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Calibration: Ammonia Determination (Nesslerization spectrophotometric method)

Calibration to determinate Ammonia concentration from a standard solution (NH4Cl) with the Nessler reaction.

Duration: 2 h Adapted from: www.irsa.cnr.it/Docs/Capitoli/4030.pdf

Materials

  • Cuvette (1 mL)
  • Glass Bottle
  • Falcon Tube 15 mL

Equipment

  • Spectrophotometer UV-VIS
  • Chemical hood
  • Pipette 1000 uL and 100 uL

Solutions

  • Nessler reagent (TOXIC)
  • Rochelle salt solution: the potassium sodium tartrate solution (1.8M)
  • Standard Solution: NH4Cl

Procedure

  1. Preparation of Rochelle salt solution:
    • Add 50 g of Potassium Sodium Tartrate to 30 mL of deionized water and boil to remove ammonia trace. Once the solution is cold again, add deionized water until 100 mL.
  2. Preparation of the Standard-1:
    • Dry NH4Cl during 1 hours in an oven at 105 °C.
    • Weight precisely 0.3819 g of NH4Cl.
    • Add the 0.3819 g of NH4Cl in a glass bottle.
    • Add deionized water until around 50 mL.
    • Stir until all is dissolved.
    • Add deionized water until exactly 100 mL.
    • Label the glass bottle with “Laboratory name; Operator initials; standard 1000 mg N/L for NH4+ ; preparation date; until end date”.
    • This standard solution has an ammoniumconcentration of 1000 mg N L-1 and can be stored for 1 month in the fridge at 4°C.
  3. Preparation of the Standard-2:
    • Dilute 1:100 Standard-1 solution: put 500 µL of Standard-1 in 49.5 mL of deionized water.
  4. Preparation of the Standards-3:
    • The Concentration Range that is possible to read correctly with the spectophotometer is between 0.5 mg/L - 4.0 mg/L, and the estabilished Vf for each solution (Standard-3) is 8.0 mL.
Cf (mg/L) Cf (mg(mL) SOL. B (mL) Vf (mL)
0.5 0.0005 0.4 7.6
1 0.001 0.8 7.2
1.5 0.0015 1.2 6.8
2 0.002 1.6 6.4
2.5 0.0025 2 6
3 0.003 2.4 5.6
3.5 0.0035 2.8 5.2
4 0.004 3.2 4.8
  1. Take 1 mL from each Standards-3 and transfer in the respective cuovette.
  2. Prepare the BLANK, adding 1 mL of deionized water in one couvette.
  3. Add to each couvette, 100 µL of Rochelle salt solution, 100 µL Nessler Reagent (DO IT UNDER THE CHEMICAL HOOD), cover all the couvett with silver paper, and wait 15 min.
  4. Set the spectrophotometer to 420 nm and use the BLANK sample to set at zero the instrument.
  5. Measure all the samples noting the OD measured.
  6. Use the OD obtained to make the calibration curve.

Expected results (quantitative information, graphics, images)

mg/L OD
0.1 0.01
0.25 0.0335
0.5 0.072
1 0.172
1.5 0.278
2 0.3245
2.5 0.4145
3 0.5035
3.5 0.5395
4 0.618

calibration_curve

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