Method for detecting p-nitroiodobenzene in water body and soil based on ultra-high performance liquid chromatography

Abstract

The invention discloses a method for detecting p-nitroiodobenzene in a water body and soil based on ultra-high performance liquid chromatography, which comprises the following steps: 1, detecting by using an ultra-high performance liquid chromatograph, and determining different detection conditions according to the water body and the soil; 2, preparing a working solution of a detected object; 3, carrying out a specific experiment on the p-nitroiodobenzene; 4, carrying out a recovery rate and repeatability experiment, 5, carrying out a detection limit and a quantification limit, and 6, analyzing an experiment result. Compared with the prior art, the method has the advantages that the retention time of p-nitroiodobenzene in the water body is about 2.9 min, baseline separation of a target peak and other components can be achieved under the detection wavelength of 296 nm, interference of other impurities does not exist, the method has good selectivity, the retention time of p-nitroiodobenzene in soil is about 6.1 min under the detection wavelength of 296 nm, and the retention time of p-nitroiodobenzene in soil is about 6.1 min. Baseline separation of a target peak can be achieved, the separation degree of the target peak and other impurity peaks is larger than 1.5, the interference phenomenon of other impurities does not exist, and the method has good selectivity.

Description

Technical Field

[0001] This invention relates to the field of water and soil detection technology, specifically a method for detecting p-nitrobenzene in water and soil based on ultra-high performance liquid chromatography. Background Technology

[0002] p-Nitroiodobenzene is an important industrial raw material, widely used in the chemical synthesis of dyes, pesticides, pharmaceuticals, and rubber. However, p-nitrobenzene is highly toxic and potentially carcinogenic. It can enter the human body through the respiratory tract, skin, and digestive tract, leading to methemoglobinemia, and in severe cases, damage to the central nervous system and hemolytic anemia. In environmental media, nitrobenzene is difficult to degrade naturally and easily accumulates in soil and water bodies, posing a long-term threat to ecosystems and human health through bioaccumulation via the food chain. Therefore, a method for detecting p-nitrobenzene in water and soil is urgently needed.

[0003] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the above-mentioned technical defects and provide a method for detecting p-nitroiodobenzene in water and soil based on ultra-high performance liquid chromatography. The retention time of p-nitroiodobenzene in water is about 2.9 min. At a detection wavelength of 296 nm, the target peak can achieve baseline separation from other components, and there is no interference from other impurities. The method has good selectivity. At a detection wavelength of 296 nm, the retention time of p-nitroiodobenzene in soil is about 6.1 min. The target peak can achieve baseline separation, and the resolution with other impurity peaks is >1.5. There is no interference from other impurities. The method has good selectivity.

[0005] To address the aforementioned problems, the technical solution of this invention is a method for detecting p-nitroiodobenzene in water and soil based on ultra-high performance liquid chromatography, comprising the following steps:

[0006] Step 1: Use ultra-high performance liquid chromatography (UHPLC) for detection, and determine different detection conditions based on the water body and soil.

[0007] Step 2: Preparation of the working solution for the analyte, including the preparation of the standard stock solution and the standard working solution;

[0008] Step 3: Specificity test of p-nitroiodobenzene. The specificity test of p-nitroiodobenzene in water is as follows: blank solvent, blank matrix sample, and standard working solution of the lowest concentration were injected for testing. The specificity test of p-nitroiodobenzene in soil is as follows: the established liquid chromatography analysis method was used to determine the blank sample of 0.01 mol / L calcium chloride solution, the recovery rate of 0.01 mol / L calcium chloride solution, blank water samples and soil samples of red soil, black soil, brown soil, alluvial soil, and paddy soil, and the recovery rate water samples and soil samples of red soil, black soil, brown soil, alluvial soil, and paddy soil. The spectra of blank samples and recovery rate samples were compared.

[0009] Step 4: Recovery and Repeatability Experiments. The recovery and repeatability experiments in water are as follows: One matrix blank control group and three spiking concentrations were set up for each of the test water, algae culture medium, and M4 culture medium for recovery and repeatability experiments. Five replicates were performed for each concentration. The precision of the analytical method was expressed as repeatability, using relative standard deviation (RSD). Repeatability was analyzed using the recovery test solution, and the RSD was calculated for five levels each of low, medium, and high concentrations. The recovery and repeatability experiments in soil are as follows: Five matrix blank control groups and two spiking concentrations were set up for soil for recovery and repeatability experiments. Five samples were prepared in parallel for each concentration. The accuracy of the analytical method was expressed as the recovery rate, using the average recovery rate. The precision of the analytical method was expressed as repeatability, using relative standard deviation (RSD).

[0010] Step 5: Limit of Detection (LOD) and Limit of Quantification (LOQ). The method's LOD is set at 3 times the baseline noise, i.e., the concentration of the target analyte at a signal-to-noise ratio (SNR) of 3. The method's LOD is set at 10 times the baseline noise, i.e., the concentration of the target analyte at a SNR of 10.

[0011] Step 6: Analysis of experimental results, including specificity, linear range and correlation, recovery and repeatability, limit of detection and limit of quantitation.

[0012] Preferably, in step one, the instrument conditions for testing p-nitrobenzene in water are as follows: Agilent 1290-6495A instrument; VWD detector; flow rate 0.500 mL / min; injection volume 20.00 μL; column temperature 30.0℃; mobile phase and its ratio (V / V) acetonitrile:0.1% phosphoric acid water (70:30); detection wavelength 296 nm; run time 8.00 min; retention time approximately 2.9 min. The instrument conditions for testing p-nitrobenzene in soil are as follows: Agilent 1290-6495A instrument; Accurasil column. C18, 250mm × 4.6mm (id), 5μm, (SN: 074263A); detector: VWD; detection wavelength: 296nm; mobile phase A: acetonitrile; mobile phase B: 0.1% phosphoric acid water; flow rate: 1.000mL / min; injection volume: 50.00μL; column temperature: 30.0℃; run time: 15.00min; retention time: approximately 6.1min.

[0013] Preferably, in step two, the standard stock solution in the water is prepared as follows: the standard sample name is p-nitroiodobenzene, the amount of standard substance weighed is 0.02006 g, the purity is 99.7%, the diluent is methanol, the volume is adjusted to 20.00 mL, the stock solution concentration is 1000 mg / L, the volume is adjusted to the mark with solvent, mixed well, and stored in a refrigerator at 2℃-8℃ in a light-proof and sealed manner for later use; the standard stock solution in the soil is prepared as follows: the test substance is weighed and dissolved in methanol to prepare the test substance stock solution (STDSTD-1000ppm). The test substance stock solution was prepared by dissolving it in calcium chloride solution to obtain a test substance stock solution (STD-CaCl2-4.00ppm), which was used for the preparation of standard working solutions. Test substance stock solutions of different concentrations were diluted with acetonitrile for spiking recovery tests. The standard working solutions in water were prepared as follows: different concentrations and different transfer volumes of mother liquor were taken and diluted with acetonitrile to obtain standard working solutions of different concentrations. The standard working solutions in soil were prepared as follows: different concentrations and different transfer volumes of mother liquor were taken and diluted with methanol to obtain standard working solutions of different concentrations.

[0014] Preferably, in step two, the linearity and range of p-nitroiodobenzene in water are as follows: A series of diluted standard working solutions are obtained, and the standard curve is set with 6 calibration points. The linear range is 10.0–500 μg / L. The standard curve is fitted using a linear regression equation with the p-nitroiodobenzene concentration as the abscissa and the average peak area as the ordinate. The linearity and range of p-nitroiodobenzene in soil are as follows: A series of diluted standard working solutions are obtained, and the standard curve is set with 6 calibration points. The linear range is 4.00–200 μg / L. Two samples are collected at each concentration. The standard curve is fitted using a linear regression equation with the analyte concentration as the abscissa and the average peak area as the ordinate.

[0015] Preferably, in step four, the recovery rate is calculated using the following formula: Where R is the recovery rate, C d C represents the measured concentration of the target analyte. a The actual concentration of the target substance added;

[0016] The formula for calculating standard deviation is: Where S is the standard deviation, x i Let i be the recovery rate obtained from the i-th measurement. is the average recovery rate, and N is the number of recovery rates included in the calculation;

[0017] The formula for calculating the relative standard deviation is: RSD stands for relative standard deviation.

[0018] Preferably, in step five, the formula for calculating the detection limit of the method is: Where LOD is the limit of detection; C is the actual concentration added in the low concentration range; R S / N The signal-to-noise ratio corresponds to the concentration.

[0019] The formula for calculating the limit of quantitation of a method is: Where LOQ is the limit of quantitation; C is the theoretical concentration for recovery at low concentration levels; R S / N This represents the signal-to-noise ratio corresponding to the concentration.

[0020] The advantages of this invention compared to existing technologies are:

[0021] 1. This invention uses ultra-high performance liquid chromatography (UHPLC) to detect p-nitroiodobenzene in water and soil. The retention time of p-nitroiodobenzene in water is approximately 2.9 min. At a detection wavelength of 296 nm, the target peak can be baseline separated from other components without interference from other impurities, and the method has good selectivity. In soil, the retention time of p-nitroiodobenzene at a detection wavelength of 296 nm is approximately 6.1 min. The target peak can be baseline separated, and the resolution with other impurity peaks is >1.5, with no interference from other impurities. The method has good selectivity.

[0022] 2. Through experiments, this invention determined that the LOQ (Limited Quantity) of p-nitrobenzene in water was 1.57 μg / L and the LOD (Limited Quantity) was 0.471 μg / L in the test water; the LOQ in algal culture medium was 3.10 μg / L and the LOD was 0.932 μg / L; and the LOQ in M4 culture medium was 1.41 μg / L and the LOD was 0.423 μg / L. The limits of quantification (LOQs) for p-nitrobenzene in 0.01 mol / L calcium chloride solutions after equilibration in red soil, black soil, brown soil, alluvial soil, and paddy soil were 2.11 μg / L, 1.93 μg / L, 2.01 μg / L, 2.25 μg / L, and 2.07 μg / L, respectively. The limits of detection for p-nitroiodobenzene in red soil, black soil, brown soil, alluvial soil, and paddy soil were 0.0365 μg / g, 0.0419 μg / g, 0.0394 μg / g, 0.0320 μg / g, and 0.0320 μg / g, respectively, and the limits of detection were 0.0110 μg / g, 0.0126 μg / g, 0.0118 μg / g, 0.00961 μg / g, and 0.00962 μg / g, respectively. Attached image description:

[0023] Figure 1 It is a calibration curve.

[0024] Figure 2 This is a typical spectrum of a standard working solution.

[0025] Figure 3 This is a typical blank spectrum of methanol.

[0026] Figure 4 It is a typical spectrum of the test water blank.

[0027] Figure 5 This is a blank spectrum of the algal culture medium.

[0028] Figure 6 This is a blank spectrum of M4 culture medium.

[0029] Figure 7 This is a typical spectrum of the standard working solution (theoretical concentration 4.00 μg / L).

[0030] Figure 8 This is a typical spectrum of a standard working solution (theoretical concentration 200 μg / L).

[0031] Figure 9 This is a blank spectrum of a 0.01 mol / L CaCl2 solution.

[0032] Figure 10 It is a blank map of red soil.

[0033] Figure 11 It is a blank map of black soil.

[0034] Figure 12 It is a blank map of brown soil.

[0035] Figure 13 It is a blank map of the tidal flats.

[0036] Figure 14 It is a blank map of paddy soil. Detailed Implementation

[0037] To make the content of this invention easier to understand, the technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings.

[0038] This invention discloses a method for detecting p-nitroboiodobenzene in water and soil based on ultra-high performance liquid chromatography, specifically including the following steps:

[0039] The substance being tested is p-nitroiodobenzene, with a purity of 99.7% and an appearance of a pale yellow powder.

[0040] The main instruments and equipment include: ultra-high performance liquid chromatograph, electronic balance, centrifuge, constant temperature incubator shaker, and pH meter.

[0041] The main reagents include: acetonitrile, phosphoric acid, methanol, ultrapure water (resistivity 18.2 MΩ×cm); test water; algal culture medium. All culture solutions were prepared using pure water. The final concentrations of various nutrients in the culture solution are shown in the table below.

[0042]

[0043] The preparation process for M4 culture medium is as follows:

[0044] First, use UP water to prepare stock solutions (I) for each trace element. Then, use stock solution (I) to prepare stock solution (II). The trace elements (mixed solutions) are shown in the table below.

[0045] Trace element preparation table

[0046]

[0047] Na2EDTA and FeSO4 were prepared separately, then sterilized immediately after being poured together. M4 medium was prepared using stock solution II; macronutrients and vitamins are listed in the table below.

[0048] Table of macroelements and vitamins

[0049]

[0050]

[0051] Prepare a mixed vitamin stock solution by adding the three vitamins to 1L of water, as shown in the table below.

[0052] Mixed Vitamin Stock Solution Preparation Table

[0053]

[0054] Mixed vitamin stock solutions should be aliquoted and refrigerated. Add the vitamins to the culture medium before use.

[0055] Note: When preparing the culture medium, in order to avoid salt precipitation, an appropriate amount of stock solution should be added to 500-800 mL of UP water and then filled to 1 L. If the preparation volume increases, the amount of UP water added should also be increased proportionally.

[0056] The soils used in the tests were alluvial soil from Langfang City, Hebei Province; paddy soil from Shaoxing City, Zhejiang Province; brown soil from Tangshan City, Hebei Province; red soil from Changsha City, Hunan Province; and black soil from Gongzhuling, Jilin Province.

[0057] Instrument conditions for testing p-nitroiodobenzene in water: the analyte is p-nitroiodobenzene, the detector is a VWD detector, the flow rate is 0.500 mL / min, the injection volume is 20.00 μL, the column temperature is 30.0℃, the mobile phase and its ratio (V / V) is acetonitrile: 0.1% phosphoric acid water (70:30), the detection wavelength is 296 nm, the running time is 8.00 min, and the retention time is approximately 2.9 min.

[0058] Instrument conditions for testing p-nitrobenzene in soil: Detector: p-nitrobenzene; Column: Accurasil C18, 250 mm × 4.6 mm (id), 5 μm, (SN: 074263A).

[0059] Detector: VWD; Detection wavelength: 296 nm; Mobile phase A: acetonitrile; Mobile phase B: 0.1% phosphoric acid water; Flow rate: 1.000 mL / min; Injection volume: 50.00 μL; Column temperature: 30.0℃; Run time: 15.00 min; Retention time: approximately 6.1 min.

[0060] Table 1 Elution gradient

[0061]

[0062] Preparation of standard stock solutions in water bodies:

[0063] Table 2. Record of Preparation of Standard Reserve Solution in Water Bodies

[0064]

[0065] Pretreatment: Dilute to the mark with solvent, mix well, prepare and store in a refrigerator at 2℃~8℃ in a sealed container away from light for later use.

[0066] Preparation of standard stock solution in soil: Weigh 0.10030 g of the test sample into a 100.00 mL volumetric flask, dissolve in methanol and dilute to the mark, shake well, and prepare a solution with a concentration of 1.00 × 10⁻⁶ g / mL. 3 The test substance stock solution (STDSTD-1000ppm) was prepared at a concentration of mg / L.

[0067] Take 0.400 mL of the test substance stock solution (STDSTD-1000ppm) into a 100.0 mL volumetric flask, dissolve it with 0.01 mol / L calcium chloride solution and dilute to the mark, shake well to prepare a test substance stock solution with a concentration of 4.00 mg / L (STD-CaCl2-4.00ppm), which will be used for the preparation of the standard working solution; take 9.00 mL of the test substance stock solution (STDSTD-1000ppm) into a 100.0 mL volumetric flask. In the experiment, acetonitrile was used to dilute to the mark, and the solution was shaken well to prepare a 90.0 mg / L test substance stock solution (STDSTD-90.0 ppm) for use in the spiking recovery test; 0.600 mL of the test substance stock solution (STDSTD-90.0 ppm) was taken into a 100.0 mL volumetric flask, diluted to the mark with acetonitrile, and shaken well to prepare a 540 μg / L test substance stock solution (STDSTD-540 ppb) for use in the spiking recovery test.

[0068] Preparation of standard working solution:

[0069] Preparation of standard working solutions in water bodies

[0070] Table 3. Standard Working Solution Preparation Record Sheet

[0071]

[0072] Preparation of standard working solution in soil: Table 4 Standard Working Solution Preparation Record Sheet

[0075] Specificity of p-nitrobenzene in water:

[0076] The above analytical method was established to test blank solvent, blank matrix sample, and standard working solution of the lowest concentration, with one sample collected from each.

[0077] Specificity of p-nitrobenzene in soil:

[0078] The established liquid chromatography method was used to determine the blank sample of 0.01 mol / L calcium chloride solution, the spiking recovery sample of 0.01 mol / L calcium chloride solution, the blank water and soil samples of red soil, black soil, brown soil, alluvial soil, and paddy soil, and the spiking recovery water and soil samples of red soil, black soil, brown soil, alluvial soil, and paddy soil. The spectra of the blank sample and the spiking recovery sample were compared.

[0079] Linearity and range of p-nitroiodobenzene in water:

[0080] The standard working solutions were obtained by stepwise dilution according to Table 3. Six calibration points were set for the standard curve, with a linear range of 10.0 to 500 μg / L. The standard curve was fitted by a linear regression equation with the concentration of p-nitroiodobenzene as the x-axis and the average peak area as the y-axis.

[0081] Linearity and range of p-nitroiodobenzene in soil:

[0082] After dilution according to Table 4, a series of standard working solutions were obtained. The standard curve was set with 6 calibration points, and the linear range was 4.00 to 200 μg / L. Two samples were collected for each concentration. The standard curve was fitted by a linear regression equation with the concentration of the test substance as the abscissa and the average peak area as the ordinate.

[0083] Recovery rate and repeatability in water:

[0084] Recovery and repeatability tests were conducted on one substrate blank control group and three different spiking concentrations in each of the experimental water, algae culture medium, and M4 culture medium. Five replicates were performed for each concentration.

[0085] The precision of an analytical method is reflected in its repeatability, expressed as the relative standard deviation (RSD).

[0086] Repeatability was analyzed using the recovery test solution, and the RSD was calculated for five levels each of low, medium, and high concentrations.

[0087] The concentrations of nitrobenzene added for recovery tests in the experimental water were 12.5 μg / L, 250 μg / L, and 6.00 mg / L, respectively; the concentrations in the algal culture medium were 50.0 μg / L, 250 μg / L, and 6.00 mg / L, respectively; and the concentrations in the M4 culture medium were 12.5 μg / L, 250 μg / L, and 6.00 mg / L, respectively. The preparation process is shown in Tables 5, 6, and 7.

[0088] The blank matrix and the p-nitroiodobenzene addition recovery test samples were filtered through a 0.45 μm filter membrane, pretreated, and tested according to the instrument operating conditions. The recovery rate and the relative standard deviation (RSD) of the recovery rate were calculated.

[0089] The formula for calculating the recovery rate is: Where R is the recovery rate, C d C represents the measured concentration of the target analyte. a The actual concentration of the target substance added;

[0090] The formula for calculating standard deviation is: Where S is the standard deviation, x i Let i be the recovery rate obtained from the i-th measurement. is the average recovery rate, and N is the number of recovery rates included in the calculation;

[0091] The formula for calculating the relative standard deviation is: RSD stands for relative standard deviation.

[0092] Table 5. Preparation of Samples for Adding Recycling to Test Water

[0093]

[0094]

[0095] Pretreatment: Take blank matrix (test water) and STDHSL-BQS-L-1~5 directly for instrumental determination of test substance concentration; dilute STDHSL-BQS-M-1~5 with methanol 5 times before instrumental determination of test substance concentration; dilute STDHSL-BQS-H-1~5 with methanol 20 times before instrumental determination of test substance concentration.

[0096] Table 6. Preparation of Samples for Recovery Test in Algal Culture Medium

[0097]

[0098] Pretreatment: Take blank substrate (algal culture medium) and directly measure the concentration of test substance; dilute STDHSL-PYJ-L-1~5 with methanol 2 times and measure the concentration of test substance; dilute STDHSL-PYJ-M-1~5 with methanol 5 times and measure the concentration of test substance; dilute STDHSL-PYJ-H-1~5 with methanol 20 times and measure the concentration of test substance.

[0099] Table 7. Preparation of Samples for Recovery Test in M4 Culture Medium

[0100]

[0101] Pretreatment: Take blank substrate (M4 medium) and STDHSL-M4PYJ-L-1~5: directly measure the concentration of the test substance on the instrument; STDHSL-M4PYJ-M-1~5: dilute with methanol 5 times; STDHSL-M4PYJ-H-1~5: dilute with methanol 20 times and then measure the concentration of the test substance on the instrument.

[0102] Recovery rate and repeatability in soil:

[0103] Five blank control groups and two spiking concentrations were set up for soil analysis to determine recovery and repeatability. Five samples were prepared in parallel for each concentration. The accuracy of the analytical method was expressed as the recovery rate, using the average recovery rate; the precision of the analytical method was expressed as the repeatability, using the relative standard deviation (RSD).

[0104] Soil addition and recovery: Weigh approximately 1.0 g of the prepared red soil, black soil, brown soil, alluvial soil, and paddy soil into 50 mL centrifuge tubes, add 0.100 mL of the working solution of the test substance with a concentration of 540 μg / L (STD-540 ppb), vortex and mix well to prepare a soil recovery sample with a low concentration of 0.0540 μg / g. Weigh approximately 1.0 g of the prepared red soil, black soil, brown soil, alluvial soil, and paddy soil into 50 mL centrifuge tubes, add 0.100 mL of the working solution of the test substance with a concentration of 90.0 mg / L (STD-90.0 ppm), vortex and mix well to prepare a soil recovery sample with a high concentration of 9.00 μg / g. Each concentration sample was prepared in duplicate, and a soil blank was also set up for the experiment. Add 10.00 mL of acetonitrile to both the recovery test sample and the soil blank sample, sonicate for 10 min, centrifuge at 10000 r / min for 5 min, and filter the supernatant through a 0.22 μm filter membrane. For low-concentration recovery test samples and soil blank samples, the concentration of the test substance was directly measured. For high-concentration recovery test samples, the concentration of the test substance was determined by diluting the sample 5 times with acetonitrile.

[0105] Soil water sample addition and recovery: Weigh approximately 4.0g of prepared red soil, black soil, brown soil, alluvial soil, and paddy soil into 50mL centrifuge tubes. Prepare 3 replicates for each soil type. Add 40.00mL of 0.01mol / L calcium chloride solution, mix and shake for 12 hours for pre-equilibration, then centrifuge at 10000r / min for 5min and separate the supernatant. Take 0.100 mL of the working solution of the test substance with a concentration of 540 μg / L (STD-540 ppb) into a 10.00 mL volumetric flask, and dilute to the mark with the supernatant of each soil sample. Mix well to prepare a soil water sample with a low concentration recovery rate of 5.40 μg / L. Take 0.100 mL of the working solution of the test substance with a concentration of 90.0 mg / L (STD-90.0 ppm) into a 10.00 mL volumetric flask, and dilute to the mark with the supernatant of each soil sample. Mix well to prepare a soil water sample with a high concentration recovery rate of 900 μg / L. Prepare five replicates for each concentration sample, and simultaneously set up a soil water blank for testing. Filter the above recovery samples through a 0.22 μm filter membrane. The low concentration recovery test samples and the soil water blank samples were directly measured, while the high concentration recovery test samples were diluted five times with acetonitrile before determining the test substance concentration.

[0106] The average recovery rate of soil and soil water samples and 0.01 mol / L calcium chloride solution samples should be between 80% and 110%.

[0107] The formula for calculating the recovery rate is: Where R is the recovery rate, C d C represents the measured concentration of the target analyte. a The actual concentration of the target substance added;

[0108] The formula for calculating standard deviation is: Where S is the standard deviation, x i Let i be the recovery rate obtained from the i-th measurement. is the average recovery rate, and N is the number of recovery rates included in the calculation;

[0109] The formula for calculating the relative standard deviation is: RSD stands for relative standard deviation.

[0110] Limit of detection and limit of quantitation:

[0111] The limit of detection (LOD) of a method is generally set to three times the baseline noise, that is, the concentration of the target analyte at a signal-to-noise ratio of 3.

[0112] The formula for calculating the detection limit of the method is: Where LOD is the limit of detection; C is the actual concentration added in the low concentration range; R S / N The signal-to-noise ratio corresponds to the concentration.

[0113] The formula for calculating the limit of quantitation of a method is: Where LOQ is the limit of quantitation; C is the theoretical concentration for recovery at low concentration levels; R S / N This represents the signal-to-noise ratio corresponding to the concentration.

[0114] Test results Specificity of p-nitrobenzene in water: The retention time of p-nitroiodobenzene is about 2.9 min. At a detection wavelength of 296 nm, the target peak can be baseline separated from other components, and there is no interference from other impurities. The method has good selectivity.

[0117] Specificity of p-nitrobenzene in soil:

[0118] At a detection wavelength of 296 nm, p-nitroiodobenzene has a retention time of approximately 6.1 min. No response peak was observed at this time point in the blank samples of 0.01 mol / L calcium chloride solution, red soil, black soil, alluvial soil, paddy soil, and brown soil. The target peak can achieve baseline separation, and the resolution with other impurity peaks is >1.5. There is no interference from other impurities, and the method has good selectivity.

[0119] Linear range and correlation:

[0120] In acetonitrile, p-nitroiodobenzene showed a linear equation of y = 0.12744x + 0.00918 in the range of 4.00–200 μg / L, with a linear correlation coefficient of 0.9999.

[0121] In methanol, p-nitrobenzene exhibits a linear equation of y = 0.1126x + 0.1760 within the range of 10.0 μg / L to 500 μg / L, with an R² of 0.9996.

[0122] Recovery rate and repeatability:

[0123] Recovery and repeatability of p-nitroboiodobenzene in water:

[0124] Recovery tests were conducted in test water at three different concentrations of p-nitrobenzene. The average recovery rate was 93.0% with an RSD of 3.13% at a concentration of 12.5 μg / L; the average recovery rate was 105% with an RSD of 2.09% at a concentration of 250 μg / L; and the average recovery rate was 94.3% with an RSD of 0.867% at a concentration of 6.00 mg / L.

[0125] Recovery tests were conducted on p-nitroiodobenzene at three different concentrations in algal culture medium. The average recovery rate was 84.2% with an RSD of 0.260% at a concentration of 50.0 μg / L; the average recovery rate was 88.7% with an RSD of 2.71% at a concentration of 250 μg / L; and the average recovery rate was 96.7% with an RSD of 4.25% at a concentration of 6.00 mg / L.

[0126] Recovery tests were conducted on p-nitroiodobenzene at three different concentrations in M4 algal culture medium. The average recovery rate was 101% with an RSD of 4.73% at a concentration of 12.5 μg / L; the average recovery rate was 95.0% with an RSD of 3.20% at a concentration of 250 μg / L; and the average recovery rate was 92.3% with an RSD of 1.83% at a concentration of 6.00 mg / L.

[0127] Recovery and repeatability of p-nitrobenzene in soil:

[0128] Recovery experiments were conducted with p-nitrobenzene at two different concentrations in 0.01 mol / L calcium chloride solutions after equilibration in red soil, black soil, brown soil, alluvial soil, and paddy soil. The average recoveries of p-nitrobenzene at a concentration of 5.40 μg / L were 97.3%, 95.3%, 94.3%, 93.4%, and 95.2%, with RSDs of 1.97%, 1.14%, 2.24%, 2.22%, and 2.33%, respectively. The average recoveries at a concentration of 900 μg / L were 104%, 102%, 104%, 103%, and 103%, with RSDs of 0.527%, 0.297%, 0.430%, 0.106%, and 0.532%, respectively.

[0129] Recovery experiments were conducted at two concentrations of p-nitrobenzene in red soil, black soil, brown soil, alluvial soil, and paddy soil. The average recoveries of p-nitrobenzene at a concentration of 0.0540 μg / g were 98.4%, 99.5%, 105%, 103%, and 97.4%, with RSDs of 6.40%, 5.52%, 4.18%, 2.42%, and 5.64%, respectively. The average recoveries of p-nitrobenzene at a concentration of 9.00 μg / g were 102%, 102%, 102%, 101%, and 99.1%, with RSDs of 0.438%, 0.537%, 0.438%, 0.542%, and 0.648%, respectively.

[0130] Limit of detection and limit of quantitation:

[0131] Limits of detection and limits of quantification for p-nitrobenzene in water:

[0132] The LOQ of 4-nitroiodobenzene in the test water was 1.57 μg / L and the LOD was 0.471 μg / L; the LOQ in the algal culture medium was 3.10 μg / L and the LOD was 0.932 μg / L; and the LOQ in the M4 medium was 1.41 μg / L and the LOD was 0.423 μg / L.

[0133] Limits of detection and limits of quantification for p-nitrobenzene in soil:

[0134] The limits of quantification (LOQs) for p-nitroiodobenzene in 0.01 mol / L calcium chloride solutions after equilibration in red soil, black soil, brown soil, alluvial soil, and paddy soil were 2.11 μg / L, 1.93 μg / L, 2.01 μg / L, 2.25 μg / L, and 2.07 μg / L, respectively, and the limits of detection (LODs) were 0.633 μg / L, 0.579 μg / L, 0.604 μg / L, 0.675 μg / L, and 0.620 μg / L, respectively.

[0135] The limits of quantification for p-nitroiodobenzene in red soil, black soil, brown soil, alluvial soil, and paddy soil were 0.0365 μg / g, 0.0419 μg / g, 0.0394 μg / g, 0.0320 μg / g, and 0.0320 μg / g, respectively, and the limits of detection were 0.0110 μg / g, 0.0126 μg / g, 0.0118 μg / g, 0.00961 μg / g, and 0.00962 μg / g, respectively.

[0136] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for detecting p-nitroboiodobenzene in water and soil based on ultra-high performance liquid chromatography, characterized in that: Includes the following steps: Step 1: Use ultra-high performance liquid chromatography (UHPLC) for detection, and determine different detection conditions based on the water body and soil. Step 2: Preparation of the working solution for the analyte, including the preparation of the standard stock solution and the standard working solution; Step 3: Specificity test of p-nitroiodobenzene. The specificity test of p-nitroiodobenzene in water is as follows: blank solvent, blank matrix sample, and standard working solution of the lowest concentration were injected for testing. The specificity test of p-nitroiodobenzene in soil is as follows: the established liquid chromatography analysis method was used to determine the blank sample of 0.01 mol / L calcium chloride solution, the recovery rate of 0.01 mol / L calcium chloride solution, blank water samples and soil samples of red soil, black soil, brown soil, alluvial soil, and paddy soil, and the recovery rate water samples and soil samples of red soil, black soil, brown soil, alluvial soil, and paddy soil. The spectra of blank samples and recovery rate samples were compared. Step 4: Recovery and Repeatability Experiments. The recovery and repeatability experiments in water are as follows: One matrix blank control group and three spiking concentrations were set up for each of the test water, algae culture medium, and M4 culture medium for recovery and repeatability experiments. Five replicates were performed for each concentration. The precision of the analytical method was expressed as repeatability, using relative standard deviation (RSD). Repeatability was analyzed using the recovery test solution, and the RSD was calculated for five levels each of low, medium, and high concentrations. The recovery and repeatability experiments in soil are as follows: Five matrix blank control groups and two spiking concentrations were set up for soil for recovery and repeatability experiments. Five samples were prepared in parallel for each concentration. The accuracy of the analytical method was expressed as the recovery rate, using the average recovery rate. The precision of the analytical method was expressed as repeatability, using relative standard deviation (RSD). Step 5: Limit of Detection (LOD) and Limit of Quantification (LOQ). The method's LOD is set at 3 times the baseline noise, i.e., the concentration of the target analyte at a signal-to-noise ratio (SNR) of 3. The method's LOD is set at 10 times the baseline noise, i.e., the concentration of the target analyte at a SNR of 10. Step 6: Analysis of experimental results, including specificity, linear range and correlation, recovery and repeatability, limit of detection and limit of quantitation.

2. The method for detecting p-nitroboiodobenzene in water and soil based on ultra-high performance liquid chromatography according to claim 1, characterized in that: In step one, The instrument conditions for testing p-nitrobenzene in water were as follows: An Agilent 1290-6495A instrument was used; a VWD detector was used; the flow rate was 0.500 mL / min; the injection volume was 20.00 μL; the column temperature was 30.0℃; the mobile phase and its ratio (V / V) were acetonitrile:0.1% phosphoric acid water (70:30); the detection wavelength was 296 nm; the run time was 8.00 min; and the retention time was approximately 2.9 min. The instrument conditions for testing p-nitrobenzene in soil were as follows: the instrument used was an Agilent 1290-6495A; the column was an Accurasil C18, 250 mm × 4.6 mm (id), 5 μm, (SN: 074263A); the detector was a VWD; the detection wavelength was 296 nm; mobile phase A was acetonitrile; mobile phase B was 0.1% phosphoric acid water; the flow rate was 1.000 mL / min; the injection volume was 50.00 μL; the column temperature was 30.0℃; the run time was 15.00 min; and the retention time was approximately 6.1 min.

3. The method for detecting p-nitroboiodobenzene in water and soil based on ultra-high performance liquid chromatography according to claim 1, characterized in that: In step two, The standard stock solution in water is prepared as follows: the standard sample name is p-nitroiodobenzene, the amount of standard substance weighed is 0.02006g, the purity is 99.7%, the diluent is methanol, the volume is 20.00mL, the stock solution concentration is 1000mg / L, the volume is adjusted to the mark with solvent, mixed well, and stored in a refrigerator at 2℃-8℃ in a sealed container away from light for later use; The standard stock solutions for soil were prepared as follows: The test sample was weighed and dissolved in methanol to prepare a test sample stock solution (STD-1000ppm); the test sample stock solution was then dissolved in calcium chloride solution to prepare a test sample stock solution (STD-CaCl2-4.00ppm), which was used for preparing the standard working solution; test sample stock solutions of different concentrations were diluted with acetonitrile for spiking recovery tests. The standard working solution in water is prepared as follows: take mother liquor of different concentrations and different transfer amounts, and dilute with acetonitrile to obtain standard working solutions of different concentrations; The standard working solution in soil was prepared as follows: different concentrations and different transfer amounts of mother liquor were taken and diluted with methanol to obtain standard working solutions of different concentrations.

4. The method for detecting p-nitroboiodobenzene in water and soil based on ultra-high performance liquid chromatography according to claim 1, characterized in that: In step two, Linearity and range of p-nitrobenzene in water: A series of standard working solutions were obtained after dilution. Six calibration points were set for the standard curve. The linear range was 10.0 to 500 μg / L. The standard curve was fitted by a linear regression equation with the concentration of p-nitrobenzene as the abscissa and the average peak area as the ordinate. Linearity and range of p-nitroiodobenzene in soil: A series of standard working solutions were obtained after dilution. Six calibration points were set for the standard curve. The linear range was 4.00 to 200 μg / L. Two samples were collected for each concentration. The standard curve was fitted by a linear regression equation with the concentration of the test substance as the abscissa and the average peak area as the ordinate.

5. The method for detecting p-nitroboiodobenzene in water and soil based on ultra-high performance liquid chromatography according to claim 1, characterized in that: In step four, the recovery rate is calculated using the following formula: Where R is the recovery rate, C d C represents the measured concentration of the target analyte. a The actual concentration of the target substance added; The formula for calculating standard deviation is: Where S is the standard deviation, x i Let i be the recovery rate obtained from the i-th measurement. is the average recovery rate, and N is the number of recovery rates included in the calculation; The formula for calculating the relative standard deviation is: RSD stands for relative standard deviation.

6. The method for detecting p-nitroboiodobenzene in water and soil based on ultra-high performance liquid chromatography according to claim 1, characterized in that: In step five, the formula for calculating the detection limit of the method is: Where LOD is the limit of detection; C is the actual concentration added in the low concentration range; R S / N The signal-to-noise ratio corresponds to the concentration. The formula for calculating the limit of quantitation of a method is: Where LOQ is the limit of quantitation; C is the theoretical concentration for recovery at low concentration levels; R S / N This represents the signal-to-noise ratio corresponding to the concentration.

Images