Method 6.0 2014

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METHOD 6:0

Printed with effect from 1st March 2014

UREA

1: Scope and Field of Application


This method is for the determination of urea in feeding stuffs.

2: Principle
The sample is suspended in water with a clarifying agent and filtered. The urea content
of the filtrate is determined after the addition of 4-dimethylaminobenzaldehyde (4-
DMAB) by measuring the optical density at a wavelength of 420nm.

3: Reagents
3:1 4-DMAB solution: dissolve 1.6g of 4-DMAB in 100ml of 96% ethanol and add 10ml of
hydrochloric acid (d=1.19g/ml).
3:2 Carrez solution I: dissolve in water 21.9g of zinc acetate dihydrate, Zn(CH3COO)2
2H2O and 3g of glacial acetic acid. Dilute to 100ml with water.
3:3 Carrez solution II: dissolve in water 10.6g potassium ferrocyanide, K4 Fe (CN)6 3H2O.
Dilute to 100ml with water.
3:4 Active carbon which does not absorb urea (check before use).
3:5 Urea, 0.1% solution (w/v).

4: Apparatus
4:1 Mixer (tumbler): approximately 35 to 40 rpm.
4:2 Test tubes: 160 x 16 mm with ground-glass stoppers.
4:3 Spectrophotometer.

5: Procedure
5:1 Dissolution of sample
Weigh out 2g of the prepared sample to the nearest mg with 1g of active carbon (3:4) and
transfer to a 500ml graduated flask. Add 400ml of water and add 5ml Carrez solution I
(3:2), followed by 5ml Carrez solution II (3:3), mixing well between additions. Dilute to
volume with water, mix well and filter.

5:2 Determination
Transfer 5ml of the transparent colourless filtrate to a ground-glass stoppered test tube,
add 5ml of 4- DMAB solution (3:1) and mix. Place the tubes in a water bath at 20 C (±
4 C) and allow to stand for 15 minutes. Measure the optical density of the sample
solution with the spectrophotometer at 420nm. Compare with the blank test solution of
the reagents.

5:3 Calibration curve


Dilute 1, 2, 4, 5 and 10ml of the urea solution (3:5) to 100ml with water. Transfer 5ml of
each solution to ground-glass stoppered test tubes and add 5ml of 4-DMAB solution (3:1)
to each, homogenise and measure the optical density as shown above in comparison with
a control solution containing 5ml of 4-DMAB and 5ml of water free from urea. Plot the
calibration curve.

6:0/1
6: Expression of Results
6:1 Determine the amount of urea in the sample using the calibration curve.

6:2 Express the result as a percentage of the sample.

7: Repeatability
The difference between the results of 2 parallel determinations must not exceed 10%
relative to the higher value.

8: Observations
8:1 In the case of contents of urea exceeding 3%, reduce the sample to 1g or dilute the
original solution so that there are not more than 50mg of urea in 500ml.
8:2 In the case of low contents of urea, increase the sample as long as the filtrate remains
transparent and colourless.
8:3 If the sample contains simple nitrogenous compounds such as amino acids, the optical
density shall be measured at 435nm.

NOTE: If the sample is highly coloured the proportion of activated charcoal must be
increased up to 5g. The final solution after filtering should be colourless.

6:0/2

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