阅读说明：本技术 土壤中苯并（α）芘的检测方法 (Method for detecting benzo (alpha) pyrene in soil ) 是由 王克超 宋鸽 杜晶晶 李慧娟 张俊龙 常建军 李梅 于 2020-03-16 设计创作，主要内容包括：本发明提出了土壤中苯并(α)芘的检测方法,所述方法包括：将土壤置于具塞三角瓶中,加入无水硫酸钠和丙酮-正己烷混合溶液,在回旋振荡器上振荡提取2～6小时,以便得到提取液；将所述提取液进行过滤处理,收集滤液；将所述滤液进行旋转蒸发处理,期间加入正己烷,以便得到浓缩液；利用层析柱对所述浓缩液进行净化处理,以便得到净化液；对所述净化液进行液相色谱检测。本发明的检测方法可以准确地检测出土壤中的苯并(α)芘含量,操作简便,用时短,适于规模化应用。(The invention provides a method for detecting benzo (alpha) pyrene in soil, which comprises the following steps: placing the soil into a triangular flask with a plug, adding a mixed solution of anhydrous sodium sulfate and acetone-n-hexane, and performing oscillation extraction on a rotary oscillator for 2-6 hours to obtain an extracting solution; filtering the extracting solution, and collecting filtrate; carrying out rotary evaporation treatment on the filtrate, and adding n-hexane during the rotary evaporation treatment so as to obtain a concentrated solution; purifying the concentrated solution by using a chromatographic column so as to obtain a purified solution; and carrying out liquid chromatography detection on the purified liquid. The detection method can accurately detect the content of the benzo (alpha) pyrene in the soil, is simple and convenient to operate, short in time consumption and suitable for large-scale application.)

1. A method for detecting benzo (alpha) pyrene in soil is characterized by comprising the following steps:

(1) weighing 10g of soil into a triangular flask with a plug, adding anhydrous sodium sulfate and 100mL of acetone-n-hexane mixed solution with the volume ratio of 1:1, and extracting for 4h by oscillation of a rotary oscillator to obtain an extracting solution;

(2) filling a layer of glass fiber filter paper on a glass funnel, adding 5g of anhydrous sodium sulfate, collecting the bottom of the glass funnel by using a rotary evaporation bottle, filtering the extracting solution by using the anhydrous sodium sulfate and the filter paper, entering the rotary evaporation bottle, washing the triangular bottle with a stopper and the funnel by using the acetone-n-hexane mixed solution, combining the liquids, filtering the liquids into the rotary evaporation bottle, carrying out rotary evaporation at 45 ℃ to 1ml, adding 5ml of n-hexane, concentrating the n-hexane and the n-hexane to 1ml, repeating the operation of adding the n-hexane and the n-hexane for 3 times, and concentrating the n-hexane and the n-hexane to 1ml again to obtain a concentrated solution;

(3) activating a florisil small column by using 6ml of n-hexane, adding the concentrated solution into the small column when the liquid level is quickly drained, collecting effluent liquid into a test tube, washing the rotary evaporation bottle for multiple times by using the n-hexane, and finally eluting the small column by using 10ml of dichloromethane-n-hexane mixed solution with the volume ratio of 1: 1; collecting the eluent in the same test tube, drying the eluent with nitrogen at 45 ℃ to 1ml, adding 3ml of acetonitrile, concentrating the eluent to 1ml, repeating the operations of adding the acetonitrile and concentrating for 2 times to completely convert the solvent into the acetonitrile, finally drying the eluent and fixing the volume of the eluent to 1ml to obtain a purified solution;

(4) and (3) carrying out liquid chromatography detection on the purified solution under the following conditions:

a chromatographic column: c18 chromatographic column with size of 250nm × 4.6mm and 5 μm;

mobile phase: acetonitrile-water solution with volume ratio of 88: 12;

flow rate: 1.0 mL/min;

a fluorescence detector: the excitation wavelength is 384nm, and the emission wavelength is 406 nm;

sample introduction amount: 20 mu L of the solution;

column temperature: 35 ℃ is carried out.

2. A method for detecting benzo (alpha) pyrene in soil is characterized by comprising the following steps:

placing the soil into a triangular flask with a plug, adding a mixed solution of anhydrous sodium sulfate and acetone-n-hexane, and performing oscillation extraction on a rotary oscillator for 2-6 hours to obtain an extracting solution;

filtering the extracting solution, and collecting filtrate;

carrying out rotary evaporation treatment on the filtrate, and adding n-hexane during the rotary evaporation treatment so as to obtain a concentrated solution;

purifying the concentrated solution by using a chromatographic column so as to obtain a purified solution;

and carrying out liquid chromatography detection on the purified liquid.

3. The method according to claim 2, wherein the acetone-n-hexane mixed solution is added in an amount of 8-12 mL based on 1g of the soil, and the volume ratio of acetone to n-hexane in the acetone-n-hexane mixed solution is 1: 1.

4. The method of claim 2, wherein the filtering process comprises:

placing filter paper on a glass funnel, placing anhydrous sodium sulfate on the filter paper, adding the extracting solution to the filter paper, and collecting filtrate filtered by the glass funnel.

5. The method of claim 2, wherein the chromatography column is selected from florisil small columns.

6. The method of claim 5, wherein the decontamination process comprises:

adding the concentrated solution into a florisil small column activated by n-hexane, and collecting effluent liquid to a test tube;

adding a dichloromethane-n-hexane solution into the Florisil small column, collecting and combining and flowing out liquid;

and blowing the combined effluent to 0.5-1.5 mL by nitrogen at 40-50 ℃, adding acetonitrile, concentrating, and continuously blowing to 0.5-1.5 mL so as to obtain the purified liquid.

7. The method according to claim 2, wherein the liquid chromatography detection conditions are as follows:

a chromatographic column: c18 chromatographic column with size of 250nm × 4.6mm and 5 μm;

mobile phase: acetonitrile-water solution with volume ratio of 88: 12;

flow rate: 1.0 mL/min;

a fluorescence detector: the excitation wavelength is 384nm, and the emission wavelength is 406 nm;

sample introduction amount: 20 mu L of the solution;

column temperature: 35 ℃ is carried out.

Technical Field

The present invention relates to the field of the environment. Specifically, the invention relates to a method for detecting benzo (alpha) pyrene in soil.

Background

Benzopyrene is also called benzo (alpha) pyrene and is a common indirect carcinogen with high activity. It is easily combined with human DNA after breathing or eating, and disturbs the synthetic process of human protein. It plays a "trigger" role in the DNA activity: the structure, direction and function of DNA can be changed only by extremely micro (nanogram level) benzo (alpha) pyrene; with the DNA bound to benzo (α) pyrene, the synthesized cells are no longer normal cells, but are tumors, which also lead to cancer. The benzo (alpha) pyrene compound is easy to remain in water, soil and crops, investigation on the content of benzo (alpha) pyrene in soil is carried out in many countries, the residue concentration depends on the nature and distance of a pollution source, and the content of benzo (alpha) pyrene in soil on two sides of a highway, soil near an oil refinery and soil polluted by coal tar and asphalt is not low.

However, the detection method of benzo (alpha) pyrene in soil still needs to be researched.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides the method for detecting the benzo (alpha) pyrene in the soil, the method can accurately detect the benzo (alpha) pyrene content in the soil, the operation is simple and convenient, the time consumption is short, and the method is suitable for large-scale application.

The invention provides a method for detecting benzo (alpha) pyrene in soil. According to an embodiment of the invention, the method comprises: placing the soil into a triangular flask with a plug, adding a mixed solution of anhydrous sodium sulfate and acetone-n-hexane, and performing oscillation extraction on a rotary oscillator for 2-6 hours to obtain an extracting solution; filtering the extracting solution, and collecting filtrate; carrying out rotary evaporation treatment on the filtrate, and adding n-hexane during the rotary evaporation treatment so as to obtain a concentrated solution; purifying the concentrated solution by using a chromatographic column so as to obtain a purified solution; and carrying out liquid chromatography detection on the purified liquid.

The inventor finds that the contact and extraction of benzo (alpha) pyrene with acetone and normal hexane are easily hindered due to more impurities in soil, and the extraction efficiency is low and the detection result is low if benzo (alpha) pyrene is directly mixed with the acetone and the normal hexane. Therefore, the inventor utilizes the cyclotron oscillator to enable the contact between the soil and the acetone and the normal hexane to be more sufficient, so that the extraction of benzo (alpha) pyrene is more facilitated, and the accuracy of a detection result is ensured. In addition, the extraction process is short in time, and benzo (alpha) pyrene can be extracted sufficiently only in 2-6 hours. Therefore, the method for detecting benzo (alpha) pyrene in soil according to the embodiment of the invention has the advantages of strong accuracy, simple operation, short time and suitability for large-scale application.

According to the embodiment of the invention, the method for detecting benzo (alpha) pyrene in soil can also have the following additional technical characteristics:

according to the embodiment of the invention, the addition amount of the acetone-n-hexane mixed solution is 8-12 mL based on 1g of soil, and the volume ratio of acetone to n-hexane in the acetone-n-hexane mixed solution is 1: 1.

According to an embodiment of the invention, the filtering process comprises: placing filter paper on a glass funnel, placing anhydrous sodium sulfate on the filter paper, adding the extracting solution to the filter paper, and collecting filtrate filtered by the glass funnel.

According to an embodiment of the invention, the chromatography column is selected from florisil pillars.

According to an embodiment of the invention, the purification process comprises: adding the concentrated solution into a florisil small column activated by n-hexane, and collecting effluent liquid to a test tube; adding a dichloromethane-n-hexane solution into the Florisil small column, collecting and combining and flowing out liquid; and blowing the combined effluent to 0.5-1.5 mL by nitrogen at 40-50 ℃, adding acetonitrile, concentrating, and continuously blowing to 0.5-1.5 mL so as to obtain the purified liquid.

According to an embodiment of the present invention, the liquid chromatography detection conditions are as follows: a chromatographic column: c18 chromatographic column with size of 250nm × 4.6mm and 5 μm; mobile phase: acetonitrile-water solution with volume ratio of 88: 12; flow rate: 1.0 mL/min; a fluorescence detector: the excitation wavelength is 384nm, and the emission wavelength is 406 nm; sample introduction amount: 20 mu L of the solution; column temperature: 35 ℃ is carried out.

In addition, the invention provides another method for detecting benzo (alpha) pyrene in soil. According to an embodiment of the invention, the method comprises: (1) weighing 10g of soil into a triangular flask with a plug, adding anhydrous sodium sulfate and 100mL of acetone-n-hexane mixed solution with the volume ratio of 1:1, and extracting for 4h by oscillation of a rotary oscillator to obtain an extracting solution; (2) filling a layer of glass fiber filter paper on a glass funnel, adding 5g of anhydrous sodium sulfate, collecting the bottom of the glass funnel by using a rotary evaporation bottle, filtering the extracting solution by using the anhydrous sodium sulfate and the filter paper, entering the rotary evaporation bottle, washing the triangular bottle with a stopper and the funnel by using the acetone-n-hexane mixed solution, combining the liquids, filtering the liquids into the rotary evaporation bottle, carrying out rotary evaporation at 45 ℃ to 1ml, adding 5ml of n-hexane, concentrating the n-hexane and the n-hexane to 1ml, repeating the operation of adding the n-hexane and the n-hexane for 3 times, and concentrating the n-hexane and the n-hexane to 1ml again to obtain a concentrated solution; (3) activating a florisil small column by using 6ml of n-hexane, adding the concentrated solution into the small column when the liquid level is quickly drained, collecting effluent liquid into a test tube, washing the rotary evaporation bottle for multiple times by using the n-hexane, and finally eluting the small column by using 10ml of dichloromethane-n-hexane mixed solution with the volume ratio of 1: 1; collecting the eluent in the same test tube, drying the eluent with nitrogen at 45 ℃ to 1ml, adding 3ml of acetonitrile, concentrating the eluent to 1ml, repeating the operations of adding the acetonitrile and concentrating for 2 times to completely convert the solvent into the acetonitrile, finally drying the eluent and fixing the volume of the eluent to 1ml to obtain a purified solution; (4) and (3) carrying out liquid chromatography detection on the purified solution under the following conditions: a chromatographic column: c18 chromatographic column with size of 250nm × 4.6mm and 5 μm; mobile phase: acetonitrile-water solution with volume ratio of 88: 12; flow rate: 1.0 mL/min; a fluorescence detector: the excitation wavelength is 384nm, and the emission wavelength is 406 nm; sample introduction amount: 20 mu L of the solution; column temperature: 35 ℃ is carried out. Therefore, the method for detecting benzo (alpha) pyrene in soil according to the embodiment of the invention has the advantages of strong accuracy, simple operation, short time and suitability for large-scale application.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a chromatogram according to an embodiment of the invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

The invention provides a method for detecting benzo (alpha) pyrene in soil. According to an embodiment of the invention, the method comprises: placing the soil into a triangular flask with a plug, adding a mixed solution of anhydrous sodium sulfate and acetone-n-hexane, and performing oscillation extraction on a rotary oscillator for 2-6 hours to obtain an extracting solution; filtering the extracting solution, and collecting filtrate; carrying out rotary evaporation treatment on the filtrate, and adding n-hexane during the rotary evaporation treatment so as to obtain a concentrated solution; purifying the concentrated solution by using a chromatographic column so as to obtain a purified solution; and carrying out liquid chromatography detection on the purified liquid.

The inventor finds that the contact and extraction of benzo (alpha) pyrene with acetone and normal hexane are easily hindered due to more impurities in soil, and the extraction efficiency is low and the detection result is low if benzo (alpha) pyrene is directly mixed with the acetone and the normal hexane. Therefore, the inventor utilizes the cyclotron oscillator to enable the contact between the soil and the acetone and the normal hexane to be more sufficient, so that the extraction of benzo (alpha) pyrene is more facilitated, and the accuracy of a detection result is ensured. In addition, the extraction process is short in time, and benzo (alpha) pyrene can be extracted sufficiently only in 2-6 hours. Therefore, the method for detecting benzo (alpha) pyrene in soil according to the embodiment of the invention has the advantages of strong accuracy, simple operation, short time and suitability for large-scale application.

According to the embodiment of the invention, the addition amount of the acetone-n-hexane mixed solution is 8-12 mL based on 1g of soil, and the volume ratio of acetone to n-hexane in the acetone-n-hexane mixed solution is 1: 1. The composition and the addition amount of the acetone-n-hexane mixed solution are obtained through a large number of experiments, so that benzo (alpha) pyrene in soil can be fully extracted, and the accuracy of a detection result is ensured.

According to an embodiment of the invention, the filtering process comprises: placing filter paper on the glass funnel, placing anhydrous sodium sulfate on the filter paper, adding the extract to the filter paper, and collecting the filtrate filtered out by the glass funnel. Therefore, impurities can be effectively removed, wherein the anhydrous sodium sulfate can effectively remove water in the sample extracting solution, and the influence of the anhydrous sodium sulfate on subsequent experiments is avoided.

According to an embodiment of the invention, the chromatography column is selected from florisil pillars. The inventor researches and analyzes various chromatographic columns based on the composition of concentrated solution, and finds that impurities can be effectively removed by using a Florisil small column, so that the influence of the Florisil small column on subsequent chromatographic detection is avoided, and the accuracy of a detection result is ensured.

According to an embodiment of the invention, the purification process comprises: adding the concentrated solution into a florisil small column activated by n-hexane, and collecting effluent liquid to a test tube; adding a dichloromethane-n-hexane solution into a Florisil small column, collecting and combining and flowing out liquid; and blowing the combined effluent to 0.5-1.5 mL by nitrogen at 40-50 ℃, adding acetonitrile, concentrating, and continuously blowing to 0.5-1.5 mL so as to obtain the purified liquid. Therefore, impurities can be effectively removed, the influence of the impurities on subsequent chromatographic detection is avoided, and the accuracy of a detection result is ensured.

According to the embodiment of the invention, the detection conditions of the liquid chromatography are as follows: a chromatographic column: c18 chromatographic column with size of 250nm × 4.6mm and 5 μm; mobile phase: acetonitrile-water solution with volume ratio of 88: 12; flow rate: 1.0 mL/min; a fluorescence detector: the excitation wavelength is 384nm, and the emission wavelength is 406 nm; sample introduction amount: 20 mu L of the solution; column temperature: 35 ℃ is carried out. The inventor obtains the detection condition of the liquid chromatogram through a large number of experiments, so that benzo (alpha) pyrene can be accurately detected, and interference caused by other impurities is avoided.

In addition, the invention provides another method for detecting benzo (alpha) pyrene in soil. According to an embodiment of the invention, the method comprises: (1) weighing 10g of soil into a triangular flask with a plug, adding anhydrous sodium sulfate and 100mL of acetone-n-hexane mixed solution with the volume ratio of 1:1, and extracting for 4h by oscillation of a rotary oscillator to obtain an extracting solution; (2) filling a layer of glass fiber filter paper on a glass funnel, adding 5g of anhydrous sodium sulfate, collecting the bottom of the glass funnel by using a rotary evaporation bottle, filtering the extracting solution by using the anhydrous sodium sulfate and the filter paper, entering the rotary evaporation bottle, washing the triangular bottle with a stopper and the funnel by using the acetone-n-hexane mixed solution, combining the liquids, filtering the liquids into the rotary evaporation bottle, carrying out rotary evaporation at 45 ℃ to 1ml, adding 5ml of n-hexane, concentrating the n-hexane and the n-hexane to 1ml, repeating the operation of adding the n-hexane and the n-hexane for 3 times, and concentrating the n-hexane and the n-hexane to 1ml again to obtain a concentrated solution; (3) activating a florisil small column by using 6ml of n-hexane, adding the concentrated solution into the small column when the liquid level is quickly drained, collecting effluent liquid into a test tube, washing the rotary evaporation bottle for multiple times by using the n-hexane, and finally eluting the small column by using 10ml of dichloromethane-n-hexane mixed solution with the volume ratio of 1: 1; collecting the eluent in the same test tube, drying the eluent with nitrogen at 45 ℃ to 1ml, adding 3ml of acetonitrile, concentrating the eluent to 1ml, repeating the operations of adding the acetonitrile and concentrating for 2 times to completely convert the solvent into the acetonitrile, finally drying the eluent and fixing the volume of the eluent to 1ml to obtain a purified solution; (4) and (3) carrying out liquid chromatography detection on the purified solution under the following conditions: a chromatographic column: c18 chromatographic column with size of 250nm × 4.6mm and 5 μm; mobile phase: acetonitrile-water solution with volume ratio of 88: 12; flow rate: 1.0 mL/min; a fluorescence detector: the excitation wavelength is 384nm, and the emission wavelength is 406 nm; sample introduction amount: 20 mu L of the solution; column temperature: 35 ℃ is carried out. Therefore, the method for detecting benzo (alpha) pyrene in soil according to the embodiment of the invention has the advantages of strong accuracy, simple operation, short time and suitability for large-scale application.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

In this example, the benzo (α) pyrene content in soil was measured according to the following method:

1. weighing 10g (accurate to 0.001g) of soil into a triangular flask with a plug, adding a proper amount of anhydrous sodium sulfate, adding 100mL of acetone-n-hexane (1:1) mixed solution, and extracting for 4h by oscillation of a rotary oscillator to obtain an extracting solution;

2. filling a layer of glass fiber filter paper on a glass funnel, adding 5g of anhydrous sodium sulfate, collecting the bottom of the glass funnel by using a rotary evaporation bottle, filtering an extracting solution by using the anhydrous sodium sulfate and the filter paper, entering the rotary evaporation bottle, washing the triangular bottle with a stopper and the funnel by using an acetone-n-hexane (1:1) mixed solution, merging the liquid into the same rotary evaporation bottle, carrying out rotary evaporation at 45 ℃ to 1ml, adding 5ml of n-hexane and concentrating to 1ml, repeating the operation of adding the n-hexane and concentrating for 3 times, and concentrating to 1ml again to obtain a concentrated solution;

3. activating a Florisil small column by using 6ml of n-hexane, adding a concentrated solution into the small column when the liquid level is quickly drained, collecting an effluent liquid into a test tube, cleaning a rotary evaporation bottle by using the n-hexane for a plurality of times, finally eluting by using 10ml of dichloromethane-n-hexane (1:1) solution, collecting an eluent into the same test tube, blowing nitrogen to 1ml at 45 ℃, adding 3ml of acetonitrile, concentrating to 1ml, repeating the operations of adding the acetonitrile and concentrating for 2 times, completely converting a solvent into the acetonitrile, continuously drying, and accurately fixing the volume to 1ml to obtain a purified solution;

4. the purified liquid is subjected to liquid chromatography detection under the following conditions, and the chromatogram is shown in figure 1.

A chromatographic column: c18 column (250 nm. times.4.6 mm, 5 μm) or other equivalent;

mobile phase: acetonitrile: water 88: 12;

flow rate: 1.0 mL/min;

a fluorescence detector: the excitation wavelength is 384nm, and the emission wavelength is 406 nm;

sample introduction amount: 20 mu L of the solution;

column temperature: 35 ℃ is carried out.

Detection limit

(1) Sucking the benzo (a) pyrene standard substance to prepare standard substances with the concentrations of 0ng/ml, 0.5ng/ml, 1ng/ml, 5ng/ml, 10ng/ml and 20 ng/ml.

(2) The method of example 1 was used to measure different concentrations of standard.

Drawing a standard curve by taking the peak area of the target peak as an ordinate (Y) and the concentration of benzo (a) pyrene as an abscissa (X), wherein Y is 67733.8581X +11531.4504, R²0.99911. And (3) obtaining the detection limit and the quantification limit of the stepwise dilution analysis method according to the peak response value with the signal-to-noise ratio of 3 and the peak response value with the signal-to-noise ratio of 10. The results are shown in Table 1, and it can be seen that example 1 can detect a lower concentration of the analyte.

TABLE 1 detection limits

	Example 1
		Detection limit (mug/kg)	0.1
Limit of quantitation (ug/kg)	0.3

Recovery rate

And adding a known amount of benzo (alpha) pyrene into a sample to be detected, detecting the benzo (alpha) pyrene by using the method of example 1, and determining the recovery rate.

As shown in Table 2, the recovery rate was in the range of 90 to 100%, and the recovery rate was in accordance with the national standard.

TABLE 2 recovery

	Additive concentration (μ g/kg)	Test results (μ g/kg)	The recovery rate is high
				Example 1	0.5	0.495	99.0
Example 2	0.1	0.0928	92.8
				Example 3	2	1.93	96.5

Precision degree

Relative standard deviations were calculated as the results of 6 parallel experiments of examples 1, 2 and 3 in the recovery assay. As shown in Table 3, it was found that the accuracy was high and the sensitivity was high. Moreover, the whole operation time is short.

TABLE 3 precision

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.