EN ISO 8968-1:2001

SLOVENSKI STANDARD
01-junij-2002
0OHNR'RORþHYDQMHGXãLNDGHO0HWRGDSR.MHOGDKOX,62
Milk - Determination of nitrogen content - Part 1: Kjeldahl method (ISO 8968-1:2001)
Milch - Bestimmung des Stickstoffgehaltes - Teil 1: Kjeldahl-Verfahren (ISO 8968-
1:2001)
Lait - Détermination de la teneur en azote - Partie 1: Méthode Kjeldahl (ISO 8968-
1:2001)
Ta slovenski standard je istoveten z: EN ISO 8968-1:2001
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 8968-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2001
ICS 67.100.10
English version
Milk - Determination of nitrogen content - Part 1: Kjeldahl
method (ISO 8968-1:2001)
Lait - Détermination de la teneur en azote - Partie 1: Milch - Bestimmung des Stickstoffgehaltes - Teil 1:
Méthode Kjeldahl (ISO 8968-1:2001) Kjeldahl-Verfahren (ISO 8968-1:2001)
This European Standard was approved by CEN on 15 December 2001.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2001 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 8968-1:2001 E
worldwide for CEN national Members.

CORRECTED  2002-03-13
Foreword
This document (ISO 8968-1:2001) has been prepared by Technical Committee ISO/TC 34
"Agricultural food products" in collaboration with Technical Committee CEN/TC 302 "Milk and
milk products - Methods of sampling and analysis", the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of
an identical text or by endorsement, at the latest by June 2002, and conflicting national
standards shall be withdrawn at the latest by June 2002.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium, Czech
Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg,
Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of the International Standard ISO 8968-1:2001 has been approved by CEN as a
European Standard without any modifications.
INTERNATIONAL ISO
STANDARD 8968-1
IDF
20-1
First edition
2001-12-15
Milk — Determination of nitrogen
content —
Part 1:
Kjeldahl method
Lait — Détermination de la teneur en azote
Partie 1: Méthode Kjeldahl
Reference numbers
ISO 8968-1:2001(E)
IDF 20-1:2001(E)
©
ISO and IDF 2001
ISO 8968-1:2001(E)
IDF 20-1:2001(E)
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©  ISO and IDF 2001
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ii © ISO and IDF 2001 – All rights reserved

ISO 8968-1:2001(E)
IDF 20-1:2001(E)
Contents Page
Foreword.iv
1 Scope .1
2 Normative reference.1
3 Term and definition .1
4 Principle.1
5 Reagents.2
6 Apparatus .3
7 Sampling.3
8 Preparation of test sample.4
9 Procedure .4
9.1 Test portion and pretreatment .4
9.2 Determination.4
9.3 Blank test.5
9.4 Recovery tests .6
10 Calculation and expression of results.6
10.1 Calculation of nitrogen content .6
10.2 Calculation of crude protein content.7
11 Precision.7
11.1 Interlaboratory test.7
11.2 Repeatability .8
11.3 Reproducibility.8
12 Test report .8
Annex A (informative) Modified procedure for analysis of other milk products when a separate
standard for that product does not exist .9
Bibliography.11

ISO 8968-1:2001(E)
IDF 20-1:2001(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 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 3.
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 part of ISO 8968IDF 20 may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO 8968-1IDF 20-1 was prepared by Technical Committee ISO/TC 34, Food products,
Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with
AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International.
ISO 8968IDF 20 consists of the following parts, under the general title Milk — Determination of nitrogen content:
— Part 1: Kjeldahl method
— Part 2: Block-digestion method (Macro method)
— Part 3: Block-digestion method (Semi-micro rapid routine method)
— Part 4: Determination of the non-protein-nitrogen content
— Part 5: Determination of the protein-nitrogen content
Annex A of this part of ISO 8968IDF 20 is for information only.
iv © ISO and IDF 2001 – All rights reserved

ISO 8968-1:2001(E)
IDF 20-1:2001(E)
Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in
every member country. Every National Committee has the right to be represented on the IDF Standing Committees
carrying out the technical work. IDF collaborates with ISO and AOAC International in the development of standard
methods of analysis and sampling for milk and milk products.
Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National
Committees for voting. Publication as an International Standard requires approval by at least 50 % of National
Committees casting a vote.
International Standard ISO 8968-1IDF 20-1 was prepared by Technical Committee ISO/TC 34, Food products,
Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with
AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International.
All work was carried out by the Joint ISO/IDF/AOAC Action Team, Nitrogen compounds, under the aegis of its
project leader, Mr D.M. Barbano (US).

ISO 8968-1:2001(E)
INTERNATIONAL STANDARD
IDF 20-1:2001(E)
Milk — Determination of nitrogen content —
Part 1:
Kjeldahl method
WARNING — The use of this part of ISO 8968ΩΩΩΩIDF 20 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 standard to establish appropriate safety and healthy
practices and determine the applicability of local regulatory limitations prior to use.
1 Scope
This part of ISO 8968ΩIDF 20 specifies a method for the determination of the nitrogen content of liquid milk, whole
or skimmed, by the Kjeldahl principle.
2 Normative reference
The following normative document contains provisions which, through reference in this text, constitute provisions of
this part of ISO 8968ΩIDF 20. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this part of ISO 8968ΩIDF 20 are encouraged
to investigate the possibility of applying the most recent edition of the normative document indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
ISO 385-1, Laboratory glassware — Burettes — Part 1: General requirements
3 Term and definition
For the purposes of this part of ISO 8968ΩIDF 20, the following term and definition apply.
3.1
nitrogen content
mass fraction of nitrogen determined by the procedure specified in this part of ISO 8968ΩIDF 20
NOTE The nitrogen content is expressed as a percentage by mass.
4 Principle
A test portion is digested with a mixture of concentrated sulfuric acid and potassium sulfate, using copper(II) sulfate
as a catalyst to thereby convert organic nitrogen present to ammonium sulfate. The function of the potassium
sulfate is to elevate the boiling point of the sulfuric acid and to provide a stronger oxidizing mixture for digestion.
Excess sodium hydroxide is added to the cooled digest to liberate ammonia.The liberated ammonia is distilled into
excess boric acid solution then titrated with hydrochloric acid. The nitrogen content is calculated from the amount of
ammonia produced.
ISO 8968-1:2001(E)
IDF 20-1:2001(E)
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water
or water of equivalent purity.
5.1 Potassium sulfate (K SO ), nitrogen free.
2 4
5.2 Copper(II) sulfate solution, c(CuSO ) = 5,0 g per 100 ml.
Dissolve 5,0 g of copper(II) sulfate pentahydrate (CuSO ⋅5H O) in water in a 100 ml one-mark volumetric flask.
4 2
Dilute to the mark with water and mix.
5.3 Sulfuric acid (H SO ), with a mass fraction of at least 95 % to 98 %, nitrogen free (р = 1,84 g/ml
2 4 20
approximately).
5.4 Sodium hydroxide solution (NaOH), nitrogen free, containing 50 g of sodium hydroxide per 100 g of
solution.
5.5 Indicator solution
Dissolve 0,1 g of methyl red in 95 % (volume fraction) ethanol. Dilute to 50 ml with the ethanol. Dissolve 0,5 g of
bromocresol green in 95 % (volume fraction) ethanol. Dilute to 250 ml with the ethanol. Mix amounts of one part of
the methyl red solution with five parts of the bromocresol green solution or combine and mix all of both solutions.
5.6 Boric acid solution, c(H BO ) = 40,0 g/l.
3 3
Dissolve 40,0 g of boric acid in 1 litre of hot water in a 1 000 ml one-mark volumetric flask. Allow the flask and its
contents to cool to 20 °C. Dilute to the mark with water, add 3 ml of the indicator solution (5.5) and mix. Store the
solution, which will be light orange in colour, in a borosilicate glass bottle. Protect the solution from light and
sources of ammonia fumes during storage.
If using the electronic pH endpoint titration, the addition of the indicator solution to the boric acid solution may be
omitted. On the other hand, the change in colour may also be used as a check of proper titration procedures.
5.7 Hydrochloric acid standard volumetric solution, c(HCI) = (0,1 ± 0,000 5) mol/l.
It is recommended that this material be purchased prestandardized by the manufacturer to meet or exceed the
above specification.
NOTE Often systematic errors (which can be avoided) introduced by an analyst diluting a concentrated stock acid and then
determining the molarity of the acid, can reduce the reproducibility of the method. The analyst should not use a solution for
titration that has a higher concentration than 0,1 mol/l, because this will reduce the total titration volume per sample and the
uncertainty in readability of the burette will become a larger percentage of the value. This will have a negative impact on the
repeatability and reproducibility of the method. The same issues and additional sources of error arise when another acid (e.g.
sulfuric acid) is substituted for hydrochloric acid. Thus, these substitutions are not recommended.
5.8 Ammonium sulfate [(NH ) SO ], minimum assay 99,9 % (mass fraction) on dried material.
4 2 4
Immediately before use, dry the ammonium sulfate at 102 °C ± 2 °C for not less than 2 h. Cool to room temperature
in a desiccator.
5.9 Tryptophan (C H N O ) or lysine hydrochloride (C H ClN O ), minimum assay 99 % (mass fraction).
11 12 2 2 6 15 2 2
Do not dry these reagents in an oven before use.
5.10 Sucrose, with a nitrogen content of not more than 0,002 % (mass fraction).
Do not dry the sucrose in an oven before use.
2 © ISO and IDF 2001 – All rights reserved

ISO 8968-1:2001(E)
IDF 20-1:2001(E)
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 Water bath, capable of being maintained at 38 °C ± 2 °C
6.2 Kjeldahl flasks, of capacity 500 ml or 800 ml.
6.3 Analytical balance, capable of weighing to the nearest 0,1 mg.
6.4 Boiling aids, e.g. glowed pumice, zinc dust, hard pieces of porcelain or high-purity amphoteric alundum (i.e.
carbarundum) granules, plain, mesh size 10.
Do not reuse the aids.
NOTE Glass beads of approximately 5 mm diameter are sometimes used, but they might not promote as efficient boiling
as the alundum granules and more foaming problems can be encountered during digestion with glass beads.
6.5 Burette or automatic pipette, capable of delivering 1,0 ml portions of the copper sulfate solution (5.2).
6.6 Graduated measuring cylinders, of capacity 50 ml, 100 ml and 500 ml.
6.7 Digestion apparatus, to hold the Kjeldahl flasks (6.2) in an inclined position (at approximately 45°), with
electric heaters or gas burners that do not heat the flasks above the level of their contents, and with a fume
extraction system.
The heater source should be adjustable to control the maximum heater setting to be used during digestion. Preheat
the heat source at the heater setting for evaluation. In the case of a gas heater, the preheated period shall be
10 min, and for an electric heater it shall be 30 min. For each of the heaters, determine the heater setting that
brings 250 ml of water including 5 to 10 boiling aids with an initial temperature of 25 °C to its boiling point in 5 min
to 6 min. This is the maximum heater setting to be used during digestion.
6.8 Distillation apparatus, made of borosilicate glass or other suitable material to which can be fitted a Kjeldahl
flask (6.2) consisting of an efficient splash-head connected to an efficient condenser with straight inner tube and an
outlet tube attached to its lower end.
The connecting tubing and stopper(s) shall be close fitting and preferably made of neoprene.
6.9 Conical flasks, of capacity 500 ml, graduated at every 200 ml.
6.10 Burette, of capacity 50 ml, graduated at least at every 0,01 ml, complying with the requirements of
ISO 385-1, class A.
Alternatively, an automatic burette may be used if it fulfils the same requirements.
6.11 Automatic titrator provided with a pH-meter
The pH-meter should be correctly calibrated in the range of pH 4 to 7 following normal laboratory pH-calibration
procedures.
7 Sampling
Sampling is not part of the method specified in this part of ISO 8968ΩIDF 20. A recommended sampling method is
given in ISO 707.
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changed during transport or storage.
ISO 8968-1:2001(E)
IDF 20-1:2001(E)
8 Preparation of test sample
Warm the test sample in the water bath (6.1) set at 38 °C. Gently mix the test sample thoroughly by repeatedly
inverting the sample bottle without causing frothing or churning. Cool the sample to room temperature immediately
prior to weighing the test portion (9.1).
NOTE For advice on sample size to apply this method to dairy products other than milk, see annex A.
9 Procedure
9.1 Test portion and pretreatment
Add to a clean and dry Kj
...