Determination of the concentration of benzo(a)pyrene in water: acetylation filter paper chromatography fluorescence spectrophotometry

Acetylation filter paper chromatography fluorescence spectrophotometry

Water quality—Determination of benzo(a)-Pyrene-Acetylated

Paper chromatography with fluorescence spectrophotometric method

GB 11895-89

1 Subject content and scope of application

This method specifies a method for measuring benzo (a) flower [hereinafter referred to as B(a)P] in water quality.

This standard applies to drinking water, surface water, domestic sewage, industrial wastewater. The minimum proof concentration is 0.004 μg/L.

Note: B(a)P is a polycyclic aromatic hydrocarbon composed of five rings, which is a strong carcinogenic representative of polycyclic aromatic hydrocarbons. Based on the strong carcinogenicity of B(a)P, gloves that are resistant to organic solvents must be worn when analyzed according to this standard method. The operation should be carried out in a white enamel pan (such as solution transfer, constant volume, spotting, etc.). The room should be protected from direct sunlight and well ventilated.

2 Principle

The polycyclic aromatic hydrocarbons and cyclohexane solubles in the water are extracted with cyclohexane (the water sample must be thoroughly shaken), the extract is dehydrated with anhydrous sodium sulfate, concentrated, and then separated by acetylation filter paper. The separated B(a)P was measured with a fluorescence spectrophotometer.

3 reagents

Analytically pure reagents and distilled water were used for the analysis unless otherwise stated.

3.1 Preparation of B(a)P standard solution: Weigh 5.00mg solid standard B(a)P in 50mL volumetric flask [Because B(a)P is a strong carcinogen, in order to reduce pollution, it is better to reduce the transfer] After dissolving with a small amount of benzene, cyclohexane was added to the mark at a concentration of 100 μg/mL. Here, the stock solution was diluted with cyclohexane to a standard use solution of 10 μg/mL, and stored in a refrigerator in the dark.

3.2 Preparation of E-cooling filter paper: 15 to 20 cm of 15×30 cm chromatography filter paper is rolled into a cylindrical shape of 15 cm in height, placed one by one into a 1000 mL high-type beaker, and the first wall of the cup wall and the cup is placed. Insert a glass rod and place a glass-sealed electromagnetic stirring iron core in the middle of the cup. In the fume hood, slowly pour the acetylating agent along the wall of the cup (made of benzoic anhydride + concentrated sulfuric acid = 750mL + 250mL + 0.5mL), the temperature of the magnetic constant temperature stirrer is maintained at 55 ± 1 ° C. Continuous reaction for 6h. The acetylated filter paper was taken out, rinsed with tap water for 3 to 4 times, rinsed with distilled water for 2 to 3 times, and air-dried. After soaking for 4 hours with absolute ethanol for the next day, the acetylated filter paper was taken out, dried and flattened, and set aside.

3.3 Cyclohexane, re-steamed. Check with a fluorescence spectrophotometer: at a fluorescence excitation wavelength of 367 nm, a slit of 10 nm; a fluorescence emission slit of 2 nm, and a wavelength of 405 nm should have no peak.

3.4 Acetone, re-steamed.

3.5 Methanol.

3.6 ether.

3.7 Benzene, re-steamed.

3.8 acetic anhydride.

3.9 Sulfuric acid, ρ = 1.84 g/mL.

3.10 anhydrous sodium sulfate. 3.11 Dimethylhydrazine (DMSO): Extract twice with cyclohexane before use (500 mL of dimethyl hydrazine plus 50 mL of cyclohexane). Discard cyclohexane and set aside.

4 instruments

Common laboratory equipment and the following instruments.

4.1 Fluorescence spectrophotometer with UV excitation and fluorescence spectrometry, quartz cuvette with an optical path of 10 mm.

4.2 UV analyzer (with 365nm or 254nm filter).

4.3 Kang's oscillator.

4.4 Magnetic constant temperature agitator.

4.5 Vertical centrifuge with a speed of 4000r/min.

4.6 separatory funnel, 1L, 3L, 100mL. The oil lubricant is disabled on the piston, and the piston can be directly lubricated with water or organic solvent.

4.7 Erlenmeyer flask, 250 mL. With ground glass stopper.

4.8 Constant temperature water bath.

4.9 Chromatography cylinder.

4.10 Centrifugal tube with a grinder stopper, 5 mL.

4.11 Spot the glass capillary (home made).

4.12 Analyze the balance and the sensitivity is 0.01mg.

5 sample storage

The water sample should be stored in a glass bottle and protected from light. It should be extracted with cyclohexane on the same day (within 24 hours), and the cyclohexane extract should be stored in the refrigerator.

6 steps

6.1 Pretreatment of samples and standards

6.1.1 Clean water and surface water extraction

Take a well-mixed clean water sample 2000mL into a 3000mL separatory funnel, extract twice with cyclohexane, use 50mL each time, shake each time for 3min on the Kelvin oscillator, remove the deflation, and let stand for half an hour. After stratification, the two cyclohexane extracts were collected in a stoppered Erlenmeyer flask and the aqueous phase portion was discarded.

6.1.2 Extraction of industrial wastewater

Take 1000 mL of mixed industrial wastewater, put it into a 1000 mL separatory funnel, extract twice with 50 mL of cyclohexane each time, shake it for 3 min each time on a Kelvin shaker, remove the venting gas, let stand for half an hour, wait for minutes After the layer, the two cyclohexane extracts were collected in a stoppered Erlenmeyer flask and the aqueous phase portion was discarded.

6.1.3 Dehydration and concentration

Anhydrous sodium sulfate (about 20 to 50 g) was added to the above-mentioned cyclohexane extract, and allowed to stand until completely dehydrated (about 1 to 2 hours) until the bottom of the stoppered conical flask was anhydrous. If the color of the cyclohexane extract is darker, the cyclohexane is made up to 100 mL after dehydration, and the volume is concentrated in a certain volume; if the color is not deep, it is completely concentrated. The mixture was concentrated to near dryness with a KD concentrator at a temperature of 70 to 75 C, and the wall was concentrated three times with benzene, three drops each time, and then concentrated to 0.05 mL for paper chromatography.

6.1.4 Paper chromatography separation

At a lower end of the acetylated filter paper at a length of 30 cm, a horizontal line was drawn with a pencil, and 1.5 cm was left at each end of the horizontal line, and the standard B(a)P and the sample concentrate were cross-shaped with a glass capillary at intervals of 2.4 cm. The spotted spot diameter does not exceed 3 to 4 mm. Blow dry with cold air during the spotting process. Wash each concentrating tube twice, using one drop of benzene each time, all on paper. The spotted chromatography paper was hung on the inner shelf of the chromatography cylinder, and a developing agent [methanol + diethyl ether + distilled water = 4 + 4 + 1 (volume ratio)] was added until the lower end of the filter paper was immersed in the developing agent 1 cm. The cover is sealed with a transparent adhesive tape. Spread in the dark room for 2 to 4 hours. The chromatographic filter paper was taken out, and the B(a)P spot of the standard and the range of purple-blue spots in the sample having the same height (Rf value) as the sample were circled with a pencil under the irradiation of the ultraviolet analyzer.

Cut out the spots circled with a pencil, cut into small strips, and place them in a 5 mL stoppered centrifuge tube. Bake in an oven at 105-110 ° C for 10 min (can also be dried in a desiccator or in clean air). After cooling in a desiccator, acetone was added to the mark. After shaking for 1 min by hand, it was centrifuged at 3000 r/min for 2 min. The supernatant is left for measurement.

6.2 Determination

The standard B(a)P spot and the acetone eluate of the sample spot were respectively injected into a 10 mm quartz cuvette. The excitation and emission slits were 10 nm and 2 nm, respectively, and the excitation wavelength was 367 nm. The emission wavelength was 402 nm. Fluorescence intensity F at 405 nm and 408 nm.

7 representation of the result

The relative fluorescence intensity of the standard B(a)P and the sample B(a)P was calculated by the narrow baseline method according to the following formula, and the content C of the B(a)P was calculated (using a relative comparison calculation method).

Where: C - water sample B (a) P content, μg / L;

M——Standard B(a)P spotting amount, μg;

F standard - the relative fluorescence intensity of standard B(a)P;

F sample - the relative fluorescence intensity of the sample spot;

V——water sample volume, L;

R - the ratio of the total volume of the cyclohexane extract to the volume of the cyclohexane extract taken during concentration.

8 Precision and accuracy

8.1 Precision

The precision of coking wastewater containing B(a)P approx. O.2μg/L prepared by five laboratories is shown in Table 1.

Table 1 Precision

8.2 Accuracy

The recoveries of the two industrial wastewaters are shown in Table 2 and Table 3.

Table 2 Coking wastewater recycling results

Table 3 Asphalt wastewater spiked recovery results

Appendix A Description of Removal of B(a)P Interferences

(Supplementary)

A1 Petroleum and oily wastewater The following (6.1.2) 100 mL cyclohexane extract was made to volume, and 20 mL was taken out and placed in a 100 mL separatory funnel. Extract 2 times with DMSO, 5 mL each time, shake by hand for 2 min, pay attention to deflation, and let stand for half an hour. After the stratification, the twice extracted DMso solution was collected in another 100 mL separatory funnel, and the cyclohexane solution was discarded. To a separatory funnel containing 10 mL of DMSo solution, 15 mL of 1:1 HCl previously cooled with ice was added, and the mixture was cooled to room temperature, and then back-extracted twice with cyclohexane, 5 ml each time, and shaken by hand for 2 min, paying attention to deflation. 10 mL of two cyclohexane extracts were combined in another 100 mL separatory funnel, washed once with 5 mL of 15% NaOH solution, shaken for two minutes, and the NaOH layer was discarded. Then, it was washed with distilled water for 2-3 times, each time 15 mL, until the distilled water after washing was pH=7, the aqueous phase was discarded, the cyclohexane extract was dehydrated with anhydrous sodium chloride, and concentrated on a KD concentrator. The following steps include acetylation filter paper chromatography separation, fluorescence spectrophotometer measurement, and calculation is the same as (7).

The B(a)P acetone eluent after A2 measurement should not be discarded at will, and it can be placed in a special large beaker in the fume hood for uniform treatment.

A5 This experiment should be carried out under direct sunlight.

The glassware used in A4 must be washed with soaking solution for 4 hours.

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