UGANDA STANDARD
 
        US ISO 5983-2
First Edition
   2009-09-04
 
Reference numbe r 
     US ISO 5983-2:2005
 © UNBS 2009
  
Animal feeding stuffs — Determination of nitrogen content and 
calculation of crude protein content — Part 2: Block 
digestion/steam distillation method 
 US ISO 5983-2:2005 
 
© 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 
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
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permission from UNBS. 
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The Executive Director 
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Fax: 256 41 286 123 
E-mail: unbs@infocom.co.ug
Web: www.unbs.go.ug  
      US ISO 5983-2:2005  
© 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 under Cap 327, of the Laws of U ganda. 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 5983-2, Animal feeding stuffs — Determination of nitrogen content and
calculation of crude protein content — Part 2: Block digestion/steam distillation method , is identical
with and has been reproduced from an International Standard, ISO 5983-2:2005, Animal feeding stuffs — 
Determination of nitrogen content and calculation of crude protein content — Part 2: Block 
digestion/steam distillation method , and adopted as a Uganda Standard.  
This standard was developed by the Food and Agricu lture Standards Technical Committee (UNBS/TC 2).  
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." 
   
 
  
 Reference numbe r
ISO 5983-2:2005(E)
© ISO 2005
  
 
 INTERNATIONAL 
STANDARD  ISO
5983-2
First edition
2005-07-01
Animal feeding stuffs — Determination of 
nitrogen content and calculation of crude protein content — 
Part 2: 
Block digestion/steam distillation method
Aliments des animaux — Détermination de la teneur en azote et calcul 
de la teneur en protéines brutes — 
Partie 1: Méthode de digestion en bloc et distillation à la vapeur 
 ISO 5983-2:2005(E) 
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ii © ISO 2005 – All rights reserved
  ISO 5983-2:2005(E) 
© ISO 2005 – All rights reserved  iiiContents Page 
Foreword....................................................................................................................... ..................................... iv  
1 Scope .......................................................................................................................... ........................... 1  
2 Normative references ........................................................................................................... ................ 1  
3 Terms and definitions.......................................................................................................... ................. 2  
4 Principle...................................................................................................................... ........................... 2  
5 Reagents....................................................................................................................... ......................... 2  
6 Apparatus ...................................................................................................................... ........................ 4  
7 Sampling....................................................................................................................... ......................... 4  
8 Preparation of test sample..................................................................................................... .............. 5  
9 Procedure ...................................................................................................................... ........................ 5  
10 Calculation and expression of results.......................................................................................... ...... 7  
11 Precision...................................................................................................................... .......................... 8  
12 Test report .................................................................................................................... ......................... 9  
Annex A  (informative)  Results of interlaboratory test.................................................................................. 10  
Annex B  (informative)  Automatic distillation with potentiometric endpoint detection............................. 13  
Bibliography ................................................................................................................... .................................. 14  
 ISO 5983-2:2005(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 5983-2 was prepared by Technical Committee ISO/TC 34, Food products , Subcommittee SC 10, Animal 
feeding stuffs . 
This first edition of ISO 5983-2, together with ISO 5983-1:2005, 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 
 INTERNATIONAL STANDARD ISO 5983-2:2005(E)
 
© ISO 2005 – All rights reserved  1Animal feeding stuffs — Determination of nitrogen content 
and calculation of crude protein content — 
Part 2: 
Block digestion/steam distillation method 
WARNING  The use of this method may involve the use of hazardous materials, operations and 
equipment. This standard does not purport to address all the safety risks associated with its use. It is 
the responsibility of the user of this method to establish appropriate health and safety practices and determine the applicability of local regulatory limitations prior to use.  
1 Scope 
This part of ISO 5983 specifies a method for the determination of nitrogen content of animal feeding stuffs 
according to the Kjeldahl method, and a method for the calculation of the crude protein content. 
It concerns a semi-micro rapid routine method using block-digestion, copper catalyst and steam distillation into 
boric acid. 
The method is applicable to the determination of greater than 0,5 % Kjeldahl nitrogen in animal feeding stuffs, 
pet foods and their raw materials. 
The method does not measure oxidized forms of nitrogen nor heterocyclic nitrogen compounds. 
The method does not distinguish between protein nitrogen and non-protein nitrogen. 
NOTE If it is of importance to determine the content of non-protein-nitrogen, an appropriate method can 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 referred document 
(including any amendments) applies. 
ISO 385:2005, Laboratory glassware — Burettes 
ISO 1871, Agricultural food products — General directions for the determination of nitrogen by the Kjeldahl 
method  
ISO 6498:1998, Animal feeding stuffs — Preparation of test samples  ISO 5983-2:2005(E) 
2 © ISO 2005 – All rights reserved
 3 Terms and definitions 
For the purposes of this document, the following terms and definitions apply. 
3.1 
nitrogen content mass fraction of nitrogen determined by the procedure specified in this document 
NOTE The nitrogen content is expressed as a percentage by mass or in grams per kilogram. 
3.2 crude protein content amount of nitrogen content (3.1) multiplied by the factor 6,25 
NOTE The crude protein content is expressed as a percentage by mass or in grams per kilogram. 
4 Principle 
The test portion is digested using a block-digestion or equivalent apparatus. Concentrated sulfuric acid is used to convert protein nitrogen to ammonium sulfate at a boiling point elevated by the addition of potassium sulfate. 
A copper catalyst is used to enhance the reaction rate. An excess of sodium hydroxide is added to the cooled 
digest to liberate ammonia. 
The liberated ammonia is distilled, using a manual, semi-automatic or fully automatic steam distillation unit. In 
the case of manual or semi-automatic steam distillation, distillation of the ammonia into an excess of boric acid solution is followed by titration with hydrochloric acid solution to a colorimetric endpoint. Where a fully automatic system is employed, automatic titration of the ammonia is carried out simultaneously with the 
distillation. 
The nitrogen content is calculated from the amount of ammonia produced. The crude protein content is 
obtained by multiplying the result by the conventional conversion factor of 6,25. 
NOTE 1 As in ISO 5983-1, the automatic titration of the ammonia can also be carried out with endpoint detection using 
a potentiometric pH system (see Annex B). 
NOTE 2 In principle, sulfuric acid could also be used for the titration. 
5 Reagents 
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized 
water or water of equivalent purity. 
5.1 Kjeldahl catalyst tablets , comprising of 3,5 g of potassium sulfate and 0,4 g of copper(II) sulfate 
pentahydrate per tablet. 
These tablets may be purchased ready prepared. Other types of tablet may be used provided that 
a) they contain a quantity of potassium sulfate such that 7 g of potassium sulfate and 0,8 g of copper(II) 
sulfate pentahydrate can be dispensed using an integer number of whole tablets, and 
b) they do not contain salts of toxic metals such as selenium or mercury. 
5.2 Sulfuric acid  (H
2SO4), with a mass fraction of at least 98 %, nitrogen-free (approximately 
ρ20 = 1,84 g/ml). ISO 5983-2:2005(E) 
© ISO 2005 – All rights reserved  35.3 Hydrogen peroxide solution , containing approximately 30 g of H2O2 per 100 ml. 
5.4 Antifoaming agent . 
A silicone preparation is recommended, for example with a mass fraction of 30 % aqueous emulsion. 
5.5 Sodium hydroxide  (NaOH) solution, approximately 40 % (mass fraction), nitrogen-free ( < 5 µg of N per 
gram). 
5.6 Indicator solutions . 
5.6.1 Methyl red solution . 
Dissolve 100 mg of methyl red (C15H15N3O2) in 100 ml of ethanol or methanol. 
5.6.2 Bromocresol green solution . 
Dissolve 100 mg of bromocresol green (C21H14Br4O5S) in 100 ml of ethanol or methanol. 
5.7 Concentrated boric acid solution , c(H3BO3) = 40,0 g/l. 
Dissolve 400 g of boric acid in about 5 l to 6 l of hot deionized water. Mix and add more hot deionized water to 
a volume of about 9 l. Allow to cool to room temperature. Add 70 ml of the methyl red solution (5.6.1) and 
100 ml of the bromocresol green solution (5.6.2) and mix. Dilute to a final volume of 10 l with water and mix well. Depending on the water used, the pH of the boric acid solution can differ from batch to batch. Often an adjustment with a small volume of alkali is necessary to obtain a positive blank (0,05 ml to 0,15 ml of titrant). The colour shall turn green when 100 ml of distilled water are added to 25 ml of the boric acid solution. If still red, titrate with 0,1 mol/l NaOH until “neutral grey” and calculate the amount of alkali needed for the 10 l batch. 
Store the solution, which will be red in colour, at room temperature and protect the solution from light and 
sources of ammonia fumes during storage. 
5.8 Dilute boric acid solution , c(H
3BO3) = 10,0 g/l (optional trapping solution for titrators that automatically 
begin titration when distillation begins). 
Dissolve 100 g of boric acid in about 5 l to 6 l of hot deionized water, mix and add more hot deionized water to 
a volume of about 9 l. Allow to cool to room temperature. Add 70 ml of the methyl red solution (5.6.1) and 
100 ml of the bromocresol green solution (5.6.2) and mix. Dilute to a final volume of 10 l. Depending on the 
water used, the pH of the boric acid solution can differ from batch to batch. Often an adjustment with a small volume of alkali is necessary to obtain a positive blank (0,05 ml to 0,15 ml of titrant). The colour shall turn green when 100 ml of distilled water are added to 25 ml of the boric acid solution. If still red, titrate with 0,1 mol/l NaOH until “neutral grey” and calculate the amount of alkali needed for the 10 l batch. 
Store the solution, which will be light green in colour, at room temperature and protect the solution from light 
and sources of ammonia fumes during storage. 
NOTE The addition of about 3 ml to 4 ml of 0,1 M NaOH into 1 l of 1 % boric acid usually gives good adjustments. 
5.9 Hydrochloric acid standard volumetric solution , c(HCl) = 0,100 0 mol/l.  
Other concentrations of HCl or sulfuric acid may be used if this is corrected for in the calculations. The 
concentrations should always be expressed to four decimal places. 
5.10 Ammonium sulfate  [(NH4)2SO4], min. 99,5 % (mass fraction), with certified purity. Dry ammonium 
sulfate at 102 °C ± 2 °C for 4 h and store in a desiccator. 
Percent nitrogen in ammonium sulfate (at 99,5 % purity) is 21,09. 
5.11 Ammonium iron(II) sulfate  [(NH4)2⋅Fe(SO4)2⋅6H2O], with certified purity.  ISO 5983-2:2005(E) 
4 © ISO 2005 – All rights reserved
 Percent nitrogen in ammonium iron(II) sulfate (at 100 % purity) is 7,145. 
5.12 Standard materials 
One of the following (5.12.1 or 5.12.2) standard materials may be used. 
In addition to the standard materials listed below, suitable reference materials with certified values for Kjeldahl 
nitrogen/protein should be used whenever possible. 
NOTE The moisture content can be checked on a separate portion. 
5.12.1 Tryptophan  (C11H12N2O2), with melting point 282 °C; nitrogen content 137,2 g/kg. Before use, dry 
the tryptophan. 
5.12.2 Acetanilide  (C8H9NO), minimum assay 99 % (mass fraction). Nitrogen content 103,6 g/kg. Do not dry 
in an oven before use. 
5.13 Sucrose  (C12H22O11), with a nitrogen content of not more than 0,002 % (mass fraction). Do not dry in 
an oven before use. 
6 Apparatus 
Usual laboratory apparatus and, in particular, the following. 
6.1 Analytical balance , capable of weighing to the nearest 0,1 mg, with a readability of 0,1 mg. 
6.2 Digestion block , aluminium alloy block or equivalent block, fitted with an adjustable temperature 
control and device for measuring block temperature, warmed up to 420 °C ± 5 °C. 
6.3 Digestion tubes , of capacity 250 ml, suitable for use with the digestion block (6.2). 
6.4 Exhaust manifold , suitable for use with the digestion tubes (6.3). 
6.5 Centrifugal scrubber apparatus, filter pump or aspirator , constructed of acid-resistant material, for 
use with mains water supply. 
6.6 Automatic pipettes  (dispensers), capable of delivering up to 25 ml portions. 
6.7 Graduated measuring cylinders , of capacity 50 ml. 
6.8 Distillation unit , capable of steam distilling, manual or semi-automatic, suitable to accept the digestion 
tubes (6.3) and the conical flasks (6.9), or capable of steam distillation and autotitration. 
6.9 Conical flasks , of capacity 250 ml. 
6.10 Burette , of capacity 25 ml or suitable capacity, with at least a readability of 0,05 ml, complying with the 
requirements of ISO 385:2005, class A. 
Alternatively, an automatic burette may be used fulfilling the same requirements. 
7 Sampling 
A representative sample should have been sent to the laboratory. It should not have been damaged or 
changed during transport or storage. ISO 5983-2:2005(E) 
© ISO 2005 – All rights reserved  5Sampling is not part of the method specified in this part of ISO 5983. A recommended sampling method is 
given in ISO 6497. 
8 Preparation of test sample 
Prepare the test sample in accordance with ISO 6498. 
9 Procedure 
9.1 General 
Usually test samples should be analysed in batches according to the described procedure. For general requirements on the application of the Kjeldahl method, see ISO 1871. 
9.2 Test portion 
As the test portion, weigh, to the nearest 0,1 mg, 
 approximately 1,0 g for materials with 3 % to 30 % protein, 
 approximately 0,5 g for materials with 30 % to 80 % protein, or 
 approximately 0,3 g for materials with more than 80 % protein. 
Do not exceed 1,2 g. 
Always perform quality control and standards as well as a reagent blank with each batch. 
9.3 Determination 
9.3.1 Digestion 
Transfer the test portion (9.2) to the digestion tube (6.3) and add two catalyst tablets (5.1) to each tube. Using 
a pipetting dispenser, add 12 ml of sulfuric acid (5.2) to each tube. Use 15 ml for high fat materials ( > 10 % 
fat). It is possible to stop at this point and continue work the following day. 
If foaming is a problem, slowly add 3 ml to 5 ml of hydrogen peroxide (5.3). Swirl gently and let the reaction 
subside. Alternatively a few drops of antifoaming agent (5.4) may be used. 
Attach the heat side shields to the tube rack. Place the exhaust manifold (6.4) tightly on the tubes and turn the 
water aspirator or scrubber (6.5) on completely. Place the rack of tubes in the pre-heated (420 °C) digestion block. 
After 10 min, turn the water aspirator down until the acid fumes are just contained within the exhaust hood. A 
condensation zone should be maintained within the tube. After the bulk of the sulfur oxide fumes are produced 
during the initial stages of digestion, the vacuum source shall be reduced to prevent loss of sulfuric acid. 
Digest for an additional 50 min. The total digestion time should be approximately 60 min. 
Turn the digestor off. Remove the rack of tubes with the exhaust still in place and put it in the stand to cool for 
10 min to 20 min. When fuming has stopped, remove the manifold and shut off the aspirator. Remove the side shields. 
Allow the tubes to cool. It is recommended to predilute samples manually prior to distilling. Wearing gloves 
and eye protection, carefully add a few millilitres of deionized water to each tube. If spattering occurs, this ISO 5983-2:2005(E) 
6 © ISO 2005 – All rights reserved
 means that the tubes are still too hot. Allow to cool for a few more minutes. Add water to each tube to a total 
volume of approximately 80 ml. 
If the sample solidifies, place the tube containing the diluted digest in the block digester and carefully warm 
with occasional swirling until salts dissolve, or distil for a further 30 s to 60 s. 
NOTE 1 Some instruments perform the addition of water automatically. The predilution before placing the tube in the 
instrument is only required if very solid cakes form. 
NOTE 2 Some distillation instruments start with the addition of steam before the addition of alkali, which leads to a 
dissolution of salt cakes and a less violent reaction during alkali addition. Crystallization during digestion can cause 
nitrogen losses. 
9.3.2 Distillation 
Transfer the digestion tube (see 9.3.1) to the distillation unit (6.8). 
Where titration of the ammonia content of the distillate is performed manually, the procedure mentioned below 
applies. Where the distillation unit is fully automated to include titration of the ammonia content of the distillate, follow the manufacturer’s instructions for operation of the distillation unit. 
Place a conical flask (6.9) containing 25 ml to 30 ml of the concentrated boric acid solution (5.7) under the 
outlet of the condenser in such a way that the delivery tube is below the surface of the excess boric acid solution. Adjust the distillation unit to dispense 50 ml of sodium hydroxide solution (5.5). Operate the distillation unit in accordance with the manufacturer's instructions and distil off the ammonia liberated by the addition of the sodium hydroxide solution. Collect the distillate in the boric acid receiving solution. The amount of distillate (time of steam distillation) depends on the amount of nitrogen in the sample. Follow the 
manufacturer's instructions. 
NOTE In a semi-automatic distillation unit, the addition of excess sodium hydroxide and the steam distillation are 
performed automatically. 
9.3.3 Titration 
Titrate the contents of the conical flask (9.3.2) with the hydrochloric acid standard volumetric solution (5.9) 
using a burette (6.10) and read the amount of titrant used. The endpoint is reached at the first trace of pink colour in the contents. Estimate the burette reading to the nearest 0,05 ml. An illuminated magnetic stirrer plate or a photometric detector may aid visualization of the endpoint. 
This can be done automatically using a steam distiller with automatic titration. 
Follow the manufacturer's instructions for operation of the specific distiller
 or distiller/titrator. 
When an automatic titration system is used, titration begins immediately after distillation starts and the 1 % 
boric acid solution (5.8) should be used. 
Where a fully automatic distillation unit is used, the automatic titration of the ammonia may also be carried out 
with endpoint detection using a potentiometric pH system (see Annex B). 
9.4 Blank test 
Carry out a blank test following the procedure described in 9.1 to 9.3.3 taking 2 ml of water and about 0,7 g of 
sucrose (5.13) instead of the test portion. Keep a record of blank values. If blank values change, identify the 
cause. 
The amount of titrant used in the blank test should always be greater than 0,0 ml. Blanks within the same 
laboratory should be consistent over time. ISO 5983-2:2005(E) 
© ISO 2005 – All rights reserved  79.5 Recovery tests 
9.5.1 General 
The regularly run recovery tests to check the accuracy of the procedure and equipment are described in 9.5.2 
to 9.5.4. 
9.5.2 Nitrogen loss 
Use 0,12 g of ammonium sulfate (5.10) and 0,67 g of sucrose (5.13) per flask. Add all other reagents as 
stated in 9.3. Digest and distil under the same conditions as for the sample. Recoveries shall be W 99 %. 
9.5.3 Digestion efficiency 
Use a test portion of at least 0,15 g of tryptophan (5.12.1) or acetanilide (5.12.2), weighed to the nearest 
0,1 mg, and with addition of about 0,7 g of sucrose (5.13). Determine the nitrogen content according to the 
procedure described in 9.1 to 9.3.3. The recoveries should be W 99,5 % for acetanilide (5.12.2) and W 98,5 % 
for tryptophan (5.12.1) [5]. 
9.5.4 Distillation and titration efficiency  
Weigh 0,10 g to 0,15 g, to the nearest 0,000 1 g of ammonium sulfate (5.10), or 0,3 g to 0,5 g, to the nearest 
0,000 1 g, of ammonium iron(II) sulfate (5.11) into a tube. Add 80 ml of distilled water and proceed according 
to 9.3.2 and 9.3.3. The recovery shall be W 99,5 %. 
9.5.5 Limits 
Recoveries less than specified or more than 101,0 % in any of the above recovery tests indicate failures in the 
procedures and/or inaccurate concentration of the standard volumetric hydrochloric acid solution (5.9). 
10 Calculation and expression of results 
10.1 Calculation 
10.1.1 Calculation of nitrogen content 
Calculate the nitrogen content of the sample, wN, as a percentage by mass: 
( ) sb s
N1 ,400 7 VV cwm−=  
where 
Vs is the numerical value of the volume of the hydrochloric acid standard volumetric solution (5.9) used 
in the determination (9.3), in millilitres, expressed to the nearest 0,05 ml; 
Vb is the numerical value of the volume of the hydrochloric acid standard volumetric solution (5.9) used 
in the blank test (9.4), in millilitres, expressed to the nearest 0,05 ml; 
cs is the numerical value of the exact concentration, in moles per litre, of the hydrochloric acid standard 
volumetric solution (5.9), expressed to four decimal places; 
m is the numerical value of the mass of the test portion (9.2), in grams. 
For reporting the result in grams per kilogram, a factor of 14,007 may be used in the above equation. ISO 5983-2:2005(E) 
8 © ISO 2005 – All rights reserved
 10.1.2 Calculation of recovery of ammonium salts 
Calculate the recovery for ammonium sulfate with 99,5 % purity level, P1, as follows: 
1 100 %21,09NP=×  
where N is the recovery of nitrogen, in percent. 
Calculate the recovery for ammonium iron(II) sulfate with 100 % purity level, P2, as follows: 
2 100 %7,145NP=×  
The above calculations shall be adjusted if other purity levels of ammonium salts are used. 
10.2 Calculation of crude protein content 
Calculate the crude protein content, wp, as a percentage by mass, using the following equation: 
pNww F=⋅  
where 
wN is the nitrogen content of the sample, expressed as a percentage by mass to four decimal places 
(10.1); 
F is the factor to convert Kjeldahl nitrogen to protein; for feedstuffs, F = 6,25. 
For reporting the crude protein content in grams per kilogram, the result according to the above equation may 
be multiplied by 10. 
10.3 Expression of the crude protein content results 
Express the results to four decimal places if needed for further calculations. For end results, express those 
obtained for the nitrogen content to three decimal places, and for the protein content to two decimal places. 
The results should not be rounded further until the final use of the test value is made. This is particularly true 
when the values are to be used for further calculations. 
EXAMPLES One example is when the individual test values obtained from the analysis of many sample materials are 
used to calculate method performance statistics for within and between laboratory variation. Another example is when the 
values are used as a reference for instrument calibration (e.g. infrared analyser), where the values from many samples will 
be used in a simple or multiple regression calculation before they are used for further calculations. 
11 Precision 
11.1 Interlaboratory test 
Details of an interlaboratory test on the precision of the method are summarized in Annex A. The values 
derived from this test may not be applicable to concentration ranges and matrices other than those given. ISO 5983-2:2005(E) 
© ISO 2005 – All rights reserved  911.2 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 exceed the repeatability limit ( r) derived from the equation: 
p 0,433 0,008 5rw=+  
where 
r is the repeatability limit, in percent protein; 
wp is the mean of the two single test results for crude protein content, in percent protein. 
11.3 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 0,414 0,012 7R w =+  
where 
R is the reproducibility limit, in percent protein; 
wp is the mean of the two single test results for crude protein content, in percent protein. 
12 Test report 
The test report shall specify: a) all information required for the complete identification of the sample; b) the sampling method used, if known; c) the 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 incident which may have influenced the results; 
e) the test results obtained, either the nitrogen content (in percent or grams per kilogram), or the crude 
protein content (in percent or grams per kilogram), combined with the conversion factor 6,25; 
f) if the repeatability has been checked, the final quoted result obtained; g) if the recovery has been checked, the final quoted result obtained. ISO 5983-2:2005(E) 
10 © ISO 2005 – All rights reserved
 Annex A  
(informative) 
 
Results of interlaboratory test 
An interlaboratory test was organized by the AOAC International in 2001 and was carried out in accordance 
with ISO 5725-2. In this test, thirteen laboratories from North America and Europe participated. Fourteen blind 
samples were investigated, including meat and bone meal, dog food, chinchilla food, bird seed, soybeans, 
corn silage green chop, grass hay, alfalfa hay, milk replacer, albumin, swine pellets, sunflower seeds, protein block (with urea) and fish meal. 
The recovery of nitrogen from standard compounds was 100,1 % for acetanilide and 98,8 % for tryptophan. 
Table A.1 — Results of interlaboratory test 
Parameter Sample  a 
 1 2 3 4 5 6 7 
Number of laboratories retained 
after elimination of outliers 11 11 13 12 12 12 10 
Mean crude protein content, % (on 
as-is basis) 40,20 37,00 7,10 7,10 64,60 24,50 18,10 
Repeatability standard deviation 
(sr), % crude protein 0,20 0,20 0,20 0,10 0,50 0,20 0,20 
Repeatability relative standard 
deviation, % 0,40 0,50 1,90 1,90 0,70 0,80 0,80 
Repeatability limit, r 
(= 2,8 sr), % crude protein 0,50 0,50 0,40 0,40 1,30 0,60 0,40 
Horrat value  b 0,3 0,3 1,0 1,0 0,5 0,5 0,5 
Reproducibility standard deviation 
(sR), % crude protein 0,30 0,20 0,20 0,10 0,70 0,20 0,20 
Reproducibility relative standard 
deviation, % 0,70 0,60 2,70 1,90 1,00 0,90 0,80 
Reproducibility limit, R (= 2,8 sR), % 
crude protein 0,80 0,60 0,50 0,40 1,90 0,60 0,40 
Horrat value b 0,3 0,2 0,9 0,6 0,5 0,4 0,3 
 ISO 5983-2:2005(E) 
© ISO 2005 – All rights reserved  11Table A.1  (continued ) 
Parameter Sample  a 
  8 9 10 11 12 13 14 
Number of laboratories retained after 
elimination of outliers 11 12 12 12 11 12 12 
Mean crude protein content, % (on as-is 
basis) 79,10 13,50 50,10 20,80 38,80 17,40 18,80 
Repeatability standard deviation ( sr), % 
crude protein 0,30 0,10 0,90 0,30 0,40 0,40 0,30 
Repeatability relative standard 
deviation, % 0,40 0,80 1,80 1,30 0,90 2,30 1,40 
Repeatability limit, r 
(= 2,8 sr), % crude protein 0,90 0,30 2,60 0,80 1,00 1,10 0,70 
Horrat value  b 0,3 0,5 1,3 0,8 0,6 1,3 0,8 
Reproducibility standard deviation ( sR), % 
crude protein 0,40 0,20 0,90 0,30 0,40 0,40 0,30 
Reproducibility relative standard 
deviation, % 0,50 1,30 1,80 1,30 1,00 2,30 1,40 
Reproducibility limit, R 
(2,8 sR), % crude protein 1,00 0,50 2,60 0,80 1,00 1,10 0,70 
Horrat value b 0,2 0,5 0,8 0,5 0,4 0,9 0,5 
a Sample 1: protein block 
Sample 2: swine pellets 
Sample 3: corn silage Sample 4: grass hay 
Sample 5: fish meal 
Sample 6: dog food Sample 7: chinchilla feed 
Sample 8: albumin 
Sample 9: bird seed Sample 10: meat and bone meal 
Sample 11: milk replacer 
Sample 12: soybeans Sample 13: sunflower seed 
Sample 14: legume hay. 
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 [5], [6]. 
 ISO 5983-2:2005(E) 
12 © ISO 2005 – All rights reserved
  
Key 
X mean value, % 
Y precision values, % 
 repeatability limit, r 
 reproducibilty limit, R 
NOTE Repeatability limit r = 0,008 5 x + 0,433  1); reproducibility limit R = 0,0127  x + 0,414  2), where x is the mean 
value. 
Figure A.1 — Relationship between precision values and the mean value 
                                                      
1) Excluding sample No. 10 (meat and bone meal). ISO 5983-2:2005(E) 
© ISO 2005 – All rights reserved  13Annex B  
(informative) 
 
Automatic distillation with potentiometric endpoint detection 
B.1 General 
Where a fully automatic distillation unit is used, the automatic titration of the ammonia may also be carried out 
with endpoint detection using a potentiometric pH system. 
In this case, the following changes in apparatus and procedure apply: 
B.2 Apparatus 
B.1.1 Automatic titrator , with a pH-meter. 
The pH-meter should be  calibrated properly in the range of pH 4 to pH 7 following normal laboratory 
pH-calibration procedures. 
B.3 Titration procedure 
Titrate the contents of the conical flask (see 9.3.2)  with the hydrochloric acid standard volumetric solution (5.9) 
using a properly calibrated automatic titrator provided with a pH-meter (B.1.1). The pH endpoint of the titration 
is reached at pH 4,6, being the steepest point in the titration curve (inflection point). Read the amount of the 
used titrant on the automatic titrator. 
NOTE Automatic titration with potentiometric endpoint detection is described in ISO 5983-1. This option has not been 
validated in the data given in Annex A. 
 ISO 5983-2:2005(E) 
14 © ISO 2005 – All rights reserved
 Bibliography 
[1] ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results — Part 1: 
General principles and definitions  
[2] ISO 5725-2, 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  
[3] ISO 5983-1, Animal feeding stuffs — Determination of nitrogen content and calculation of crude 
protein content — Part 1: Kjeldahl method  
[4] ISO 6497:2002, Animal feeding stuffs — Sampling  
[5] T HIEX, N.J., M ANSON , H., A NDERSON , S. and P ERSSON , J.A. Determination of Crude Protein in Animal 
Feed, Forage, Grain and Oilseeds by Using Block Digestion with a Copper Catalyst and Steam 
Distillation into Boric Acid: Collaborative Study . Journal of AOAC International , 85, No 2, 2002, 
pp. 309-317 
[6] H ORWITZ  W. Evaluation of methods used for regulation of foods and drugs . Analytical Chemistry , 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 . Journal of AOAC , 72, No 5, 1989, pp. 784-806 
 ISO 5983-2:2005(E) 
ICS  65.120 
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© ISO 2005 – All rights reserved