UGANDA STANDARD  
      US ISO 6654
First Edition
   2009 -09-04
 
Reference numbe r 
   US ISO 6654:1991
 © UNBS 200 9 
  
Animal feeding stuffs — Determination of urea content 
 US ISO 6654:1991  
 
© UNBS  2009 – All rights reserved Compliance with this standard does not, of itself confer immunity from legal obligations 
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct application  
© UNBS 2009  
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permission from UNBS. 
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     US ISO 6654:1991  
© UNBS  2009 - All rights reserved National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry  of Tourism, Trade and 
Industry established by the Act of Parliament of 198 3, of the Laws of Uganda.  UNBS is mandated to co-
ordinate the elaboration of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex A limentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT/SPS Agr eements of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a gi ven field or area and consists of representatives of 
consumers, traders, academician s, manufacturers, governme nt and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committ ee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council. 
This Uganda Standard, US ISO 6654:1991,  Animal feeding stuffs — Determination of urea content, is 
identical with and  has been reproduced from an International Standard ISO 6654:1991,  Animal feeding stuffs
 — Determination of urea content, and has been adopted as a Uganda Standard . 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." INTERNATIONAL 
STANDARD 
First edition 
1991-03-15 
Animal feeding stuffs - Determination of Urea 
content 
Aliments des animaux - DHermination de la teneur en ur6e 
Reference number 
___.------ ISO 6654:1991(E) ISO 6654:1991(E) 
Foreword 
ISO (the International Organization for Standardization) is a worldwide’ 
federation of national Standards bodies (ISO member bodies). The work 
of preparing International Standards is normaily carried out through ISO 
technical committees. Esch member body interested in a subject for 
which a technical committee has been established has the right to be 
represented on that committee. International organizations, govern- 
mental and non-governmental, in liaison with ISO, also take part in the 
work. ISO collaborates closely with the International Electrotechnical 
Commission (IEC) on all matters of electrotechnical standardization. 
Draft International Standards adopted by the technical committees are 
circulated to the member bodies for voting. Publication as an tnter- 
national Standard requires approval by at least 75 % of the member 
bodies casting a vote. 
International Standard ISO 6654 was prepared by Technical Committee 
ISO/TC 34, Agricultural food products. 
0 ISO 1991 
All rights reserved. No part of this pubiication may be reproduced or utiiized in any form 
or by any means, eiectronic or mechanical, inciuding photocopying and microfilm, without 
Permission in writing from the pubiisher. 
international Organkation for Standardization 
Case Postaie 56 * CH-1211 Geneve 20 l Switzer 
Printed in Switzeriand 
ii iand _ .~~ 
INTERNATIONAL STANDARD ISO 6654:1991 (E) 
Animal feeding stuffs - Determination of Urea content 
1 Scope 5 Reagents 
This International Standard specifres a spectro- All reagents shall be of recognized analytical grade. 
metric method for the determination of the Urea The water used shall be distilled water or water of 
content of animal feeding stuffs. at least equivalent purity. 
2 Normative reference 
The following Standard contains provisions which, 
through reference in this text, constitute provisions 
of this International Standard. At the time of publi- 
cation, the edition indicated was valid. All Standards 
are subject to revision, and Parties to agreements 
based on this International Standard are encour- 
aged to investigate the possibility of applying the 
most recent edition of the Standard indicated below. 
Members of IEC and ISO maintain registers of cur- 
rently valid International Standards. 
ISO 6498:1983, Animal feeding stuffs - Preparation 
of test samples. 
3 Definition 
For the purposes of this International Standard, the 
following definition applies. 
Urea content: The mass fraction of substances de- 
termined using the procedure specified in this lnter- 
national Standard. 
lt is expressed as a percentage by mass. 
4 Principle 
Suspension in water of the test Portion in the pres- 
ence of a decolorant. Stirring of the Suspension, 
then filtration. Addition to the filtrate of 
4-dimethyl-amino-benzaldehyde (4-DMAB) and 
spectrometric measurement at 420 nm of the 
absorbance of the Solution thus obtained. 5.1 Activated carbon, which does not adsorb Urea. 
5.2 4-dlmethyl-amino-benzaldehyde (4-DMAB), sol- 
ution prepared as follows. 
Dissolve 1,6 g of 4-DMAB in 100 ml of 96 % (V/v) 
ethanol, add 10 ml of concentrated hydrochloric acid 
(P 20 = 1,19 g/ml) and mix. 
This Solution may be stored for a maximum of 
2 weeks. 
5.3 Carrez 1 solution. 
Dissolve In water 24 g of zinc acetate dihydrate 
[Zn(CH,C00)2.2H20] and 3 g of glacial acetic acid. 
Make up to 100 ml with water and mix. 
5.4 Carrez ll solution. 
Dissolve in water IO,6 g of potassium hexa- 
cyanoferrate(ll) trihydrate (potassium ferrocyanide 
trihydrate) {K4[Fe(CN)J.3H20}. Make up to 100 ml 
with water and mix. 
5.5 Urea, Standard Solution corresponding to 1 g 
of Urea per Iitre. 
6 Apparatus 
Usual laboratory apparatus and, in particular, the 
following. 
6,l Rotary shaker, capable of being operated at a 
rotational frequency of about 30 min-1 to 40 min-l. 
6.2 Spectrometer, suitable for measuring 
absorbance at a wavelength of 420 nm, and 
equipped with cells of thickness IO mm. ISO 6654:1991(E) 
6.3 Test tubes, 160 mm x 16 mm, fitted with ground 
glass Stoppers. 9.4 Blank test 
Carry out a blank test in parallel with the determi- 
nation using the Same procedure and the Same 
quantities of all reagents, but omitting the test por- 
tion. 6.4 Volumetric flasks, of 100 ml and 500 ml capac- 
ity. 
65 Water-bath, capable of being controlled at 
20 “C. 9.5 Preparatlon of fhe calibration graph 
7 Sampling 
Sampling will be the subject of a future International 
Standard. 
8 Preparation of the test Sample 
Prepare the test Sample in accordance with 
ISO 6498. 
9 Procedure 
9.1 Test Portion 
Weigh, to the nearest 1 mg, about 2 g of the test 
Sample (clause 8). 
For Urea contents greater than 3 % (m/m), reduce 
the test Portion to 1 g or dilute the test Solution 
(see 9.2) so that it does not exceed a concentration 
of 50 mg of Urea per 500 ml. 
For low Urea contents, the test Portion may be in- 
creased provided that the filtrate remains clear and 
colourless. 
9.2 Preparation of the test solution 
9.2.1 Transfer the test Portion (9.1) together with 
1 g of the activated carbon (5.1) into a 500 ml 
volumetric flask (6.4). Add 400 ml of water, 5 ml of 
the Carrez I Solution (5.3) and 5 ml of the Carrez II 
Solution (5.4). Mix for 30 min using a rotary shaker 
(6.1). Make up to the mark with water, mix and filter 
on a dense, slow, qualitative filter Paper. 
9.2.2 lf the filtrate is coloured, prepare a fresh test 
Solution in accordance with 9.2.1 but increase the 
quantity of activated carbon added. 
9.3 Colour development 
Transfer, by means of a pipette, 5 ml of the clear 
colourless filtrate (9.2) into a test tube (6.3) and add, 
by means of a pipette, 5 ml of the 4-DMAB solution 
(5.2). 
Mix and leave to stand for 15 min in a water-bath 
(6.5) controlled at 20 OC. 9.5.1 Pipette into a series of five 100 ml volumetric 
flasks (6.4), 3 ml, 2 ml, 4 ml, 5 ml and IO ml of the 
Urea Standard Solution (5.5). Make each flask up to 
the mark with water. One millilitre of the Standard 
solutions cohtains IO pg, 20 pg, 40 pg, 50 pg and 
100 pg of Urea respectively. 
9.5.2 Pipette into a series of five test tubes (6.3) 
5 ml of each of these solutions (9.5.1) (one dilution 
per test tube). Add to each test tube, by means of a 
pipette, 5 ml of the 4-DMAB Solution (5.2) and mix. 
Transfer the solutions to spectrometer cells and 
measure their absorbances at 420 nm, using the 
spectrometer (6.2), against a compensation Solution 
containing 5 ml of 4-DMAB and 5 ml of water. 
9.5.3 Plot the calibration graph, with the 
absorbance values on the Ordinate and the corre- 
sponding concentrations of Urea, in micrograms per 
millilitre, on the abscissa. 
9.6 Spectrometric measurement 
Transfer the Solution obtained in 9.3 to a 
spectrometer cell and measure its absorbance at 
420 nm, using the spectrometer (6.2), against the 
blank test (9.4). 
NOTE 1 If the Sample contains simple nitrogenous 
compounds, such as amino acids, carry out the measure- 
ment of absorbance at a wavelength of 435 nm. 
9.7 Number of determinations 
Carry 
from t s on test portions taken out two determination 
he Same test Sample. 
10 Expression of results 
The Urea content, expressed as a percentage by 
mass, of the Sample is equal to 
C 
20 x m 
where 
c is the Urea content, in micrograms per 
millilltre, of the filtrate of the test Solution, 
determined from the calibration graph 
(9.5.3); 
I 
/ 2 m is the mass, in grams, of the test Portion ISO 6654:1991(E) 
dard, or regarded as optional, together with details 
(9 1) . . of any incidents which may have influenced the re- 
sult. 
13 Test report 
The test report shall specify the method used and 
the result obtained. lt shall also mention all operat- 
ing details not specifred in this International Stan- The test report shall include all information necess- 
ary for the complete identification of the Sample. 
3 This page intentionally left blank This page intentionally ieft blank ISO 6654:1991(E) 
UL9C b36.085:543.8:547.495.2 
Descriptors: agricultural products, animal feeding products, Chemical analysis, determlnation of content, ureas. 
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