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Analytical Method for Selenium in Water

This method for measuring selenium in water uses the Spectroquant® Photometer and a clear, filtered sample to evaluate the results at 445 nm.

Instruments and Reagents Required

Spectroquant® Photometer NOVA 30 (Product No. 109748) or
Spectroquant® Photometer NOVA 60 (Product No. 109751)
Rectangular cells 50 mm (Product No. 114944)
Formic acid 98-100% for analysis (Product No. 100264 )
3,3‘-Diaminobenzidine tetrahydrochloride (CAS 868272-85-9) (Product No. 32750)
TitriplexIII® solution 0.1 mol/l (Product No. 109992)
Ammonia solution 25% for analysis (Product No. 105432)
Toluene for gas chromatography (Product No. 108389)
pH-indicator strips, pH 2.0 - 9.0 (Product No. 1.09584)
Sodium sulfate anhydrous for analysis (Product No. 106649)
Water for analysis (Product No. 116754)
Membrane filter with 0.45 μm pore diameter

Measuring Range

0.05 – 2.00 mg/L

Reagent 1

Fill 9.4 mL formic acid 100% in a calibrated or conformity-checked 100-mL volumetric flask. Make up to volume with water for analysis and mix.

Reagent 2

Dissolve 0.5 g 3,3’-diaminobenzidine tetrahydrochloride in 100 mL chilled water for analysis. Prepare freshly each day and store in an amber glass bottle.

Reagent 3

Fill 48 mL ammonia solution 25% for analysis in a calibrated or conformity-checked 100-mL volumetric flask. Make up to volume with water for analysis and mix.

Sample Preparation

The sample must be clear; if not, filter through a membrane filter with 0.45 μm pore diameter.


  1. Mix 30 mL of nearly neutral sample solution with 2 mL reagent 1, 2 mL Titriplex III® solution 0.1 mol/l and 2 mL reagent 2.
  2. Adjust the pH value of the mixture with reagent 3 to 2.5 and set aside for 45 min in the dark.
  3. Adjust the pH value of the mixture again with reagent 3 to 7.0, transfer into a 100-mL separation funnel, add 15 mL water for analysis and 7 mL toluene for analysis. Shake the mixture in the closed funnel for 1 min.
  4. Allow to separate and discharge the lower water phase.
  5. Fill the toluene extract in a test tube, add some anhydrous sodium sulfate and shake.
  6. Fill the clear, dried toluene extract in a 50 mm rectangular cell and measure at 445 nm against pure toluene.

Parameter List

Calculation when measuring the absorbance value using NOVA photometers without programming the method in the memory of the instrument:

Concentration [mg/l] = Absorbance x 0.654 – 0.037


Result = measured value in mg/l Se4+

Statistical Data:

Standard deviation: 0.019 mg/l

Coefficient of variation: 2.9 %



Fries J, Getrost H. 1975. Organische Reagenzien für die Spurenanalyse. Darmstadt: E. Merck.
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