take it out of the glove box. Heat to 170°C with stirring (1800 rpm) and keep the temperature constant for 3 hours.

[0109] S2. Cool to room temperature with running tap water. Open the lid and quickly add 200 microliters of 2M NaOH solution of 2-bromo-4-hydroxybenzoic acid (2-bromo-4-hydroxybenzoic acid concentration 50ng / μL). After tightening the lid, put it into the centrifuge for solid-liquid separation (7000rpm, 1min). Prepare a brown centrifuge tube with 1.5 ml of concentrated hydrochloric acid and a magnetic stirrer in advance, and place the centrifuge tube in an ice water bath. Pour the supernatant into the centrifuge tube. Add 2 ml of 1M NaOH solution to the reactor, cover the lid tightly, and stir vigorously for 2min. Centrifuge again and pour the supernatant into the centrifuge tube. Repeat the washing operation twice. Acidify the aqueous solution in the centrifuge tube with 6M HCl to a pH of 1-1.5. Add 11 ml of ether, stir vigorously for 2min and let it stand. After the stratification is complete, use a glass dropper to add the upper ether solution to the top of the purification column. The purification column consists of a drying column and a silica gel column connected in series. The upper drying column contains 5 g of sodium sulfate, the lower silica gel column contains 0.5 g of silica gel, and the silica gel is covered with glass wool. Pressurize the ether to flow through the purification column, and collect the effluent in a brown flask. Repeat the extraction operation twice. Wash the purification column with 6 ml of ether, remove the drying column, and wash the silica gel column with 3 ml of ether / methanol solution (9:1, v / v). Concentrate the ether solution using a rotary evaporator and flush with nitrogen at the end. Transfer the residue in the flask to a 5 ml reaction bottle with ether and concentrate to dryness again.

[0110] S3. After adding the stirring bar, dry under vacuum for 5-20 minutes until no water droplets are visible. Transfer the reaction bottle to a nitrogen glove box, add 20 mg of ammonium ferrous sulfate, drip 500 μl of tetrabutylammonium hydroxide (40%, w / w), 0.5 ml of dichloromethane, and 100 μl of iodomethane along the bottle wall, cover the lid and take it out of the glove box. Stir at 30°C for 120 minutes.

[0111] S4. Open the lid, add 50 μl of 1M HCl, stir for 10 seconds, then add 1.5 ml of ether and stir for 1 min. Prepare a purification column in advance. The lower part of the column is 0.5 g of silica gel, and the upper part is a mixture of 0.8 g of silica gel and 0.8 g of magnesium sulfate. Add the slurry in the reaction bottle to the upper part of the purification column, wash the reaction bottle with 1 ml of ether, add the washing liquid to the upper part of the purification column, and repeat the washing operation once. Pressurize the ether solution to flow through the purification column, and use a 12 ml brown glass bottle to receive the solution. Wash the purification column with 4.5 ml of ether, and combine the effluent in the brown bottle. Concentrate the ether solution to dryness using a rotary evaporator.

[0112] S5. Add 2-iodobenzoic acid ethyl ester toluene solution and mix well. Analyze using GC-MS SIM mode. Injection volume 1 μl, split ratio set to 50:1.

[0113] The above experiment was repeated 9 times, and the results are shown in Table 4.

[0114] Table 4

[0115]

[0116] Example 3: Characterization of lignin in cultivated soil samples

[0117] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 1.00 g of CuO, 0.5000 g of soil, and 10 mg of glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove from the glove box. Heat to 170°C with stirring (1800 rpm) and keep the temperature constant for 3 h.

[0118] S2-S5 are the same as in Example 2.

[0119] The above experiment was repeated 3 more times. The results are shown in Table 5.

[0120] Table 5

[0121]

[0122]

[0123] Example 4: Characterization of lignin in freshwater sediment samples

[0124] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 1.00 g of CuO, 0.5000 g of soil, and 20 mg of glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove from the glove box. Heat to 170°C with stirring (1800 rpm) and keep the temperature constant for 3 h.

[0125] S2-S5 are the same as in Example 3.

[0126] The above experiment was repeated 3 more times. The results are shown in Table 6.

[0127] Table 6

[0128]

[0129]

[0130] Example 5: Characterization of lignin in grassland soil sample #1

[0131] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 0.500 g of CuO, 0.5000 g of grassland soil sample #1 (organic carbon content of 8.62 g / kg), and 20 mg of glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove from the glove box. Heat to 170°C with stirring (1800 rpm) and keep the temperature constant for 2 h.

[0132] S2-S5 are the same as in Example 2.

[0133] The above experiment was repeated once more, and the results are shown in Table 7.

[0134] Table 7

[0135]

[0136] Note: "V+S+C" represents the sum of the concentrations of the first eight compounds (i.e., VAL+VON+VAD+SAL+SON+SAD+CAD+FAD).

[0137] Example 6: Traditional method for lignin in grassland soil sample #1 (C 18 -SPE) characterization

[0138] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 0.500 g of CuO, 0.5000 g of grassland soil sample #1 (organic carbon content of 8.62 g / kg), and 20 mg of glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove from the glove box. Heat to 170°C with stirring (1800 rpm) and keep the temperature constant for 2 h.

[0139] S2. Open the lid of the reactor, add 0.5 ml of 2M NaOH solution of ethyl vanillin (50 μg / mL), stir for 5 min and transfer the mixture to a 25 ml brown centrifuge tube. Wash the reactor with 3 ml of distilled water and pour it into the centrifuge tube. Repeat the washing operation 3 times. Use a centrifuge to separate the solid and liquid (3500 rpm, 15 min), and pour the supernatant into another 50 mL centrifuge tube. Wash the reactor again with 3 ml of distilled water, pour the washing liquid into a 25 mL centrifuge tube, centrifuge and separate, and transfer the supernatant to a 50 mL centrifuge tube. Repeat this operation once more. Neutralize the aqueous solution in the centrifuge tube to pH ~ 2 with 12M HCl. Let stand in the dark for 1 hour. Centrifuge for solid-liquid separation (8000 rpm, 25 min), and transfer the supernatant to a 100 mL brown volumetric flask. Wash the precipitate with distilled water at pH 2, centrifuge again (8000 rpm, 25 min), and transfer the supernatant to a volumetric flask. Make up to 100 mL with distilled water.

[0140] S3. Prepare a C 18 For the solid phase extraction column, soak the filler with 3 mL of ethyl acetate for 2 min, then remove it under reduced pressure, soak the filler with 3 mL of anhydrous ethanol for 2 min, then remove it under reduced pressure, soak it with 5 mL of distilled water for 2 min, then remove it under reduced pressure.

[0141] S4. Use a large-capacity sampler to slowly pass the aqueous solution in the volumetric flask through C under reduced pressure. 18 Column. After all the solution has passed through the column, use nitrogen to blow dry the water inside the column. Elute the column with 0.5 mL of ethyl acetate and collect the effluent in a test tube wrapped in tin foil. Repeat the above elution operation 6 times. The 1st-2nd times flow out under reduced pressure, the 3rd-4th times flow out under gravity, and the 5th-7th times flow out under reduced pressure. Transfer the effluent in the test tube to a derivatization bottle. Use a pipette to remove the water droplets at the bottom of the derivatization bottle. Add 0.5 mL of methanol solution of phenylacetic acid (50 μg / mL) to the derivatization bottle. Mix well and blow dry with nitrogen (avoid light).

[0142] S5. Add 50 μl of pyridine and 100 μl of derivatization reagent (bis(trimethylsilyl)trifluoroacetamide + 1% trimethylsilyl chloride) into the derivatization bottle, and derivatize at 60°C for 15 min, shaking the bottle every 5 min.

[0143] GC-FID analysis was used. The injection volume was 1 μL and the split ratio was set to 10:1. The test results are shown in Table 8.

[0144] Table 8

[0145]

[0146]

[0147] Example 7: Characterization of lignin in grassland soil sample #2

[0148] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 0.500 g of CuO, 0.5000 g of grassland soil sample #2 (organic carbon content of 12.66 g / kg), and 20 mg of glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove from the glove box. Heat to 170°C with stirring (1800 rpm) and keep the temperature constant for 2 h.

[0149] S2-S5 are the same as in Example 2.

[0150] The above experiment was repeated once more, and the results are shown in Table 9.

[0151] Table 9

[0152]

[0153]

[0154] Note: n is the number of repetitions.

[0155] Example 8: Traditional method for lignin in grassland soil sample #2 (C 18 -SPE) characterization

[0156] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 0.500 g of CuO, 0.5000 g of grassland soil sample #2 (organic carbon content of 12.66 g / kg), and 20 mg of glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove from the glove box. Heat to 170°C with stirring (1800 rpm) and keep the temperature constant for 2 h.

[0157] S2-S5 are the same as in Example 6. The test results are shown in Table 10.

[0158] Table 10

[0159] Compound <![CDATA[Concentration of lignophenol (μg g -1 )]]> VAL 8.30 VON 4.19 VAD 6.56 SAL 1.90 SON 0.85 SAD 4.77 CAD 19.79 FAD 16.62 RS 83.3％ V+S+C 62.98

[0160] Example 9: Characterization of lignin in forest soil sample #1

[0161] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 0.500 g of CuO, 0.5000 g of forest soil sample #1 (organic carbon content of 13.92 g / kg), and 20 mg of glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove from the glove box. Heat to 170°C with stirring (1800 rpm) and keep the temperature constant for 2 h.

[0162] S2-S5 are the same as in Example 2.

[0163] The above experiment was repeated once more, and the results are shown in Table 11.

[0164] Table 11

[0165]

[0166] Example 10: Characterization of Lignin in Forest Soil Sample #1 by Traditional Method (Ethyl Acetate Extraction)

[0167] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 0.500 g of CuO, 0.5000 g of forest soil sample #1 (organic carbon content 13.92 g / kg), and 20 mg of glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove from the glove box. Heat to 170°C with stirring (1800 rpm) and keep the temperature constant for 2 h. Cool to room temperature with tap water.

[0168] S2. Open the lid of the reactor and add 400 μL of 2M NaOH solution of ethyl vanillin (100 μg / mL). After stirring for 30 seconds, transfer the mixture in the reactor to a 50mL centrifuge tube and use a centrifuge for solid-liquid separation (4000rpm, 3min). Transfer the supernatant to another 50mL centrifuge tube. Wash the precipitate twice with 5mL of distilled water and combine the supernatant. Acidify the aqueous solution with 6M HCl to pH <1. Let stand for 1h in the dark.

[0169] S3. Extract the aqueous solution with 10 mL of ethyl acetate, repeat 3 times, combine the extracts in a 30 mL centrifuge tube, and blow dry with nitrogen at 38°C.

[0170] S4. Prepare 3 standard samples at the same time. Add 100 μL of 2M NaOH solution (1 mg / mL) of the standard mixture (including 8 standard samples) and 400 μL of 2M NaOH solution (0.1 mg / mL) of ethyl vanillin into a 2mL derivatization bottle. Mix well and blow dry with nitrogen.

[0171] S5. Add 100 μL of pyridine and 400 μL of derivatization reagent (bis(trimethylsilyl)trifluoroacetamide+1% trimethylsilyl chloride) into a 2 mL derivatization bottle, mix well, react at 70° C. for 3 h, and then cool to room temperature.

[0172] GC-MS analysis was performed with an injection port temperature of 250°C, an injection volume of 1 μL, and a split ratio of 5:1. The test results are shown in Table 12.

[0173] Table 12

[0174] Compound <![CDATA[Concentration of lignophenol (μg g -1 )]]> VAL 17.80 VON 5.16 VAD 7.65 SAL 15.31 SON 3.83 SAD 5.49 CAD 2.33 FAD 12.48 RS 87.5％ V+S+C 70.05

[0175] Example 11: Characterization of lignin in forest soil sample #2

[0176] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 0.500 g of CuO, 0.5000 g of forest soil sample #2 (organic carbon content 14.35 g / kg), and 20 mg of glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove from the glove box. Heat to 170°C with stirring (1800 rpm) and keep the temperature constant for 2 h.

[0177] S2-S5 are the same as in Example 2.

[0178] The above experiment was repeated once more, and the results are shown in Table 13.

[0179] Table 13

[0180]

[0181]

[0182] Example 12: Characterization of Lignin in Forest Soil Sample #2 Using Traditional Methods (Ethyl Acetate Extraction)

[0183] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ammonium ferrous sulfate, 0.500 g of CuO, 0.5000 g of forest soil sample #2 (organic carbon content 14.35 g / kg), and 20 mg of glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove from the glove box. Heat to 170°C with stirring (1800 rpm) for 2 h. Cool to room temperature with tap water.

[0184] S2-S5 are the same as in Example 10, and the test results are shown in Table 14.

[0185] Table 14

[0186] Compound <![CDATA[Concentration of lignophenol (μg g -1 )]]> VAL 48.46 VON 21.33 VAD 18.31 SAL 31.55 SON 24.85 SAD 10.42 CAD 4.62 FAD 17.53 RS 85.4％ V+S+C 177.52

[0187] Example 13: Relative response factor RRf stability determination

[0188] Accurately weigh 5 mg of each of 11 lignin phenols, methyl 2-bromo-4-methoxybenzoate and ethyl 2-iodobenzoate in a 100 mL volumetric flask, add toluene to dissolve, dilute to 100 mL, the concentration of each compound is about 50 ng / μL, and the solution is sealed and stored in a refrigerator at -20°C. When determining the relative response factor, first take out the solution and warm it to near room temperature, take about 100 μL and add it to the inner liner of the chromatographic sample bottle, analyze it in the SIM mode of GC-MS, the injection volume is 1 μL, and the split ratio is set to 50:1. Analyze and calculate the relative response factor 18 times continuously or at intervals of 1 day, repeat the analysis 3 times each time, take the average value, calculate the average value and standard deviation of the 18 average values, and the calculation results are shown in Table 15.

[0189] Table 15

[0190]

[0191] Example 14: Analysis of sample stability (after 20 h storage at room temperature)

[0192] The sample solution of Example 2 (Characterization of lignin in forest soil samples) was placed at room temperature for 20 hours and then analyzed once using the SIM mode of GC-MS. The analysis results are shown in Table 16.

[0193] Table 16

[0194]

[0195] Example 15: Analysis of sample stability (after 30 days storage at -20°C)

[0196] The sample solution of Example 2 (Characterization of lignin in forest soil samples) was stored at -20°C for 30 days and then taken out and re-analyzed using the SIM mode of GC-MS. The analysis was repeated 4 times, and the average value and standard deviation were calculated. The analysis results are shown in Table 17.

[0197] Table 17

[0198]

[0199]

[0200] The present invention proposes a new procedure for characterizing lignin in environmental samples. First, the sample is oxidized using the conventional CuO / NaOH method. A specially designed stainless steel reaction vessel is used and the reaction is conveniently carried out with magnetic stirring. The second step is the purification of the CuO oxidation product. The acidified aqueous solution is extracted with ether and the evaporated sample is further purified using a pretreated silica gel column. The third step is the methylation reaction of the sample using tetrabutylammonium hydroxide / methyl iodide in dichloromethane. Although we found that trace amounts of ethyl acetate can reduce the conversion of lignin phenols to the methylated form, impurities in the studied soil samples did not interfere with the methylation reaction. In contrast, the BSTFA / TMCS lignin phenol derivatization reaction is sensitive to compounds containing active protons, such as water and alcohols (4). Finally, the methylation product mixture is purified by extraction with ether, and the concentrated sample is subsequently purified using a silica gel column. The use of ether is crucial to shorten the overall operation time because it can be quickly and safely removed by rotary evaporation.

[0201] Ether extraction and silica gel column purification removed most of the impurities in the sample and gave a significantly simplified chromatogram. Therefore, the time consumed for GC maintenance will be significantly reduced. Except for the CuO oxidation reaction and methylation reaction, which were performed under nitrogen protection, all other operations were performed without argon or nitrogen protection. Therefore, the complexity of the operation was greatly reduced. Methylated lignin phenol is stable to air and trace water; in contrast, the efficiency of lignin phenol derivatization by BSTFA / TMCS is sensitive to water and alcohol. Due to concerns that the TMS derivative of lignin phenol will decompose due to the gradual introduction of water during storage, it is necessary to perform the analysis within 24 hours. In contrast, we did not observe decomposition of the toluene solution of methylated lignin phenol after one week of storage in a freezer (~20°C). These new features of this method make it suitable for lignin characterization in geochemical samples with high lignin content.< / unk>

Claims

1. A method for characterizing lignin in an environmental sample, characterized in that: The method comprises the step of alkylating the environmental sample after the purification step with tetraalkylammonium hydroxide / alkylating agent in an organic solvent, and further comprising the following steps: (1) oxidizing the environmental sample with a CuO / NaOH reaction system; (2) purifying: extracting the acidified aqueous solution with ether, and further purifying the ether extract with a pretreated silica gel column to obtain a purified environmental sample; (3) diluting the alkylated product mixture with ether, and then purifying the reaction mixture with a silica gel column to prepare a sample solution; (4) injecting the sample solution into a gas chromatograph or a gas chromatograph-mass spectrometer for analysis and detection; wherein the alkylation reaction is between the step (2) and the step (3); The organic solvent is selected from dichloromethane or 1,2-dichloroethane; the alkylating agent is selected from one of methyl iodide, ethyl iodide, ethyl bromide, dimethyl sulfate and diethyl sulfate; the tetraalkylammonium hydroxide is selected from one of tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, tetrapentylammonium hydroxide and tetrahexylammonium hydroxide.

2. A method for characterizing lignin in an environmental sample, characterized in that: The method comprises the step of methylating the environmental sample after the purification step by using tetrabutylammonium hydroxide / methyl iodide in dichloromethane, and further comprising the following steps: (1) oxidizing the environmental sample by using a CuO / NaOH reaction system; (2) purifying: extracting the acidified aqueous solution with ether, and further purifying the evaporated sample with a pretreated silica gel column to obtain a purified environmental sample; (3) extracting and purifying the methylated product mixture with ether, and then purifying the concentrated sample with a silica gel column to prepare a sample solution; and (4) injecting the sample solution into a gas chromatograph for analysis and detection.

3. The method for characterizing lignin in an environmental sample according to claim 1, characterized in that: The environmental sample in step (1) is one of water, plants, soil, sediment, sewage and sludge.

4. The method for characterizing lignin in an environmental sample according to claim 2, characterized in that: The conditions for the gas chromatograph analysis in step (4) are as follows: the temperature of the injection port and the detector is 300° C., the chromatographic column is a capillary chromatographic column, the capillary chromatographic column is an SE-54 capillary column, the specification is 50 m long, 0.25 mm inner diameter, 0.25 mm film thickness, the stationary phase is 5% phenyl and 95% methyl polysiloxane, the detector is a hydrogen flame ionization detector, the carrier gas is nitrogen with a purity of >99.999%, the carrier gas flow rate is 1 mL / min, the injection volume is 1.0-2.0 μL, and the split ratio is 2:

1.

5. The method for characterizing lignin in an environmental sample according to claim 2, characterized in that: The gas chromatograph in step (4) is a Shimadzu GC-2014C gas chromatograph.

6. The method for characterizing lignin in an environmental sample according to claim 2, characterized in that: The heating program of the gas chromatograph in step (4) is as follows: starting temperature 50°C, heating to 170°C at a heating rate of 4°C / min and maintaining for 20 min, then heating to 270°C at a heating rate of 10°C / min and maintaining for 20 min.

7. The method for characterizing lignin in an environmental sample according to claim 1, characterized in that: The oxidation reaction in step (1) is carried out in a cylindrical stainless steel reaction container with a total capacity of 10 mL, wherein the reaction container is equipped with a polytetrafluoroethylene magnetic stirrer, 500 mg of copper oxide, 200 mg of ammonium ferric sulfate hexahydrate and 500 mg of an environmental sample, the reaction container is transferred into a plastic bag, connected to a vacuum nitrogen pipeline and the plastic bag is sealed, evacuated and backfilled with nitrogen four times, 7 mL of a 2 mol / L NaOH solution is added into the reaction container, the mixture in the reaction container is stirred at 1800 rpm, the temperature is maintained at 40° C. for 30 min, increased to 170° C. at 5° C. / min, and maintained for 180 min, and then the reaction container is cooled with tap water for 10 min.

8. The method for characterizing lignin in an environmental sample according to claim 1, characterized in that: The step (2) includes opening the reaction container, immediately injecting 200 μL of an aqueous solution of ethyl vanillin in 2 mol / L NaOH therein, sealing the reaction container and magnetically stirring at 1800 rpm for 30 s, centrifuging each reaction container at 9000 rpm for 60 s, quickly pouring the supernatant into a 50 mL plastic centrifuge tube, pre-adding 1 mL of concentrated hydrochloric acid and a stirrer to the centrifuge tube, and immersing the bottom 1 / 3 of the centrifuge tube in ice water, injecting 2 mL of a 0.5 mol / L NaOH solution along the inner wall of each reaction container, tightening the lid by hand and rotating horizontally for 30 s, stirring the mixture in the container at 1800 rpm for 60 s, then centrifuging each container again, adding the supernatant to the centrifuge tube, repeating the washing process twice, and acidifying the solution in the centrifuge tube to a pH of 6.

2. Extract the acidified solution with 8 mL of ether and centrifuge at 4500 rpm for 60 s. Transfer the ether layer to a 50 mL conical flask with a glass pipette. Repeat the extraction process three times. Dry the ether solution with anhydrous sodium sulfate for 10 min. Filter the ether extract through a layer of glass wool into a 50 mL evaporating flask. Wash the conical flask with 2 mL of ether three times and concentrate to dryness on a rotary evaporator at room temperature. During evaporation, the flask can be immersed in a water bath below 25°C. Purify the residue in the flask with a 0.5 g silica gel column. Wash the residue in the flask with ether and add the solution to the top of the silica gel column. Repeat the washing three more times. Wash the column with an additional 7 mL of ether. Collect the eluate in a 25 mL flask and concentrate it on a rotary evaporator at room temperature. Transfer the concentrated residue to a 5 mL Hungate reaction vial with ether. Concentrate the solution and further dry it under vacuum for 20 min after adding a stir bar.

9. The method for characterizing lignin in an environmental sample according to claim 1, characterized in that: The step (2) opens the reaction vessel, immediately injects 200 μL of a solution containing 2 mol / L NaOH and 50 ng / μL 2-bromo-4-hydroxybenzoic acid therein, seals the reaction vessel and stirs magnetically at 7000 rpm for 1 min, pre-adds 1.5 mL of concentrated hydrochloric acid and a stirrer into a centrifuge tube, immerses the bottom 1 / 3 of the centrifuge tube in ice water, adds the supernatant into the centrifuge tube, injects 2 mL of 1 mol / L NaOH solution along the inner wall of each reaction vessel, tightens the lid and stirs magnetically for 2 min, centrifuges again, adds the supernatant into the centrifuge tube, repeats the washing process twice, and acidifies the solution in the centrifuge tube to a pH of 6 with 6 mol / L hydrochloric acid. 1-1.5, extract the acidified solution with 11 mL of ether, stir for 2 minutes and let stand, transfer the ether layer to the upper part of the purification column with a glass pipette after the stratification is complete, pressurize the ether to flow through the purification column, and use a brown flask to receive the effluent, repeat the extraction process twice, wash the purification column with 6 mL of ether, remove the drying column, and wash the silica gel column with 3 mL of ether / methanol solution (9:1, v / v); concentrate the ether solution with a rotary evaporator, and flush with nitrogen at the end; transfer the residue in the flask to a 5 mL reaction bottle with ether, and concentrate to dryness again; After adding a stir bar, the mixture was further dried under vacuum for 5-20 min.

10. The method for characterizing lignin in an environmental sample according to claim 1, characterized in that: The specific operation process of step (3) is as follows: adding ether to the reaction mixture after alkylation, stirring and then adding the slurry mixture to the upper part of the purification column; the upper part of the purification column is a desiccant, and the lower part is silica gel or diatomaceous earth; the desiccant is selected from one of magnesium sulfate, sodium sulfate, and calcium chloride; when preparing the sample solution, a toluene solution of a quantitative internal standard is used, and the quantitative internal standard is ethyl 2-iodobenzoate.

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