Method for detecting lignin content in purple soil

Abstract

This invention discloses a method for detecting lignin content in purple soil, belonging to the field of soil component detection technology. The method includes: dividing the purple soil sample to be tested into three sub-sample groups; establishing a background baseline for the first sub-sample group to obtain physical state reference parameters; performing lignin-directed capture on the second sub-sample group to obtain a first state response parameter; performing standard addition and lignin-directed capture on the third sub-sample group to obtain a second state response parameter; and determining the lignin content based on the logical comparison results of the differences between the physical state reference parameter, the first state response parameter, and the second state response parameter. This method utilizes magnetic nanoparticles for specific capture of lignin, combined with standard addition and difference comparison calculations, to achieve rapid, non-dissipative determination of soil lignin while eliminating interference from the complex matrix of purple soil.

Description

Technical Field

[0001] This invention relates to the field of soil composition detection technology, specifically a method for detecting lignin content in purple soil. Background Technology

[0002] Accurate determination of lignin content in purple soils is of significant reference value for assessing soil organic matter composition and stability. Current techniques for quantitative detection of soil lignin largely rely on chemical degradation or wet chemical oxidation methods. These methods decompose lignin into characteristic monomeric products, which are then indirectly estimated using gas chromatography or liquid chromatography. However, these methods require treatment of purple soil samples with strong acids, strong alkalis, or high temperature and pressure, making the process lengthy and disrupting the original physical colloidal state of the soil. Consequently, they fail to reflect the true occurrence and behavior of lignin in the natural suspension system of purple soils.

[0003] For the rapid detection of lignin in soil, direct analytical methods based on spectral or turbidity responses have been attempted. However, purple soils are rich in iron and aluminum oxides, clay minerals, and highly reactive humic acids, which generate strong background scattering and absorption interference in the liquid phase system. Existing direct analytical methods generally adopt a full-volume measurement mode, equating the overall optical response of the soil suspension with the target signal of lignin. This lack of selective identification and separation steps for lignin leads to a large number of false positive contributions from inorganic clay particles and non-target organic matter in the measured total turbidity or absorbance, resulting in a significant deviation between the detection results and the true lignin content.

[0004] Purple soil matrix has a complex composition and significant batch-to-batch physicochemical variations. Even with the same processing procedures, the initial turbidity background of soil suspensions from different sampling points exhibits natural fluctuations. Existing methods lack effective correction mechanisms for this fluctuation, typically relying solely on absolute measurements from a single sample or simple blank subtraction, making it difficult to extract the unique signal fraction of lignin from a dynamically changing matrix background.

[0005] The technical problem to be solved by this invention is how to achieve selective identification and directional enrichment of lignin components in a purple soil suspension system with high background interference, and how to establish a comparative analysis logic that can adaptively correct matrix turbidity differences without relying on full digestion treatment. Summary of the Invention

[0006] This invention provides a method for detecting lignin content in purple soil, aiming to solve the problems of selective detection of lignin under high background interference in purple soil matrix and the inability to effectively correct differences in turbidity background between different samples, and to achieve rapid determination of lignin content in purple soil under non-digestion conditions.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The present invention discloses a method for detecting lignin content in purple soil. This method establishes a triple-parameter system that distinguishes between a physical state baseline, a lignin-oriented capture response, and a standard addition capture response, and accurately quantifies lignin in purple soil using differential logic comparison. The method first obtains a purple soil sample to be tested and divides it into a first subsample group, a second subsample group, and a third subsample group with equal mass.

[0008] A background baseline establishment operation is performed on the first subsample group to obtain physical state reference parameters reflecting the soil's own physical state. Specifically, the first subsample group is mixed with deionized water at a preset mass-to-volume ratio to form a first mixed suspension. This suspension is then ultrasonically dispersed at a frequency of 40 kHz for a specified duration. After settling, the supernatant is collected, and its turbidity value is measured using a turbidimeter. This turbidity value is used as the physical state reference parameter. Preferably, the ultrasonic dispersion time is 10 to 20 minutes to ensure sufficient separation of soil particles without damaging the intrinsic morphology of organic matter.

[0009] A lignin-directed capture operation was performed on the second subsample group to obtain the first-state response parameter after lignin capture. This operation employed magnetic nanoparticles surface-modified with boric acid affinity ligands. The boric acid ligands can specifically and reversibly bind to the cis-dihydroxy groups in the lignin structure, achieving highly selective directional capture. Preferably, the magnetic nanoparticles had a particle size of 50 to 200 nanometers, and the boric acid affinity ligands were selected from 3-aminophenylboronic acid or 4-mercaptophenylboronic acid to maintain capture efficiency under high background interference in purple soil. In the operation, the second subsample group was prepared into a second dispersion suspension at the same mass-to-volume ratio. The dispersed magnetic bead stock solution was added, and the mixture was incubated at 25 to 37 degrees Celsius with shaking at 200 rpm for 30 to 60 minutes to form a capture mixture. After separating the magnetic nanoparticles using an external magnetic field, the residual liquid was collected and its turbidity was measured. The turbidity value of the residual liquid was recorded as the first-state response parameter.

[0010] A standard addition and lignin-directed capture operation is performed on the third subsample group to obtain the second-state response parameter after standard addition and lignin capture. In this step, the third subsample group is mixed with deionized water, and a lignin standard solution of known mass concentration is added to form a standard addition suspension. This suspension is then ultrasonically dispersed, incubated with magnetic nanoparticles for capture, magnetically separated, and the turbidity of the residual liquid is measured. The resulting turbidity value is used as the second-state response parameter. As a preferred embodiment of the invention, the third subsample group can be equally divided into at least three parallel standard addition subsamples, and lignin standard solutions with mass concentrations distributed in an arithmetic sequence are added to each subsample to obtain a multi-gradient standard addition suspension, which can be used to construct a more robust ratio-content mapping relationship.

[0011] Based on the logical comparison results of the differences between the aforementioned physical state baseline parameters, the first state response parameters, and the second state response parameters, the lignin content in the purple soil sample to be tested is determined. Specifically, the difference between the first state response parameter and the physical state baseline parameter is calculated to obtain the first net turbidity increment; the difference between the second state response parameter and the physical state baseline parameter is calculated to obtain the second net turbidity increment. The turbidity inhibition ratio is obtained by the ratio of the second net turbidity increment to the first net turbidity increment. This ratio has a clear correspondence with the lignin content and can shield the influence of soil matrix differences and uneven particle distribution. When the first net turbidity increment is zero, the turbidity inhibition ratio is directly assigned to zero; otherwise, it is obtained by division. By comparing the turbidity inhibition ratio with the preset ratio-content mapping relationship, the lignin content can be determined. The ratio-content mapping relationship can be obtained by using a ratio-content standard curve pre-constructed with a homologous blank purple soil matrix. This involves adding at least five different mass concentrations of lignin standards to the blank matrix, measuring the turbidity inhibition ratio at each concentration, and fitting the standard curve with the added concentration as the x-axis and the turbidity inhibition ratio as the y-axis. Alternatively, it can be directly calculated using a pre-constructed linear regression equation with the added standard concentration as the independent variable and the turbidity inhibition ratio as the dependent variable.

[0012] This invention involves collecting topsoil from the cultivated layer of purple soil in the field, removing plant debris and gravel, air-drying to constant weight, grinding and sieving, collecting the sieved soil particles, and then using a rotary sampler to equally divide the sample, ensuring the consistency of the subsamples. The entire detection process uses turbidity change as the core sensing signal, combined with magnetic separation technology to achieve homogeneous reaction and heterogeneous separation. The operation is simple and does not require large chromatographic or spectroscopic instruments, significantly reducing detection costs and operational complexity. The specific capture of lignin by boric acid-affinity magnetic nanoparticles effectively eliminates interference from other organic matter and minerals in purple soil. The calculation method of turbidity inhibition ratio further eliminates differences in soil background turbidity between batches, giving the method good reproducibility and resistance to matrix interference, making it particularly suitable for rapid batch determination of lignin content in purple soil cultivated areas.

[0013] The technical effects and advantages provided by the present invention in the above technical solution are as follows:

[0014] Magnetic nanoparticles with boric acid affinity ligands on their surface were used as the directional capture medium for lignin. In the liquid phase, the boric acid ligands can reversibly covalently bind with the ortho-diol groups in the lignin molecule, forming a specific cyclic ester structure. The inorganic clay particles, iron and aluminum oxides, and non-target humic substances abundant in the purple soil lack ortho-diol functional groups and are not captured by the magnetic nanoparticles. After applying an external magnetic field, the lignin-magnetic bead complex is rapidly removed from the suspension, leaving only unbound non-lignin components in the residual liquid. The turbidity of this residual liquid was measured, and the resulting signal value, after subtracting the turbidity contribution of lignin itself, yielded a first-state response parameter reflecting the system change after the selective removal of lignin. This method bypasses the traditional path of full-component digestion of purple soil, directly physically separating lignin from the complex matrix and eliminating spectral interference from inorganic colloids and non-target organic matter.

[0015] To address the inherent differences in turbidity background among different purple soil samples, a standard addition and differential logic comparison mechanism is introduced. A third independent subsample group is extracted from the same sample to be tested. Under consistent operating conditions, a lignin standard of known concentration is pre-added, and then the same directional capture procedure as the second subsample group is executed. The resulting second-state response parameter contains information on the turbidity change after the combined effect of added lignin and captured background lignin. By incorporating the second-state response parameter, physical state reference parameter, and first-state response parameter into a unified differential calculation, the turbidity inhibition ratio caused by standard addition is calculated. This ratio depends on the signal change amplitude after the added lignin participates in competitive adsorption, rather than a single measured absolute turbidity value. Therefore, during the calculation, the initial turbidity differences caused by variations in mineral composition and organic matter abundance within the purple soil samples are automatically canceled out. Based on the mapping relationship between this ratio and lignin content, the actual lignin concentration in the sample to be tested is inferred, making the measurement results of different batches of purple soil samples comparable and avoiding systematic measurement bias caused by fluctuations in soil matrix background. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0017] Figure 1 This is a flowchart of the method for detecting lignin content in purple soil;

[0018] Figure 2 This is a flowchart of the lignin-directed capture operation;

[0019] Figure 3 This is a flowchart for determining lignin content based on logical comparison of turbidity differences;

[0020] Figure 4 This is a curve showing the change in turbidity of the supernatant over the settling time of the purple soil suspension.

[0021] Figure 5 This is a graph showing the standard curve and inference results of lignin content in purple soil samples;

[0022] Figure 6 This is a statistical chart showing the quality distribution of purple soil samples. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] See Figure 1 This invention provides a method for detecting lignin content in purple soil, comprising the following steps: acquiring a purple soil sample to be tested, dividing the purple soil sample to be tested into a first sub-sample group, a second sub-sample group, and a third sub-sample group; performing a background baseline establishment operation on the first sub-sample group to obtain the physical state reference parameters of the purple soil sample to be tested; performing a lignin-directed capture operation on the second sub-sample group to obtain the first state response parameter of the second sub-sample group after lignin capture; performing a standard addition and lignin-directed capture operation on the third sub-sample group to obtain the second state response parameter of the third sub-sample group after standard addition and lignin capture; and determining the lignin content in the purple soil sample to be tested based on the logical comparison results of the differences between the physical state reference parameters, the first state response parameter, and the second state response parameter.

[0025] Example 1:

[0026] In practice, after obtaining the purple soil sample to be tested, the purple soil sample to be tested is divided into the first subsample group, the second subsample group and the third subsample group, and the background baseline establishment operation is performed on the first subsample group.

[0027] In some embodiments, the background baseline establishment operation includes: mixing a first subsample group with deionized water at a preset mass-to-volume ratio to form a first mixed suspension. The preset mass-to-volume ratio is selected based on the texture type of the purple soil, which is classified into four types according to the Chinese Soil Texture Classification Standard: sandy loam, loam, silty loam, and clay loam. When the purple soil texture type is sandy loam, the preset mass-to-volume ratio is set to 1:5 because sandy loam has larger pores between particles, requiring less volume of deionized water to form a uniform suspension; when the purple soil texture type is loam, the preset mass-to-volume ratio is set to 1:8; when the purple soil texture type is silty loam, the preset mass-to-volume ratio is set to 1:10; and when the purple soil texture type is clay loam, the preset mass-to-volume ratio is set to 1:20 because clay loam has a high clay content, requiring more volume of deionized water to achieve sufficient dispersion. Taking a typical purple silty loam as an example, the preset mass-to-volume ratio is 1:10. The specific operation is as follows: weigh 10.00 grams of the first subsample group, measure 100 ml of deionized water, put the first subsample group and deionized water into a 250 ml glass beaker, stir with a glass rod for 2 minutes to form the first mixed suspension.

[0028] The first mixed suspension is placed in an ultrasonic disperser and subjected to ultrasonic dispersion treatment for a specified duration to obtain a first dispersed suspension. Optionally, a trough-type ultrasonic disperser is used, and the operating frequency of the ultrasonic disperser is set to 40 kHz. The 40 kHz operating frequency is set based on the fact that, for secondary mineral particles with a particle size distribution of 2 to 50 micrometers in purple soil, the microjets generated by the ultrasonic cavitation effect at 40 kHz can overcome the van der Waals forces between particles without damaging the structural integrity of the organic matter macromolecules. The specified duration is related to the preset mass-to-volume ratio and the texture type of the purple soil. When the preset mass-to-volume ratio is in the range of 1:5 to 1:8, the specified duration is set to 10 minutes; when the preset mass-to-volume ratio is 1:10, the specified duration is set to 15 minutes; and when the preset mass-to-volume ratio is in the range of 1:15 to 1:20, the specified duration is set to 20 minutes. In the implementation scenario where the preset mass-to-volume ratio is 1:10, a beaker containing the first mixed suspension is placed in the water tank of the ultrasonic disperser, with the water level 2 cm above the surface of the suspension in the beaker. The timer of the ultrasonic disperser is set to 15 minutes, and the ultrasonic disperser is started to perform ultrasonic dispersion treatment. During the treatment, the suspension is stirred with a glass rod once every 5 minutes to ensure uniform dispersion. After the ultrasonic dispersion treatment is completed, the beaker is removed to obtain the first dispersed suspension.

[0029] It is understandable that after ultrasonic dispersion treatment, the first dispersed suspension is allowed to stand for a specified time. The specified standing time is uniformly set at 30 minutes. This 30-minute setting is based on sedimentation tests: in the purple soil suspension system, soil particles larger than 5 micrometers in diameter basically settle within 30 minutes under gravity, while clay particles and organic colloids smaller than 5 micrometers in diameter remain suspended. At this time, the turbidity of the supernatant is mainly contributed by colloidal particles, which can reflect the physical dispersion state of the soil sample and avoid turbidity fluctuations caused by the sedimentation of coarse particles. During the standing process, the beaker is placed on a vibration-free platform, and the mouth of the beaker is covered with a watch glass to prevent dust from falling in. The ambient temperature is controlled between 20 and 25 degrees Celsius.

[0030] After the specified settling time, the turbidity value of the supernatant of the first dispersed suspension is measured using a turbidimeter, and the turbidity value of the supernatant is recorded as a physical state reference parameter. In some embodiments, the turbidimeter is calibrated using formalin standard solution before measurement. The specific calibration steps are as follows: prepare a formalin standard stock solution with a concentration of 400 NTU, and then dilute it to obtain a series of standard solutions of 0 NTU, 0.2 NTU, 1 NTU, 10 NTU, 100 NTU, and 400 NTU. The turbidity values ​​of the standard series solutions are measured sequentially using a turbidimeter. A calibration curve is established with the standard value on the x-axis and the turbidimeter reading on the y-axis. The linear correlation coefficient of the calibration curve must reach above 0.999 before it can be used for sample measurement. The turbidimeter measurement wavelength is selected as 860 nm. The selection of the wavelength of 860 nm is based on the fact that dissolved organic matter in the supernatant of purple soil has strong absorption interference in the short-wavelength region of visible light, while the absorption of organic matter at 860 nm is extremely weak. The turbidity response signal mainly originates from particle light scattering, which can more accurately reflect the concentration of colloidal particles. When measuring the turbidity of the supernatant of the first dispersion suspension, use a pipette to draw 20 ml of supernatant from 2 cm below the liquid surface in the beaker and transfer it into the turbidimeter sample cell. Take three consecutive turbidimeter readings for each sample; these three readings are designated as Reading 1, Reading 2, and Reading 3. Take the arithmetic mean of the three readings as the final reading. The arithmetic mean is calculated using the formula:

[0031]

[0032] in, Represents the physical state reference parameter, with the unit being NTU; , , The figures represent the first, second, and third turbidimeter readings, respectively, all in NTU. Record the calculated physical state reference parameters in the experimental record table. After the turbidimeter measurements are completed, immediately clean the sample cell and rinse it three times with deionized water. Collect and dispose of the remaining first dispersion suspension according to laboratory waste management regulations.

[0033] See Figure 4 The figure shows the curve of turbidity of the supernatant of the first dispersed suspension of purple soil sample as a function of settling time, obtained from the background baseline establishment operation in Example 1, and its sedimentation double exponential fitting result. The horizontal axis represents the settling time in minutes, ranging from 0 to 30 minutes; the vertical axis represents turbidity in NTUs, ranging from approximately 40 to 280 NTUs. The scatter points in the figure represent the actual measured values ​​of supernatant turbidity, and the curve is the fitting curve based on the sedimentation double exponential model.

[0034] As shown in the figure, the initial turbidity was approximately 280 NTU. With increasing settling time, the turbidity showed a significant decreasing trend, with a faster rate of decrease in the first 10 minutes, dropping from approximately 280 NTU to about 80 NTU. Afterward, the rate of turbidity decrease slowed, gradually stabilizing between 15 and 30 minutes, eventually settling at approximately 45 to 50 NTU. This trend reflects the sedimentation process of larger particles in the purple soil suspension under gravity. Soil particles larger than 5 micrometers in diameter basically completed sedimentation within 30 minutes, and the remaining supernatant turbidity was mainly contributed by colloidal particles smaller than 5 micrometers in diameter.

[0035] The fitted curves closely match the measured values, indicating that the sedimentation double-exponential model can accurately describe the turbidity variation of the purple soil suspension, and demonstrating the stability and reliability of the physical state reference parameters of the suspension system after ultrasonic dispersion treatment. These fitting results provide a theoretical basis for establishing a subsequent turbidity baseline and help to accurately obtain the physical state reference parameters. This lays the foundation for the logical comparison analysis of the difference in lignin content in the examples.

[0036] Example 2:

[0037] In specific implementation, please refer to Figure 2 After obtaining the purple soil samples to be tested, the purple soil samples to be tested were divided into the first subsample group, the second subsample group and the third subsample group. The lignin-directed capture operation was performed on the second subsample group.

[0038] In some embodiments, the lignin-directed capture operation includes: mixing a second subsample group with deionized water at a preset mass-to-volume ratio to form a second mixed suspension. The preset mass-to-volume ratio is selected based on the texture type of the purple soil and is numerically consistent with the preset mass-to-volume ratio used in the background baseline establishment operation. When the purple soil texture type is sandy loam, the preset mass-to-volume ratio is set to 1:5; when the purple soil texture type is loam, the preset mass-to-volume ratio is set to 1:8; when the purple soil texture type is silty loam, the preset mass-to-volume ratio is set to 1:10; and when the purple soil texture type is clay loam, the preset mass-to-volume ratio is set to 1:20. In an embodiment of purple silty loam, the preset mass-to-volume ratio is 1:10. Specifically, 10.00 grams of the second subsample group and 100 ml of deionized water are weighed, and the second subsample group and deionized water are placed in a 250 ml glass beaker. The mixture is stirred with a glass rod for 2 minutes to initially mix the soil particles with the deionized water, forming the second mixed suspension.

[0039] Optionally, the second mixed suspension is placed in an ultrasonic disperser and subjected to ultrasonic dispersion treatment for a specified duration to obtain a second dispersed suspension. A trough-type ultrasonic disperser is selected, and its operating frequency is set to 40 kHz. The 40 kHz operating frequency setting is based on the same logic as the setting in the background baseline establishment operation, namely, for secondary mineral particles with a particle size distribution of 2 to 50 micrometers in purple soil, the microjets generated by the 40 kHz ultrasonic cavitation effect can overcome the van der Waals forces between particles without damaging the structural integrity of the lignin macromolecules. The specified duration is determined as follows: when the preset mass-to-volume ratio is in the range of 1:5 to 1:8, the specified duration is set to 10 minutes; when the preset mass-to-volume ratio is 1:10, the specified duration is set to 15 minutes; and when the preset mass-to-volume ratio is in the range of 1:15 to 1:20, the specified duration is set to 20 minutes. In the implementation scenario where the preset mass-to-volume ratio is 1:10, a beaker containing the second mixed suspension is placed in the water tank of the ultrasonic disperser, with the water level in the tank exceeding the surface of the suspension in the beaker by 2 cm. The timer of the ultrasonic disperser is set to 15 minutes, and the ultrasonic disperser is started to perform ultrasonic dispersion treatment. During the treatment, the suspension is stirred with a glass rod once every 5 minutes. After the ultrasonic dispersion treatment is completed, the beaker is removed to obtain the second dispersed suspension.

[0040] It is understood that after obtaining the second dispersion suspension, magnetic nanoparticles with boric acid affinity ligands modified on their surface are added to the second dispersion suspension, and the mixture is incubated with shaking at a specified temperature for a specified incubation time to form a capture-mix system. The particle size range of the magnetic nanoparticles with boric acid affinity ligands modified on their surface is 50 nm to 200 nm. The lower limit of the magnetic nanoparticle particle size is set at 50 nm because the magnetic responsiveness of the magnetic nanoparticles is significantly reduced when the particle size is smaller than 50 nm, making it difficult to achieve efficient separation under the action of an external magnetic field; the upper limit of the magnetic nanoparticle particle size is set at 200 nm because the specific surface area of ​​the magnetic nanoparticles decreases when the particle size exceeds 200 nm, and the grafting density of the surface boric acid affinity ligands decreases, resulting in insufficient capture capacity for lignin. The boric acid affinity ligands are 3-aminophenylboronic acid or 4-mercaptophenylboronic acid. When 3-aminophenylboronic acid was selected, the surface of the magnetic nanoparticles was modified with 3-aminophenylboronic acid via glutaraldehyde crosslinking. The crosslinking reaction was carried out in a phosphate buffer system with a pH of 8.5 for 4 hours. When 4-mercaptophenylboronic acid was selected, the surface of the magnetic nanoparticles was modified with 4-mercaptophenylboronic acid via gold thiol self-assembly monolayer. The self-assembly reaction was carried out in an anhydrous ethanol system for 12 hours. Both boric acid affinity ligands can reversibly covalently bond with the ortho-diol groups in the lignin molecule under alkaline conditions to form a five-membered cyclic borate ester structure.

[0041] In some embodiments, the addition of magnetic nanoparticles with boric acid affinity ligands to the second dispersion suspension is performed as follows: the magnetic nanoparticles with boric acid affinity ligands are pre-dispersed in a phosphate buffer to form a magnetic bead stock solution. The phosphate buffer has a pH of 8.5 and a concentration of 0.1 mol / L. The pH of 8.5 is set because the pKa of the boric acid group is approximately 8.8. In a buffer system with a pH of 8.5, the boric acid group mainly exists in a non-dissociated state, resulting in the highest efficiency of nucleophilic binding with ortho-diols. Simultaneously, under this pH condition, carbonate minerals in the purple soil do not undergo significant dissolution. The solid content of the magnetic bead stock solution is set at 10 mg / mL. This solid content represents a balance between the dispersion stability of the magnetic beads and the accuracy of the transfer volume. When the solid content exceeds 10 mg / mL, the magnetic bead stock solution is prone to sedimentation during settling. When the solid content is below 10 mg / mL, the volume of the magnetic bead stock solution added to the suspension is too large, which would significantly dilute the soil suspension system. According to the preset ratio of magnetic beads to soil mass, the magnetic bead stock solution was added to the second dispersion suspension. The preset ratio of magnetic beads to soil mass was set at 1:5. The 1:5 ratio was determined based on the saturated adsorption capacity of magnetic nanoparticles for lignin in purple soil, which was measured through isothermal adsorption experiments. When the ratio of magnetic beads to soil mass is 1:5, the molar amount of boric acid ligands on the surface of the magnetic beads is approximately 10 times the molar amount of ortho-diol groups in the lignin in the soil, ensuring that lignin is fully captured.

[0042] The second dispersion suspension, after adding the magnetic bead stock solution, was placed in a thermostatic shaker and incubated at a specified temperature, speed, and duration. A water bath thermostatic shaker was used, with a temperature control accuracy of ±0.5 degrees Celsius. The specified temperature range was set from 25 to 37 degrees Celsius. When the ambient temperature was above 20 degrees Celsius, the specified temperature was 25 degrees Celsius; when the ambient temperature was below 20 degrees Celsius, the specified temperature was 37 degrees Celsius to ensure the incubation system temperature remained stable near the set value. The 25-degree Celsius setting was chosen because at this temperature, the reversible covalent reaction between boric acid and ortho-diol exhibited a moderate binding rate, preventing non-specific adsorption. The 37-degree Celsius upper limit was chosen because above 37 degrees Celsius, the Brownian motion of the magnetic nanoparticles in the suspension intensified, and the increased collision frequency might lead to the adsorption of some non-target components onto the magnetic bead surface. The specified rotation speed was set to 200 revolutions per minute (rpm). This speed ensures that the solid particles in the second dispersion suspension remain suspended and do not settle, while preventing collision and wear between magnetic nanoparticles due to excessive rotation speed. The specified incubation time ranged from 30 to 60 minutes. The incubation time was set to 30 minutes when the organic matter content of the purple soil was below 20 g / kg, 45 minutes when the organic matter content was between 20 g / kg and 40 g / kg, and 60 minutes when the organic matter content was above 40 g / kg. The thermostatic shaker was continuously operated during incubation. After incubation, the beaker was removed from the thermostatic shaker to obtain the captured mixture system.

[0043] Understandably, after incubation, an external magnetic field is used to separate magnetic nanoparticles from the captured mixture. The external magnetic field is provided by a neodymium iron boron (NdFeB) permanent magnet with a surface magnetic field strength of at least 0.5 Tesla. The magnet is placed in contact with the outer wall of the beaker. The magnetic separation time is set to 5 minutes. This 5-minute setting is based on magnetic separation kinetics experiments, which determined that under a 0.5 Tesla external magnetic field, magnetic nanoparticles with diameters of 50 to 200 nanometers are completely attracted to the beaker wall in 100 ml of reaction system within 5 minutes. During the magnetic separation operation, the NdFeB permanent magnet is attached to one side of the beaker's outer wall. After standing for 5 minutes, while maintaining the magnet's attachment, the beaker is tilted, and a pipette is used to transfer a portion of the liquid to another clean container. During the transfer, the pipette tip is kept away from the beaker wall on the side where the magnet is attached. The collected liquid is the separated residual liquid. The turbidity value of the residual liquid is measured using a turbidimeter, and this value is recorded as the first-state response parameter. Before measurement, the turbidity meter was calibrated using formalin standard solution according to the calibration method described in Example 1, with a measurement wavelength of 860 nm selected. When measuring the turbidity value of the residual liquid, the residual liquid was transferred to the turbidity meter sample cell, and the turbidity meter readings were taken three times consecutively. These three turbidity meter readings were recorded as follows: , , The arithmetic mean of the three readings is taken as the final reading. The arithmetic mean is calculated using the following formula:

[0044]

[0045] in, This represents the first-state response parameter, in NTU. This indicates the first turbidimeter reading when measuring the residual liquid, in NTU units. This indicates the second turbidimeter reading when measuring the residual liquid, in NTU units. This indicates the third turbidimeter reading during residual liquid measurement, in NTU. Record the calculated first-state response parameter values ​​in the experimental record table. After measurement, rinse the turbidimeter sample cell three times with deionized water. Dispose of the remaining residual liquid according to laboratory waste management regulations.

[0046] Example 3:

[0047] In practice, after obtaining the purple soil sample to be tested, the purple soil sample is divided into a first subsample group, a second subsample group, and a third subsample group. The third subsample group is subjected to standard addition and lignin-directed capture operations.

[0048] In some embodiments, the standard addition and lignin-directed capture operation includes: mixing a third subsample group with deionized water at a preset mass-to-volume ratio to form a third mixed suspension. The preset mass-to-volume ratio is selected based on the texture type of the purple soil and is numerically consistent with the preset mass-to-volume ratio used in the background baseline establishment operation and the lignin-directed capture operation. When the purple soil texture type is sandy loam, the preset mass-to-volume ratio is set to 1:5; when the purple soil texture type is loam, the preset mass-to-volume ratio is set to 1:8; when the purple soil texture type is silty loam, the preset mass-to-volume ratio is set to 1:10; and when the purple soil texture type is clay loam, the preset mass-to-volume ratio is set to 1:20. In an implementation scenario using purple silty loam, the preset mass-to-volume ratio is 1:10. Specifically, 10.00 grams of the third subsample group is weighed, and 100 ml of deionized water is measured. The third subsample group and deionized water are placed in a 250 ml glass beaker and stirred with a glass rod for 2 minutes to initially mix the soil particles with the deionized water, forming a third mixed suspension.

[0049] Optionally, after forming the third mixed suspension, the third subsample group is equally divided into at least three standard addition parallel subsamples. The equal division is performed as follows: transfer the entire third mixed suspension to a clean aliquot container, weigh the total mass of the third mixed suspension using an electronic balance, and divide the total mass by the number of standard addition parallel subsamples to obtain the mass to be taken for each standard addition parallel subsample. The number of standard addition parallel subsamples is at least three. This number is based on the minimum requirement for data points in linear regression analysis using the standard addition method; fewer than three data points make it impossible to determine whether a linear relationship exists between the response value and the spiked concentration. Taking the case of dividing three parallel subsamples of standard addition as an example, the total mass of the third mixed suspension is 110.00 grams. Then, the mass of each parallel subsample of standard addition should be 36.67 grams. Use a pipette to transfer the third mixed suspension into three 100 ml Erlenmeyer flasks, and take 36.67 grams from each Erlenmeyer flask. After the transfer is completed, seal the mouth of the Erlenmeyer flask with sealing film.

[0050] It is understood that lignin standard solutions of different mass concentrations are added to each parallel subsample of the standard addition, and the mass concentrations of the lignin standard solutions corresponding to the different parallel subsamples of the standard addition exhibit an arithmetic progression. The lignin standard solution is an alkali lignin standard solution, extracted from the deep parent material layer of a purple soil profile homologous to the purple soil samples to be tested. The extraction method employed is dioxane-water acidic hydrolysis extraction. The extracted alkali lignin standard was freeze-dried and stored in a phosphorus pentoxide desiccator. The mass concentration of the lignin standard solution stock solution was prepared to be 100 mg / L, using a phosphate buffer solution with a pH of 8.5 and a concentration of 0.1 mol / L. The tolerance of the arithmetic progression was set based on the estimated range of lignin content in the purple soil samples to be tested, which was indirectly estimated by the soluble organic carbon determination results of the second subsample group. When the estimated content ranges from 0.5 mg / L to 5.0 mg / L, the tolerance is set at 2.5 mg / L; when the estimated content ranges from 5.0 mg / L to 20.0 mg / L, the tolerance is set at 5.0 mg / L; and when the estimated content ranges from 20.0 mg / L to 50.0 mg / L, the tolerance is set at 10.0 mg / L. Taking three parallel subsamples of standard addition with estimated content ranging from 5.0 mg / L to 20.0 mg / L as an example, the mass concentrations of the added lignin standard solutions are 5.0 mg / L, 10.0 mg / L, and 15.0 mg / L, respectively. The specific addition procedure was as follows: 1.83 mL of lignin standard solution stock solution was added to the first conical flask to achieve a concentration of 5.0 mg / L; 3.67 mL of lignin standard solution stock solution was added to the second conical flask to achieve a concentration of 10.0 mg / L; and 5.50 mL of lignin standard solution stock solution was added to the third conical flask to achieve a concentration of 15.0 mg / L. After addition, the mixture in each conical flask was stirred with a glass rod for 1 minute. Each parallel subsample containing the added lignin standard solution was labeled as a standard addition suspension with a different concentration gradient.

[0051] In some embodiments, each standard addition suspension is placed in an ultrasonic disperser and subjected to ultrasonic dispersion for a specified duration to obtain a corresponding third dispersion suspension. A trough-type ultrasonic disperser is used, with its operating frequency set to 40 kHz. The 40 kHz operating frequency setting follows the same logic as the settings in the background baseline establishment and lignin-directed capture operations; that is, for secondary mineral particles with a particle size distribution of 2 to 50 micrometers in purple soil, the microjets generated by the 40 kHz ultrasonic cavitation effect can overcome the van der Waals forces between particles without damaging the structural integrity of the lignin macromolecules and the lignin standard. The specified duration is determined as follows: when the preset mass-to-volume ratio is in the range of 1:5 to 1:8, the specified duration is set to 10 minutes; when the preset mass-to-volume ratio is 1:10, the specified duration is set to 15 minutes; and when the preset mass-to-volume ratio is in the range of 1:15 to 1:20, the specified duration is set to 20 minutes. In the implementation scenario with a preset mass-to-volume ratio of 1:10, the conical flask containing the first standard addition suspension is placed in the water tank of the ultrasonic disperser, with the water level in the tank exceeding the surface of the suspension in the conical flask by 2 cm. The ultrasonic disperser timer is set to 15 minutes, and the ultrasonic disperser is started for ultrasonic dispersion treatment. During the treatment, the suspension is stirred with a glass rod once every 5 minutes. After the ultrasonic dispersion treatment is completed, the conical flask is removed to obtain the first third dispersion suspension. The same operation is followed to perform ultrasonic dispersion treatment on the second and third standard addition suspensions in sequence to obtain the second and third third dispersion suspensions, respectively.

[0052] Optionally, after obtaining each fraction of the third dispersion suspension, magnetic nanoparticles with boric acid affinity ligands modified on their surface are added to each fraction of the third dispersion suspension. The particle size range of the magnetic nanoparticles with boric acid affinity ligands modified on their surface is 50 nm to 200 nm. The lower limit of the magnetic nanoparticle particle size is set at 50 nm because the magnetic responsiveness of the magnetic nanoparticles is significantly reduced when the particle size is smaller than 50 nm, making it difficult to achieve efficient separation under an external magnetic field; the upper limit of the magnetic nanoparticle particle size is set at 200 nm because the specific surface area of ​​the magnetic nanoparticles decreases when the particle size exceeds 200 nm, and the grafting density of the surface boric acid affinity ligands decreases, resulting in insufficient capture capacity for lignin and lignin standards. The boric acid affinity ligand is 3-aminophenylboronic acid or 4-mercaptophenylboronic acid. When 3-aminophenylboronic acid was selected, the surface of the magnetic nanoparticles was modified with 3-aminophenylboronic acid via glutaraldehyde crosslinking. The crosslinking reaction was carried out in a phosphate buffer system with a pH of 8.5 for 4 hours. When 4-mercaptophenylboronic acid was selected, the surface of the magnetic nanoparticles was modified with 4-mercaptophenylboronic acid via gold thiol self-assembly monolayer. The self-assembly reaction was carried out in an anhydrous ethanol system for 12 hours. Both boric acid affinity ligands can reversibly covalently bond with the ortho-diol groups in the lignin molecule and lignin standard molecule under alkaline conditions to form a five-membered cyclic borate ester structure.

[0053] In some embodiments, the addition of magnetic nanoparticles with boric acid affinity ligands to each third dispersion suspension is performed as follows: the magnetic nanoparticles with boric acid affinity ligands are pre-dispersed in a phosphate buffer to form a magnetic bead stock solution. The phosphate buffer has a pH of 8.5 and a concentration of 0.1 mol / L. The pH of 8.5 is set because the pKa of the boric acid group is approximately 8.8. In a buffer system with a pH of 8.5, the boric acid group mainly exists in a non-dissociated state, resulting in the highest efficiency of nucleophilic binding reaction with ortho-diols. Simultaneously, under this pH condition, carbonate minerals in purple soil do not undergo significant dissolution. The solid content of the magnetic bead stock solution is set at 10 mg / mL. This solid content of 10 mg / mL represents a balance between the dispersion stability of the magnetic beads and the accuracy of the transfer volume. When the solid content exceeds 10 mg / mL, the magnetic bead stock solution is prone to sedimentation during standing. When the solid content is below 10 mg / mL, the volume of the magnetic bead stock solution added to the suspension is too large, which will significantly dilute the standard addition suspension system. According to the preset ratio of magnetic beads to soil mass, the magnetic bead stock solution was added to each portion of the third dispersion suspension. The preset ratio of magnetic beads to soil mass was set at 1:5. This 1:5 ratio was determined based on the saturated adsorption capacity of magnetic nanoparticles for lignin in purple soil, as measured by isothermal adsorption experiments. When the ratio of magnetic beads to soil mass is 1:5, the molar amount of boric acid ligands on the surface of the magnetic beads is approximately 10 times the total molar amount of ortho-diol groups in the lignin in the soil and the added lignin standard, ensuring that lignin and lignin standard are fully captured. The specific addition procedure was as follows: 2.00 ml of magnetic bead stock solution was added to the first portion of the third dispersion suspension, 2.00 ml of magnetic bead stock solution was added to the second portion of the third dispersion suspension, and 2.00 ml of magnetic bead stock solution was added to the third portion of the third dispersion suspension.

[0054] It is understood that after adding the magnetic bead stock solution, each portion of the third dispersion suspension is incubated at a specified temperature, speed, and duration to form a standard capture mixture system. A water bath isothermal shaker is used, with a temperature control accuracy of ±0.5 degrees Celsius. The specified temperature range is set from 25 to 37 degrees Celsius. When the ambient temperature is above 20 degrees Celsius, the specified temperature is 25 degrees Celsius; when the ambient temperature is below 20 degrees Celsius, the specified temperature is 37 degrees Celsius to ensure the incubation system temperature remains stable near the set value. The 25-degree Celsius setting is because at this temperature, the reversible covalent reaction between boric acid and ortho-diol has a moderate binding rate, which can avoid non-specific adsorption. The 37-degree Celsius upper limit is because above 37 degrees Celsius, the Brownian motion of the magnetic nanoparticles in the suspension intensifies, and the increased collision frequency may lead to the adsorption of some non-target components onto the magnetic bead surface. The specified rotation speed was set to 200 revolutions per minute (RPM). This speed ensures that the solid particles in the third dispersion suspension remain suspended and do not settle, while preventing collision and wear between magnetic nanoparticles due to excessive rotation speed. The specified incubation time ranged from 30 to 60 minutes. The incubation time was set to 30 minutes when the organic matter content of the purple soil was below 20 g / kg, 45 minutes when the organic matter content was between 20 g / kg and 40 g / kg, and 60 minutes when the organic matter content was above 40 g / kg. The thermostatic shaker was continuously operated during incubation. After incubation, the conical flasks were removed from the shaker to obtain the first, second, and third standard sample capture mixture systems.

[0055] In some embodiments, an external magnetic field is used to separate magnetic nanoparticles from each standard sample capture mixture. The external magnetic field is provided by a neodymium iron boron (NdFeB) permanent magnet with a surface magnetic field strength of not less than 0.5 Tesla, and the magnet is placed in contact with the outer wall of the conical flask. The magnetic separation time is set to 5 minutes, based on magnetic separation kinetics experiments, which determined that under a 0.5 Tesla external magnetic field, magnetic nanoparticles with a particle size of 50 to 200 nanometers are completely attracted to the flask wall within 5 minutes in the reaction system inside the conical flask. During the magnetic separation operation, the NdFeB permanent magnet is attached to one side of the outer wall of the conical flask. After standing for 5 minutes, while maintaining the magnet's attachment, the conical flask is tilted, and a pipette is used to transfer a portion of the liquid to another clean container. During the transfer, the pipette tip is kept away from the conical flask wall on the side where the magnet is attached. The collected liquid is the residual liquid after standard capture. Magnetic separation was performed sequentially on the first, second, and third standard capture mixture systems to obtain the residual liquid after the capture of the first, second, and third standard samples, respectively.

[0056] It is understood that after collecting the residual liquid from each standard sample capture, a turbidimeter is used to measure the turbidity value of the residual liquid from each standard sample capture, and the turbidity value of the residual liquid from each standard sample capture is recorded as the second state response parameter. Before measurement, the turbidimeter is calibrated using formalin standard solution according to the calibration method described in Example 1, and the measurement wavelength is selected as 860 nm. When measuring the turbidity value of the residual liquid from each standard sample capture, the residual liquid is transferred to the turbidimeter sample cell, and the turbidimeter reading is read three times consecutively. For the residual liquid from the first standard sample capture, the three turbidimeter readings are recorded as follows: , , For the residual liquid after the capture of the second standard sample, the three turbidimeter readings were recorded as follows: , , For the residual liquid after the capture of the third standard sample, the three turbidimeter readings were recorded as follows: , , The arithmetic mean of three readings for each residual solution is taken as the final reading at the corresponding spiked concentration. The arithmetic mean is calculated using the formula:

[0057]

[0058] in, This represents the second-state response parameter, in NTU. Indicates the serial number of the parallel subsample added to the standard. The value can be 1, 2 or 3, which correspond to the residual liquid after the capture of the first standard sample, the residual liquid after the capture of the second standard sample and the residual liquid after the capture of the third standard sample, respectively. Indicates the first The first turbidity meter reading when measuring the residual liquid after capturing a standard sample, in NTU; Indicates the first The second turbidimeter reading during the measurement of the residual liquid after capturing a standard sample, in NTU; Indicates the first The turbidimeter reading was taken for the third time when the residual liquid after capturing the standard sample was measured. The unit is NTU. The calculated values ​​of each second-state response parameter and the corresponding standard addition concentration were recorded in the experimental record table. After the measurement, the turbidimeter sample cell was rinsed three times with deionized water. The remaining residual liquid was disposed of in accordance with the laboratory waste liquid management regulations.

[0059] Example 4:

[0060] In specific implementation, please refer to Figure 3The process of determining the lignin content in the purple soil sample to be tested based on the logical comparison results of the differences between the physical state reference parameters, the first state response parameters, and the second state response parameters is completed through a series of difference calculations, ratio calculations, and mapping comparisons.

[0061] In some embodiments, the first step of the differential logic comparison is to calculate the difference between the first state response parameter and the physical state reference parameter to obtain the first net turbidity increment. The first net turbidity increment reflects the difference between the turbidity contribution of soil particulate matter in the second subsample group and the turbidity of the residual liquid after lignin capture. This difference is mainly caused by the reduction of colloidal lignin in the supernatant due to the capture of lignin by magnetic nanoparticles with boric acid affinity ligands on their surfaces. The physical state reference parameter is obtained by the background baseline establishment operation described in Example 1, and is denoted as... The first state response parameter is obtained by the lignin-directed capture operation described in Example 2, and is denoted as... The net increase in the first turbidity is then recorded as , The calculation method is as follows ,in and All units are NTU. The unit is NTU.

[0062] The second step of the difference logic comparison can be understood as follows: calculating the difference between the second-state response parameter corresponding to each standard addition concentration and the physical state baseline parameter, thus obtaining the net increase in second turbidity corresponding to each standard addition concentration. The net increase in second turbidity reflects the difference between the contribution of soil particulate matter turbidity and the turbidity of the residual liquid after standard capture in the third sub-sample group after the addition of a lignin standard solution of known mass concentration. This difference is jointly determined by the capture of background lignin in the soil by magnetic nanoparticles with boric acid affinity ligands on their surface and the reduction in lignin in the supernatant after the addition of the lignin standard. The physical state baseline parameter is denoted as... The second-state response parameters corresponding to the addition concentrations of each standard were obtained from the standard addition and lignin-directed capture operations described in Example 3, and are denoted in ascending order of standard addition concentration as follows: , , The net increase in second turbidity corresponding to the addition concentration of each standard is denoted as follows: , , The calculation method for the net increase in turbidity is as follows: , , All the quantities mentioned above are in NTU.

[0063] In some embodiments, when calculating the ratio of the second net increase in turbidity to the first net increase in turbidity, it is first determined whether the first net increase in turbidity is zero. The threshold for zero value determination is set to 0.01 NTU. When the absolute value of the first net increase in turbidity is less than 0.01 NTU, the first net increase in turbidity is determined to be zero. A zero first net increase in turbidity indicates that the lignin content in the tested purple soil sample is lower than the detection limit of magnetic nanoparticles with boric acid affinity ligands on the surface. The turbidity of the residual liquid in the second subsample group is consistent with the turbidity of the supernatant in the first subsample group within the measurement error range, and the magnetic nanoparticles failed to capture detectable lignin from the second subsample group. When the first net increase in turbidity is zero, the turbidity inhibition ratio is directly assigned to zero. Assigning zero means that there is no contribution of detectable lignin to the turbidity signal. The turbidity inhibition effect produced by the added lignin standard in the third subsample group is entirely attributed to the added standard itself, and there is no competitive inhibition effect from the soil background lignin.

[0064] When the net increase in the first turbidity is not zero, the net increase in each of the second turbidity values ​​is divided by the net increase in the first turbidity value, and the result of the division is taken as the turbidity inhibition ratio. The turbidity inhibition ratio reflects the degree of competition between the added lignin standard and the capture capacity of the magnetic nanoparticles. When the concentration of the added standard increases, the proportion of ortho-diol binding sites of the boric acid affinity ligands on the surface of the magnetic nanoparticles that are competitively occupied by the added standard increases, while the proportion of sites capturing background lignin in the soil decreases, manifested as a decrease in the net increase in the second turbidity relative to the net increase in the first turbidity. The turbidity inhibition ratio is calculated as follows: for each of the three standard addition concentrations, the first turbidity inhibition ratio is obtained. Second turbidity suppression ratio Third turbidity suppression ratio . The calculation method is as follows , The calculation method is as follows , The calculation method is as follows . , , All are dimensionless quantities, and their values ​​are within a closed interval between 0 and 1.

[0065] Optionally, the turbidity inhibition ratio can be compared with a preset ratio-content mapping relationship. The ratio-content mapping relationship is calculated by substituting the turbidity inhibition ratio into a pre-constructed linear regression equation, where the standard addition concentration is the independent variable and the turbidity inhibition ratio is the dependent variable. The linear regression equation is constructed as follows: the standard addition concentrations corresponding to the parallel subsamples of the three standards in Example 3 are denoted as... , , The units are all mg / L. The calculated turbidity inhibition ratio is denoted as . , , .by , , As the independent variable, with , , Using as the dependent variable, the least squares method is used to fit a linear regression equation, which takes the form of: ,in This indicates the standard concentration, expressed in milligrams per liter. This represents the turbidity suppression ratio, which is a dimensionless quantity. This indicates the slope, expressed in liters per milligram. The intercept is a dimensionless quantity. Slope The calculation method is as follows:

[0066]

[0067] in, Indicates the serial number of the parallel subsample added to the standard. The value can be 1, 2, or 3. Indicates the first The concentration of the standard added to each parallel subsample is given in mg / L. Indicates the first The turbidity suppression ratio corresponding to each parallel subsample added to the standard is a dimensionless quantity. Intercept The calculation method is as follows ,in express , , The arithmetic mean, in milligrams per liter. express , , The arithmetic mean of is a dimensionless quantity.

[0068] It is understandable that another pre-defined form of the ratio-content mapping relationship is the ratio-content standard curve. The construction method of the ratio-content standard curve is independent of the linear regression equation construction method described above and is completed before the testing of the purple soil samples to be tested. A blank purple soil matrix from the same source as the purple soil samples to be tested is used. The blank purple soil matrix is ​​prepared as follows: purple soil from the same plot and soil layer as the purple soil samples to be tested is collected, air-dried, ground, and sieved. Then, it is ignited in a muffle furnace at 550°C for 6 hours to remove all organic matter. After cooling, deionized water is added to 60% of the field capacity, and the soil is placed in a 25°C constant temperature incubator for 48 hours to restore the soil's physical structure. At least five different mass concentrations of lignin standards are added to the blank purple soil matrix. The five mass concentrations are 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 15.0 mg / L, and 20.0 mg / L, respectively. The five mass concentrations cover the common range of variation in lignin content in purple soil. The turbidity inhibition ratio was measured at each additive concentration, following the same procedures as those used for the purple soil samples in Examples 1 to 3. A ratio-content standard curve was obtained by fitting the additive concentration on the x-axis and the turbidity inhibition ratio on the y-axis using the least squares method. The regression equation for the ratio-content standard curve is as follows: ,in This indicates the concentration added, in milligrams per liter. This represents the turbidity suppression ratio, which is a dimensionless quantity. This represents the slope of the ratio-content standard curve, expressed in liters per milligram. This represents the intercept of the ratio-content standard curve, and is a dimensionless quantity.

[0069] In some embodiments, the calculated turbidity inhibition ratio is substituted into the ratio-content mapping relationship. If a linear regression equation is used, then let... , , Zhongyu The concentration corresponding to the closest value was used as a preliminary estimate of the lignin content in the purple soil sample to be tested, and then... The absolute value of the intercept of the linear regression equation on the horizontal axis is taken as the lignin content in the purple soil sample to be tested, i.e., the lignin content in the purple soil sample to be tested. , The unit is mg / L. If a ratio-content standard curve is used, the first turbidity inhibition ratio will be... Second turbidity suppression ratio Third turbidity suppression ratio Substitute the values ​​into the ratio-content standard curve equation to obtain the corresponding apparent content values. Take the arithmetic mean of the three apparent content values ​​as the lignin content in the purple soil sample to be tested.

[0070] See Figure 5 In the figure, the horizontal axis represents the concentration of lignin standard added, in mg / L, ranging from 0 to approximately 32.7 mg / L; the vertical axis represents the turbidity inhibition ratio, a dimensionless quantity ranging from 0 to 1. The curve in the figure is the result of linear regression of the ratio-content standard curve, showing a clear linear negative correlation trend. As the concentration of the standard added increases, the turbidity inhibition ratio gradually decreases.

[0071] The solid black line in the figure represents the ratio-content linear regression equation obtained by least squares fitting. This equation is based on measured data points (gray scattering points) from five different concentrations of standards added to a blank matrix. The points marked with black plus signs represent the turbidity inhibition ratio of the purple soil sample to be tested, distributed at different locations with different standard concentrations. These ratios are significantly lower than the linear standard curve values ​​for the corresponding concentrations, indicating the lignin capture effect in the actual sample. The black crosses indicated by the dashed line on the right side of the figure correspond to the inferred lignin content in the purple soil sample, which is 32.7 mg / L.

[0072] This figure illustrates the mapping relationship established in Example 4 between the turbidity inhibition ratio and the concentration of the added standard. A linear regression equation was used to compare the turbidity inhibition ratio of the test sample, thereby accurately inferring the lignin content in the purple soil. The trend of the turbidity inhibition ratio shows a negative linear relationship with the concentration of the added standard, indicating that the lignin capture capacity of the boric acid affinity ligands on the surface of the magnetic nanoparticles gradually saturates with increasing lignin concentration, and competitive binding weakens the turbidity inhibition effect. The turbidity inhibition ratios corresponding to the test samples are distributed below the standard curve, indicating the presence of a certain lignin content in the samples and a significant competitive effect on capture. The inferred lignin content of 32.7 mg / L falls within the data distribution range shown in the figure, meeting the quantitative determination requirements of the detection method.

[0073] Overall, the figure clearly demonstrates the construction and application process of the ratio-content mapping relationship, verifying the effectiveness and accuracy of the difference logic comparison and ratio-content mapping method in Example 4 for the quantitative detection of lignin content in purple soil, and providing theoretical and experimental basis for the rapid and accurate determination of lignin content in purple soil.

[0074] Example 5:

[0075] In practice, the process of obtaining the purple soil sample to be tested and dividing it into the first subsample group, the second subsample group, and the third subsample group is the basic sample preparation step of the entire detection method.

[0076] In some embodiments, the operation of obtaining the purple soil sample to be tested includes: collecting topsoil from the purple soil field. The sampling plot is selected from a representative purple soil distribution area, with the parent material of the purple soil being weathered purple sandstone and shale from the Jurassic Penglaizhen Formation. The sampling depth is set to 0 cm to 20 cm, which corresponds to the thickness of the topsoil layer of the purple soil. This layer is the main distribution area of ​​crop roots, and the sources of lignin input include crop residues, root exudates, and organic fertilizer input. The lignin content level can reflect the dynamic changes in organic matter in the topsoil layer of the purple soil. A stainless steel soil drill with an inner diameter of 5 cm is used as the sampling tool. Ten sampling points are arranged in an S-shaped pattern at each sampling plot. One soil column is collected from each sampling point using the stainless steel soil drill. The ten soil columns are combined into a mixed soil sample, with a mass of approximately 2 kg. The collected mixed soil sample is placed in a clean cotton bag, and the opening of the cotton bag is tied tightly with cotton rope to avoid compression during transportation.

[0077] Understandably, after collecting the topsoil from the purple soil field, plant debris and gravel are removed. The mixed soil sample is poured into a clean white enamel dish, and visible plant debris, including root fragments, crop stem and leaf remnants, and undecomposed organic matter particles, is picked out one by one with stainless steel tweezers. This process is conducted under natural light and takes at least fifteen minutes, until no visible plant tissue remains in the enamel dish. After the plant debris is removed, the mixed soil sample is dry-sieved using a 2mm nylon sieve. The upper portion is retained, and gravel particles larger than 2mm are picked out from this portion with stainless steel tweezers. Gravel particles are defined as rock fragments that cannot be crushed by fingers and are not covered by a soil film. The removed plant debris and gravel are collected and discarded separately.

[0078] Optionally, after removing plant debris and gravel, the soil sample is placed in a ventilated area to air dry until constant weight. The air-drying site should be a well-ventilated indoor environment free from direct sunlight, ammonia, and volatile acidic or alkaline gases. The selected soil sample is evenly spread on a stainless steel tray, with a thickness not exceeding 2 cm. This 2 cm thickness limit ensures sufficient airflow within the soil sample, preventing anaerobic microenvironment from altering lignin content due to microbial activity. During air drying, the soil sample is turned over every eight hours using a stainless steel spatula to ensure even air contact. The air-drying time is determined based on ambient temperature and relative humidity. When the ambient temperature is between 20°C and 25°C and the relative humidity is between 40% and 60%, the air-drying time is generally 48 to 72 hours. Constant weight is determined when the difference between two consecutive weighings does not exceed 0.3% of the total soil sample mass, with a four-hour interval between the two weighings. After reaching the constant weight standard, the air-dried soil samples were collected and temporarily stored in polyethylene self-sealing bags.

[0079] In some embodiments, the air-dried soil sample is ground and sieved, and the soil particles that pass through the sieve are collected as the purple soil sample to be tested. A ceramic mortar and pestle are used as the grinding tools. The ceramic material is chosen to avoid the introduction of metal ions such as iron and manganese during the grinding process, as these metal ions may compete with boric acid ligands for coordination in the subsequent lignin-directed capture operation. The air-dried soil sample is added to the ceramic mortar in batches, with each addition not exceeding one-third of the mortar's capacity. The sample is ground with a ceramic pestle at a uniform pressure, alternating between clockwise and counterclockwise directions. After grinding, the soil sample is passed through a 2 mm nylon sieve. During the sieving operation, the nylon sieve is placed above the receiving tray, and the edges of the sieve frame are gently tapped with the palm of the hand to allow the soil particles to pass through the sieve holes. Larger particles remaining on the sieve are returned to the ceramic mortar for further grinding until all the soil sample passes through the 2 mm nylon sieve. The soil particles that pass through the sieve are collected as the purple soil sample to be tested. The purple soil sample is placed in a clean polyethylene self-sealing bag, labeled with the sample number and preparation date, and stored in a desiccator with a color-changing silica gel desiccant at the bottom.

[0080] It is understandable that after obtaining the purple soil sample to be tested, a rotary sampler is used to equally divide the sample into three sub-sample groups of equal mass: a first sub-sample group, a second sub-sample group, and a third sub-sample group. A grid-type rotary sampler is used, which has twenty-four equally divided grids. The sampler hopper has a volume of 500 ml, and the rotation is driven by a manual crank. The sampling principle of the rotary sampler is that the purple soil sample to be tested rotates at a constant speed in the rotating hopper. Under the combined action of centrifugal force and gravity, the soil particles fall evenly into the twenty-four grids. Collecting the samples from the symmetrical grids yields equally divided sub-samples. The specific operating steps are as follows: pour all the purple soil sample to be tested into the rotary sampler hopper, close the hopper lid, and crank the handle at a constant speed of 30 revolutions per minute. The speed is controlled at 30 revolutions per minute because at this speed, the uniformity coefficient of soil particle falling reaches above 0.95, and the sampling deviation is less than 2%. After all the purple soil samples to be tested have fallen from the hopper into the grid cells, the shaking is stopped, and the samples collected from the twenty-four grid cells are combined according to a preset combination. The preset combination is as follows: samples from grid cells 1 to 8 are combined into the first subsample group, samples from grid cells 9 to 16 are combined into the second subsample group, and samples from grid cells 17 to 24 are combined into the third subsample group. Theoretically, the masses of the first, second, and third subsample groups obtained after merging are equal.

[0081] In some embodiments, to ensure the uniformity of the mass of the three sub-sample groups, an analytical balance is used to weigh each of the merged sub-sample groups. The analytical balance has a sensitivity of 0.01 grams and a maximum weighing range of 200 grams. The mass of the first sub-sample group is recorded as follows: The mass of the second subgroup of samples is recorded as . The mass of the third subgroup of samples is recorded as . Calculate the arithmetic mean of the quality of the three sub-sample groups. The arithmetic mean is calculated using the formula:

[0082]

[0083] in, The arithmetic mean of the masses of the first, second, and third subgroups is expressed in grams. This represents the mass of the first subsample group obtained by weighing using an analytical balance, in grams. This indicates the mass of the second subsample group obtained by weighing using an analytical balance, in grams. This represents the mass of the third subsample obtained by weighing using an analytical balance, in grams. , , The sampling is considered acceptable if the relative deviation between any two of the three factors does not exceed 3%. The relative deviation is calculated as the absolute value of the difference in quality between the two subsample groups divided by... Multiply by 100%. Each subsample group must weigh at least 10 grams. This minimum 10-gram requirement is based on the assumption that, in the lignin-directed capture operation with a preset mass-to-volume ratio of 1:10, the suspension volume formed by adding 10 grams of soil sample to 100 ml of deionized water must be compatible with the capacity of a 250 ml beaker, while ensuring sufficient sample volume for turbidity measurement. Subsample groups meeting the quality requirements should be sealed and stored separately in clean polyethylene self-sealing bags. The subsample group category and number should be marked on the outside of the bags with a marker. The first subsample group is used for background baseline establishment, the second for lignin-directed capture, and the third for standard addition and lignin-directed capture. Each sealed subsample group must be analyzed within 24 hours, with the polyethylene self-sealing bags placed in a desiccator during storage.

[0084] See Figure 6 The figure shows a frequency distribution histogram of the masses of the first, second, and third sub-sample groups after the purple soil samples to be tested were subdivided in Example 5. The horizontal axis represents the mass of the sub-sample group in grams, with a scale range of approximately 98.5 grams to 101.5 grams. The vertical axis represents the frequency of the corresponding mass range, with the maximum frequency exceeding 20. In the figure, the mass of the first sub-sample group is represented by a diagonal line, the mass of the second sub-sample group is represented by dots, and the mass of the third sub-sample group is represented by a cross-shaped diagonal line.

[0085] As shown in the figure, the masses of the three sub-sample groups are concentrated between 99.5 g and 100.5 g, exhibiting a relatively symmetrical bell-shaped distribution. Furthermore, the masses of the three sub-sample groups show a high degree of overlap, with no significant deviation. This indicates that after sampling using the grid-type rotary sampler, the sub-sample groups have balanced masses, meeting the requirements for mass uniformity control in Example 5, i.e., the relative deviation between the masses of each sub-sample group is controlled within 3%. The mass peak in the figure is concentrated at approximately 100.2 g, consistent with the theoretically expected equal division after sampling, ensuring the consistency of sample mass in subsequent background baseline establishment, lignin-oriented capture, and standard addition operations.

[0086] Overall, the mass distribution histogram verifies the effectiveness of using a rotating sampler in Example 5 to uniformly divide the purple soil sample into three sub-sample groups of equal mass. This provides a reliable sample basis for obtaining accurate and comparable physical state reference parameters, first state response parameters, and second state response parameters in subsequent steps, ensuring the accuracy and repeatability of the test results.

[0087] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A method for detecting lignin content in purple soil, characterized in that, Includes the following steps: Obtain purple soil samples to be tested, and divide the purple soil samples to be tested into a first subsample group, a second subsample group, and a third subsample group; In the step of performing a background baseline establishment operation on the first sub-sample group to obtain the physical state reference parameters of the purple soil sample to be tested: The first subsample group is mixed with deionized water at a preset mass-volume ratio to form a first mixed suspension; the first mixed suspension is placed in an ultrasonic disperser and subjected to ultrasonic dispersion treatment for a specified duration to obtain a first dispersed suspension; after the first dispersed suspension has been allowed to stand for a specified duration, the turbidity value of the supernatant of the first dispersed suspension is measured using a turbidimeter, and the turbidity value of the supernatant is recorded as the physical state reference parameter; In the step of performing a lignin-directed capture operation on the second sub-sample group to obtain the first-state response parameter of the second sub-sample group after lignin capture: The second subsample group is mixed with deionized water at the preset mass-volume ratio to form a second mixed suspension; the second mixed suspension is placed in the ultrasonic disperser and subjected to ultrasonic dispersion treatment for the specified duration to obtain a second dispersed suspension; magnetic nanoparticles with boric acid affinity ligands on their surface are added to the second dispersed suspension and incubated at a specified temperature for a specified duration to form a capture-mix system; the magnetic nanoparticles are separated from the capture-mix system using an external magnetic field, the residual liquid after separation is collected, the turbidity value of the residual liquid is measured using the turbidimeter, and the turbidity value of the residual liquid is recorded as the first state response parameter; Perform standard addition and lignin-directed capture operations on the third sub-sample group to obtain the second state response parameter of the third sub-sample group after standard addition and lignin capture. Based on the logical comparison results of the difference between the physical state reference parameter, the first state response parameter, and the second state response parameter, the lignin content in the purple soil sample to be tested is determined.

2. The method for detecting lignin content in purple soil according to claim 1, characterized in that, The ultrasonic diffuser operates at a frequency of 40 kHz, and the specified duration is 10 to 20 minutes.

3. The method for detecting lignin content in purple soil according to claim 2, characterized in that, The magnetic nanoparticles with surface-modified boric acid affinity ligands have a particle size of 50 nm to 200 nm, and the boric acid affinity ligands are 3-aminophenylboronic acid or 4-mercaptophenylboronic acid.

4. The method for detecting lignin content in purple soil according to claim 3, characterized in that, In the step of performing standard addition and lignin-oriented capture operations on the third sub-sample group to obtain the second-state response parameter of the third sub-sample group after standard addition and lignin capture: The third subsample group is mixed with deionized water according to the preset mass-volume ratio to form a third mixed suspension. A lignin standard solution of known mass concentration is added to the third mixed suspension to obtain a standard addition suspension; The standard addition suspension is placed in the ultrasonic disperser and ultrasonic dispersion is performed for the specified duration to obtain a third dispersion suspension. The magnetic nanoparticles with boric acid affinity ligands on their surface are added to the third dispersion suspension, and the mixture is incubated at the specified temperature for the specified incubation time to form a standard capture mixture system. The magnetic nanoparticles are separated from the standard capture mixture using an external magnetic field. The residual liquid after standard capture is collected, and the turbidity value of the residual liquid after standard capture is measured using the turbidimeter. The turbidity value of the residual liquid after standard capture is recorded as the second state response parameter.

5. The method for detecting lignin content in purple soil according to claim 4, characterized in that, In the step of determining the lignin content in the purple soil sample to be tested based on the logical comparison results of the difference between the physical state reference parameter, the first state response parameter, and the second state response parameter: Calculate the difference between the first state response parameter and the physical state reference parameter to obtain the first net turbidity increase; Calculate the difference between the second state response parameter and the physical state reference parameter to obtain the second net turbidity increment; Calculate the ratio of the second net increase in turbidity to the first net increase in turbidity to obtain the turbidity suppression ratio; The turbidity inhibition ratio is compared with the preset ratio-content mapping relationship, and the preset content value that matches the turbidity inhibition ratio is determined as the lignin content in the purple soil sample to be tested.

6. The method for detecting lignin content in purple soil according to claim 5, characterized in that, The ratio-content mapping relationship is calculated by substituting the turbidity inhibition ratio into a pre-constructed linear regression equation, which uses the standard added concentration as the independent variable and the turbidity inhibition ratio as the dependent variable.

7. The method for detecting lignin content in purple soil according to claim 6, characterized in that, In the step of calculating the ratio of the second net increase in turbidity to the first net increase in turbidity to obtain the turbidity suppression ratio: Determine whether the net increase in the first turbidity is zero. When the first net increase in turbidity is zero, the turbidity suppression ratio is directly assigned to zero; When the first net increase in turbidity is not zero, the second net increase in turbidity is divided by the first net increase in turbidity, and the result of the division operation is taken as the turbidity suppression ratio.

8. The method for detecting lignin content in purple soil according to claim 7, characterized in that, In the step of adding a lignin standard solution of known mass concentration to the third mixed suspension to obtain a standard addition suspension: The third subsample group is equally divided into at least three standard addition parallel subsamples; Different mass concentrations of lignin standard solutions were added to each standard addition parallel subsample, and the mass concentrations of the lignin standard solutions corresponding to the different standard addition parallel subsamples showed an arithmetic sequence distribution. Each parallel subsample containing lignin standard solution was labeled as a standard addition suspension with different concentration gradients.

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