UGANDA STANDARD  
       US ISO 5983-1
First Edition
   2009 -09-04
 
Reference numbe r 
  US ISO 5983-1:2005
 © UNBS 2008
  
Animal feeding stuffs — Determination of nitrogen content and 
calculation of crude protein content — Part 1: Kjeldahl method 
 US ISO 5983-1:2005  
 
© UNBS  2008 – 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 2008  
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
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P.O. Box 6329 
Kampala  
Uganda 
Tel: 256 41 505 995 
Fax: 256 41 286 123 
E-mail: unbs@infocom.co.ug
   US ISO 5983-1: 2005  
© UNBS  2008 - 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 5983-1:2005, Animal feeding stuffs — Determination of nitrogen content
and calculation of crude protei n content — Part 1: Kjeldahl method, is identical with and  has been
reproduced from an International Standard ISO 5983-1:2005,  Animal feeding stuffs — Determination of 
nitrogen content and calculation of crude pr otein content — Part 1: Kjeldahl method, and is has been adopted
as a Uganda Standard . 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard."   
 
  
 Reference numbe r
ISO 5983-1:2005(E)
© ISO 2005
  
 
 INTERNATIONAL 
STANDARD  ISO
5983-1
First edition
2005-07-01
Animal feeding stuffs — Determination of 
nitrogen content and calculation of crude protein content — 
Part 1: 
Kjeldahl method 
Aliments des animaux — Détermination de la teneur en azote et calcul 
de la teneur en protéines brutes — 
Partie 1: Méthode Kjeldahl 
 ISO 5983-1:2005(E) 
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ii © ISO 2005 – All rights reserved
  ISO 5983-1:2005(E) 
© ISO 2005 – All rights reserved  iiiContents Page 
Foreword....................................................................................................................... ..................................... iv  
1 Scope .......................................................................................................................... ........................... 1  
2 Normative references ........................................................................................................... ................ 1  
3 Principle...................................................................................................................... ........................... 1  
4 Reagents and materials ......................................................................................................... .............. 1  
5 Apparatus ...................................................................................................................... ........................ 2  
6 Sampling....................................................................................................................... ......................... 2  
7 Preparation of test sample..................................................................................................... .............. 3  
8 Procedure ...................................................................................................................... ........................ 3  
9 Calculation and expression of results.......................................................................................... ...... 5  
10 Precision...................................................................................................................... .......................... 6  
11 Test report .................................................................................................................... ......................... 7  
Annex A  (informative)  Results of interlaboratory test.................................................................................... 8  
Bibliography ................................................................................................................... .................................. 10  
 ISO 5983-1:2005(E) 
iv © ISO 2005 – All rights reserved
 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 normally carried out through ISO 
technical committees. Each 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, governmental 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. 
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards 
adopted by the technical committees are circulated to the member bodies for voting. Publication as an 
International Standard requires approval by at least 75 % of the member bodies casting a vote. 
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent 
rights. ISO shall not be held responsible for identifying any or all such patent rights. 
ISO 5983-1 was prepared by Technical Committee ISO/TC 34, Food products , Subcommittee SC 10, Animal 
feeding stuffs . 
This first edition of ISO 5983-1, together with ISO 5983-2:2005, cancels and replaces ISO 5983:1997, which 
has been technically revised. 
ISO 5983 consists of the following parts, under the general title Animal feeding stuffs — Determination of 
nitrogen content and calculation of crude protein content : 
 Part 1: Kjeldahl method 
 Part 2: Block digestion/steam distillation method 
 INTERNATIONAL STANDARD ISO 5983-1:2005(E)
 
© ISO 2005 – All rights reserved  1Animal feeding stuffs — Determination of nitrogen content 
and calculation of crude protein content — 
Part 1: 
Kjeldahl method 
1 Scope 
This part of ISO 5983 specifies a method for the determination of the nitrogen content of animal feeding stuffs 
by the Kjeldahl process, and a method for the calculation of the crude protein content. 
The method does not measure oxidized forms of nitrogen or heterocyclic nitrogen compounds. 
This method does not distinguish between protein nitrogen and non-protein nitrogen. If it is important to 
determine the content of non-protein nitrogen, an appropriate method should be used. 
2 Normative references 
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced 
document (including any amendments) applies. 
ISO 6498,  Animal feeding stuffs — Preparation of test samples 
3 Principle 
The organic matter is digested by sulfuric acid in the presence of a catalyst. The reaction product is rendered 
alkaline, then the liberated ammonia is distilled and titrated. The nitrogen content is calculated and the result is multiplied by the conventional factor to obtain the crude protein content. 
4 Reagents and materials 
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or deionized water 
or water of equivalent purity. 
The reagents [except the standard materials (4.6)] shall be practically free from nitrogenous compounds. 
4.1 Potassium sulfate . 
4.2 Catalyst , either 4.2.1 or 4.2.2. 
4.2.1 Copper(II) oxide  (CuO). 
4.2.2 Copper(II) sulfate pentahydrate  (CuSO4·5H2O). 
4.3 Sulfuric acid , c(H2SO4) = 18 mol/l, ρ20(H2SO4) = 1,84 g/ml. ISO 5983-1:2005(E) 
2 © ISO 2005 – All rights reserved
 4.4 Paraffin wax . 
4.5 Saccharose . 
4.6 Standard materials , either 4.6.1 or 4.6.2. 
4.6.1 Acetanilide , with melting point 114 °C; nitrogen (N) content 103,6 g/kg. 
4.6.2 Tryptophan , with melting point 282 °C; nitrogen (N) content 137,2 g/kg. 
Dry before use. 
4.7 Sodium hydroxide solution , w(NaOH) = 33 % (mass fraction). 
4.8 Collecting liquid , either 4.8.1 or 4.8.2. 
4.8.1 Sulfuric acid , standard volumetric solution, c(H2SO4) = 0,05 mol/l or c(H2SO4) = 0,125 mol/l. 
4.8.2 Boric acid , ρ(H3BO3) = 40 g/l. 
4.9 Solutions for titration . 
4.9.1 Sodium hydroxide , standard volumetric solution, c(NaOH) 0,1 mol/l or c(NaOH) = 0,25 mol/l. 
4.9.2 Sulfuric acid , standard volumetric solution, c(H2SO4) = 0,05 mol/l or c(H2SO4) = 0,125 mol/l. 
The molarity of standard volumetric solutions should be known to the fourth decimal point. 
4.10 Mixed indicator , neutral point at pH 4,4 to 5,8. 
Dissolve 2 g of methyl red and 1 g of methylene blue in 1 000 ml of ethanol [ ϕ(C2H5OH) = 95 % (volume 
fraction)]. 
4.11 pH indicator paper . 
4.12 Boiling aids , such as granulated pumice stone, or glass beads of diameter 5 mm to 7 mm, or 
carborundum chips, washed in hydrochloric acid and in distilled water, and ashed. 
5 Apparatus 
Usual laboratory apparatus and, in particular, the following. 
5.1 Analytical balance . 
5.2 Digestion, distillation and titration apparatus . 
6 Sampling 
A representative sample should have been sent to the laboratory. It should not have been damaged or 
changed during transport or storage. 
Sampling is not part of the method specified in this part of ISO 5893. A recommended sampling method is 
given in ISO 6497. 
Store the sample in such a way that deterioration and change in its composition are prevented. ISO 5983-1:2005(E) 
© ISO 2005 – All rights reserved  37 Preparation of test sample 
Prepare the test sample in accordance with ISO 6498. 
8 Procedure 
WARNING — The operations described in 8.3.1 and 8.3.2 should be carried out under a well-ventilated 
hood or in a fume cupboard which is resistant to sulfuric acid. 
8.1 General 
For general directions on the application of the Kjeldahl method, see ISO 1871. 
8.2 Test portion 
Weigh, to the nearest 1 mg, a mass of the test sample chosen according to the expected nitrogen content so 
that the test portion contains between 0,005 g and 0,2 g of nitrogen and, preferably, more than 0,02 g. 
The mass of the test portion of homogeneous air-dry samples should be between 0,5 g and 2,0 g. The mass 
of the test portion of wet and/or inhomogeneous samples should be between 2,5 g and 5,0 g. 
8.3 Determination 
8.3.1 Digestion of organic matter 
Transfer the test portion quantitatively into a Kjeldahl digestion flask of suitable size (usually 800 ml). 
Add 15 g of potassium sulfate (4.1). Add an appropriate quantity of catalyst as follows: 0,3 g of copper(II) oxide (4.2.1) or 0,9 g to 1,2 g of 
copper(II) sulfate pentahydrate (4.2.2). 
Add 25 ml of sulfuric acid (4.3) for the first gram of dry matter of the test portion and 6 ml to 12 ml for each 
additional gram of dry matter. Mix thoroughly, ensuring complete wetting of the test portion. 
Support the flask so that its axis is inclined at an angle of 30° to 45° to the vertical. Maintain the flask in this 
position throughout heating. 
Heat the flask moderately at first to prevent foam from rising into the neck of the flask or escaping from the 
flask. 
NOTE 1 It may be advisable to add an anti-foaming agent such as paraffin wax (4.4). 
Heat moderately, swirling from time to time, until the mass has carbonized and the foam has disappeared. 
Then heat more intensively until the liquid is boiling steadily. 
NOTE 2 Heating is adequate if the boiling acid condenses towards the middle of the neck of the Kjeldahl flask. 
Avoid overheating of the walls of the flask not in contact with liquid. 
NOTE 3 If a naked flame is used, such overheating can be prevented by placing the flask on a sheet of heat-resistant 
material with an aperture of diameter slightly less than that of the flask at the liquid level. 
After the liquid has become clear with a light green-blue colour, heat for another 2 h. 
Leave to cool. If the digest starts to solidify, add some water and mix by swirling. ISO 5983-1:2005(E) 
4 © ISO 2005 – All rights reserved
 8.3.2 Distillation of ammonia 
8.3.2.1 Carefully add 250 ml to 350 ml of water to dissolve the sulfates completely. If necessary, facilitate 
dissolving by heating the flask in warm water. Mix by swirling and allow to cool.  
Add a few boiling aids (4.12). 
For some specific samples, the sulfates may not completely dissolve in the added water. In that case, it is 
recommended to repeat the digestion with a reduced mass of potassium sulfate (4.1). 
8.3.2.2 Pipette, into the collecting flask of the distillation apparatus, 25 ml of the sulfuric acid (4.8.1), 
choosing the concentration according to the expected nitrogen content of the test portion. Add 100 ml to 
150 ml of water. Add a few drops of the mixed indicator (4.10). Proceed in accordance with 8.3.2.4. 
8.3.2.3 Alternatively, transfer into the collecting flask 100 ml to 250 ml of boric acid (4.8.2). Add a few 
drops of mixed indicator (4.10). 
Simultaneous titration of the ammonia (see 8.3.3.3) during distillation is recommended since it facilitates 
verification of the end of distillation. 
8.3.2.4 Immerse the end of the condenser in the liquid contained in the collecting flask, to a depth of at 
least 1 cm. 
Slowly pour 100 ml of sodium hydroxide solution (4.7) into the digestion flask along the wall. Immediately connect the flask to the distillation apparatus. Heat the flask in such a manner that approximately 150 ml of distillate is collected in 30 min. At the end of this 
time, check the pH of the distillate at the tip of the condenser using litmus paper (4.11). If the reaction is 
alkaline, continue distillation. 
IMPORTANT — Lift the condenser from the liquid just before the end of the distillation, to prevent 
backflow. 
If, during distillation using sulfuric acid as collecting liquid, the contents of the collecting flask become alkaline, 
recommence the determination, making appropriate adjustments. 
8.3.3 Titration 
8.3.3.1 Titration with automatic endpoint indication using a pH-meter is recommended. Otherwise, the 
endpoint is indicated by the change in colour of the mixed indicator (4.10) added in 8.3.2. 
8.3.3.2 If sulfuric acid is used as the collecting liquid, titrate, in the collecting flask, the excess sulfuric 
acid with sodium hydroxide solution (4.9.1), c(NaOH) = 0,1 mol/l or c(NaOH) = 0,25 mol/l as appropriate, until 
the endpoint is indicated by the pH-meter or until the colour changes from violet to green. 8.3.3.3 If boric acid is used as the collecting liquid, titrate the ammonia with sulfuric acid (4.9.2), 
c(H
2SO 4) = 0,05 mol/l or c(H2SO 4) = 0,125 mol/l as appropriate, until the endpoint is indicated by the pH-meter 
or the colour changes from green to violet. 
If simultaneous titration is not possible (see 8.3.2.3), the titration should be carried out as soon as possible 
after the distillation is complete, ensuring that the temperature of the distillate does not exceed 25 °C. Under 
these conditions, losses of ammonia are avoided. 
8.4 Blank test 
Perform a blank test using about 1 g of saccharose (4.5) in place of the test portion. ISO 5983-1:2005(E) 
© ISO 2005 – All rights reserved  58.5 Check test 
Perform a check test by determining the nitrogen content of acetanilide (4.6.1) or tryptophan (4.6.2) after 
addition of 1 g of saccharose (4.5). 
The choice of the substance for the check test should be related to the digestibility of the samples to be 
analysed. Acetanilide is easily digested, whereas the digestion of tryptophan is more difficult. 
The recovery of nitrogen from acetanilide or tryptophan should be at least 99,5 % for acetanilide and at least 
99,0 % for tryptophan. 
9 Calculation and expression of results 
9.1 Calculation of nitrogen content 
9.1.1 Distillate collected in sulfuric acid 
Provided that the volumes of sulfuric acid used to collect the ammonia for the determination (8.3) and for the 
blank test (8.4) are equal, calculate the nitrogen content, wn1, in grams per kilogram of the test sample, by the 
following equation: 
( ) 01 1
n1VV cMwm−× ×=  
where 
V0 is the volume, in millilitres, of the sodium hydroxide solution (4.9.1) required for the blank test; 
V1  is the volume, in millilitres, of the sodium hydroxide solution (4.9.1) required for the determination; 
c1 is the concentration, in moles per litre, of the sodium hydroxide solution (4.9.1) used for the titrations; 
M is the molar mass, in grams per mole, of nitrogen ( M = 14 g/mol); 
m is the mass, in grams, of the test portion. 
Report the result to the nearest 0,01 g/kg. 
9.1.2 Distillate collected in boric acid 
Calculate the nitrogen content of the test sample by the equation: 
( ) 32 2
n22VV cMwm−× ×=  
where 
wn2 is the nitrogen content, in grams per kilogram, of the test sample; 
V2 is the volume, in millilitres, of the sulfuric acid (4.9.2) required for the blank test; 
V3 is the volume, in millilitres, of the sulfuric acid (4.9.2) required for the determination; 
M is the molar mass, in grams per mole, of nitrogen ( M 14 g/mol); 
c2 is the concentration, in moles per litre, of the sulfuric acid (4.9.2) used for the titrations; 
m is the mass, in grams, of the test portion. 
Report the result to the nearest 0,01 g/kg. ISO 5983-1:2005(E) 
6 © ISO 2005 – All rights reserved
 9.1.3 Calculation of crude protein content 
The crude protein content may be reported in percent or in grams per kilogram.  Calculate the crude protein 
content of the test sample: 
np6,25 g/kg ww=  
or 
pn0,625 % ww=  
where 
wp is the crude protein content, expressed in grams per kilogram or in percent; 
wn is the nitrogen content, in grams per kilogram, of the test sample (either wn1 or wn2, see 9.1); 
Report the result to the nearest 0,01 g/kg or 0,1 %. 
10 Precision 
10.1 Interlaboratory test 
Details of an interlaboratory test on the precision of the method are summarized in Annex A. The values 
derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those given. 
10.2 Repeatability 
The absolute difference between two independent single test results, obtained using the same method on 
identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5 % of cases be greater than the repeatability limit (
r) derived from the 
equation: 
p 0,3 % 0,008rw=+  
where 
r is the repeatability limit, in percent; 
wp is the mean of the two single test results for crude protein content, in percent. 
10.3 Reproducibility 
The absolute difference between two single test results, obtained using the same method on identical test 
material in different laboratories with different operators using different equipment, will in not more than 5 % of 
cases be greater than the reproducibility limit ( R) derived from the equation: 
p 1,3 % 0,027R w =+  
where 
R is the reproducibility limit, in percent; 
wp is the mean of the two single test results for crude protein content, in percent. ISO 5983-1:2005(E) 
© ISO 2005 – All rights reserved  711 Test report 
The test report shall specify: 
a) all information necessary for the complete identification of the sample; b) the sampling method used, if known; 
c) the test method used, with reference to this part of ISO 5983; 
d) all operating details not specified in this part of ISO 5983, or regarded as optional, together with details of 
any incidents occurred when performing the method, which may have influenced the test result(s); 
e) the test result obtained, either the nitrogen content, or the crude protein content, in grams per kilogram or 
in percent, combined with the conversion factor used (i.e. 6,25), or, if the repeatability has been checked, 
the final quoted result obtained. 
 ISO 5983-1:2005(E) 
8 © ISO 2005 – All rights reserved
 Annex A  
(informative) 
 
Results of interlaboratory test 
An interlaboratory test was organized by ISO/TC 34/SC 10 in 1987 and carried out in accordance with 
ISO 5725:1986. The final statistical analysis was carried out in accordance with ISO 5725-2. In this test 
25 laboratories participated; samples of corn gluten feed, finished mixed feed stuff, fish meal, mixed feed stuff 
concentrate (2 types), premixed feedstuff and yeast were investigated. 
Table A.1 — Statistical results of interlaboratory test (recalculated 2002) 
Parameter Sample  a 
 1 2 3 4 5 6 7 
Number of laboratories retained after 
elimination of outliers 23 23 22 17 23 23 23 
Mean crude protein content, % (based on dry matter) 70,5 81,1 45,9 3,0 39,6 47,5 25,3 
Repeatability standard deviation ( sr), % 
crude protein 0,29 0,33 0,34 0,06 0,28 0,20 0,24 
Repeatability relative standard deviation, % 0,41 0,41 0,75 1,85 0,7 0,42 0,93 
Repeatability limit ( r) 
[r = 2,8 × sr], % crude protein 0,82 0,92 0,96 0,16 0,78 0,56 0,66 
Horrat value  b 0,3 0,3 0,5 0,9 0,5 0,3 0,7 
Reproducibility standard deviation ( sR), % 
crude protein 1,00 1,22 0,90 0,37 1,03 0,97 0,74 
Reproducibility relative standard 
deviation, % 1,42 1,5 1,96 12,1 2,59 2,03 2,94 
Reproducibility limit ( R) 
[R = 2,8 × sR], % crude protein 2,80 3,42 2,53 1,02 2,88 2,78 2,08 
Horrat value b 0,7 0,7 1,2 3,8 1,2 0,9 1,3 
a Sample 1: fish meal 
 Sample 2: corn gluten feed 
 Sample 3: yeast 
 Sample 4: premixed feed stuff   Sample 5: mixed feed stuff concentrate 
 Sample 6: mixed feed stuff concentrate 
 Sample 7: finished mixed feed stuff. 
b A Horrat value of 1 usually indicates satisfactory precision, while a value > 2 indicates unsatisfactory precision; i.e. a precision that 
is too variable for most analytical purposes or where the variation obtained is greater than expected for the type of method 
employed  [6], [7]. 
 ISO 5983-1:2005(E) 
© ISO 2005 – All rights reserved  9 
Key 
X mean, wm, g/kg 
Y precision values, g/kg 
 Repeatability limit r (% crude protein) = 0,008 wm + 0,3 
 Reproducibility limit R (% crude protein) = 0,027 wm + 1,3 
Figure A.1 — Relationship between precision values (r, R) and the mean  (wm) 
 ISO 5983-1:2005(E) 
10 © ISO 2005 – All rights reserved
 Bibliography 
[1] ISO 1871, Agricultural food products — General directions for the determination of nitrogen by the 
Kjeldahl method  
[2] ISO 5725:1986, Precision of test methods — Determination of repeatability and reproducibility for a 
standard test method by inter-laboratory tests  (now withdrawn) 
[3] ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results — Part 1: 
General principles and definitions  
[4] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results — Part 2: 
Basic method for the determination of repeatability and reproducibility of a standard measurement 
method  
[5] ISO 6497:2002, Animal feeding stuffs  — Sampling  
[6] H ORWITZ  W. Evaluation of methods used for regulation of foods and drugs,  Anal. Chem. , 57, 1982, 
pp. 67A-76A 
[7] P EELER  J. T., H ORWITZ  W. and A LBERT  R. Precision parameters of standard methods of analysis of 
dairy products, J. AOAC , 72 (5), 1989, pp. 784-806 
 ISO 5983-1:2005(E) 
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