A method for identifying organic free radicals in advanced oxidation

Abstract

A method for identifying organic free radicals in advanced oxidation processes. The method involves sampling the effluent after ozone oxidation of ciprofloxacin, and combining the sample with the free radical scavenger 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to form free radical adducts. Subsequently, electron paramagnetic resonance (EPR) spectroscopy is used to detect the presence and evolution of organic free radicals in the reaction system. High-resolution mass spectrometry (HRMS) is used to analyze both the original effluent sample without DMPO and the sample reacted with DMPO, screening and identifying the organic free radical-DMPO adducts and polymerization products, and deducing their chemical structures.

Description

Technical Field

[0001] This invention belongs to the field of environmental analysis technology, and specifically relates to a method for identifying organic free radicals in advanced oxidation processes. Background Technology

[0002] Advanced oxidation processes (AEs) utilize highly reactive oxidizing species such as hydroxyl radicals, sulfate radicals, and ozone to efficiently degrade organic pollutants. During the degradation and oxidation of pollutants, the generation of organic free radical intermediates (such as alkoxy radicals and carbon-center radicals) is common. These free radicals possess unpaired electrons, exhibit high reactivity, and have extremely short lifespans in the environment. Their generation and transformation influence the reaction pathway and treatment efficiency of AE systems. In AE systems, organic free radicals play a crucial role in both mineralization and oxidative polymerization pathways. During mineralization, organic free radicals act as degradation intermediates, participating in and promoting free radical chain reactions, driving the gradual degradation of pollutants until complete mineralization. In the oxidative polymerization pathway, organic free radicals can also serve as precursors for polymer formation, actively driving intermolecular bonding through coupling reactions between free radicals, ultimately forming polymerization conversion products.

[0003] In advanced oxidation processes, oxidants such as hydroxyl radicals and ozone molecules react with pollutants through hydrogen extraction, electron transfer, addition, and Criegee mechanisms. During oxidation, the degradation of piperazines, phenols, and aromatic compounds is accompanied by the generation of ammonia radicals, nitroxide radicals, phenoxy radicals, semiquinone radicals, and carbon-centered radicals.

[0004] However, due to the short lifetime and high reactivity of organic free radicals, their accurate detection and structural characterization remain a technical bottleneck in the study of advanced oxidation processes. The difficulty in directly and effectively capturing and identifying these transient free radicals severely restricts a deeper understanding of the mechanisms of advanced oxidation reactions, intermediate transformation pathways, and product formation mechanisms. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies in directly detecting short-lived, highly reactive organic free radicals during the reaction process, and to provide a method that combines EPR in situ capture with high-resolution mass spectrometry (HRMS) for precise molecular weight determination. By screening and identifying the chemical structures of organic free radical-DMPO adducts and polymerization products, the accurate identification of organic free radicals in advanced oxidation can be indirectly achieved.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for identifying organic free radicals in advanced oxidation processes, the method comprising:

[0008] Step 1: Perform advanced oxidation treatment on the solution containing the target pollutant to obtain the test solution;

[0009] Step 2: Mix the test solution and the free radical scavenger to obtain a mixed solution containing a stable adduct;

[0010] Step 3: Perform EPR and HRMS tests on the mixed solution sequentially to obtain relevant information about the organic free radical adduct.

[0011] Furthermore, in step one, the target pollutant is an antibiotic pollutant, and the concentration of the target pollutant in the solution containing the target pollutant is 0.1~6 mg / L.

[0012] Furthermore, in step one, the advanced oxidation treatment is ozone oxidation, with an ozone concentration of 2-10 mg / L in the solution, an oxidation reaction time of 0-300 s, and a reaction temperature of 0-30 ℃.

[0013] Furthermore, in step two, the free radical scavenger must be kept at a low temperature of 0~5 ℃ before mixing and must not come into contact with oxygen.

[0014] Furthermore, in step two, the free radical scavenger is a commercially available conventional DMPO solution, with a final concentration of 100-500 mmol / L. DMPO refers to 5,5-dimethyl-1-pyrrolline-N-oxide.

[0015] Further, in step three, the EPR test method is as follows: the diode current is stabilized at 200 μA; the measurement is performed at room temperature; the central magnetic field is 3505 G; the scan width is 70.0 G; the microwave power is 20 mW; the scan time is 573.48 s / s; the time constant is 163.84 ms; the conversion time is 163.85 ms; and 12 scans are performed.

[0016] Further, in step three, the HRMS testing method is as follows: the sample is filtered through a 0.22 μm filter membrane before measurement; the chromatographic conditions are as follows: the mobile phase consists of acetonitrile and an aqueous solution containing 0.1% formic acid, the flow rate is 0.3 mL / min, and the sample injection volume is 10 μL; the gradient elution program is as follows: maintain an acetonitrile ratio of 10% for 0-2 minutes, linearly increase the acetonitrile ratio to 100% for 2-11 minutes, maintain it at 100% for 11-13 minutes, rapidly drop back to 10% for 13-13.1 minutes, and maintain this ratio for 20 minutes to allow the column to reequilibrate; mass spectrometry detection uses an electrospray ionization source in positive ion mode (ESI+), and MS spectra with a mass-to-charge ratio (m / z) range of 50-1200 are acquired in full scan mode.

[0017] Furthermore, in step three, the specific methods for obtaining information related to organic free radical adducts include:

[0018] (1) EPR analysis and free radical category identification: The mixed solution containing stable adducts after free radical capture was transferred to a quartz capillary glass tube, and electron paramagnetic resonance spectroscopy analysis was performed under EPR test conditions; the analysis was conducted by using hyperfine splitting parameters (such as the hyperfine coupling constant A of nitrogen). N The coupling constant A of hydrogen Hβ Analysis of the g-factor value and the type of free radical captured can determine the type of free radical captured.

[0019] (2) Identification of free radical adducts: Compare the total ion chromatograms of samples with and without added DMPO, identify the characteristic peaks belonging to free radical-DMPO adducts, and record their precise mass-to-charge ratios;

[0020] (3) Determination of free radical molecular formula: The mass-to-charge ratio of the captured free radical is obtained by subtracting the exact mass number of the DMPO portion from the charge-to-mass ratio of the adduct; the most likely molecular formula of the free radical is determined by using HRMS fitting software within the set mass error range, combined with the constraints of the type and number of heteroatoms in the contamination.

[0021] (4) Free radical structure prediction: Starting from the original molecular structure of the target pollutant, combined with the degradation reaction type of the advanced oxidation process, the molecular structure is systematically pruned and rearranged. By controlling the number of elements such as C, H, O, and N in the target pollutant, the organic free radical structure that is completely matched with the molecular formula and has a reasonable chemical structure is gradually predicted.

[0022] The identification method provided by this invention uses DMPO as a scavenging agent, which can capture and oxidize organic free radicals in water. The resulting organic free radical-DMPO adduct can be clearly identified by MS, and EPR and MS information can be obtained simultaneously to identify the presence, type, and structure of organic free radicals. This method can rapidly analyze the structural information of advanced fluoroquinolone antibiotics and has broad application prospects in environmental catalysis, environmental analysis, and other fields.

[0023] Compared with existing methods, the present invention has the following advantages:

[0024] (1) This invention combines EPR and high-resolution mass spectrometry, which can verify the paramagnetic properties of organic free radicals and accurately determine their molecular weight and elemental composition, thus achieving a complete analysis from existence confirmation to structural identification.

[0025] (2) The present invention has simple steps, clear deduction process, strong applicability, and can quickly screen out organic free radical adducts that meet the requirements from mass spectrometry data and extend it to other pollutant systems. Attached Figure Description

[0026] Figure 1 EPR spectrum of organic free radical-DMPO adduct;

[0027] Figure 2 The mass spectrum of the organic free radical-DMPO adduct;

[0028] Figure 3 This is the mass spectrum of the dimer organic free radical-DMPO adduct. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but this should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made to the present invention by those skilled in the art based on the above description should be included within the scope of protection of the present invention.

[0030] Example 1: EPR detection and mass spectrometric identification of organic radical adducts

[0031] Step 1: Ozone oxidation treatment was performed on a 6 mg / L ciprofloxacin solution. The ozone concentration in the solution was 6 mg / L to obtain the test solution. The oxidation reaction time was 300 s and the reaction temperature was 25~30 ℃.

[0032] Step 2: Mix 50 μl of the test solution and 200 μl of 1 mol / L DMPO solution in a centrifuge tube to obtain a mixed solution containing a stable adduct; the DMPO solution should be kept at a low temperature of 0~5 ℃ and not in contact with oxygen before mixing.

[0033] Step 3: The mixed solution is transferred to the EPR quartz glass capillary tube via capillary action. After transfer, the other end is sealed with Vaseline, and then the tube is fixed in the resonant cavity of the EPR instrument for measurement. The EPR detection method is as follows: The instrument is tuned before measurement, and the diode current is maintained at approximately 200 μA during data acquisition; the measurement is performed at room temperature; the central magnetic field is 3505 G; the scan width is 70.0 G; the microwave power is 20 mW; the scan time is 573.48 s / s; the time constant is 163.84 ms; the conversion time is 163.85 ms; and 12 scans are performed.

[0034] Figure 1 To analyze the EPR test results, the detected peaks were fitted using the SpinFit function in Xenon software. Under a reaction condition of 1 min, four free radical signals were identified. Among them, three exhibited a six-fold characteristic peak signal with an intensity ratio of 1:1:1:1:1:1. All six-fold peak signals showed similar hyperfine splitting parameters, A N =15.4 G, AHβ =22.623.0 G, slightly different. The g-factor value is 2.0054~2.0068. The above parameters indicate that the measured free radical is a carbon-centered free radical with electron-donating properties, similar to alkyl free radicals.

[0035] High-resolution mass spectrometry (HMS) was used to analyze the reaction samples captured by DMPO. Samples were filtered through a 0.22 μm filter before analysis. Chromatographic conditions were as follows: the mobile phase consisted of acetonitrile and an aqueous solution containing 0.1% formic acid, with a flow rate of 0.3 mL / min and an injection volume of 10 μL. The gradient elution program was as follows: 0–2 minutes to maintain a 10% acetonitrile concentration; 2–11 minutes to linearly increase the acetonitrile concentration to 100%; 11–13 minutes to maintain at 100%; 13–13.1 minutes to rapidly decrease to 10%, and this concentration was maintained for 20 minutes to allow the column to reequilibrate. Mass spectrometry detection was performed using an electrospray ionization source in positive ion mode (ESI+), acquiring MS spectra with a mass-to-charge ratio (m / z) ranging from 50 to 1200 in full scan mode.

[0036] Comparing the total ion chromatograms of DMPO-added and DMPO-free samples, a prominent characteristic peak was identified in the DMPO-added sample during the detection time, with a mass-to-charge ratio (M / C ratio) of 475.1993. This characteristic peak corresponds to the ion peak of the organic radical-DMPO adduct. Subtracting the M / C ratio of DMPO (115.0997) from its M / C ratio yields the corresponding organic radical M / C ratio of 360.0996. The elemental composition of the ion at m / z 360.0996 was fitted using Thermo Xcalibur Qual Browser software, limiting the element types and atomic numbers to the following ranges: C 0–17, H 0–40, O 0–10, N 0–3, F 0–1, with a mass error controlled within 10 ppm. Under these conditions, two possible molecular formulas were obtained, namely C 17 H 15 O5N3F and C 14 H 17 O8N2F. Using the molecular skeleton of CIP as a reference, considering its number of nitrogen atoms and basic structure, and under the constraints of the unsaturation degree corresponding to the number of carbon and hydrogen atoms and the number of oxygen atoms, the original structure of CIP was derived through reduction and reconstruction. The results show that C... 14 H 17 In the hypothesized structure corresponding to O8N2F, the benzene ring undergoes hydroxylation, and the piperazine ring is reduced to only one methylamino group, which differs significantly from the CIP core skeleton; while C 17 H 15 The quality error of O5N3F is 9.04 ppm, which is within the set limits, and the structure is more reasonable. Figure 2The chemical structure of the corresponding organic free radical-DMPO adduct is deduced.

[0037] In addition, a quasi-molecular ion peak of another captured adduct was detected at a retention time of 4.08 min, with m / z = 517.2495. After subtracting the DMPO portion, the m / z of the captured radical ion was obtained as 402.1498, which is significantly larger than the m / z of the protonated CIP molecule (332.14261). Based on this, it is inferred that this species belongs to the dimer organic radical of CIP. To determine its elemental composition, the "Elemental Composition" function was called in Thermo Xcalibur Qual Browser software. The measured mass number 402.1498 was entered, the mass error was set to 10 ppm, and the range of element types and atomic numbers was limited as follows: C 0~45, H 0~60, O 0~15, N 0~6, F 0~2, while constraining with the number of nitrogen atoms and a reasonable degree of unsaturation. The software then provided a large number of candidate molecular formulas. Because the core structure of CIP is a nitrogen-containing heterocycle, the number of nitrogen atoms strongly constrains the carbon skeleton. During degradation, the benzene ring structure is usually preserved intact, resulting in products with high unsaturation, characterized by similar numbers of C and H atoms. Based on this chemical logic, we can further screen the output results for highly unsaturated chemical formulas that meet the C-N dependency relationship and have a C≈H number, such as C... 20 H 20 O3N4F2, C 23 H 19 O2N4F, C 17 H 22 O6N3F2, C 20 H 21 O5N3F, etc. The structural estimation of the dimer compound proceeds as follows: First, two CIP molecules are covalently linked by a C-C bond to construct the initial dimer framework. Then, prioritizing the number of nitrogen atoms in the target molecular formula, excess nitrogen-containing bonds are cleaved or excess nitrogen-containing segments are removed to prune the framework; because the positions of nitrogen atoms on the piperazine ring and the quinolone ring are different, multiple structural isomers under the same chemical formula can be generated. After determining the number of nitrogen atoms, the remaining carbon skeleton is further reduced or rearranged until the number of carbon atoms matches, thereby locking in the general structural framework of the dimer. Finally, while ensuring a reasonable overall unsaturation, oxygen atoms are introduced as needed: for regions with high local saturation, they are added in the form of hydroxyl groups; for regions with high local unsaturation, they are preferentially introduced in the form of carbonyl groups, while preserving as many original fluorine substitution positions as possible from the CIP. Through this estimation, a dimer structure that conforms to both high-resolution mass spectrometry data and chemical rationality can be obtained.

[0038] C 20 H 21 The specific process for deducing the chemical formula of O5N3F is as follows:

[0039] For a dimer candidate chemical formula with a total of 3 nitrogen atoms, the nitrogen atoms in the two CIP units can be allocated into two forms: (3+0) and (2+1). The reasoning process for the (3+0) pattern is outlined below:

[0040] On one side of the CIP fragment containing 3 nitrogen atoms, the original connection between the quinolone ring and the piperazine ring can be maintained. On the other side, the CIP fragment with 0 nitrogen atoms completely loses the nitrogen-containing heterocycle, retaining only the benzene ring structure. Since there is only one fluorine atom in the molecule, it means that the fluorine on the nitrogen-free side of the benzene ring has likely been removed or replaced by a hydroxyl group. Hydroxyl substitution satisfies both the absence of fluorine and helps meet the molecule's requirement for the number of oxygen atoms. At this point, the total number of carbon atoms is still 23. To match this number, the quinolone unit retaining 3 nitrogen atoms needs to be modified: the quinolone ring will open, removing 3 carbon atoms. During this decarbonization process, the number of oxygen atoms attached to the ring will also decrease accordingly; this process can be temporarily excluded as a reservation. Subsequently, the cyclopropyl group attached to the nitrogen atom of the quinolone ring is further considered for oxidative removal. Finally, a hydroxyl group is introduced at the ring-opening site to replenish the number of hydrogen and oxygen atoms, while maintaining a reasonable overall unsaturation. From this, we can deduce a candidate structure under the (3+0) allocation method, corresponding to Figure 3 The chemical structure is shown.

[0041] The reasoning for the (2+1) model can be developed using similar logic: one side of the CIP unit retains two N atoms, and the other side retains one N atom. Similarly, by adjusting the carbon skeleton (e.g., ring opening of quinolones, removal of piperazine rings), the substitution or removal of fluorine atoms, and the targeted addition of oxygen atoms through hydroxyl or carbonyl additions, the number of carbon, hydrogen, oxygen, nitrogen, and fluorine atoms can be made consistent with the measured high-resolution mass spectrometry data. Finally, for the chemical structure prediction of the dimer polymerization product, the structure of each CIP degradation product should be similar to the product structure described in relevant literature. Figure 3 The results are inferred from the organic radical-DMPO adduct of the dimer polymer.

Claims

1. A method for identifying organic free radicals in advanced oxidation processes, characterized in that: The method is as follows: Step 1: Perform advanced oxidation treatment on the solution containing the target pollutant to obtain the test solution; Step 2: Mix the test solution and the free radical scavenger to obtain a mixed solution containing a stable adduct; Step 3: Perform EPR and HRMS tests on the mixed solution sequentially to obtain relevant information about the organic free radical adduct.

2. The method according to claim 1, characterized in that: In step one, the target pollutant is an antibiotic pollutant, and the concentration of the target pollutant in the solution containing the target pollutant is 0.1~6 mg / L.

3. The method according to claim 1, characterized in that: In step one, the advanced oxidation treatment is ozone oxidation, with an ozone concentration of 2-10 mg / L in the solution, an oxidation reaction time of 0-300 s, and a reaction temperature of 0-30 ℃.

4. The method according to claim 1, characterized in that: In step two, the free radical scavenger must be kept at a low temperature of 0~5 ℃ before mixing and must not come into contact with oxygen.

5. The method according to claim 1, characterized in that: In step two, the free radical scavenger is a DMPO solution, and the final concentration of DMPO is 100~500 mmol / L.

6. The method according to claim 1, characterized in that: In step three, the EPR test method is as follows: the diode current is stabilized at 200 μA; the measurement is performed at room temperature; the central magnetic field is 3505 G; the scan width is 70.0 G; the microwave power is 20 mW; the scan time is 573.48 s / s; the time constant is 163.84 ms; the conversion time is 163.85 ms; and 12 scans are performed.

7. The method according to claim 1, characterized in that: In step three, the HRMS testing method is as follows: the sample is filtered through a 0.22 μm filter membrane before measurement; the chromatographic conditions are as follows: the mobile phase consists of acetonitrile and an aqueous solution containing 0.1% formic acid, the flow rate is 0.3 mL / min, and the sample injection volume is 10 μL; the gradient elution program is as follows: maintain an acetonitrile ratio of 10% for 0-2 minutes, linearly increase the acetonitrile ratio to 100% for 2-11 minutes, maintain it at 100% for 11-13 minutes, rapidly drop back to 10% for 13-13.1 minutes, and maintain this ratio for 20 minutes to allow the column to reequilibrate; mass spectrometry detection uses an electrospray ionization source in positive ion mode (ESI+), and MS spectra with a mass-to-charge ratio (m / z) range of 50-1200 are acquired in full scan mode.

8. The method according to claim 1, characterized in that: Step three, specifically the methods for obtaining information about organic free radical adducts, includes: (1) EPR analysis and free radical category identification: The mixed solution containing stable adducts after free radical capture was transferred to a quartz capillary glass tube, and electron paramagnetic resonance spectroscopy analysis was performed under EPR test conditions; the analysis was conducted by using hyperfine splitting parameters (such as the hyperfine coupling constant A of nitrogen). N The coupling constant A of hydrogen Hβ Analysis of the g-factor value and the type of free radical captured can determine the type of free radical captured. (2) Identification of free radical adducts: Compare the total ion chromatograms of samples with and without added DMPO, identify the characteristic peaks belonging to free radical-DMPO adducts, and record their precise mass-to-charge ratios; (3) Determination of free radical molecular formula: The mass-to-charge ratio of the captured free radical is obtained by subtracting the exact mass number of the DMPO portion from the charge-to-mass ratio of the adduct; the most likely molecular formula of the free radical is determined by using HRMS fitting software within the set mass error range, combined with the constraints of the type and number of heteroatoms in the contamination. (4) Free radical structure prediction: Starting from the original molecular structure of the target pollutant, combined with the degradation reaction type of the advanced oxidation process, the molecular structure is systematically pruned and rearranged. By controlling the number of elements such as C, H, O, and N in the target pollutant, the organic free radical structure that is completely matched with the molecular formula and has a reasonable chemical structure is gradually predicted.

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