Method for pre-enriching humic acid in lake water body and determining molecular weight

Abstract

The invention relates to the technical field of environment detection, in particular to a method for pre-enriching humic acid in a lake water body and determining molecular weight. The method comprises the following steps: (a) extracting and purifying humic acid in a lake water body; (b) preparing a lake water humic acid measured solution; (c) selecting a fluorescence peak position of a titration experiment; (d) fluorescence quenching titration of humic acid; and (e) calculating the molecular weight of humic acid. Tyrosine is used as a model compound and a humic acid standard sample for verification, and the accuracy and reliability of the method are proved.

Description

Technical Field

[0001] The invention relates to the technical field of environmental detection, and in particular to a method for pre-enrichment and molecular weight determination of humic acid in lake water. Background Art

[0002] Humic acid is a kind of natural organic macromolecular compound widely present in water bodies. They are formed by long-term decomposition and transformation of plant and animal residues under the action of microorganisms. These molecules have complex structures, with aromatic rings and fatty chains as the basic skeletons, and contain a variety of active functional groups, such as carboxyl, hydroxyl, carbonyl, etc. These functional groups make humic acid have strong adsorption, complexation, chelation and redox capabilities, and can interact with metal ions, organic pollutants, etc. in water bodies, affecting their migration, transformation and degradation processes. In water bodies, the migration and transformation of humic acid is mainly manifested as diffusion, and its migration ability increases with the increase of initial concentration. The migration behavior of humic acid in different media shows that they mainly show diffusion in fine sand, while they may undergo transformation in marlstone, being degraded by microorganisms and transformed into soluble microbial metabolites. This transformation not only affects the environmental behavior of humic acid itself, but may also affect other substances in water bodies. The impact of humic acid on the water environment is multifaceted. They can reduce the concentration of heavy metal ions in water bodies through adsorption and complexation, and reduce their toxicity and mobility. Humic acid can also adsorb organic pollutants through coordination exchange, hydrophobic interaction and hydrogen bonding, enhance their solubility and limit their migration in water bodies. In addition, humic acid has active photochemical properties, which can trigger free radical reactions, affect the photodegradation process of organic pollutants, and reduce the content of organic pollutants in water bodies through redox reactions. These characteristics make humic acid play an important role in water pollution prevention and control and environmental quality improvement, especially in reducing the environmental risks of heavy metals and organic pollutants. Therefore, humic acid has important application potential in water environment protection and restoration.

[0003] However, the complexity of the molecular structure of humic acid and the wide distribution of molecular weight (ranging from hundreds to hundreds of thousands) pose challenges to the accurate determination of its molecular weight. This heterogeneity and polydispersity limit the accuracy of traditional experimental techniques in studying the molecular weight of humic acid. At present, the methods for determining the molecular weight of humic acid in water bodies mainly include size exclusion chromatography, asymmetric field flow analysis, high performance liquid chromatography, ultraviolet-visible spectrophotometry, and XAD macroporous resin adsorption-total organic carbon coupling. Size exclusion chromatography is a chromatographic method that separates solute molecules according to the volume size in the mobile phase solution. By controlling the size of the gel pores, the macromolecules in the sample cannot enter the gel pores and are completely excluded, thereby achieving separation. Asymmetric field flow analysis is a general technology that takes into account both analytical and semi-preparative separations. It is suitable for a variety of analytes, including proteins, polymers, viruses, etc., and its detection size range is from 1nm to 10μm. High performance liquid chromatography analyzes humic acid samples through chromatographic modules and detectors to identify humic acid components with ultraviolet and fluorescence characteristic absorption peaks. UV-visible spectrophotometry can be used for qualitative analysis through the analysis of molecular UV-visible molecular absorption spectroscopy, and can be used for quantitative analysis based on the Lambert-Beer law. XAD macroporous resin adsorption-total organic carbon coupling method uses macroporous resin to adsorb humic acid, and then uses a total organic carbon analyzer to measure the total organic carbon value of the adsorbed humic acid, thereby analyzing the molecular weight of humic acid. These methods have their own characteristics and are suitable for different research purposes and conditions, and can provide important data for understanding the environmental behavior and ecological effects of humic acid. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for pre-enrichment and molecular weight determination of humic acid in lake water.

[0005] To achieve the above object, the technical solution of the present invention is implemented as follows: a method for pre-enrichment and molecular weight determination of humic acid in lake water, characterized in that the method for pre-enrichment and molecular weight determination of humic acid in lake water comprises the following steps:

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

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

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

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

[0010] (e) Calculation of molecular weight of humic acid.

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

[0012] a1) Collect lake water samples, filter them, adjust the lake water samples to a specific pH value with strong alkali and strong acid, stir them continuously, let them stand, and centrifuge the resulting solids to mark as crude humic acid solids;

[0013] a2) dissolving the mixture with a solution containing a strong base and a strong acid and strong base salt under nitrogen protection, and centrifuging at high speed to obtain a supernatant;

[0014] a3) acidifying the supernatant with hydrochloric acid to a specific pH value, stirring continuously and then allowing to stand, and centrifuging to obtain a solid which is marked as primary purified humic acid;

[0015] a4) adding strong acid and concentrated hydrofluoric acid solution to the first purified humic acid in sequence to a specific concentration, stirring continuously and then standing, centrifuging to obtain a solid marked as silicon-free humic acid;

[0016] a5) adding a strong acid to the silicon-free humic acid, stirring continuously, allowing to stand, and centrifuging to obtain a solid which is marked as secondary purified humic acid;

[0017] a6) adding a strong acid to the second purified humic acid, stirring continuously and then allowing to stand, and centrifuging to obtain a solid which is marked as the third purified humic acid;

[0018] a7) adding a strong acid to the tertiary humic acid, placing the mixture in a suction filtration device for filtration, continuously adding ultrapure water to the filter hole to wash the solid, and freeze-drying and grinding the obtained solid to obtain a humic acid solid powder.

[0019] Preferably, the preparation process of the humic acid test solution in step b is:

[0020] b1) Weighing humic acid solid powder m1, dissolving it with a strong base, adjusting it to a specific pH value with a strong acid and a strong base solution, the pH value range is between 4 and 9, stirring and keeping the pH stable, filtering, adding a salt solution to adjust the ionic strength, and fixing the volume to V, and marking it as the humic acid test solution;

[0021] b2) After drying, weigh the filter membrane before and after filtration respectively, and mark the mass difference as m2.

[0022] b3) The concentration of the humic acid solution is calculated by subtracting the amount of humic acid intercepted by the filter membrane m2 from the mass of humic acid added m1, and the total volume V of the humic acid solution, using the following formula (1):

[0023] Humic acid test solution concentration

[0024] Preferably, in step c, the process of selecting the fluorescence peak position of the titration experiment is as follows:

[0025] c1) Measure the humic acid test solution;

[0026] c2) adjusting the humic acid test solution to a specific pH value, stirring and maintaining a stable pH value;

[0027] c3) scanning the three-dimensional fluorescence spectrum of the humic acid test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0028] c4) After deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and emission wavelength at which the fluorescence intensity is the maximum in the three-dimensional fluorescence spectrum of the humic acid test solution are selected as the fluorescence peak position of the titration experiment.

[0029] Preferably, the step d comprises the following steps:

[0030] d1) measuring the humic acid test solution prepared in step b;

[0031] d2) adjusting the measured humic acid test solution to a specific pH value, stirring and maintaining a stable pH value;

[0032] d3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0033] d4) measuring the fluorescence light scattering intensity I0 of the humic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

[0034] 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;

[0035] d6) adjusting the pH value of the humic acid test solution to the same as that in step d2, stirring and maintaining the pH value stable;

[0036] d7) measuring the fluorescence intensity F value of the humic acid test solution at the fluorescence peak position of the titration experiment determined in step c;

[0037] d8) measuring the light scattering intensity I of the humic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

[0038] d9) Repeat steps d5 to d8 until I>2I0 and then stop step d.

[0039] Preferably, in step e, the specific process of calculating the molecular weight of humic acid is:

[0040] e1) Use formula (2) to calculate F end :

[0041]

[0042] e2) F end Substitute into formula (3) to calculate the concentration of humic acid in lake water: C L :

[0043]

[0044] e3) C L Substitute into formula (4) to obtain the molecular weight M of humic acid:

[0045]

[0046] Among them, C Q F is the concentration of the quenching substance solution added in step d5 during the titration process; end is the fluorescence intensity fitted by humic acid when titrated to saturation; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of humic acid in the test solution in step d1; C L The molar concentration of the humic acid solution prepared in step b; M is the molecular weight of humic acid.

[0047] Preferably, the error of the specific pH value in steps a1 and a3 is ±0.1, and the pH value range is between 1-3; the continuous stirring time in the continuous stirring and standing operation in step a is 15 minutes, and the standing time is not less than 24 hours; steps a2, a5, a6, and a7 make the solid-liquid ratio of the solution 1:10; the concentration of the strong acid used in steps a4, a5, a6, and a7 is a specific concentration, and the concentration range is 0.05-0.2 mol / L; the concentration of hydrofluoric acid in step a4 is a fixed value, and the concentration range is between 0.05-0.1 mol / L.

[0048] Preferably, after adding 100 ml of ultrapure water in step a7, the chloride ion content in the filtrate of step a7 is determined by the silver nitrate method. If chloride ions are present, 100 ml of ultrapure water is continuously added, and the chloride ion content in the filtrate is determined again until no chloride ions are detected in the filtrate;

[0049] Preferably, the humic acid solid powder obtained in step a7 is dried at 80-100°C for 20-24h, and then calcined at 550°C for 5h, and then the ash content of the humic acid solid powder is measured. If the ash content of the humic acid solid powder is greater than 1.0% (by dry weight), repeat steps a2-a7 until the ash content is less than 1.0%.

[0050] Preferably, the salt concentration in the humic acid test solution in step b is a specific value, and the concentration is between 0.1-1000mmol / L; the humic acid concentration in the humic acid test solution in step b is a specific value, and the concentration range should be controlled between 5-20mg / L.

[0051] Preferably, the cumulative volume of the acid and alkaline solutions added to adjust the pH value in step c is not greater than 1‰ of the volume of the humic acid solution tested in step c1.

[0052] Preferably, the cumulative volume of the acid and / or alkaline solution added for adjusting the pH value in step d is not greater than 1‰ of the volume of the background solution in step d1; the solution containing the quenching substance added in step d includes but is not limited to copper ions, mercury ions, etc.; the cumulative volume of the solution containing the quenching substance added during the titration process in step d is not greater than 1‰ of the volume of the humic acid solution to be tested in step c1.

[0053] Preferably, the pH value of the solution in steps b, c and d is a fixed value (with an error of ±0.02), and the pH value range is between 5-7; the concentration of the strong base in steps a2 and b1 is a specific value, between 0.05-0.2M; the salt used to adjust the ionic strength in steps a2 and b is any one or more of potassium perchlorate, sodium perchlorate, sodium chloride, potassium chloride and potassium nitrate solution.

[0054] 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.

[0055] Preferably, the strong acid is one of hydrochloric acid, perchloric acid, dilute nitric acid, dilute sulfuric acid or a mixture thereof; the strong base is one of sodium hydroxide and potassium hydroxide or a mixture thereof; maintaining the pH value stable means that the pH value of the solution changes by less than 0.02 pH units within no less than 30 minutes. The fluorescence determinations in steps c and d are both carried out under the protection of an inert gas such as nitrogen, helium or argon.

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

[0057] (1) The method for determining the molecular weight of humic acid provided by the present invention is stable, the measurement method is simple to operate, and the equipment requirements are low. In addition, the method results are proved to be accurate and reliable using model compounds of tyrosine molecules and humic acid standards.

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

[0059] (3) In the process of preparing the humic acid test solution provided by the present invention, a strong base is used to dissolve the humic acid solid, which can speed up the dissolution process and save the total measurement time. At the same time, deionized water can also be used to oscillate and dissolve the humic acid (generally the oscillation time is 12-24 hours), which reduces the impact on the test results, but the dissolution amount may not be enough to carry out the experiment.

[0060] (4) In the method for determining the molecular weight of humic acid provided by the present invention, controlling the pH value can not only ensure that humic acid can exist in the solution in a free form, but also avoid the situation that when the acidity is too strong, that is, when the pH value is less than 3, humic acid will exist in the form of acid, and when the pH value is less than 1, humic acid will precipitate. At the same time, when the alkalinity is too strong, that is, when the pH value is greater than 10, copper ions will combine with a large number of free hydroxyl groups in the solution, thereby affecting the determination results, thereby improving the accuracy of the test results.

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

[0062] (6) Tyrosine used in the present invention is an organic substance with a fixed molecular weight. When it combines with ions such as copper ions and mercury ions, fluorescence quenching occurs. Therefore, it can be used as a reference substance to test the reliability of the method for determining the molecular weight of humic acid by fluorescence quenching titration provided by the present invention. DETAILED DESCRIPTION

[0063] The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the comparative examples. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0064] Example 1

[0065] A method for pre-enrichment and molecular weight determination of humic acid in lake water, the method comprising the following steps:

[0066] a: Extraction and purification of humic acid from lake water

[0067] A water sample from a certain lake was collected, filtered, and then adjusted to pH = 1.0 ± 0.02 with strong alkali and strong acid. The crude humic acid solid was obtained by continuous stirring and standing, and then centrifuged. Under nitrogen protection, the solid was dissolved with solutions containing strong alkali and strong acid and strong base salts, respectively, and separated by high-speed centrifugation to obtain the supernatant.

[0068] The supernatant was acidified to pH 1.0±0.02 with hydrochloric acid, allowed to stand after continuous stirring, and centrifuged to obtain a purified humic acid. Strong acid and concentrated hydrofluoric acid were added in sequence to a concentration of 0.1 M, allowed to stand after continuous stirring, and centrifuged to obtain silicon-free humic acid.

[0069] Add strong acid to the silicon-free humic acid, keep stirring and let it stand, centrifuge to obtain secondary purified humic acid, add strong acid again, keep stirring and let it stand, centrifuge to obtain tertiary purified humic acid, finally add strong acid, place it in a suction filtration device for filtration, continuously add ultrapure water to the filter hole to wash the solid, and freeze-dry the obtained solid, grind it to obtain humic acid solid powder.

[0070] b: Preparation of the test solution of humic acid in lake water;

[0071] Weigh 111.19 mg of humic acid solid powder, dissolve it with 0.1M strong alkali, adjust the pH to 6.0±0.02 with strong acid and strong alkali solution, stir and keep the pH stable for 60 minutes, filter, add KClO4 solution to adjust the ionic strength, and adjust the volume to 10L to obtain the humic acid test solution. The mass difference of the glass fiber membrane before and after filtration is 5.2 mg.

[0072] c: Selection of the fluorescence peak position of the titration experiment; Measure the humic acid test solution, and adjust the humic acid test solution to pH = 6.0 ± 0.02, stir and keep the pH value stable for 60 minutes.

[0073] The three-dimensional fluorescence spectrum of the humic acid test solution was scanned, wherein the excitation wavelength and emission wavelength scanning range were both 200-600 nm. After deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and emission wavelength at which the fluorescence intensity was the maximum in the three-dimensional fluorescence spectrum of the humic acid test solution were selected as the fluorescence peak position of the titration experiment.

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

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

[0076] 2) Adjust the pH of the measured humic acid solution to 6.0±0.02, stir and keep the pH value stable for 60 minutes;

[0077] 3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

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

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

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

[0082] 8) Determine the light scattering intensity I of the humic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

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

[0084] e: Calculation of molecular weight of humic acid.

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

[0086]

[0087] F end Substitute into formula (2) to calculate the concentration of humic acid in lake water: C L :

[0088]

[0089] C L Substitute into formula (3) to find the molecular weight M of humic acid:

[0090]

[0091] Among them, C Q F is the concentration of the quenching substance solution added in step d5 during the titration process; end is the fluorescence intensity fitted by humic acid when titrated to saturation; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of humic acid in the test solution in step d1; C L The concentration of the substance in the humic acid test solution prepared in step b; M is the molecular weight of humic acid. By changing the concentration value C of the copper added Q Substituting the corresponding series of fluorescence intensity values ​​F (such as F1, F2, F3...Fn) into formula (2), C L , and then substitute it into formula (3) to obtain M, where the mass volume concentration of humic acid in step d) in the test liquid is m = (111.17 mg - 5.2 mg) / 10 L = 10.60 mg / L.

[0092] Using the above method, the molecular weight M of humic acid was obtained to be 3276±153 g / mol.

[0093] Example 2

[0094] A method for pre-enrichment and molecular weight determination of humic acid in lake water, which is basically the same as Example 1, except that:

[0095] a: Extraction and purification of humic acid from lake water

[0096] A water sample from a lake was collected and extracted and purified using the above method to obtain humic acid solid powder.

[0097] b: Preparation of the test solution of humic acid in lake water;

[0098] Prepare a humus test solution with a concentration of 10 mg / L.

[0099] c: Selection of fluorescence peak position in titration experiment;

[0100] 1) Measure the humic acid test solution, adjust the pH of the humic acid test solution to 4.0±0.02, stir and keep the pH value stable for 20 minutes.

[0101] 2) Scanning the three-dimensional fluorescence spectrum of the humic acid test solution, wherein the excitation wavelength and the emission wavelength scanning range are both 200-600 nm, and after deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and the emission wavelength at which the fluorescence intensity is the maximum in the three-dimensional fluorescence spectrum of the humic acid test solution are selected as the fluorescence peak position of the titration experiment.

[0102] d: Fluorescence quenching titration of humic acid

[0103] 1) Measure the prepared humic acid test solution;

[0104] 2) Adjust the pH of the measured humic acid solution to 4.0±0.02, stir and keep the pH value stable for 20 minutes;

[0105] 3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

[0106] 4) Determine the fluorescence light scattering intensity I0 of the humic acid test solution when the excitation wavelength and emission wavelength are both 500nm;

[0107] 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;

[0108] 6) Adjust the pH value of the humic acid test solution to the same as that in step d2, stir and keep the pH value stable for 20 minutes;

[0109] 7) Determine the fluorescence intensity F value of the humic acid test solution at the fluorescence peak position of the titration experiment determined in step c;

[0110] 8) Determine the light scattering intensity I of the humic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

[0111] 9) Repeat steps d5 to d8 until I>2I0 and then stop step d.

[0112] Using the above method, the molecular weight M of humic acid was obtained to be 2737±78.6 g / mol.

[0113] Example 3

[0114] A method for pre-enrichment and molecular weight determination of humic acid in lake water, which is basically the same as Example 1, except that:

[0115] a: Extraction and purification of humic acid from lake water

[0116] A water sample from a lake was collected and extracted and purified using the above method to obtain humic acid solid powder.

[0117] b: Preparation of the test solution of humic acid in lake water;

[0118] Prepare a humus test solution with a concentration of 10 mg / L.

[0119] c: Selection of fluorescence peak position in titration experiment;

[0120] 1) Measure the humic acid test solution, adjust the pH of the humic acid test solution to 8.2±0.02, stir and keep the pH value stable for 45 minutes.

[0121] 2) Scanning the three-dimensional fluorescence spectrum of the humic acid test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600nm,

[0122] After deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and emission wavelength at which the fluorescence intensity is the maximum in the three-dimensional fluorescence spectrum of the humic acid test solution are selected as the excitation wavelength and emission wavelength of the titration experiment.

[0123] d: Fluorescence quenching titration of humic acid

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

[0125] 2) The measured humic acid solution was adjusted to pH = 8.2 ± 0.02, stirred and the pH value was kept stable for 45 minutes;

[0126] 3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

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

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

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

[0131] 8) Determine the light scattering intensity I of the humic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

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

[0133] Using the above method, the molecular weight M of humic acid was obtained to be 2987±83.1 g / mol.

[0134] Example 4

[0135] A method for determining the molecular weight of tyrosine, 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 selected in step b;

[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 of 6 specified in step c 2), stirring and maintaining the pH value stable for 60 minutes;

[0154] 7) measuring the fluorescence intensity F' of the tyrosine test solution at the fluorescence peak position of the titration experiment determined in step c;

[0155] 8) Determine the light scattering intensity I of the tyrosine test solution when the excitation wavelength and the emission wavelength are both 400 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 6, 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 c;

[0161] 4) Add copper ions to make the concentration of the quenching substance solution in the background solution the same as the concentration of the quenching substance solution in step c5, repeat steps d2 to d4, and measure the fluorescence intensity Fr' at the fluorescence peak position of the titration experiment determined in step c.

[0162] e: Calculation of molecular weight 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 molecular weight M of tyrosine:

[0167]

[0168] Among them, C Q F is the total concentration of the quenching substance solution 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 molecular weight of tyrosine.

[0169] Different concentrations of Cu 2+ The corresponding measured fluorescence intensity value F is shown in Table 1.

[0170] Table 1 Different concentrations of Cu 2+ Corresponding to the measured fluorescence intensity value F result statistics

[0171]

[0172] Wherein, the mass concentration of the tyrosine test solution in step c 1) is m = (52.79 mg - 1.20 mg) / 10 L = 5.16 mg / L. The molecular weight of tyrosine calculated by formula (3) and (4) is 183.5 ± 10.0.

[0173] By using the fluorescence quenching titration method provided by the present invention, the pH value of the measured liquid, the concentration of the cumulatively added copper ions, the concentration of the measured liquid and other parameters are changed during the titration process, and the measured molecular weight of tyrosine is 178.1-185.4 g / mol, which has an error of less than 5% compared with the actual molecular weight of tyrosine (181.20 g / mol).

[0174] Example 5

[0175] A method for pre-enrichment and molecular weight determination of humic acid in lake water is basically the same as Example 1, except that: 27 mg of an International Humic Acid Association standard sample (Suwannee River HA, 3S101H) is weighed, dissolved with 2 mL of a 0.1 M potassium hydroxide strong alkali 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 fixed to 2 L with deionized water to simulate lake water for experiments.

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

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

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

[0179] The humic acid test solution was prepared with a concentration of 10.0 mg / L. The mass difference of the glass fiber membrane before and after filtration was 3.7 mg.

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

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

[0182] 2) Scanning the three-dimensional fluorescence spectrum of the humic acid test solution, wherein the excitation wavelength and the emission wavelength scanning range are both 200-600 nm, and after deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and the emission wavelength at which the fluorescence intensity is the maximum in the three-dimensional fluorescence spectrum of the humic acid test solution are selected as the fluorescence peak position of the titration experiment.

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

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

[0185] 2) Adjust the pH value of the measured humic acid solution to 6.0±0.02, stir and keep the pH value stable for 20 minutes;

[0186] 3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c;

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

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

[0190] 7) Determine the fluorescence intensity F value of the humic acid test solution at the fluorescence peak position of the titration experiment determined in step c;

[0191] 8) Determine the light scattering intensity I of the humic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm;

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

[0193] Using the above method, the molecular weight M of humic acid was obtained to be 1737 g / mol.

[0194] By using the fluorescence quenching titration method provided by the present invention, the pH value of the measured liquid, the concentration of the cumulatively added copper ions, the concentration of the measured liquid and other parameters are changed during the titration process, and the measured molecular weight of humic acid is 1737 g / mol, which is less than 5% compared with the actual molecular weight of humic acid (1813 g / mol).

[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 pre-enrichment and molecular weight determination of humic acid in lake water, characterized in that: The method for pre-enrichment and molecular weight determination of humic acid in lake water includes the following steps: (a) Extraction and purification of humic acid from lake water; (b) Preparation of the test solution of humic acid in lake water; (c) Selection of fluorescence peak position in titration experiment; (d) Fluorescence quenching titration of humic acid; (e) Calculation of molecular weight of humic acid.

2. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 1, characterized in that: The extraction and purification process of humic acid from lake water in step a is as follows: a1) Collect lake water samples, filter them, adjust the lake water samples to a specific pH value with strong alkali and strong acid, stir them continuously, let them stand, and centrifuge the resulting solids to mark as crude humic acid solids; a2) dissolving the mixture with a solution containing a strong base and a strong acid and strong base salt under nitrogen protection, and centrifuging at high speed to obtain a supernatant; a3) acidifying the supernatant with hydrochloric acid to a specific pH value, stirring continuously and then allowing to stand, and centrifuging to obtain a solid which is marked as primary purified humic acid; a4) adding strong acid and concentrated hydrofluoric acid solution to the first purified humic acid in sequence to a specific concentration, stirring continuously and then standing, centrifuging to obtain a solid marked as silicon-free humic acid; a5) adding a strong acid to the silicon-free humic acid, stirring continuously, allowing to stand, and centrifuging to obtain a solid which is marked as secondary purified humic acid; a6) adding a strong acid to the second purified humic acid, stirring continuously and then allowing to stand, and centrifuging to obtain a solid which is marked as the third purified humic acid; a7) adding a strong acid to the tertiary humic acid, placing the mixture in a suction filtration device for filtration, continuously adding ultrapure water to the filter hole to wash the solid, and freeze-drying and grinding the obtained solid to obtain a humic acid solid powder.

3. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 2, characterized in that: The preparation process of the humic acid test solution in step b is: b1) Weighing humic acid solid powder m1, dissolving it with a strong base, adjusting it to a specific pH value with a strong acid and a strong base solution, the pH value range is between 4 and 9, stirring and keeping the pH stable, filtering, adding a salt solution to adjust the ionic strength, and fixing the volume to V, and marking it as the humic acid test solution; b2) Weigh the filter membrane before and after filtration after drying, and mark the mass difference as m2; b3) The concentration of the humic acid solution is calculated by subtracting the amount of humic acid intercepted by the filter membrane m2 from the mass of humic acid added m1, and the total volume V of the humic acid solution, using the following formula (1): Humic acid test solution concentration 4. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 3, characterized in that: The process of selecting the fluorescence peak position of the titration experiment in step c is: c1) Measure the humic acid test solution; c2) adjusting the humic acid test solution to a specific pH value, stirring and maintaining a stable pH value; c3) scanning the three-dimensional fluorescence spectrum of the humic acid test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm; c4) After deducting the Rayleigh scattering and Raman scattering peaks, the excitation wavelength and emission wavelength at which the fluorescence intensity is the maximum in the three-dimensional fluorescence spectrum of the humic acid test solution are selected as the fluorescence peak position of the titration experiment.

5. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 4, characterized in that: Step d comprises the following steps: d1) measuring the humic acid test solution prepared in step b; d2) adjusting the measured humic acid test solution to a specific pH value, stirring and maintaining a stable pH value; d3) measuring the fluorescence intensity F0 of the humic acid test solution at the fluorescence peak position of the titration experiment selected in step c; d4) measuring the fluorescence light scattering intensity I0 of the humic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm; d5) adding a solution containing a quenching substance so that the concentration of the solution containing the quenching substance is between 0 and 1000 μmol / L; d6) adjusting the pH value of the humic acid test solution to the same as that in step d2, stirring and maintaining the pH value stable; d7) measuring the fluorescence intensity F value of the humic acid test solution at the fluorescence peak position of the titration experiment determined in step c; d8) measuring the light scattering intensity I of the humic acid test solution when the excitation wavelength and the emission wavelength are both 500 nm; d9) Repeat steps d5 to d8 until I>2I0 and then stop step d.

6. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 5, characterized in that: In step e, the specific process of calculating the molecular weight of humic acid is: e1) Use formula (2) to calculate F end : e2) F end Substitute into formula (3) to calculate the concentration of humic acid in lake water: C L : e3) C L Substitute into formula (4) to find the molecular weight M of humic acid: Among them, C Q F is the concentration of the quenching substance solution added in step d5 during the titration process; end is the fluorescence intensity fitted by humic acid when titrated to saturation; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of humic acid in the test solution in step d1; C L The molar concentration of the humic acid solution prepared in step b; M is the molecular weight of humic acid.

7. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 2, characterized in that: The error of the specific pH value in steps a1 and a3 is ±0.1, and the pH value range is between 1-3; the continuous stirring time in the continuous stirring and standing operation in step a is 15 minutes, and the standing time is not less than 24 hours; steps a2, a5, a6, and a7 make the solid-liquid ratio of the solution 1:10; the concentration of the strong acid used in steps a4, a5, a6, and a7 is a specific concentration, and the concentration range is 0.05-0.2 mol / L; the concentration of hydrofluoric acid in step a4 is a fixed value, and the concentration range is between 0.05-0.1 mol / L.

8. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 2, characterized in that: After adding 100 ml of ultrapure water in step a7, the chloride ion content in the filtrate of step a7 is determined by the silver nitrate method. If chloride ions are present, 100 ml of ultrapure water is continued to be added, and the chloride ion content in the filtrate is determined again until no chloride ions are detected in the filtrate.

9. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 2, characterized in that: The humic acid solid powder obtained in step a7 is dried at 80-100°C for 20-24h, and then calcined at 550°C for 5h. The ash content of the humic acid solid powder is then measured. If the ash content of the humic acid solid powder is greater than 1.0%, based on dry weight, steps a2-a7 are repeated until the ash content is less than 1.0%.

10. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 1, characterized in that: The salt concentration in the humic acid test solution in step b is a specific value, and the concentration is between 0.1-1000mmol / L; the humic acid concentration in the humic acid test solution in step b is a specific value, and the concentration range should be controlled between 5-20mg / L.

11. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 4, characterized in that: The cumulative volume of the acid and alkali solutions added to adjust the pH value in step c is not greater than 1‰ of the volume of the humic acid solution tested in step c1.

12. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 5, characterized in that: The cumulative volume of the acid and / or alkaline solution added to adjust the pH value in step d is not greater than 1‰ of the volume of the background solution in step d1; the solution containing the quenching substance added in step d includes but is not limited to copper ions, mercury ions, etc.; the cumulative volume of the solution containing the quenching substance added during the titration process in step d is not greater than 1‰ of the volume of the humic acid solution to be tested in step c1.

13. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 5, characterized in that: The pH value of the solution in steps b, c and d is a fixed value with an error of ±0.02, and the pH value range is between 5 and 7; the concentration of the strong base in steps a2 and b1 is a specific value, between 0.05 and 0.2 M; the salt used to adjust the ionic strength in step b is any one or more of potassium perchlorate, sodium perchlorate, sodium chloride, potassium chloride and potassium nitrate solution.

14. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 3, characterized in that: The filter membranes used for filtration in steps a and b are both glass fiber filter membranes with a pore size of 0.22-0.7 μm, and the glass fiber filter membranes are calcined at 450° C.-550° C. for 5-8 hours before use. The glass fiber filter membranes are dried at 60° C.-80° C. for 8-12 hours before and after filtration, and cooled to room temperature in a drying dish.

15. The method for pre-enrichment and molecular weight determination of humic acid in lake water according to claim 3, characterized in that: The strong acid is one of hydrochloric acid, perchloric acid, dilute nitric acid, dilute sulfuric acid or a mixture thereof; the strong base is one of sodium hydroxide and potassium hydroxide or a mixture thereof; maintaining a stable pH value means that the pH value of the solution changes by less than 0.02 pH units within no less than 20 minutes.

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