Fluorescence spectrometry for measuring molecular weight of naturally dissolved organic matters

Abstract

The invention relates to the technical field of environment detection, in particular to a fluorescence spectrometry for measuring the molecular weight of naturally dissolved organic matters. The fluorescence spectrometry method for measuring the molecular weight of the naturally dissolved organic matter comprises the following steps: (a) preparing a naturally dissolved organic matter measured solution and a background solution; (b) selecting a fluorescence peak position of a titration experiment; (c) carrying out fluorescence quenching titration on naturally dissolved organic matters; (d) fluorescence determination of the background solution; and (e) calculating the molecular weight of the naturally dissolved organic matters. The method is simple to operate and low in requirements on equipment and operation skills, and results of the method are accurate and reliable according to model compounds of natural dissolved organic matter molecules such as tyrosine and natural dissolved organic matter standard samples.

Description

Technical Field

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

[0002] Natural dissolved organic matter is a macromolecular organic mixture formed by animal or plant residues through complex physical, chemical and biological processes. It is widely distributed in environmental media such as water, soil and air. Natural dissolved organic matter is the main environmental factor that affects and controls the toxicity and biological effectiveness of the migration and transformation of pollutants and nutrients in water and soil. The structure of natural dissolved organic matter is very complex, and the molecular weight ranges from hundreds to hundreds of thousands. Generally speaking, natural dissolved organic matter is heterogeneous and polydisperse, making it difficult to accurately determine the molecular weight of natural dissolved organic matter.

[0003] Due to the limitations of experimental technical conditions, there are relatively few studies on the molecular weight and distribution of natural dissolved organic matter. The main measurement technologies include size exclusion chromatography and ultrafiltration membrane technology, which have high instrument requirements and certain limitations. Taking size exclusion chromatography as an example, this method mainly uses the unique properties of porous gel stationary phase to estimate the molecular weight of the measured object based on the difference in molecular size. Specifically, the large molecules in the sample cannot enter the gel pores and are completely excluded. They are eluted by the mobile phase through the gaps between the porous gel particles through the chromatographic column; medium-sized molecules can enter some medium-sized pores in the gel, are retained in the column, and elute from the column more slowly; small molecules enter the vast majority of pores in the gel, are more strongly retained in the column, and will elute more slowly, thereby achieving the separation of samples with different molecular sizes. Since size exclusion chromatography can directly measure the molecular size of natural dissolved organic matter, the pretreatment process is simple, and the amount of sample required is small, it has been widely used. 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 affects the molecular weight determination of natural dissolved organic matter to a certain extent. In addition, size exclusion chromatography estimates the molecular weight of organic molecules based on molecular size or volume, which is obviously unreasonable 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, cumbersome pretreatment operations, and is difficult to promote.

[0004] The above contents are only used to assist in understanding the technical solution of the present invention, and do not constitute an admission that the above contents are the closest prior art. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a fluorescence spectroscopy 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 in step a;

[0014] c2) adjusting the measured natural dissolved organic matter test solution to a specific pH value, the pH range is between 3-10, stirring and keeping 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) Determine the light scattering intensity I0 of the natural dissolved organic matter test solution when the excitation wavelength and emission wavelength are both 400nm;

[0017] c5) adding a solution containing a quenching substance so that the concentration of the quenching substance is between 0 and 1000 μmol / L;

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

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

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

[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 value of the measured background solution to make the pH value of the background solution the same as the pH value of the natural dissolved organic matter test solution in step c2, stirring and keeping the pH value stable 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 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 of the titration experiment 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) Substitute the F calculated in formula (1) into 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; F end is the fluorescence intensity fitted by the natural dissolved organic matter when the titration is saturated; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of the natural dissolved organic matter in the measured liquid 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 the solid sample of natural dissolved organic matter, dissolve it with a strong base under inert gas protection, adjust it to a specific pH value with a strong acid and strong alkali solution, the pH value range is between 3-10, filter it with a glass fiber filter membrane, adjust the ionic strength with a salt solution, and 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 value of the background solution to the same as the pH value of the natural dissolved organic matter test solution in b2, stir and keep 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 the 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, determine the excitation wavelength and emission wavelength when the fluorescence intensity is maximum, and use them as the excitation wavelength and emission wavelength during 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, and 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 5-20mg / 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 0.1-1000mmol / 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 substances added during the titration of step c is not greater than 1‰ of the volume of the natural dissolved organic matter solution tested in step c1; the cumulative volume of the quenching substances added during the titration of step d is not greater than 1‰ of the volume of the background solution in step d1, and the concentration of the quenching substances in step c5 is 0-400 μmol / L, preferably 0-100 μmol / L.

[0051] Preferably, the solution in steps b, c and d is adjusted to a specific pH value (error is ±0.02), the pH value range is between 5-7, and the acid solution used to adjust the pH value is one solution of perchloric acid, hydrochloric acid, sulfuric acid, nitric acid solution or a mixed solution of more than one; the alkaline solution used to adjust the pH value is one solution of sodium hydroxide and potassium hydroxide solution or a mixed solution of more than one; 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 natural dissolved organic matter solution 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. The temperature during the titration process of steps c and d is 20-35°C; the fluorescence determination of steps b, c and d is carried out 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 has low requirements on equipment. 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 ion strength during the fluorescence determination of natural dissolved organic matter, and the influence of its concentration and ion type on the determination result can be basically ignored, thereby improving the detection effect.

[0055] (3) In the process of preparing the natural dissolved organic matter test liquid provided by the present invention, a strong base is used to dissolve the natural dissolved organic matter solid under the protection of inert gas, 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, and at the same time avoid the phenomenon that the quenching substance combines with the free hydroxyl groups existing in large quantities 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 detection results.

[0057] (5) The method of measuring the molecular weight of natural dissolved organic matter by fluorescence spectroscopy provided by the present invention requires that a natural dissolved organic matter molecule is combined with a quenching substance in the reaction system. When the quenching substance causes the natural dissolved organic matter molecules to further flocculate, it will cause a sharp increase in the scattering intensity. Therefore, the present invention requires that I<2I0 in step c.

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

[0060] Example 1

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

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

[0063] Weigh 100.17 mg of natural dissolved organic matter solid sample, dissolve it with 2 mL of 0.1 M potassium hydroxide strong base solution, dilute it with deionized water to about 1 L, adjust it to a neutral pH of 5-7 with hydrochloric acid, filter it with a glass fiber membrane with a pore size of 0.45 μm, add 138.55 g of potassium perchlorate solid after filtration and dissolve it, and dilute it to 10 L with deionized water to obtain the natural dissolved organic matter test solution. The mass difference of the glass fiber membrane before and after filtration is 1.50 mg.

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

[0065] The glass fiber filter membrane is burned at 450°C-550°C for 5-8 hours before use; it is dried at 60°C-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 keep 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 200-600nm;

[0070] b4) measuring the background solution;

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

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

[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, determining the excitation wavelength and emission wavelength when the fluorescence intensity is maximum, and using them as the excitation wavelength and emission wavelength during fluorescence quenching titration.

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

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

[0076] c2) Adjust the pH of the natural dissolved organic matter test solution to 6, stir and keep the pH value 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) Determine the light scattering intensity I0 of the natural dissolved organic matter test solution when the excitation wavelength and emission wavelength are both 400nm;

[0079] c5) adding a solution containing a quenching substance so that the concentration of the quenching substance is between 0 and 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 keeping the pH value stable for 60 minutes;

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

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

[0083] c9) Repeat the above steps c5) to 8) until I>2I0 and stop step c.

[0084] d: Background solution fluorescence measurement

[0085] d1) measuring the background solution;

[0086] d2) adjusting the pH value of the background solution to 6±0.02 to the same as step c, stirring and keeping the pH value 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 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 of the titration experiment 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) Substitute the F calculated in formula (1) end Substitute into formula (2) to find 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; F endis the fluorescence intensity fitted by the natural dissolved organic matter when the titration is saturated; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of the natural dissolved organic matter in the measured liquid 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 dissolved organic matter. Wherein, the mass volume concentration of natural dissolved organic matter in the test liquid in step c1) is m=(100.17mg-1.50mg) / 10L=9.87mg / 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 spectroscopic method for determining the molecular weight of natural dissolved organic matter, the method is basically the same as Example 1, the difference being that:

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

[0101] The natural dissolved organic matter test solution was prepared with a concentration of 19 mg / L, and the background solution was 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 200-600nm;

[0106] b4) measuring the background solution;

[0107] b5) adjusting the pH value of the background solution to 8.2, stirring and keeping the pH value stable for 20 minutes;

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

[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, determining the excitation wavelength and emission wavelength when the fluorescence intensity is maximum, and using them as the excitation wavelength and emission wavelength during fluorescence quenching titration.

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

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

[0112] c2) Adjust the pH value 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) Determine the light scattering intensity I0 of the natural dissolved organic matter test solution when the excitation wavelength and emission wavelength are both 400nm;

[0115] c5) adding a solution containing a quenching substance so that the concentration of the quenching substance is between 0 and 200 μmol / L;

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

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

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

[0119] c9) repeating steps c5) to 8) until I>2I0 and stopping step c;

[0120] d: Background solution fluorescence measurement

[0121] d1) measuring the background solution;

[0122] d2) adjusting the pH value of the background solution to 8.2 to the same as step c, stirring and keeping the pH value stable 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 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 of the titration experiment 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 spectroscopic method for determining the molecular weight of natural dissolved organic matter, the method is basically the same as Example 1, the difference being that:

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

[0129] Prepare a natural dissolved organic matter test solution with a concentration of 3 mg / L, and the background solution is a 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 liquid,

[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, determining the excitation wavelength and emission wavelength when the fluorescence intensity is maximum, and using them as the excitation wavelength and emission wavelength during fluorescence quenching titration.

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

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

[0140] c2) Adjust the pH value of the natural dissolved organic matter test solution to 4.5, stir and keep the pH value 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) Determine the light scattering intensity I0 of the natural dissolved organic matter test solution when the excitation wavelength and emission wavelength are both 400nm;

[0143] c5) adding a solution containing a quenching substance so that the concentration of the quenching substance is between 0 and 400 μmol / L;

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

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

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

[0147] c9) repeating steps c5) to 8) until I>2I0 and stopping step c;

[0148] d: Background solution fluorescence measurement

[0149] d1) measuring the background solution;

[0150] d2) adjusting the pH value of the background solution to 4.5, the same as step c, stirring and keeping the pH value stable 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 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 of the titration experiment 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. After 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 935g / mol (International Humic Acid Association standard 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 tyrosine solid sample, dissolve it with deionized water, filter it with a glass fiber membrane with a pore size of 0.45 μm, prepare a 1L tyrosine concentrated solution, filter the tyrosine concentrated solution, and the mass difference of the glass fiber membrane before and after filtration is 1.20 mg; add 138.55 g of potassium perchlorate solid to the filtered tyrosine concentrated solution and dissolve it, and dilute it to 10L with deionized water to obtain the tyrosine test solution.

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

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

[0161] b1) measuring the tyrosine test solution;

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

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

[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, setting the Raman and Rayleigh scattering intensities to zero, determining the excitation wavelength and emission wavelength when the fluorescence intensity is maximum, and using them as the excitation wavelength and emission wavelength of 350 nm, respectively, during fluorescence quenching titration.

[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 a stable pH value 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 the excitation wavelength and the emission wavelength are both 400 nm;

[0173] c5) adding a quenching substance to make the concentration of the quenching substance between 0 and 1000 μmol / L;

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

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

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

[0177] c9) Repeat steps c5) to 8) until I>2I0 and stop step c.

[0178] d: Background solution fluorescence measurement

[0179] d1) measuring the background solution;

[0180] d2) adjusting the pH value of the background solution to 6, the same as step c, stirring and keeping the pH value stable 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 to make the concentration of the quenching substance in the background solution 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 of the titration experiment 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) Substitute the F calculated in formula (1) end Substitute into formula (2) to find 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; F end is the fluorescence intensity fitted by the natural dissolved organic matter when the titration is saturated; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of the natural dissolved organic matter in the measured liquid 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] Wherein, the mass concentration of the tyrosine test solution in the above 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 provided by the present invention, the pH value of the measured liquid, the concentration of the cumulatively added copper ions, the concentration of the measured liquid and other parameters are changed during the titration process, and the measured molecular weight of tyrosine is 178.1-185.4 g / mol, which has an error of less than 5% compared with the actual molecular weight of tyrosine (181.20 g / mol).

[0195] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter, characterized in that: The fluorescence spectroscopic method for determining the molecular weight of natural 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; (d) Fluorescence measurement of background solution; (e) Calculation of molecular weight of natural dissolved organic matter.

2. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 1, characterized in that: Step c comprises the following steps: c1) measuring the natural dissolved organic matter test solution in step a; c2) adjusting the measured natural dissolved organic matter test solution to a specific pH value, the pH range is between 3-10, stirring and keeping 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) Determine the light scattering intensity I0 of the natural dissolved organic matter test solution when the excitation wavelength and emission wavelength are both 400nm; c5) adding a solution containing a quenching substance so that the concentration of the quenching substance is between 0 and 1000 μmol / L; c6: adjusting the pH value of the natural dissolved organic matter test solution to the same value as that 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 fluorescence peak position of the titration experiment determined in step b; c8) Determine the light scattering intensity I of the natural dissolved organic matter test solution when the excitation wavelength and emission wavelength are both 400nm; c9) Repeat steps c5 to c8 until I>2I0 and stop step c.

3. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 2, characterized in that: The background solution fluorescence measurement process in step d is: d1) measuring the background solution in step a; d2) adjusting the pH value of the measured background solution to make the pH value of the background solution the same as the pH value of the natural dissolved organic matter test solution in step c2, stirring and keeping the pH value stable for 15-60 minutes; d3) measuring the fluorescence intensity Fr0' of the background solution at the fluorescence peak position 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 of the titration experiment determined in step b.

4. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 3, characterized in that: In step e, the specific process of calculating the molecular weight of natural dissolved organic matter is: e1) Calculate F0 = F0' - Fr0' and calculate F = F' - Fr' e2) Use formula (1) to calculate F end : e3) Use the F calculated in formula (1) end Substitute into formula (2) to find the molecular weight M of natural dissolved organic matter: Among them, C Q is the total concentration of the quenching substance added in step c5 during the titration process; F end is the fluorescence intensity fitted by the natural dissolved organic matter when the titration is saturated; α is a constant greater than 0; K is the conditional equilibrium constant; m is the mass volume concentration of the natural dissolved organic matter in the measured liquid in step c1; M is the molecular weight of the natural dissolved organic matter.

5. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 3 or 4, characterized in that: The preparation process of the natural dissolved organic matter test solution in step a is: a1) Weigh a solid sample of natural dissolved organic matter, dissolve it with a strong base under inert gas protection, adjust it to a specific pH value with a strong acid and a strong alkali solution, the pH value range is between 3-10, filter it with a glass fiber filter membrane, adjust the ionic strength with a salt solution, and prepare a natural dissolved organic matter 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: a3) Take 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.

6. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 5, characterized in that: The process of selecting the fluorescence peak position of the titration experiment in step b is: b1) Measure the natural dissolved organic matter test solution; b2) Adjust the pH value of the natural dissolved organic matter test solution to a specific value, the pH range is between 3-10, stir and keep 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 200-600nm; 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 keep the pH value stable for 15-60 minutes; b6) scanning the three-dimensional fluorescence spectrum of the background solution, wherein the excitation wavelength and the emission wavelength scanning range are both 200-600 nm; 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, determine the excitation wavelength and emission wavelength when the fluorescence intensity is maximum, and use them as the excitation wavelength and emission wavelength during fluorescence quenching titration.

7. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 6, 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.

8. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 7, 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 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 5-20mg / 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 solution, and the salt concentration of the natural dissolved organic matter test solution and the background solution is 0.1-1000mmol / L.

9. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 8, characterized in that: The quenching substances added in steps c and d include but are not limited to copper, mercury ions, etc.; the cumulative volume of the solution containing the quenching substance added during the titration process of step c is not greater 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 process of step d is not greater than 1‰ of the volume of the background solution in step d1, and the concentration of the quenching substance in step c5 is generally 0-400μmol / L.

10. A fluorescence spectrometry method for determining the molecular weight of natural dissolved organic matter according to claim 9, characterized in that: The solutions in steps b, c and d have the same specific pH value (with an error of ±0.02), the pH value range is between 5-7, and the solution used to adjust the pH value is one solution or a mixed solution of more than one of 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 in step c to adjust the pH value is not greater than 1‰ of the volume of the natural dissolved organic matter solution to be tested in step c1; the cumulative volume of the acid and / or alkaline solution added in step d to adjust the pH value is not greater than 1‰ of the background solution volume in step d1.

Images