Method for pre-enrichment and molecular weight determination of humic acid in lake water body

Abstract

The present application relates to the technical field of environmental detection, and more particularly to a method for pre-enrichment and molecular weight determination of humic acid in lake water, which comprises the following steps: (a) extraction and purification of humic acid in lake water; (b) preparation of a humic acid sample 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 molecular weight of humic acid. The accuracy and reliability of the method are verified by using tyrosine as a model compound and humic acid standard samples.

Description

Technical Field

[0001] This invention relates to the field of environmental testing technology, and in particular to a method for pre-enriching and determining the molecular weight of humic acid in lake water. Background Technology

[0002] Humic acids are a class of naturally occurring organic macromolecules widely found in water bodies. They are formed from the long-term decomposition and transformation of plant and animal remains by microorganisms. These molecules have complex structures, with aromatic rings and aliphatic chains as the basic framework, and contain various active functional groups, such as carboxyl, hydroxyl, and carbonyl groups. These functional groups give humic acids strong adsorption, complexation, chelation, and redox capabilities, enabling them to interact with metal ions and organic pollutants in water bodies, influencing their migration, transformation, and degradation processes. In water bodies, the migration and transformation of humic acids mainly manifest as dispersion, and their migration capacity increases with increasing initial concentration. The transport behavior of humic acids in different media indicates that they mainly exhibit dispersion in fine sand, while in marl, they may undergo transformation, being degraded by microorganisms and converted into soluble microbial metabolites. This transformation not only affects the environmental behavior of humic acids themselves but may also affect other substances in the water body. The impact of humic acids on the aquatic environment is multifaceted. Humic acids can reduce the concentration of heavy metal ions in water through adsorption and complexation, thereby decreasing their toxicity and mobility. They can also adsorb organic pollutants through coordination exchange, hydrophobic interactions, and hydrogen bonding, enhancing their solubility and limiting their migration in water. Furthermore, humic acids are highly photochemically active, initiating free radical reactions that influence the photodegradation process of organic pollutants and reducing their concentration in water through redox reactions. These properties make humic acids crucial for water pollution control and environmental quality improvement, particularly in reducing the environmental risks posed by heavy metals and organic pollutants. Therefore, humic acids have significant application potential in water environment protection and remediation.

[0003] However, the complexity of humic acid molecular structure and the wide distribution of molecular weights (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 acids. Currently, methods for determining the molecular weight of humic acids in water mainly include size exclusion chromatography, asymmetric field flow analysis, high performance liquid chromatography (HPLC), ultraviolet-visible spectrophotometry, and XAD macroporous resin adsorption-total organic carbon coupling method. Size exclusion chromatography is a chromatographic method that separates solute molecules according to their volume in the mobile phase solution. By controlling the size of the gel pores, large molecules in the sample are completely excluded from the gel pores, thus achieving separation. Asymmetric field flow analysis is a versatile technique that combines analytical and semi-preparative separation, applicable to a variety of analytes, including proteins, polymers, and viruses, with a detection size range from 1 nm to 10 μm. High performance liquid chromatography (HPLC) analyzes humic acid samples using a chromatographic module and detector to identify humic acid components with characteristic ultraviolet and fluorescence absorption peaks. Ultraviolet-visible spectrophotometry, using molecular ultraviolet-visible molecular absorption spectroscopy, can perform qualitative analysis and quantitative analysis based on the Lambert-Beer law. The XAD macroporous resin adsorption-total organic carbon (TOC) coupled method uses macroporous resin to adsorb humic acid, and then measures the total organic carbon value of the adsorbed humic acid using a TOC analyzer, thereby analyzing the molecular weight of humic acid. These methods each have their own characteristics, 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 aforementioned objective, 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 includes the following steps:

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

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

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

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

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

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

[0012] a1) Collect lake water samples, filter them, adjust the lake water samples to a specific pH using strong alkali and strong acid, stir continuously, let stand, and centrifuge to obtain solids, which are labeled as crude humic acid solids.

[0013] a2) Under nitrogen protection, dissolve the sample in a solution containing a strong base and a strong acid-strong base salt, then centrifuge at high speed to obtain the supernatant.

[0014] a3) Acidify the supernatant to a specific pH value with hydrochloric acid, stir continuously and let stand, centrifuge to obtain solid and label it as purified humic acid;

[0015] a4) Add strong acid and concentrated hydrofluoric acid solution to the first purified humic acid in sequence to a specific concentration, stir continuously and let stand, centrifuge to separate the solid and label it as silicon-free humic acid.

[0016] a5) Add strong acid to silicon-free humic acid, stir continuously, let stand, and centrifuge to obtain solid, which is labeled as secondary purified humic acid.

[0017] a6) Add a strong acid to the secondary purified humic acid, stir continuously, let stand, and centrifuge to obtain a solid, which is labeled as tertiary purified humic acid.

[0018] a7) Add strong acid to humic acid three times, filter it in a vacuum filter, and continuously add ultrapure water into the filter hole to wash the solid. The resulting solid is freeze-dried and ground to obtain humic acid solid powder.

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

[0020] b1) Weigh out m1 of humic acid solid powder and dissolve it with a strong base. Adjust the pH value to a specific value with strong acid and strong base solution. The pH value range is between 4 and 9. After stirring and keeping the pH stable, filter the solution, add salt solution to adjust the ionic strength, and make up to V volume. Then label it as humic acid test solution.

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

[0022] b3) The concentration of humic acid in the test 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 test solution, as shown in the following formula (1):

[0023] Humic acid concentration in the test solution

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

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

[0026] c2) Adjust the humic acid test solution to a specific pH value, stir and keep the pH value stable;

[0027] c3) Scan 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 subtracting the Rayleigh and Raman scattering peaks, select the excitation and emission wavelengths at which the fluorescence intensity is at its maximum in the three-dimensional fluorescence spectrum of the humic acid test solution as the positions of the fluorescence peaks in the titration experiment.

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

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

[0031] d2) Adjust the measured humic acid test solution to a specific pH value, stir and keep the pH value stable;

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

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

[0034] 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;

[0035] d6) Adjust the pH of the humic acid test solution to be the same as that in step d2, stir and keep the pH stable;

[0036] d7) Measure the fluorescence intensity F value of the humic acid test solution at the position of the fluorescence peak determined in step c.

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

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

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

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

[0041]

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

[0043]

[0044] e3) C L Substituting into formula (4), the molecular weight M of humic acid can be calculated:

[0045]

[0046] Among them, C Q F represents the concentration of the solution containing the quenching agent added in step d5 of the titration process. end α is the fluorescence intensity fitted by humic acid at titration saturation; K is the conditional equilibrium constant; m is the mass-volume concentration of humic acid in its test solution during step d1; C L M represents the molar concentration of the humic acid in the test 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 and 3; in step a, the continuous stirring time is 15 min and the standing time is not less than 24 h; in steps a2, a5, a6, and a7, the solid-liquid ratio of the solution is 1:10; the concentration of the strong acid used in steps a4, a5, a6, and a7 is a specific concentration, with a concentration range of 0.05-0.2 mol / L; and the concentration of hydrofluoric acid in step a4 is a fixed value, with a concentration range of 0.05-0.1 mol / L.

[0048] Preferably, after adding 100ml 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, another 100ml of ultrapure water is 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 20-24 hours, then calcined at 550°C for 5 hours, and the ash content of the humic acid solid powder is then determined. If the ash content of the humic acid solid powder is greater than 1.0% (on a dry weight basis), steps a2-a7 are repeated until the ash content is less than 1.0%.

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

[0051] Preferably, the cumulative volume of the acid and alkali solutions added in step c to adjust the pH value is no greater than 1‰ of the volume of the humic acid solution being tested in step c1.

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

[0053] Preferably, the pH value of the solution in steps b, c and d is a constant (with an error of ±0.02), and the pH value ranges 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 steps a2 and b is any one or more of potassium perchlorate, sodium perchlorate, sodium chloride, potassium chloride and potassium nitrate solutions.

[0054] Preferably, the filter membranes used in steps a and b are glass fiber filter membranes with a pore size of 0.22-0.7μm. The glass fiber filter membranes are calcined at 450℃-550℃ for 5-8 hours before use and dried at 60℃-80℃ for 8-12 hours before and after filtration, and then cooled to room temperature in a desiccator.

[0055] Preferably, the strong acid is one or a mixture of strong acids such as hydrochloric acid, perchloric acid, dilute nitric acid, and dilute sulfuric acid; the strong base is one or a mixture of sodium hydroxide and potassium hydroxide; maintaining pH stability means that the pH value of the solution changes by less than 0.02 pH units within at least 30 minutes; the fluorescence measurements in steps c and d are performed under the protection of inert gases such as nitrogen, helium, or argon.

[0056] The beneficial effects of this invention are reflected 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. Furthermore, the method results are proven to be accurate and reliable by using tyrosine molecule model compounds and humic acid standard samples.

[0058] (2) The salt used in this invention can provide a certain ionic strength during the fluorescence determination of humic acid. Its concentration and ion type have negligible influence on the determination results, thereby improving the detection effect.

[0059] (3) In the preparation of the humic acid test solution provided by the present invention, the humic acid solid is dissolved by strong alkali, which can speed up the dissolution process and save the total measurement time. At the same time, deionized water can be used to dissolve the humic acid by shaking (generally shaking time is 12-24h), which reduces the impact on the detection results. However, the amount of dissolution may not be sufficient to carry out the experiment.

[0060] (4) The method for determining the molecular weight of humic acid provided by this invention controls the pH value, ensuring that humic acid exists in a free form in the solution. This avoids the situation where, when the acidity is too strong (pH value less than 3), humic acid exists in an acidic form, and when the pH value is less than 1, humic acid precipitates. Simultaneously, it avoids the situation where, when the alkalinity is too strong (pH value greater than 10), copper ions combine with the abundant free hydroxyl groups in the solution, affecting the measurement results, thereby improving the accuracy of the detection results.

[0061] (5) The method for determining the molecular weight of humic acid using fluorescence quenching titration provided by this invention requires that one humic acid molecule binds to one copper ion in the reaction system. When the copper ion causes further flocculation of the humic acid molecule, it leads to a sharp increase in scattering intensity. Therefore, this invention requires that I < 2I0 in step d.

[0062] (6) Tyrosine used in this invention is an organic compound with a fixed molecular weight. When it combines with ions such as copper ions and mercury ions, it will undergo fluorescence quenching. Therefore, it can be used as a reference to verify the reliability of the method for determining the molecular weight of humic acid by fluorescence quenching titration provided by this invention. Detailed Implementation

[0063] 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.

[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 was collected from a lake. After filtration, the pH of the lake water sample was adjusted to 1.0 ± 0.02 using strong alkali and strong acid. After continuous stirring and standing, the crude humic acid solid was obtained by centrifugation. Under nitrogen protection, the solid was dissolved in solutions containing strong alkali and strong acid-strong alkali 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, stirred continuously, and allowed to stand. After centrifugation, purified humic acid was obtained. Strong acid and concentrated hydrofluoric acid solution were added sequentially to a concentration of 0.1 M. After stirring continuously and allowed to stand, the humic acid was separated by centrifugation to obtain silica-free humic acid.

[0069] A strong acid was added to the silica-free humic acid, and after continuous stirring and standing, the mixture was centrifuged to obtain secondary purified humic acid. Then, a strong acid was added again, and after continuous stirring and standing, the mixture was centrifuged to obtain tertiary purified humic acid. Finally, a strong acid was added, and the mixture was placed in a vacuum filtration device for filtration. Ultrapure water was continuously added to the holes of the vacuum filter to wash the solid. The resulting solid was freeze-dried and ground to obtain humic acid solid powder.

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

[0071] Weigh 111.19 mg of humic acid solid powder and dissolve it in 0.1 M strong alkali. Adjust the pH to 6.0 ± 0.02 with strong acid and strong alkali solutions. Stir and maintain pH stability for 60 min, then filter. Add KClO4 solution to adjust the ionic strength, and bring the volume to 10 L 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 fluorescence peak position in titration experiment; measure the humic acid test solution and adjust the pH of the humic 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 humic 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 the highest in the three-dimensional fluorescence spectrum of the humic acid test solution were selected as the positions of the fluorescence peaks in 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 test solution to 6.0 ± 0.02, stir and keep the pH stable for 60 min;

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

[0078] 4) Measure the fluorescence scattering intensity I0 of the humic 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 humic 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 value of the humic acid test solution at the position of the fluorescence peak determined in step c.

[0082] 8) Measure the light scattering intensity I of the humic 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: Molecular weight calculation of humic acid.

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

[0086]

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

[0088]

[0089] C L Substituting into formula (3), the molecular weight M of humic acid can be calculated:

[0090]

[0091] Among them, C Q F represents the concentration of the solution containing the quenching agent added in step d5 of the titration process. end α is the fluorescence intensity fitted by humic acid at titration saturation; K is the conditional equilibrium constant; m is the mass-volume concentration of humic acid in its test solution during step d1; C L The molar concentration of humic acid in the test solution prepared in step b is given by M, where M is the molecular weight of humic acid. The concentration of added copper, C, will be adjusted by changing the concentration value of C. 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 humic acid in step d1) in the test solution is m = (111.17mg - 5.2mg) / 10L = 10.60mg / L.

[0092] Using the above method, the molecular weight M of humic acid was determined 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 is basically the same as in Example 1, except that:

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

[0096] A water sample was collected from a lake, and humic acid solid powder was obtained by extraction and purification using the method described above.

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

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

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

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

[0101] 2) Scan the three-dimensional fluorescence spectrum of the humic 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 humic acid test solution as the positions of the fluorescence peaks in 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 test solution to 4.0 ± 0.02, stir and keep the pH stable for 20 min;

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

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

[0107] 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;

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

[0109] 7) Measure the fluorescence intensity F value of the humic acid test solution at the position of the fluorescence peak determined in step c.

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

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

[0112] Using the above method, the molecular weight M of humic acid was determined 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 is basically the same as in Example 1, except that:

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

[0116] A water sample was collected from a lake, and humic acid solid powder was obtained by extraction and purification using the method described above.

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

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

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

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

[0121] 2) Scan the three-dimensional fluorescence spectrum of the humic acid test solution, where the excitation and emission wavelengths are both in the range of 200-600 nm.

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

[0123] d: Fluorescence quenching titration of humic acid

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

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

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

[0127] 4) Measure the fluorescence scattering intensity I0 of the humic 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 humic acid test solution to be the same as that in step d2, stir and keep the pH stable for 45 min;

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

[0131] 8) Measure the light scattering intensity I of the humic 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 molecular weight M of humic acid was determined to be 2987±83.1 g / mol.

[0134] Example 4

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

[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 tyrosine test solution to the pH value specified in step c2) 6, stir and keep the pH value 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 c;

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

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

[0162] e: Molecular weight 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 molecular weight 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 molecular weight of tyrosine.

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

[0170] Table 1 Cu at different concentrations 2+ Statistical analysis of the corresponding measured fluorescence intensity values ​​F

[0171]

[0172] 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. Formulas (3) and (4) calculate the molecular weight of tyrosine to be 183.5 ± 10.0.

[0173] Using the fluorescence quenching titration 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 molecular weight of tyrosine was measured to be 178.1-185.4 g / mol, with an error of less than 5% compared to 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 in Example 1, except that: 27 mg of the International Humic Acid Association standard (Suwannee River HA, 3S101H) is 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 humic acid from lake water;

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

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

[0179] A humic 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 3.7 mg.

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

[0181] 1) Measure the humic acid test solution and adjust the pH of the humic 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 humic 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 humic acid test solution as the positions of the fluorescence peaks in the titration experiment.

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

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

[0185] 2) Adjust the measured humic 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 humic acid test solution at the position of the fluorescence peak selected in step c of the titration experiment;

[0187] 4) Measure the fluorescence scattering intensity I0 of the humic 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 humic 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 humic acid test solution at the position of the fluorescence peak determined in step c.

[0191] 8) Measure the light scattering intensity I of the humic 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 molecular weight M of humic acid was determined to be 1737 g / mol.

[0194] Using the fluorescence quenching titration 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 molecular weight of humic acid was measured to be 1737 g / mol, which has an error of 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 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 pre-enrichment and molecular weight determination of humic acid in a lake water body, characterized in that, The method for pre-enriching and determining the molecular weight of humic acid in a lake water body comprises the following steps: (a) extracting and purifying humic acid in a lake water body; (b) preparing a humic acid sample solution for a lake water body; (c) selecting the fluorescence peak position in a titration experiment; (d) fluorescence quenching titration of humic acid; (e) calculating the molecular weight of humic acid; In step c, the process of selecting the fluorescence peak position in the titration experiment is as follows: c1) measuring the humic acid sample solution; c2) adjusting the pH value of the humic acid sample solution, stirring and keeping the pH value stable; c3) scanning the three-dimensional fluorescence spectrum of the humic acid sample solution, wherein the scanning range of the excitation wavelength and the emission wavelength is 200-600 nm; c4) after removing the Rayleigh scattering and Raman scattering peaks, selecting the excitation wavelength and the emission wavelength at which the fluorescence intensity is the highest in the three-dimensional fluorescence spectrum of the humic acid sample solution as the fluorescence peak position in the titration experiment; Step d comprises the following steps: d1) measuring the humic acid sample solution prepared in step b; d2) adjusting the pH value of the measured humic acid sample solution, stirring and keeping the pH value stable; d3) measuring the fluorescence intensity F0 of the humic acid sample solution at the fluorescence peak position selected in step c; d4) measuring the fluorescence light scattering intensity I0 of the humic acid sample solution when the excitation wavelength and the emission wavelength are both 500 nm; d5) adding a solution containing a quenching substance, the solution containing the quenching substance comprising copper ions or mercury ions; the concentration of the solution containing the quenching substance is between 0-1000 µmol / L; d6) adjusting the pH value of the humic acid sample solution to be the same as that in step d2, stirring and keeping the pH value stable; d7) measuring the fluorescence intensity F of the humic acid sample solution at the fluorescence peak position determined in step c; d8) measuring the light scattering intensity I of the humic acid sample solution when the excitation wavelength and the emission wavelength are both 500 nm; d9) repeating steps d5 to d8 until I>2I0, and then stopping step d; In step e, the specific process of calculating the molecular weight of humic acid is as follows: e1) F is calculated using equation (2) end : (2) e2) F end is brought into the following formula to find the molecular weight M of humic acid in the water body of the lake: 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 fluorescence intensity fitted for humic acid at the end of the titration; a is a constant greater than 0; K is the conditional equilibrium constant; m is the mass by volume concentration of humic acid in the sample under test in step d1 ; M is the molecular weight of humic acid.

2. The method according to claim 1, wherein the method is characterized by, In step a, the process of extracting and purifying humic acid in a lake water body is as follows: a1) collecting a lake water body sample, filtering, adjusting the pH of the lake water body sample with strong alkali and strong acid, continuously stirring, standing and then centrifuging to obtain a solid labeled as crude humic acid solid; a2) dissolving with a solution containing strong alkali and strong acid under nitrogen protection, high-speed centrifugal separation to obtain an upper clear liquid; a3) acidifying the upper clear liquid with hydrochloric acid, continuously stirring, standing, and then centrifuging to obtain a solid labeled as first-purified humic acid; a4) adding strong acid and concentrated hydrofluoric acid to the first-purified humic acid, continuously stirring, standing, and then centrifuging to obtain a solid labeled as silicon-free humic acid; a5) adding strong acid to the silicon-free humic acid, continuously stirring, standing, and then centrifuging to obtain a solid labeled as second-purified humic acid; a6) adding strong acid to the second-purified humic acid, continuously stirring, standing, and then centrifuging to obtain a solid labeled as third-purified humic acid; a7) adding strong acid to the third-purified humic acid, filtering in a suction filtration device, continuously adding ultrapure water to the suction filter leak to wash the solid, and then freeze-drying and grinding the obtained solid to obtain a humic acid solid powder.

3. The method according to claim 2, wherein the method is characterized by, The preparation process of the humic acid test solution in step b is as follows: b1) weigh humic acid solid powder m1, dissolve with strong base, adjust pH value with strong acid and strong base solution, the pH value range is between 4-9, filter after stirring and keeping pH stable, adjust ionic strength by adding salt solution, mark as humic acid test solution after constant volume V; b2) weigh the filter membrane before and after filtration after drying treatment, the mass difference is marked as m2; b3) the concentration of humic acid test solution is calculated by adding the mass of humic acid m1, subtracting the amount of humic acid intercepted by the filter membrane m2, and the total volume V of humic acid test solution, the formula is as follows (1): Humic acid measured solution concentration (1).

4. The method according to claim 2, wherein the method is characterized by, The error of 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 min, and the standing time is not less than 24 h; the solid-liquid ratio of the solution in steps a2, a5, a6 and a7 is 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 range of hydrofluoric acid in step a4 is 0.05-0.1 mol / L.

5. The method according to claim 2, wherein the method is characterized by, After adding 100 ml of ultrapure water in step a7, the content of chloride ions in the filtrate of step a7 is determined by silver nitrate method, if chloride ions exist, continue to add 100 ml of ultrapure water, and determine the content of chloride ions in the filtrate again until no chloride ions are detected in the filtrate.

6. The method according to claim 2, wherein the method is characterized by, The humic acid solid powder obtained in step a7 is dried at 80-100℃ for 20-24h, then calcined at 550℃ for 5h, and then the ash content of the humic acid solid powder is determined, if the ash content of the humic acid solid powder is greater than 1.0%, calculated by dry weight, repeat the operation of steps a2-a7 until the ash content is less than 1.0%.

7. The method according to claim 3, wherein the method is characterized by, The salt concentration in the humic acid test solution in step b is a specific value, and the concentration is between 0.1-1000 mmol / 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 5-20 mg / L.

8. The method according to claim 3, wherein the method is characterized by, The cumulative volume of acid and base solution added in step c to adjust the pH value is not more than 1‰ of the volume of the humic acid test solution in step c1.

9. The method according to claim 3, wherein the method is characterized by, The cumulative volume of acid and / or base solution added in step d to adjust the pH value is not more than 1‰ of the volume of the background solution in step d1; 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 humic acid test solution in step c1.

10. The method according to claim 3, wherein the method is characterized by, The pH value error of the solution in steps b, c and d is ±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, which is between 0.05-0.2 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.

11. The method according to claim 3, wherein the method is characterized by, The filter membrane used for filtration in steps a and b is glass fiber filter membrane with pore size of 0.22-0.7 µm, and the glass fiber filter membrane is calcined at 450℃-550℃ for 5-8h before use, and the glass fiber filter membrane is dried at 60℃-80℃ for 8-12h before and after filtration, and cooled to room temperature in a drying dish.

12. The method according to claim 3, wherein the method is characterized by, The strong acid is one of hydrochloric acid, perchloric acid, dilute nitric acid, dilute sulfuric acid or a mixed acid thereof; the strong base is one of sodium hydroxide and potassium hydroxide or a mixture thereof; the stable pH value means that the pH value of the solution changes less than 0.02 pH units within not less than 20 min.

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