SIST-TS CEN ISO/TS 16634-2:2010

SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 16634-2:2010
01-januar-2010
äLYLOD'RORþHYDQMHVNXSQHJDGXãLNDVVHåLJRPSR'XPDVXLQL]UDþXQRPGHOHåD
VXURYLKEHOMDNRYLQGHOäLWRVWURþQLFHLQåLWQLPOHYVNLSURL]YRGL,6276

Food products - Determination of the total nitrogen content by combustion according to
the Dumas principle and calculation of the crude protein content - Part 2: Cereals, pulses
and milled cereal products (ISO/TS 16634-2:2009)
Lebensmittelerzeugnisse - Bestimmung des Gehaltes an Gesamtstickstoff mit dem
Verbrennungsverfahren nach Dumas und Berechnung des Gehaltes an Rohprotein - Teil
2: Getreide, Hülsenfrüchte und gemahlene Getreideerzeugnisse (ISO/TS 16634-2:2009)
Produits alimentaires - Détermination de la teneur en azote total par combustion selon le
principe Dumas et calcul de la teneur en protéines brutes - Partie 2: Céréales,
légumineuses et produits céréaliers de mouture (ISO/TS 16634-2:2009)
Ta slovenski standard je istoveten z: CEN ISO/TS 16634-2:2009
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.060 äLWDVWURþQLFHLQSURL]YRGLL] Cereals, pulses and derived
QMLK products
SIST-TS CEN ISO/TS 16634-2:2010 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN ISO/TS 16634-2:2010

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SIST-TS CEN ISO/TS 16634-2:2010


TECHNICAL SPECIFICATION
CEN ISO/TS 16634-2

SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION
October 2009
ICS 67.050; 67.060
English Version
Food products - Determination of the total nitrogen content by
combustion according to the Dumas principle and calculation of
the crude protein content - Part 2: Cereals, pulses and milled
cereal products (ISO/TS 16634-2:2009)
Produits alimentaires - Détermination de la teneur en azote Lebensmittelerzeugnisse - Bestimmung des Gehaltes an
total par combustion selon le principe Dumas et calcul de la
Gesamtstickstoff mit dem Verbrennungsverfahren nach
teneur en protéines brutes - Partie 2: Céréales, Dumas und Berechnung des Gehaltes an Rohprotein - Teil
légumineuses et produits céréaliers de mouture (ISO/TS 2: Getreide, Hülsenfrüchte und gemahlene
16634-2:2009) Getreideerzeugnisse (ISO/TS 16634-2:2009)
This Technical Specification (CEN/TS) was approved by CEN on 24 August 2009 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.






EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2009 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 16634-2:2009: E
worldwide for CEN national Members.

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SIST-TS CEN ISO/TS 16634-2:2010
CEN ISO/TS 16634-2:2009 (E)
Contents Page
Foreword .3
2

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SIST-TS CEN ISO/TS 16634-2:2010
CEN ISO/TS 16634-2:2009 (E)
Foreword
This document (CEN ISO/TS 16634-2:2009) has been prepared by Technical Committee CEN/TC 338
“Cereal and cereal products", the secretariat of which is held by AFNOR, in collaboration with Technical
Committee ISO/TC 34 "Food products".
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
3

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SIST-TS CEN ISO/TS 16634-2:2010

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SIST-TS CEN ISO/TS 16634-2:2010

TECHNICAL ISO/TS
SPECIFICATION 16634-2
First edition
2009-10-01

Food products — Determination of the
total nitrogen content by combustion
according to the Dumas principle and
calculation of the crude protein
content —
Part 2:
Cereals, pulses and milled cereal
products
Produits alimentaires — Détermination de la teneur en azote total par
combustion selon le principe Dumas et calcul de la teneur en protéines
brutes —
Partie 2: Céréales, légumineuses et produits céréaliers de mouture




Reference number
ISO/TS 16634-2:2009(E)
©
ISO 2009

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SIST-TS CEN ISO/TS 16634-2:2010
ISO/TS 16634-2:2009(E)
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ii © ISO 2009 – All rights reserved

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SIST-TS CEN ISO/TS 16634-2:2010
ISO/TS 16634-2:2009(E)
Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Normative references.1
3 Terms and definitions .1
4 Principle .2
5 Reagents .2
6 Apparatus.3
7 Sampling .3
8 Preparation of test sample .3
9 Procedure.4
9.1 General .4
9.2 Test portion.4
9.3 Control of oxygen supply .4
9.4 Calibration.5
9.5 Determination .5
9.6 Detection and integration .6
10 Calculation and expression of results .6
10.1 Calculation .6
10.2 Expression of results.6
11 Precision .7
11.1 Interlaboratory tests.7
11.2 Repeatability .7
11.3 Reproducibility .7
11.4 Critical difference .7
11.5 Uncertainty.8
12 Test report.8
Annex A (informative) Flowchart for a basic Dumas apparatus .9
Annex B (informative) Schematic diagrams of suitable types of Dumas apparatus .10
Annex C (informative) Equipment calibration.13
Annex D (informative) Examples of factors for converting nitrogen content to protein content.15
Annex E (informative) Results of interlaboratory tests.16
Bibliography.23

© ISO 2009 – All rights reserved iii

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SIST-TS CEN ISO/TS 16634-2:2010
ISO/TS 16634-2:2009(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 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.
In other circumstances, particularly when there is an urgent market requirement for such documents, a
technical committee may decide to publish other types of document:
⎯ an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in
an ISO working group and is accepted for publication if it is approved by more than 50 % of the members
of the parent committee casting a vote;
⎯ an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical
committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting
a vote.
An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a
further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is
confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an
International Standard or be withdrawn.
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/TS 16634-2 was prepared by the European Committee for Standardization (CEN) in collaboration with
ISO Technical Committee TC 34, Food products, in accordance with the Agreement on technical cooperation
between ISO and CEN (Vienna Agreement).
ISO 16634 consists of the following parts, under the general title Food products — Determination of the total
nitrogen content by combustion according to the Dumas principle and calculation of the crude protein content:
⎯ Part 1: Oilseeds and animal feeding stuffs
⎯ Part 2: Cereals, pulses and milled cereal products [Technical Specification]
iv © ISO 2009 – All rights reserved

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SIST-TS CEN ISO/TS 16634-2:2010
ISO/TS 16634-2:2009(E)
Introduction
For a long time, the Kjeldahl method has been the most frequently used method for the determination of the
protein content of food products. However, in recent years, the Kjeldahl method has increasingly been
replaced by the Dumas method, which is faster and does not use dangerous chemicals. Although the
principles of the two methods are different, both measure the nitrogen content of the product. Nitrogen content
can be converted into protein content by using an appropriate factor. The value of this factor varies depending
on the relative amounts of different proteins and their amino-acid composition in a given product.
Neither the Dumas nor the Kjeldahl method distinguishes between protein and non-protein nitrogen. In most
cases, results obtained by the Dumas method are slightly higher than those of the Kjeldahl method. This is
due to the fact that the Dumas method measures almost all of the non-protein nitrogen, whereas the Kjeldahl
method measures only a part of it.
Taking into consideration the fact that the protein content of a product calculated by both methods only
approximates to the true value, it is a matter of discretion which one is accepted. The most appropriate
solution should be the use of a second factor for the elimination of the systematic error caused by the non-
protein nitrogen content of the different products. However, this second factor has to be determined for each
product, like the existing factors which indicate the ratio of the protein content to the nitrogen content.
© ISO 2009 – All rights reserved v

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SIST-TS CEN ISO/TS 16634-2:2010

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SIST-TS CEN ISO/TS 16634-2:2010
TECHNICAL SPECIFICATION ISO/TS 16634-2:2009(E)

Food products — Determination of the total nitrogen content by
combustion according to the Dumas principle and calculation
of the crude protein content —
Part 2:
Cereals, pulses and milled cereal products
1 Scope
This part of ISO 16634 specifies a method for the determination of the total nitrogen content and the
calculation of the crude protein content of cereals, pulses and milled cereal products.
This method, like the Kjeldahl method (see References [1] and [6]), does not distinguish between protein
nitrogen and non-protein nitrogen. For the calculation of the protein content, various conversion factors are
used (see Annex D).
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 712, Cereals and cereal products — Determination of moisture content — Reference method
ISO 6540, Maize — Determination of moisture content (on milled grains and on whole grains)
ISO 24557, Pulses — Determination of moisture content — Air-oven method
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
nitrogen content
mass fraction of the total nitrogen determined by the procedure specified in this part of ISO 16634
NOTE The mass fraction is expressed as a percentage.
3.2
crude protein content
nitrogen content (3.1) multiplied by a factor, usually 5,7 for wheat, rye and their milled products and 6,25 for
others products falling within the scope of this part of ISO 16634
NOTE The factors for calculation of the crude protein content from the total nitrogen content are derived from the
Kjeldahl method, which is the reference method for the determination of total nitrogen content. As the method specified in
this part of ISO 16634 uses the same factors as the Kjeldahl method, the validity of these factors has to be verified due to
the slight difference in the results obtained with the Kjeldahl and Dumas methods.
© ISO 2009 – All rights reserved 1

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SIST-TS CEN ISO/TS 16634-2:2010
ISO/TS 16634-2:2009(E)
4 Principle
Samples are converted to gases by heating in a combustion tube. Interfering components are removed from
the resulting gas mixture. The nitrogen compounds in the gas mixture, or a representative part of them, are
converted to molecular nitrogen which is quantitatively determined by a thermal-conductivity detector. The
nitrogen content is then calculated by a microprocessor.
5 Reagents
Use only reagents of recognized analytical grade or reagents of equivalent purity as specified by instrument
manufacturers. Except for the reference materials (see 5.12), all reagents shall be free from nitrogen.
5.1 Carrier gas(es): use either 5.1.1 or 5.1.2.
5.1.1 Carbon dioxide, as pure as possible, but with a minimum CO volume fraction of 99,99 %.
2
5.1.2 Helium, as pure as possible, but with a minimum He volume fraction of 99,99 %.
5.2 Oxygen, as pure as possible, but with a minimum O volume fraction of 99,99 %.
2
5.3 Sulfur dioxide and halogen absorbent, to eliminate any sulfur from the sample [e.g. lead chromate
(PbCrO ) or steel wool].
4
5.4 Copper oxide/platinum catalyst, for the post-combustion tube.
Platinum catalyst [5 % of Pt on alumina (Al O )] is blended with CuO in the ratio 1 part:7 parts or
2 3
1 part:8 parts in accordance with the manufacturer's recommendations.
To prevent separation as a result of the different bulk densities of the two materials, it is recommended not to
prepare the mixture before filling the tube but to pour the platinum catalyst and copper oxide simultaneously
into the post-combustion tube using a suitable funnel.
5.5 Silver or copper wool.
This shall be disaggregated before being inserted into the post-combustion or reduction tube.
5.6 Silica (quartz) or glass wool or cotton wool, as recommended by the instrument manufacturer.
5.7 Copper or tungsten (wire, cuttings, turnings or powder), for the reduction tube.
The use of copper or tungsten in one of these forms can improve the precision of analytical results for
samples with low nitrogen contents (about 1 % mass fraction).
5.8 Diphosphorus pentoxide (P O ) or granulated magnesium perchlorate [Mg(ClO ) ], or another
2 5 4 2
suitable drying agent, to fill the drying tubes.
5.9 Hollow corundum spheres or aluminium oxide pellets, for the combustion tube.
5.10 Copper oxide (CuO), as filling material for the combustion tube.
5.11 Sodium hydroxide (NaOH), on a support material.
5.12 Aspartic acid (C H NO ) or ethylenediaminetetraacetic acid (C H N O ) or glutamic acid
4 7 4 10 16 2 8
(C H NO ) or hippuric acid (C H NO ) standard, or other suitable reference materials with a known,
5 9 4 9 9 3
constant, certified nitrogen content.
The minimum recovery should preferably be 99 % mass fraction.
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ISO/TS 16634-2:2009(E)
5.13 Light petroleum, with a boiling range between 30 °C and 60 °C, or acetone or ethanol.
6 Apparatus
Usual laboratory equipment and, in particular, the following:
6.1 Analytical balance, capable of weighing to the nearest 0,000 1 g.
6.2 Grinding device, appropriate to the nature of the sample.
6.3 Sieve, of nominal opening size 800 µm or 1 mm, made of non-ferrous material.
6.4 Crucibles (e.g. made of stainless steel, quartz, ceramic material or platinum) or tin capsules or
nitrogen-free filter paper, suitable for the Dumas apparatus used.
NOTE 1 Several instruments provided with an automatic sampler are commercially available.
NOTE 2 Some solid samples (e.g. powders) can be pressed to form pellets.
1)
6.5 Dumas apparatus , fitted with a furnace able to maintain a given temperature greater than or equal to
850 °C, with a thermal-conductivity detector and suitable device for signal integration.
Suitable Dumas apparatus operates according to the general flowchart given in Annex A, although different
arrangements and components may be used.
NOTE Schematic diagrams of three commercially available instruments are shown as examples in Figures B.1, B.2
and B.3.
To avoid leaks, the sealing O-rings shall be slightly lubricated with high-vacuum grease prior to installation.
Experience has shown that it is important to clean all pieces of silicaware and glassware carefully and to
remove fingerprints from tubes, using a suitable solvent (e.g. acetone), before inserting them in the furnace.
7 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 16634. Recommended sampling methods are
[7]
given in ISO 24333 for cereals and cereal products.
8 Preparation of test sample
The test sample shall be prepared from the laboratory sample in such a way that a homogeneous test sample
is obtained.
Using a suitable grinding device (6.2), grind the laboratory sample. Generally, pass the ground material
through a sieve (6.3) of nominal opening size 800 µm for small sample sizes (under 300 mg) or a sieve of
nominal opening size 1 mm for larger sample sizes (300 mg or more). Mills that produce particle sizes
meeting the specifications given in Table 1 will give acceptable results.

1) Elementar Analysensysteme, Sumika Chemical Analysis Service and LECO Instruments produce suitable equipment
available commercially. This information is given for the convenience of users of this Technical Specification and does not
constitute an endorsement by ISO of this equipment. Equivalent products may be used if they can be shown to lead to the
same results.
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SIST-TS CEN ISO/TS 16634-2:2010
ISO/TS 16634-2:2009(E)
Table 1 — Required particle size
Nominal size of sieve openings Amount passing through sieve
µm % mass fraction
710 100
500 95 to 100
200 85 or less

Grinding may result in moisture loss and therefore the moisture content of the ground sample should
preferably also be determined when reporting nitrogen or protein contents on a dry-matter or constant-
moisture basis. Determination of the moisture content shall be carried out in accordance with ISO 712 for
cereals other than maize, ISO 6540 for maize and ISO 24557 for pulses.
The grinder efficiency can be checked by replicate preparation of ground samples of a 2+1 mixture of maize
and soya seeds. The expected coefficient of variation should be less than 2 % mass fraction.
9 Procedure
9.1 General
Carefully follow the manufacturer's instructions for instrument set-up, optimization, calibration and operation.
Switch the instrument on and allow it to stabilize as defined in local procedures.
An instrument performance test should be carried out daily, using the reference material (5.12). The recovery
of nitrogen should be > 99,0 % mass fraction.
9.2 Test portion
Weigh, to the nearest 0,000 1 g, at least 0,1 g of the test sample into a crucible or tin capsule or nitrogen-free
filter paper (6.4). For samples low in protein (< 1 % mass fraction), the amount of the test portion can be
increased up to 3,5 g, depending on the type of Dumas equipment being used and on the nature of the
sample.
Depending on the type of equipment used, if the sample contains over 17 % mass fraction of moisture, it may
be necessary to dry it before analysis.
Lower masses may be necessary for very high protein content samples or when only very small amounts of
sample are available. In the case of masses less than 0,1 g, a second (validation) determination shall be
performed.
9.3 Control of oxygen supply
Control the oxygen supply, in particular the flow, in accordance with the instructions of the material supplier.
With each series of nitrogen content determinations, conduct as many blank runs as necessary to stabilize the
equipment, using for each run an equivalent mass of sucrose in place of the test portion. The sucrose blank
provides the amount of nitrogen that is introduced in the form of atmospheric air trapped within a powdered
organic material. Use the mean value of the blank determinations as an error correction in the calculation of
the nitrogen content of each test sample.
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SIST-TS CEN ISO/TS 16634-2:2010
ISO/TS 16634-2:2009(E)
9.4 Calibration
For long-term instrument calibration, use pure compounds with a known, constant nitrogen content, e.g.
aspartic acid (see 5.12), as standards. Analyse in duplicate three pure compounds, each in three different
amounts chosen as a function of the measurement range for the actual samples.
To prepare a calibration curve, carry out at least five determinations with different amounts of the same
compound, choosing the compound and the amounts used in such a way that the curve obtained will cover
the range of nitrogen contents in the samples to be analysed.
If the test portion contains more than 200 mg of nitrogen, the calibration curve is likely to be non-linear. In the
non-linear section, short segments can nevertheless be used for calibration purposes. To ensure the reliability
of the curve in these segments, the amount of standard used shall be increased in steps corresponding to
1 mg to 5 mg of nitrogen over the segments.
Calibration can also be performed using standard aqueous solutions.
Check the calibration at least three times at the beginning of a series of analyses and then after every 15 to 25
samples, analysing either one of the standards (see 5.12) or a sample of known value. The value obtained for
the nitrogen mass fraction shall differ by less than 0,05 % from the expected value. If it does not, repeat the
calibration check after checking instrument performance.
9.5 Determination
With the instrument operating in the stable s
...