Method for simultaneously determining four amide compounds in soil

Abstract

The invention discloses a method for simultaneously determining four amide compounds in soil, an extracting agent selected in a sample pretreatment process comprises 3%-10% acetonitrile and / or 0.1%-1% ammonia water, the extraction efficiency is high, in addition, 3%-10% acetonitrile is selected for gradient elution in an elution procedure of chromatographic analysis, and 0-6 min, 3%-10% acetonitrile is selected; the reaction time is 6-7.5 min, the reaction time is 10% acetonitrile, and the reaction time is 7.5-9 min, the reaction time is 10%-3% acetonitrile The flow rate is 1.0 mL / min; the column temperature is 30 DEG C; the detection wavelength is 198 nm; the sample injection volume is 10.0 [mu] L; the method disclosed by the invention is simple, stable, sensitive, accurate, relatively low in cost, simple and convenient to operate and easy to popularize.

Description

Technical Field

[0001] The present invention relates to a method for determining toxic substances in soil, in particular to a method for simultaneously determining four amide compounds in soil. Background Art

[0002] Formamide, N,N-dimethylformamide and N,N-dimethylacetamide are mainly used as solvents and synthetic raw materials. Acrylamide is an important product in the acrylamide and methacrylamide series products, and is widely used in the film, fiber, coating, pharmaceutical synthetic leather and clothing industries. Production companies will discharge amide pollutants into the environment in the form of exhaust pipes, wastewater or solid waste. Amide compounds are easily soluble in water, and pollutants can be enriched in the soil through rainfall, surface runoff or solid waste leaching. Their hydrophilic nature makes them easy to migrate in the soil, which can further contaminate groundwater. Such substances can be absorbed through the skin, mucous membranes, respiratory tract and intestines, and enriched in the human body, endangering human health.

[0003] Currently, the main methods used for the detection of amides in China and abroad include liquid chromatography, gas chromatography, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). These methods primarily target the atmosphere, water, and products. GC-MS and LC-MS are expensive, making them difficult to use. Furthermore, there are currently no methods for the simultaneous determination of all four amides in soil. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for simultaneously determining four amide compounds in soil. The method is simple, stable, sensitive, accurate, low-cost, easy to operate and easy to popularize, providing technical support for monitoring methods for the prevention and control of pollution by four amides in soil. In particular, the pretreatment is simple and efficient, solving the problems of high cost and inability to simultaneously determine four amide compounds by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry.

[0005] To solve the above technical problems, the technical solution of the present invention is: a method for simultaneously determining four amide compounds in soil, comprising the following steps:

[0006] 1) Sample pretreatment: Weigh 5.00 g of fresh soil sample and place it in a 50 mL plastic centrifuge tube. Add 5 mL of an extractant containing 3% to 10% acetonitrile and / or 0.1% to 1% aqueous ammonia. Tighten the cap of the tube and extract at 1000 rpm in a multitube mixer for 5 to 30 min. Centrifuge at 3500 rpm for 10 min. Transfer the extract to a volumetric flask. Repeat this process twice. Combine the extracts and dilute to 10 mL. Filter the supernatant through a syringe filter.

[0007] 2) Chromatographic Analysis: A high performance liquid chromatograph was used, and the filtrate from step 1) was added to the HPLC. The chromatographic column was ZORBAX C18 reverse phase chromatography column, 4.6 mm × 150 mm × 5 μm; mobile phase A was acetonitrile; mobile phase B was ultrapure water; elution program was: 3% to 10% acetonitrile for 0-6 min; 10% acetonitrile for 6-7.5 min; 10% to 3% acetonitrile for 7.5-9 min; flow rate was 1.0 mL / min; column temperature was 30°C; detection wavelength was 198 nm; injection volume was 10.0 μL;

[0008] 3) Calibration curve and detection limit: 2.5 μL of the standard solution was added to each of several blank matrices, and the calibration curve was prepared using a mixed standard solution consisting of 500 μg / ml acrylamide, 500 μg / ml N,N-dimethylformamide, 1000 μg / ml formamide, and 1000 μg / ml N,N-dimethylacetamide.

[0009] The concentrations of formamide and N,N-dimethylacetamide were selected as follows: 0.10 mg / L, 0.50 mg / L, 1.00 mg / L, 2.00 mg / L, 5.00 mg / L, and 10.0 mg / L; the concentrations of acrylamide and N,N-dimethylformamide were selected as follows: 0.05 mg / L, 0.25 mg / L, 0.50 mg / L, 1.00 mg / L, 2.50 mg / L, and 5.00 mg / L. The solvent was pure water. The correlation coefficients of the standard curves of the four substances were all above 0.999. The detection limits ranged from 0.05 mg / kg to 0.21 mg / kg, and the quantification limits ranged from 0.20 mg / kg to 0.84 mg / kg.

[0010] 4) Precision and Accuracy: Actual soil samples were spiked with low, medium, and high concentrations. Formamide and N,N-dimethylacetamide were spiked at the same concentrations: 1.00 mg / L, 5.00 mg / L, and 20.0 mg / L, respectively. Acrylamide and N,N-dimethylformamide were spiked at the same concentrations: 0.50 mg / L, 2.50 mg / L, and 10.00 mg / L, respectively. Six consecutive measurements were performed for each spiked concentration. The recoveries and precision of the spiked samples were calculated. The recoveries of the actual soil samples ranged from 74.4% to 119%, with standard deviations of 1.4% to 10.6%.

[0011] Preferably, the extractant added in step 1) is a mixed solution of 10% acetonitrile and 0.5% ammonia water.

[0012] Preferably, the needle filter selected in step 1) is a 0.22 μm polytetrafluoroethylene needle filter.

[0013] Preferably, in step 1), the extraction is performed by oscillation in a multi-tube mixer for 10 minutes.

[0014] After adopting the above technical scheme, the effect of the present invention is as follows: since the present invention applies for a method for simultaneously determining four amide compounds in soil, the sample pretreatment process is simple to operate, the extraction agent selected includes 3% to 10% acetonitrile and / or 0.1% to 1% ammonia water, and the extraction efficiency is high. In addition, the elution procedure of the chromatographic analysis selects 3%-10% acetonitrile gradient elution, wherein, 0-6min, 3%-10% acetonitrile; 6-7.5min, 10% acetonitrile, 7.5-9min, 10%-3% acetonitrile; flow rate: 1.0mL / min; column temperature: 30℃; detection wavelength: 198nm; injection volume: 10.0μL; elution effect is good, the method applied for by the present invention is simple, stable, sensitive and accurate, low cost, simple to operate and easy to popularize, providing technical support for monitoring methods for the prevention and control of pollution of four amides in soil.

[0015] Furthermore, since the extraction agent added in step 1) is a mixed solution of 10% acetonitrile and 0.5% ammonia water, and the extraction is performed in a multi-tube mixer for 10 minutes, the extraction effect is better under the above conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the chromatogram of 3% acetonitrile isocratic elution;

[0017] Figure 2 This is the chromatogram of 10% acetonitrile isocratic elution;

[0018] Figure 3 The chromatogram is a gradient elution chromatogram of 3%-10% acetonitrile;

[0019] Figure 4 The effect of different extraction agents on the recovery rate;

[0020] Figure 5 The effects of different oscillation time and ultrasonic time on the average recovery of the four components. DETAILED DESCRIPTION

[0021] The present invention is described in further detail below.

[0022] 1 Experimental section

[0023] 1.1 Instruments and Reagents

[0024] High performance liquid chromatograph (Agilent, 1260); ultrasonic cleaner (Kunshan Hechuang, HCS5404), power 0.4kW; multi-tube mixer (Nantong Hubei Instrument, HB-DMT-2500), centrifuge (Anhui Zhongke Zhongjia, SC-3610), chromatographic column (Agilent, C18,

[0025] 4.6mm×150mm×5μm).

[0026] Four amide standard substances (acrylamide and N,N-dimethylformamide 500 μg / ml, formamide and N,N-dimethylacetamide 1000 μg / ml, quality control by Tanmo), acetonitrile (chromatographic grade), ammonia water (analytical grade), ultrapure water (Millipore, Synergy UV, USA), phosphoric acid (analytical grade), and a 0.22 μm polytetrafluoroethylene syringe filter.

[0027] 1.2 Instrumental analysis conditions

[0028] Chromatographic column: ZORBAX C18 reverse phase chromatography, 4.6 mm × 150 mm × 5 μm; mobile phase A: acetonitrile; mobile phase B: ultrapure water; elution program: 0-6 min, 3%-10% acetonitrile; 6-7.5 min, 10% acetonitrile, 7.5-9 min, 10%-3% acetonitrile; flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 198 nm; injection volume: 10.0 μL.

[0029] 1.3 Sample pretreatment

[0030] Weigh 5.00 g of fresh soil sample, place it in a 50 mL plastic centrifuge tube, add 5 mL of extractant, the extractant includes 3% to 10% acetonitrile and / or 0.1% to 1% ammonia water, tighten the bottle cap, shake and extract at 1000 rpm in a multi-tube mixer for 5 to 30 minutes, then place it in a centrifuge at 3500 rpm for 10 minutes, transfer the first extract to a volumetric flask, repeat the above process, continue to add 5 mL of extractant, shake and extract at 1000 rpm in a multi-tube mixer for 5 to 30 minutes, then place it in a centrifuge at 3500 rpm for 10 minutes, transfer the second extract to a volumetric flask, combine the first extract and the second extract, and adjust the volume to 10 mL. Take the supernatant and filter it through a syringe filter.

[0031] 2 Results and discussion

[0032] 2.1 Optimization of chromatographic conditions

[0033] In liquid chromatography, this paper selected the most commonly used C18 chromatographic column for testing and research, and investigated the effect of the proportion of acetonitrile in the mobile phase on the separation effect of four substances.

[0034] The first scheme used 3% acetonitrile / water system isocratic elution, and it was found that the four components could be effectively separated, and all components could be completely eluted within 8 minutes. Figure 1It can be seen that the peaks of N,N-dimethylformamide and N,N-dimethylacetamide in this system are relatively broad, with the maximum peak width reaching more than 0.2 min, and the column efficiency is not high.

[0035] The second method uses a 10% acetonitrile / water system for isocratic elution. Figure 2 It can be seen that under this system, the maximum peak width is reduced to about 0.1 min, but the peaks of the four substances are relatively close together, and the negative peak of water is not completely separated from acrylamide.

[0036] The third option requires both a large enough separation and a high column efficiency, using a gradient elution of 3%-10% acetonitrile. Figure 3 It can be seen that the acrylamide and water peaks are completely separated, and the maximum peak width is controlled at about 0.15 min. The specific peak width information is shown in Table 1. Therefore, a 3%-10% acetonitrile system gradient elution was selected as the optimal mobile phase system.

[0037] Table 1 Peak width of different mobile phase systems

[0038]

[0039] 2.2 Selection of extraction agent

[0040] The four amides studied in this paper are all water-soluble compounds. We initially considered using pure water as the extractant to investigate the extraction efficiency of the four amides. 10 μL of standard solution was added to 5 g of fresh soil, and 10 mL of pure water was added as the extractant. After oscillation extraction for 30 minutes, the supernatant was centrifuged for testing. The results showed that the extraction efficiency of formamide was 19.8%, and the extraction efficiency of other components reached 70%-90%. Because formamide has strong polarity and small molecular weight, it is easy to combine with polar sites in the soil, and pure water is difficult to elute it. The acetonitrile water system has a high extraction efficiency for amides, and the extraction efficiency is even higher after adding alkaline substances to the extractant. In this experiment, the competitiveness of polar sites in the soil was increased by adding acids and bases, and the extraction efficiency was studied using acetonitrile aqueous solution as the extractant. The experimental data are shown in Table 2.

[0041] like Figure 4 The experimental results show that when the proportion of phosphoric acid increases from 0.1% to 1%, the recovery rate of formamide increases to 52.6%, while the recovery rate of acrylamide decreases; when the proportion of ammonia increases from 0.1% to 1%, the recovery rate of formamide increases to 61.8%, and the recovery rates of other components remain above 80%. When the proportion of ammonia increases to 1%, the peak of N,N-dimethylacetamide has severe tailing; when the proportion of acetonitrile increases from 5% to 50%, the recovery rate of formamide increases to 70.7%. However, the increase in the proportion of acetonitrile will also increase the detection of impurities and affect the elution of the target peak. When the proportion of acetonitrile is controlled within 10%, the detection rate of impurities is low, and the purification step can be omitted.

[0042] From the above results, it can be seen that when the proportion of ammonia water is 0.5%, the extraction efficiency of the four components is optimal and the interference is minimal. When the proportion of phosphoric acid is 1%, the extraction efficiency of the four components is better. Considering the optimal extraction ratio of ammonia water and phosphoric acid, and the good extraction efficiency of acetonitrile for formamide, this experiment selected 10% acetonitrile-0.5% ammonia water and 10% acetonitrile-1% phosphoric acid systems as sample extractants. It was determined that the one-time extraction efficiency of the four components reached more than 70.0%.

[0043] In order to further improve the extraction efficiency, this experiment finally adopted a two-step extraction method, adding 5.0 mL of extractant each time, oscillating and centrifuging, collecting the extractant, repeating the above process, combining the extractants and making the volume up to 10 mL. The average recovery rates of the four components were measured on the machine and all reached more than 80.0%.

[0044] Both 10% acetonitrile-0.5% ammonia water and 10% acetonitrile-1% phosphoric acid extraction agents had good extraction efficiency for the four components in this experiment, and the 10% acetonitrile-0.5% ammonia water system was selected for subsequent research.

[0045] Table 2 Recovery rates of different extraction agents

[0046]

[0047] 2.3 Optimization of extraction method

[0048] This application selected ultrasonic extraction and oscillation extraction for comparative study.

[0049] 10 μL of standard solution was added to 5 g of fresh soil, and 10% acetonitrile-0.5% ammonia water was selected as the extraction solvent. Under ultrasonic and oscillation conditions, the effects of different extraction times on the average extraction efficiency of four amide compounds were investigated. The ultrasonic and oscillation times were set at 5, 10, 20, 30 and 40 min.

[0050] like Figure 5 As shown in the figure, when the extraction time was 10 min, the recovery rate of oscillation extraction reached the highest and then tended to be stable. The recovery rate of ultrasonic extraction tended to be stable after 20 min. The recovery rates of the two extraction methods could reach more than 80.0%.

[0051] Considering that the temperature of the sample solution begins to rise under ultrasonic conditions for 20 minutes, which is not conducive to the stability of the sample, and for sticky soil or soil that has formed lumps, the use of high-frequency oscillation can better loosen the soil so that it can fully contact with the extractant, it is preferred to perform sample extraction by oscillation extraction for 10 minutes.

[0052] 2.4 Calibration curve and detection limit

[0053] Add 2.5 μL (volume unit) of standard solution to each of the seven blank matrices. According to the Technical Guidelines for the Development and Revision of Environmental Monitoring Analytical Method Standards (HJ 168-2020), the detection limit of the method was calculated using the following formula 1.

[0054] A mixed standard solution (acrylamide and N,N-dimethylformamide 500 μg / ml, formamide and N,N-dimethylacetamide 1000 μg / ml) was used to configure the curve. The concentrations of formamide and N,N-dimethylacetamide were the same: 0.10, 0.50, 1.00, 2.00, 5.00 and 10.0 mg / L, and the concentrations of acrylamide and N,N-dimethylformamide were the same: 0.05, 0.25, 0.50, 1.00, 2.50 and 5.00 mg / L. The solvent was pure water.

[0055] As shown in Table 3, the correlation coefficients of the standard curves of the four substances were all above 0.999, and the detection limits and quantification limits were between 0.05 mg / kg and 0.21 mg / kg and 0.20 mg / kg and 0.84 mg / kg respectively.

[0056] MLD=3.143×S (Formula 1)

[0057] Where: MLD - method detection limit;

[0058] 3.143 – the t-distribution value at a 99% confidence level;

[0059] S is the relative standard deviation of n parallel measurements.

[0060] Table 3 Linear equation and detection limit

[0061]

[0062] 2.5 Precision and Accuracy

[0063] Actual soil samples were spiked with high, medium and low concentrations, and each spiked concentration was measured six times in a row. The recovery and precision of the spiked samples were calculated. As shown in Table 4, the spiked recoveries of the actual soil samples ranged from 74.4% to 119%, and the relative standard deviations were from 1.4% to 10.6%. The higher the spiked recovery, the higher the precision. The precision and accuracy of this method can meet the needs of soil environmental monitoring.

[0064] Table 4 Contents, recoveries and RSDs of 16aniline compounds in real samples

[0065]

[0066]

[0067] 3 Conclusion

[0068] In this paper, oscillation extraction-high performance liquid chromatography was used to determine four amide substances in soil. The instrument conditions and pretreatment process were optimized and improved. This method is simple and efficient, does not require complex extraction processes and purification steps, and can effectively avoid interference from impurities. This method has high sensitivity, and the detection limit reaches 0.05mg / kg~0.21mg / kg, which can meet the requirements of simultaneous determination of four amide substances in soil. The establishment of this method can make up for the current lack of relevant detection methods for four amide substances in soil, and provide technical support for the investigation of soil amide characteristic pollutant concentrations.

[0069] The above embodiments are descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Various modifications and alterations to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for simultaneously determining four amide compounds in soil, characterized by: The following steps are involved: 1) Sample pretreatment: Weigh 5.00 g of fresh soil sample and place it in a 50 mL plastic centrifuge tube. Add 5 mL of an extractant containing 3% to 10% acetonitrile and / or 0.1% to 1% aqueous ammonia. Tighten the cap of the tube and extract at 1000 rpm in a multitube mixer for 5 to 30 min. Centrifuge at 3500 rpm for 10 min. Transfer the extract to a volumetric flask. Repeat this process twice. Combine the extracts and dilute to 10 mL. Filter the supernatant through a syringe filter. 2) Chromatographic Analysis: A high performance liquid chromatograph was used, and the filtrate from step 1) was added to the HPLC. The chromatographic column was ZORBAX C18 reverse phase chromatography column, 4.6 mm × 150 mm × 5 μm; mobile phase A was acetonitrile; mobile phase B was ultrapure water; elution program was: 3% to 10% acetonitrile for 0-6 min; 10% acetonitrile for 6-7.5 min; 10% to 3% acetonitrile for 7.5-9 min; flow rate was 1.0 mL / min; column temperature was 30°C; detection wavelength was 198 nm; injection volume was 10.0 μL; 3) Calibration curve and detection limit: 2.5 μL of the standard solution was added to each of several blank matrices, and the calibration curve was prepared using a mixed standard solution consisting of 500 μg / ml acrylamide, 500 μg / ml N,N-dimethylformamide, 1000 μg / ml formamide, and 1000 μg / ml N,N-dimethylacetamide. The concentrations of formamide and N,N-dimethylacetamide were the same, selected as 0.10 mg / L, 0.50 mg / L, 1.00 mg / L, 2.00 mg / L, 5.00 mg / L, and 10.0 mg / L, respectively. The concentrations of acrylamide and N,N-dimethylformamide were the same, selected as 0.05 mg / L, 0.25 mg / L, 0.50 mg / L, 1.00 mg / L, 2.50 mg / L, and 5.00 mg / L, respectively. The solvent was pure water. The correlation coefficients of the standard curves of the four substances were all above 0.

999. The detection limits ranged from 0.05 mg / kg to 0.21 mg / kg, and the quantification limits ranged from 0.20 mg / kg to 0.84 mg / kg. 4) Precision and Accuracy: Actual soil samples were spiked with low, medium, and high concentrations. Formamide and N,N-dimethylacetamide were spiked at the same concentrations: 1.00 mg / L, 5.00 mg / L, and 20.0 mg / L, respectively. Acrylamide and N,N-dimethylformamide were spiked at the same concentrations: 0.50 mg / L, 2.50 mg / L, and 10.00 mg / L, respectively. Six consecutive measurements were performed at each spike concentration. The recoveries and precision of the spiked samples were calculated. The recoveries of the actual soil samples ranged from 74.4% to 119%, with standard deviations of 1.4% to 10.6%.

2. The method for simultaneously determining four amide compounds in soil according to claim 1, wherein: The extractant added in step 1) is a mixed solution of 10% acetonitrile and 0.5% ammonia water.

3. The method for simultaneously determining four amide compounds in soil according to claim 1, wherein: In the step 1), the needle filter selected is a 0.22 μm polytetrafluoroethylene needle filter.

4. The method for simultaneously determining four amide compounds in soil according to claim 1, wherein: The step 1) was performed by oscillating and extracting in a multi-tube mixer for 10 minutes.

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