Method for measuring molar mass of fulvic acid in soil

Abstract

The invention relates to the technical field of environment detection, in particular to a method for measuring the molar mass of fulvic acid in soil. The method comprises the following steps: (a) extracting and purifying fulvic acid in soil; (b) preparing a soil fulvic acid liquid to be detected; (c) selecting a fluorescence peak position of a titration experiment; (d) fluorescence quenching titration of fulvic acid; and (e) calculating the molar mass of fulvic acid. According to the method, only conventional laboratory equipment is used, and the requirement for the skill of an experiment operator is not high. Model compounds such as tyrosine and fulvic acid standard samples are used 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, and in particular to a method for determining the molar mass of fulvic acid in soil. Background Art

[0002] Fulvic acid is an important organic substance in the soil. It is a type of humic acid and is known for its small molar mass and high water solubility. Fulvic acid molecules contain a variety of functional groups, including carboxyl, alcoholic hydroxyl, etc. These functional groups give fulvic acid strong binding and adsorption capabilities. Due to its structural characteristics, fulvic acid has a strong migration ability in the soil and can form complexes with metal ions in the soil, affecting the migration and fixation of metal ions. It can passivate heavy metal ions through chelation with carboxyl and phenolic hydroxyl groups, or form soil organic-inorganic complexes through complexation with carboxyl and other groups, and adsorb and fix soil heavy metal ions. The environmental impact of fulvic acid is mainly reflected in its active participation and promotion of soil fertility, as well as its promotion and restriction of the migration, fixation and leaching of soil metal ions and trace elements. As a stabilizer of soil structure, fulvic acid affects the base exchange capacity and water holding capacity of the soil, and is also a warehouse for plant materials. It can also regulate the material circulation, energy conversion and information transmission between biological systems, soil systems and environmental systems, and maintain the stability of 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. At present, 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 through 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 vapor pressure of the solution; and high performance liquid chromatography combines UV and fluorescence detectors to quantify and analyze the molar mass distribution of fulvic acid. The combined use of these methods not only improves the accuracy of the determination, but also deepens our understanding of the chemical properties of fulvic acid, including its function as an electrolyte, its role in supplementing the minerals and trace elements needed by 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 technology of fulvic acid plays a vital role in the fields of 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 determining the molar mass of fulvic acid in soil.

[0005] To achieve the above object, the technical solution of the present invention is implemented 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, step a comprises the following steps:

[0012] a1) Collect soil samples, weigh the soil, remove tree 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 strong acidity with strong acid and strong base, then adding hydrochloric acid thereto to make the solution have a specific solid-liquid ratio, and stirring to obtain a solid-liquid mixture; stirring the solid-liquid mixture continuously and allowing it to stand, and centrifuging 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 a sodium hydroxide solution to the mixed solution under nitrogen protection to make the solution have a specific solid-liquid ratio, continuously stirring and standing, and centrifuging to obtain an upper clear liquid and a lower precipitate 1;

[0015] Then, under nitrogen protection, hydrochloric acid solution was added to the upper clear liquid 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 a sodium hydroxide solution to the solution under nitrogen protection to make the solution have a specific solid-liquid ratio, stirring and standing, and centrifuging to obtain a supernatant;

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

[0018] a5) combining the supernatants 1, 2 and 3, and passing them through a resin column for adsorption at a flow rate of 5 column volumes / h, wherein the resin column is filled with XAD-8, wherein each gram of dry soil sample corresponds to 2 ml of resin; after the adsorption is completed, the resin column is eluted with 1 column volume of deionized water, and finally, the resin is eluted with 1 column volume of sodium hydroxide solution and 3 column volumes of deionized water, the effluent is immediately acidified to strong acidity, and 0.1 M hydrofluoric acid is added to the effluent and allowed to stand, and the effluent is marked as effluent 1;

[0019] a6) passing the effluent 1 through a resin column for adsorption at a flow rate of 3 column volumes / h, wherein the resin column is filled with XAD-8, wherein each gram of dry soil sample corresponds to 0.5 ml of resin; after the adsorption is completed, the resin column is eluted with 1 column volume of deionized water, the effluent is discarded, and then the resin is eluted with 1 column volume of sodium hydroxide solution and 2 column volumes of deionized water, which is marked 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 a 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 it to a specific pH value with a strong acid and a strong base solution, the pH value range is between 4-9, stir and keep the pH stable, then filter, add a salt solution to adjust the ionic strength, and dilute to V, then mark it as the fulvic acid test solution;

[0024] b2) After drying, weigh the filter membrane before and after filtration respectively, 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 m1 of the added fulvic acid 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 test liquid;

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

[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 a stable pH value;

[0034] d3) measuring the fluorescence intensity F of the fulvic acid test solution at the fluorescence peak position of the titration experiment selected in step c 0 ;

[0035] d4) Determine the fluorescence light scattering intensity I of the fulvic acid test solution when the excitation wavelength and emission wavelength are both 500nm 0 ;

[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 value of the fulvic acid test solution to the same as that of step d2, stirring and maintaining the pH value 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 the excitation wavelength and the emission wavelength are both 500 nm;

[0040] d9) Repeat steps d5 to d8 until I>2I 0 Stop step d.

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

[0042] e1 Use formula (1) to find F end :

[0043]

[0044] e2Use formula (2) to calculate the 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 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; 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, and the concentration range is between 0.1-0.5mol / L;

[0050] Preferably, the fulvic acid solid powder obtained in step a7 is dried at 80-100°C for 20-24h, and then calcined at 550°C for 5h, 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 with 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 of 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; 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 model compounds of tyrosine molecules and fulvic acid standards.

[0058] (2) The salt used in the present invention can provide a certain ion strength during the fluorescence determination of fulvic acid, and the influence of its concentration and ion type on the determination result 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 that when the acidity is too strong, that is, when the pH value is less than 3, fulvic acid will exist in the form of acid. At the same time, it can also avoid the situation that when the alkalinity is too strong, that is, when the pH value is greater than 10, copper ions combine with a large number of free hydroxyl groups in the solution, 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 by fluorescence quenching titration provided by the present invention requires that a fulvic acid molecule is combined with a copper ion in the reaction system. When the copper ion causes the fulvic acid molecules to further flocculate, the scattering intensity will increase sharply. Therefore, the present invention requires that in step d, I<2I 0 .

[0062] (6) Tyrosine used in the present invention is an organic substance with a fixed molar mass. When combined with copper ions, fluorescence quenching occurs. Therefore, it can be used as a reference substance to further verify 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 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.

[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 place, remove tree roots, stones and other debris, 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, and 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 an upper clear liquid 1 and a lower precipitate;

[0069] Deionized water was added to the lower precipitate, and the pH 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 make the final solid-liquid ratio of the NaOH solution 1:10. The solution was stirred for 4 hours and then allowed to stand for 24 hours, and then centrifuged to obtain an upper clear liquid and a lower precipitate 1;

[0070] Then, under nitrogen protection, HCl was added to the upper supernatant to make its pH = 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 precipitation 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 make the final NaOH concentration 0.1 mol / L and the solid-liquid ratio 1:10. The solution was stirred for 4 hours and then allowed to stand for 24 hours, and then centrifuged to obtain the supernatant;

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

[0073] The supernatants 1, 2, and 3 were combined and adsorbed through a resin column at a flow rate of 5 column volumes / h. The resin column was filled with XAD-8, wherein each gram of dry soil sample corresponded to 2 ml of resin; the resin column was 50 cm long and 10 cm in diameter. After the adsorption was completed, the resin column was eluted with 1 column volume of deionized water, and finally 1 column volume of NaOH solution and 3 column volumes of deionized water. The effluent was immediately acidified to pH = 1.0 ± 0.02, and 0.1 mol / L hydrofluoric acid was added to the effluent and allowed to stand for 24 hours, which was marked as effluent 1;

[0074] The effluent 1 was adsorbed through a resin column at a flow rate of 3 column volumes / h. The resin column was filled with XAD-8, wherein each gram of dry soil sample corresponded to 0.5 ml of resin, and the resin column was 50 cm long and 5 cm in diameter. After the adsorption was completed, the resin column was eluted with 1 column volume of deionized water, and finally, the resin was eluted with 1 column volume of NaOH solution and 2 column volumes of deionized water, which was marked as 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 a 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 with 0.1 M strong alkali. Adjust the pH value to 6.0 ± 0.02 with strong acid and strong alkali solution. Stir and keep the pH stable for 60 min, then filter. Add KClO 4 The salt solution was used to adjust the ionic strength, and the volume was fixed to 10 L and marked as the fulvic acid test solution. The mass difference of the glass fiber membrane before and after filtration was 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 the maximum in the three-dimensional fluorescence spectrum of the fulvic acid test solution were selected as the fluorescence peak position 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 value of the measured fulvic acid solution to 6.0±0.02, stir and keep the pH value stable for 60 minutes;

[0085] 3) Determine the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment selected in step c 0 ;

[0086] 4) Determine the fluorescence light scattering intensity I of the fulvic acid test solution when the excitation wavelength and emission wavelength are both 500nm0 ;

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

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

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

[0091] 9) Repeat steps d5 to d8 until I>2I 0 Stop step d.

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

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

[0094]

[0095] Use formula (2) to calculate the concentration of fulvic acid in soil: 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), C L , and then substitute it into formula (3) to obtain M, where the mass volume concentration of fulvic acid in step d) in the test liquid 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, the method is basically the same as 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, 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 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 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 value stable for 20 minutes;

[0114] 3) Determine the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment selected in step c 0 ;

[0115] 4) Determine the fluorescence light scattering intensity I of the fulvic acid test solution when the excitation wavelength and emission wavelength are both 500nm 0 ;

[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) adjusting the pH value of the fulvic acid test solution to the same as that in step d2, stirring and keeping the pH value stable for 20 minutes;

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

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

[0120] 9) Repeat steps d5 to d8 until I>2I 0 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, the method is basically the same as 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, 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, and 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 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 value of the measured fulvic acid solution to 8.2±0.02, stir and keep the pH value stable for 45 minutes;

[0134] 3) Determine the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment selected in step c 0 ;

[0135] 4) Determine the fluorescence light scattering intensity I of the fulvic acid test solution when the excitation wavelength and emission wavelength are both 500nm 0 ;

[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) adjusting the pH value of the fulvic acid test solution to the same as that in step d2, stirring and keeping the pH value stable for 20 minutes;

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

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

[0140] 9) Repeat steps d5 to d8 until I>2I 0 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 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.

[0146] 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.

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

[0148] 1) Measure the tyrosine test solution;

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

[0150] 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;

[0151] 4) Measure the background solution;

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

[0153] 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;

[0154] 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 350 nm and 445 nm, respectively, during the fluorescence quenching titration.

[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 value stable for 60 minutes;

[0158] 3) Determine the fluorescence intensity F 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. 0 ;

[0159] 4) Determine the light scattering intensity I of the tyrosine test solution when the excitation wavelength and emission wavelength are both 400nm 0 ;

[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 tyrosine test solution to the pH value specified in step c 2), stirring and keeping 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 the excitation wavelength and the emission wavelength are both 500 nm;

[0164] 9) Repeat step c 5) to 8) until I>2I 0 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 value stable for 60 minutes;

[0168] 3) Measure the fluorescence intensity Fr of the background solution at the fluorescence peak position of the titration experiment determined in step c 0 ;

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

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

[0171] 1) Calculate F 0 =F 0 '-Fr 0 '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) above is m=(52.79 mg-1.20 mg) / 10 L=5.16 mg / L.

[0180] F 0 , m and Cu in Table 1 2+ Concentration C Q , the fluorescence intensity value F was substituted into formula (1) to calculate the molar mass of tyrosine 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 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 molar mass of tyrosine is 178.1-185.4 g / mol, which is less than 5% of 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, the method is basically the same as 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), 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 10.0 mg / L fulvic acid test solution. The mass difference of the glass fiber membrane before and after filtration is 0.73 mg.

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

[0187] 1) 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 20 minutes;

[0188] 2) Scanning the three-dimensional fluorescence spectrum of the fulvic 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 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 value of the measured fulvic acid solution to 6.0±0.02, stir and keep the pH value stable for 20 minutes;

[0192] 3) Determine the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment selected in step c 0 ;

[0193] 4) Determine the fluorescence light scattering intensity I of the fulvic acid test solution when the excitation wavelength and emission wavelength are both 500nm 0 ;

[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) adjusting the pH value of the fulvic acid test solution to the same as that in step d2, stirring and keeping the pH value stable for 20 minutes;

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

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

[0198] 9) Repeat steps d5 to d8 until I>2I 0 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 cumulatively added copper ions, the concentration of the measured liquid and other parameters are changed during the titration process, and the measured molar mass of fulvic acid is 996.13 g / mol, which is less than 5% compared with 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 protection scope 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 fulvic acid from soil; (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.

2. A method for determining 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 tree 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 strong acidity with strong acid and strong base, then adding hydrochloric acid thereto to make the solution have a specific solid-liquid ratio, and stirring to obtain a solid-liquid mixture; stirring the solid-liquid mixture continuously and allowing it to stand, and centrifuging 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 a sodium hydroxide solution to the mixed solution under nitrogen protection to make the solution have a specific solid-liquid ratio, continuously stirring and standing, and centrifuging to obtain an upper clear liquid and a lower precipitate 1; Then, under nitrogen protection, hydrochloric acid solution was added to the upper clear liquid 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 neutrality with a strong acid and a strong base, adding a sodium hydroxide solution to the solution under nitrogen protection to make the solution have a specific solid-liquid ratio, stirring and standing, and centrifuging to obtain a supernatant; Then, under nitrogen protection, hydrochloric acid is added to the upper clear liquid to make its pH = 1.0-3.0, stirred for 15-30 minutes, allowed to stand for 20-28 hours, and centrifuged to obtain supernatant 3; a5) combining the supernatants 1, 2 and 3, and passing them through a resin column for adsorption at a flow rate of 5 column volumes / h, wherein the resin column is filled with XAD-8, wherein each gram of dry soil sample corresponds to 2 ml of resin; after the adsorption is completed, the resin column is eluted with 1 column volume of deionized water, and finally, the resin is eluted with 1 column volume of sodium hydroxide solution and 3 column volumes of deionized water, the effluent is immediately acidified to strong acidity, and 0.1 M hydrofluoric acid is added to the effluent and allowed to stand, and the effluent is marked as effluent 1; a6) passing the effluent 1 through a resin column for adsorption at a flow rate of 3 column volumes / h, wherein the resin column is filled with XAD-8, wherein each gram of dry soil sample corresponds to 0.5 ml of resin; after the adsorption is completed, the resin column is eluted with 1 column volume of deionized water, the effluent is discarded, and then the resin is eluted with 1 column volume of sodium hydroxide solution and 2 column volumes of deionized water, which is marked as effluent 2; 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 a solution of fulvic acid in the soil; a7) freeze-drying the effluent 3 in step a6 to obtain a purified fulvic acid sample.

3. A method for determining 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, dissolve it with a strong base, adjust it to a specific pH value with a strong acid and a strong base solution, the pH value range is between 4-9, stir and keep the pH stable, then filter, add a salt solution to adjust the ionic strength, and dilute to V, then mark it 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 liquid is calculated by subtracting the amount of fulvic acid intercepted by the filter membrane m2 from the mass m1 of the added fulvic acid, and the total volume V of the fulvic acid test liquid.

4. A method for determining 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 liquid to be tested; c2) adjusting the fulvic acid test solution to a specific pH value, stirring and maintaining a stable pH value; 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 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.

5. The method for measuring the molar mass of fulvic acid in soil according to claim 4, characterized in that: Step d comprises the following steps: d1) measuring the fulvic acid test solution prepared in step b; d2) adjusting the measured fulvic acid test solution to a specific pH value, stirring and maintaining a stable pH value; 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 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 fulvic acid test solution to the same as that of step d2, stirring and maintaining the pH value stable; d7) measuring the fluorescence intensity F value of the fulvic 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 fulvic 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. A method for determining the molar mass of fulvic acid in soil according to claim 5, characterized in that: In step e, the specific process of calculating the molar mass of fulvic acid is: e1 Use formula (1) to find F end : e2Use formula (2) to calculate the concentration of fulvic acid in the soil C L : e3Use formula (3) to calculate the molar mass M of fulvic acid: 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.

7. A method for determining 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; 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 times the column volume per hour, and the flow rate in step a6 is 1-3 times the column volume per hour; in step a5, the hydrofluoric acid has a specific concentration, and the concentration range is between 0.1-0.5mol / L.

8. A method for determining 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 20-24h, calcine it at 550°C for 5h, 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%, based on dry weight, dissolve the fulvic acid solid powder with 0.1mol / L hydrofluoric acid, and repeat steps a6 and a7 until its ash content is less than 5.0%.

9. A method for determining the molar mass of fulvic acid in soil according to claim 2, 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.

10. A method for determining the molar mass of fulvic acid in soil according to claim 2, 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.

11. A method for determining the molar mass of fulvic acid in soil according to claim 5, characterized in that: 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 of step d is not greater than 1‰ of the volume of the fulvic acid solution to be tested in step c1.

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

13. The method for determining the molar mass of fulvic acid in soil according to claim 5, 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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