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

Abstract

The present application relates to the technical field of environmental detection, and particularly relates to a method for determining the molar mass of humic acid in lake water based on three-dimensional fluorescence spectrum, which comprises the following steps: (a) extraction and purification of humic acid in lake water; (b) preparation of a measured solution of humic acid in lake water; (c) selection of the fluorescence peak position in titration experiment; (d) fluorescence quenching titration of humic acid; and (e) calculation of the molar mass of humic acid. The method of the present application only uses conventional laboratory equipment and has low skill requirements for experiment operators. The accuracy and reliability of the method are verified by using model compounds such as tyrosine and standard samples of humic acid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of environmental detection, and particularly relates to a method for determining the molar mass of fulvic acid in a lake water body by three-dimensional fluorescence spectrum. BACKGROUND

[0002] Fulvic acid is a kind of low-molar-mass and high-water-solubility organic matter widely distributed in water bodies. Its structure is complex, mainly composed of carbon, oxygen and hydrogen elements, and contains a small amount of nitrogen, sulfur and phosphorus. Fulvic acid molecules contain condensed benzene rings, aliphatic rings and the like, and are connected to each other through functional groups such as carboxyl, hydroxyl and carbonyl. These functional groups endow fulvic acid with strong complexing and adsorbing capacity, so that it forms organic colloid with various metal oxides in water bodies, and migrates with water bodies, affecting the adsorption of phosphate in soil and the availability of phosphorus. The photochemical properties of fulvic acid enable it to undergo rapid photochemical reactions under ultraviolet light or sunlight radiation, affecting the migration and transformation of organic pollutants and heavy metals in water, and thus 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 and distribution of water body dissolved organic matter, and thus affects the ecological health and water quality of water bodies. Studies have shown that the content of fulvic acid has a strong influence on the decrease of soil phosphorus availability induced by microplastics, and increasing the content of fulvic acid can improve the availability of phosphorus by reducing the adsorption capacity of microplastics for phosphorus. The electron transfer capacity of fulvic acid is related to its degradation capacity for pollutants such as pentachlorophenol, and the reduction and conversion capacity of landfill fulvic acid is due to its electron transfer capacity, and these functions are beneficial to promote reductive dechlorination. Therefore, fulvic acid plays an important role in water bodies, and its structure, migration and transformation and environmental influence have a profound impact on the chemical behavior and ecological health of water environment.

[0003] There are various methods to determine the molar mass of humic acids in water bodies, including gel chromatography, vapor pressure osmometry, 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, and ultraviolet-visible spectrophotometry and total organic carbon analysis. Gel chromatography separates substances of different molar mass through a gel chromatography column to provide relative information on the molar mass distribution; vapor pressure osmometry determines the average molar mass based on osmotic pressure, but the results are affected by the degree of dissociation of humic acids; 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 humic acid samples, but may cause fragmentation and / or adduct formation of molecules during ionization and detection; the solution equilibrium method can directly determine the average molar mass of humic acids in aqueous solution, calculate the equilibrium constant and stoichiometric ratio; the ultrafiltration membrane separation technology estimates the molar mass range of humic acids by separating humic acids of different molar mass; and the ultraviolet-visible spectrophotometry and total organic carbon analysis indirectly infer the molar mass of humic acids by measuring the absorbance value and total organic carbon content of the solution. These methods have their own characteristics and limitations, but they collectively provide multiple ways to determine the molar mass of humic acids in water bodies, which helps to better understand the chemical properties and environmental behavior of humic acids. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method for determining the molar mass of humic acids in lake water based on three-dimensional fluorescence spectroscopy.

[0005] To achieve the above purpose, the technical scheme of the present application is as follows: a method for determining the molar mass of humic acids in lake water based on three-dimensional fluorescence spectroscopy, the method comprising the following steps:

[0006] (a) extraction and purification of humic acids in lake water;

[0007] (b) preparation of a measured solution of humic acids in lake water;

[0008] (c) selection of the fluorescence peak position for titration experiment;

[0009] (d) fluorescence quenching titration of humic acids;

[0010] (e) calculation of the molar mass of humic acids.

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

[0012] a1) collecting a lake water sample, adjusting the lake water sample to a specific pH with strong alkali and strong acid, and filtering the resulting filtrate after standing to mark it as a filtered water sample;

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

[0014] a3) under nitrogen protection, elute the resin column with strong base solution, monitor the absorbance of the effluent in real time, the absorbance of the effluent first increases and then decreases, when the absorbance of the effluent is less than 0.1, stop elution, and combine the effluent and mark as total eluate;

[0015] a4) under nitrogen protection, immediately acidify the total eluate to a specific pH value, stir, stand still, and then filter, and mark the filtrate as crude leonardite extract;

[0016] a5) add hydrofluoric acid to the crude leonardite extract, stir, stand still, and then centrifugal separation to obtain supernatant, and mark as silicon-free crude leonardite extract;

[0017] a6) pass the silicon-free crude leonardite extract through a resin column, and discard the effluent;

[0018] a7) after the silicon-free crude leonardite extract is adsorbed on the resin column, wash the resin column with 2 times column volume of deionized water, and discard the effluent;

[0019] a8) under nitrogen protection, sequentially wash the resin column with 1 times column volume of strong base solution and 2 times column volume of deionized water, and collect the effluent;

[0020] a9) under nitrogen protection, ion exchange the collected effluent through a hydrogen ion saturated hydrogen type cation exchange resin, and the final effluent is a salt-free leonardite high concentration solution;

[0021] a10) freeze-dry and grind the salt-free leonardite high concentration solution to obtain leonardite solid powder.

[0022] Preferably, the preparation process of the leonardite test solution in step b is as follows:

[0023] b1) weigh leonardite solid powder m1, dissolve with strong base, adjust to a specific pH value with strong acid and strong base solution, the pH value ranges from 4 to 9, filter after stirring and keeping the pH stable, add a salt solution to adjust the ionic strength, and mark as leonardite test solution after constant volume to V;

[0024] b2) dry the filter membrane before and after filtration, and weigh respectively, and mark the mass difference as m2;

[0025] b3) the concentration of the leonardite test solution is calculated by subtracting the amount of leonardite filtered by the filter membrane m2 from the mass of leonardite m1, and the total volume V of the leonardite test solution.

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

[0027] c1) measure the leonardite test solution;

[0028] c2) adjust the fulvic acid sample solution to a specific pH value, stir and keep the pH value stable;

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

[0030] c4) select the excitation wavelength and the emission wavelength at which the fluorescence intensity is the largest in the three-dimensional fluorescence spectrum of the fulvic acid sample solution after deducting the Rayleigh scattering and Raman scattering peaks, as the fluorescence peak position for titration experiment.

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

[0032] d1) measure the fulvic acid sample solution prepared in step b;

[0033] d2) adjust the measured fulvic acid sample solution to a specific pH value, stir and keep the pH value stable;

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

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

[0036] d5) add a solution containing a quencher, so that the concentration of the solution containing the quencher is between 0-1000 μmol / L;

[0037] d6) adjust the fulvic acid sample solution to the same pH value as in step d2, stir and keep the pH value stable;

[0038] d7) measure the fluorescence intensity F of the fulvic acid sample solution at the fluorescence peak position determined in step b for titration experiment;

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

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

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

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

[0043]

[0044] e2) calculate the amount-of-substance concentration C of the fulvic acid in the lake water body using formula (2) L :

[0045]

[0046] e3 The molar mass M of fulvic acid is calculated using formula (3):

[0047]

[0048] wherein C Q is the concentration of the solution containing the quenching substance added in step d5 during titration; F end is the normalized fluorescence intensity of fulvic acid at saturation; K is the conditional equilibrium constant; C L is the concentration of the substance in the fulvic acid sample prepared in step b; and M is the molar mass of fulvic acid.

[0049] Preferably, the filter membrane used in steps a and b is a glass fiber filter membrane with a pore size of 0.22-0.7 μm, and the glass fiber filter membrane is calcined at 450-550 °C for 5-8 h before use. The glass fiber filter membrane is dried at 60-80 °C for 8-12 h 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 20-24 h, then calcined at 550 °C for 5 h, and then the ash content of the fulvic acid solid powder is determined. 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 0.1 mol / L hydrochloric acid, and the 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 one of sodium hydroxide and potassium hydroxide, or a mixture thereof; the concentration of the strong base in steps a3 and a8 is 0.01-0.1 M; the resin column used in steps a2, a3, a6, a7, and a8 is one or more of DAX-8 or XAD-7 resin columns; the flow rate in steps a2 and a7 is 10-15 times the column volume per hour, and the flow rate in steps a3, a6, and a8 is 5-10 times the column volume per hour; the concentration of the hydrofluoric acid in step a5 is a specific value, and the concentration range is 0.1-0.5 mol / L; the concentration of fulvic acid in the fulvic acid sample in step b is a specific value, and the concentration range should be controlled to be 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 sample 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 and the like; the cumulative volume of the solution containing the quenching substance added during the titration process in step d is not more than 1‰ of the volume of the furfuranic acid measured solution in step c1.

[0053] Preferably, the pH value of the solution in step a is a constant value (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 value (with an error of ±0.02), and the pH value range is between 5-7; the stirring time is 30 min; the standing time is 24 h; the pH value is kept stable, that is, the pH value of the solution changes by less than 0.02 pH units within not less than 20 min; the fluorescence determination in steps c and d is carried out under the protection of inert gases such as nitrogen, helium or argon.

[0054] Preferably, the cumulative volume of the acid and base solution added for adjusting the pH value in step c is not more than 1‰ of the volume of the furfuranic acid measured solution in step c1; the cumulative volume of the acid and / or base solution added for adjusting the pH value in step d is not more than 1‰ of the volume of the background solution in step d1.

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

[0056] (1) The method for determining the molar mass of furfuranic acid provided by the present application is stable, the measurement method is simple to operate, the equipment requirement is low, and the method is proved to be accurate and reliable by using tyrosine molecules as model compounds and furfuranic acid standards.

[0057] (2) The salt used in the present application can provide a certain ionic strength during the fluorescence determination of furfuranic acid, and the concentration and ion species have basically negligible influence on the determination results, thereby improving the detection effect.

[0058] (3) In the preparation process of the furfuranic acid measured solution provided by the present application, the furfuranic acid solid is dissolved with a strong base, which can speed up the dissolution process, save the total determination time, and also can use deionized water to oscillate and dissolve the furfuranic acid (generally, the oscillation time is 12-24 h), thereby reducing the influence on the detection results.

[0059] (4) In the method for determining the molar mass of furfuranic acid provided by the present application, the pH value is controlled, which can not only ensure that the furfuranic acid exists in the form of free acid in the solution, avoid the phenomenon that the furfuranic acid exists in the form of acid when the acidity is too strong, that is, the pH value is less than 3, and also avoid the phenomenon that the copper ions combine with a large number of free hydroxyl groups in the solution when the alkalinity is too strong, that is, the pH value is greater than 10, thereby affecting the determination results, thereby improving the accuracy of the detection results.

[0060] (5) The method for determining the molar mass of fulvic acid using three-dimensional fluorescence spectroscopy provided by this invention requires that one fulvic acid molecule binds to one copper ion in the reaction system. When the copper ion causes further flocculation of the fulvic acid molecule, it leads to a sharp increase in scattering intensity. Therefore, this invention requires that I < 2I0 in step d.

[0061] (6) Tyrosine used in this invention is an organic compound with a fixed molar mass. When it binds with copper ions, it will undergo fluorescence quenching. Therefore, it can be used as a reference standard to further verify the reliability of the method for determining the molar mass of fulvic acid by three-dimensional fluorescence spectroscopy provided by this invention. Detailed Implementation

[0062] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments and comparative examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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] Water samples were collected from a lake and adjusted to pH 2.0 ± 0.1 using strong alkali and strong acid. After settling, the samples were filtered. The filtered water samples were then passed through a resin column, and under nitrogen protection, the column was eluted with a 0.1 M strong alkali solution. The absorbance of the effluent was monitored in real time. When the absorbance of the effluent was less than 0.1, elution was stopped, and the effluents were combined.

[0067] Under nitrogen protection, the total eluent was immediately acidified to pH 2.0, stirred, allowed to stand, and then filtered to obtain crude fulvic acid extract. Hydrofluoric acid was then added to the crude fulvic acid extract, stirred, allowed to stand, and centrifuged to obtain the supernatant, which was the silica-free crude fulvic acid extract. After adsorption was completed, the resin column was rinsed with 2 column volumes of deionized water and the eluent was discarded.

[0068] Under nitrogen protection, the resin column was rinsed sequentially with 1 column volume of strong alkali solution and 2 column volumes of deionized water. The effluent was collected and then passed through a hydrogen-saturated hydrogen-form cation exchange resin for ion exchange, ultimately yielding a high-concentration salt-free fulvic acid solution. Finally, the high-concentration salt-free fulvic acid solution was freeze-dried and ground to obtain fulvic acid solid powder.

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

[0070] 100.5 mg of fulvic acid solid powder was weighed and dissolved in 0.1 M strong alkali. The pH was adjusted to 6.0 ± 0.02 with strong acid and strong alkali solutions. After stirring and maintaining pH stability for 60 min, the mixture was filtered. KClO4 solution was added to adjust the ionic strength, and the volume was brought to 10 L. This solution was then labeled as the fulvic acid test solution. The mass difference of the glass fiber membrane before and after filtration was 2.1 mg.

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

[0072] Measure the fulvic acid test solution and adjust the pH of the fulvic acid test solution to 6.0 ± 0.02. Stir and keep the pH stable for 60 min.

[0073] The three-dimensional fluorescence spectrum of the fulvic acid test solution was scanned, with the excitation and emission wavelengths scanning range of 200-600 nm. After subtracting the Rayleigh scattering and Raman scattering peaks, the excitation and emission wavelengths at which the fluorescence intensity was at its maximum in the three-dimensional fluorescence spectrum of the fulvic acid test solution were selected as the positions of the fluorescence peaks in the titration experiment.

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

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

[0076] 2) Adjust the measured fulvic acid test solution to pH = 6.0 ± 0.02, stir and keep the pH stable for 60 min;

[0077] 3) Measure the fluorescence intensity F0 of the fulvic acid test solution at the fluorescence peak position selected in step c.

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

[0079] 5) Add a solution containing the quenching substance, so that the concentration of the solution containing the quenching substance is between 0-1000 μmol / L;

[0080] 6) Adjust the pH of the fulvic acid test solution to be the same as that in step d2, stir and keep the pH stable;

[0081] 7) Measure the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak determined in step b.

[0082] 8) Measure the light scattering intensity I of the fulvic acid test solution when both the excitation and emission wavelengths are 500 nm;

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

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

[0085] F can be obtained using formula (1) end :

[0086]

[0087] The concentration C of fulvic acid in the lake water can be calculated using formula (2). L :

[0088]

[0089] The molar mass M of fulvic acid can be calculated using formula (3):

[0090]

[0091] Among them, C Q F represents the concentration of the solution containing the quenching substance added in step c3 of the titration process. end The normalized fluorescence intensity fitted to fulvic acid at titration saturation; K is the conditional equilibrium constant; C L M represents the molar concentration of fulvic acid in the test solution prepared in step b; M is the molar mass of fulvic acid.

[0092] The concentration C of added copper will be changed. Q Substituting the corresponding series of fluorescence intensity values ​​F (e.g., F1, F2, F3…Fn) into formula (2) yields C. L Then, by substituting into formula (3), M can be calculated, where the mass volume concentration of fulvic acid in step d1) in the test solution is m = (100.5mg - 2.1mg) / 10L = 9.84mg / L.

[0093] Using the above method, the molar mass M of fulvic acid was determined 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 is basically the same as in Example 1, except that:

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

[0097] Lake water samples were collected, and fulvic acid solid powder was obtained by extraction and purification using the above method.

[0098] b: Preparation of the 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 the position of the fluorescence peak in the titration experiment;

[0101] 1) Measure the fulvic acid test solution and adjust the pH of the fulvic acid test solution to 4.0±0.01, stir and keep the pH stable for 20 min;

[0102] 2) Scan the three-dimensional fluorescence spectrum of the fulvic acid test solution. The excitation and emission wavelengths are both in the range of 200-600 nm. After subtracting the Rayleigh scattering and Raman scattering peaks, select the excitation and emission wavelengths with the highest fluorescence intensity in the three-dimensional fluorescence spectrum of the fulvic acid test solution as the positions of the fluorescence peaks in the titration experiment.

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

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

[0105] 2) Adjust the measured fulvic acid test solution to pH = 4.0 ± 0.01, stir and keep the pH stable for 20 min;

[0106] 3) Measure the fluorescence intensity F0 of the fulvic acid test solution at the fluorescence peak position selected in step c.

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

[0108] 5) Add a solution containing the quenching substance, so that the concentration of the solution containing the quenching substance is between 0-1000 μmol / L;

[0109] 6) Adjust the pH of the fulvic acid test solution to be the same as that in step d2, stir and keep the pH stable for 20 min;

[0110] 7) Measure the fluorescence intensity F value of the fulvic acid test solution at the position of the fluorescence peak determined in step b.

[0111] 8) Measure the light scattering intensity I of the fulvic acid test solution when both the excitation and emission wavelengths are 500 nm;

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

[0113] Using the above method, the molar mass M of fulvic acid was determined 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 is basically the same as in Example 1, except that:

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

[0117] A water sample was collected from a lake, and fulvic acid solid powder was obtained by extraction and purification using the method described above.

[0118] b: Preparation of the 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 the position of the fluorescence peak in the titration experiment;

[0121] 1) Measure the fulvic acid test solution and adjust the pH of the fulvic acid test solution to 8.2±0.02, stir and keep the pH stable for 45 min;

[0122] 2) Scan the three-dimensional fluorescence spectrum of the fulvic acid test solution. The excitation and emission wavelengths are both in the range of 200-600 nm. After subtracting the Rayleigh scattering and Raman scattering peaks, select the excitation and emission wavelengths at which the fluorescence intensity is the highest in the three-dimensional fluorescence spectrum of the fulvic acid test solution as the excitation and emission wavelengths for the titration experiment.

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

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

[0125] 2) Adjust the measured fulvic acid test solution to pH = 8.2 ± 0.02, stir and keep the pH stable for 45 min;

[0126] 3) Measure the fluorescence intensity F0 of the fulvic acid test solution at the fluorescence peak position selected in step c.

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

[0128] 5) Add a solution containing the quenching substance, so that the concentration of the solution containing the quenching substance is between 0-1000 μmol / L;

[0129] 6) Adjust the pH of the fulvic acid test solution to be the same as that in step d2, stir and keep the pH stable for 20 min;

[0130] 7) Measure the fluorescence intensity F of the fulvic acid test solution at the position of the fluorescence peak determined in step b.

[0131] 8) Measure the light scattering intensity I of the fulvic acid test solution when both the excitation and emission wavelengths are 500 nm;

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

[0133] Using the above method, the molar mass M of fulvic acid was determined 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, comprising the following steps:

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

[0137] Weigh 52.79 mg of solid tyrosine sample, dissolve it in deionized water, filter it through a glass fiber membrane with a pore size of 0.45 μm, and prepare a 1 L concentrated tyrosine solution. Filter the concentrated tyrosine 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 concentrated tyrosine solution and dissolve it. Make up the volume to 10 L with deionized water to obtain the tyrosine test solution.

[0138] Weigh 138.55 g of potassium perchlorate solid sample, dissolve it in deionized water, filter it through a glass fiber membrane with a pore size of 0.45 μm, and prepare a 10 L background solution.

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

[0140] 1) Measure the tyrosine test solution;

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

[0142] 3) Scan the three-dimensional fluorescence spectrum of the tyrosine test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0143] 4) Measure the background solution;

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

[0145] 6) Scan the three-dimensional fluorescence spectrum of the background solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0146] 7) Subtract the three-dimensional fluorescence spectrum data of the background solution from the obtained three-dimensional fluorescence spectrum data of the tyrosine test solution, and set the Raman and Rayleigh scattering intensities to zero. Determine the excitation and emission wavelengths at which the fluorescence intensity is maximum, and use them as the excitation and emission wavelengths for fluorescence quenching titration, which are 350 nm and 445 nm, respectively.

[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 maintain the pH value for 60 min;

[0150] 3) Measure the fluorescence intensity F0 of the tyrosine test solution at the position of the fluorescence peak determined in step b, i.e., when the excitation wavelength is 350 nm and the emission wavelength is 440 nm.

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

[0152] 5) Add a solution containing the quenching substance, so that the concentration of the solution containing the quenching substance is between 0-1000 μmol / L;

[0153] 6) Adjust the pH of the tyrosine test solution to the value specified in step c2), stir and keep the pH stable for 60 min;

[0154] 7) Measure the fluorescence intensity F of the tyrosine test solution at the position of the fluorescence peak determined in step b.

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

[0156] 9) Repeat steps 5) to 8) of c until I>2I0, then stop step c.

[0157] d: Background solution fluorescence measurement

[0158] 1) Measure the background solution;

[0159] 2) Adjust the pH of the background solution to be the same as in step c, stir and keep the pH stable for 60 minutes;

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

[0161] 4) Add a solution containing a quenching substance, and when the concentration of the quenching substance in the background solution is the same as the concentration in step c (5), repeat steps d (2) to 4), and measure the fluorescence intensity Fr at the position of the fluorescence peak of the titration experiment determined in step b.

[0162] e: Molar mass calculation 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) Calculate the molar mass M of tyrosine using formula (2):

[0167]

[0168] Among them, C Q F represents the total concentration of the solution containing the quenching substance added in step c3 of the titration process; end α is the fluorescence intensity fitted by tyrosine at titration saturation; K is a constant greater than 0; 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 (subtracting blank) are shown in Table 1. Table 1: Cu at different concentrations 2+ Statistical analysis of the corresponding measured fluorescence intensity values ​​F (excluding blank)

[0170] Cu 2+ Concentration C Q (mol / L) Tyrosine fluorescence intensity values 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] In step c) above, the mass concentration of the tyrosine test solution 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 Substituting the fluorescence intensity value F into formula (1), the molar mass of tyrosine is calculated to be 183.5 ± 10.0.

[0173] Using the three-dimensional fluorescence spectroscopy method provided by this invention, by changing parameters such as the pH value of the test solution, the concentration of cumulatively added copper ions, and the concentration of the test solution during the titration process, the measured molar mass of tyrosine is 178.1-185.4 g / mol, and the error is less than 5% compared with the actual molar mass of tyrosine (181.20 g / mol).

[0174] Example 5

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

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

[0177] Fulvic acid solid powder was obtained by collecting water samples from a simulated lake and extracting and purifying them using the methods described above.

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

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

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

[0181] 1) Measure the fulvic acid test solution and adjust the pH of the fulvic acid test solution to 6.0±0.02, stir and keep the pH stable for 20 min;

[0182] 2) Scan the three-dimensional fluorescence spectrum of the fulvic acid test solution. The excitation and emission wavelengths are both in the range of 200-600 nm. After subtracting the Rayleigh scattering and Raman scattering peaks, select the excitation and emission wavelengths with the highest fluorescence intensity in the three-dimensional fluorescence spectrum of the fulvic acid test solution as the positions of the fluorescence peaks in the titration experiment.

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

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

[0185] 2) Adjust the measured fulvic acid test solution to pH = 6.0 ± 0.02, stir and keep the pH stable for 20 min;

[0186] 3) Measure the fluorescence intensity F0 of the fulvic acid test solution at the fluorescence peak position selected in step c.

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

[0188] 5) Add a solution containing the quenching substance, so that the concentration of the solution containing the quenching substance is between 0-1000 μmol / L;

[0189] 6) Adjust the pH of the fulvic acid test solution to be the same as that in step d2, stir and keep the pH stable for 20 min;

[0190] 7) Measure the fluorescence intensity F value of the fulvic acid test solution at the position of the fluorescence peak determined in step b.

[0191] 8) Measure the light scattering intensity I of the fulvic acid test solution when both the excitation and emission wavelengths are 500 nm;

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

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

[0194] Using the three-dimensional fluorescence spectroscopy method provided by this invention, by changing parameters such as the pH value of the test solution, the concentration of the cumulative added copper ions, and the concentration of the test solution during the titration process, the measured molar mass of fulvic acid is 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 substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the molar mass of fulvic acid in a lake water body based on three-dimensional fluorescence spectroscopy, characterized in that, The method for determining the molar mass of fulvic acid in a lake water body by three-dimensional fluorescence spectrum comprises the following steps: (a) fulvic acid extraction and purification of the lake water body; (b) preparation of the fulvic acid sample solution of the lake water body; The preparation process of the fulvic acid sample solution in step b is as follows: b1) weigh the fulvic acid solid powder m1, dissolve it with a strong base, adjust the pH value of the solution with a strong acid and a strong base solution, the pH value range is 4-9, filter after stirring and keeping the pH value stable, add a salt solution to adjust the ionic strength, and mark as the fulvic acid sample solution after constant volume V; b2) dry the filter membrane before and after filtration, respectively, and mark the mass difference as m2; b3) the concentration of the fulvic acid sample solution is calculated by subtracting the amount of fulvic acid intercepted by the filter membrane m2 from the mass of the added fulvic acid m1, and dividing by the total volume V of the fulvic acid sample solution; (c) selection of the fluorescence peak position in the titration experiment; The selection process of the fluorescence peak position in the titration experiment in step c is as follows: c1) measure the fulvic acid sample solution; c2) adjust the pH value of the fulvic acid sample solution, stir and keep the pH value stable; c3) scan the three-dimensional fluorescence spectrum of the fulvic acid sample 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 the maximum fluorescence intensity in the three-dimensional fluorescence spectrum of the fulvic acid sample solution are selected as the fluorescence peak position in the titration experiment; (d) fluorescence quenching titration of fulvic acid; Step d comprises the following steps: d1) measure the fulvic acid sample solution prepared in step b; d2) adjust the pH value of the measured fulvic acid sample solution, stir and keep the pH value stable; d3) measure the fluorescence intensity F0 of the fulvic acid sample solution at the fluorescence peak position selected in step c; d4) measure the fluorescence light scattering intensity I0 of the fulvic acid sample solution when the excitation wavelength and emission wavelength are both 500 nm; d5) add a solution containing a quenching substance, so that the concentration of the solution containing the quenching substance is between 0-1000 µmol / L; d6) adjust the pH value of the fulvic acid sample solution to be the same as that in step d2, stir and keep the pH value stable; d7) measure the fluorescence intensity F of the fulvic acid sample solution at the fluorescence peak position determined in step b; d8) measure the light scattering intensity I of the fulvic acid sample solution when the excitation wavelength and emission wavelength are both 500 nm; d9) repeat steps d5 to d8 until I>2I0, then stop step d; (e) calculation of the molar mass of fulvic acid; In step e, the specific process of calculating the molar mass of fulvic acid is as follows: e1) F is calculated using equation (1) end : (1) e2) use formula (2) to calculate the molar mass M of fulvic acid in the lake water body: (2) wherein C Q is the concentration of the solution containing the quenching substance added in step d5 of the titration process; F end is the normalized fluorescence intensity fitted out for furilic acid at the end of the titration; a is a constant greater than 0; m is the mass by volume concentration of furilic acid in the solution in which it is measured in step cl; K is the conditional equilibrium constant; M is the molar mass of furilic acid.

2. The method for determining the molar mass of fulvic acid in a lake water body based on three-dimensional fluorescence spectroscopy according to claim 1, characterized in that, Step a, the extraction and purification process of fulvic acid in the lake water body is as follows: a1) collect the lake water body sample, adjust the pH of the lake water body sample with a strong base and a strong acid, and filter the obtained filtrate to mark as the filtered water body sample; a2) make the filtered water body sample flow through the resin column, and discard the effluent; a3) Eluting the resin column with a strong base solution under nitrogen protection, monitoring the absorbance of the effluent in real time, the absorbance of the effluent first increases and then decreases, when the absorbance of the effluent is less than 0.1, stop eluting, and the effluent is marked as total eluate; a4) Immediately acidifying the total eluate under nitrogen protection, stirring, standing and filtering, and the filtrate is marked as crude furfuranic acid extract; a5) Adding hydrofluoric acid to the crude furfuranic acid extract, stirring, standing and centrifuging to obtain the supernatant, which is marked as silicon-free crude furfuranic acid extract; a6) Passing the silicon-free crude furfuranic acid extract through a resin column, and discarding the effluent; a7) After the silicon-free crude furfuranic acid extract is adsorbed on the resin column, washing the resin column with 2 column volumes of deionized water, and discarding the effluent; a8) Washing the resin column with 1 column volume of strong base solution and 2 column volumes of deionized water under nitrogen protection, and collecting the effluent; a9) Passing the collected effluent through a hydrogen ion saturated hydrogen type cation exchange resin under nitrogen protection to exchange ions, and the final effluent is the high-concentration salt-free furfuranic acid solution; a10) Freeze-drying and grinding the high-concentration salt-free furfuranic acid solution to obtain furfuranic acid solid powder.

3. The method for determining the molar mass of fulvic acid in a lake water body based on three-dimensional fluorescence spectroscopy according to claim 2, characterized in that, The filter membranes used in steps a and b are glass fiber filter membranes with a pore size of 0.22-0.7 µm, and the glass fiber filter membranes are calcined at 450-550 °C for 5-8 h before use. The glass fiber filter membranes are dried at 60-80 °C for 8-12 h before and after filtration, and cooled to room temperature in a drying dish.

4. The method for determining the molar mass of fulvic acid in a lake water body based on three-dimensional fluorescence spectroscopy according to claim 2, characterized in that, The furfuranic acid solid powder obtained in step a10 is dried at 80-100 °C for 20-24 h, then calcined at 550 °C for 5 h, and the ash content of the furfuranic acid solid powder is determined. When the ash content of the furfuranic acid solid powder is greater than 1.0% by dry weight, the furfuranic acid solid powder is dissolved with 0.1 mol / L hydrochloric acid, and the steps a5-a10 are repeated until the ash content is less than 1.0%.

5. The method for determining the molar mass of fulvic acid in a lake water body 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 one of sodium hydroxide and potassium hydroxide or a mixture thereof; the concentration of the strong base in steps a3 and a8 is 0.01-0.1 M; the resin column in steps a2, a3, a6, a7 and a8 is one or both of DAX-8 or XAD-7 resin columns; the flow rate in steps a2 and a7 is 10-15 column volumes per hour, and the flow rate in steps a3, a6 and a8 is 5-10 column volumes per hour; the concentration of hydrofluoric acid in step a5 is 0.1-0.5 mol / L; the concentration of furfuranic acid in the furfuranic acid test solution in step b should be controlled between 5-20 mg / L; and the salt used to adjust the ionic strength in step b is any one or more of potassium perchlorate, sodium perchlorate, sodium chloride, potassium chloride and potassium nitrate solution, and the salt concentration in the furfuranic acid test solution is 0.1-1000 mmol / L.

6. The method for determining the molar mass of fulvic acid in a lake water body based on three-dimensional fluorescence spectroscopy according to claim 5, characterized in that, The solution containing the quenching substance added in step d includes copper ions or mercury ions; and the volume of the solution containing the quenching substance added during the titration process in step d is not more than 1‰ of the volume of the furfuranic acid test solution in step c1.

7. The method for determining the molar mass of fulvic acid in a lake water body based on three-dimensional fluorescence spectroscopy according to claim 6, characterized in that, The pH value of the solution in step a is a constant value, 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 value, with an error of ±0.02, and the pH value range is between 5-7; the stirring time is 30 min; the standing time is 24 h; and keeping the pH value stable means that the solution pH value changes less than 0.02 pH value units within not less than 20 min.

8. The method for determining the molar mass of humic acid in a lake water body by three-dimensional fluorescence spectroscopy according to claim 7, characterized in that, The cumulative volume of the acid and base solution added for adjusting the pH value in step c is not more than 1‰ of the volume of the fulvic acid test solution in step c1; and the cumulative volume of the acid and / or base solution added for adjusting the pH value in step d is not more than 1‰ of the volume of the background solution in step d1.

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