Velp Scientifica
– UDK139 Operating Manual
10004762/B5
104
33 - Method to determine urea nitrogen in feed and roughages
During ensiling of green forages some urea can be added. Some feed can be
supplemented with urea in order to increase raw protein content. Urea content can
be determined separately from other nitrogen compounds by the enzyme urease
which hydrolizes urea to ammonia and carbon dioxide. The method is based on
steam distillation of ammonia before and after enzymatic treatment with urease and
its determination by titration.
Procedure
1) Reagents for anzymatic incubation:
Urease 1 International Unit (I.U.) produces 1,0 mg of ammonia nitrogen from urea
in 5 min at pH 7,0 at 20 °C. Commercial urease powders show activities between
17000 and 200000 units per gram. It’s necessary to prepare a standardized
solution of enzyme containing 0,5-1 I.U./ml, able to free completely ammonia from
100 mg of urea (46,6 mg as N) in 1 hour at room temperature.
Silicone antifoam.
Calcium chloride solution: 25 g of CaCl
2
in 100 ml of water.
2) Standardization of urease solution.
Urease solutions are alkaline and should be neutralized to pH 7 after dilution in water
of a known amount (e.g. 0,1 g in 50 ml) using hydrochloric acid 0,1 N and methyl red
indicator. The solutions cannot be stored.
The determination of the enzymatic activity is performed by adding increasing known
volumes of neutralized urease solution to 0,1 g of pure urea. After 1 hour incubation
at room temperature, ammonia is steam distilled.
It is to be used the volume of urease solution which frees completely ammonia from
0.1g of urea during 1 hour.
3) Incubation:
2 g of homogeneous sample finely grinded and weighed are
introduced into a test tube with 100 ml of water and the adopted volume of urease
solution, close and incubate 1 hour at room temperature shaking at intervals. If the
urea content of the examined material is more than 5% use more urease.
4) Distillation and titration:
at the end of incubation introduce in the test tube 2 g of
magnesium oxide, 5 ml of calcium chloride solution and some drops of antifoam.
Place in position in the steam distilling unit the test tube with the sample.
Set the following analytical parameters:
H
2
O=50ml. H
3
BO
3
=30ml. NaOH=50 ml.
Titrant = HCl 0,2 N
Distillation time = 4 min
Steam power: 100%
The nitrogen content of urea is 46,65%. The amount of ammonia freed from 0,1 g of
urea requires 16,64 ml of acid of this normality (1 ml HCl 0,2 N = 2,803 mg N - NH
4
).
If the original sample contains ammonia , it is to be determined separately and
substracted from the value obtained after incubation with urease.
References:
AOAC. Official Methods of Analysis (1984).
291
