A method for determining the molar mass of fulvic acid in soil

Abstract

The present invention relates to the field of environmental testing technology, and in particular to a method for determining the molar mass of fulvic acid in soil. The method comprises the following steps: (a) extraction and purification of soil fulvic acid; (b) preparation of a soil fulvic acid test solution; (c) selection of a fluorescence peak position in a titration experiment; (d) fluorescence quenching titration of fulvic acid; and (e) calculation of the molar mass of fulvic acid. The method of the present invention uses only conventional laboratory equipment and does not require high skill from the experimental operator. The accuracy and reliability of the method were demonstrated by using model compounds such as tyrosine and fulvic acid standard samples for verification.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental detection, in particular to a method for measuring the molar mass of fulvic acid in soil. Background Art

[0002] Fulvic acid is a key organic substance in soil, a type of humic acid known for its low molar mass and high water solubility. Fulvic acid molecules contain multiple functional groups, including carboxyl and alcoholic hydroxyl groups, which impart powerful binding and adsorption capabilities. Due to its structural characteristics, fulvic acid has a strong mobility in soil and can form complexes with metal ions, affecting their migration and fixation. It can passivate heavy metal ions through chelation with carboxyl and phenolic hydroxyl groups, or form organic-inorganic complexes with carboxyl and other groups, adsorbing and fixing heavy metal ions. The environmental impact of fulvic acid is primarily reflected in its active participation and promotion of soil fertility, as well as its facilitation and restriction of the migration, fixation, and leaching of metal ions and trace elements in the soil. As a stabilizer of soil structure, fulvic acid influences the soil's base exchange capacity and water retention, and also serves as a reservoir for plant matter. It also regulates material circulation, energy conversion, and information transmission between biological, soil, and environmental systems, maintaining stable soil functions. In addition, fulvic acid has obvious reducing ability and is closely related to the redox reactions of metals and organic matter in the environment. In particular, the quinone group plays an important role in electron transfer. Its redox properties enable it to play an important role in the migration, transformation and degradation of pollutants, and have a profound impact on environmental quality and ecological balance.

[0003] As a key organic component in soil, the accurate determination of the molar mass of fulvic acid is of great significance for revealing its environmental behavior and ecological effects. Currently, researchers use a variety of methods to determine the molar mass of fulvic acid, including gel chromatography, ultrafiltration membrane separation technology, UV-visible spectrophotometry, vapor pressure osmosis, high-performance liquid chromatography, and a combination of UV / visible spectroscopy and partial least squares modeling. Gel chromatography uses gel columns of different pore sizes to separate fulvic acid molecules and determine their molar mass using fluorescence and UV detectors; ultrafiltration membrane technology estimates the molar mass range by separating fulvic acid of different molar masses; UV-visible spectrophotometry indirectly infers the molar mass by measuring the absorbance value and total organic carbon content of the solution; vapor pressure osmosis determines the molar mass by measuring the change in the vapor pressure of the solution; and high-performance liquid chromatography combines UV and fluorescence detectors to quantify fulvic acid and analyze its molar mass distribution. The combined use of these methods not only improves measurement accuracy but also deepens our understanding of the chemical properties of fulvic acid, including its function as an electrolyte, its role in replenishing essential minerals and trace elements for the human body, and its important environmental functions in promoting acid-base balance, scavenging free radicals, complexing heavy metals, detoxifying pollutants, and enhancing immunity. Therefore, the molar mass determination of fulvic acid plays a vital role in soil science and environmental research. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for measuring the molar mass of fulvic acid in soil.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows: a method for determining the molar mass of fulvic acid in soil, the method comprising the following steps:

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

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

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

[0009] (d) Fluorescence quenching titration of fulvic acid;

[0010] (e) Calculation of the molar mass of fulvic acid.

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

[0012] a1) Collect soil samples: weigh the soil, remove roots and stones, air-dry, grind, and sieve to obtain soil samples;

[0013] a2) adding deionized water to the soil sample, adjusting the pH thereof to a strongly acidic state with a strong acid and a strong base, then adding hydrochloric acid to the solution to achieve a specific solid-liquid ratio, and stirring to obtain a solid-liquid mixture; the solid-liquid mixture is allowed to stand with continuous stirring, and centrifuged to obtain a supernatant 1 and a lower precipitate;

[0014] a3) adding deionized water to the lower precipitate in step a2, adjusting the pH thereof to near neutrality with a strong acid and a strong base to obtain a mixed solution, adding sodium hydroxide solution to the mixed solution under nitrogen protection to adjust the solution to a specific solid-liquid ratio, continuously stirring and allowing the solution to stand, and centrifuging to obtain a supernatant and a lower precipitate 1;

[0015] Then, under nitrogen protection, hydrochloric acid solution was added to the upper supernatant until it became strongly acidic, stirred and allowed to stand, and centrifuged again to obtain supernatant 2;

[0016] a4) adding deionized water to the lower precipitate 1 in step c, adjusting the pH of the solution to near neutrality with a strong acid and a strong base, adding sodium hydroxide solution to the solution under nitrogen protection to adjust the solution to a specific solid-liquid ratio, stirring, standing, and centrifuging to obtain a supernatant;

[0017] Then, under nitrogen protection, hydrochloric acid was added to the upper supernatant to adjust its pH to 1.0-3.0, stirred for 15-30 minutes, allowed to stand for 20-28 hours, and centrifuged to obtain supernatant 3;

[0018] a5) Combine supernatants 1, 2, and 3 and pass them through a resin column packed with XAD-8 at a flow rate of 5 column volumes / h, with 2 ml of resin corresponding to each gram of dry soil sample. After adsorption, rinse the resin column with 1 column volume of deionized water, and finally with 1 column volume of sodium hydroxide solution and 3 column volumes of deionized water. Immediately acidify the effluent to a strong acidity, add 0.1 M hydrofluoric acid to the effluent, and let it stand. Label this effluent as effluent 1.

[0019] a6) passing effluent 1 through a resin column packed with XAD-8 at a flow rate of 3 column volumes / h for adsorption, wherein 0.5 ml of resin corresponds to each gram of dry soil sample. After adsorption, the resin column is rinsed with 1 column volume of deionized water, the effluent is discarded, and the resin is then rinsed with 1 column volume of sodium hydroxide solution and 2 column volumes of deionized water, which is labeled as effluent 2.

[0020] The effluent 2 is immediately passed through a hydrogen-type cationic resin saturated with hydrogen ions, and the effluent is marked as effluent 3, which is the solution of fulvic acid in the soil.

[0021] a7) freeze-drying the effluent 3 in step a6 to obtain a purified fulvic acid sample.

[0022] Preferably, the preparation process of the fulvic acid test solution in step b is:

[0023] b1) Weigh fulvic acid solid powder m1, dissolve it with a strong base, adjust the pH to a specific value with a strong acid and strong base solution, the pH value range is 4-9, stir and maintain the pH stable, then filter, add salt solution to adjust the ionic strength, and dilute to V, which is marked as the fulvic acid test solution;

[0024] b2) After drying, weigh the filter membrane before and after filtration, and mark the mass difference as m2.

[0025] b3) The concentration of the fulvic acid solution to be tested is calculated by subtracting the amount of fulvic acid intercepted by the filter membrane m2 from the mass of fulvic acid added m1, and the total volume V of the fulvic acid solution to be tested;

[0026] Preferably, the process of selecting the fluorescence peak position of the titration experiment in step c is:

[0027] c1) Measure the fulvic acid liquid to be tested;

[0028] c2) adjusting the fulvic acid test solution to a specific pH value, stirring and maintaining the pH value stable;

[0029] c3) scanning the three-dimensional fluorescence spectrum of the fulvic acid test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0030] c4) After deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum in the three-dimensional fluorescence spectrum of the fulvic acid test solution are selected as the fluorescence peak position of the titration experiment.

[0031] Preferably, the step d comprises the following steps:

[0032] d1) measuring the fulvic acid test solution prepared in step b;

[0033] d2) adjusting the measured fulvic acid test solution to a specific pH value, stirring and maintaining the pH value stable;

[0034] d3) measuring the fluorescence intensity F0 of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment selected in step c;

[0035] d4) measuring the fluorescence light scattering intensity I0 of the fulvic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0036] 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;

[0037] d6) adjusting the pH of the fulvic acid test solution to the same as that in step d2, stirring and maintaining the pH stable;

[0038] d7) measuring the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment determined in step c;

[0039] d8) measuring the light scattering intensity I of the fulvic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0040] d9) Repeat steps d5 to d8 until I>2I0, then stop step d.

[0041] Preferably, the specific process of calculating the molar mass of fulvic acid in step e is:

[0042] e1 uses formula (1) to calculate F end :

[0043]

[0044] e2Use formula (2) to calculate the molar concentration of fulvic acid in the soil C L :

[0045]

[0046] e3Use formula (3) to calculate the molar mass M of fulvic acid:

[0047]

[0048] Among them, C Q F is the concentration of the solution containing the quenching substance added in step d5 during the titration process; end is the normalized fluorescence intensity of fulvic acid fitted at saturated titration; K is the conditional equilibrium constant; C L The molar concentration of fulvic acid in the test solution prepared in step b; M is the molar mass of fulvic acid.

[0049] Preferably, the strong acid is any one of perchloric acid, sulfuric acid, hydrochloric acid and nitric acid, or a mixture thereof; the strong base is any one of sodium hydroxide and potassium hydroxide, or a mixture thereof; the concentration of the strong base in steps a2, a3 and a4 is between 0.01-0.1M; the resin column in steps a5 and a6 adopts one or more resin columns such as DAX-8 or XAD-7; the flow rate in step a5 is 3-5 column volumes per hour, and the flow rate in step a6 is 1-3 column volumes per hour; the hydrofluoric acid in step a5 is a specific concentration, ranging from 0.1-0.5 mol / L;

[0050] Preferably, the fulvic acid solid powder obtained in step a7 is dried at 80-100°C for 20-24 hours, calcined at 550°C for 5 hours, and then the ash content of the fulvic acid solid powder is measured. If the ash content of the fulvic acid solid powder is greater than 5.0% (by dry weight), the fulvic acid solid powder is dissolved in 0.1 mol / L hydrofluoric acid and steps a6 and a7 are repeated until the ash content is less than 5.0%.

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

[0052] Preferably, 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.

[0053] Preferably, 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 fulvic acid solution to be tested in step c1.

[0054] Preferably, the pH value of the solution in step a is a constant value (with an error of ±0.1), the strongly acidic pH value range is between 1-3, and the near-neutral pH value range is between 6-8; 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 5-7; the stirring time is not less than 15 minutes; the standing time is 20-24 hours; and maintaining the pH value stable means that the pH value of the solution changes by less than 0.02 pH units in not less than 20 minutes.

[0055] Preferably, the cumulative volume of the strong acid and strong base solutions added to adjust the pH value in step c is not greater than 1‰ of the volume of the fulvic acid solution to be tested in step c1; the cumulative volume of the acid and / or base solutions added to adjust the pH value in step d is not greater than 1‰ of the volume of the background solution in step d1.

[0056] The beneficial effects of the present invention are embodied in:

[0057] (1) The method for determining the molar mass of fulvic acid provided by the present invention is stable, the measurement method is simple to operate, and has low requirements on equipment. In addition, the method results are proved to be accurate and reliable using a model compound of tyrosine molecule and a fulvic acid standard sample.

[0058] (2) The salt used in the present invention can provide a certain ionic strength during the fluorescence determination of fulvic acid, and the influence of its concentration and ion type on the determination results can be basically ignored, thereby improving the detection effect.

[0059] (3) In the preparation process of the fulvic acid test solution provided by the present invention, a strong base is used to dissolve the fulvic 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 fulvic acid (generally the oscillation time is 12-24 hours), which reduces the impact on the test results.

[0060] (4) In the method for determining the molar mass of fulvic acid provided by the present invention, controlling the pH value can not only ensure that fulvic acid can exist in the solution in a free form, but also avoid the situation where fulvic acid exists in the form of acid when the acidity is too strong, that is, when the pH value is less than 3, but also avoid the situation where copper ions combine with a large amount 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 measurement results, thereby improving the accuracy of the test results.

[0061] (5) The method for determining the molar mass of fulvic acid using fluorescence quenching titration provided by the present invention requires that one fulvic acid molecule be combined with one copper ion in the reaction system. When the copper ion causes further flocculation of the fulvic acid molecules, the scattering intensity increases sharply. Therefore, the present invention requires that in step d, I<2I0.

[0062] (6) Tyrosine used in the present invention is an organic substance with a fixed molar mass. When combined with copper ions, it will produce fluorescence quenching phenomenon. Therefore, it can be used as a reference substance to further test the reliability of the method for determining the molar mass of fulvic acid by fluorescence quenching titration provided by the present invention. DETAILED DESCRIPTION

[0063] 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.

[0064] Example 1

[0065] A method for determining the molar mass of fulvic acid in soil, the method comprising the following steps:

[0066] a: Extraction and purification of soil fulvic acid;

[0067] Weigh a soil sample from a certain location, remove debris such as tree roots and stones, air-dry it, grind it, and pass it through a 2.0 mm sieve to obtain 1000 g of soil sample;

[0068] Deionized water was added to the soil sample, and its pH was adjusted to 1.0±0.02 with HCl and NaOH. Then, 0.1 mol / L HCl solution was added thereto and stirred evenly to obtain a mixed solution with a solid-liquid ratio of 1:10. The mixed solution was stirred continuously for 4 h, allowed to stand for 24 h, and centrifuged to obtain a supernatant 1 and a lower precipitate.

[0069] Deionized water was added to the lower precipitate, and the pH thereof was adjusted to 6.0±0.02 with HCl and NaOH to obtain a mixed solution. Under nitrogen protection, 0.2 mol / L NaOH solution was added to the mixed solution, and the solution was diluted with deionized water to a final solid-liquid ratio of 1:10. The solution was stirred for 4 h and then allowed to stand for 24 h, followed by centrifugation to obtain a supernatant and a lower precipitate 1;

[0070] Then, under nitrogen protection, HCl was added to the supernatant to adjust its pH to 1.0 ± 0.02, stirred for 15 min, allowed to stand for 24 h, and centrifuged again to obtain supernatant 2;

[0071] Deionized water was added to the lower layer of precipitate 1, and the pH of the solution was adjusted to 6.0 ± 0.02 with HCl and NaOH. Under nitrogen protection, 0.2 mol / L NaOH solution was added to the solution, and the solution was diluted with deionized water to a final NaOH concentration of 0.1 mol / L and a solid-liquid ratio of 1:10. The solution was stirred for 4 h, allowed to stand for 24 h, and then centrifuged to obtain the supernatant.

[0072] Then, under nitrogen protection, HCl was added to the supernatant to adjust its pH to 1.0 ± 0.02, stirred for 15 min, allowed to stand for 24 h, and centrifuged to obtain supernatant 3;

[0073] Supernatants 1, 2, and 3 were combined and passed through a resin column packed with XAD-8 at a flow rate of 5 column volumes / h, with 2 ml of resin per gram of dry soil sample. The column was 50 cm long and 10 cm in diameter. After adsorption, the column was rinsed with 1 column volume of deionized water, followed by 1 column volume of NaOH solution and 3 column volumes of deionized water. The effluent was immediately acidified to a pH of 1.0 ± 0.02, and 0.1 mol / L hydrofluoric acid was added to the effluent and allowed to stand for 24 hours. This was labeled effluent 1.

[0074] Effluent 1 was adsorbed through a resin column packed with XAD-8 at a flow rate of 3 column volumes / h. The column was 50 cm long and 5 cm in diameter, with 0.5 ml of resin per gram of dry soil sample. After adsorption, the column was rinsed with 1 column volume of deionized water, followed by 1 column volume of NaOH solution and 2 column volumes of deionized water. This was labeled Effluent 2.

[0075] The effluent 2 is immediately passed through a hydrogen-type cationic resin saturated with hydrogen ions, and the effluent is marked as effluent 3, which is the solution of fulvic acid in the soil.

[0076] The entire effluent 3 was freeze-dried to obtain 0.375 g of a purified fulvic acid sample.

[0077] b: Preparation of soil fulvic acid test solution;

[0078] Weigh 100.7 mg of fulvic acid solid powder and dissolve it in 0.1 M strong base. Adjust the pH to 6.0 ± 0.02 using strong acid and strong base solutions. Stir and maintain a stable pH for 60 minutes before filtering. Add KClO₄ salt solution to adjust the ionic strength. The volume is then fixed to 10 L and labeled as the fulvic acid test solution. The mass difference between the glass fiber membrane before and after filtration is 1.3 mg.

[0079] c: Selection of fluorescence peak position in titration experiment;

[0080] Measure the fulvic acid test solution, adjust the pH of the fulvic acid test solution to 6.0 ± 0.02, stir and keep the pH value stable for 60 minutes;

[0081] The three-dimensional fluorescence spectrum of the fulvic acid test solution was scanned, wherein the excitation wavelength and emission wavelength scanning ranges 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 fulvic acid test solution were selected as the fluorescence peak positions of the titration experiment.

[0082] d: Fluorescence quenching titration of fulvic acid;

[0083] 1) Measure the prepared fulvic acid test solution;

[0084] 2) Adjust the pH of the measured fulvic acid solution to 6.0 ± 0.02, stir, and maintain a stable pH value for 60 minutes;

[0085] 3) measuring the fluorescence intensity F0 of the fulvic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0086] 4) measuring the fluorescence light scattering intensity I0 of the fulvic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0087] 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;

[0088] 6) adjusting the pH of the fulvic acid test solution to the same as that in step d2, stirring and maintaining a stable pH value;

[0089] 7) measuring the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment determined in step c;

[0090] 8) measuring the light scattering intensity I of the fulvic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0091] 9) Repeat steps d5 to d8 until I>2I0 and stop step d.

[0092] e: Calculation of molar mass of fulvic acid.

[0093] Use formula (1) to calculate F end :

[0094]

[0095] Use formula (2) to calculate the molar concentration of fulvic acid in soil C L :

[0096]

[0097] Use formula (3) to calculate the molar mass M of fulvic acid:

[0098]

[0099] Among them, C Q F is the concentration of the solution containing the quenching substance added in step c3 during the titration process; end is the normalized fluorescence intensity of fulvic acid fitted at saturated titration; K is the conditional equilibrium constant; C L The molar concentration of fulvic acid in the test solution prepared in step b; M is the molar mass of fulvic acid.

[0100] By changing the concentration 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 , then substitute into formula (3) to calculate M, where the mass volume concentration of fulvic acid in the test liquid in step d) is m = (100.7 mg - 1.3 mg) / 10 L = 9.94 mg / L.

[0101] Using the above method, the molar mass M of fulvic acid was obtained to be 1873±72 g / mol.

[0102] Example 2

[0103] A method for determining the molar mass of fulvic acid in soil is basically the same as that in Example 1, except that:

[0104] a: Extraction and purification of soil fulvic acid;

[0105] Soil samples were collected and extracted and purified using the above method to obtain fulvic acid solid powder.

[0106] b: Preparation of soil fulvic acid test solution;

[0107] Prepare a fulvic acid test solution with a concentration of 10.0 mg / L.

[0108] c: Selection of fluorescence peak position in titration experiment;

[0109] 1) Measure the fulvic acid test solution and adjust the pH of the fulvic acid test solution to 4.0 ± 0.01, stir and keep the pH value stable for 20 minutes;

[0110] 2) Scanning the three-dimensional fluorescence spectrum of the fulvic acid test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm, and after deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum in the three-dimensional fluorescence spectrum of the fulvic acid test solution are selected as the fluorescence peak position of the titration experiment.

[0111] d: Fluorescence quenching titration of fulvic acid;

[0112] 1) Measure the prepared fulvic acid test solution;

[0113] 2) Adjust the pH of the measured fulvic acid solution to 4.0 ± 0.01, stir and keep the pH stable for 20 minutes;

[0114] 3) measuring the fluorescence intensity F0 of the fulvic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0115] 4) measuring the fluorescence light scattering intensity I0 of the fulvic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0116] 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;

[0117] 6) Adjust the pH of the fulvic acid test solution to the same value as in step d2, stir, and maintain the pH stable for 20 minutes;

[0118] 7) measuring the fluorescence intensity F value of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment determined in step c;

[0119] 8) measuring the light scattering intensity I of the fulvic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0120] 9) Repeat steps d5 to d8 until I>2I0 and stop step d.

[0121] Using the above method, the molar mass M of fulvic acid was obtained to be 1642±87.1 g / mol.

[0122] Example 3

[0123] A method for determining the molar mass of fulvic acid in soil is basically the same as that in Example 1, except that:

[0124] a: Extraction and purification of soil fulvic acid;

[0125] A soil sample was collected and extracted and purified using the above method to obtain fulvic acid solid powder.

[0126] b: Preparation of soil fulvic acid test solution;

[0127] Prepare a fulvic acid test solution with a concentration of 10.0 mg / L.

[0128] c: Selection of fluorescence peak position in titration experiment;

[0129] 1) Measure the fulvic acid test solution and adjust the pH of the fulvic acid test solution to 8.2±0.02, stir and keep the pH value stable for 45 minutes;

[0130] 2) Scanning the three-dimensional fluorescence spectrum of the fulvic acid test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm. After deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum in the three-dimensional fluorescence spectrum of the fulvic acid test solution are selected as the excitation wavelength and emission wavelength of the titration experiment.

[0131] d: Fluorescence quenching titration of fulvic acid;

[0132] 1) Measure the prepared fulvic acid test solution;

[0133] 2) Adjust the pH of the measured fulvic acid solution to 8.2 ± 0.02, stir, and maintain a stable pH value for 45 minutes;

[0134] 3) measuring the fluorescence intensity F0 of the fulvic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0135] 4) measuring the fluorescence light scattering intensity I0 of the fulvic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0136] 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;

[0137] 6) Adjust the pH of the fulvic acid test solution to the same value as in step d2, stir, and maintain the pH stable for 20 minutes;

[0138] 7) measuring the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment determined in step c;

[0139] 8) measuring the light scattering intensity I of the fulvic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0140] 9) Repeat steps d5 to d8 until I>2I0 and stop step d.

[0141] Using the above method, the molar mass M of fulvic acid was obtained to be 2007±103.2 g / mol.

[0142] Example 4

[0143] A method for measuring the molar mass of tyrosine, the method comprising the following steps:

[0144] a: Preparation of tyrosine test solution and background solution

[0145] 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, and 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. Then dilute to 10 L with deionized water to prepare the tyrosine test solution.

[0146] 138.55 g of potassium perchlorate solid sample was weighed, dissolved in deionized water, and filtered through a glass fiber membrane with a pore size of 0.45 μm to prepare a background solution with a volume of 10 L.

[0147] b: Selection of fluorescence peak position in tyrosine titration experiment

[0148] 1) Measure the tyrosine test solution;

[0149] 2) Adjust the pH of the tyrosine solution to 6, stir and maintain the pH stable for 60 minutes;

[0150] 3) scanning the three-dimensional fluorescence spectrum of the tyrosine test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0151] 4) Measure the background solution;

[0152] 5) Adjust the pH value of the background solution to 6, stir and keep the pH stable for 60 minutes;

[0153] 6) scanning the three-dimensional fluorescence spectrum of the background solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0154] 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, and use them as the excitation wavelength and emission wavelength for the fluorescence quenching titration, which are 350 nm and 445 nm, respectively.

[0155] c: Fluorescence quenching titration of tyrosine

[0156] 1) Measure the tyrosine test solution;

[0157] 2) Adjust the pH of the tyrosine test solution to 6, stir and keep the pH stable for 60 minutes;

[0158] 3) measuring the fluorescence intensity F0 of the tyrosine test solution at the fluorescence peak position of the titration experiment determined in step c, i.e., at an excitation wavelength of 350 nm and an emission wavelength of 440 nm;

[0159] 4) Determine the light scattering intensity I0 of the tyrosine test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0160] 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;

[0161] 6) adjusting the pH of the tyrosine test solution to the specified pH value in step c) 2), stirring and maintaining the pH value stable for 60 minutes;

[0162] 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;

[0163] 8) Determine the light scattering intensity I of the tyrosine test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0164] 9) Repeat step c from 5) to 8) until I>2I0 and stop step c.

[0165] d: Background solution fluorescence measurement

[0166] 1) Measure the background solution;

[0167] 2) Adjust the pH value of the background solution to the same as step c, stir and keep the pH stable for 60 minutes;

[0168] 3) measuring the fluorescence intensity Fr0 of the background solution at the position of the fluorescence peak of the titration experiment determined in step c;

[0169] 4) adding a solution containing a quenching substance so that the concentration of the solution containing the quenching substance in the background solution is the same as the concentration in step c) 5) and repeating steps d) 2) to 4) to measure the fluorescence intensity Fr of the background solution at the fluorescence peak position determined in the titration experiment in step c.

[0170] e: Calculation of the molar mass of tyrosine

[0171] 1) Calculate F0 = F0' - Fr0' and calculate F = F' - Fr'

[0172] 2) Use formula (1) to calculate F end :

[0173]

[0174] 3) Use formula (2) to calculate the molar mass M of tyrosine:

[0175]

[0176] Among them, C Q F is the total concentration of the solution containing the quenching substance 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 molar mass of tyrosine.

[0177] Different concentrations of Cu 2+ The corresponding measured fluorescence intensity values ​​F (blank deducted) are shown in Table 1. Table 1 Different concentrations of Cu 2+ The corresponding fluorescence intensity value F (minus blank) is statistically analyzed.

[0178] <![CDATA[Cu 2+ Concentration C Q (mol / L) Tyrosine fluorescence intensity value F(AU) 0 283.9±5.6 0.000028 157.1±4.7 0.00008 73.34±2.3 0.0004 23.69±1.7

[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.

[0180] F0, m and Cu in Table 1 2+ Concentration C Q Substituting the fluorescence intensity value F into formula (1), the molar mass of tyrosine was calculated to be 183.5±10.0.

[0181] By using the fluorescence quenching titration method provided by the present invention, the pH value of the measured solution, the cumulative concentration of added copper ions, the concentration of the measured solution, and other parameters are changed during the titration process. The measured molar mass of tyrosine is 178.1-185.4 g / mol, which has an error of less than 5% compared with the actual molar mass of tyrosine (181.20 g / mol).

[0182] Example 5

[0183] A method for determining the molar mass of fulvic acid in soil is basically the same as that in Example 1, except that an International Humic Acid Association standard sample (Suwannee River FA, 3S101F) is used for the experiment.

[0184] b: Preparation of soil fulvic acid test solution;

[0185] Weigh 20.2 mg of the International Humic Acid Association standard (Suwannee River FA, 3S101F) 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 10.0 mg / L fulvic acid test solution. The difference in mass between the glass fiber membrane before and after filtration was 0.73 mg.

[0186] b: Selection of fluorescence peak position in titration experiment;

[0187] 1) Measure the fulvic acid test solution and adjust the pH of the fulvic acid test solution to 6.0 ± 0.02, stir and keep the pH value stable for 20 minutes;

[0188] 2) Scanning the three-dimensional fluorescence spectrum of the fulvic acid test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm, and after deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum in the three-dimensional fluorescence spectrum of the fulvic acid test solution are selected as the fluorescence peak position of the titration experiment.

[0189] d: Fluorescence quenching titration of fulvic acid;

[0190] 1) Measure the prepared fulvic acid test solution;

[0191] 2) Adjust the pH of the measured fulvic acid solution to 6.0 ± 0.02, stir and keep the pH stable for 20 minutes;

[0192] 3) measuring the fluorescence intensity F0 of the fulvic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0193] 4) measuring the fluorescence light scattering intensity I0 of the fulvic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0194] 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;

[0195] 6) Adjust the pH of the fulvic acid test solution to the same value as in step d2, stir, and maintain the pH stable for 20 minutes;

[0196] 7) measuring the fluorescence intensity F value of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment determined in step c;

[0197] 8) measuring the light scattering intensity I of the fulvic acid test solution when both the excitation wavelength and the emission wavelength are 500 nm;

[0198] 9) Repeat steps d5 to d8 until I>2I0 and stop step d.

[0199] Using the above method, the molar mass M of fulvic acid was obtained to be 996.13 g / mol.

[0200] By using the fluorescence quenching titration method provided by the present invention, the pH value of the measured liquid, the concentration of the cumulative added copper ions, the concentration of the measured liquid and other parameters were changed during the titration process. The measured molar mass of fulvic acid was 996.13 g / mol, which is less than 5% of the actual molar mass of fulvic acid (1028 g / mol).

[0201] 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 molar mass of fulvic acid in soil, characterized in that: The method for determining the molar mass of fulvic acid in soil comprises the following steps: (a) Extraction and purification of soil fulvic acid; (b) Preparation of soil fulvic acid test solution; (c) Selection of fluorescence peak position in titration experiment; (d) Fluorescence quenching titration of fulvic acid; (e) Calculation of the molar mass of fulvic acid; Step d comprises the following steps: d1) measuring the fulvic acid test solution prepared in step b; d2) adjusting the pH value of the measured fulvic acid test solution, stirring and maintaining the pH value stable; d3) measuring the fluorescence intensity F0 of the fulvic 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 fulvic 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 added solution containing a 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 fulvic 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 fulvic 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 fulvic 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 molar mass of fulvic acid is: e1 uses formula (1) to calculate F end : (1) e2Use formula (2) to calculate the molar mass M of fulvic acid: (2) Among them, C Q F is the concentration of the solution containing the quenching substance added in step d5 during the titration process; end is the normalized fluorescence intensity fitted by fulvic 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 fulvic acid in the test solution in step d1; M is the molar mass of fulvic acid.

2. A method for measuring the molar mass of fulvic acid in soil according to claim 1, characterized in that, The soil fulvic acid extraction and purification process in step a is: a1) Collect soil samples: weigh the soil, remove roots and stones, air-dry, grind, and sieve to obtain soil samples; a2) adding deionized water to the soil sample, adjusting the pH thereof to a strongly acidic state with a strong acid and a strong base, then adding hydrochloric acid thereto and stirring uniformly to obtain a solid-liquid mixture; the solid-liquid mixture is allowed to stand with continuous stirring, and centrifuged to obtain a supernatant 1 and a lower precipitate; a3) adding deionized water to the lower precipitate in step a2, adjusting the pH thereof to near neutrality with a strong acid and a strong base to obtain a mixed solution, adding sodium hydroxide solution to the mixed solution under nitrogen protection, stirring continuously, allowing the mixture to stand, and centrifuging to obtain a supernatant and a lower precipitate 1; Then, under nitrogen protection, hydrochloric acid solution was added to the upper supernatant until it became strongly acidic, stirred and allowed to stand, and centrifuged again to obtain supernatant 2; a4) adding deionized water to the lower precipitate 1 in step c, adjusting the pH of the solution to near neutral with a strong acid and a strong base, adding sodium hydroxide solution to the solution under nitrogen protection, stirring, allowing to stand, and centrifuging to obtain a supernatant; Then, under nitrogen protection, hydrochloric acid was added to the upper supernatant to adjust the pH to 1.0-3.0, stirred for 15-30 minutes, allowed to stand for 20-28 hours, and centrifuged to obtain supernatant 3; a5) Combine supernatants 1, 2, and 3 and pass them through a resin column packed with XAD-8 at a flow rate of 5 column volumes / h, with 2 ml of resin corresponding to each gram of dry soil sample. After adsorption, rinse the resin column with 1 column volume of deionized water, and finally with 1 column volume of sodium hydroxide solution and 3 column volumes of deionized water. Immediately acidify the effluent to a strong acidity, add 0.1 M hydrofluoric acid to the effluent, and let it stand. Label it as effluent 1. a6) Pass effluent 1 through a resin column packed with XAD-8 at a flow rate of 3 column volumes / h for adsorption, wherein 0.5 ml of resin corresponds to each gram of dry soil sample. After adsorption, rinse the resin column with 1 column volume of deionized water, discard the effluent, and then rinse the resin with 1 column volume of sodium hydroxide solution and 2 column volumes of deionized water, marking it as effluent 2; The effluent 2 is immediately passed through a hydrogen-type cationic resin saturated with hydrogen ions, and the effluent is labeled as effluent 3, which is the solution of fulvic acid in the soil; a7) The effluent 3 in step a6 is completely freeze-dried to obtain a purified fulvic acid sample.

3. A method for measuring the molar mass of fulvic acid in soil according to claim 2, characterized in that, The preparation process of the fulvic acid test solution in step b is: b1) Weigh fulvic 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 fulvic 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 fulvic acid test solution is calculated by subtracting the amount of fulvic acid intercepted by the filter membrane m2 from the mass of fulvic acid added m1, and the total volume V of the fulvic acid test solution.

4. A method for measuring the molar mass of fulvic acid in soil 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 fulvic acid test liquid; c2) Adjust the pH value of the fulvic acid test solution, stir and maintain the pH value stable; c3) scanning the three-dimensional fluorescence spectrum of the fulvic 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 fulvic acid test solution as the fluorescence peak positions for the titration experiment.

5. A method for measuring the molar mass of fulvic acid in soil according to claim 3, characterized in that, The strong acid is any one of perchloric acid, sulfuric acid, hydrochloric acid and nitric acid, or a mixture thereof; the strong base is sodium hydroxide, potassium hydroxide, or a mixture thereof; the concentration of the strong base in steps a2, a3 and a4 is between 0.01-0.1M.

6. A method for measuring the molar mass of fulvic acid in soil according to claim 2, characterized in that, Take the fulvic acid sample obtained in step a7, dry it at 80-100°C for 20-24 hours, calcine it at 550°C for 5 hours, and then determine the ash content of the fulvic acid solid powder; if the ash content of the fulvic acid solid powder is greater than 5.0% by dry weight, dissolve the fulvic acid solid powder in 0.1 mol / L hydrofluoric acid and repeat steps a6 and a7 until the ash content is less than 5.0%.

7. A method for measuring the molar mass of fulvic acid in soil according to claim 3, characterized in that, The fulvic acid concentration in the fulvic acid test solution in step b is a specific value, and the concentration range should be controlled between 5-20 mg / L; the salt used for adjusting the ionic strength in step b is any one or more of potassium perchlorate, sodium perchlorate, sodium chloride, potassium chloride and potassium nitrate solution, and the salt concentration in the fulvic acid test solution is 0.1-1000 mmol / L.

8. A method for measuring the molar mass of fulvic acid in soil according to claim 3, 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.

9. A method for measuring the molar mass of fulvic acid in soil according to claim 4, characterized in that, The cumulative volume of the solution containing the quenching substance added during the titration process of step d is not greater than 1‰ of the volume of the fulvic acid solution to be measured in step c1.

10. A method for measuring the molar mass of fulvic acid in soil according to claim 4, characterized in that, The pH value of the solution in step a is a constant value with an error of ±0.1, the strongly acidic pH value range is between 1-3, and the near-neutral pH value range is between 6-8; 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 5-7; the stirring time is not less than 15 minutes; the standing time is 20-24 hours; maintaining the pH value stable means that the pH value of the solution changes by less than 0.02 pH units in not less than 20 minutes.

11. A method for measuring the molar mass of fulvic acid in soil according to claim 4, characterized in that: The cumulative volume of the strong acid and strong base solutions added to adjust the pH value in step c is not greater than 1‰ of the volume of the fulvic acid solution to be tested in step c1; the cumulative volume of the acid and / or base solutions added to adjust the pH value in step d is not greater than 1‰ of the volume of the background solution in step d1.

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