Method for measuring molecular weight of soil humic acid through heavy metal titration

Abstract

The invention relates to the technical field of environment detection, in particular to a method for measuring soil humic acid molecular weight through heavy metal titration. The method comprises the following steps: (a) extracting and purifying soil humic acid; (b) preparing a soil humic acid measured solution; (c) selecting a fluorescence peak position of a titration experiment; (d) carrying out heavy metal titration on humic acid; and (e) calculating the molecular weight of humic acid. Tyrosine is used as a model compound and a humic acid standard sample for verification, and the accuracy and reliability of the method are proved.

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 acid is a class of high molecular organic compounds widely found in soil and sediments. They are formed by the decomposition and transformation of plant and animal residues under the action of microorganisms. The migration and transformation ability of humic acid is closely related to its composition and structure. They can form chelates with metal ions, affect metals and organic matter in the environment through redox reactions, and even act as electron transfer in electron transfer. This ability enables humic acid to play an important role in regulating the material cycle, energy conversion and information transfer between biological systems, soil systems and environmental systems, and is crucial to maintaining the stability of soil functions. These complex molecular structures are rich in aromatic rings and fatty chains, as well as a variety of active functional groups such as carboxyl, hydroxyl, carbonyl, quinone and methoxy groups, which give humic acid strong complexation, adsorption and redox capabilities. These characteristics enable humic acid to have complex interactions with metal ions, oxides, hydroxides, minerals, organic matter and toxic active pollutants in the environment, affecting the migration, transformation and degradation of these substances in the environment. In terms of environmental impact, humic acid plays a positive role by improving soil fertility, protecting soil ecology and participating in the earth's carbon cycle. They can passivate heavy metal ions by binding to them, reducing their activity in the environment, while providing 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 in the 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, diffusion method, 13 C nuclear magnetic resonance spectroscopy analysis and tetramethylammonium hydroxylate gas chromatography / mass spectrometry, etc. Size exclusion chromatography uses the characteristics of porous gel stationary phase to separate solute molecules according to their size; liquid chromatography technology calculates the molecular weight distribution by separating humic acid molecules and detecting the peak area or peak height of each molecular weight segment; solution viscosity determination method calculates its average molecular weight through the viscosity of humic acid solution; small angle X-ray scattering method can determine the size of humic molecules; diffusion law infers its molecular weight by measuring the fluid radius of humic acid molecules; 13C nuclear magnetic resonance spectroscopy analysis provides structural characteristic information of humic acid samples; the tetramethylammonium hydroxylate thermochemical law 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 the soil, and thus better understand its environmental behavior and ecological effects in the soil. The molecular weight of humic acid has an important influence on its properties and functions. The larger the molecular weight, the more complex the cross-linking structure inside the molecule, and its solubility and activity are correspondingly reduced. At the same time, the stronger the binding ability to soil particles, the more stable soil organic matter structure can be formed, thereby affecting the ecological and environmental effects of humic acid in the 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, step a comprises the following steps:

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

[0013] 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, adding NaOH solution to the solid-liquid mixed solution, and diluting it with deionized water to adjust the NaOH to a specific solid-liquid ratio, stirring continuously and then standing, and centrifuging to obtain a supernatant and a solid soil sample 2;

[0014] Under nitrogen protection, HCl was added to the supernatant to adjust the pH value of the solution to strong acidity, 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 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 to adjust the NaOH to a specific solid-liquid ratio, stirring continuously and then standing, 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 highly acidic and reach a specific solid-liquid ratio, stirring continuously and then standing, and centrifuging to obtain a humic acid solid sample;

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

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

[0021] b1) Weighing humic acid solid powder m1, dissolving it with a strong base, adjusting it to a specific pH value with a strong acid and a strong base solution, the pH value range is between 4 and 9, stirring and keeping the pH stable, filtering, adding a salt solution to adjust the ionic strength, and fixing the volume to V, and marking it as the humic acid test solution;

[0022] b2) After drying, weigh the filter membrane before and after filtration respectively, 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

[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) adjusting the humic acid test solution to a specific pH value, stirring and maintaining 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 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 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 the excitation wavelength and the emission wavelength are both 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 value of the humic acid test solution to the same as that in step d2, stirring and maintaining the pH value stable;

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

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

[0039] d9) Repeat steps d5 to d8 until I>2I0 and 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]

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

[0044]

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

[0046]

[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 strong acid pH value range is between 1-3, and the near-neutral pH value range is between 6-8. In step a, stirring is continued for 4-8 hours, and the stirring time during the standing operation is not less than 15 minutes, and the standing time is not less than 20 hours. In step a, the solution reaches a specific solid-liquid ratio of about 1:10; the strong acid concentration range used in steps a4 and a5 is 0.05-0.2 mol / L; the strong base concentration range in step a2 is 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 20-24h, and then calcined at 550°C for 5h, 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% (by dry weight), repeat steps a4 and a5 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 0.1-1000mmol / 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 5-20mg / L.

[0052] Preferably, the cumulative volume of the acid and alkaline 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.

[0053] Preferably, the cumulative volume of the acid and / or alkaline solution added for adjusting the pH value in step d is not greater 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 not greater than 1‰ of the volume of the humic acid solution to be tested in step c1.

[0054] Preferably, the pH value of the solution in steps b, c and d is a fixed value (with an error of ±0.02), and the pH value range is between 5-7; the concentration of the strong base in steps a2 and b1 is a specific value, between 0.05-0.2M; 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 and the like, 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 determinations 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, the measurement method is simple to operate, and the equipment requirements are low. In addition, the method results are proved to be accurate and reliable using model compounds of tyrosine molecules and humic acid standards.

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

[0060] (3) In the process of preparing 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 20-24h), which reduces the impact on the test results, but the dissolution amount may not be enough 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 can not only ensure that humic acid can exist in the solution in a free form, but also avoid the situation that when the acidity is too strong, that is, when the pH value is less than 3, humic acid will exist in the form of acid, and when the pH value is less than 1, humic acid will precipitate. At the same time, when the alkalinity is too strong, that is, when the pH value is greater than 10, copper ions will combine with a large number of free hydroxyl groups in the solution, thereby affecting the determination results, thereby improving the accuracy of the test results.

[0062] (5) The method for determining the molecular weight of soil humic acid by heavy metal titration provided by the present invention requires that one humic acid molecule is 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, fluorescence quenching occurs. 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 combination 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 in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0065] Example 1

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

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

[0068] A soil sample was collected from a certain place, and tree roots and stones in the soil were removed. After air drying, the soil was ground through a 100-mesh sieve to obtain a soil sample for use; 1 kg of the soil sample was weighed and added to deionized water, and the pH of the solution was adjusted to 1.0±0.02 with HCl and NaOH, and then 0.1 M HCl solution was added thereto to a solid-liquid ratio of 1:10. After continuous stirring for 4 hours, the soil was allowed to stand for 24 hours, and solid soil sample 1 was obtained by centrifugation;

[0069] Deionized water was added to the solid soil sample 1, and the pH value of the solution was adjusted to 7±0.02 with HCl and NaOH to obtain a solid-liquid mixed solution; under nitrogen protection, 0.2M NaOH solution was added to the solid-liquid mixed solution, and it was diluted with deionized water to make the solid-liquid ratio of the NaOH solution 1:10, and the solution was stirred for 4 hours and then allowed to stand for 24 hours, and then centrifuged to obtain a supernatant and a solid soil sample 2;

[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) adding deionized water to the solid soil sample 2, adjusting the pH value to 7±0.02 with HCl and NaOH to obtain a solid-liquid mixture; under nitrogen protection, adding 0.2M NaOH solution to the solid-liquid mixture, and diluting it with deionized water to make the NaOH solid-liquid ratio in the solution 1:10, stirring the solution for 1 hour and then standing it for 24 hours, and centrifuging to obtain a supernatant and soil residue;

[0072] Under nitrogen protection, concentrated HCl was added to the supernatant to adjust the solution pH to 1.0±0.02, stirred for 15 min, then allowed to stand for 24 h, 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 0.1M HCl and HF solution to the crude humic acid sample to make the solid-liquid ratio 1:10, stirring continuously for 4 hours, standing for 24 hours, and centrifuging to obtain a humic acid solid sample;

[0075] a5) washing the purified humic acid solid sample repeatedly, freeze-drying it, and grinding it for standby use 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, dissolve it with 0.1M strong alkali, adjust the pH to 6.0±0.02 with strong acid and strong alkali solution, stir and keep the pH stable for 60 minutes, filter, add KClO4 solution to adjust the ionic strength, and adjust the volume to 10L to obtain the humic acid test solution. The mass difference of the glass fiber membrane before and after filtration is 3.6 mg.

[0078] c: Selection of the fluorescence peak position of the titration experiment; Measure the humic acid test solution, and 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 the 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 value 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) Determine the fluorescence light scattering intensity I0 of the humic acid test solution when the excitation wavelength and emission wavelength are both 500nm;

[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 value of the humic acid test solution to the same as that in step d2, stirring and maintaining the pH value stable;

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

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

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

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

[0091] Use formula (1) to find F end :

[0092]

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

[0094]

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

[0096]

[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 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 C of the copper added 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 obtain M, where the mass volume concentration of humic acid in step d) in the test liquid 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, 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) Scanning the three-dimensional fluorescence spectrum of the humic acid test solution, wherein the excitation wavelength and the emission wavelength scanning range are both 200-600 nm, and after deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and the 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 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) Determine the fluorescence light scattering intensity I0 of the humic acid test solution when the excitation wavelength and emission wavelength are both 500nm;

[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 value of the humic acid test solution to the same as that in step d2, stir and keep the pH value stable for 20 minutes;

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

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

[0117] 9) Repeat steps d5 to d8 until I>2I0 and 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, 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) Scanning the three-dimensional fluorescence spectrum of the humic acid test solution, wherein 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) The measured humic acid solution was adjusted to pH = 8.2 ± 0.02, stirred and the pH value was kept stable 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) Determine the fluorescence light scattering intensity I0 of the humic acid test solution when the excitation wavelength and emission wavelength are both 500nm;

[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 value of the humic acid test solution to the same as that in step d2, stir and keep the pH value stable for 45 minutes;

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

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

[0138] 9) Repeat steps d5 to d8 until I>2I0 and 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] The method for determining the molecular weight of tyrosine by heavy metal titration comprises the following steps:

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

[0143] Weigh 52.79 mg of tyrosine solid sample, dissolve it with deionized water, filter it with a glass fiber membrane with a pore size of 0.45 μm, prepare a 1L tyrosine concentrated solution, filter the tyrosine concentrated solution, and the mass difference of the glass fiber membrane before and after filtration is 1.20 mg; add 138.55 g of potassium perchlorate solid to the filtered tyrosine concentrated solution and dissolve it, and dilute it to 10L with deionized water to obtain the tyrosine test solution.

[0144] 138.55 g of potassium perchlorate solid sample was weighed, dissolved with deionized water, filtered with a glass fiber membrane with a pore size of 0.45 μm, and then a background solution with a volume of 10 L was prepared.

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

[0146] 1) Measure the tyrosine test solution;

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

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

[0149] 4) Measure the background solution;

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

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

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

[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 value stable for 60 minutes;

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

[0157] 4) Determine the light scattering intensity I0 of the tyrosine test solution when the excitation wavelength and emission wavelength are both 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) adjusting the tyrosine test solution to the pH value of 6 specified in step c 2), stirring and maintaining the pH value stable for 60 minutes;

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

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

[0162] 9) Repeat step c from 5) to 8) until I>2I0 and 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 step c, stir and keep the pH value 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 to make the concentration of the quenching substance solution in the background solution the same as that of the quenching substance solution in step c5, repeat steps d2 to d4, and measure the fluorescence intensity Fr' at the fluorescence peak position of the titration experiment 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]

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

[0173]

[0174] Among them, C Q F is the total concentration of the quenching substance solution added in step c3 during the titration process; endis 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]

[0179] Wherein, 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 formula (3) and (4) is 183.5 ± 10.0.

[0180] By using the heavy metal titration method provided by the present invention, the pH value of the measured liquid, the concentration of the cumulatively added copper ions, the concentration of the measured liquid and other parameters are changed during the titration process, and the measured molecular weight of tyrosine is 178.1-185.4 g / mol, which has an error of less than 5% compared with the actual molecular weight of tyrosine (181.20 g / mol).

[0181] Example 5

[0182] 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.

[0183] b: Preparation of soil humic acid test solution;

[0184] Weigh 27 mg of the International Humic Acid Association standard (Suwannee River HA, 3S101H), dissolve it with 2 mL of 0.1 M potassium hydroxide strong base solution, dilute it with deionized water to about 1 L, filter it with a glass fiber membrane with a pore size of 0.45 μm, and then dilute it with deionized water to 2 L to prepare a humic acid test solution with a concentration of 10.0 mg / L. The mass difference of the glass fiber membrane before and after filtration is 3.7 mg.

[0185] c: Selection of fluorescence peak position in titration experiment;

[0186] 1) Measure the humic acid test solution, adjust the pH of the humic acid test solution to 6.0±0.02, stir and keep the pH value stable for 20 minutes;

[0187] 2) Scanning the three-dimensional fluorescence spectrum of the humic acid test solution, wherein the excitation wavelength and the emission wavelength scanning range are both 200-600 nm, and after deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and the 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 fluorescence peak position of the titration experiment.

[0188] d: Heavy metal titration of humic acid;

[0189] 1) Measure the prepared humic acid test solution;

[0190] 2) Adjust the pH value of the measured humic acid solution to 6.0±0.02, stir and keep the pH value stable for 20 minutes;

[0191] 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;

[0192] 4) Determine the fluorescence light scattering intensity I0 of the humic acid test solution when the excitation wavelength and emission wavelength are both 500nm;

[0193] 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;

[0194] 6) Adjust the pH value of the humic acid test solution to the same as that in step d2, stir and keep the pH value stable for 20 minutes;

[0195] 7) Determine the fluorescence intensity F value of the humic acid test solution at the fluorescence peak position of the titration experiment determined in step c;

[0196] 8) Determine the light scattering intensity I of the humic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

[0197] 9) Repeat steps d5 to d8 until I>2I0 and then stop step d.

[0198] Using the above method, the molecular weight M of humic acid was obtained to be 1737 g / mol.

[0199] By using the heavy metal titration method provided by the present invention, the pH value of the measured liquid, the concentration of the cumulatively added copper ions, the concentration of the measured liquid and other parameters are changed during the titration process, and the measured molecular weight of humic acid is 1737 g / mol, which is less than 5% compared with the actual molecular weight of humic acid (1813 g / mol).

[0200] 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 protection scope 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.

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 tree roots and stones from the soil, air-drying the soil, grinding and sieving the soil sample to obtain a soil sample for later use; adding deionized water to the soil sample, adjusting the solution to strong acidity with HCl and NaOH, and then adding HCl solution to a specific solid-liquid ratio, stirring continuously and then standing, 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 to adjust the NaOH to a specific solid-liquid ratio. After continuous stirring, the solution was allowed to stand, and centrifuged to obtain a supernatant and a solid soil sample 2; Under nitrogen protection, HCl was added to the supernatant to adjust the pH value of the solution to strong acidity, 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, adjusting 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 to adjust the NaOH to a specific solid-liquid ratio, stirring continuously and then standing, centrifuging 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 highly acidic and reach a specific solid-liquid ratio, stirring continuously and then standing, 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 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) Weighing humic acid solid powder m1, dissolving it with a strong base, adjusting it to a specific pH value with a strong acid and a strong base solution, the pH value range is between 4 and 9, stirring and keeping the pH stable, filtering, adding a salt solution to adjust the ionic strength, and fixing the volume to V, and marking it 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 4. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 3, characterized in that: The process of selecting the fluorescence peak position of the titration experiment in step c is: c1) Measure the humic acid test solution; c2) adjusting the humic acid test solution to a specific pH value, stirring and maintaining a stable pH value; 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 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 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 4, characterized in that: Step d comprises the following steps: d1) measuring the humic acid test solution prepared in step b; d2) adjusting the measured humic acid test solution to a specific pH value, stirring and maintaining a stable pH value; 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 the excitation wavelength and the emission wavelength are both 500 nm; 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; d6) adjusting the pH value of the humic acid test solution to the same as that in step d2, stirring and maintaining the pH value stable; d7) measuring the fluorescence intensity F value of the humic acid test solution at the fluorescence peak position of the titration experiment determined in step c; d8) measuring the light scattering intensity I of the humic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm; d9) Repeat steps d5 to d8 until I>2I0 and then stop step d.

6. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 5, characterized in that: In step e, the specific process of calculating the molecular weight of humic acid is: e1) Use formula (2) to calculate F end : e2) F end Substitute into formula (3) to calculate the concentration of humic acid in the soil C L : e3) C L Substitute into formula (4) to find the molecular weight M of humic acid: 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.

7. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 2, characterized in that: The error of the specific pH value in step a is ±0.1; the strong acid 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 not less than 15 minutes, and the standing time is not less than 20 hours; the solution in step a reaches a specific solid-liquid ratio of about 1:10; the strong acid concentration range used in steps a4 and a5 is 0.05-0.2 mol / L; the strong base concentration range in step a2 is between 0.1-0.2 mol / L.

8. 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, and use the silver nitrate method to determine the chloride ion content in the filtrate of step a5. 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.

9. 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, and then 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, repeat steps a4 and a5 until the ash content is less than 5.0%.

10. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 1, characterized in that: The salt concentration in the humic acid test solution in step b is a specific value, and the concentration is between 0.1-1000mmol / 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 5-20mg / L.

11. 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.

12. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 5, 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 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 not greater than 1‰ of the volume of the humic acid solution to be tested in step c1.

13. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 5, characterized in that: The pH value of the solution in steps b, c and d is a fixed value (with an error of ±0.02), and the pH value ranges from 5 to 7; the concentration of the strong base in steps a2 and b1 is a specific value, ranging from 0.05 to 0.2 M; 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.

14. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 3, characterized in that: 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.

15. The method for determining the molecular weight of soil humic acid by heavy metal titration according to claim 3, 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; 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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