Analytical Method: Urea in water and wastewater, enzymatic determination

Photometric determination using the indophenol blue method after cleavage of the urea

Instruments and Reagents:

Cat.No. 173016  Spectroquant® VIS Spectrophotometer Prove 100 or

Cat.No. 173017  Spectroquant® UV/VIS Spectrophotometer Prove 300 or

Cat.No. 173018  Spectroquant® UV/VIS Spectrophotometer Prove 600 or

Cat.No. 109751  Spectroquant® Photometer NOVA 60
Cat.No. 114544  Spectroquant® Ammonium Cell Test
Cat.No. 106578  tri-Sodium phosphate dodecahydrate for analysis
Cat.No. 106346  Sodium dihydrogen phosphate monohydrate for analysis
Cat.No. 108418  Titriplex® III for analysis
Cat.No. 116754  Water for analysis
Cat.No. 116493  Urease
Reagent 1:
Dissolve 1.5 g of tri-sodium phosphate dodecahydrate, 0.5 g sodium dihydrogenphosphate monohydrate, and
1.5 g Titriplex® III in 100 ml water for analysis.
Reagent 2:
Urease causes ammonium to be released from the urea; the ammonium can be photometrically determined according to the indophenol blue method. When ammonium is present in the sample, the sum determination and the determination of the ammonium concentration can be used to calculate the urea concentration.

Measuring range:
0.5 - 25.0 mg/l urea at 690 nm in the 16-mm round cell

To 5.0 ml of the sample solution add 2 drops of reagent 1 as well as 1 drop of reagent 2 and mix; leave to stand for 5 min. Take 0.50 ml of this solution for the determination of ammonium by means of the Spectroquant® Ammonium Cell Test.

Parameter list for programming the photometer Spectroquant® NOVA 60, Pharo 100, and
Pharo 300


Method number -------
Method-designation Urea
Wavelength 690 nm
Cititation Urea
Units mg/l
Resolution 0.1
Zero point (E0) 0.050
Slope (E/C) 0.0556
Meas. range begin 0.5
Meas. range end 25.0
Reference cell 16 mm round


Parameter list for programming the photometer Spectroquant® Prove 100, Prove 300 and
Prove 600


Method-designation Urea
Units mg/l
Cititation Urea
Wavelength 690 nm
Cell 16 mm round
Resolution 0.1
Meas. range 0.05 - 25.0
User range user defined
Zero point (E0) 0.050
calibr. coefficient (A1) 18.0


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


Measured absorbance
of sample in A
Measured absorbance
of blank in A
Result in mg/l
[(Asample-Ablank) * 1 / 0.0556]
0.850 0.050 14.4


Differentiation between urea and ammonium:

A. Measurement of the concentration of ammonium without the addition of reagent 1 and 2: Measurement value A in mg/l NH4+ (using method for Spectroquant® Ammonium Cell Test)
= Measuring value A in mg/l NH4
B. Measurement of the sum of ammonium + urea after the addition of reagent 1 and 2: Measurement value B in mg/l NH4+ (using parameter list for programming photometer NOVA 60, Prove and Pharo instruments
= Measuring value B in mg/l urea
C. Calculation of the urea concentration:
urea concentration in mg/l urea = B - (A x 1.66)


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