Method for determining trace hydroxyl radicals based on sodium nitrite color development and application

Abstract

The invention provides a trace hydroxyl radical determination method based on sodium nitrite color development and application, and belongs to the field of chemometrics. The method comprises the following steps: constructing two reaction systems, namely a system based on a mixed solution of 2, 3-DHBA and 2, 5-DHBA and a. OH sample determination reaction system based on H2O2 concentration regulation, reacting under specific conditions, and adding an HCl solution to terminate the reaction; the method comprises the following steps: measuring the absorbance, establishing a calibration equation according to the absorbance of a mixed solution system of 2, 3-DHBA and 2, 5-DHBA, establishing a calculation equation according to the absorbance of a H2O2 concentration regulation and control system, establishing a reference equation in combination with an electron spin resonance measurement result, verifying the accuracy of the method through data crosslinking, and finally substituting the accuracy into a related equation to obtain the concentration of. OH in a sample to be detected. According to the method, the nitrosation capacity of NaNO2 is combined with hydroxylation capture of DHBA, and a unique color development path is constructed. The method has the characteristics of simplicity and convenience in operation, low cost and high sensitivity, and is suitable for quantitative analysis of. OH in stoichiometry.

Description

Technical Field

[0001] The present invention relates to a method for determining trace hydroxyl radicals based on the color reaction of sodium nitrite and its application, belonging to the field of chemometrics. Background Art

[0002] As one of the most reactive oxidative species, the detection of the concentration of hydroxyl radicals (·OH) has important application values in many fields such as environmental catalysis, advanced oxidation processes, and biomedicine. Currently, the detection of ·OH in the industry mainly relies on techniques such as electron spin resonance (ESR), high-performance liquid chromatography (HPLC), laser-induced fluorescence (LIF), gas chromatography (GC), and fluorophotometry. However, due to the following inherent characteristics of ·OH: (1) special activity with a standard oxidation potential as high as 2.8 V; (2) the concentration in the liquid phase system is usually in the order of μmol / L to nmol / L; (3) the half-life of free radicals is generally less than 1 μs, there are still significant technical bottlenecks in the test sensitivity, operation economy, and detection reliability of existing detection methods.

[0003] Specifically, the conventional detection schemes face three challenges: First, although ESR detection can directly capture the signals of paramagnetic free radicals, it is limited by the complexity of the pretreatment of cryogenic capturers and the high cost of superconducting magnet equipment; second, chromatographic coupling techniques rely on multi-step derivatization reactions to indirectly determine the end products, and quantitative errors often occur due to uncertain secondary reaction paths; third, the fluorescence detection system is easily interfered by the quenching effect in the test matrix, and the basic signal-to-noise ratio is difficult to meet the requirements of trace detection.

[0004] In view of the above technical pain points, it is very necessary to provide a new determination method. Summary of the Invention

[0005] In order to achieve the above object, the present invention provides a method for determining trace hydroxyl radicals based on the color reaction of sodium nitrite (NaNO2) and its application.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The present invention provides a method for determining trace hydroxyl radicals based on the color reaction of sodium nitrite. The determination method includes the following steps: (1) Mix an Fe 2+ solution, a C7H6O3 solution, and a NaNO2 solution, then add H2O2 solutions with different concentrations to initiate the reaction, and terminate the reaction with an HCl solution after the reaction is completed to obtain system A; Another Fe 2+The solution, C7H6O3 solution and NaNO2 solution are mixed, and after adding different concentrations of DHBA standard solution, HCl solution is added to obtain system B; wherein, the DHBA standard solution is composed of a mixture of 2,3-DHBA solution and 2,5-DHBA solution; (2) Measure the absorbance of the system A and system B respectively, establish a calibration equation according to the absorbance data of the system B, establish a calculation equation according to the absorbance data of the system A, and establish a reference equation in combination with the measurement results of electron spin resonance; (3) Combine data cross-linking verification, substitute the absorbance of the sample solution to be measured into the corresponding equation (specifically, fitting equation II or fitting equation III) to obtain the concentration of ·OH in the sample solution to be measured. It should be noted that data cross-linking is to compare the relationship between the ·OH concentrations obtained by ultraviolet spectrophotometry and ESR method under the same H2O2 concentration. When the relative error satisfies P<0.05, it proves that the method is accurate and reliable.

[0008] Further, in step (1), the concentration range of the Fe 2+ solution is greater than 0 and less than or equal to 2 mM; the concentration of the C7H6O3 solution is 3 to 8 times that of the Fe 2+ solution; the concentration of the NaNO2 solution is 20 to 30 times that of the Fe 2+ solution.

[0009] Further, in step (1), the concentration range of the H2O2 solution is 0 to 2 mM; different concentrations are obtained by gradient dilution, specifically including a series of concentrations of 0 mM, 0.125 mM, 0.250 mM, 0.500 mM, 1.000 mM, 2.000 mM; the concentration range of the DHBA standard solution is 0 to 2 mM; wherein, the DHBA standard solution includes 2,3-DHBA solution and 2,5-DHBA solution with a volume ratio of 1:1; the concentration of the HCl solution is 0.4 to 0.6 M.

[0010] Further, the measurement method specifically includes: sequentially adding 2 mL of 2 mM Fe 2+ solution, 2 mL of 50 mM NaNO2 solution, 2 mL of 8 mM C7H6O3 solution and 2 mL of H2O2 solution with different concentrations into a reaction flask, and reacting for 35 to 45 min in a nitrogen-light-shielded environment at 35 to 45 °C to obtain the system A; sequentially adding 2 mL of 2 mM Fe 2+ solution, 2 mL of 50 mM NaNO2 solution, 2 mL of 8 mM C7H6O3 solution and 2 mL of DHBA standard solution with different concentrations into a reaction flask, and reacting for 35 to 45 min in a nitrogen-light-shielded environment at 35 to 45 °C to obtain the system B; measuring the absorbance of the system A and system B, and the sample solution to be measured at a wavelength of 405 to 415 nm by ultraviolet spectrophotometry.

[0011] Further, the calibration equation in step (2) is y = 1.81x + 0.045, R 2 = 0.998; where x is the concentration of the DHBA standard solution, in mM, and y is the absorbance measured at a wavelength of 410 nm.

[0012] Further, the calculation equation in step (2) is y = 0.042x + 0.046, R 2 = 0.998; where x is the concentration of the H2O2 solution, in mM, and y is the absorbance measured at a wavelength of 410 nm.

[0013] Further, the reference equation in step (2) is y = 1.85×10 -5 x - 2.8×10 -7 , R 2 = 0.997; where x is the concentration of the H2O2 solution, in mM, and y is the ·OH concentration, in mol / L.

[0014] Further, the determination method further includes: separately preparing solutions of different concentrations of Fe 2+ solution, C7H6O3 solution, NaNO2 solution, H2O2 solution, HCl solution, 2,3-DHBA and 2,5-DHBA solution.

[0015] Among them, in the determination method, in step (2), for a fixed Fe 2+ concentration of 2 mM, for different concentrations of H2O2 solutions (0.5 mM, 1.0 mM, and 2.0 mM), electron spin resonance (ESR) measurements are respectively carried out to obtain the corresponding ESR spectra and ·OH concentrations for verifying the accuracy of the results.

[0016] In the determination method, in the DHBA standard solution in step (2), the 2,3-DHBA and 2,5-DHBA solutions should be accurately prepared by diluting an appropriate amount of the stock solution and uniformly mixed strictly according to a volume ratio of 1:1 to ensure the uniform distribution of the two compounds. This mixed standard solution is used for the calibration of spectrophotometric determination.

[0017] Further, the sample to be measured includes samples in the fields of environmental monitoring, biomedical research, food safety assessment, and industrial process control.

[0018] The present invention also provides the application of the above-mentioned method for determining trace hydroxyl radicals based on sodium nitrite color development in quantitatively determining the content of ·OH in a sample or during the sample production process.

[0019] The principle of a method for determining ·OH using NaNO2 as a color developer provided by the present invention is shown as follows:

[0020] Using H2O2 and Fe 2+ The interaction of OH and its reaction products:

[0021] H2O2+Fe 2+ → OH+OH - +Fe 3+

[0022] OH reacts with C7H6O3 to produce 2,3-DHBA and 2,5-DHBA:

[0023] OH+C7H6O3→2,3-DHBA

[0024] OH+C7H6O3→2,5-DHBA

[0025] When NaNO2 is introduced, the substance hydrolyzes to generate HNO2 and OH - :

[0026]

[0027] In 4 <pH<7条件下,HNO2与2,3-DHBA和2,5-DHBA反应生成N-nitroso-2,3-DHBA和N-nitroso-2,5-DHBA:

[0028] HNO2+2,3-DHBA→N-nitroso-2,3-DHBA

[0029] HNO2+2,5-DHBA→N-nitroso-2,5-DHBA

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention provides a method and application for the determination of trace hydroxyl free radicals based on sodium nitrite color development. This method combines the nitrosation ability of NaNO2 with the capture of hydroxylation by DHBA to construct a unique color development pathway. In this method, C7H6O3 is used to capture ·OH to generate 2,3-DHBA and 2,5-DHBA. NaNO2 reacts with 2,3-DHBA and 2,5-DHBA to generate N-nitroso-2,3-DHBA and N-nitroso-2,5-DHBA. The absorbance of the test solution is measured using an ultraviolet spectrophotometer, thereby achieving a method for quantitatively determining the ·OH concentration.

[0032] 2. The method of the present invention has simple operation steps, does not require complicated pre-treatment steps, is suitable for rapid analysis, can accurately determine ·OH in a short time, and is suitable for routine detection in laboratories and industries.

[0033] 3. The method of the present invention can significantly improve the efficiency and reliability of ·OH determination, meet the requirements of various industries for ·OH detection, be applicable to the determination of ·OH in different concentration ranges, and have broad application prospects. Description of the Drawings

[0034] One or more embodiments are exemplarily illustrated by the pictures in the corresponding drawings. These exemplary illustrations do not constitute limitations on the embodiments. Unless otherwise stated, the figures in the drawings do not constitute a proportional limitation.

[0035] Figure 1 is the ESR spectrum of ·OH produced by the reaction of 0.5 mM H2O2 with 2 mM Fe 2+ reaction;

[0036] Figure 2 is the ESR spectrum of ·OH in the reaction of 1.0 mM H2O2 with 2 mM Fe 2+ reaction;

[0037] Figure 3 is the ESR spectrum of ·OH in the reaction of 2.0 mM H2O2 with 2 mM Fe 2+ reaction. Detailed Embodiments

[0038] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention is provided in conjunction with specific examples. In the following specific embodiments, for the parts where specific experimental steps or conditions are not specified, the standard experimental steps or conditions described in the conventional literature in the field can be referred to for operation. For the reagents or instruments whose manufacturers are not specified, they are all commercially available conventional reagent products. Unless otherwise stated, in the following embodiments of the present invention, the reagent drugs used are all of analytical grade.

[0039] 1. Theoretical Explanation of the Reaction Mechanism

[0040] The determination of ·OH in the present invention is based on the synergistic effect of a series of chemical reactions. First, the generation of ·OH is mainly achieved through the classical Fenton reaction, and the generated ·OH can be captured by salicylic acid (C7H6O3) to generate 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA).

[0041] The color reagent NaNO2 used in the present invention will undergo hydrolysis reaction in aqueous solution to generate nitrous acid (HNO2) and hydroxide ions (OH -) Under acidic conditions (4 < pH < 7), HNO2 has a certain activity and can react with the generated 2,3-DHBA and 2,5-DHBA to form nitroso derivatives, mainly nitroso-2,3-dihydroxybenzoic acid (N-nitroso-2,3-DHBA) and nitroso-2,5-dihydroxybenzoic acid (N-nitroso-2,5-DHBA).

[0042] 2. Specific measurement method

[0043] The present invention provides a simple, rapid, and accurate method for measuring the concentration of trace ·OH. This method achieves precise quantification of the ·OH concentration through the following steps.

[0044] First, ·OH is generated through the Fenton reaction, and its reaction equation is:

[0045] H2O2 + Fe 2+ → ·OH + OH - + Fe 3+

[0046] The generated ·OH can be captured by C7H6O3 to produce 2,3-DHBA and 2,5-DHBA, and its reaction equation is:

[0047] ·OH + C7H6O3 → 2,3-DHBA

[0048] ·OH + C7H6O3 → 2,5-DHBA

[0049] During this process, the color reagent NaNO2 used in the present invention will hydrolyze in aqueous solution to generate HNO2 and OH - , and its reaction equation is:

[0050]

[0051] Under ordinary acidic conditions (4 < pH < 7), HNO2 reacts with the generated 2,3-DHBA and 2,5-DHBA to form N-nitroso-2,3-DHBA and N-nitroso-2,5-DHBA:

[0052] HNO2 + 2,3-DHBA → N-nitroso-2,3-DHBA

[0053] HNO2 + 2,5-DHBA → N-nitroso-2,5-DHBA

[0054] Finally, after using HCl to terminate the reaction, the absorbance of the generated nitroso derivatives is measured by ultraviolet spectrophotometry and compared with the established standard curve, thereby achieving the quantification of the trace ·OH concentration.

[0055] Example 1:

[0056] 1. Prepare the ·OH detection reagent:

[0057] (1) Prepare the Fe 2+ solution: Accurately weigh an appropriate amount of ferrous salt (using FeSO4·7H2O) and prepare a 2 mM Fe 2+ solution, and make up the volume with a suitable volumetric flask.

[0058] (2) Prepare the C7H6O3 solution: According to the given ratio, the concentration of the C7H6O3 solution should be 3 - 8 times that of the Fe 2+ solution. In this example, prepare an 8 mM C7H6O3 solution. Accurately weigh C7H6O3, dissolve it in an appropriate amount of absolute ethanol, and then make up the volume with absolute ethanol. Salicylic acid has better solubility in ethanol and has no special influence on subsequent reactions.

[0059] (3) Prepare the NaNO2 solution: The concentration of the NaNO2 solution should be 20 - 30 times that of the Fe 2+ solution. In this example, prepare a 50 mM NaNO2 solution, dissolve the NaNO2 solid in deionized water, and then make up the volume.

[0060] (4) Prepare the H2O2 solution: The maximum concentration is the same as that of the Fe 2+ solution, and the other concentrations are obtained by gradient dilution. Prepare H2O2 solutions with concentrations of 0 mM, 0.125 mM, 0.250 mM, 0.500 mM, 1.000 mM, and 2.000 mM. Accurately measure the concentrated hydrogen peroxide solution, dilute it with deionized water, and accurately calculate the concentration.

[0061] (5) Prepare the HCl solution: Prepare an HCl solution with a concentration of 0.4 - 0.6 M. In this example, prepare a 0.5 M HCl solution. Measure the concentrated HCl, slowly add it to deionized water, and make up the volume after cooling.

[0062] (6) Prepare the 2,3 - DHBA and 2,5 - DHBA solutions: Prepare solutions with concentrations of 0 mM, 0.125 mM, 0.250 mM, 0.500 mM, 1.000 mM, and 2.000 mM. Accurately prepare them by diluting an appropriate amount of the stock solution, and strictly mix them evenly according to a volume ratio of 1:1 to obtain the DHBA standard solution.

[0063] 2. Construct a reaction system based on 2,3 - DHBA and 2,5 - DHBA:

[0064] (1) Take a reaction flask and successively add 2 mL of 2 mM Fe 2+ solution, 2 mL of 50 mM NaNO2 solution, 2 mL of 8 mM C7H6O3 solution, and 2 mL of DHBA standard solutions with different concentrations.

[0065] (2) Place the reaction flask in a nitrogen environment at 40 °C and react for 40 min. Finally, add 2 mL of 0.5 M HCl solution.

[0066] 3. Reaction system for the determination of ·OH samples based on the regulation of H2O2 concentration:

[0067] (1) Take a reaction flask and sequentially add 2 mL of 2 mM Fe 2+ solution, 2 mL of 50 mM NaNO2 solution, 2 mL of 8 mM C7H6O3 solution, and 2 mL of H2O2 solutions with different concentrations (such as 0 mM, 0.125 mM, 0.25 mM, 0.5 mM, 1.0 mM, and 2.0 mM).

[0068] (2) Place the reaction flask in a nitrogen environment at 40 °C and react for 40 min. After the reaction is completed, add 2 mL of 0.5 M HCl solution to terminate the reaction.

[0069] 4. Absorbance measurement:

[0070] (1) Use a UV-visible spectrophotometer to measure the absorbance of the sample solution and the standard solution at a wavelength of 410 nm, respectively.

[0071] (2) Establish a fitting equation II (calibration) based on the absorbance data of the standard solution (Table 1).

[0072] (3) Establish a fitting equation III (calculation) based on the absorbance data of the sample solution (Table 2).

[0073] Table 1 Data of mixed standard solutions of 2,3-DHBA and 2,5–DHBA with different concentrations

[0074]

[0075] Table 2 Data of hydrogen peroxide with different concentrations

[0076]

[0077]

[0078] 5. ESR measurement and result comparison:

[0079] (1) ESR measurement and data acquisition:

[0080] Detect the above reaction schemes of different H2O2 concentrations (0.5 mM, 1.0 mM, and 2.0 mM) reacting with 2 mM ferrous ions using an electron spin resonance (ESR) spectrometer. According to the standard ESR detection procedure, measure each sample under appropriate instrument parameter conditions to obtain the corresponding ESR spectra. Among them,Figure 1 is the ESR spectrum of ·OH generated by the reaction of 0.5 mM H2O2 with 2 mM Fe 2+ ; Figure 2 is the ESR spectrum of ·OH generated by the reaction of 1.0 mM H2O2 with 2 mM Fe 2+ ; Figure 3 is the ESR spectrum of ·OH generated by the reaction of 2.0 mM H2O2 with 2 mM Fe 2+ . By analyzing the ESR spectrum and calculating according to the standard method of ESR quantitative analysis, the corresponding ·OH concentration data are obtained. These data include the ESR spectrum of each sample and the corresponding ·OH concentration value. The specific content is as follows:

[0081] Table 3 Hydrogen peroxide at different concentrations

[0082]

[0083] (2) Explanation of method verification:

[0084] In the study of the ·OH determination method of the present invention, the key to ensuring the accuracy and reliability of the new method lies in comparing and verifying the results obtained by different methods, especially comparing the ultraviolet spectrophotometry method with the electron spin resonance (ESR) method. This process involves in-depth analysis of two different reaction systems in the measurement process of ultraviolet spectrophotometry. Each system has unique variables and measurement purposes, and these measurement results can provide a data basis for the establishment of subsequent fitting equations to comprehensively evaluate the accuracy and reliability of the determination method of the present invention.

[0085] The concentration of the 2,3-DHBA and 2,5-DHBA mixed solution as a variable in step 2 of this example is of great significance. This system is independent of the Fenton reaction and uses externally configured 2,3-DHB A and 2,5-DHBA mixed solutions with different concentrations (ranging from 0 to 2 mM). In this system, its concentration is intrinsically related to the amounts of 2,3-DHBA and 2,5-DHBA generated by the capture of ·OH by salicylic acid in the sample determination reaction system.

[0086] When using ultraviolet spectrophotometry to detect the absorbance at a wavelength of 410 nm, it can be observed that the absorbance shows a significant linear law with the change of the concentration of the 2,3-DHBA and 2,5-DHBA mixed solution. This change lays a foundation for constructing the quantitative relationship between the concentration of the 2, and 5-DHBA mixed solution and the absorbance. This quantitative relationship is a key link in calculating the ·OH concentration. The principle is that through this quantitative relationship, the amounts of free radicals captured by salicylic acid to generate 2,3-DHBA and 2,5-DHBA in the sample determination reaction system can be inversely calculated based on the absorbance value.

[0087] In the reaction system of step 3 of this embodiment, the change in the concentration of the H2O2 solution is the core factor. As the key reactant for the generation of ·OH in the Fenton reaction, the concentration of H2O2 directly determines the amount of ·OH generated. By measuring the absorbance of the reaction solution at different H2O2 concentrations at a wavelength of 410 nm using ultraviolet spectrophotometry, a clear correlation between the absorbance and the H2O2 concentration can be observed. This correlation provides a key perspective for understanding the internal relationship between the amount of ·OH generated and the experimental conditions, and constitutes an important basis for calculating the concentration of ·OH. Accordingly, the amount of ·OH generated under different experimental conditions can be inferred reversely based on the absorbance value, and then the accurate calculation of the ·OH concentration can be achieved.

[0088] (3) Data cross-linking and method verification

[0089] Samples after the reaction of different H2O2 concentrations with 2 mM ferrous ions were detected using an ESR instrument. The ESR technique detects free radicals with high precision based on the electron spin characteristics, obtains the ESR spectrum according to strict procedures and appropriate parameters, calculates the ·OH concentration data through analysis and the standard method of ESR quantitative analysis, and establishes a fitting equation Ⅰ (i.e., the reference equation described above) with the H2O2 concentration as the independent variable and the ·OH concentration as the dependent variable. This is an important reference for measuring the accuracy of the new method.

[0090] For the reaction system based on 2,3-DHBA and 2,5-DHBA, the concentration of the mixed solution of 2,3-DHBA and 2,5-DHBA is used as the independent variable, and the absorbance measured by ultraviolet spectrophotometry is used as the dependent variable. Multiple groups of experimental data were collected and sorted out, and a fitting equation Ⅱ (i.e., the calibration equation described above) was established by least squares linear regression. It is the calibration basis for subsequently inferring the concentration of related substances from the absorbance, and is indirectly used for the calculation of the ·OH concentration.

[0091] In the reaction system for the determination of ·OH samples regulated by the H2O2 concentration, with the concentration of the H2O2 solution as the independent variable and the absorbance as the dependent variable, a fitting equation Ⅲ (i.e., the calculation equation described above) was established through the data processing process. This equation reflects the relationship between the concentration of the H2O2 solution and the absorbance. Since there is a direct causal relationship between the H2O2 concentration and the amount of ·OH generated, the relationship between the ·OH concentration and the absorbance can be indirectly established through equation Ⅲ, so as to calculate the ·OH concentration in the sample based on the absorbance.

[0092] Cross-link the data obtained from fitting Equation Ⅱ, fitting Equation Ⅲ, and the fitting Equation Ⅰ obtained by ESR measurement to comprehensively evaluate the performance of the new measurement method based on ultraviolet spectrophotometry. Data cross-linking is to compare the relationship of the dependent variable (·OH concentration) obtained by the two methods under the same independent variable (H2O2 concentration). ESR measurement is a high-precision method in the field of free radical detection, and its result can be used as an absolute benchmark. By comparing the ·OH concentrations predicted by different fitting equations at the same H2O2 concentration, statistical indicators such as relative error are calculated. The relative errors of the two methods at each concentration point are reasonable (P < 0.05), which proves that the method of the present invention based on NaN O2 using ultraviolet spectrophotometry is accurate and reliable in ·OH measurement.

[0093] Fitting Equation Ⅰ (reference) based on ESR detection

[0094] y = 1.85×10 -5 x - 2.8×10 -7 (R 2 = 0.997) Ⅰ

[0095] Variable meaning: (Data according to Table 3): The concentration x of the H2O2 solution (unit: mM) is used as the abscissa, and the ·OH concentration y is used as the ordinate (unit: mol / L).

[0096] Fitting Equation Ⅱ based on the reaction system of 2,3-DHBA and 2,5-DHBA

[0097] y = 1.81x + 0.045 (R 2 = 0.998) Ⅱ

[0098] Variable meaning: (Data according to Table 1): The concentration x of the mixed solution of 2,3-DHBA and 2,5-DHBA (unit: mM) is used as the abscissa, and the absorbance y measured at a wavelength of 410 nm is used as the ordinate.

[0099] Fitting Equation Ⅲ based on the ·OH sample determination reaction system regulated by H2O2 concentration

[0100] y = 0.042x + 0.046 (R 2 = 0.998) Ⅲ

[0101] Variable meaning: (Data according to Table 2): The concentration x of the H2O2 solution (unit: mM) is used as the abscissa, and the absorbance y measured at a wavelength of 410 nm is also used as the ordinate.

[0102] 6. OH concentration calculation formula and derivation:

[0103] (1) Fitting Equation Ⅰ based on ESR (reference)

[0104] Equation: y = 1.85×10-5 x - 2.8×10 -7 (R 2 = 0.997)

[0105] Meaning of variables: x is the concentration of H2O2 solution (unit: mM), and y is the concentration of ·OH (unit: mol / L).

[0106] Derivation: Obtained by using an electron spin resonance (ESR) spectrometer to detect samples after the reaction of different H2O2 concentrations with 2 mM ferrous ions. The ESR technique is based on the electron spin characteristics. According to strict procedures and appropriate parameters, an ESR spectrum is obtained. Through professional analysis and the standard method of ESR quantitative analysis, the concentration data of ·OH is calculated, and an equation is established based on this, which serves as an important reference for measuring the accuracy of the new method.

[0107] (2) Fitting equation Ⅱ (calibration) based on the 2,3 - DHBA and 2,5 - DHBA systems

[0108] Equation: y = 1.81x + 0.045 (R 2 = 0.998)

[0109] Meaning of variables: x is the concentration of the mixed solution of 2,3 - DHBA and 2,5 - DHBA (unit: mM), and y is the absorbance measured at a wavelength of 410 nm.

[0110] Derivation: In this system, the concentration of the mixed solution of 2,3 - DHBA and 2,5 - DHBA is used as a variable. In the experiment, the absorbance is detected by ultraviolet spectrophotometry at a wavelength of 410 nm, and it is found that the absorbance shows a significant linear pattern with the change of the mixed solution concentration. This is because there is an internal relationship between the concentration of the mixed solution in this system and the amount of 2,3 - DHBA and 2,5 - DHBA generated by the capture of ·OH by salicylic acid in the sample determination reaction system. After collecting multiple sets of experimental data, an equation is established by linear regression using the least - squares method. This equation can be used to reverse - calculate the concentration of the mixed solution based on the absorbance, and then infer the amount of 2,3 - DHBA and 2,5 - DHBA generated in the sample determination reaction system based on their relationship, providing a basis for calculating the concentration of ·OH.

[0111] (3) Fitting equation Ⅲ (calculation) for the ·OH sample determination reaction system based on the regulation of H2O2 concentration

[0112] Equation: y = 0.042x + 0.046 (R 2 = 0.998)

[0113] Meaning of variables: x is the concentration of H2O2 solution (unit: mM), and y is the absorbance measured at a wavelength of 410 nm.

[0114] Derivation: In this reaction system, H2O2 is the key reactant for generating ·OH in the Fenton reaction, and its concentration directly determines the amount of ·OH generated. By measuring the absorbance of the reaction solution at different H2O2 concentrations at a wavelength of 410 nm using ultraviolet spectrophotometry, a clear correlation was found between the absorbance and the H2O2 concentration. An equation was established through data processing, and this equation reflects the relationship between the concentration of the H2O2 solution and the absorbance. Since there is a direct causal relationship between the H2O2 concentration and the amount of ·OH generated, when the absorbance y is known, the concentration x of the H2O2 solution can be deduced through the equation, and then based on the Fenton reaction H2O2 + Fe 2+ →·OH + OH - + Fe 3+ the stoichiometric relationship is used to calculate the amount of ·OH generated, thereby obtaining the ·OH concentration.

[0115] (4) Data cross-linking to verify the accuracy of the new method

[0116] Principle: The data of the fitting equation II, the fitting equation III and the fitting equation I obtained by ESR measurement are cross-linked to comprehensively evaluate the performance of the new measurement method based on ultraviolet spectrophotometry. Data cross-linking is to compare the relationship between the dependent variables (·OH concentration) obtained by two methods (ultraviolet spectrophotometry and ESR method) under the same independent variable (H2O2 concentration).

[0117] Verification process: ESR measurement is a high-precision method in the field of free radical detection, and its results can be used as an absolute benchmark. By comparing the ·OH concentrations predicted by different fitting equations at the same H2O2 concentration, statistical indicators such as relative error are calculated. When the relative errors of the two methods at each concentration point are reasonable (P < 0.05), it is proved that the present invention based on NaNO2 using ultraviolet spectrophotometry is accurate and reliable in the determination of trace ·OH. When actually calculating the ·OH concentration, according to the specific measurement system (whether it is related to the concentration measurement of the 2,3-DHBA and 2,5-DHBA mixed solution or the H2O2 concentration measurement), the corresponding fitting equation can be used for calculation, and the reliability of the results can be verified by comparing with the ESR method. For example, if the absorbance y and the information related to the concentration of the 2,3-DHBA and 2,5-DHBA mixed solution are known, the relevant intermediate quantities can be deduced first through equation II, and then the ·OH concentration can be calculated in combination with other information; if the absorbance y and the information related to the H2O2 concentration are known, the ·OH concentration can be calculated through equation III and compared with the ESR method (equation I) for verification. The key point here is to accurately calculate the ·OH concentration and verify the reliability of the method through the synergistic effect of the three equations, using the relationship between different reaction systems and measurement data, especially utilizing the causal relationship between H2O2 and ·OH generation and the linear relationship between absorbance and the concentration of related substances in different systems. At the same time, the key role of ESR as a reference method in the verification is emphasized.

[0118] 7. Precautions for experiments:

[0119] (1) Precision of solution preparation: When preparing various solutions, it is necessary to ensure accurate measurement of the mass of the solute and the volume of the solution. Especially during the gradient dilution process, the concentration should be accurately calculated to avoid dilution errors.

[0120] (2) Control of reaction conditions: Strictly control reaction conditions such as temperature, time, and nitrogen environment to ensure complete and stable reactions. Especially keep the conditions consistent when making standard curves and measuring samples.

[0121] (3) Standard operation of instruments: Correctly use instruments such as ultraviolet spectrophotometers and ESR instruments, and calibrate them regularly to ensure the accuracy of measurement results.

[0122] Those of ordinary skill in the art can understand that the above embodiments are specific examples for implementing the present invention. In actual applications, various changes can be made in form and details without departing from the spirit and scope of the present invention. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for determining trace hydroxyl radicals based on the color reaction of sodium nitrite, characterized in that, The determination method includes the following steps: (1) Mix the Fe 2+ solution, C7H6O3 solution and NaNO2 solution, then add H2O2 solutions with different concentrations to initiate the reaction. After the reaction is completed, terminate the reaction with HCl solution to obtain system A; Another, mix the Fe 2+ solution, C7H6O3 solution and NaNO2 solution, add different concentrations of DHBA standard solution and then add HCl solution to obtain System B; among them, the DHBA standard solution is formed by mixing 2,3-DHBA solution and 2,5-DHBA solution; (2) Measure the absorbance of the system A and the system B respectively, establish a calibration equation according to the absorbance data of the system B, establish a calculation equation according to the absorbance data of the system A, and establish a reference equation in combination with the determination results of electron spin resonance; (3) In combination with data cross-linking verification, substitute the absorbance of the sample solution to be measured into the corresponding equation to obtain the concentration of ·OH in the sample to be measured.

2. The method for determining trace hydroxyl radicals based on the color development of sodium nitrite according to claim 1, wherein The Fe described in step (1) 2+ The concentration range of the solution is greater than 0 and less than or equal to 2 mM; The concentration of the C7H6O3 solution is 3 to 8 times that of the Fe 2+ solution concentration; The concentration of the NaNO2 solution is 20 to 30 times that of the Fe 2+ solution concentration.

3. The method for determining trace hydroxyl radicals based on the color reaction of sodium nitrite as described in claim 2, characterized in that, In step (1), the concentration range of the H2O2 solution is 0 to 2 mM; different concentrations are obtained by gradient dilution, specifically including a series of concentrations of 0 mM, 0.125 mM, 0.250 mM, 0.500 mM, 1.000 mM, and 2.000 mM; The concentration range of the DHBA standard solution is 0 to 2 mM; wherein, the DHBA standard solution includes a 2,3-DHBA solution and a 2,5-DHBA solution with a volume ratio of 1:1; The concentration of the HCl solution is 0.4 to 0.6 M.

4. The method for determining trace hydroxyl radicals based on the color reaction of sodium nitrite as claimed in claim 1, wherein, The determination method specifically includes: sequentially adding 2 mL of 2 mM Fe 2+ solution, 2 mL of 50 mM NaNO2 solution, 2 mL of 8 mM C7H6O3 solution, and 2 mL of H2O2 solution with different concentrations into a reaction flask, and reacting for 35 - 45 min in a nitrogen-light-shielded environment at 35 - 45 °C to obtain the system A; Add 2 mL of 2 mM Fe 2+ solution, 2 mL of 50 mM NaNO2 solution, 2 mL of 8 mM C7H6O3 solution and 2 mL of DHBA standard solution with different concentrations into the reaction flask, and react for 35 - 45 min in a nitrogen-light-shielded environment at 35 - 45 °C to obtain the system B; The absorbance of the system A and the system B, and the sample solution to be measured are measured by ultraviolet spectrophotometry at a wavelength of 405 to 415 nm.

5. The method for determining trace hydroxyl radicals based on the color development of sodium nitrite according to claim 4, characterized in that, The calibration equation in step (2) is y = 1.81x + 0.045, R 2 = 0.998; where x is the concentration of the DHBA standard solution in mM and y is the absorbance measured at a wavelength of 410 nm.

6. The method for determining trace hydroxyl radicals based on the color reaction of sodium nitrite according to claim 4, wherein The calculation equation in step (2) is y = 0.042x + 0.046, R 2 = 0.998; where x is the concentration of the H2O2 solution, in mM, and y is the absorbance measured at a wavelength of 410 nm.

7. The method for determining trace hydroxyl radicals based on the color reaction of sodium nitrite as claimed in claim 4, wherein The reference equation in step (2) is y = 1.85×10 -5 x - 2.8×10 -7 , R 2 = 0.997; where x is the concentration of the H2O2 solution, in mM, and y is the ·OH concentration, in mol / L.

8. The method for determining trace hydroxyl radicals based on the color reaction of sodium nitrite according to claim 1, characterized in that, The determination method also includes: separately preparing Fe solutions with different concentrations 2+ solutions, C7H6O3 solutions, NaNO2 solutions, H2O2 solutions, HCl solutions, and DHBA standard solutions.

9. The method for determining trace hydroxyl radicals based on the color development of sodium nitrite according to claim 1, wherein The sample to be measured includes samples in the fields of environmental monitoring, biomedical research, food safety assessment, and industrial process control.

10. Use of the method for determining trace hydroxyl radicals based on sodium nitrite color development according to any one of claims 1 to 9 in quantitatively determining the content of ·OH in a sample or during the sample production process.

Images