A method for characterizing lignin in an environmental sample

Abstract

The application discloses a method for characterizing lignin in environmental samples, and belongs to the technical field of lignin detection. The method comprises the following steps: oxidizing a sample by using a CuO / NaOH system; acidifying supernatant; extracting the supernatant by using diethyl ether; further purifying the diethyl ether extract by using a pretreated silica gel column; performing a methylation reaction on the sample by using tetrabutylammonium hydroxide / iodomethane in dichloromethane; diluting the methylation product mixture after neutralization by using diethyl ether; and purifying the reaction mixture by using a silica gel column. The combination of diethyl ether extraction and silica gel column chromatography removes most of the impurities in the sample, and the solvent can be quickly removed under reduced pressure, so that the operation complexity is greatly reduced. The time consumed for gas chromatography maintenance is greatly reduced. The main operation is performed under the protection of gas, so that the operation complexity is greatly reduced. The methylated lignin phenol is stable to air and water, and no obvious change is found after being stored in a refrigeration chamber for one week. The method has no limitation on the number of simultaneous analysis, and is suitable for the conventional lignin characterization of geochemical samples with high lignin content.

Description

Technical Field

[0001] This invention belongs to the field of lignin detection technology, specifically relating to a method for characterizing lignin in environmental samples. Background Technology

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

[0003] Copper oxide oxidizes lignin to produce 11 lignin phenol monomers. These are classified into four types: vanillin (V-type), eugenol (S-type), p-hydroxyphenol (H-type), and cinnamylphenol (C-type). Each of the V-, S-, and H-types contains one aldehyde, one ketone, and one acid; generally, aldehyde phenols are more abundant than ketone phenols and acid phenols within the same type. The relative content of each type of phenol in a sample is determined by its plant origin (Fate of lignins in soils: A review). For example, gymnosperm wood contains approximately 80% V-type phenols and a small amount of C-type phenols; in contrast, angiosperm wood primarily produces roughly equal amounts of V-type and S-type phenols. Therefore, the relative contributions of the three types of phenols (e.g., S / V and C / V ratios) are often used to characterize lignin in a sample. On the other hand, the phenolic composition in a sample changes with alterations in lignin (e.g., biodegradation). Compared to aldehyde-containing phenols in the V- and S-types, the increase in phenols containing carboxylic acid groups leads to an increase in the acid / aldehyde ratio in these two types of phenols. During lignin degradation, eugenol and cinnamyl units (S- and C-type) are lost more rapidly than guaiacyl units (V-type), leading to a decrease in the S / V and C / V ratios. Since H-type phenols are not a lignin source, they are rarely used as geochemical tracers. Therefore, accurate characterization of lignin provides important information for the properties of geochemical samples. CuO / NaOH aqueous solutions have traditionally been used as oxidizing agents to depolymerize lignin into phenols, such as... Figure 1As shown. The resulting lignin-phenol 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, derivatized the phenols into trimethylsilyl derivatives using a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 1% trimethylchlorosilane (TMCS), and analyzed their composition by capillary GC equipped with a flame ionization detector (FID). Bochter (Characterization of lignin in forest humus layers by high-performance liquid chromatography of cupric oxide oxidation products) improved this method by using solid-phase extraction (SPE) with a C18 column instead of ether extraction for phenols. Many research groups now use C18 columns to extract phenols because the chromatograms produced are simpler than those obtained by solvent extraction.

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

[0005] Extraction with organic solvents can lead to the concentration of other soluble compounds along with the lignin. BSTFA / TMCS is a highly reactive derivatizing reagent that reacts not only with the phenolic and carboxyl groups of lignin but also with the alcoholic hydroxyl groups of impurities, resulting in complex volatile mixtures and complex chromatograms. These complex chromatograms pose challenges to product identification and quantification; the complex components can contaminate the gas chromatograph's injection port, capillary column, and FID detector, as well as the vacuum chamber and ion source of the mass spectrometer. Furthermore, trimethylsilane (TMS) derivatives of lignin are highly sensitive to moisture and undergo isomerization and decomposition at room temperature. Typically, samples should be analyzed immediately after derivatization by GC or GC-MS. This immediacy limits the analytical throughput of samples, as leaving samples on the autosampler rack overnight can lead to 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 monomers, some of which differ from those produced by CuO oxidation. For most of these methylated monomers, there are no commercially available standards, making the determination of retention times and response factors difficult. High temperatures also produce complex chromatograms (Characterization of wood by pyrolysis derivatization - gas chromatography / mass spectrometry), and only an MS detector can be used to characterize lignin. Furthermore, many factors in the THM process, including the concentration of TMAH, solvent, and temperature, affect the product distribution (Influence of parameters on pyrolysis - GC / MS of lignin in the presence of tetramethylammonium 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 method of thermochemolysis with tetramethylammonium hydroxide (TMAH)). There is an urgent need in the art to develop a high-throughput, low-maintenance method for characterizing lignin in environmental samples. 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 novel derivatization method for converting lignin monomers obtained from CuO oxidation. These monomers are quantitatively methylated in dichloromethane using tetrabutylammonium hydroxide / iodomethane. Stable methylated products can be purified by silica gel column chromatography, simplifying the gas chromatogram of the sample and enabling and reliably performing GC-FID or GC-MS analysis. Dissolving the sample in toluene as a solvent and setting appropriate temperature programming conditions can significantly reduce peak tailing and alleviate the burden of instrument maintenance. Furthermore, stable products can be analyzed at any time, thereby increasing sample throughput.

[0008] Copper oxide oxidizes lignin to produce 11 lignin phenol monomers. These are classified into four types: vanillylphenol (V type), eugenol (S type), p-hydroxyphenol (H type), and cinnamylphenol (C type), as detailed in the appendix. Figure 1 As shown.

[0009] A method for characterizing lignin in environmental samples includes a step of alkylating the purified environmental sample in an organic solvent using tetraalkylammonium hydroxide / alkylating reagent, and further includes the following steps: (1) oxidizing the environmental sample using a CuO / NaOH reaction system; (2) purification: extracting the acidified aqueous solution with diethyl ether and further purifying the diethyl ether extract with a pretreated silica gel column to obtain a purified environmental sample; (3) diluting the alkylation product mixture with diethyl ether and then further 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 steps (2) and (3).

[0010] The organic solvent is selected from dichloromethane or 1,2-dichloroethane; the alkylating agent is selected from iodomethane, iodoethane, bromoethane, dimethyl sulfate, and diethyl sulfate; these alkylating agents can all undergo quantitative alkylation reactions with lignin phenols, but iodomethane has the fastest alkylation reaction rate and relatively low toxicity, so iodomethane 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 all play the same phase transfer catalyst role in the alkylation reaction to realize the alkylation reaction of lignin phenols, 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 environmental samples, in addition to the step of methylating the purified environmental sample in dichloromethane using tetrabutylammonium hydroxide / iodomethane, further includes the following steps:

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

[0015] Furthermore, environmental samples were methylated in dichloromethane using tetrabutylammonium hydroxide / iodomethane. The methylation of lignin phenols was carried out in 5 mL Hungett vials. After drying under vacuum, the vials were purged with nitrogen. 250-500 μL of a 40% (w / w) tetrabutylammonium hydroxide aqueous solution, 0.5 mL of dichloromethane, and 100 μL of iodomethane were added sequentially to the vials. The reaction was carried out at 28-30 °C for 90 min-12 h. The pressure in the vials was released by inserting a syringe through a diaphragm. After neutralization with 50 μL of 1 mol / L hydrochloric acid, the mixture was concentrated by evaporation using a rotary evaporator. 1 mL of water was added to the vial, and the mixture was extracted with 1 mL of diethyl ether. The extract was passed through an anhydrous magnesium sulfate column, and the extraction process was repeated four times. Rinse the desiccator with 5 mL of diethyl ether. Collect the diethyl ether solution in a 25 mL flask and concentrate the diethyl ether solution to dryness under reduced pressure using a rotary evaporator at room temperature. Purify the residue in the flask by passing it through a 1 g silica gel column. The silica gel column is pre-washed with water, methanol, diethyl 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 and discard the eluent. Then, wash the flask with 1 mL of diethyl ether and pass the diethyl ether solution through the column. Repeat the diethyl ether washing four times. Then, wash the silica gel column with an additional 6 mL of diethyl ether. Collect the eluent in a 25 mL flask and concentrate it to approximately 0.5 mL using a rotary evaporator. Transfer the residue to a 5 mL vial with diethyl ether and concentrate it to dryness again. Dissolve the residue in the vial with 200 μL of internal standard solution and mix thoroughly.

[0016] As another preferred method, environmental samples were methylated in dichloromethane using tetrabutylammonium hydroxide / iodomethane. The methylation of lignin phenols was carried out in 5 mL Hengette vials. After drying under vacuum, the vials were purged with nitrogen. 100-500 μL of 40% tetrabutylammonium hydroxide aqueous solution, 0.3-1.5 mL of dichloromethane, and 50-100 μL of iodomethane were added sequentially to the vials, and the reaction was carried out at 26-30 °C for 90 min-12 h. After neutralizing with 50 μL of 1 mol / L hydrochloric acid, add 1.5 mL of diethyl ether, stir for 30 s, and transfer the slurry to the upper part of the purification column. The packing material of the purification column consists of a desiccant at the top and silica gel at the bottom. Collect the eluent in a brown glass bottle. Wash the reaction flask twice with 2 mL of diethyl ether, and add the washings to the upper part of the purification column as well. Apply pressure to make the solution flow through the column. Wash the purification column with 4.5 mL of diethyl ether. Concentrate the solution in the receiving flask to dryness using a rotary evaporator. Add a certain volume of toluene solution of ethyl 2-iodobenzoate to the bottle and mix well. This sample can be analyzed by GC-MS or GC-FID. If sample impurities interfere with the GC-FID chromatographic peaks, the concentrated sample can be further purified and concentrated using the following method before adding the toluene solution of ethyl 2-iodobenzoate and then analyzing by GC-FID: Prepare a column packed with 2g of silica gel. Wash the concentrated sample four times with 4mL of n-hexane, passing the washings through the silica gel column. Then wash the silica gel column with 4mL of n-hexane and discard all the eluent. Wash the concentrate three times with 1.5mL of diethyl ether, passing the washings through the silica gel column. Finally, wash the silica gel column with 4mL of diethyl ether and collect all the diethyl ether solution in a dark brown glass bottle. Concentrate the diethyl ether solution to dryness under reduced pressure. Add the toluene solution of ethyl 2-iodobenzoate and then analyze by GC-FID.

[0017] Furthermore, the environmental sample in step (1) is one of the following: plant, water, soil, sediment, sewage, or silt.

[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 stir bar to 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 inside a nitrogen glove box. Add 7-20 mL of 2M NaOH solution to the reactor using a pipette. Remove the reactor from the glove box and place it on a stirrer. Heat the reactor to 150-170°C with stirring for 30 minutes. Maintain the temperature for 120-180 minutes after reaching the set temperature. After the set time, cool the reactor with running tap water for 5 minutes, or remove the heating element and allow it to cool naturally with stirring overnight (operating time 40 minutes).

[0022] 2. Purification

[0023] Open the lid of a reaction vessel and quickly add 250 μL (48 ng / μL) of 2M NaOH solution for recovering the internal standard (2-bromo-4-methoxybenzoic acid), then tighten the lid. Centrifuge the reaction vessel (7000 rpm, 1 min) and quickly pour the supernatant into a centrifuge tube containing concentrated hydrochloric acid, immersing one-third of the tube in an ice-water bath. Add 2 mL of 0.5-2M NaOH solution or water to the inner wall of the reaction vessel, tighten the lid, and stir vigorously for 1-5 min. Centrifuge and pour the supernatant into the centrifuge tube. Repeat the above washing process twice (for a total of 3 washes).

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

[0025] Extract the aqueous solution with 11 mL of diethyl ether. If emulsification occurs at the interface, centrifugation can be used to accelerate separation. Add the ether extract to the purification column. Repeat the ether extraction twice (for a total of three times).

[0026] The purification column consists of two columns connected in series. The upper column contains 3-5 g of anhydrous sodium sulfate, and the lower column contains 0.5-1 g of silica gel or diatomaceous earth. The surface of the silica gel or diatomaceous earth is covered with glass wool. The effluent from the purification column is collected in a brown flask.

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

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

[0029] The concentrated residue was transferred to a reaction tube using diethyl ether and concentrated again until it was dry.

[0030] Add a stir bar and about 20 mg of ferrous sulfate to the reaction tube, and dry under vacuum at 26-30°C for 5-20 minutes.

[0031] (Operation time: 3 hours)

[0032] 3. Methylation derivatives

[0033] Transfer the reaction flask to a nitrogen glove box and add 100-500 μL of tetramethylammonium hydroxide (TBAOH) along the glass wall.

[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] (600 rpm)

[0036] 4. Isolation of methylation products

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

[0038] The bottom of the purification column contains 0.5g of silica gel, and the top contains a desiccant, which can be magnesium sulfate, sodium sulfate, or calcium chloride. Wash the reaction flask twice with 2mL of diethyl ether, passing the washings through the purification column as well. Wash the purification column with 4-5mL of diethyl ether. Combine all eluents.

[0039] Concentrate the diethyl ether solution under reduced pressure until dry (operation time 30 min).

[0040] Furthermore, the conditions for gas chromatograph analysis in step (5) are as follows: the temperature of the injection port and detector is 300℃, the chromatographic column is a capillary column, the capillary column is an SE-54 capillary column with a length of 50m, an inner diameter of 0.25mm, a film thickness of 0.25mm, the stationary phase is 5% phenyl and 95% methyl polysiloxane, the detector is a flame ionization detector, the carrier gas is nitrogen with a purity of >99.999%, the carrier gas flow rate is 1mL / min, the injection volume is 1.0~2.0μL, 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 temperature program of the gas chromatograph in step (5) is as follows: starting temperature 50℃, heating to 170℃ at a heating rate of 4℃ / min and holding for 20min, then heating to 270℃ at a heating rate of 10℃ / min and holding for 20min.

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

[0044] Further, 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 into it. The reaction vessel is then sealed 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 stir bar are added to the centrifuge tube beforehand, and the bottom 1 / 3 of the centrifuge tube is immersed in ice water. 2 mL of 0.5 mol / L NaOH solution is injected along the inner wall of each reaction vessel. The cap is tightened by hand and rotated horizontally for 30 s. The mixture in the vessel is stirred at 1800 rpm for 60 s. Each vessel is then centrifuged again, and the supernatant is added to the centrifuge tube. The washing process is repeated twice. The solution in the centrifuge tube is acidified to pH 6 mol / L hydrochloric acid. 2. Extract the acidified solution with 8 mL of diethyl ether and centrifuge at 4500 rpm for 60 s. Transfer the ether layer to a 50 mL Erlenmeyer flask using 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 Erlenmeyer flask three times with 2 mL of diethyl ether. 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 using a 0.5 g silica gel column. Wash the residue in the flask with diethyl ether and add the solution to the top of the silica gel column. Repeat the washing three times. Wash the column with an additional 7 mL of diethyl ether. Receive the eluent in a 25 mL flask and concentrate on a rotary evaporator at room temperature. Transfer the concentrated residue to a 5 mL Hungate reaction vial with diethyl ether. Concentrate the solution and further dry under vacuum for 20 min after adding a stir bar.

[0045] As another preferred embodiment, in step (2), the reaction vessel is opened, and 200 μL of a solution containing 2 mol / L NaOH and 50 ng / μL 2-bromo-4-hydroxybenzoic acid is immediately injected into it. The reaction vessel is then sealed and magnetically stirred at 7000 rpm for 1 min. 1.5 mL of concentrated hydrochloric acid and a stir bar are added to a centrifuge tube beforehand, and the bottom 1 / 3 of the centrifuge tube is immersed in ice water. The supernatant is added to the centrifuge tube, and 2 mL of 1 mol / L NaOH solution is injected along the inner wall of the reaction vessel. The cap is tightened by hand, and the mixture is magnetically stirred for 2 min. The mixture is then centrifuged again, and the supernatant is added to the centrifuge tube. The washing process is repeated twice. The solution in the centrifuge tube is acidified to pH 6 with 6 mol / L hydrochloric acid. 1-1.5 Extract the acidified solution with 11 mL of diethyl ether, stir for 2 min and let stand until the layers are completely separated. Then, use a glass pipette to transfer the ether layer to the top of the purification column. Pressurize the column to allow the ether to flow through it, and collect the eluent in a brown flask. Repeat the extraction process twice. Wash the purification column with 6 mL of diethyl ether to remove the dried column. Wash the silica gel column with 3 mL of diethyl ether / methanol solution (9:1, v / v). Concentrate the ether solution using a rotary evaporator, and purge with nitrogen at the end. Transfer the residue in the flask to a 5 mL reaction flask with diethyl ether and concentrate it to dryness again. After adding a stir bar, dry under vacuum for 5-20 min.

[0046] Furthermore, the methylation reaction of the lignin phenol was carried out in a 5 mL Hengette vial. After drying under vacuum, the vial was purged with nitrogen. Then, 250 μL of a 40% (w / w) tetrabutylammonium hydroxide aqueous solution, 0.5 mL of dichloromethane, and 100 μL of iodomethane were added sequentially to the vial. The reaction was carried out at 28 °C for 12 h. The pressure generated in the vial was released by inserting a syringe through a 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, and the mixture was extracted with 1 mL of diethyl ether. The extract was passed through an anhydrous magnesium sulfate column, and the extraction was repeated four times. The desiccant was washed with an additional 5 mL of diethyl ether. The eluent was collected 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 it through a 1 g silica gel column. The silica gel column was pre-washed with water, methanol, diethyl 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. This process was repeated seven times, and the eluent was discarded. The flask was then washed with 1 mL of diethyl ether, and the ether solution was passed through the column. This washing was repeated four times, and the column was eluted with an additional 6 mL of diethyl ether. The eluent was collected in a 25 mL flask and concentrated using a rotary evaporator. The residue was transferred to a 5 mL vial with diethyl ether and concentrated again. The residue in the vial was dissolved and mixed with 200 μL of internal standard solution.

[0047] The beneficial effects of this invention are:

[0048] (1) The characterization method of the present invention has good repeatability and reliability: the relative deviation of the seven repeated characterizations, except for CAD and FAD (10%), is 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 rapid: the reaction takes only 90-120 minutes at room temperature.

[0051] (4) High yield of derivatization reaction: The sample impurities have a very small effect on the yield of derivatization reaction.

[0052] (5) Applicable to 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 take only 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). In contrast, the price of each lignin standard used for determining the relative response factor by traditional methods is 1440 yuan / 10mg (Sigma-Aldrich).

[0055] (8) The derivative reagents are inexpensive: the prices of tetrabutylammonium hydroxide and iodomethane are much lower than the price of bis(trimethylsilyl)trifluoroacetamide (25mL, price ~1500 yuan).

[0056] (9) Extremely weak chromatographic peak tailing: Analytical interference and instrument maintenance problems caused by sample matrix contamination at the gas chromatograph injection port are common challenges that traditional lignin characterization methods need to address. However, the quality chromatograms obtained by characterization methods based on lignin phenol methyl derivatives are minimally affected by matrix contamination.

[0057] Comparative studies with traditional lignin characterization methods show that the lignin content measured by the new method is higher than that measured by the traditional method, reaching 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 traditional lignin characterization methods is unreliable.

[0058] The methylation reaction of samples in dichloromethane using tetrabutylammonium hydroxide / iodomethane yielded the best results. Specifically, the sample was first oxidized using CuO / NaOH. A specially designed stainless steel reaction vessel was used, and the reaction was facilitated by magnetic stirring. The second step was the purification of the CuO oxidation product. The acidified aqueous solution was extracted with diethyl ether, and the evaporated sample was further purified using a pre-treated silica gel column. Excessive silica gel was used to prevent the loss of carboxyl-containing lignin due to tailing effects. The third step was the methylation reaction of the sample in dichloromethane using tetrabutylammonium hydroxide / iodomethane. Although we found that trace amounts of ethyl acetate could hinder the conversion of lignin to its methylated form, the remaining impurities in the purified soil samples did not interfere with the methylation reaction. In contrast, the derivatization reaction of lignin using conventional BSTFA / TMCS was sensitive to compounds containing reactive protons, such as water and alcohols. Finally, the methylation product mixture was purified by diethyl ether extraction, followed by further purification of the concentrated sample using a silica gel column. The use of ether is crucial for shortening the overall operation time, as it can be removed quickly and safely via rotary evaporation.

[0059] Ether extraction and silica gel column purification removed most impurities from the sample, yielding a simplified chromatogram. This significantly reduced the time required for gas chromatography maintenance. All operations, except for CuO oxidation and methylation, were performed without argon or nitrogen protection. This greatly reduced operational complexity. Methylated lignin phenols were stable to air and water; after one week of storage in a freezer (-20°C), no decomposition of the toluene solution of methylated lignin phenols was observed. In contrast, TMS derivatives of lignin phenols decomposed during storage due to the gradual introduction of water, necessitating analysis within 24 hours. These novel features of the method make it suitable for routine lignin characterization of geochemical samples with high lignin content. Attached Figure Description

[0060] The present invention will now be described in detail with reference to the accompanying drawings:

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

[0062] Figure 2 The chromatograms of the methylated phenol standard mixtures shown in Table 1 are as follows;

[0063] Figure 3 This is a GC chromatogram of a forest soil sample obtained after CuO oxidation. The mixture of acidified aqueous solution of lignin phenol and methylation products was purified by extraction with diethyl ether only. The peaks marked with vertical short lines are the internal standard and the lignin phenol peaks.

[0064] Figure 4 This is a chromatogram of a forest soil sample. The sample was oxidized with CuO / NaOH, acidified with aqueous solution, extracted with diethyl ether, and purified by silica gel column chromatography. The mixture of methylated products was also extracted with diethyl ether and purified by silica gel column chromatography. Detailed Implementation

[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. Samples were diluted with 200 μL of internal standard (1,4-diisopropylbenzene) toluene solution and thoroughly mixed. Injection volumes were 1 μL and 2 μL for samples with concentrations >0.1 mg / mL and <0.1 mg / mL, respectively, with a split ratio of 2:1 for all samples. The syringe and detector temperatures were set to 300 °C. The diaphragm purification flow rate was 3 mL / min. Straight quartz liners filled with non-deactivated glass wool were used. Separation was performed using an SE-54 capillary column (50 m long, 0.25 mm inner diameter, 0.25 mm film thickness) with a stationary phase of 5% phenyl and 95% methylpolysiloxane. Nitrogen gas with a purity >99.999% was used as the carrier gas (flow rate 1 mL / min). The gas chromatograph temperature program was: 50°C to 170°C, 4°C / min (hold for 20 min), to 270°C, 10°C / min (hold for 20 min). Product identification was performed by comparing its retention time in a standard mixture. Quantification was 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 based on the following equation:

[0068]

[0069] Where C STD and C x A represents the concentration of the internal standard and component x, respectively. STD and A x These are the peak areas of the internal standard and component x, respectively. Therefore, the concentration C of component x in the sample is... x It is calculated using the following equation:

[0070]

[0071] The parameters here have the same meaning as in the previous equation. R f (x) is typically determined at the start of GC analysis by injecting a standard mixture.

[0072] (2) Copper oxide oxidation:

[0073] The oxidation reaction was performed using a specially designed cylindrical stainless steel reaction vessel. The vessel lid contained a PTFE gasket (20 mm in diameter, 2 mm thick). The total capacity of the vessel was 10 mL. The reaction vessel was equipped with a magnetic stir bar (14 × 7 mm) covered with PTFE. 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, then evacuated and refilled with nitrogen four times. 7 mL of 2 mol / L NaOH solution was injected into the vessel, the lid was replaced, 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, then increased to 170 °C at a rate of 5 °C / min and maintained for 180 min. The reaction vessel was cooled with tap water for 10 min. After each oxidation reaction, the PTFE gasket of the reaction vessel was replaced with a new one.

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

[0075] Open the reaction vessel and immediately add 200 μL of a 2 mol / L NaOH aqueous solution of ethyl vanillin. Seal the vessel and magnetically stir at 1800 rpm for 30 s. Centrifuge the vessel at 9000 rpm for 60 s. Quickly pour the supernatant into a 50 mL plastic centrifuge tube containing 1 mL of concentrated hydrochloric acid and a stir bar (7 × 14 mm). Immerse the bottom 1 / 3 of the centrifuge tube in ice water. Pour 2 mL of 0.5 M NaOH solution along the inner wall of the vessel. Tighten the cap by hand and rotate horizontally for 30 s. Stir the mixture in the vessel at 1800 rpm for 60 s. Then centrifuge each vessel again and add the supernatant to the centrifuge tube. Repeat the washing process twice. Acidify the solution in the centrifuge tube to pH 2 with 6 M hydrochloric acid. Extract the acidified solution with 8 mL of diethyl ether and centrifuge at 4500 rpm for 60 s. Transfer the ether layer to a 50 mL Erlenmeyer flask using a glass pipette. Repeat the extraction process three times. The ether solution was dried with anhydrous sodium sulfate for 10 min. The ether extract was filtered through a layer of glass wool into a 50 mL evaporating flask, and the Erlenmeyer flask was washed three times with 2 mL of ether. The ether solution was concentrated to dryness on a rotary evaporator at room temperature. During evaporation, the flask could be immersed in a water bath below 25°C.

[0076] The residue in the flask was purified using a 0.5 g silica gel column. The column was washed consecutively with water (2 mL × 2), methanol (2 mL × 3), and diethyl ether (2 mL × 3). The residue in the flask was washed with 0.7 mL of diethyl ether, and this solution was added to the top of the silica gel column. The washing of the flask was repeated three times. The column was then eluted with an additional 7 mL of diethyl ether. The eluent was collected in a 25 mL flask and concentrated using a rotary evaporator at room temperature. The residue was transferred to a 5 mL Hungate vial with diethyl ether. The solution was concentrated and further dried under vacuum (100 Pa) for 20 min after adding a stir bar (5 × 10 mm) to remove any volatile components that might interfere with subsequent methylation reactions.

[0077] (4) Purification with tetrabutylammonium hydroxide / iodomethane methylation and silica gel column:

[0078] The methylation of lignin phenols 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 sequentially to the reaction vial. The reaction was carried out at 28 °C for 12 h. The pressure in the vial was released by inserting a syringe through the diaphragm. After neutralization with 50 μL of 1M HCl, the mixture was concentrated using a rotary evaporator. 1 mL of water was added to the vial, and the mixture was extracted five times with 1 mL of diethyl ether. Each extract was dried over a 0.6 g column of anhydrous magnesium sulfate. The drying agent was then rinsed with an additional 5 mL of diethyl 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 a 1 g silica gel column. The silica gel column was pre-washed with water (3 mL × 2), methanol (3 mL × 3), diethyl ether (3 mL × 3), and n-hexane (3 mL × 3). First, wash the flask with 1 mL of n-hexane and pass the hexane solution through a silica gel column. Repeat the n-hexane washing process seven times. Discard the eluent. Then wash the flask four times with 1 mL of diethyl ether, passing the ether solution through the silica gel column each time. Elute the column with an additional 6 mL of diethyl ether. Collect the ether eluent in a 25 mL flask and concentrate the ether solution using a rotary evaporator at room temperature. Transfer the residue to a 5 mL vial with diethyl ether and concentrate again. Dissolve the residue in the vial with 200 μL of toluene solution (internal standard) and mix well.

[0079] Example 2

[0080] Experimental results

[0081] 1. Methylation of a standard mixture of lignin phenols

[0082] Although we have previously reported that single lignin monomers can be quantitatively methylated in dichloromethane using iodomethane tetrabutylammonium hydroxide / iodomethane at 28 °C (58), a lignin phenol standard mixture (see Figure 1 The methylation conditions for ) remain unknown. We prepared methanol solutions of a mixture of 14 compounds, including 3-hydroxybenzoic acid and 3,5-dihydroxybenzoic acid, frequently found in CuO oxidation products in environmental samples, and the commonly used recovery standard 3-ethoxy-4-hydroxybenzaldehyde, at concentrations of ~0.2 mg / mL for each compound. 200 μL of the solution was injected into a 5 mL reaction flask equipped with a diaphragm and screw cap using a syringe. The solvent was removed using a rotary evaporator, and after adding a stir bar, the mixture was further dried under vacuum (~100 Pa) for 5 min. After purging the flask with nitrogen, 100 μL of tetrabutylammonium hydroxide, 300 μL of dichloromethane, and 100 μL of iodomethane were added sequentially. 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 diethyl ether (1.0 mL × 4). The extract was passed sequentially through a 0.6 g magnesium sulfate column and a 1.0 g silica gel column. The ether solution was dried and purified. The column system was washed with 6.0 mL of ether. The eluent was concentrated, and the residue was transferred to a 5 mL vial. The methylated products were analyzed by GC-FID using an SE-54 capillary column (50 m × 0.25 mm × 0.25 μm) after adding 200 μL of a toluene solution containing the internal standard (1,4-diisopropylbenzene). The symbols and conversions of the compounds are listed in Table 1. The chromatograms of the methylated product standards (~0.07 mg / mL) are shown below. Figure 2 As shown.

[0083] Except for 3,5-dihydroxybenzoic acid (DiOHBA), the average conversion of the other monomers (three experiments) ranged from 91.6% to 105.2% (Table 1). The relatively low conversion of PAL and PON is likely due to mass loss during vacuum evaporation caused by their lower boiling points (249 °C and 264 °C for methylated PAL and PON, respectively). The two hydroxyl groups in DiOHBA, or the corresponding methoxy groups in the intermediates, have a strong inducible electron-withdrawing effect on the carboxylate ions. Therefore, the stability of the esters formed from DiOHBA is reduced. We also investigated the conversion of ethyl acetate solutions of a standard mixture of lignin phenols. 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 indicates that even trace amounts of some components can have a significant impact on conversion. The conversion and recovery of the compounds and their methylation reactions are shown in Table 1. A standard mixture of 13 compounds (approximately 40 μg of each compound) was used to determine the conversion. The methylation reaction was carried out at 28°C for 10 hours using 0.3 mL of dichloromethane, 100 μL of tetrabutylammonium hydroxide, and iodomethane. The mean deviation was calculated.

[0084] Table 1. Compound symbols and their conversion and recovery rates in methylation reactions.

[0085]

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

[0087] The composition of CuO oxidation products in typical geochemical samples is often quite complex, potentially containing impurities that affect methylation reactions. Furthermore, non-volatile components also contribute to the deterioration of capillary column performance. Silica gel column chromatography is a widely used technique for purifying organic chemicals. By eluting the mixture in the column with different solvents using silica gel column chromatography, impurities with polarity less than or greater than lignin or methylated lignin can be partially removed. However, to our knowledge, silica gel column chromatography has not yet been applied to purify lignin obtained from CuO oxidation in geochemical samples. Therefore, we tested the feasibility of purifying phenols and methylated lignin using silica gel column chromatography.

[0088] We first analyzed the recoveries of the lignin phenol standard mixture (containing approximately 0.1 mg of each compound). A 200 μL solution of the mixture in diethyl ether was loaded onto the top of a 0.5 g silica gel column and eluted with 10 mL of diethyl ether. The eluent was concentrated, and the residue was transferred to a reaction vial. The residue was further dried under vacuum for 20 min and methylated under the previously described conditions. The reaction mixture was neutralized with 1 M HCl and dichloromethane was removed by rotary evaporation. 1 mL of water was added to the residue, and the methylated product was extracted with diethyl ether (1 mL × 5). Each extract was passed sequentially through a 0.6 g anhydrous magnesium sulfate column and a 1 g silica gel column. The column system was then washed with an additional 5 mL of diethyl ether. The received diethyl ether solution was concentrated using a rotary evaporator, and the residue was transferred to a 5 mL vial with diethyl ether. After removing the diethyl ether by rotary evaporation, a toluene solution as an internal standard was added, and the product was analyzed by GC-FID. The analytical recoveries for each phenol are listed in column 4 of Table 1.

[0089] The average recoveries of ligninols in the two tests ranged from 87.9% for SAD to 105.5% for VAL. The relatively low recovery of SAD is likely due to partial loss during the first silica gel column chromatography. SAD is the most polar of the 11 ligninols. Separate silica gel column chromatography of SAD indicated a tailing effect, which may explain its lower recovery. The methylated compound standard mixture in Table 1 (each compound...) <unk>When a 1 mg toluene solution was loaded onto a 1 g silica gel column, we found that 5 mL of diethyl ether completely eluted all compounds from the column. These results indicate that silica gel column chromatography is suitable for purifying lignins and methylated lignins.

[0090] 3. CuO oxidation in forest soil samples

[0091] We first selected forest soil samples (organic carbon content 13.6 g / kg) to study a novel method for CuO oxidation and methylation. We used the reaction conditions reported by Hedge(2). CuO, Fe(NH4)2(SO4)2·6H2O, a stir bar, and 0.5 g of soil were added to the reaction vessel. Atmosphere exchange was easily achieved by placing the reaction vessel in a plastic bag, sealing it, evacuating it, and filling it with nitrogen four times. We first maintained the reaction at 40 °C for 30 minutes to remove trace amounts of molecular oxygen by reacting with Fe(NH4)2(SO4)2·6H2O. Then we reacted at 170 °C for 3 hours.

[0092] 4. Purification using only ether extraction.

[0093] To clarify the purification effect of silica gel column chromatography, we did not purify the ether extract using silica gel column chromatography. The ether extract was concentrated, and the residue was transferred to a reaction vial after washing the flask five times with 1 mL of ether. The ether solution in the vial was concentrated again to obtain approximately 3 mg of residue. After methylation with tetrabutylammonium hydroxide / iodomethane at room temperature for 10 h, the reaction mixture was neutralized, concentrated, and extracted with ether. The extract was dried over magnesium sulfate only, but not purified using silica gel column chromatography. After adding 200 μL of toluene solution as an internal standard, the sample solution was analyzed by GC-FID, and the chromatogram is shown below. Figure 3 As shown. Besides the peaks of the 13 analyzed compounds (marked with blue vertical bars), numerous impurity peaks illustrate the compositional complexity of a typical soil sample. The content of aldehyde phenols in vanillyl and eugenyl lignins is higher than that of their corresponding ketones and carboxylic acids (...). Figure 2 The characteristic of CuO oxidation products in soil samples is wavy (2). Furthermore, the baseline is wavy and cannot be restored to a smooth level even after 150 min. This situation raises significant concerns about GC maintenance. Kaiser et al. reported that even if samples are purified via polymer SPE columns, the packing material in the injection liner should be replaced frequently (once every 24 samples), and the tip of the capillary column should be trimmed after approximately 300 samples to avoid peak tailing effects (4). Quantitative results for 11 lignins and DiOHBA are shown in column 2 of Table 2. The concentrations of lignins obtained from oxidized forest soil samples are shown in Table 2, where the error is the standard deviation.

[0094] Table 2. Concentrations of lignin obtained from oxidized forest soil samples.

[0095]

[0096] 5. Diethyl ether extraction and dual silica gel column purification

[0097] The above results indicate that purifying CuO oxidation products solely through ether extraction produces a rather complex chromatogram. Figure 3 The successful validation of the purification of a mixture of lignin phenol standards and subsequent mixtures of methylated products using silica gel column chromatography encouraged us to investigate the effectiveness of purifying mixtures of CuO oxidation products and methylated products. The ether extract obtained from CuO oxidation products of forest soil samples was concentrated by rotary evaporation. The residues were purified using a pretreated 0.5 g silica gel column. The silica gel in the used column was pale orange, 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 measured chromatograms are shown below. Figure 4 As shown in the figure, the content of each lignin phenol was analyzed and listed in the fourth column of Table 2.

[0098] and Figure 3 In comparison, the chromatograms were significantly simplified, indicating that after purification of lignin and methylated lignin using silica gel column chromatography, most volatile byproducts in the sample were removed, and all volatile components eluted from the capillary column within 75 minutes, with the baseline stabilizing within 90 minutes. After 50 injections, the packing material in the injection port liner turned somewhat gray, but no tailing or deposition of non-volatile components on the liner wall was observed. Comparing the data for the same lignin 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 ether extraction alone. In column 2, the yields of PON, CAD, and FAD were 30%, 133%, and 132% higher than those in column 4, respectively. This may be due to the co-elution of byproducts and lignin. On the other hand, in column 2, the yields of VON, DiOHBA, SON, and SAD were lower than those obtained by ether extraction and silica gel column purification. We infer that the reduced yield is due to impurities in the mixture hindering the conversion of these lignins in the methylation reaction. These results indicate that the use of ether extraction and silica gel column chromatography is crucial for achieving simplified chromatograms and quantitative conversion of lignins to their methylated forms. To further validate the efficiency of the new analytical method, we determined the recoveries of 11 lignins, DiOHBA, CiAD, and EVAL.

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

[0100] The recoveries of EVAL, CiAD, and all lignin phenols were determined by adding 200 μL of a standard mixture of these compounds in 2M NaOH (0.1 mg / mL for each compound) to the reaction mixture immediately after CuO oxidation of the forest soil samples was stopped and the mixture was cooled to room temperature. Recovery was a comprehensive indicator of the efficiency of multiple sample transfers, ether extraction, silica gel column purification, and methylation reactions. Recovery was calculated by subtracting the corresponding analytical value for each lignin phenol from the value shown in column 4 of Table 2, dividing by the spiked value of that lignin phenol, and multiplying by 100%. The recoveries of these compounds are shown in column 5 of Table 2. The recoveries of all compounds (except DiOHBA) ranged from 81% to 103%. These results indicate that the two silica gel column purifications and methylation reactions did not result in a significant loss of lignin phenols. 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 this method using precision and reproducibility. The relative standard deviation (% deviation) of individual lignins in forest soil samples ranged from 3.2% to 16% (n=5) (Table 2, column 5). An inverse correlation existed between lignin content and % deviation: the higher the lignin content, the smaller the % deviation, and vice versa. Higher % deviations at lower lignin contents may be due to two factors: interference from byproducts eluted before or after the target peak in the sample, and lower sensitivity of the FID detector. Higher % deviations for PAL, PON, and PAD may also be related to their higher volatility, especially at lower concentrations. Therefore, excessive drying of methylated lignins should be avoided. The % deviation of individual lignin recoveries ranged from 4% to 10% (Table 2, column 7). Significant improvements in % deviation were due to the addition of a standard mixture, which increased the content of each individual lignin.

[0103] To further investigate the precision and reproducibility of the new analytical method, and the interference of soil impurities on the lignin conversion rate in the methylation reaction, we characterized lignin in three different soils on different dates: tomato field soil, grassland soil, and freshwater sediment. Due to the different sample sources, the impurities in these samples should vary considerably. Quantitative analysis results are shown in Table 3. A similar pattern of variation was found in the average % deviation (n = 2–4) of these three samples: the average % deviation for lignin > 10 μg / g was 2–8%, and the average % deviation for lignin < 10 μg / g was 12–19%. The chromatograms of these samples showed similarities to those of the forest soil samples. The concentrations of CuO oxidation products in grassland soil, tomato field soil, and freshwater sediment are shown in Table 3. These products were obtained through… Figure 4 Purification was performed using the methods described in the text.

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

[0105]

[0106]

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

[0108] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg of ferrous ammonium 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 remove the reactor from the glove box. Heat to 170°C with stirring (1800 rpm) and maintain the temperature for 3 hours.

[0109] S2. Cool to room temperature with running tap water. Open the cap and quickly add 200 μL of 2M NaOH solution containing 2-bromo-4-hydroxybenzoic acid (2-bromo-4-hydroxybenzoic acid concentration 50 ng / μL). Tighten the cap and place in a centrifuge for solid-liquid separation (7000 rpm, 1 min). Prepare a brown centrifuge tube containing 1.5 mL of concentrated hydrochloric acid and a magnetic stir bar, 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 reaction vessel, tighten the cap, and stir vigorously with a magnetic stir bar for 2 min. 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 diethyl ether, stir vigorously for 2 min, and let stand. After complete separation, use a glass dropper to add the upper layer of diethyl ether solution to the top of the purification column. The purification column consisted of a drying column and a silica gel column connected in series. The upper drying column contained 5 g of sodium sulfate, and the lower silica gel column contained 0.5 g of silica gel, with glass wool covering the top of the silica gel. Ether was pressurized and flowed through the purification column, with the eluent collected in a brown flask. The extraction was repeated twice. The purification column was washed with 6 mL of ether to remove the drying column, and the silica gel column was washed with 3 mL of ether / methanol solution (9:1, v / v). The ether solution was concentrated using a rotary evaporator, and nitrogen was introduced at the end. The residue in the flask was transferred to a 5 mL reaction flask and concentrated again to dryness.

[0110] S3. After adding the stir bar, dry under vacuum for 5-20 minutes until no water droplets are visible. Transfer the reaction flask to a nitrogen glove box, add 20 mg of ferrous ammonium sulfate, and dropwise add 500 μL of tetrabutylammonium hydroxide (40%, w / w), 0.5 mL of dichloromethane, and 100 μL of iodomethane along the flask wall. Seal the flask and remove it from the glove box. Stir at 30°C for 120 minutes.

[0111] S4. Open the cap, add 50 μL of 1M HCl, stir for 10 seconds, then add 1.5 mL of diethyl ether and stir for 1 minute. Prepare a purification column in advance, with 0.5 g of silica gel at the bottom and a mixture of 0.8 g of silica gel and 0.8 g of magnesium sulfate at the top. Add the slurry from the reaction flask to the top of the purification column, wash the reaction flask with 1 mL of diethyl ether, add the washings to the top of the purification column, and repeat the washing operation once. Pressurize to force the diethyl ether solution through the purification column, and collect the solution in a 12 mL amber glass bottle. Wash the purification column with 4.5 mL of diethyl ether, and combine the eluents in the amber bottle. Concentrate the diethyl ether solution to dryness using a rotary evaporator.

[0112] S5. Add the toluene solution of ethyl 2-iodobenzoate and mix well. Analyze using GC-MS in SIM mode. Injection volume: 1 μL; split ratio: 50:1.

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

[0114] Table 4

[0115]

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

[0117] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg ferrous ammonium sulfate, 1.00 g CuO, 0.5000 g soil, and 10 mg 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 maintain the temperature for 3 hours.

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

[0119] The experiment was repeated three more times, and 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 ferrous ammonium sulfate, 1.00 g CuO, 0.5000 g soil, and 20 mg 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 maintain the temperature for 3 hours.

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

[0126] The experiment was repeated three more times, and 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 ferrous ammonium sulfate, 0.500 g CuO, 0.5000 g grassland soil sample #1 (organic carbon content 8.62 g / kg), and 20 mg 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 maintain the temperature for 2 hours.

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

[0133] The 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: Conventional 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 ferrous ammonium sulfate, 0.500 g CuO, 0.5000 g grassland soil sample #1 (organic carbon content 8.62 g / kg), and 20 mg 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 maintain the temperature for 2 hours.

[0139] S2. Open the reactor lid, 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 the solution into the centrifuge tube. Repeat the washing operation 3 times. Centrifuge to separate the solid and liquid components (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 washings into a 25 mL centrifuge tube, centrifuge, and transfer the supernatant into 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 h. Centrifuge to separate the solid and liquid components (8000 rpm, 25 min), and transfer the supernatant into 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 into a volumetric flask. Dilute to 100 mL with distilled water.

[0140] S3. Prepare a C 18 For solid-phase extraction, the packing material was wetted with 3 mL of ethyl acetate for 2 min, then removed under reduced pressure. The packing material was then wetted with 3 mL of anhydrous ethanol for 2 min, then removed under reduced pressure. Finally, the packing material was wetted with 5 mL of distilled water for 2 min, then removed 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, dry the inside of the column with nitrogen gas. Elute the column with 0.5 mL of ethyl acetate, collecting the eluent in a test tube wrapped with aluminum foil. Repeat the above elution operation 6 times. Elute under reduced pressure for the first 1-2 times, under gravity for the 3-4 times, and under reduced pressure for the 5-7 times. Transfer the eluent in the test tube to a derivatization flask. Remove the water droplets at the bottom of the derivatization flask with a pipette. Add 0.5 mL of a methanol solution of phenylacetic acid (50 μg / mL) to the derivatization flask. Mix well and dry with nitrogen gas (protected from light).

[0142] S5. Add 50 μL of pyridine and 100 μL of derivatizing reagent (bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane) to the derivatization flask. Derivatize at 60 °C for 15 min, shaking the flask every 5 min.

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

[0144] Table 8

[0145]

[0146]

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

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

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

[0150] The 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: Conventional method for lignin in grassland soil sample #2 (C 18 -SPE) characterization

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

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

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

[0163] The 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 using conventional methods (ethyl acetate extraction)

[0167] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg ferrous ammonium sulfate, 0.500 g CuO, 0.5000 g forest soil sample #1 (organic carbon content 13.92 g / kg), and 20 mg 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 maintain the temperature for 2 hours. Cool to room temperature with tap water.

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

[0169] S3. Extract the aqueous solution with 10 mL of ethyl acetate, repeating three times. Combine the extracts in a 30 mL centrifuge tube. Dry the tube under nitrogen at 38 °C.

[0170] S4. Prepare three standard samples simultaneously. Add 100 μL of a standard mixture (containing 8 standard samples) in 2M NaOH solution (1 mg / mL) and 400 μL of ethyl vanillin in 2M NaOH solution (0.1 mg / mL) to a 2 mL derivatization flask. Mix well and then dry with nitrogen gas.

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

[0172] GC-MS analysis was performed. The injection port temperature was 250℃, the injection volume was 1 μL, and the split ratio was set to 5:1. The detection 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. Add a magnetic stir bar to a stainless steel reactor, along with 100 mg ferrous ammonium sulfate, 0.500 g CuO, 0.5000 g forest soil sample #2 (organic carbon content 14.35 g / kg), and 20 mg glucose. Transfer the reactor to a nitrogen glove box and add 8 mL of 2M NaOH solution. Tighten the lid and remove the reactor from the glove box. Heat to 170°C with stirring (1800 rpm) and maintain the temperature for 2 hours.

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

[0178] The 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 conventional methods (ethyl acetate extraction)

[0183] S1. Place a magnetic stir bar in a stainless steel reactor, add 100 mg ferrous ammonium sulfate, 0.500 g CuO, 0.5000 g forest soil sample #2 (organic carbon content 14.35 g / kg), and 20 mg 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 maintain the temperature for 2 hours. 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: Determination of the stability of the relative response factor (RRf)

[0188] Accurately weigh 5 mg each of 11 lignins, methyl 2-bromo-4-methoxybenzoate, and ethyl 2-iodobenzoate into a 100 mL volumetric flask, dissolve in toluene, and dilute to 100 mL. The concentration of each compound is approximately 50 ng / μL. Store the solution in a sealed container at -20°C. For the determination of the relative response factor, first remove the solution and warm it to near room temperature. Add approximately 100 μL to the inner liner of the chromatographic sample vial. Analyze using GC-MS in SIM mode with an injection volume of 1 μL and a split ratio of 50:1. Analyze and calculate the relative response factor 18 times consecutively or at intervals of 1 day, with each analysis repeated 3 times. Take the average value, calculate the mean and standard deviation of the 18 average values, and the results are shown in Table 15.

[0189] Table 15

[0190]

[0191] Example 14: Stability determination of analytical samples (after 20 hours of storage at room temperature)

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

[0193] Table 16

[0194]

[0195] Example 15: Stability determination of analytical samples (after storage at -20°C for 30 days)

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

[0197] Table 17

[0198]

[0199]

[0200] This invention proposes a novel 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 was used, and the reaction was facilitated by magnetic stirring. The second step is the purification of the CuO oxidation products. The acidified aqueous solution was extracted with diethyl ether, and the evaporated sample was further purified using a pretreated silica gel column. The third step is the methylation of the sample in dichloromethane using tetrabutylammonium hydroxide / iodomethane. Although we found that trace amounts of ethyl acetate can reduce the conversion of lignin phenols to methylated forms, impurities in the soil samples studied did not interfere with the methylation reaction. In contrast, the lignin derivatization reaction of BSTFA / TMCS is sensitive to compounds containing active protons, such as water and alcohols (4). Finally, the mixture of methylated products was purified by extraction with diethyl ether, followed by subsequent purification of the concentrated sample using a silica gel column. The use of diethyl ether is crucial for shortening the overall operation time, as it can be removed quickly and safely by rotary evaporation.

[0201] Ether extraction and silica gel column purification removed most impurities from the sample, yielding a significantly simplified chromatogram. Therefore, the time required for gas chromatography maintenance is significantly reduced. Except for the CuO oxidation and methylation reactions, which were carried out under nitrogen protection, all other operations were performed without argon or nitrogen protection. This greatly reduces operational complexity. Methylated lignin phenols are stable to air and trace amounts of water; in contrast, the lignin derivatization efficiency of BSTFA / TMCS is sensitive to water and alcohol. Due to concerns about the decomposition of TMS derivatives of lignin phenols during storage due to gradual introduction of water, analysis within 24 hours is necessary. In contrast, after one week of storage in a freezer (~20°C), we did not observe decomposition of the toluene solution of methylated lignin phenols. These novel characteristics of the method make it suitable for lignin characterization of geochemical samples with high lignin content.< / unk>

Claims

1. A method for characterizing lignin in environmental samples, characterized in that, Includes the following steps: (1) Oxidation of copper oxide: The oxidation reaction was carried out in a cylindrical stainless steel reaction vessel containing a PTFE magnetic stir bar, 500 mg of copper oxide, 200 mg of Fe(NH4)2(SO4)2∙6H2O, and 500 mg of environmental sample. The reaction vessel was then transferred to a plastic bag and connected to a vacuum nitrogen line. The bag was sealed, evacuated, and refilled with nitrogen four times. 7 mL of 2 mol / L NaOH solution was injected into the reaction vessel, the lid was closed, and the bag was removed. The mixture in the reaction vessel was stirred at 1800 rpm. The temperature was maintained at 40 °C for 30 min, increased to 170 °C at a rate of 5 °C / min, and maintained for 180 min. The reaction vessel was then cooled with tap water for 10 min. (2) Extraction with diethyl ether and purification with silica gel column chromatography Open the reaction vessel and immediately inject an aqueous solution of ethyl vanillin in NaOH (2 mol / L). Seal the reaction vessel and stir magnetically at 1800 rpm. Centrifuge the reaction vessel at 9000 rpm. Quickly pour the supernatant into a plastic centrifuge tube containing 1 mL of concentrated hydrochloric acid and a stir bar. Immerse the bottom 1 / 3 of the centrifuge tube in ice water. Inject 2 mL of 0.5 M NaOH solution along the inner wall of the reaction vessel. Tighten the cap by hand and rotate horizontally. Stir the mixture in the reaction vessel at 1800 rpm. Centrifuge the reaction vessel again and add the supernatant to the centrifuge tube. Repeat the washing process twice; acidify the solution in the centrifuge tube to pH 2 with 6M hydrochloric acid; extract the acidified solution with 8 mL of diethyl ether and centrifuge at 4500 rpm; transfer the ether layer to an Erlenmeyer flask using a glass pipette; repeat the extraction process three times; dry the ether solution with anhydrous sodium sulfate; filter the ether extract through a layer of glass wool into an evaporating flask, and wash the Erlenmeyer flask three times with 2 mL of diethyl ether; concentrate the ether solution to dryness on a rotary evaporator at room temperature; during evaporation, immerse the flask in a water bath below 25°C; The residue in the flask was purified using a 0.5 g silica gel column; the column was pre-washed with water, methanol, and diethyl ether; the residue in the flask was washed with 0.7 mL of diethyl ether, and this solution was added to the top of the silica gel column; the washing of the flask was repeated three times; the column was then eluted with an additional 7 mL of diethyl ether; the eluent was collected in a flask and concentrated using a rotary evaporator at room temperature; the residue was transferred to a Hungate reaction vial with diethyl ether; the solution was concentrated and further dried under vacuum for 20 min after adding a stir bar; (3) Methylation with tetrabutylammonium hydroxide / iodomethane and purification with silica gel column: The methylation of lignin phenols was carried out in a Hungate reaction flask; after drying under vacuum, the flask was filled with nitrogen; 250 μL of 40% w / w tetrabutylammonium hydroxide, 0.5 mL of dichloromethane, and 100 μL of iodomethane were added sequentially to the reaction flask; the reaction was carried out at 28 °C for 12 h; the pressure in the flask was released by inserting a syringe through a diaphragm; the mixture was neutralized with 50 μL of 1M HCl and concentrated using a rotary evaporator; 1 mL of water was added to the flask, and the mixture was extracted five times with 1 mL of diethyl ether; each extract was dried through a 0.6 g column of anhydrous magnesium sulfate; the drying agent was then rinsed with an additional 5 mL of diethyl ether; the ether solution was received in a flask and concentrated to dryness using a rotary evaporator at room temperature; the residue in the flask was purified by a 1 g silica gel column; the silica gel column was pre-washed with water, methanol, diethyl ether, and n-hexane; the flask was first washed with 1 mL of n-hexane, and the n-hexane solution was then purified by passing the silica gel column through a 1 g column. Silica gel column; repeat the hexane washing process seven times; discard the eluent; then wash the flask four times with 1 mL of diethyl ether, passing the ether solution through the silica gel column each time; elute the column with an additional 6 mL of diethyl ether; collect the ether eluent in a flask and concentrate the ether solution using a rotary evaporator at room temperature; transfer the residue to a vial with diethyl ether and concentrate again; dissolve the residue in the vial with 200 μL of toluene solution containing internal standard and mix well; analyze the methylation products using GC-FID with an SE-54 capillary column; (4) Gas chromatography analysis: The conditions for gas chromatography analysis were as follows: the temperature of the injection port and detector was 300℃; the chromatographic column was an SE-54 capillary column with a length of 50m, an inner diameter of 0.25mm, and a film thickness of 0.25μm; the detector was a flame ionization detector; the carrier gas was nitrogen with a purity >99.999%; the carrier gas flow rate was 1mL / min; the injection volume was 1.0-2.0μL; and the split ratio was 2:

1. The temperature program for the gas chromatograph is as follows: starting temperature 50℃, increasing to 170℃ at a rate of 4℃ / min and holding for 20min, then increasing to 270℃ at a rate of 10℃ / min and holding for 20min.

2. The method for characterizing lignin in environmental samples according to claim 1, characterized in that, The environmental sample in step (1) is one of water, plants, soil, sediment, sewage or silt.

3. The method for characterizing lignin in environmental samples according to claim 1, characterized in that, The gas chromatograph used in step (4) is a Shimadzu GC-2014C gas chromatograph.

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