Method for determining the molecular weight of soil humic acid by heavy metal titration

Abstract

The present invention relates to the field of environmental testing technology, and in particular to a method for determining the molecular weight of soil humic acid by heavy metal titration. The method comprises the following steps: (a) extracting and purifying soil humic acid; (b) preparing a soil humic acid test solution; (c) selecting the position of a fluorescence peak in the titration experiment; (d) performing heavy metal titration of humic acid; and (e) calculating the molecular weight of humic acid. The accuracy and reliability of the method were demonstrated by using tyrosine as a model compound and a humic acid standard sample for validation.

Description

Technical Field

[0001] The invention relates to the technical field of environmental detection, in particular to a method for determining the molecular weight of soil humic acid by heavy metal titration. Background Art

[0002] Humic acids are a class of high-molecular-weight organic compounds widely found in soils and sediments. They are formed by the decomposition and transformation of plant and animal residues by microorganisms. The transport and transformation capabilities of humic acids are closely related to their composition and structure. They can form chelates with metal ions, affecting metals and organic matter in the environment through redox reactions, and even acting as electron transporters in electron transfer. This ability enables humic acids to play a vital role in regulating the circulation of substances, energy conversion, and information transfer between biological systems, soil systems, and the environment, and is crucial for maintaining soil functional stability. These complex molecular structures are rich in aromatic rings and aliphatic chains, as well as various active functional groups such as carboxyl, hydroxyl, carbonyl, quinone, and methoxy groups, endowing humic acids with powerful chelating, adsorption, and redox abilities. These properties enable humic acids to interact in complex ways with metal ions, oxides, hydroxides, minerals, organic matter, and toxic and active pollutants in the environment, influencing their transport, transformation, and degradation. Humic acids play a positive role in the environment by improving soil fertility, protecting soil ecology, and participating in the global carbon cycle. They can passivate heavy metal ions by binding to them, reducing their mobility in the environment. They also provide substrates and energy for soil microbial activity, indirectly affecting the activity of heavy metal ions in the soil. Therefore, humic acid is not only an important component of soil, but also a key factor in environmental quality and ecological balance.

[0003] The molecular structure of humic acid is extremely complex, and its molecular weight is also extremely wide, ranging from a few hundred to hundreds of thousands, which makes it difficult to accurately measure its molecular weight and limits the accuracy of traditional experimental methods in studying the molecular weight of humic acid. There are many methods for determining the molecular weight of humic acid in soil, including size exclusion chromatography, liquid chromatography technology, average molecular weight method for solution viscosity determination, small angle X-ray scattering method, diffusion method, 13 C nuclear magnetic resonance spectroscopy and tetramethylammonium hydroxylate gas chromatography / mass spectrometry, etc. Size exclusion chromatography utilizes the characteristics of a porous gel stationary phase to separate solute molecules based on their size; liquid chromatography separates humic acid molecules and measures the peak area or peak height of each molecular weight segment to calculate the molecular weight distribution; solution viscosity determination calculates the average molecular weight of humic acid solution based on its viscosity; small-angle X-ray scattering can determine the size of humic substances; and the diffusion method infers the molecular weight of humic acid molecules by measuring their fluid radius. 13C nuclear magnetic resonance spectroscopy provides structural information about humic acid samples; the tetramethylammonium hydroxylate thermochemical method determines the composition of humic acid by analyzing organic carbon samples. These methods each have their own advantages and can complement each other, helping us to more accurately determine the molecular weight of humic acid in soil and, in turn, better understand its environmental behavior and ecological effects in soil. The molecular weight of humic acid has a significant impact on its properties and functions. The larger the molecular weight, the more complex the cross-linking structure within the molecule, and its solubility and activity are correspondingly reduced. At the same time, the ability to bind to soil particles is also stronger, which can form a more stable soil organic matter structure, thereby affecting the ecological and environmental effects of humic acid in soil. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for determining the molecular weight of soil humic acid by heavy metal titration.

[0005] To achieve the above object, the technical solution of the present invention is implemented as follows: a method for determining the molecular weight of soil humic acid by heavy metal titration, characterized in that the method for determining the molecular weight of soil humic acid by heavy metal titration comprises the following steps:

[0006] (a) Extraction and purification of soil humic acid;

[0007] (b) Preparation of soil humic acid test solution;

[0008] (c) Selection of fluorescence peak position in titration experiment;

[0009] (d) Heavy metal titration of humic acid;

[0010] (e) Calculation of molecular weight of humic acid.

[0011] Preferably, the step a comprises the following steps:

[0012] a1) collecting a soil sample, removing roots and stones from the soil, air-drying the soil, grinding and sieving the soil sample for later use; adding deionized water to the soil sample, adjusting the solution to a strongly acidic state with HCl and NaOH, then adding HCl solution to a specific solid-to-liquid ratio, stirring continuously, allowing the solution to stand, and centrifuging to obtain a solid soil sample 1;

[0013] a2) Deionized water is added to the solid soil sample 1, and the solution is adjusted to near neutrality with HCl and NaOH to obtain a solid-liquid mixed solution. Under nitrogen protection, NaOH solution is added to the solid-liquid mixed solution, and the solution is diluted with deionized water to adjust the NaOH to a specific solid-liquid ratio. After continuous stirring, the mixture is allowed to stand and centrifuged to obtain a supernatant and solid soil sample 2;

[0014] Under nitrogen protection, HCl was added to the supernatant to adjust the pH of the solution to a strongly acidic state, and the solution was allowed to stand after continuous stirring, and centrifuged to obtain humic acid sample 1;

[0015] a3) adding deionized water to the solid soil sample 2 of step a2, adjusting the mixture to near neutrality with HCl and NaOH to obtain a solid-liquid mixture; adding NaOH solution to the solid-liquid mixture under nitrogen protection, and diluting the mixture with deionized water to adjust the NaOH to a specific solid-liquid ratio, stirring continuously, allowing the mixture to stand, and centrifuging to obtain a supernatant and soil residue;

[0016] Under nitrogen protection, concentrated HCl was added to the supernatant to make the solution strongly acidic, and the solution was allowed to stand after continuous stirring, and centrifuged to obtain humic acid sample 2;

[0017] Humic acid sample 1 and humic acid sample 2 were combined and marked as crude humic acid sample;

[0018] a4) adding HCl and HF solutions to the crude humic acid sample obtained in step a3 to make it strongly acidic and reach a specific solid-liquid ratio, stirring continuously, allowing it to stand, and centrifuging to obtain a humic acid solid sample;

[0019] a5) repeatedly washing the purified humic acid solid sample, freeze-drying it, and grinding it to obtain a humic acid solid powder;

[0020] Preferably, the preparation process of the humic acid test solution in step b is:

[0021] b1) Weigh humic acid solid powder m1 and dissolve it with a strong base. Adjust the pH to a specific value (4-9) with a strong acid and strong base solution. Stir and maintain a stable pH before filtering. Add salt solution to adjust the ionic strength. Constantly adjust the volume to V and mark as the humic acid test solution.

[0022] b2) After drying, weigh the filter membrane before and after filtration and mark the mass difference as m2.

[0023] b3) The concentration of the humic acid solution is calculated by subtracting the amount of humic acid intercepted by the filter membrane m2 from the mass of humic acid added m1, and the total volume V of the humic acid solution, using the following formula (1):

[0024] Humic acid test solution concentration (1).

[0025] Preferably, in step c, the process of selecting the fluorescence peak position of the titration experiment is as follows:

[0026] c1) Measure the humic acid test solution;

[0027] c2) Adjust the humic acid test solution to a specific pH value, stir and maintain a stable pH value;

[0028] c3) scanning the three-dimensional fluorescence spectrum of the humic acid test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0029] c4) After deducting the Rayleigh and Raman scattering peaks, select the excitation and emission wavelengths at which the fluorescence intensity is maximum in the three-dimensional fluorescence spectrum of the humic acid test solution as the fluorescence peak position of the titration experiment.

[0030] Preferably, the step d comprises the following steps:

[0031] d1) measuring the humic acid test solution prepared in step b;

[0032] d2) adjusting the measured humic acid test solution to a specific pH value, stirring and maintaining a stable pH value;

[0033] d3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0034] d4) measuring the fluorescence light scattering intensity I0 of the humic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0035] d5) adding a solution containing a quenching substance so that the concentration of the solution containing the quenching substance is between 0 and 1000 µmol / L;

[0036] d6) adjusting the pH of the humic acid test solution to the same value as in step d2, stirring and maintaining a stable pH value;

[0037] d7) measuring the fluorescence intensity F value of the humic acid test solution at the position of the fluorescence peak of the titration experiment determined in step c;

[0038] d8) measuring the light scattering intensity I of the humic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0039] d9) Repeat steps d5 to d8 until I>2I0, then stop step d.

[0040] Preferably, in step e, the specific process of calculating the molecular weight of humic acid is:

[0041] e1) Use formula (2) to calculate F end :

[0042] (2)

[0043] e2) F end Substitute into formula (3) to calculate the concentration of humic acid in the soil C L :

[0044] (3)

[0045] e3) C L Substitute into formula (4) to calculate the molecular weight M of humic acid:

[0046] (4)

[0047] Among them, C Q F is the concentration of the quenching substance solution added in step d5 during the titration process; end is the fluorescence intensity fitted by humic acid when titrated to saturation; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of humic acid in the test solution in step d1; C L The molar concentration of the humic acid solution prepared in step b; M is the molecular weight of humic acid.

[0048] Preferably, the error of the specific pH value in step a is ±0.1; the strongly acidic pH value range is between 1-3, and the near-neutral pH value range is between 6-8; the stirring in step a is continued for 4-8 hours, the stirring time in the standing operation is continued for not less than 15 minutes, and the standing time is not less than 20 hours; the solution in step a is made to reach a specific solid-liquid ratio of about 1:10; the strong acid concentration used in steps a4 and a5 is in the range of 0.05-0.2 mol / L; the strong base concentration in step a2 is in the range of 0.1-0.2 mol / L.

[0049] Preferably, the cleaning process in step a5 is to place the purified humic acid solid sample in a triangular filter and add 3 times the volume of deionized water, and determine the chloride ion content in the filtrate of step a5 by the silver nitrate method. If chloride ions are present, continue to add about 3 times the volume of deionized water, and then determine the chloride ion content in the filtrate again until no chloride ions are detected in the filtrate.

[0050] Preferably, the humic acid solid powder obtained in step a5 is dried at 80-100°C for 20-24 hours, then calcined at 550°C for 5 hours, and the ash content of the humic acid solid powder is measured. If the ash content of the humic acid solid powder is greater than 5.0% (by dry weight), steps a4 and a5 are repeated until the ash content is less than 5.0%.

[0051] Preferably, the salt concentration in the humic acid test solution in step b is a specific value, and the concentration is between 0.1-1000 mmol / L; the humic acid concentration in the humic acid test solution in step b is a specific value, and the concentration range should be controlled between 5-20 mg / L.

[0052] Preferably, the cumulative volume of the acid and alkaline solutions added to adjust the pH value in step c is no more than 1‰ of the volume of the humic acid solution tested in step c1.

[0053] Preferably, the cumulative volume of the acid and / or alkaline solution added for adjusting the pH value in step d is no more than 1‰ of the volume of the background solution in step d1; the solution containing the quenching substance added in step d includes but is not limited to copper ions, mercury ions, etc.; the cumulative volume of the solution containing the quenching substance added during the titration process in step d is no more than 1‰ of the volume of the humic acid test solution in step c1.

[0054] Preferably, the pH value of the solution in steps b, c and d is a constant value (with an error of ±0.02), and the pH value range is between 5 and 7; the concentration of the strong base in steps a2 and b1 is a specific value, ranging from 0.05 to 0.2 M; and the salt used to adjust the ionic strength in steps a2 and b is any one or more of potassium perchlorate, sodium perchlorate, sodium chloride, potassium chloride and potassium nitrate solution.

[0055] Preferably, the filter membranes used for filtration in steps a and b are both glass fiber filter membranes with a pore size of 0.22-0.7 μm, and the glass fiber filter membranes are calcined at 450°C-550°C for 5-8 hours before use. The glass fiber filter membranes are dried at 60°C-80°C for 8-12 hours before and after filtration, and cooled to room temperature in a drying dish.

[0056] Preferably, the strong acid is one of hydrochloric acid, perchloric acid, dilute nitric acid, dilute sulfuric acid or a mixture thereof; the strong base is one of sodium hydroxide and potassium hydroxide or a mixture thereof; maintaining the pH value stable means that the pH value of the solution changes by less than 0.02 pH units within not less than 30 minutes; the fluorescence measurements in steps c and d are both carried out under the protection of an inert gas such as nitrogen, helium or argon.

[0057] The beneficial effects of the present invention are embodied in:

[0058] (1) The method for determining the molecular weight of humic acid provided by the present invention is stable, simple to operate, and has low requirements on equipment. In addition, the method is verified to be accurate and reliable using a model compound of tyrosine molecules and a humic acid standard sample.

[0059] (2) The salt used in the present invention can provide a certain ionic strength during the humic acid fluorescence determination process, and the influence of its concentration and ion type on the determination results can be basically ignored, thereby improving the detection effect.

[0060] (3) In the preparation process of the humic acid test solution provided by the present invention, a strong base is used to dissolve the humic acid solid, which can speed up the dissolution process and save the total measurement time. At the same time, deionized water can also be used to oscillate and dissolve the humic acid (generally, the oscillation time is 20-24 hours), which reduces the impact on the test results. However, the dissolution amount may not be sufficient to carry out the experiment.

[0061] (4) In the method for determining the molecular weight of humic acid provided by the present invention, controlling the pH value not only ensures that humic acid can exist in the solution in a free form, but also avoids the situation where humic acid exists in the form of acid when the acidity is too strong, that is, when the pH value is less than 3, and humic acid precipitates when the pH value is less than 1. At the same time, it also avoids the situation where copper ions combine with a large number of free hydroxyl groups in the solution when the alkalinity is too strong, that is, when the pH value is greater than 10, thereby affecting the determination results, thereby improving the accuracy of the test results.

[0062] (5) The method provided by the present invention for determining the molecular weight of soil humic acid using heavy metal titration requires that one humic acid molecule be combined with one copper ion in the reaction system. When the copper ion causes further flocculation of the humic acid molecules, the scattering intensity increases sharply. Therefore, the present invention requires that I<2I0 in step d.

[0063] (6) Tyrosine used in the present invention is an organic substance with a fixed molecular weight. When it combines with ions such as copper ions and mercury ions, it will produce fluorescence quenching phenomenon. Therefore, it can be used as a reference substance to test the reliability of the method provided by the present invention for determining the molecular weight of soil humic acid by heavy metal titration. DETAILED DESCRIPTION

[0064] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the comparative examples. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0065] Example 1

[0066] A method for determining the molecular weight of soil humic acid by heavy metal titration, the method comprising the following steps:

[0067] a: Extraction and purification of soil humic acid

[0068] A soil sample was collected from a certain location, and tree roots and stones were removed from the soil. After air drying, the soil was ground through a 100-mesh sieve to obtain a soil sample for later use. 1 kg of the soil sample was weighed and added to deionized water. The pH of the solution was adjusted to 1.0 ± 0.02 with HCl and NaOH. Then, 0.1 M HCl solution was added to the solution to a solid-liquid ratio of 1:10. The sample was stirred continuously for 4 hours, allowed to stand for 24 hours, and centrifuged to obtain a solid soil sample 1.

[0069] Deionized water was added to the solid soil sample 1, and the pH of the solution was adjusted to 7±0.02 with HCl and NaOH to obtain a solid-liquid mixed solution. Under nitrogen protection, 0.2 M NaOH solution was added to the solid-liquid mixed solution, and the solution was diluted with deionized water to make the solid-liquid ratio of the NaOH solution 1:10. The solution was stirred for 4 h and then allowed to stand for 24 h. The supernatant and solid soil sample 2 were obtained by centrifugation.

[0070] Under nitrogen protection, HCl was added to the supernatant to adjust the solution pH to 1.0±0.02, and the solution was stirred for 15 min, allowed to stand for 24 h, and centrifuged to obtain humic acid sample 1;

[0071] a3) Deionized water was added to the solid soil sample 2, and the pH was adjusted to 7 ± 0.02 with HCl and NaOH to obtain a solid-liquid mixture. Under nitrogen, 0.2 M NaOH solution was added to the solid-liquid mixture, and the mixture was diluted with deionized water to a NaOH solid-to-liquid ratio of 1:10. The solution was stirred for 1 hour and then allowed to stand for 24 hours. The supernatant and soil residue were separated by centrifugation.

[0072] Under nitrogen protection, concentrated HCl was added to the supernatant to adjust the solution pH to 1.0 ± 0.02, stirred for 15 minutes, allowed to stand for 24 hours, and centrifuged to obtain humic acid sample 2;

[0073] Humic acid sample 1 and humic acid sample 2 were combined and marked as crude humic acid sample;

[0074] a4) adding 0.1 M HCl and HF solutions to the crude humic acid sample to a solid-to-liquid ratio of 1:10, stirring continuously for 4 h, allowing to stand for 24 h, and centrifuging to obtain a humic acid solid sample;

[0075] a5) repeatedly washing the purified humic acid solid sample, freeze-drying it, and grinding it to obtain a humic acid solid powder;

[0076] b: Preparation of soil humic acid test solution;

[0077] Weigh 110.73 mg of humic acid solid powder and dissolve it in 0.1 M strong base. Adjust the pH to 6.0 ± 0.02 with strong acid and strong base solutions. Stir and maintain a stable pH for 60 minutes before filtering. Add KClO₄ solution to adjust the ionic strength and adjust the volume to 10 L to prepare the humic acid test solution. The mass difference between the glass fiber membrane before and after filtration is 3.6 mg.

[0078] c: Selection of the fluorescence peak position in the titration experiment; Measure the humic acid test solution, adjust the humic acid test solution to pH = 6.0 ± 0.02, stir and keep the pH value stable for 60 minutes.

[0079] The three-dimensional fluorescence spectrum of the humic acid test solution was scanned, wherein the excitation wavelength and emission wavelength scanning range were both 200-600 nm. After deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and emission wavelength at which the fluorescence intensity was maximum in the three-dimensional fluorescence spectrum of the humic acid test solution were selected as the fluorescence peak position of the titration experiment.

[0080] d: Heavy metal titration of humic acid;

[0081] 1) Measure the prepared humic acid test solution;

[0082] 2) Adjust the pH of the measured humic acid solution to 6.0 ± 0.02, stir and keep the pH stable for 60 minutes;

[0083] 3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0084] 4) Measure the fluorescence light scattering intensity I0 of the humic acid test solution when both the excitation wavelength and emission wavelength are 500 nm;

[0085] 5) adding a solution containing a quenching substance so that the concentration of the solution containing the quenching substance is between 0 and 1000 µmol / L;

[0086] 6) Adjusting the pH of the humic acid test solution to the same value as in step d2, stirring and maintaining a stable pH value;

[0087] 7) measuring the fluorescence intensity F value of the humic acid test solution at the fluorescence peak position determined in step c;

[0088] 8) Measure the light scattering intensity I of the humic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0089] 9) Repeat steps d5 to d8 until I>2I0, then stop step d.

[0090] e: Calculation of molecular weight of humic acid.

[0091] Use formula (1) to calculate Fend :

[0092] (1)

[0093] F end Substitute into formula (2) to calculate the concentration of humic acid in the soil C L :

[0094] (2)

[0095] C L Substitute into formula (3) to calculate the molecular weight M of humic acid:

[0096] (3)

[0097] Among them, C Q F is the concentration of the quenching substance solution added in step d5 during the titration process; end is the fluorescence intensity fitted by humic acid when titrated to saturation; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of humic acid in the test solution in step d1; C L The molar concentration of the substance in the humic acid test solution prepared in step b; M is the molecular weight of humic acid. By changing the concentration value of copper added C Q Substituting the corresponding series of fluorescence intensity values ​​F (such as F1, F2, F3…Fn) into formula (2) can calculate C L , and then substitute it into formula (3) to calculate M, where the mass volume concentration of humic acid in the test liquid in step d) is m = (111.73 mg - 3.6 mg) / 10 L = 10.71 mg / L.

[0098] Using the above method, the molecular weight M of humic acid was obtained to be 2866±130 g / mol.

[0099] Example 2

[0100] The method for determining the molecular weight of soil humic acid by heavy metal titration is basically the same as that in Example 1, except that:

[0101] a: Extraction and purification of soil humic acid

[0102] Soil samples were collected from a certain place, and humic acid solid powder was obtained by extraction and purification using the above method.

[0103] b: Preparation of soil humic acid test solution;

[0104] Prepare a humus test solution with a concentration of 10 mg / L.

[0105] c: Selection of fluorescence peak position in titration experiment;

[0106] 1) Measure the humic acid test solution and adjust the pH of the humic acid test solution to 4.0 ± 0.02. Stir and keep the pH value stable for 20 minutes.

[0107] 2) Scan the three-dimensional fluorescence spectrum of the humic acid test solution, where the excitation wavelength and emission wavelength scanning range are both 200-600 nm. After deducting the Rayleigh scattering and Raman scattering peaks, select the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum in the three-dimensional fluorescence spectrum of the humic acid test solution as the fluorescence peak position of the titration experiment.

[0108] d: Heavy metal titration of humic acid

[0109] 1) Measure the prepared humic acid test solution;

[0110] 2) Adjust the pH of the measured humic acid solution to 4.0 ± 0.02, stir and keep the pH value stable for 20 minutes;

[0111] 3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0112] 4) Measure the fluorescence light scattering intensity I0 of the humic acid test solution when both the excitation wavelength and emission wavelength are 500 nm;

[0113] 5) adding a solution containing a quenching substance so that the concentration of the solution containing the quenching substance is between 0 and 1000 µmol / L;

[0114] 6) Adjust the pH of the humic acid test solution to the same value as in step d2, stir, and maintain a stable pH for 20 minutes;

[0115] 7) measuring the fluorescence intensity F value of the humic acid test solution at the fluorescence peak position determined in step c;

[0116] 8) Measure the light scattering intensity I of the humic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0117] 9) Repeat steps d5 to d8 until I>2I0, then stop step d.

[0118] Using the above method, the molecular weight M of humic acid was obtained to be 1998±101.3 g / mol.

[0119] Example 3

[0120] The method for determining the molecular weight of soil humic acid by heavy metal titration is basically the same as that in Example 1, except that:

[0121] a: Extraction and purification of soil humic acid

[0122] Soil samples were collected from a certain place, and humic acid solid powder was obtained by extraction and purification using the above method.

[0123] b: Preparation of soil humic acid test solution;

[0124] Prepare a humus test solution with a concentration of 10 mg / L.

[0125] c: Selection of fluorescence peak position in titration experiment;

[0126] 1) Measure the humic acid test solution and adjust the pH of the humic acid test solution to 8.2±0.02. Stir and keep the pH value stable for 45 minutes.

[0127] 2) Scan the three-dimensional fluorescence spectrum of the humic acid test solution, where the excitation wavelength and emission wavelength scanning range are both 200-600nm,

[0128] After deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and emission wavelength at which the fluorescence intensity is the maximum in the three-dimensional fluorescence spectrum of the humic acid test solution are selected as the excitation wavelength and emission wavelength of the titration experiment.

[0129] d: Heavy metal titration of humic acid

[0130] 1) Measure the prepared humic acid test solution;

[0131] 2) Adjust the pH of the measured humic acid solution to 8.2 ± 0.02, stir, and maintain a stable pH value for 45 minutes;

[0132] 3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0133] 4) Measure the fluorescence light scattering intensity I0 of the humic acid test solution when both the excitation wavelength and emission wavelength are 500 nm;

[0134] 5) adding a solution containing a quenching substance so that the concentration of the solution containing the quenching substance is between 0 and 1000 µmol / L;

[0135] 6) Adjust the pH of the humic acid test solution to the same value as in step d2, stir, and maintain a stable pH for 45 minutes;

[0136] 7) measuring the fluorescence intensity F value of the humic acid test solution at the fluorescence peak position determined in step c;

[0137] 8) Measure the light scattering intensity I of the humic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0138] 9) Repeat steps d5 to d8 until I>2I0, then stop step d.

[0139] Using the above method, the molecular weight M of humic acid was obtained to be 3037±92.6 g / mol.

[0140] Example 4

[0141] A method for determining the molecular weight of tyrosine by heavy metal titration, the method comprising the following steps:

[0142] a: Preparation of tyrosine test solution and background solution

[0143] Weigh 52.79 mg of a solid tyrosine sample, dissolve it in deionized water, and filter it through a glass fiber membrane with a pore size of 0.45 µm. Prepare a 1 L concentrated tyrosine solution. Filter the concentrated tyrosine solution. The difference in mass between the glass fiber membrane before and after filtration is 1.20 mg. Add 138.55 g of solid potassium perchlorate to the filtered concentrated tyrosine solution and dissolve it. Dose the solution up to 10 L with deionized water to prepare the tyrosine test solution.

[0144] Weigh 138.55 g of potassium perchlorate solid sample, dissolve it in deionized water, and filter it through a glass fiber membrane with a pore size of 0.45 µm to prepare a background solution with a volume of 10 L.

[0145] b: Selection of fluorescence peak position in tyrosine titration experiment

[0146] 1) Measure the tyrosine test solution;

[0147] 2) Adjust the pH of the tyrosine solution to 6, stir and maintain a stable pH for 60 minutes;

[0148] 3) scanning the three-dimensional fluorescence spectrum of the tyrosine test solution, wherein the excitation wavelength and emission wavelength scanning range are 200-600nm;

[0149] 4) Measure the background solution;

[0150] 5) Adjust the pH value of the background solution to 6, stir and keep the pH stable for 60 minutes;

[0151] 6) Scanning the three-dimensional fluorescence spectrum of the background solution, wherein the excitation wavelength and emission wavelength scanning range are 200-600nm;

[0152] 7) Subtract the three-dimensional fluorescence spectrum data of the background solution from the obtained three-dimensional fluorescence spectrum data of the tyrosine test solution, and simultaneously set the Raman and Rayleigh scattering intensities to zero. Determine the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum. These wavelengths are used as the excitation wavelength and emission wavelength for heavy metal titration, which are 350 nm and 445 nm, respectively.

[0153] c: Heavy metal titration of tyrosine

[0154] 1) Measure the tyrosine test solution;

[0155] 2) Adjust the pH of the tyrosine test solution to 6, stir and keep the pH stable for 60 minutes;

[0156] 3) measuring the fluorescence intensity F0' of the tyrosine test solution at the fluorescence peak position selected in step b;

[0157] 4) Measure the light scattering intensity I0 of the tyrosine test solution when both the excitation wavelength and emission wavelength are 400 nm;

[0158] 5) adding a solution containing a quenching substance so that the concentration of the solution containing the quenching substance is between 0 and 1000 µmol / L;

[0159] 6) Adjust the tyrosine test solution to the pH value of 6 specified in step c) 2), stir and maintain the pH value stable for 60 minutes;

[0160] 7) measuring the fluorescence intensity F' of the tyrosine test solution at the position of the fluorescence peak of the titration experiment determined in step c;

[0161] 8) Measure the light scattering intensity I of the tyrosine test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0162] 9) Repeat step c from 5) to 8) until I>2I0, then stop step c.

[0163] d: Background solution fluorescence measurement

[0164] 1) Measure the background solution;

[0165] 2) Adjust the pH value of the background solution to 6, the same as in step c, stir and keep the pH stable for 60 minutes;

[0166] 3) measuring the fluorescence intensity Fr0' of the background solution at the fluorescence peak position of the titration experiment determined in step c;

[0167] 4) Add copper ions so that the concentration of the quenching substance solution in the background solution is the same as the concentration of the quenching substance solution in step c5, repeat steps d2 to d4, and measure the fluorescence intensity Fr' at the fluorescence peak position determined in step c.

[0168] e: Calculation of molecular weight of tyrosine

[0169] 1) Calculate F0=F0'-Fr0' and calculate F=F'-Fr'

[0170] 2) Use formula (1) to calculate F end :

[0171] (1)

[0172] 3) Use formula (2) to calculate the molecular weight M of tyrosine:

[0173] (2)

[0174] Among them, C Q F is the total concentration of the quenching substance solution added in step c3 during the titration process; end is the fluorescence intensity fitted by tyrosine when the titration is saturated; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of tyrosine in the test solution in step c1; and M is the molecular weight of tyrosine.

[0175] Different concentrations of Cu 2+ The corresponding measured fluorescence intensity value F is shown in Table 1.

[0176] Table 1 Different concentrations of Cu 2+ Corresponding to the measured fluorescence intensity value F result statistics

[0177]

[0178] The mass concentration of the tyrosine test solution in step c) 1) is m = (52.79 mg - 1.20 mg) / 10 L = 5.16 mg / L. The molecular weight of tyrosine calculated by formulas (3) and (4) is 183.5 ± 10.0.

[0179] Using the heavy metal titration method provided by the present invention, the pH value of the measured solution, the cumulative concentration of copper ions added, and the concentration of the measured solution were changed during the titration process. The measured molecular weight of tyrosine was 178.1-185.4 g / mol, which is less than 5% of the actual molecular weight of tyrosine (181.20 g / mol).

[0180] The method for determining the molecular weight of soil humic acid by heavy metal titration is basically the same as that in Example 1, except that the International Humic Acid Association standard sample (Suwannee River HA, 3S101H) is used for the experiment.

[0181] b: Preparation of soil humic acid test solution;

[0182] Weigh 27 mg of the International Humic Acid Association standard (Suwannee River HA, 3S101H) and dissolve it in 2 mL of 0.1 M potassium hydroxide solution. Dilute the solution to approximately 1 L with deionized water and filter through a 0.45 µm pore glass fiber membrane. After filtration, dilute the volume to 2 L with deionized water to prepare a humic acid test solution with a concentration of 10.0 mg / L. The difference in mass between the glass fiber membrane before and after filtration was 3.7 mg.

[0183] c: Selection of fluorescence peak position in titration experiment;

[0184] 1) Measure the humic acid test solution and adjust the pH of the humic acid test solution to 6.0 ± 0.02, stir and keep the pH value stable for 20 minutes;

[0185] 2) Scan the three-dimensional fluorescence spectrum of the humic acid test solution, where the excitation wavelength and emission wavelength scanning range are both 200-600 nm. After deducting the Rayleigh scattering and Raman scattering peaks, select the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum in the three-dimensional fluorescence spectrum of the humic acid test solution as the fluorescence peak position of the titration experiment.

[0186] d: Heavy metal titration of humic acid;

[0187] 1) Measure the prepared humic acid test solution;

[0188] 2) Adjust the pH of the measured humic acid solution to 6.0 ± 0.02, stir and keep the pH stable for 20 minutes;

[0189] 3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0190] 4) Measure the fluorescence light scattering intensity I0 of the humic acid test solution when both the excitation wavelength and emission wavelength are 500 nm;

[0191] 5) adding a solution containing a quenching substance so that the concentration of the solution containing the quenching substance is between 0 and 1000 µmol / L;

[0192] 6) Adjust the pH of the humic acid test solution to the same value as in step d2, stir, and maintain a stable pH for 20 minutes;

[0193] 7) measuring the fluorescence intensity F value of the humic acid test solution at the fluorescence peak position determined in step c;

[0194] 8) Measure the light scattering intensity I of the humic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0195] 9) Repeat steps d5 to d8 until I>2I0, then stop step d.

[0196] Using the above method, the molecular weight M of humic acid was obtained to be 1737 g / mol.

[0197] Using the heavy metal titration method provided by the present invention, by changing the pH value of the measured liquid during the titration process, the cumulative concentration of added copper ions, the concentration of the measured liquid and other parameters, the measured molecular weight of humic acid was 1737 g / mol, which is less than 5% of the actual molecular weight of humic acid (1813 g / mol).

[0198] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining the molecular weight of soil humic acid by heavy metal titration, characterized in that: The method for determining the molecular weight of soil humic acid by heavy metal titration includes the following steps: (a) Extraction and purification of soil humic acid; (b) Preparation of soil humic acid test solution; (c) Selection of fluorescence peak position in titration experiment; (d) Heavy metal titration of humic acid; (e) Calculation of molecular weight of humic acid; Step d comprises the following steps: d1) measuring the humic acid test solution prepared in step b; d2) adjusting the pH value of the measured humic acid test solution, stirring and maintaining the pH value stable; d3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c; d4) measuring the fluorescence light scattering intensity I0 of the humic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm; d5) adding a solution containing a quenching substance, wherein the solution containing the quenching substance comprises copper ions or mercury ions, so that the concentration of the solution containing the quenching substance is between 0 and 1000 µmol / L; d6) adjusting the pH of the humic acid test solution to the same value as in step d2, stirring and maintaining a stable pH value; d7) measuring the fluorescence intensity F value of the humic acid test solution at the position of the fluorescence peak of the titration experiment determined in step c; d8) measuring the light scattering intensity I of the humic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm; d9) Repeat steps d5 to d8 until I>2I0, then stop step d; In step e, the specific process of calculating the molecular weight of humic acid is: e1) Use formula (2) to calculate F end : (2) e2) F end Substitute the following formula to calculate the molecular weight M of humic acid in the soil: Among them, C Q F is the concentration of the quenching substance solution added in step d5 during the titration process; end is the fluorescence intensity fitted by humic acid when titrated to saturation; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of humic acid in the test solution in step d1; and M is the molecular weight of humic acid.

2. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 1, characterized in that: The soil humic acid extraction and purification process in step a is: a1) collecting a soil sample, removing roots and stones from the soil, air-drying the soil, grinding and sieving the soil sample to obtain a solid soil sample for later use; adding deionized water to the soil sample, adjusting the solution to a strongly acidic state with HCl and NaOH, then adding HCl solution to the solution, stirring continuously, allowing the solution to stand, and centrifuging to obtain a solid soil sample 1; a2) adding deionized water to the solid soil sample 1 and adjusting the solution to near neutrality with HCl and NaOH to obtain a solid-liquid mixed solution; Under nitrogen protection, NaOH solution was added to the solid-liquid mixed solution, and the solution was diluted with deionized water. After continuous stirring, the solution was allowed to stand and centrifuged to obtain a supernatant and solid soil sample 2. Under nitrogen protection, HCl was added to the supernatant to adjust the pH of the solution to a strongly acidic state, and the solution was allowed to stand after continuous stirring, and centrifuged to obtain humic acid sample 1; a3) adding deionized water to the solid soil sample 2 of step a2, and adjusting the mixture to near neutrality with HCl and NaOH to obtain a solid-liquid mixture; under nitrogen protection, adding NaOH solution to the solid-liquid mixture, and diluting it with deionized water. After continuous stirring, the mixture was allowed to stand and centrifuged to obtain a supernatant and soil residue; Under nitrogen protection, concentrated HCl was added to the supernatant to make the solution strongly acidic, and the solution was allowed to stand after continuous stirring, and centrifuged to obtain humic acid sample 2; Humic acid sample 1 and humic acid sample 2 were combined and marked as crude humic acid sample; a4) adding HCl and HF solutions to the crude humic acid sample obtained in step a3 to make it strongly acidic, stirring continuously, allowing it to stand, and centrifuging to obtain a humic acid solid sample; a5) The purified humic acid solid sample is repeatedly washed, freeze-dried, and ground to obtain a humic acid solid powder.

3. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 2, characterized in that: The preparation process of the humic acid test solution in step b is: b1) Weigh humic acid solid powder m1 and dissolve it with a strong base. Adjust the pH to a specific value (4-9) with a strong acid and strong base solution. Stir and maintain a stable pH before filtering. Add salt solution to adjust the ionic strength. Constantly adjust the volume to V and mark as the humic acid test solution. b2) Weigh the filter membrane before and after filtration after drying, and mark the mass difference as m2; b3) The concentration of the humic acid solution is calculated by subtracting the amount of humic acid intercepted by the filter membrane m2 from the mass of humic acid added m1, and the total volume V of the humic acid solution, using the following formula (1): Humic acid test solution concentration (1).

4. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 3, wherein: The process of selecting the fluorescence peak position of the titration experiment in step c is: c1) Measure the humic acid test solution; c2) Adjust the pH value of the humic acid test solution, stir and keep the pH value stable; c3) scanning the three-dimensional fluorescence spectrum of the humic acid test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm; c4) After deducting the Rayleigh and Raman scattering peaks, select the excitation and emission wavelengths at which the fluorescence intensity is maximum in the three-dimensional fluorescence spectrum of the humic acid test solution as the fluorescence peak position of the titration experiment.

5. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 2, wherein: The pH value range of strong acid is between 1-3, and the pH value range of near neutral is between 6-8; the continuous stirring time in step a is not less than 15 minutes, and the standing time is not less than 20 hours; the solution in step a is made to reach a specific solid-liquid ratio of 1:10; the concentration range of strong acid used in steps a4 and a5 is 0.05-0.2 mol / L; the concentration range of strong base in step a2 is 0.1-0.2 mol / L.

6. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 2, characterized in that: The cleaning process in step a5 is to place the purified humic acid solid sample in a triangular filter and add 3 times the volume of deionized water. The chloride ion content in the filtrate of step a5 is determined by the silver nitrate method. If chloride ions are present, approximately 3 times the volume of deionized water is added and the chloride ion content in the filtrate is determined again until no chloride ions are detected in the filtrate.

7. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 2, characterized in that: The humic acid solid powder obtained in step a5 is dried at 80-100°C for 20-24 hours, calcined at 550°C for 5 hours, and then the ash content of the humic acid solid powder is measured; if the ash content of the humic acid solid powder is greater than 5.0%, based on dry weight, steps a4 and a5 are repeated until the ash content is less than 5.0%.

8. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 3, characterized in that: The salt concentration in the humic acid test solution in step b is a specific value, and the concentration is between 0.1-1000 mmol / L; the humic acid concentration in the humic acid test solution in step b is a specific value, and the concentration range should be controlled between 5-20 mg / L.

9. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 4, characterized in that: The cumulative volume of the acid and alkali solutions added to adjust the pH value in step c is not greater than 1‰ of the volume of the humic acid solution tested in step c1.

10. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 4, characterized in that: The cumulative volume of the acid and / or alkaline solution added to adjust the pH value in step d is not greater than 1‰ of the volume of the background solution in step d1; the cumulative volume of the quenching substance-containing solution added during the titration process in step d is not greater than 1‰ of the volume of the humic acid test solution in step c1.

11. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 4, characterized in that: The pH value of the solution in steps b, c and d is a constant value with an error of ±0.02, and the pH value range is between 5 and 7; the concentration of the strong base in steps a2 and b1 is a specific value, ranging from 0.05 to 0.2 M; and the salt used to adjust the ionic strength in step b is any one or more of potassium perchlorate, sodium perchlorate, sodium chloride, potassium chloride and potassium nitrate solution.

12. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 4, characterized in that: The filter membranes used for filtration in step b are all glass fiber filter membranes with a pore size of 0.22-0.7 μm, and the glass fiber filter membranes are calcined at 450°C-550°C for 5-8 hours before use. The glass fiber filter membranes are dried at 60°C-80°C for 8-12 hours before and after filtration and cooled to room temperature in a drying dish.

13. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 4, characterized in that: The strong acid is one of hydrochloric acid, perchloric acid, dilute nitric acid, dilute sulfuric acid or a mixture thereof; the strong base is one of sodium hydroxide and potassium hydroxide or a mixture thereof; and maintaining a stable pH value means that the pH value of the solution changes by less than 0.02 pH units within no less than 20 minutes.

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