Method for quantitatively detecting hydroxyl radicals in soil suspension

Abstract

The invention discloses a method for quantitatively detecting hydroxyl radicals in a soil suspension. Benzoic acid is used as a trapping agent of hydroxyl radicals, is captured at a benzene ring para-position within extremely short reaction time and is stably stored in a benzene ring-OH form, and the concentration of the hydroxyl radicals in the soil suspension to be detected can be accurately quantified through a standard curve of a multi-point external standard method by detecting the peak integral area of a sample. Compared with the existing method, the method has the advantages that the result reproducibility is good, and the detection limit can be as low as 0.58 mu g / L. And the adopted HPLC-MS / MS is used for quantitative analysis of p-hydroxybenzoic acid, has high response and can improve the detection accuracy.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a method for quantitatively detecting hydroxyl radicals in soil suspension. Background Art

[0002] Hydroxyl radicals (·OH) are composed of oxygen atoms and hydrogen atoms bonded together. They are highly active oxidants that can undergo different chemical reactions with organic or inorganic substances. They have a very high reaction rate constant and their oxidizing ability far exceeds that of ordinary chemical oxidants. ·OH can react with carbon-carbon unsaturated bonds and aromatic rings in organic molecules through addition, substitution, electron transfer, etc., oxidizing and decomposing them into carbon dioxide, water, and inorganic substances. Since free radicals are very active, have low concentrations, and exist for a very short time, their detection methods are particularly important in research involving free radicals.

[0003] The current quantitative detection methods for hydroxyl radicals in soil suspensions include: electron spin resonance, chemiluminescence, spectrophotometry and high performance liquid chromatography-mass spectrometry. However, the precision of electron spin resonance, chemiluminescence and spectrophotometry is low and the quantitative determination is not accurate enough. The study of high performance liquid chromatography-mass spectrometry method for accurate quantitative detection of hydroxyl radical concentration in soil suspension has practical environmental significance for promoting research progress in related fields. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for quantitatively detecting hydroxyl radicals in soil suspension.

[0005] The purpose of the present invention is achieved through the following technical solutions: A method for quantitatively detecting hydroxyl radicals in a soil suspension comprises the following steps: (1) Mix the soil sample to be tested with ultrapure water under an inert atmosphere to obtain a soil suspension; (2) Add benzoic acid solution to soil suspension under inert atmosphere and mix well, then expose to air for a period of time, add methanol to terminate the reaction, centrifuge, and filter to obtain the sample solution to be tested; (3) Using a high performance liquid chromatography-mass spectrometer, the p-hydroxybenzoic acid in the sample solution obtained in step (2) is detected, and the content of hydroxyl radicals in the soil suspension is calculated.

[0006] The ratio of the soil sample to ultrapure water in step (1) is 1 g: 1-5 mL; preferably 1 g: 2.5 mL.

[0007] The ultrapure water described in step (1) needs to be deoxygenated with a high-purity nitrogen flow of N2>99.9% for more than 1 h.

[0008] The inert atmosphere in step (1) or (2) is a nitrogen atmosphere.

[0009] The volume ratio of the benzoic acid solution and the soil suspension in step (2) is 1:1.

[0010] The concentration of the benzoic acid solution in step (2) is 10 to 30 mmol / L, preferably 20 mmol / L.

[0011] The benzoic acid solution described in step (2) is prepared using ultrapure water, and after preparation, it needs to be deoxygenated using a high-purity nitrogen flow of N2>99.9% for more than 1 hour.

[0012] The reaction time of step (2) is 10 to 24 h, preferably 24 h.

[0013] The methanol in step (2) is mass spectrometry grade methanol.

[0014] The centrifugation condition in step (2) is 12000 rpm for 3 min.

[0015] The filtration in step (2) is performed by filtering through a 0.22 μm hydrophobic PTFE needle filter.

[0016] The high performance liquid chromatography-mass spectrometer described in step (3) is a Thermo Scientific Vanquish high performance liquid chromatograph connected in series with a TSQ Quantis triple quadrupole mass spectrometer.

[0017] The chromatographic column model used in the high performance liquid chromatography-mass spectrometry instrument described in step (3) is Acclaim TM RSLC 120C18 2.2 μm 120Å (2.1×100 mm).

[0018] The chromatographic parameters of the high performance liquid chromatography-mass spectrometry instrument in step (3) are: The injection volume was 2 μL, and the autosampler temperature was set to 4 °C. Mobile phases A and B contained ultrapure water and acetonitrile, respectively, with a flow rate of 0.35 mL / min. The gradient elution program was: 0 min, 40% B; 0-1 min, 40-80% B; 1-2 min, 80-100% B; 2-3 min, 100% B; 3-3.01 min, 100%-40% B; 3.01-5 min, 40% B. Selected reaction monitoring (SRM) mode was used to detect and quantify p-hydroxybenzoic acid.

[0019] The mass spectrometry parameters of the high performance liquid chromatography-mass spectrometry instrument in step (3) are: The positive ion spray voltage was 3500 V, the sheath gas and auxiliary gas pressures were 35 and 15 Arb, respectively, the ion transfer tube temperature was 320 °C, and the ion source temperature was 350 °C.

[0020] The calculation described in step (3) is performed using a regression equation obtained by calculating a standard curve obtained by detecting a standard solution of p-hydroxybenzoic acid with a gradient concentration.

[0021] The p-hydroxybenzoic acid content calculated in step (3) can be converted to the hydroxyl radical content in the soil sample. The conversion factor is 5.87, that is, the concentration of hydroxyl radicals is 5.87 times the concentration of p-hydroxybenzoic acid.

[0022] The above method for quantitatively detecting hydroxyl radicals in soil suspension is applied to detecting the content of hydroxyl radicals in soil samples.

[0023] Compared with the prior art, the present invention has the following advantages and effects: The present invention provides a method for quantitatively detecting the concentration of hydroxyl radicals in a soil suspension. Benzoic acid is used as a trapping agent for hydroxyl radicals. The hydroxyl radicals are captured at the para position of a benzene ring in a very short reaction time and stably stored in the form of benzene ring-OH. The concentration of hydroxyl radicals in the soil suspension to be tested can be accurately quantified by detecting the integrated area of ​​the sample peak and using a multi-point external standard method standard curve. Compared with the existing methods, the advantages of this method include good result reproducibility and low detection limit. The HPLC-MS / MS used for the quantitative analysis of p-hydroxybenzoic acid has a high response and can improve the accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The chromatographic result diagram of the 5 μg / L hydroxybenzoic acid standard solution detected by high performance chromatography-mass spectrometry in the example.

[0025] Figure 2 The chromatographic result diagram of the 500 μg / L hydroxybenzoic acid standard solution detected by high performance chromatography-mass spectrometry in the example.

[0026] Figure 3 This is the chromatographic result diagram of detecting p-hydroxybenzoic acid in soil suspension using high performance chromatography-mass spectrometry in Comparative Example 1.

[0027] Figure 4 This is the chromatographic result diagram of detecting p-hydroxybenzoic acid in soil suspension using high performance chromatography-mass spectrometry in Comparative Example 2.

[0028] Figure 5 This is the chromatographic result diagram of detecting p-hydroxybenzoic acid in soil suspension using high performance chromatography-mass spectrometry in Comparative Example 3. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0030] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0031] Example 1 A method for quantitatively detecting the concentration of hydroxyl radicals in soil suspension (1) Preparation of soil suspension In a nitrogen glove box (N2>99.999%, O2<0.01ppm), 50 mL of oxygen-free ultrapure water (dissolved oxygen concentration less than 0.01 mg / L) was added to 20 g of 100-mesh dry soil and then sealed. After anaerobically shaking at 200 rpm in a shaker for 20 days, a soil suspension was obtained.

[0032] (2) Adding hydroxyl radical scavenger 5 mL of the soil suspension obtained in step (1) was mixed with 5 mL of 20 mmol / L benzoic acid solution (prepared with ultrapure water and deoxygenated with a high-purity nitrogen flow of N2>99.9% for more than 1 h after preparation) in a nitrogen glove box (N2>99.999%, O2<0.01ppm) and then removed and exposed to air to generate hydroxyl radicals. After 24 h, 0.5 mL of the mixed solution was taken into a 1.5 mL centrifuge tube. 0.5 mL of mass spectrometry grade methanol was added to the centrifuge tube in advance to terminate the reaction between benzoic acid and hydroxyl radicals. The supernatant was aspirated after centrifugation at 12000 rpm for 3 min, and the sample solution to be tested was obtained after passing through a 0.22 μm hydrophobic PTFE needle filter and stored at -20 °C until detection.

[0033] The reaction equation involved is: OH + BA → p -HBA (3) Preparation of standard curve Since it is difficult to prepare a standard solution containing hydroxyl radicals, and it is generally recognized in current research that the content of p-hydroxybenzoic acid can be detected by capturing hydroxyl radicals in a sample with p-hydroxybenzoic acid, and the conversion factor is used to achieve the quantification of hydroxyl radicals. Therefore, the present invention directly prepares a standard curve of p-hydroxybenzoic acid for detection, and the specific steps are as follows: The p-hydroxybenzoic acid standard solution was added to six containers numbered ① to ⑥, wherein ① added 250 μL of p-hydroxybenzoic acid standard solution with a mass concentration of 2 mg / L, ② added 100 μL of p-hydroxybenzoic acid standard solution with a mass concentration of 2 mg / L, ③ added 50 μL of p-hydroxybenzoic acid standard solution with a mass concentration of 2 mg / L, ④ added 12.5 μL of p-hydroxybenzoic acid standard solution with a mass concentration of 2 mg / L, ⑤ added 50 μL of p-hydroxybenzoic acid standard solution with a mass concentration of 200 μg / L, and ⑥ added 25 μL of p-hydroxybenzoic acid standard solution with a mass concentration of 200 μg / L. The p-hydroxybenzoic acid standard solutions described in ① to ⑥ were all diluted to 1 mL with acetonitrile to obtain p-hydroxybenzoic acid standard solutions with concentrations of 500, 200, 100, 25, 10, and 5 μg / L, respectively.

[0034] (4) Detection of p-hydroxybenzoic acid standard solution Thermo Scientific Vanquish high performance liquid chromatograph coupled with TSQ Quantis triple quadrupole mass spectrometer (HPLC-MS / MS) was used for detection. The chromatographic column model was Acclaim TM RSLC 120 C18 2.2 μm 120Å (2.1×100 mm), injection volume was 2 μL, and the autosampler temperature was set to 4 °C. Mobile phases A and B contained ultrapure water and acetonitrile, respectively, with a flow rate of 0.35 mL / min. The gradient elution program was: 0 min, 40% B; 0-1 min, 40-80% B; 1-2 min, 80-100% B; 2-3 min, 100% B; 3-3.01 min, 100%-40% B; 3.01-5 min, 40% B. Selected reaction monitoring (SRM) mode was used to detect and quantify p-hydroxybenzoic acid. The mass spectrometry parameters were set as follows: positive ion spray voltage 3500 V, sheath gas and auxiliary gas pressures 35 and 15 Arb, respectively. The ion transfer tube temperature was 320 °C, and the ion source temperature was 350 °C. Peak area acquisition was performed using Xcalibur software.

[0035] Table 1 Ion pair information and retention time of p-hydroxybenzoic acid Compound CAS Number Precursor ion Quantitative ion Qualifier ions Retention time p-HBA 99-96-7 137.02 93.10 65.04 0.81 (5) Draw a standard curve The peak areas of ① to ⑥ in step (3) were detected using the method described in step (4), and the background peak area of ​​acetonitrile used was deducted. The standard curve was obtained with the integrated area as the ordinate and the concentration as the abscissa. The regression equation Y=1699.22X, r was obtained based on the standard curve. 2=0.9918; where Y is the peak area; X is the concentration of p-hydroxybenzoic acid, in ug / L; (6) Quantification of hydroxyl radical concentration The sample solution obtained in step (2) was detected using the method described in step (4), and the peak area of ​​the sample was 55371. Using the regression equation described in step (5), the concentration of p-hydroxybenzoic acid was calculated to be 32.59 ug / L. The conversion factor for calculating the concentration of hydroxyl radicals based on the formation rate of p-hydroxybenzoic acid was 5.87, that is, the concentration of hydroxyl radicals was 5.87 times that of p-hydroxybenzoic acid, which was 191.28 ug / L. The conversion factor was obtained by mutual calibration of pure hydroxyl radical generating source hydrogen peroxide and methanol probe, and was very close to the generation coefficient G (~6) of the reaction equation described in step (2). The conversion factor and probe calibration described in this process were cited from the literature Zhou X, Mopper K. Determination ofphotochemically produced hydroxyl radicals in seawater and freshwater[J].Marine chemistry, 1990, 30: 71-88.

[0036] The chromatogram of p-hydroxybenzoic acid in the standard solution detected by high performance liquid chromatography-mass spectrometry in the embodiment is as follows: Figure 1 , as shown in 2. From Figure 1 It can be seen that when the concentration is 5 μg / L, the signal-to-noise ratio of the chromatographic peak is 26. Taking the concentration when the signal-to-noise ratio is 3 as the detection limit, the detection limit can reach 0.58 μg / L. Figure 2 It can be seen that the peak of the chromatogram is sharp and symmetrical, the baselines on both sides are stable, there is no tailing or front extension, and there are no extra impurities or baseline peaks in the chromatogram, so the peak area can be accurately calculated.

[0037] Comparative Example 1 The experimental steps are the same as those in Example 1, except that the flow rate in step (4) is changed to 0.3 mL / min. Figure 3 shown.

[0038] Comparative Example 2 The experimental steps are the same as those in Example 1, except that the flow rate in step (4) is changed to 0.2 mL / min. Figure 4 shown.

[0039] from Figure 3 and Figure 4 It can be seen that the chromatographic peak is split and accompanied by severe tailing, and the peak area cannot be accurately calculated.

[0040] Comparative Example 3 The experimental steps are the same as those in Example 1, except that the mobile phase A in step (4) is replaced with a 0.1% formic acid aqueous solution (pH ≈ 2.5). The test results are as follows: Figure 5 As shown. Figure 5 It can be seen that the chromatographic peaks show serious wrapping and tailing phenomena, and the peak area cannot be accurately calculated.

[0041] The above experimental results show that when detecting p-hydroxybenzoic acid in a sample, different instrument parameters will have a significant impact on the detection results. Since quantification is required by calculating the peak area, accurate quantification is not possible when the peak shape is poor. Therefore, the detection method of Example 1 of the present invention achieves accurate quantification of hydroxyl radicals in soil, and has a low detection limit and good separation effect.

[0042] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for quantitatively detecting hydroxyl radicals in soil suspension, characterized in that The steps include: (1) Mix the soil sample to be tested with ultrapure water under an inert atmosphere to obtain a soil suspension; (2) Add benzoic acid solution to soil suspension under inert atmosphere and mix well, then expose to air for a period of time, add methanol to terminate the reaction, centrifuge, and filter to obtain the sample solution to be tested; (3) Using a high performance liquid chromatography-mass spectrometer, the p-hydroxybenzoic acid in the sample solution obtained in step (2) is detected, and the content of hydroxyl radicals in the soil suspension is calculated.

2. The method according to claim 1, characterized in that: The ratio of the soil sample to ultrapure water in step (1) is 1 g: 2.5 mL; The ultrapure water in step (1) needs to be deoxygenated with a high-purity nitrogen flow of N2 > 99.9% for more than 1 h; The inert atmosphere in step (1) or (2) is a nitrogen atmosphere.

3. The method according to claim 1, characterized in that: The volume ratio of the benzoic acid solution and the soil suspension in step (2) is 1:1; The concentration of the benzoic acid solution in step (2) is 10 to 30 mmol / L; The benzoic acid solution described in step (2) is prepared using ultrapure water, and after preparation, it needs to be deoxygenated with a high-purity nitrogen flow of N2 > 99.9% for more than 1 hour.

4. The method according to claim 1, characterized in that: The reaction time of step (2) is 24 hours.

5. The method according to claim 1, characterized in that: The methanol in step (2) is mass spectrometry grade methanol; The centrifugation condition in step (2) is 12000 rpm for 3 min; The filtration in step (2) is performed by filtering through a 0.22 μm hydrophobic PTFE needle filter.

6. The method according to claim 1, characterized in that: The high performance liquid chromatography-mass spectrometer in step (3) is a Thermo Scientific Vanquish high performance liquid chromatograph coupled to a TSQ Quantis triple quadrupole mass spectrometer; The chromatographic column model used in the high performance liquid chromatography-mass spectrometry instrument described in step (3) is Acclaim TM RSLC 120 C182.2 μm 120Å, 2.1×100 mm.

7. The method according to claim 1, characterized in that: The chromatographic parameters of the high performance liquid chromatography-mass spectrometry instrument in step (3) are: The injection volume was 2 μL, and the autosampler temperature was set to 4 ℃; the mobile phases A and B contained ultrapure water and acetonitrile, respectively, with a flow rate of 0.35 mL / min; the gradient elution program was: 0 min, 40% B; 0-1 min, 40-80% B; 1-2 min, 80-100% B; 2-3 min, 100% B; 3-3.01 min, 100%-40% B; 3.01-5 min, 40% B, and the selected reaction monitoring mode was used to detect and quantify p-hydroxybenzoic acid.

8. The method according to claim 1, characterized in that: The mass spectrometry parameters of the high performance liquid chromatography-mass spectrometry instrument in step (3) are: The positive ion spray voltage was 3500 V, the sheath gas and auxiliary gas pressures were 35 and 15 Arb, respectively, the ion transfer tube temperature was 320 °C, and the ion source temperature was 350 °C.

9. The method according to claim 1, characterized in that: The calculation in step (3) is performed using a regression equation obtained by calculating a standard curve obtained by testing a p-hydroxybenzoic acid standard solution with a gradient concentration; The p-hydroxybenzoic acid content calculated in step (3) can be converted to the hydroxyl radical content in the soil sample. The conversion factor is 5.87, that is, the concentration of hydroxyl radicals is 5.87 times the concentration of p-hydroxybenzoic acid.

10. Use of the method for quantitatively detecting hydroxyl radicals in a soil suspension according to any one of claims 1 to 9 in detecting the content of hydroxyl radicals in a soil sample.

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