A fluorescence spectrometric method for determining the molecular weight of natural dissolved organic matter

Abstract

The present invention relates to the field of environmental testing technology, and in particular to a fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter. The fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter comprises the following steps: (a) preparation of a natural dissolved organic matter test solution and a background solution; (b) selection of the fluorescence peak position in a titration experiment; (c) fluorescence quenching titration of the natural dissolved organic matter; (d) fluorescence measurement of the background solution; and (e) calculation of the molecular weight of the natural dissolved organic matter. This method is not only simple to operate, but also has low requirements for equipment and operating skills. Model compounds of natural dissolved organic matter molecules such as tyrosine and natural dissolved organic matter standard samples have been used to prove that the method results are accurate and reliable.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental detection, and in particular to a fluorescence spectroscopy method for measuring the molecular weight of natural dissolved organic matter. Background Art

[0002] Naturally dissolved organic matter (NDOM) is a mixture of macromolecules formed from animal or plant residues through complex physical, chemical, and biological processes. It is widely distributed in environmental media such as water, soil, and air. It is a major environmental factor influencing and controlling the migration, transformation, toxicity, and bioavailability of pollutants and nutrients in water and soil. NDO has a highly complex structure, with molecular weights ranging from a few hundred to several hundred thousand. The heterogeneity and polydispersity of NDO generally make accurate molecular weight determination challenging.

[0003] Due to limitations in experimental techniques, relatively little research has been conducted on the molecular weight and distribution of naturally occurring dissolved organic matter (DMO). The main measurement techniques include size exclusion chromatography and ultrafiltration membrane technology, which are instrumentally demanding and have certain limitations. For example, size exclusion chromatography (SEC) leverages the unique properties of porous gel stationary phases to estimate the molecular weight of analytes based on differences in molecular size. Specifically, large molecules in the sample are completely excluded from the gel pores and are transported through the interstices between the porous gel particles through the chromatographic column by the mobile phase. Medium-sized molecules can enter some of the medium-sized pores in the gel, where they are retained and elute more slowly. Small molecules enter the vast majority of the gel pores, are more strongly retained, and elute more slowly, thus enabling the separation of samples of varying molecular sizes. SEC has gained widespread application due to its ability to directly measure the molecular size of DMO, its simple pretreatment process, and the small sample size required. However, there is a certain interaction between the gel stationary phase and natural dissolved organic matter. Natural dissolved organic matter does not have a corresponding model molecule as a standard, which to a certain extent affects the determination of the molecular weight of natural dissolved organic matter. In addition, the size exclusion chromatography method estimates the molecular weight of organic molecules based on molecular size or volume, which is obviously irrational and has large errors. Ultrafiltration membrane technology can only estimate the molecular weight range of natural dissolved organic matter and cannot obtain accurate molecular weight values. Ultracentrifugation uses ultra-high-speed centrifugal force to estimate molecular weight, which requires complex and expensive equipment and cumbersome pretreatment operations, making it difficult to promote.

[0004] The above content is only used to assist in understanding the technical solution of the present invention and does not mean that the above content is the closest prior art. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter.

[0006] To achieve the above object, the technical solution of the present invention is implemented as follows: a fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter comprises the following steps:

[0007] (a) Preparation of natural dissolved organic matter test solution and background solution;

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

[0009] (c) Fluorescence quenching titration of natural dissolved organic matter;

[0010] (d) Fluorescence measurement of background solution;

[0011] (e) Calculation of molecular weight of natural dissolved organic matter.

[0012] Preferably, step c comprises the following steps:

[0013] c1) measuring the natural dissolved organic matter test solution prepared in step a;

[0014] c2) adjusting the measured natural dissolved organic matter solution to a specific pH value within the pH range of 3-10, stirring and maintaining the pH value stable for 15-60 minutes;

[0015] c3) measuring the fluorescence intensity F0′ of the natural dissolved organic matter test solution at the fluorescence peak position of the titration experiment selected in step b;

[0016] c4) measuring the light scattering intensity I0 of the natural dissolved organic matter test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0017] c5) adding a solution containing a quenching substance to a concentration of 0-1000 μmol / L;

[0018] c6) adjusting the pH of the natural dissolved organic matter test solution to the same value as in step c2, stirring and maintaining the pH stable for 15-60 minutes;

[0019] c7) measuring the fluorescence intensity F' of the natural dissolved organic matter test solution at the position of the fluorescence peak of the titration experiment determined in step b;

[0020] c8) measuring the light scattering intensity I of the natural dissolved organic matter test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0021] c9) Repeat steps c5 to c8 until I>2I0 and stop step c.

[0022] Preferably, the background solution fluorescence measurement process in step d is:

[0023] d1) measuring the background solution in step a;

[0024] d2) adjusting the pH of the measured background solution to the same pH as the natural dissolved organic matter solution in step c2, stirring and maintaining a stable pH for 15-60 minutes;

[0025] d3) measuring the fluorescence intensity Fr0′ of the background solution at the fluorescence peak position of the titration experiment determined in step b;

[0026] d4) adding a solution containing a quenching substance so that the concentration of the quenching substance in the background solution is the same as that in step c5, repeating steps d2 to d4, and measuring the fluorescence intensity Fr' at the fluorescence peak position determined in step b.

[0027] Preferably, in step e, the specific process of calculating the molecular weight of natural dissolved organic matter is:

[0028] e1) Calculate F0=F0'-Fr0' and calculate F=F'-Fr'

[0029] e2) Use formula (1) to calculate F end :

[0030]

[0031] e3) The F calculated in formula (1) end The molecular weight M of natural dissolved organic matter is calculated using formula (2):

[0032]

[0033] Among them, C Q is the total concentration of the quenching substance added in step c5 during the titration process; end is the fluorescence intensity fitted by the natural dissolved organic matter at saturation titration; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of the natural dissolved organic matter in the test solution in step c1; M is the molecular weight of the natural dissolved organic matter.

[0034] Preferably, the preparation process of the natural dissolved organic matter test solution in step a is:

[0035] a1 Weigh a solid sample of natural dissolved organic matter and dissolve it with a strong base under inert gas protection. Adjust the pH to a specific value with a strong acid and strong base solution, the pH value range is between 3-10. Filter through a glass fiber filter membrane and adjust the ionic strength with a salt solution to prepare a natural dissolved organic matter test solution with a concentration of 2-20 mg / L.

[0036] a2. Weigh the glass fiber filter membranes before and after filtration after drying.

[0037] Preferably, the preparation process of the background solution in step a is:

[0038] a3 Measure ultrapure water, adjust the ionic strength with salt solution, adjust to a specific pH value with strong acid and strong alkali solution, the pH value range is between 3-10, and filter with a glass fiber filter membrane.

[0039] Preferably, the process of selecting the fluorescence peak position of the titration experiment in step b is:

[0040] b1) Measure the natural dissolved organic matter test solution;

[0041] b2) Adjust the pH value of the natural dissolved organic matter test solution to 3-10, stir and keep the pH value stable for 15-60 minutes;

[0042] b3) Scanning the three-dimensional fluorescence spectrum of the natural dissolved organic matter test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600nm,

[0043] b4) measuring the background solution;

[0044] b6) Adjust the pH of the background solution to the same pH as the natural dissolved organic matter solution in b2, stir and maintain the specific pH value stable for 15-60 minutes;

[0045] b7) scanning the three-dimensional fluorescence spectrum of the background solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0046] b8) Subtracting the three-dimensional fluorescence spectrum data of the background solution from the obtained three-dimensional fluorescence spectrum data of the natural dissolved organic matter test solution to determine the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum, and using the wavelength as the excitation wavelength and emission wavelength for fluorescence quenching titration.

[0047] Preferably, the pore size of the glass fiber filter membrane in steps a1 and a3 is 0.22-0.7 μm, and the glass fiber filter membrane is calcined at 450°C-550°C for 5-8 hours before use. The glass fiber filter membrane is dried at 60°C-80°C for 8-12 hours before and after filtration, and cooled and dried in a drying dish.

[0048] Preferably, the strong acid solution is one solution or a mixed solution of more than one of perchloric acid, sulfuric acid, hydrochloric acid and nitric acid solution; the strong base is 0.01-0.1M sodium hydroxide or potassium hydroxide; the concentration of the natural dissolved organic matter in the measured solution is 5-20 mg / L;

[0049] The salt used to adjust the ionic strength in step a is one or more of potassium perchlorate, sodium perchlorate, sodium chloride, potassium chloride and potassium nitrate, and the concentration of the natural dissolved organic matter test solution and the background solution is 0.1-1000 mmol / L.

[0050] Preferably, the quenching substances added in steps c and d include, but are not limited to, copper, mercury ions, etc.; the cumulative volume of the quenching substance added during the titration in step c is no more than 1‰ of the volume of the natural dissolved organic matter solution tested in step c1; the cumulative volume of the quenching substance added during the titration in step d is no more than 1‰ of the volume of the background solution in step d1; and the concentration of the quenching substance in step c5 is 0-400 μmol / L, preferably 0-100 μmol / L.

[0051] Preferably, the solutions in steps b, c, and d are adjusted to a specific pH value (with an error of ±0.02), with a pH range of 5-7. The acid solution used to adjust the pH value is one or a mixture of perchloric acid, hydrochloric acid, sulfuric acid, or nitric acid; the alkaline solution used to adjust the pH value is one or a mixture of sodium hydroxide and potassium hydroxide; the cumulative volume of the acid and / or alkaline solution added to adjust the pH value in step c is no more than 1‰ of the volume of the natural dissolved organic matter solution tested in step c1; and the cumulative volume of the acid and / or alkaline solution added to adjust the pH value in step d is no more than 1‰ of the volume of the background solution in step d1. The temperature during the titration in steps c and d is 20-35°C; and the fluorescence measurements in steps b, c, and d are all performed under the protection of an inert gas such as nitrogen, helium, or argon.

[0052] The beneficial effects of the present invention are embodied in:

[0053] (1) The method for determining the molecular weight of natural dissolved organic matter 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 natural dissolved organic matter molecules such as tyrosine and natural dissolved organic matter standards.

[0054] (2) The salt used in the present invention can provide a certain ionic strength during the fluorescence determination of natural dissolved organic matter, and the influence of its concentration and ion type on the determination results can be basically ignored, thereby improving the detection effect.

[0055] (3) During the preparation of the natural dissolved organic matter test solution provided by the present invention, a strong base is used to dissolve the natural dissolved organic matter solid under inert gas protection conditions, which can speed up the dissolution process and save the total measurement time.

[0056] (4) In the method for determining the molecular weight of natural dissolved organic matter provided by the present invention, controlling the pH value can not only ensure that the natural dissolved organic matter can exist in the solution in a free form, but also avoid the natural dissolved organic matter existing in the form of acid when the acidity is too strong, that is, when the pH value is less than 3. At the same time, it avoids the phenomenon that the quenching substance combines with the free hydroxyl groups existing in a large number in the solution when the alkalinity is too strong, that is, when the pH value is greater than 10, thereby affecting the measurement results, thereby improving the accuracy of the test results.

[0057] (5) The present invention provides a method for measuring the molecular weight of natural dissolved organic matter using fluorescence spectroscopy. The method requires that a natural dissolved organic matter molecule be combined with a quenching substance in the reaction system. When the quenching substance causes further flocculation of the natural dissolved organic matter molecules, the scattering intensity increases dramatically. Therefore, the present invention requires that in step c, I<2I0.

[0058] (6) Tyrosine used in the present invention is an organic substance with a fixed molecular weight. When combined with a quenching substance, fluorescence quenching occurs. Therefore, it can be used as a reference substance to test the reliability of the fluorescence spectroscopy method provided by the present invention to further prove the molecular weight of natural dissolved organic matter. DETAILED DESCRIPTION

[0059] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction 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 of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0060] Example 1

[0061] A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter comprises the following steps:

[0062] a: Preparation of natural dissolved organic matter test solution and background solution

[0063] Weigh 100.17 mg of a natural dissolved organic matter (NDOM) solid sample and dissolve it in 2 mL of a 0.1 M potassium hydroxide solution. Dilute the sample to approximately 1 L with deionized water, adjust the pH to a neutral pH of 5-7 with hydrochloric acid, and filter through a 0.45 μm pore glass fiber membrane. After filtration, add 138.55 g of solid potassium perchlorate and dissolve it. The volume is then adjusted to 10 L with deionized water to prepare the NDOM test solution. The difference in mass between the glass fiber membrane before and after filtration is 1.50 mg.

[0064] 138.55 g of potassium perchlorate solid sample was weighed, dissolved in deionized water, and filtered through a glass fiber membrane with a pore size of 0.45 μm to prepare a background solution with a volume of 10 L.

[0065] The glass fiber filter membrane is calcined at 450-550°C for 5-8 hours before use; it is dried at 60-80°C for 8-12 hours before and after filtration, and then dried and cooled in a drying dish.

[0066] b: Selection of fluorescence peak position in titration experiment

[0067] b1) Measure the natural dissolved organic matter test solution;

[0068] b2) Adjust the pH of the natural dissolved organic matter test solution to 6 ± 0.02, stir and maintain the pH stable for 60 minutes;

[0069] b3) scanning the three-dimensional fluorescence spectrum of the natural dissolved organic matter test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0070] b4) measuring the background solution;

[0071] b5) adjusting the background solution to pH 6 ± 0.02, stirring and maintaining a stable pH for 60 min;

[0072] b6) scanning the three-dimensional fluorescence spectrum of the background solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0073] b7) Subtracting the three-dimensional fluorescence spectrum data of the background solution from the obtained three-dimensional fluorescence spectrum data of the natural dissolved organic matter test solution, while setting the Raman and Rayleigh scattering intensities to zero, and determining the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum, and using these wavelengths as the excitation wavelength and emission wavelength for the fluorescence quenching titration.

[0074] c: Fluorescence quenching titration of natural dissolved organic matter

[0075] c1) Measure the natural dissolved organic matter test solution;

[0076] c2) Adjust the pH of the natural dissolved organic matter solution to 6, stir and keep the pH stable for 60 minutes;

[0077] c3) measuring the fluorescence intensity F0′ of the natural dissolved organic matter test solution at the fluorescence peak position of the titration experiment selected in step b;

[0078] c4) measuring the light scattering intensity I0 of the natural dissolved organic matter test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0079] c5) adding a solution containing a quenching substance to a concentration of 0-1000 μmol / L;

[0080] c6) adjusting the pH value of the natural dissolved organic matter test solution to 6 as specified in step c2), stirring and maintaining the pH value stable for 60 minutes;

[0081] c7) measuring the fluorescence intensity F' of the natural dissolved organic matter test solution at the position of the fluorescence peak of the titration experiment determined in step b;

[0082] c8) measuring the light scattering intensity I of the natural dissolved organic matter test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0083] c9) Repeat the above steps c5) to c8) until I>2I0 and stop step c.

[0084] d: Background solution fluorescence measurement

[0085] d1) measuring the background solution;

[0086] d2) adjusting the pH of the background solution to 6±0.02 to the same as in step c, stirring and maintaining the pH stable for 60 min;

[0087] d3) measuring the fluorescence intensity Fr0′ of the background solution at the fluorescence peak position of the titration experiment determined in step b;

[0088] d4) adding a solution containing a quenching substance so that the concentration of the quenching substance in the background solution is the same as that in step c5), repeating steps d2 to d4, and measuring the fluorescence intensity Fr' at the fluorescence peak position determined in step b.

[0089] e: Calculation of molecular weight of natural dissolved organic matter

[0090] e1) Calculate F0=F0'-Fr0' and calculate F=F'-Fr'

[0091] e2) Use formula (1) to calculate F end :

[0092]

[0093] e3) The F calculated in formula (1) end Substitute into formula (2) to calculate the molecular weight M of natural dissolved organic matter:

[0094]

[0095] Among them, C Q is the total concentration of the quenching substance added in step c5 during the titration process; endis the fluorescence intensity fitted by the natural dissolved organic matter at saturation titration; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of the natural dissolved organic matter in the test solution in step c1; M is the molecular weight of the natural dissolved organic matter.

[0096] The quenching substance C will be added by changing Q (For example, C Q1 、C Q2 、C Q3 …C Qn ) and the corresponding series of fluorescence intensity values ​​F (such as F1, F2, F3...Fn) are substituted into formula (1) to obtain F end。 C Q Substitute F, Fend into formula (2) to calculate the molecular weight M of the dissolved organic matter. Here, the mass volume concentration of the natural dissolved organic matter in the test solution in step c1) is m = (100.17 mg - 1.50 mg) / 10 L = 9.87 mg / L.

[0097] Using the above method, the molecular weight M of natural dissolved organic matter was obtained to be 970±31 g / mol.

[0098] Example 2

[0099] A fluorescence spectrometric method for determining the molecular weight of natural dissolved organic matter is basically the same as Example 1, except that:

[0100] a: Preparation of natural dissolved organic matter test solution and background solution

[0101] Prepare a test solution with a concentration of 19 mg / L of natural dissolved organic matter, and the background solution is 800 mmol / L potassium chloride solution.

[0102] b: Selection of fluorescence peak position in titration experiment

[0103] b1) Measure the natural dissolved organic matter test solution;

[0104] b2) adjusting the pH value of the natural dissolved organic matter solution to 8.2, stirring and maintaining the pH value stable for 20 minutes;

[0105] b3) scanning the three-dimensional fluorescence spectrum of the natural dissolved organic matter test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0106] b4) measuring the background solution;

[0107] b5) Adjust the pH value of the background solution to 8.2, stir and keep the pH stable for 20 minutes;

[0108] b6) scanning the three-dimensional fluorescence spectrum of the background solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0109] b7) Subtracting the three-dimensional fluorescence spectrum data of the background solution from the obtained three-dimensional fluorescence spectrum data of the natural dissolved organic matter test solution, while setting the Raman and Rayleigh scattering intensities to zero, and determining the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum, and using these wavelengths as the excitation wavelength and emission wavelength for the fluorescence quenching titration.

[0110] c: Fluorescence quenching titration of natural dissolved organic matter

[0111] c1) Measure the natural dissolved organic matter test solution;

[0112] c2) Adjust the pH of the natural dissolved organic matter test solution to 8.2, stir and keep the pH value stable for 20 minutes;

[0113] c3) measuring the fluorescence intensity F0′ of the natural dissolved organic matter test solution at the fluorescence peak position of the titration experiment selected in step b;

[0114] c4) measuring the light scattering intensity I0 of the natural dissolved organic matter test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0115] c5) adding a solution containing a quenching substance to a concentration of 0-200 μmol / L;

[0116] c6) adjusting the pH value of the natural dissolved organic matter test solution to 8.2 as specified in step c2), stirring and maintaining the pH value at 8.2 for 15-60 minutes;

[0117] c7) measuring the fluorescence intensity F' of the natural dissolved organic matter test solution at the position of the fluorescence peak of the titration experiment determined in step b;

[0118] c8) measuring the light scattering intensity I of the natural dissolved organic matter test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0119] c9) Repeat steps c5) to c8) until I>2I0 and stop step c;

[0120] d: Background solution fluorescence measurement

[0121] d1) measuring the background solution;

[0122] d2) adjusting the pH of the background solution to 8.2, the same as in step c, stirring and maintaining a stable pH for 20 minutes;

[0123] d3) measuring the fluorescence intensity Fr0′ of the background solution at the fluorescence peak position of the titration experiment determined in step b;

[0124] d4) adding a solution containing a quenching substance so that the concentration of the quenching substance in the background solution is the same as that in step c5, repeating steps d2 to d4, and measuring the fluorescence intensity Fr' at the fluorescence peak position determined in step b.

[0125] Using the above method, the molecular weight M of natural dissolved organic matter was obtained to be 991±45 g / mol.

[0126] Example 3

[0127] A fluorescence spectrometric method for determining the molecular weight of natural dissolved organic matter is basically the same as Example 1, except that:

[0128] a: Preparation of natural dissolved organic matter test solution and background solution

[0129] Prepare a test solution with a concentration of 3 mg / L of natural dissolved organic matter and a background solution of 10 mmol / L potassium nitrate solution.

[0130] b: Selection of fluorescence peak position in titration experiment

[0131] b1) Measure the natural dissolved organic matter test solution,

[0132] b2) Adjust the pH value of the natural dissolved organic matter solution to 4.5, stir and keep the pH value stable for 45 minutes,

[0133] b3) Scanning the three-dimensional fluorescence spectrum of the natural dissolved organic matter test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600nm,

[0134] b4) Measure the background solution,

[0135] b5) Adjust the pH value of the background solution to 4.5, stir and keep the pH value stable for 45 minutes,

[0136] b6) scanning the three-dimensional fluorescence spectrum of the background solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm,

[0137] b7) Subtracting the three-dimensional fluorescence spectrum data of the background solution from the obtained three-dimensional fluorescence spectrum data of the natural dissolved organic matter test solution, while setting the Raman and Rayleigh scattering intensities to zero, and determining the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum, and using these wavelengths as the excitation wavelength and emission wavelength for the fluorescence quenching titration.

[0138] c: Fluorescence quenching titration of natural dissolved organic matter

[0139] c1) Measure the natural dissolved organic matter test solution;

[0140] c2) Adjust the pH of the natural dissolved organic matter solution to 4.5, stir and keep the pH stable for 45 minutes;

[0141] c3) measuring the fluorescence intensity F0′ of the natural dissolved organic matter test solution at the fluorescence peak position of the titration experiment selected in step b;

[0142] c4) measuring the light scattering intensity I0 of the natural dissolved organic matter test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0143] c5) adding a solution containing a quenching substance to a concentration of 0-400 μmol / L;

[0144] c6) adjusting the pH of the natural dissolved organic matter test solution to the specified pH value of 4.5 in step c2), stirring and maintaining the pH value stable for 45 minutes;

[0145] c7) measuring the fluorescence intensity F' of the natural dissolved organic matter test solution at the position of the fluorescence peak of the titration experiment determined in step b;

[0146] c8) measuring the light scattering intensity I of the natural dissolved organic matter test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0147] c9) Repeat steps c5) to c8) until I>2I0 and stop step c;

[0148] d: Background solution fluorescence measurement

[0149] d1) measuring the background solution;

[0150] d2) adjusting the pH of the background solution to 4.5, the same as in step c, stirring and maintaining a stable pH for 45 minutes;

[0151] d3) measuring the fluorescence intensity Fr0′ of the background solution at the fluorescence peak position of the titration experiment determined in step b;

[0152] d4) adding a solution containing a quenching substance so that the concentration of the quenching substance in the background solution is the same as that in step c5, repeating steps d2 to d4, and measuring the fluorescence intensity Fr' at the fluorescence peak position determined in step b.

[0153] Using the above method, the molecular weight M of natural dissolved organic matter was obtained to be 870±41 g / mol.

[0154] The molecular weight of the natural dissolved organic matter sample used in Examples 1-3 is 944±65. By comparison, it can be seen that the molecular weight of the natural dissolved organic matter measured in Examples 1-3 of the present invention is basically consistent with the molecular weight of the natural dissolved organic matter standard of 935 g / mol (International Humic Acid Association standard sample Suwannee River NOM, 2R101N).

[0155] Example 4

[0156] A fluorescence spectrometry method for measuring the molecular weight of tyrosine, the method comprising the following steps:

[0157] a: Preparation of tyrosine test solution and background solution

[0158] Weigh 52.79 mg of a solid tyrosine sample, dissolve it in deionized water, and filter it through a glass fiber membrane with a pore size of 0.45 μm. Prepare a 1 L concentrated tyrosine solution, filter the concentrated tyrosine solution, and the difference in mass between the glass fiber membrane before and after filtration is 1.20 mg. Add 138.55 g of solid potassium perchlorate to the filtered concentrated tyrosine solution and dissolve it. Then dilute to 10 L with deionized water to prepare the tyrosine test solution.

[0159] 138.55 g of potassium perchlorate solid sample was weighed, dissolved in deionized water, and filtered through a glass fiber membrane with a pore size of 0.45 μm to prepare a background solution with a volume of 10 L.

[0160] b: Selection of fluorescence peak position in tyrosine titration experiment

[0161] b1) measuring the tyrosine test solution;

[0162] b2) adjusting the pH of the tyrosine solution to 6, stirring and maintaining a stable pH for 60 minutes;

[0163] b3) scanning the three-dimensional fluorescence spectrum of the tyrosine test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0164] b4) measuring the background solution;

[0165] b5) adjusting the pH of the background solution to 6, stirring and maintaining a stable pH for 60 minutes;

[0166] b6) scanning the three-dimensional fluorescence spectrum of the background solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm;

[0167] b7) Subtracting the three-dimensional fluorescence spectrum data of the background solution from the obtained three-dimensional fluorescence spectrum data of the tyrosine test solution, while setting the Raman and Rayleigh scattering intensities to zero, and determining the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum. The excitation wavelength and emission wavelength used for the fluorescence quenching titration are 350 nm, respectively.

[0168] c: Fluorescence quenching titration of tyrosine

[0169] c1) measuring the tyrosine test solution;

[0170] c2) adjusting the pH of the tyrosine test solution to 6, stirring and maintaining the pH stable for 60 minutes;

[0171] c3) measuring the fluorescence intensity F0′ of the natural dissolved organic matter test solution at the fluorescence peak position of the titration experiment selected in step b;

[0172] c4) measuring the light scattering intensity I0 of the tyrosine test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0173] c5) adding a quenching substance to a concentration of 0-1000 μmol / L;

[0174] c6) adjusting the tyrosine test solution to the pH value of 6 specified in step c2), stirring and maintaining the pH value stable for 60 minutes;

[0175] c7) measuring the fluorescence intensity F' of the natural dissolved organic matter test solution at the position of the fluorescence peak of the titration experiment determined in step b;

[0176] c8) measuring the light scattering intensity I of the tyrosine test solution when both the excitation wavelength and the emission wavelength are 400 nm;

[0177] c9) Repeat steps c5) to c8) until I>2I0 and stop step c.

[0178] d: Background solution fluorescence measurement

[0179] d1) measuring the background solution;

[0180] d2) adjusting the pH of the background solution to 6, the same as in step c, stirring and maintaining a stable pH for 60 minutes;

[0181] d3) measuring the fluorescence intensity Fr0′ of the background solution at the fluorescence peak position of the titration experiment determined in step b;

[0182] d4) adding copper ions so that the concentration of the quenching substance in the background solution is the same as that in step c5, repeating steps d2 to d4, and measuring the fluorescence intensity Fr' at the fluorescence peak position determined in step b.

[0183] e: Calculation of molecular weight of tyrosine

[0184] e1) Calculate F0=F0'-Fr0' and calculate F=F'-Fr'

[0185] e2) Use formula (1) to calculate F end :

[0186]

[0187] e3) The F calculated in formula (1) end Substitute into formula (2) to calculate the molecular weight M of natural dissolved organic matter:

[0188]

[0189] Among them, C Q is the total concentration of the quenching substance added in step c5 during the titration process; end is the fluorescence intensity fitted by the natural dissolved organic matter at saturation titration; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of the natural dissolved organic matter in the test solution in step c1; M is the molecular weight of the natural dissolved organic matter.

[0190] Different concentrations of Cu 2+ The corresponding measured fluorescence intensity value F is shown in Table 1.

[0191] Table 1 Different concentrations of Cu 2+ Corresponding to the measured fluorescence intensity value F result statistics

[0192] <![CDATA[Cu 2+ Concentration C Q (mol / L) Tyrosine fluorescence intensity value F'(AU) 0 283.9±5.6 0.000028 157.1±4.7 0.00008 73.34±2.3 0.0004 23.69±1.7

[0193] The mass concentration of the tyrosine test solution in step c1) is m = (52.79 mg - 1.20 mg) / 10 L = 5.16 mg / L. The molecular weight of tyrosine calculated by formulas (3) and (4) is 183.5 ± 10.0.

[0194] By using the fluorescence spectroscopy method provided by the present invention, the pH value of the measured solution, the cumulative concentration of added copper ions, the concentration of the measured solution, and other parameters are changed during the titration process. 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).

[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 scope of protection of the present invention.

Claims

1. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter, characterized in that: The fluorescence spectrometric method for determining the molecular weight of naturally occurring dissolved organic matter comprises the following steps: (a) Preparation of natural dissolved organic matter test solution and background solution; (b) Selection of fluorescence peak position in titration experiment; (c) Fluorescence quenching titration of natural dissolved organic matter; Step (c) comprises the following steps: c1) measuring the natural dissolved organic matter test solution prepared in step (a); c2) adjusting the measured natural dissolved organic matter test solution to a specific pH value within the pH range of 3-10, stirring and maintaining the specific pH value stable for 15-60 minutes; c3) measuring the fluorescence intensity F0' of the natural dissolved organic matter test solution at the fluorescence peak position of the titration experiment selected in step (b); c4) measuring the light scattering intensity I0 of the natural dissolved organic matter test solution when both the excitation wavelength and the emission wavelength are 400 nm; c5) adding a solution containing a quenching substance to a concentration of the quenching substance between 0 and 1000 µmol / L; c6) Adjusting the pH of the natural dissolved organic matter test solution to the same value as in step c2), stirring and maintaining the specific pH value stable for 15-60 minutes; c7) measuring the fluorescence intensity F' of the natural dissolved organic matter test solution at the position of the fluorescence peak determined in the titration experiment in step (b); c8) measuring the light scattering intensity I of the natural dissolved organic matter test solution when both the excitation wavelength and the emission wavelength are 400 nm; c9) Repeat steps c5 to c8 until I>2I0 and stop step (c); (d) Fluorescence measurement of background solution; The background solution fluorescence measurement process in step (d) is as follows: d1) measuring the background solution from step (a); d2) adjusting the pH of the measured background solution to the same pH as the pH of the natural dissolved organic matter test solution in step c2), stirring and maintaining a stable pH for 15-60 minutes; d3) measuring the fluorescence intensity Fr0' of the background solution at the position of the fluorescence peak of the titration experiment determined in step (b); d4) adding a solution containing a quenching substance so that the concentration of the quenching substance in the background solution is the same as the concentration of the quenching substance in step c5), repeating steps d2) to d4), and measuring the fluorescence intensity Fr' at the fluorescence peak position determined in the titration experiment in step (b); The quenching substance added in steps (c) and (d) includes copper ions or mercury ions; (e) Calculation of molecular weight of naturally occurring dissolved organic matter; In step (e), the specific process of calculating the molecular weight of natural dissolved organic matter is as follows: e1) Calculate F0 = F0' - Fr0' and calculate F = F' - Fr' e2) Use formula (1) to calculate F end : (1)； e3) Use the F calculated in formula (1) end Substitute into formula (2) to calculate the molecular weight M of natural dissolved organic matter: (2)； Among them, C Q is the total concentration of the quenching substance added in step c5) during the titration process; end is the fluorescence intensity fitted by the natural dissolved organic matter at saturation titration; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of the natural dissolved organic matter in the test solution in step c1); and M is the molecular weight of the natural dissolved organic matter.

2. The fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 1, characterized in that: The preparation process of the natural dissolved organic matter test solution in step (a) is as follows: a1) Weigh a solid sample of natural dissolved organic matter (NDOM) and dissolve it with a strong base under inert gas. Adjust the pH to a specific value (pH range 3-10) with a strong acid and strong base solution. Filter the sample through a glass fiber filter and adjust the ionic strength with a salt solution to prepare a NDOM test solution with a concentration of 2-20 mg / L. a2) Drying and weighing the glass fiber filter membrane before and after filtration; The preparation process of the background solution in step (a) is as follows: a3) Take ultrapure water, adjust the ionic strength with saline solution, and adjust to a specific pH value with strong acid and strong alkali solution (pH range is 3-10), and filter through a glass fiber filter membrane.

3. The fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 2, characterized in that: The process of selecting the fluorescence peak position of the titration experiment in step (b) is as follows: b1) Measure the natural dissolved organic matter test solution; b2) adjusting the pH value of the natural dissolved organic matter test solution to a specific value, preferably within the pH range of 3-10, and stirring and maintaining the pH value stable for 15-60 minutes; b3) scanning the three-dimensional fluorescence spectrum of the natural dissolved organic matter test solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm; b4) measuring the background solution; b5) Adjust the pH value of the background solution to the same as the pH value of the natural dissolved organic matter test solution in b2), stir and maintain the pH value stable for 15-60 minutes; b6) scanning the three-dimensional fluorescence spectrum of the background solution, wherein the excitation wavelength and emission wavelength scanning range are both 200-600 nm; b7) subtracting the three-dimensional fluorescence spectrum data of the background solution from the obtained three-dimensional fluorescence spectrum data of the natural dissolved organic matter test solution to determine the excitation wavelength and emission wavelength at which the fluorescence intensity is maximum, and using the wavelength as the excitation wavelength and emission wavelength for fluorescence quenching titration.

4. The fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 3, characterized in that: The pore size of the glass fiber filter membrane in steps a1) and a3) is 0.22-0.7 µm, and the glass fiber filter membrane is calcined at 450°C-550°C for 5-8 hours before use. The glass fiber filter membrane is dried at 60°C-80°C for 8-12 hours before and after filtration, and cooled and dried in a drying dish.

5. The fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 4, characterized in that: The strong acid solution is one solution or a mixed solution of more than one of perchloric acid, sulfuric acid, hydrochloric acid and nitric acid; the strong base is 0.01-0.1M sodium hydroxide or potassium hydroxide; the concentration of the natural dissolved organic matter in the measured solution is 5-20 mg / L; The salt used to adjust the ionic strength in step (a) is one or more of potassium perchlorate, sodium perchlorate, sodium chloride, potassium chloride and potassium nitrate solutions, and the salt concentration of the natural dissolved organic matter test solution and the background solution is 0.1-1000 mmol / L.

6. The fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 5, characterized in that: The cumulative volume of the solution containing the quenching substance added during the titration in step (c) is no more than 1‰ of the volume of the natural dissolved organic matter solution to be measured in step c1); the cumulative volume of the quenching substance added during the titration in step (d) is no more than 1‰ of the volume of the background solution in step d1). The concentration of the quenching substance in step c5) is generally 0-400 µmol / L.

7. The fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 1, characterized in that: The solutions in steps (b), (c) and (d) have the same specific pH value with an error of ±0.02, and the pH value range is between 5 and 7. The solution used to adjust the pH value is one solution or a mixture of more than one solution selected from perchloric acid, hydrochloric acid, sulfuric acid, nitric acid, sodium hydroxide and potassium hydroxide solutions; the cumulative volume of the acid and / or alkaline solution added to adjust the pH value in step (c) is not greater than 1‰ of the volume of the solution of natural dissolved organic matter to be measured in step c1); 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).

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