Water quality Determination of benzo (a) pyrene
Acetylated 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 the method for the determination of benzo (a) flowers [hereinafter referred to as B (a) P] in water quality.
This standard applies to drinking water, surface water, domestic sewage, and industrial wastewater. The minimum calibration concentration is 0.004μg / L.
Note: B (a) P is a polycyclic aromatic hydrocarbon composed of five rings, which is a strong carcinogen representative of polycyclic aromatic hydrocarbons. Based on the strong carcinogenicity of B (a) P, organic solvent-resistant gloves must be worn when analyzed according to this standard method, and the operation should be performed in a white enamel tray (such as solution transfer, constant volume, spotting, etc). Indoors should avoid direct sunlight and have good ventilation.
2 Principle
The polycyclic aromatic hydrocarbons and cyclohexane solubles in water are extracted with cyclohexane (the water sample must be fully 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 Reagent
Unless otherwise stated, analytical reagents and distilled water are used for analysis.
3.1 Preparation of B (a) P standard solution: Weigh 5.00mg of solid standard B (a) P in a 50mL volumetric flask [because B (a) P is a strong carcinogen, in order to reduce pollution, it is better to transfer less], After dissolving with a small amount of benzene, add cyclohexane to the mark, its concentration is 100μg / mL. Hereby, the stock solution was diluted with cyclohexane to a standard use solution of 10 μg / mL, and stored in a refrigerator protected from light.
3.2 Preparation of E-cooling filter paper: roll 15 to 30 sheets of 15 × 30cm chromatographic filter paper into a 15cm high cylindrical shape and place them one by one in a 1000mL high-shaped beaker, between the cup wall and the first sheet of paper against the cup Insert a glass rod with a glass-sealed electromagnetic stirring iron core in the middle of the glass. In the fume hood, slowly pour the acetylating agent (made from benzene decaacetic anhydride + concentrated sulfuric acid = 750mL + 250mL + 0.5mL mixed) along the wall of the cup, the temperature of the magnetic thermostatic stirrer is maintained at 55 ± 1 ℃. Continuous reaction for 6h. Take out the acetylated filter paper, rinse with tap water 3 to 4 times, rinse with distilled water 2 to 3 times, and dry. The next day after soaking in absolute ethanol for 4h, the acetylated filter paper was taken out, dried and flattened, and set aside.
3.3 Cyclohexane, redistilled. Check with a fluorescence spectrophotometer: at a fluorescence excitation wavelength of 367 nm and a slit of 10 nm; a fluorescence emission slit of 2 nm and a wavelength of 405 nm, there should be no peaks.
3.4 Acetone, redistilled.
3.5 Methanol.
3.6 Ether.
3.7 Benzene, redistilled.
3.8 Acetic anhydride.
3.9 Sulfuric acid, Ï = 1.84g / mL.
3.10 Anhydrous sodium sulfate. 3.11 Dimethyl sulfite (DMSO): Extract twice with cyclohexane before use (500mL dimethyl sulfite plus 50mL cyclohexane extraction). Discard cyclohexane and set aside.
4 Instruments
Commonly used laboratory equipment and the following instruments.
4.1 Fluorescence spectrophotometer with ultraviolet excitation and fluorescence spectrometry, quartz cuvette with optical path of 10mm.
4.2 UV analyzer (with 365nm or 254nm filter).
4.3 Conrad oscillator.
4.4 Magnetic constant temperature stirrer.
4.5 Vertical centrifuge with a speed of 4000r / min.
4.6 Separation funnel, 1L, 3L, 100mL. Oil-based lubricants are forbidden on the piston, and the piston can be lubricated directly with water or organic solvents.
4.7 Erlenmeyer flask, 250mL. With ground glass stopper.
4.8 Constant temperature water bath.
4.9 Chromatography cylinder.
4.10 Centrifuge tube with grind plug scale, 5mL.
4.11 Glass capillary for sample application (self-made).
4.12 Analytical balance, 0.01mg.
5 Sample storage
The water sample should be stored in a glass bottle and protected from light, and extracted with cyclohexane on the same day (within 24h), and the cyclohexane extract should be kept in the refrigerator.
6 steps
6.1 Pretreatment of samples and standards
6.1.1 Clean water and surface water extraction
Take 2000mL of well-mixed clean water sample into a 3000mL separatory funnel, extract twice with cyclohexane, 50mL each time, shake for 3min each time on a Kang's shaker, remove the vent, and let stand for half an hour, After the layers were separated, the two cyclohexane extracts were collected in a conical flask with a stopper, and the aqueous phase portion was discarded.
6.1.2 Extraction of industrial wastewater
Take 1000mL of the mixed industrial wastewater sample, put it into a 1000mL separatory funnel, extract twice with 50mL cyclohexane each time, shake for 3min each time on a Kang's shaker, remove the outgassing, and let it stand for half an hour After layering, the two cyclohexane extracts were collected in a stoppered conical flask, and the aqueous phase portion was discarded.
6.1.3 Dehydration and concentration
Add anhydrous sodium sulfate (approximately 20-50g) to the cyclohexane extraction solution (above) and let stand until it is completely dehydrated (approximately 1-2h) until the bottom of the conical flask with plug is dry. If the cyclohexane extract is darker in color, set the volume of cyclohexane to 100 mL after dehydration, divide it to a certain volume and concentrate it; if the color is not dark, concentrate it all. At a temperature of 70-75C, use a KD concentrator to reduce the concentration to near dryness. Wash the wall of the concentration tube three times with benzene, using 3 drops each time, and then concentrate to 0.05 mL to prepare for paper chromatography.
6.1.4 Paper chromatography separation
At the lower end of the 30cm long acetylated filter paper, 3cm, draw a horizontal line with a pencil, leaving 1.5cm at each end of the horizontal line, and cross the standard B (a) P and the sample concentrate with a glass capillary at 2.4cm intervals. The spot diameter does not exceed 3 to 4 mm. Dry with cold air during the spotting process. Wash each concentration tube twice, one drop of benzene at a time, all on paper. Hang the spotted chromatography filter paper on the shelf in the chromatography tank, add the developing agent [methanol + ether + distilled water = 4 + 4 + 1 (volume ratio)] until the lower end of the filter paper is immersed in the developing agent 1cm. The cover is sealed with transparent tape. Unfold in a dark room for 2 ~ 4h. Take out the chromatography filter paper, circle the standard sample B (a) P spots and the range of purple-blue spots with the same height (Rf value) in the sample under the irradiation of the ultraviolet analyzer with a pencil.
Cut out the spots circled with a pencil, cut them into small strips, and put them into 5mL centrifuge tubes with stoppers. Bake in 105 ～ 110 ℃ oven for 10min (it can also be dried in a dryer or clean air). After cooling in the desiccator, add acetone to the mark. After shaking by hand for 1min, centrifuge at 3000r / min for 2min. The supernatant is reserved for measurement.
6.2 Determination
The acetone eluent of the standard B (a) P spot and the sample spot were injected into a 10 mm quartz cuvette. The excitation and emission slits were 10 nm and 2 nm, and the excitation wavelength was 367 nm. Fluorescence intensity F at 405nm and 408nm.
7 Representation of results
Calculate the relative fluorescence intensity of standard B (a) P and sample B (a) P according to the following formula using the narrow baseline method, and then calculate the content C of B (a) P (using the comparative comparison method).
In the formula: C——B (a) P content of water sample, μg / L;
M——Standard B (a) P spot size, 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 cyclohexane extract to the volume of cyclohexane extract taken during concentration.
8 Precision and accuracy
8.1 Precision
The precision of coking wastewater containing B (a) P approximately 0.2 μg / L prepared by five laboratories is shown in Table 1.
Table 1 Precision
Lab number
X1
CV,%
1
0.230
6.9
2
0.199
8.0
3
0.213
13.0
4
0.202
6.6
5
0.179
4.2
8.2 Accuracy
See Table 2 and Table 3 for the recovery rates of the two types of industrial wastewater.
Table 2 Standardization and recovery results of coking wastewater
Lab number
η
CV,%
1
107.0
6.9
2
93.3
8.0
3
121.0
13.0
4
99.0
6.6
5
73.3
4.2
Table 3 Asphalt wastewater standard recovery results
Lab number
η
CV,%
1
79.8
4.4
2
83.0
12
3
81.4
7.6
4
93.3
6.2
Appendix A Instructions for removing B (a) P interferers
(Supplementary)
A1 Petroleum and oily waste water shall be prepared as follows: After the above (6.1.2) 100mL cyclohexane extract is made up to volume, 20mL shall be taken out and put into a 100mL separating funnel. Extract twice with DMSO, 5mL each time, shake by hand for 2min, pay attention to deflation, and let stand for half an hour. After the layers were separated, the two extracted DMso liquids were collected in another 100 mL separating funnel, and the cyclohexane liquid was discarded. Into a separatory funnel containing 10 mL of DMSo solution, add 15 mL of 1: 1 HCl previously cooled with ice, cool to room temperature, and then back-extract twice with cyclohexane, 5 mL each time, shaking by hand for 2 min, and pay attention to gassing. Combine 10 mL of cyclohexane extract twice into another 100 mL separatory funnel, add 5 mL of 15% NaOH solution to wash once, shake for two minutes, and discard the NaOH liquid layer. Wash with distilled water 2-3 times, 15mL each time, until the pH of the distilled water after washing = 7, discard the aqueous phase, dehydrate the cyclohexane extract with anhydrous sodium flow, and concentrate on the KD concentrator. The following steps include acetylation filter paper chromatography separation, fluorescence spectrophotometer measurement, calculation is the same as (7).
A2 The measured B (a) P acetone eluent should not be discarded at will, but can be put into a special large beaker in a fume hood for uniform treatment.
A5 This experiment should be conducted under direct sunlight.
A4 The glassware used must be soaked in washing liquid for 4 hours and then washed.
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