Steps to detect methylmercury and ethylmercury in domestic sewage

    Mercury exists in various forms in the natural environment and is very variable. For example, mercury compounds in the environment are converted to methylmercury by microorganisms. This organic compound of methyl and mercury is not easily decomposed and accumulates in living organisms. Methylmercury and ethylmercury in water can be determined by liquid chromatography-atomic fluorescence spectrometry.
   The principle is to use an activated C18 solid phase extraction column to enrich methylmercury and ethylmercury in water, and use mobile phase to elute the enriched methylmercury and ethylmercury into a smaller volume of solution. . The eluent is separated by liquid chromatography to separate methylmercury and ethylmercury in turn, mixed with oxidant, and successively oxidized to inorganic mercury under the irradiation of ultraviolet light. to measure.

Reagents required for testing

1. Laboratory first-grade pure water
2. Hyun acetate: analytically pure.
3. Sodium hydroxide: analytical grade.
4. L-cysteine: biochemical reagent, purity ≥99.0%.
5. Sodium diethyldithiocarbamate: analytically pure.
6. Potassium hydroxide: analytically pure.
7. Hydrochloric acid: analytically pure.
8. Potassium persulfate: analytical grade.
9. Potassium borohydride: analytically pure.
10. Methylmercury standard stock solution 100ug/mL: a commercially available certified standard solution with methanol as a solvent, which can be stored frozen at -20°C after opening, protected from light and sealed for 12 months.
11. Ethylmercury standard stock solution 100ug/mL: a commercially available certified standard solution with methanol as a solvent, which can be stored frozen at -20°C after opening, protected from light and sealed for 12 months.
12. Methanol solution: Mix methanol and experimental water in a volume ratio of 1:9 to obtain a methanol solution with a volume fraction of 10%.
13. DDTC solution: Weigh 0.1 g of sodium diethyldithiocarbamate and dissolve it in 100 g of experimental water to obtain a DDTC modified solution with a mass fraction of 0.1%, which is used for the activation of the solid phase extraction column.
14. Methanol mixed solution of 60 mmol of Xuan acetate, 10 mmol of cysteine, and 5% volume fraction: measure 500 mL of methanol solution with a volume fraction of 10%, 4.62 g of Xuan acetate, 1.21 g of L-cysteine, and use experimental water Dilute the volume to 1000mL, filter through a microporous membrane through a solvent filter, and sonicate for more than 30min, and use it as the mobile phase of liquid chromatography.
15. Hydrochloric acid solution: Measure 70 mL of concentrated hydrochloric acid, dissolve it in experimental water and dilute to 1000 mL to obtain a 7% hydrochloric acid solution, which is used as a carrier solution.
16. Potassium hydroxide-potassium borohydride solution:
  Weigh 3.5g potassium hydroxide and 0.2g potassium borohydride, dissolve them in experimental water and dilute to 1000mL to obtain a mixed solution of potassium hydroxide and potassium borohydride, which is used as a reducing agent and used on the same day.
17. Potassium persulfate-potassium hydroxide solution:
  Weigh 1.0g of potassium persulfate and 3.5g of potassium hydroxide, dissolve in experimental water and dilute to 1000mL to prepare a mixed solution of potassium persulfate and potassium hydroxide, which is used as an oxidant and used on the same day.
18. Methylmercury and ethylmercury mixed standard intermediate solution:
  Accurately pipette 1.00mL of methylmercury and ethylmercury standard stock solutions into a 100mL volumetric flask, add methanol solution to dilute to the mark, and obtain 1000.0ug/L methylmercury and ethylmercury mixed standard intermediate solution.
19. Methylmercury and Ethylmercury Mixed Standard Solution 1:
  Accurately pipette 5.00mL of methylmercury and ethylmercury mixed standard intermediate solution with a concentration of 1000.0ug/L into a 50mL volumetric flask, add methanol solution to dilute to the mark, and obtain methylmercury and ethylmercury with a concentration of 100.0ug/mL. A mixed standard intermediate for mercury. Then accurately pipette 5.00mL of the above standard intermediate solution of methylmercury and ethylmercury with a concentration of 100.0ug/mL into a 100mL volumetric flask, add methanol solution to dilute to the mark, and obtain a concentration of 5.0ug/mL methylmercury and ethylmercury. Ethylmercury mixed standard solution.
20. Methylmercury and ethylmercury mixed standard solution 2:
  Accurately pipette 5.G0mL of methylmercury and ethylmercury mixed standard intermediate solution with a concentration of 1000.0ug/mL into a 100mL volumetric flask, add methanol solution to dilute to the mark, and obtain methylmercury and ethylmercury with a concentration of 50.0ug/mL. Amalgam standard use liquid.

Materials and equipment required for testing

1. C18 solid phase extraction cartridge.
2. Microporous membrane: 0.45um water/organic hybrid membrane.
3. Solid phase extraction device: both manual and automatic.
4. Liquid chromatography-atomic fluorescence spectrometer.
5. Micro syringe: 100 uL.
6. Analytical balance: Sensitivity 0.0001g.

water sample collection

   The volume of the collected water sample is not less than 1500mL. The collected water samples should be placed in a polyethylene container, adjusted to pH 3-7 with dilute hydrochloric acid, and sealed for storage. If the water sample cannot be analyzed immediately, it should be stored in a cool and dark place, and the storage time should not exceed 3d. If only methylmercury is measured, it can be stored for 7d under this condition.

Detection steps

Preparation of water samples

  The preparation of water samples is divided into three steps: solid phase extraction column activation, water sample enrichment and elution. First, the C18 solid phase extraction column was activated, washed with 4 mL of methanol solution and 4 mL of deionized water respectively, then modified with 4 mL of DDTC modification solution, and rinsed with 4 mL of deionized water. A 1000 mL water sample was passed through a C18 solid-phase extraction column at a flow rate of 5 mL/min, and then the column was rinsed with 4 mL of deionized water, and then washed with methanol solution. Take 10 mL of a mixed solution of hydrogen acetate, cysteine, and methanol at a flow rate of 1 mL/min to elute the SPE cartridge, and collect the eluate for determination.

Instrument conditions

  Liquid chromatography conditions: reverse C18 column, 150mmX4.6mm, 5um; mobile phase is a mixed solution of acetic acid, cysteine, methanol solution; liquid pump pump speed: 1mL/min; injection volume: 100uL. Atomic fluorescence spectrometer conditions: lamp current 30mA; negative high voltage 270V.

Drawing of the standard curve

  Accurately pipette 0.00mL, 0.10mL, 0.20mL, 1.00mL, 2.00mL of the mixed standard solution with a concentration of 5.0ug/L into five 10mL volumetric flasks, dilute with methanol solution to the mark, and shake well to obtain the concentration Low concentration series curves of 0.00ug/L, 0.05ug/L, 0.10ug/L, 0.50ug/L and 1.00ug/L. Accurately pipette 0.40mL, 0.80mL, 1.20mL, 1.60mL, 2.00mL of the mixed standard solution with a concentration of 50.0ug/L into 5 10mL volumetric flasks, dilute with methanol solution to the mark, shake well, and obtain the concentration High concentration series curves of 2.00ug/L, 4.00ug/L, 6.00ug/L, 8.00ug/L and 10.00ug/L.

water sample testing

  The enriched water samples were detected according to the set instrument conditions, and the retention time and peak area were recorded. Blank experiments can be performed with first-grade pure water according to the water sample preparation steps. The mass concentrations of methylmercury and ethylmercury in the final water sample can be calculated according to the corresponding formula.
  The above content comes from 《DB65/T 4367-2021 Water Quality-Determination of Methylmercury and Ethylmercury-Liquid Chromatography-Atomic Fluorescence Spectrometry》