Method for determining molar mass of fulvic acid in lake water body based on three-dimensional fluorescence spectrum

Abstract

The invention relates to the technical field of environment detection, in particular to a method for determining the molar mass of fulvic acid in a lake water body based on a three-dimensional fluorescence spectrum. The method comprises the following steps: (a) extracting and purifying fulvic acid in lake water; (b) preparing a tested solution of fulvic acid in the lake water body; (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 measuring the molar mass of fulvic acid in lake water by three-dimensional fluorescence spectroscopy. Background Art

[0002] Fulvic acid is a low-molar mass, highly water-soluble organic substance widely distributed in water bodies. It has a complex structure, mainly composed of carbon, oxygen, and hydrogen elements, and contains a small amount of nitrogen, sulfur, phosphorus, etc. Fulvic acid molecules contain condensed benzene rings, alicyclic rings, etc., and are interconnected by functional groups such as carboxyl, hydroxyl, and carbonyl. These functional groups give fulvic acid strong complexation and adsorption capabilities, allowing it to form organic colloids with various metal oxides in water bodies and migrate with the water body, affecting the adsorption of phosphates in the soil and the effectiveness of phosphorus. The photochemical properties of fulvic acid enable it to undergo rapid photochemical reactions under ultraviolet or solar radiation, affecting the migration and transformation of organic pollutants and heavy metals in water, and then controlling the environmental chemical behavior of these substances, including chemical degradation, photolysis, biological absorption, migration, and volatilization. In addition, as the main photoactive substance in water bodies, fulvic acid has a great influence on the absorption and utilization of radiation light, the generation and fate of active oxygen, and has an important influence on the composition structure and distribution law of dissolved organic matter in water bodies, thereby affecting the ecological health and water quality of water bodies. Studies have shown that fulvic acid content has a strong effect on the reduction of soil phosphorus availability induced by microplastics. Increasing fulvic acid content can improve phosphorus availability by reducing the adsorption capacity of microplastics for phosphorus. The electron transfer ability of fulvic acid is related to its ability to degrade pollutants such as pentachlorophenol. Landfill fulvic acid has reduction and transformation capabilities, which is attributed to its electron transfer ability. These functions are conducive to promoting reduction and dechlorination. Therefore, fulvic acid plays an important role in water bodies, and its structural composition, migration and transformation, and environmental impact have a profound impact on the chemical behavior and ecological health of the aquatic environment.

[0003] There are various methods for determining the molar mass of fulvic acid in water, including gel chromatography, vapor pressure osmosis, laser desorption Fourier transform ion cyclotron resonance mass spectrometry and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, solution equilibrium method, ultrafiltration membrane separation technology, UV-visible spectrophotometry and total organic carbon analysis. Gel chromatography separates substances of different molar masses through gel chromatography columns and provides relative information on molar mass distribution; vapor pressure osmosis determines the average molar mass based on osmotic pressure, but the result is affected by the degree of dissociation of fulvic acid; laser desorption Fourier transform ion cyclotron resonance mass spectrometry and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry are mass spectrometry techniques used to characterize fulvic acid samples, but may cause molecular fragmentation and / or adduct formation during ionization and detection; solution equilibrium method can directly determine the average molar mass of fulvic acid from aqueous solution, calculate equilibrium constant and reaction stoichiometry; ultrafiltration membrane separation technology estimates the molar mass range of fulvic acid by separating fulvic acid of different molar masses; UV-visible spectrophotometry and total organic carbon analysis indirectly infer the molar mass of fulvic acid by measuring the absorbance value and total organic carbon content of the solution. These methods have their own characteristics and limitations, but together they provide multiple ways to determine the molar mass of fulvic acid in water bodies, which helps to more comprehensively understand the chemical properties and environmental behavior of fulvic acid. 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 lake water based on three-dimensional fluorescence spectroscopy.

[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 lake water based on three-dimensional fluorescence spectroscopy, the method comprising the following steps:

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

[0007] (b) Preparation of fulvic acid test solution in lake water;

[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 lake water samples, adjust the lake water samples to a specific pH with strong alkali and strong acid, let them stand, filter the resulting filtrate and mark it as the filtered water sample;

[0013] a2) passing the filtered water sample through the resin column and discarding the effluent;

[0014] a3) Under nitrogen protection, the resin column is eluted with a strong alkaline solution, and the absorbance of the effluent is monitored in real time. The absorbance value of the effluent increases first and then decreases. When the absorbance value of the effluent is less than 0.1, the elution is stopped, and the effluent is combined and marked as the total eluent;

[0015] a4) under nitrogen protection, the total eluent is immediately acidified to a specific pH value, stirred, allowed to stand, and then filtered, and the filtrate is marked as a crude fulvic acid extract;

[0016] a5) adding hydrofluoric acid to the crude fulvic acid extract, stirring, standing, and then centrifuging to obtain a supernatant, which is marked as the silicon-free crude fulvic acid extract;

[0017] a6) passing the crude silicon-free fulvic acid extract through a resin column and discarding the effluent;

[0018] a7) After the crude extract of silicon-free fulvic acid is adsorbed on the resin column, the resin column is rinsed with 2 column volumes of deionized water and the effluent is discarded;

[0019] a8) Under nitrogen protection, rinse the resin column with 1 column volume of strong alkaline solution and 2 column volumes of deionized water in sequence, and collect the effluent;

[0020] a9) Under nitrogen protection, the collected effluent is passed through a hydrogen-type cation exchange resin saturated with hydrogen ions for ion exchange, and the final effluent obtained is a salt-free fulvic acid high-concentration solution;

[0021] a10) freeze-drying and grinding the salt-free fulvic acid high-concentration liquid to obtain fulvic acid solid powder.

[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) Weigh the filter membrane before and after filtration after drying, and mark the mass difference as m2;

[0025] 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.

[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, 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 F0 of the fulvic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0035] 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;

[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 b;

[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>2I0 and then 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 lake water 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 filter membranes used for filtration in steps a and b are both glass fiber filter membranes with a pore size of 0.22-0.7 μm, and the glass fiber filter membranes are calcined at 450°C-550°C for 5-8 hours before use. The glass fiber filter membranes are dried at 60°C-80°C for 8-12 hours before and after filtration, and cooled to room temperature in a drying dish.

[0050] Preferably, the fulvic acid solid powder obtained in step a10 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 1.0% (by dry weight), the fulvic acid solid powder is dissolved with 0.1mol / L hydrochloric acid, and steps a5-a10 are repeated until the ash content is less than 1.0%.

[0051] 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 a3 and a8 is between 0.01 and 0.1 M; the resin column in steps a2, a3, a6, a7 and a8 adopts one or more resin column species such as DAX-8 or XAD-7; the flow rate in steps a2 and a7 is 10 to 15 times the column volume per hour, and the flow rate in steps a3, a6, a8 flow rate is 5-10 column volumes per hour; in step a5, hydrofluoric acid is of a specific concentration, and the concentration range is between 0.1-0.5 mol / L; in step b, the fulvic acid concentration in the fulvic acid test solution 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.

[0052] 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.

[0053] Preferably, the pH value of the solution in step a is a constant value (error is ±0.1), and the pH value range is between 1-3; the pH value of the solution in steps b, c and d is a constant value (error is ±0.02), and the pH value range is between 5-7; the stirring time is 30 minutes; the standing time is 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; the fluorescence determinations in steps c and d are both carried out under the protection of an inert gas such as nitrogen, helium or argon.

[0054] Preferably, the cumulative volume of the acid and alkaline solutions added to adjust the pH value in step c is not greater than 1‰ of the volume of the fulvic acid solution to be tested in step c1; the cumulative volume of the acid and / or alkaline 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.

[0055] The beneficial effects of the present invention are embodied in:

[0056] (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.

[0057] (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.

[0058] (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.

[0059] (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.

[0060] (5) The method for determining the molar mass of fulvic acid by three-dimensional fluorescence spectroscopy provided by the present invention requires that a fulvic acid molecule is combined with a copper ion in the reaction system. When the copper ions cause further flocculation of the fulvic acid molecules, the scattering intensity increases sharply, so the present invention requires that I<2I0 in step d.

[0061] (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 three-dimensional fluorescence spectroscopy provided by the present invention. DETAILED DESCRIPTION

[0062] 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 creative work are within the scope of protection of the present invention.

[0063] Example 1

[0064] A method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy, the method comprising the following steps:

[0065] a: Extraction and purification of fulvic acid from lake water;

[0066] Collect a water sample from a lake, adjust the lake water sample to pH = 2.0 ± 0.1 with strong alkali and strong acid, and filter after standing. Pass the filtered water sample through the resin column, and under nitrogen protection, elute the resin column with 0.1M strong alkali solution, monitor the absorbance of the effluent in real time, and stop eluting when the absorbance value of the effluent is less than 0.1, and combine the effluent.

[0067] Under nitrogen protection, the total eluent is immediately acidified to pH = 2.0, stirred, allowed to stand, and then filtered to obtain a crude fulvic acid extract, and then hydrofluoric acid is added to the crude fulvic acid extract, stirred, allowed to stand, and then centrifuged to obtain a supernatant as a silicon-free fulvic acid crude extract, and passed through a resin column. After adsorption is completed, the resin column is rinsed with 2 times the column volume of deionized water, and the effluent is discarded;

[0068] Under nitrogen protection, the resin column is rinsed with 1 column volume of strong alkaline solution and 2 column volumes of deionized water in sequence, and the effluent is collected. At the same time, the collected effluent is ion exchanged through a hydrogen-type cation exchange resin saturated with hydrogen ions to finally obtain a salt-free fulvic acid high-concentration liquid. Finally, the salt-free fulvic acid high-concentration liquid is freeze-dried and ground to obtain a fulvic acid solid powder.

[0069] b: Preparation of fulvic acid test solution in lake water;

[0070] Weigh 100.5 mg of fulvic acid solid powder, dissolve it with 0.1M strong alkali, adjust the pH to 6.0±0.02 with strong acid and strong alkali solution, stir and keep the pH stable for 60 minutes, filter, add KClO4 solution to adjust the ionic strength, and mark it as fulvic acid test solution after the volume is fixed to 10L. The mass difference of the glass fiber membrane before and after filtration is 2.1 mg.

[0071] c: Selection of fluorescence peak position in titration experiment;

[0072] 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;

[0073] 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.

[0074] d: Fluorescence quenching titration of fulvic acid;

[0075] 1) Measure the prepared fulvic acid test solution;

[0076] 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;

[0077] 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;

[0078] 4) Determine the fluorescence light scattering intensity I0 of the fulvic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

[0079] 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;

[0080] 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;

[0081] 7) Determine the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment determined in step b;

[0082] 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;

[0083] 9) Repeat steps d5 to d8 until I>2I0 and then stop step d.

[0084] e: Calculation of molar mass of fulvic acid.

[0085] Use formula (1) to find F end :

[0086]

[0087] Use formula (2) to calculate the concentration of fulvic acid in lake water: L :

[0088]

[0089] Use formula (3) to calculate the molar mass M of fulvic acid:

[0090]

[0091] 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.

[0092] 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 = (100.5 mg - 2.1 mg) / 10 L = 9.84 mg / L.

[0093] Using the above method, the molar mass M of fulvic acid was obtained to be 1346±107 g / mol.

[0094] Example 2

[0095] A method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy, which is basically the same as Example 1, except that:

[0096] a: Extraction and purification of fulvic acid from lake water;

[0097] Lake water samples were collected and extracted and purified using the above method to obtain fulvic acid solid powder.

[0098] b: Preparation of fulvic acid test solution in lake water;

[0099] Prepare a fulvic acid test solution with a concentration of 10.0 mg / L.

[0100] c: Selection of fluorescence peak position in titration experiment;

[0101] 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;

[0102] 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.

[0103] d: Fluorescence quenching titration of fulvic acid;

[0104] 1) Measure the prepared fulvic acid test solution;

[0105] 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;

[0106] 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;

[0107] 4) Determine the fluorescence light scattering intensity I0 of the fulvic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

[0108] 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;

[0109] 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;

[0110] 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 b;

[0111] 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;

[0112] 9) Repeat steps d5 to d8 until I>2I0 and then stop step d.

[0113] Using the above method, the molar mass M of fulvic acid was obtained to be 1098±66 g / mol.

[0114] Example 3

[0115] A method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy, which is basically the same as Example 1, except that:

[0116] a: Extraction and purification of fulvic acid from lake water;

[0117] A water sample from a lake was collected and extracted and purified using the above method to obtain fulvic acid solid powder.

[0118] b: Preparation of fulvic acid test solution in lake water;

[0119] Prepare a fulvic acid test solution with a concentration of 10.0 mg / L.

[0120] c: Selection of fluorescence peak position in titration experiment;

[0121] 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;

[0122] 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.

[0123] d: Fluorescence quenching titration of fulvic acid;

[0124] 1) Measure the prepared fulvic acid test solution;

[0125] 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;

[0126] 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;

[0127] 4) Determine the fluorescence light scattering intensity I0 of the fulvic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

[0128] 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;

[0129] 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;

[0130] 7) Determine the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak of the titration experiment determined in step b;

[0131] 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;

[0132] 9) Repeat steps d5 to d8 until I>2I0 and then stop step d.

[0133] Using the above method, the molar mass M of fulvic acid was obtained to be 1466±53 g / mol.

[0134] Example 4

[0135] A method for determining the molar mass of tyrosine based on three-dimensional fluorescence spectroscopy, the method comprising the following steps:

[0136] a: Preparation of tyrosine test solution and background solution

[0137] 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.

[0138] 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.

[0139] b: Selection of fluorescence peak position in tyrosine titration experiment

[0140] 1) Measure the tyrosine test solution;

[0141] 2) Adjust the pH value of the tyrosine solution to 6, stir and keep the pH value stable for 60 minutes;

[0142] 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;

[0143] 4) Measure the background solution;

[0144] 5) Adjust the pH value of the background solution to 6, stir and keep the pH value stable for 60 minutes;

[0145] 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;

[0146] 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.

[0147] c: Fluorescence quenching titration of tyrosine

[0148] 1) Measure the tyrosine test solution;

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

[0150] 3) measuring the fluorescence intensity F0 of the tyrosine test solution at the fluorescence peak position of the titration experiment determined in step b, i.e., at an excitation wavelength of 350 nm and an emission wavelength of 440 nm;

[0151] 4) Determine the light scattering intensity I0 of the tyrosine test solution when the excitation wavelength and emission wavelength are both 400 nm;

[0152] 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;

[0153] 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;

[0154] 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 b;

[0155] 8) Determine the light scattering intensity I of the tyrosine test solution when the excitation wavelength and the emission wavelength are both 500 nm;

[0156] 9) Repeat step c from 5) to 8) until I>2I0 and stop step c.

[0157] d: Background solution fluorescence measurement

[0158] 1) Measure the background solution;

[0159] 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;

[0160] 3) measuring the fluorescence intensity Fr0 of the background solution at the fluorescence peak position of the titration experiment determined in step b;

[0161] 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 b.

[0162] e: Calculation of the molar mass of tyrosine

[0163] 1) Calculate F0 = F0' - Fr0' and calculate F = F' - Fr'

[0164] 2) Use formula (1) to calculate F end :

[0165]

[0166] 3) Use formula (2) to calculate the molar mass M of tyrosine:

[0167]

[0168] 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.

[0169] 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.

[0170] <![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

[0171] Wherein, the mass concentration of the tyrosine test solution in the above step c 1) is m=(52.79 mg-1.20 mg) / 10 L=5.16 mg / L.

[0172] F0, 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.

[0173] By using the three-dimensional fluorescence spectroscopy 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 molar mass of tyrosine is measured to be 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).

[0174] Example 5

[0175] A method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy is basically the same as Example 1, except that: 20.2 mg of the International Humic Acid Association standard (Suwannee River FA, 3S101F) is weighed, dissolved with 2 mL of 0.1 M potassium hydroxide strong alkaline solution, diluted to about 1 L with deionized water, filtered with a glass fiber membrane with a pore size of 0.45 μm, and after filtration, the volume is adjusted to 2 L with deionized water to simulate lake water for experiments.

[0176] a: Extraction and purification of fulvic acid from lake water;

[0177] Simulated lake water samples were collected, and fulvic acid solid powder was obtained by extraction and purification using the above method.

[0178] b: Preparation of fulvic acid test solution in lake water;

[0179] The concentration of fulvic acid test solution was 10.0 mg / L. The mass difference of the glass fiber membrane before and after filtration was 0.73 mg.

[0180] b: Selection of fluorescence peak position in titration experiment;

[0181] 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;

[0182] 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.

[0183] d: Fluorescence quenching titration of fulvic acid;

[0184] 1) Measure the prepared fulvic acid test solution;

[0185] 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;

[0186] 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;

[0187] 4) Determine the fluorescence light scattering intensity I0 of the fulvic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

[0188] 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;

[0189] 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;

[0190] 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 b;

[0191] 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;

[0192] 9) Repeat steps d5 to d8 until I>2I0 and then stop step d.

[0193] Using the above method, the molar mass M of fulvic acid was obtained to be 996.13 g / mol.

[0194] By using the three-dimensional fluorescence spectroscopy provided by the present invention, the pH value of the measured liquid during the titration, the concentration of the cumulatively added copper ions, the concentration of the measured liquid and other parameters were changed, and the molar mass of fulvic acid was measured to be 996.13 g / mol, which has an error of less than 5% compared with the actual molar mass of fulvic acid (1028 g / mol).

[0195] 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 lake water based on three-dimensional fluorescence spectroscopy, characterized in that: The method for determining the molar mass of fulvic acid in lake water by three-dimensional fluorescence spectroscopy comprises the following steps: (a) Extraction and purification of fulvic acid from lake water; (b) Preparation of fulvic acid test solution in lake water; (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. The method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy according to claim 1, characterized in that: The extraction and purification process of fulvic acid from lake water in step a is as follows: a1) Collect lake water samples, adjust the lake water samples to a specific pH with strong alkali and strong acid, filter the resulting filtrate after standing and mark it as the filtered water sample; a2) passing the filtered water sample through the resin column and discarding the effluent; a3) Under nitrogen protection, the resin column is eluted with a strong alkaline solution, and the absorbance of the effluent is monitored in real time. The absorbance value of the effluent increases first and then decreases. When the absorbance value of the effluent is less than 0.1, the elution is stopped, and the effluent is combined and marked as the total eluent; a4) under nitrogen protection, the total eluent is immediately acidified to a specific pH value, stirred, allowed to stand, and then filtered, and the filtrate is marked as a crude fulvic acid extract; a5) adding hydrofluoric acid to the crude fulvic acid extract, stirring, standing, and then centrifuging to obtain a supernatant, which is marked as the silicon-free crude fulvic acid extract; a6) passing the crude silicon-free fulvic acid extract through a resin column and discarding the effluent; a7) After the crude extract of silicon-free fulvic acid is adsorbed on the resin column, the resin column is rinsed with 2 column volumes of deionized water and the effluent is discarded; a8) Under nitrogen protection, rinse the resin column with 1 column volume of strong alkaline solution and 2 column volumes of deionized water in sequence, and collect the effluent; a9) Under nitrogen protection, the collected effluent is passed through a hydrogen ion saturated hydrogen type cation exchange resin for ion exchange, and the final effluent obtained is a salt-free fulvic acid high concentration solution; a10) freeze-drying and grinding the salt-free fulvic acid high-concentration liquid to obtain fulvic acid solid powder.

3. The method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy 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. The method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy 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 determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy 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 b; 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. The method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy 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 calculate F end : e2) Use formula (2) to calculate the concentration of fulvic acid in lake water C L : e3) Use 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. The method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy according to claim 3, characterized in that: The filter membranes used for filtration in steps a and b are both glass fiber filter membranes with a pore size of 0.22-0.7 μm, and the glass fiber filter membranes are calcined at 450° C.-550° C. for 5-8 hours before use. The glass fiber filter membranes are dried at 60° C.-80° C. for 8-12 hours before and after filtration, and cooled to room temperature in a drying dish.

8. The method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy according to claim 2, characterized in that: Take the fulvic acid solid powder obtained in step a10, dry it at 80-100°C for 20-24h, calcine it at 550°C for 5h, and then measure the ash content of the fulvic acid solid powder; if the ash content of the fulvic acid solid powder is greater than 1.0% (by dry weight), dissolve the fulvic acid solid powder with 0.1mol / L hydrochloric acid, and repeat steps a5-a10 until the ash content is less than 1.0%.

9. The method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy 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 any one of sodium hydroxide and potassium hydroxide or a mixture thereof; the concentration of the strong base in steps a3 and a8 is between 0.01-0.1M; the resin columns in steps a2, a3, a6, a7 and a8 are one or more resin columns such as DAX-8 or XAD-7; the flow rate of steps a2 and a7 is 10-15 times the column volume per hour, and the flow rate of steps a3, a6 and a8 is 5-10 times the column volume per hour; the hydrofluoric acid in step a5 is a specific concentration, and the concentration range is between 0.1-0.5mol / L; 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 5-20mg / 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 0.1-1000mmol / L.

10. The method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy 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.

11. The method for determining the molar mass of fulvic acid in lake water based on three-dimensional fluorescence spectroscopy according to claim 5, characterized in that: The pH value of the solution in step a is a constant (with an error of ±0.1), and the pH value range is between 1-3; the pH value of the solution in steps b, c and d is a constant (with an error of ±0.02), and the pH value range is between 5-7; the stirring time is 30 minutes; the standing time is 24 hours; maintaining a stable pH value means that the pH value of the solution changes by less than 0.02 pH units in not less than 20 minutes.

12. The method for measuring the molar mass of fulvic acid in lake water by three-dimensional fluorescence spectroscopy according to claim 5, characterized in that: The cumulative volume of the acid and alkaline solutions added to adjust the pH value in step c is not greater than 1‰ of the volume of the fulvic acid solution to be tested in step c1; the cumulative volume of the acid and / or alkaline 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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