EN ISO 6498:2012

SLOVENSKI STANDARD
01-oktober-2012
Krma - Smernice za pripravo vzorca (ISO 6498:2012)
Animal feeding stuffs - Guidelines for sample preparation (ISO 6498:2012)
Futtermittel - Leitfaden für die Probenvorbereitung (ISO 6498:2012)
Aliments des animaux - Lignes directrices pour la préparation d'échantillons (ISO
6498:2012)
Ta slovenski standard je istoveten z: EN ISO 6498:2012
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 6498
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2012
ICS 65.120
English Version
Animal feeding stuffs - Guidelines for sample preparation (ISO
6498:2012)
Aliments des animaux - Lignes directrices pour la Futtermittel - Leitfaden für die Probenvorbereitung (ISO
préparation des échantillons (ISO 6498:2012) 6498:2012)
This European Standard was approved by CEN on 31 May 2012.

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 CEN-CENELEC 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 CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, 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, Turkey and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 6498:2012: E
worldwide for CEN national Members.

Contents Page
Foreword .3
Foreword
This document (EN ISO 6498:2012) has been prepared by Technical Committee CEN/TC 327 “Animal feeding
stuffs - Methods of sampling and analysis", the secretariat of which is held by NEN, in collaboration with
Technical Committee ISO/TC 34 "Food products".
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 December 2012, and conflicting national standards shall be withdrawn
at the latest by December 2012.
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 organisations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, 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, Turkey and the United Kingdom.
INTERNATIONAL ISO
STANDARD 6498
Third edition
2012-06-01
Corrected version
2012-07-15
Animal feeding stuffs — Guidelines for
sample preparation
Aliments des animaux — Lignes directrices pour la préparation des
échantillons
Reference number
ISO 6498:2012(E)
©
ISO 2012
ISO 6498:2012(E)
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
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Tel. + 41 22 749 01 11
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Published in Switzerland
ii © ISO 2012 – All rights reserved

ISO 6498:2012(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Terms and definitions . 1
2.1 Definitions concerning “sample” . 1
2.2 Definitions concerning “parameters” . 2
2.3 Examples of animal feeding stuffs characteristics . 3
2.4 Definitions concerning “sample preparation procedure” . 5
3 Principle . 6
4 Consideration of sample preparation errors . 7
4.1 Subsampling and other errors . 7
4.2 Minimum mass . 8
4.3 Errors associated with division techniques . 9
5 Safety precautions .10
6 Apparatus .10
7 Procedure .12
7.1 General .12
7.2 Sample check .12
7.3 Mass reduction .14
7.4 Particle size reduction .17
7.5 Partial drying .20
7.6 Coarse grinding .22
7.7 Special sample preparation procedures .22
7.8 Storage .22
8 Performance tests (quality control) .22
8.1 General .22
8.2 Performance test for mass reduction (division) .23
8.3 Performance test for particle size reduction (grinding) .24
8.4 Performance test for mixing .25
9 Categories of feeds — Special remarks and flow charts .25
9.1 General .25
9.2 Birdseed .26
9.3 Whole cottonseed .27
9.4 Mineral mix .29
9.5 Dry feeds .29
9.6 Forages including silage, hay, haylage, TMR and byproducts .30
9.7 Oilseeds and high-fat feeds .32
9.8 Large block and molasses block feeds .33
9.9 Liquid feeds .35
9.10 Canned pet food .35
9.11 Semi-moist pet food and dog chews.36
9.12 Premixtures .37
9.13 Range and alfalfa hay pellets .38
9.14 Texturized and sticky feed .39
9.15 Aquatic feeds .40
Annex A (informative) Calculations, examples and tables for minimum mass .42
Bibliography .46
ISO 6498:2012(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.
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 6498 was prepared by the European Committee for Standardization (CEN) Technical Committee TC 327,
Animal feeding stuffs — Methods of sampling and analysis, in collaboration with ISO Technical Committee
TC 34, Food products, Subcommittee SC 10, Animal feeding stuffs, in accordance with the Agreement on
technical cooperation between ISO and CEN (Vienna Agreement).
This third edition cancels and replaces the second edition (ISO 6498:1998), which has been technically revised.
This corrected version of ISO 6498:2012 incorporates the following correction: in 7.1, paragraph 4, the phrase
“particle sizes below 4 mm ± 2 mm (4 mm to 6 mm) can be” has been substituted by “particle sizes of 4 mm to
6 mm can be”.
iv © ISO 2012 – All rights reserved

INTERNATIONAL STANDARD ISO 6498:2012(E)
Animal feeding stuffs — Guidelines for sample preparation
1 Scope
This International Standard specifies guidelines for the preparation of test samples from laboratory samples of
animal feeding stuffs, including pet foods.
NOTE 1 The guidelines mostly derive from those developed by AAFCO (see Reference [7]).
The guidelines are overruled by special instructions and regulations for sample preparation demanded by
specific analysis methods.
NOTE 2 Such analysis methods are developed by ISO and CEN.
NOTE 3 This International Standard does not include special guidelines for sample preparation for microbiological
analysis of microorganisms like yeasts, bacteria and moulds. Nonetheless, for microorganisms which are used as feed
additives (probiotics), some important aspects of sample preparation are addressed.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1 Definitions concerning “sample”
2.1.1
lot
quantity of material that is assumed to be of the same production process and represented by specified
sampling rules
NOTE For the purposes of this International Standard, the rules are those of Commission Regulation (EC)
[3]
No. 152/2009.
2.1.2
laboratory sample
sample as prepared (from the lot) for sending to the laboratory and intended for inspection or testing
2.1.3
test sample
subsample or sample prepared from the laboratory sample and from which test portions will be taken
2.1.4
test portion
quantity of material drawn from the test sample (or from the laboratory sample if both are the same)
2.1.5
reserve sample
material left over from the laboratory sample when divided or subsampled test samples have been taken and
on which no further particle size reduction is done
NOTE If, for example, mycotoxin or genetically modified organism analyses are done on the whole laboratory sample,
then the reserve sample is also reduced to the corresponding particle sizes. The reserve sample should be stored under
conditions maintaining integrity.
ISO 6498:2012(E)
2.2 Definitions concerning “parameters”
2.2.1
parameter
analyte or constituent or microorganism for which the feeding stuff is to be analysed by microscopic,
microbiological, biological or chemical procedures
2.2.1.1
stable parameter
analyte or constituent or microorganism which does not degrade during sample preparation on common
handling or storage at room temperatures of 20 °C to 25 °C
2.2.1.2
unstable parameter
analyte or constituent or microorganism which degrades during sample preparation on common handling
or storage at room temperatures of 20 °C to 25 °C because they are volatile, degradable, or sensitive to
temperature, light, enzymatic degradation or chemical oxidation
NOTE Stability of parameters in this context refers only to the influence of sample preparation, such as intensive
grinding, and not to a minimum shelf-life specified by producers or on the label, e.g. for a feed (additive).
Table 1 — Classification (in general) of stable or unstable parameters
and reasons for degradation with a view to sample preparation
Reason(s) for
Origin Stable parameters Unstable parameters
degradation/change
(Crude) protein, fat, ash, fibre Moisture Temperature (volatile)
Starch, sugar, lactose Ammonia Temperature (volatile)
Gas production and enzyme- Organic acids (e.g. lactic
soluble organic substance acid, acetic acid, butyric acid, Temperature (volatile)
Nutrients
production in in vitro tests fumaric acid, formic acid)
Air oxidation (can result in
Minerals
Unsaturated fatty acids production of short-chain fatty
(e.g. Ca, P, Mg, Na, K, Cl)
acids)
Trace elements Vitamins Temperature, ultraviolet (UV)
(e.g. Cu, Zn, Mn, Fe, Se, Co) (e.g. vitamin A, C, D, E) light, air oxidation (sensitive)
Amino acids (e.g. lysine, 1,2-Propanediol, ethylene
Temperature (volatile)
methionine, tryptophan) glycol
Feed additives
Temperature (freezing),
Microorganisms like probiotics
Enzymes (e.g. phytases, pressure (sensitive to
(e.g. Saccharomyces
non-starch polysaccharide grinding); moisture/dryness
cerevisiae, Enterococcus
enyzmes) (influences growth of
faecium)
microorganisms)
Mycotoxins (e.g. aflatoxin B ,
Mould growth and change
deoxynivalenol, fumonisins,
Heavy metals of mycotoxins possible at
ochratoxin A, T-2 toxin, HT-2
(e.g. As, Pb, Cd, Hg) room temperature; UV light
toxin, zearalenone, ergot
(sensitive – aflatoxin B )
Undesirable
alkaloids)
substances
Dioxins and polychlorinated
biphenyls (PCBs) with similar Drugs, antibiotics, pesticides Temperature (sensitive)
effects to dioxins
Hydrocyanic acid Temperature (volatile)
Banned Banned drugs, banned
Proteins of animal origin Temperature (sensitive)
substances antibiotics
Temperature (sensitive),
(Other)
Yeasts, bacteria, moulds dryness, influx of oxygen
Microorganisms
(anaerobiosis)
2 © ISO 2012 – All rights reserved

ISO 6498:2012(E)
2.3 Examples of animal feeding stuffs characteristics
Some examples of animal feeding stuffs characteristics are given here to assist with the identification and
grouping of a laboratory sample based on the terms and annexes used in these guidelines.
NOTE Definitions of animal feeding stuffs are given in legislation worldwide. Sample definitions from European
directives and, for straight feeds, in an alphabetical list from a German committee are given in References [4][5][6][8].
2.3.1
birdseed
seeds that are intended to feed birds
EXAMPLES Grains and oilseeds.
2.3.2
whole cottonseed
unprocessed cottonseed product, including the hulls, lint, and meat
2.3.3
mineral mix
supplementary feed that mainly consists of mineral ingredients in either granular, bead or small pellet form and
which is free flowing as an entire mix
NOTE Mineral pellets are an agglomerated mineral mix formed by a mechanical process (in general).
2.3.4
dry feeds
feed ingredient or complete animal feed which typically contains a moisture mass fraction of not more than 15 %
NOTE Dry feed pellets are an agglomerated dry feed produced by a mechanical process (in general).
2.3.5
green fodder
edible parts of plants, other than separated grain, that can provide feed for grazing animals or that can be
harvested for feeding, including browse, herbage, and mast
NOTE Generally, the term refers to more digestible material in contrast to less-digestible plant material, known as roughage.
2.3.6
silage
forage preserved in a succulent condition by organic acids produced by anaerobic fermentation of sugars
in the forage
2.3.7
roughage
fibrous, coarsely textured parts of plants
EXAMPLES Stovers, straws, hulls, cobs, and stalks.
2.3.8
hay
aerial portion of grass especially cut and dried for animal feeding
2.3.9
haylage
forage preserved in a succulent condition by organic acids produced by anaerobic fermentation of sugars in
the forage with a moisture mass fraction of about 45 %
ISO 6498:2012(E)
2.3.10
total mixed ration
TMR
single mixture of all feed ingredients (forages, grains, and supplements) that is supplied to an animal for a 24 h period
NOTE In practice, the 24 h allotment of the mixture may be offered in one or more feedings.
2.3.11
byproduct
product which remains after processes for the production of ingredients from plant material
EXAMPLE Dried distillers grains with solubles (DDGSs) from fermentation.
2.3.12
oilseed
any seed from which oil is extracted
EXAMPLE Sunflower seeds.
2.3.13
large block feed
molasses block feed
agglomerated feed compressed into a solid mass that is cohesive enough to hold its form
NOTE Large block feed weighs over 1 kg, generally about 20 kg. It may be marketed as a mineral block or a
“caramelized” molasses drum, containing various minerals and nutrients. Samples may be received by the laboratory as
large chunks, cores or “sticky clumps”.
2.3.14
liquid feed
feed product not solid and not aeriform
NOTE A liquid feed contains sufficient moisture to flow readily and may contain molasses.
2.3.15
canned pet food
feed product for pets which has been processed, packaged, sealed and sterilized for preservation in cans or
similar containers
2.3.16
semi-moist feed
meat-based feed product for pets or aquatic animals that has been partially dried to prevent microbial
decomposition
NOTE The moisture mass fraction may range from 15 % to 40 %. The product is generally in the form of strips or
cubes and is designed to be stored at room temperature.
2.3.17
dog chew
rawhide bone
meat and skin or peel strip that has been nearly completely dried to a leather-like consistency
2.3.18
premixture
mixture of one or more micro-ingredients with diluent or carrier
NOTE Premixtures are used to facilitate uniform dispersion of the micro-ingredients (e.g. vitamins, probiotics, drugs
or antibiotics) into a final feed.
4 © ISO 2012 – All rights reserved

ISO 6498:2012(E)
2.3.19
range and alfalfa hay pellet
agglomerated feed formed by compacting and forcing the mix through, for example, square openings by a
mechanical process
NOTE The pellets are mostly about 2 cm in diameter and 5 cm in length (volume about 16 cm ) and may contain
molasses; this definition also applies to alfalfa cubes (chopped alfalfa hay) of larger dimensions.
2.3.20
texturized feed
sticky feed
mix of assorted grains and commercial feed (generally pelleted), all of which has been treated with a coating
of, for example, molasses
NOTE Some of the grains may have been steam heated or rolled prior to incorporation into the texturized feed.
2.3.21
aquatic feed
feed which is fed to aquatic animals and which has been mechanically processed into encapsulated pellets,
flakes, crumble, and as packaged sealed powder
2.4 Definitions concerning “sample preparation procedure”
2.4.1
homogeneity
degree to which a property or a constituent is uniformly distributed throughout a quantity of material
NOTE Homogeneity may be considered to have been achieved in a practical sense when the sampling error of the
processed portion is negligible compared to the total error of the measurement system. Since homogeneity depends
on the size of the units under consideration, a mixture of two materials may be inhomogeneous at the molecular or
atomic level, but sufficiently homogeneous at the particulate level. However, uniform visual appearance does not ensure
compositional homogeneity.
2.4.2
partial drying
part of the sample preparation procedure for feedstuff samples with a high moisture content (dry mass fraction
<85 %), in which the sample is carefully dried to allow subsequent sample preparation procedures to be
applied, such as particle size reduction by grinding with a mill
NOTE 1 The partial drying procedure depends on the feeding stuff [e.g. at temperatures below 55 °C to 60 °C for
silages], and on the heat stability of the parameters (e.g. 70 °C ± 10 °C for drugs and antibiotics).
NOTE 2 Samples for microbiological analysis should not be dried (at temperatures above 40 °C).
NOTE 3 Partial drying can also be achieved by a freeze-drying procedure, which is a careful drying process using a
vacuum to allow moisture to evaporate.
2.4.3
coarse grinding
first grinding step of the whole sample when the laboratory sample contains large lumps or when its particle
size is above about 6 mm before mass reduction
NOTE Coarse grinding is a special kind of particle size reduction that ensures homogeneity of the laboratory sample
for subsampling purposes.
2.4.4
mass reduction
part of the sample preparation procedure to reduce the mass of a laboratory sample by dividing or
subsampling it using (stationary or rotary) dividers or fractional (alternate) shovelling, without changing the
consistency of the sample
NOTE After mass reduction, all subsamples should have the same properties as the original laboratory sample.
ISO 6498:2012(E)
2.4.5
particle size reduction
part of the sample preparation procedure achieved by chopping, crushing, cutting, blending (homogenizing),
macerating, milling (grinding), pressing, pulverizing to obtain a homogeneous test sample for further analysis
NOTE In general, particle size reduction follows the mass reduction step of the sample preparation procedure with
different sieve size options to ensure integrity of the test sample(s).
sampling: choice from lot
sample preparation:
laboratory sample(s) (≥500 g) with:
dry matter < 15 % mass fraction
lumps or big particles (> 6 mm)
dry matter ≥85% mass fraction
partial (or freeze) drying (optionally) coarse grinding
mass reduction (subsampling/splitting) reserve sample
test sample(s) (100 g) for:
particle size reduction
– microscopy
with different sieve-size options
– analysis of stable parameters
(1,0 mm, 0,5 mm, <0,5 mm, no
– analysis of unstable parameters
grinding)
analysis: test portion(s) (0,05 g to 25 g)
Figure 1 — Illustration of definitions concerning “sample”, “substances”
and “sample preparation procedure”
3 Principle
All sample preparation steps depend on the different properties of the feedstuffs and on the parameters to
be analysed. In each case, any special instructions concerning sample preparation in the analysis methods
require consideration.
The guidelines describe the procedure for preparing — from a sample arriving at a laboratory (in general with
a minimum mass of 0,5 kg) — a homogeneous test sample (having a minimum mass of 100 g) with the same
constitution and composition and free from contamination.
6 © ISO 2012 – All rights reserved

ISO 6498:2012(E)
In some cases, the laboratory sample size can be less than 500 g (i.e. in standards for feed additives), but it
is necessary to follow statutory regulations and, in every case, the sample size should be large enough to be
representative.
In general, the whole laboratory sample is reduced in mass and in particle size to obtain one or more test
samples for the analysis of stable and unstable parameters, for microscopy analysis and for reserve. If the
analysis protocol and the intended proceeding of the reserve sample permit it, the laboratory sample should
preferably be pre-ground completely to an adequate coarse particle size before being reduced further, in order
to ensure homogeneity of the test samples.
From a test portion (0,05 g to 25 g and above) prepared for weighing in the feedstuff analysis, representative
results should be achieved on the laboratory sample and finally on the whole lot from which the sample was drawn.
Consequently, all steps for sample preparation should be performed quickly, under convenient and very clean
conditions, so that there can be no degradation of sensitive analytes, no contamination and no oxidation due
to the influence of excessive temperature, daylight, air or residues on the apparatus used or from the samples
prepared previously or simultaneously. In particular, contamination from sample to sample should be prevented.
A loss or a change of moisture mass fraction (“content”) during sample preparation should be avoided. In
any case, it is necessary to take into account that, in order to be suitable for official control, results require
correction (to origin moisture content, dry mass fraction 88 % or 100 %).
For feedstuffs with a higher moisture content (dry matter <85 % mass fraction), partial drying or freeze-drying
before mass reduction can be necessary.
For feedstuffs with lumps or particle sizes >6 mm, coarser grinding of the whole laboratory sample to a particle
size of <6 mm before mass reduction or subsampling is absolutely necessary.
The samples have to be stored at every stage of the sample preparation under adequate conditions (e.g. at
room temperature, refrigerated, frozen, in an airtight container, protected from light or in the dark) to maintain
their integrity.
For microbiological analyses, all sample preparation steps need to be done under aseptic conditions. Laboratory
samples should be neither frozen nor heated (>40 °C), nor subjected to vacuum or oxygen levels higher than
those present in atmospheric air.
4 Consideration of sample preparation errors
Sample preparation steps have been shown to be some of the largest sources of laboratory error, a fact
which is generally overlooked. This type of error can prove much larger than that arising from subsequent
analytical procedures.
4.1 Subsampling and other errors
4.1.1 General
Errors deriving from sample heterogeneity may add to the total subsampling error (TSE) on two levels
(Reference [12]).
4.1.2 Constitutional heterogeneity
On a first level, constitutional heterogeneity is a measure of the fact that not all the particles of the laboratory
sample have the same composition (shape, size, density, etc.). If a large overall difference between the individual
fragments exists, the constitutional heterogeneity is large, but if the fragments are more homogeneous,
constitutional heterogeneity is lower. The total contribution to heterogeneity is never zero, however, as that
would imply that all fragments are strictly identical. Mixing and blending does not change constitutional
heterogeneity. The only ways to alter the constitutional heterogeneity of any given material are by comminution
(crushing or cutting) or other methods which alter the physical properties of a sample. The reduction of the
average grain size is the dominant factor in reducing constitutional heterogeneity by such means.
ISO 6498:2012(E)
Therefore, an initial coarse grinding (pre-grinding) of the whole laboratory sample is necessary before
subsampling or division to reduce constitutional heterogeneity.
This fundamental subsampling error (FSE) can be controlled by selecting the test sample mass (see 4.2)
appropriately. Therefore, collect enough mass to ensure that particles of all different compositions are contained
in the subsample or division. The larger the particle size of a material, the larger the subsample mass has to
be to minimize error.
4.1.3 Distributional heterogeneity
On a second level, distributional heterogeneity is a measure of the non-random distribution of particles in
the sample, as a result mainly of the action of gravitational force on particles of different densities, sizes and
shapes, which leads to a grouping and segregation of all particles. Particles with large differences in size or
density tend to segregate or stratify heavily, with the smallest or densest particles sinking to the bottom of the
sample. For the sake of illustration, imagine a laboratory sample consisting of black and white spheres and with
significantly different grain size distributions. If all the black spheres are found at the bottom of the sample and
the white spheres are more to the top, the system displays a very high distributional heterogeneity. If, on the
other hand, the spheres were well mixed (homogenized), the distributional heterogeneity of the system would
be significantly reduced.
To reduce this grouping and segregation error (GSE), mix or blend the sample before subsampling and collect
many increments at random from the laboratory sample (see 4.3).
Mixing is not adequate for many materials. For some materials and circumstances, mixing may actually
increase segregation instead of reducing the grouping and segregation error. As long as gravity exists, there
will be segregation. Many materials always display an innate propensity for segregation, even immediately after
mixing suspensions, e.g. highly density-fractionated materials. Such systems require constant monitoring and
treatment but, once this feature has been duly recognized, it can always be dealt with satisfactory.
Incrementing (i.e. the collection of many random increments from the laboratory sample to make up the
subsample or division) always works by reducing error from distributional heterogeneity and takes less time
and equipment to implement. Thirty increments are generally adequate. More increments are required for very
heterogeneous materials and, if little segregation is known to exist, fewer increments can be used, but in no
case can fewer than 10 be recommended.
4.1.4 Other errors
Other errors that arise from sample preparation include the loss or gain in analyte content arising from such
mechanisms as grinding, excessive heat, loss of fines, contamination, and electrostatic separation. These
errors can be large and are usually a result of carelessness or lack of knowledge.
4.2 Minimum mass
To be properly representative of a laboratory sample, the subsample or division shall have adequate mass with
a view to fundamental subsampling error (FSE) and maximum particle size (“minimum mass”) (see Table 2).
The mass required depends on the acceptable error in the subsample or division, on the density, heterogeneity,
and content of the analyte particles in the matrix, and on the largest particle size (see calculations in Annex A,
Examples 1 to 3 and Tables A.1 to A.3).
8 © ISO 2012 – All rights reserved

ISO 6498:2012(E)
Table 2 — Minimum mass: expected coefficient of variation (CV)
from laboratory subsampling; assumed density, 1 g/cm
FSE (expected CV)
%
Maximum particle size
mm
15 10 5 2 1
d
Minimum mass
g
0,5 0,06 0,13 0,5 3 12,5
0,75 0,2 0,4 2 10,5 42
1 0,4 1 4 25 100
2 4 8 32 200 400
5 56 125 500 3 130 12 500
NOTE For materials with densities other than 1 g/cm , the entries can be multiplied by the density of the material of interest;
for example, the subsampling of a material with a largest particle size of 2 mm, a tolerable subsampling CV of 5 % and a density of
0,5 g/cm would require 16 g.
4.3 Errors associated with division techniques
The data in Table 3 demonstrate the error associated with various division techniques for a model mixture of
sand particles. Figure 2 demonstrates the representativity (i.e sum of the sampling error related to precision
and accuracy) of 17 different mass reduction devices for a model mixture containing mass fractions of 89,9 %
wheat, 10,0 % rapeseed, and 0,10 % glass (see References [11][12]). The primary difference in the mass
reduction methods is the number of increments selected. For this to be true, structurally correct use of the
mass reduction devices is required (e.g. equal probability for the selection of all particles, no loss of particles,
centre of gravity rule obeyed, parallel cuts) which is difficult or impossible to obtain with shovelling and grab
sampling methods. Therefore mass reduction methods based on grab sampling or shovelling methods can
have substantial problems with precision and accuracy on trace components present as separate particles,
which may be due to selective loss or poor sampling of smaller particles (see References [11][12]). It can be
concluded from Table 6 and Figure 2 that more increments lead to improvements for the mass reduction in the
laboratory by reducing the sampling error. In general, a rotational divider reaches several hundred increments,
a stationary riffle divider about 10 to 34 increments, and coning and quartering only two increments. Therefore,
coning and quartering are not recommended in the critical mass reduction step in the laboratory, i.e. the mass
reduction step with the largest contribution to the total error. The preparation of the final test portion, where
the ratio between the mass of the laboratory sample and the mass of the final test portion is 100 to 10 000,
can usually be considered the critical step of the mass reduction of the laboratory sample. Grab sampling is to
be totally avoided for the critical mass reduction step unless it has been established that the sampling error is
insignificant compared to the total analytical error.
Table 3 — Test results from division of a mixture containing mass fractions
[1]
of 60 % coarse sand with 40 % fine sand, P = 0,6 (ISO 664 )
Standard deviation Estimated maximum
Variance
of samples sample error
%
Number of
a
% %
Method
increments
s
s
r
r
Coning and quartering 2 6,81 46,4 22,7
Stationary riffling 10 to 12 1,01 1,02 3,4
Rotary riffling >100 0,125 0,016 0,42
Random variation 0,076 0,005 8 0,25
a
Stationary rifflers with a higher number of increments and less subsampling error are available (see Reference [11]).
ISO 6498:2012(E)
NOTE Representativity should be as low as possible. Higher sums thus mean lower reliability. RK n indicates a riffle
divider with n chutes (see Reference [11]).
Figure 2 — Pooled representativity, r , equal to the square of the bias plus the square of the
precision, for a model mixture of wheat, rapeseed, and glass
5 Safety precautions
The mills for crushing, cutting and grinding have sharp moving blades. Never put hands or fingers past the
edges of the introduction chamber. Never open the mills until they have completely stopped. Check to see that
safety interlocks on all equipment are operating properly.
Wear appropriate personnel protective equipment as required in the laboratory. Safety is of great importance
during the sample preparation phase of the analysis.
Operate the dust ventilation system during dust generation procedures. To minimize dust, use a vacuum
cleaner to clean the hood area, mills, and work area.
Check that all electrical equipment is properly earthed and maintained. Do not place metal items or aluminium
foil in the microwave oven when using it for drying samples.
6 Apparatus
Usual laboratory equipment and in particular the following. All equipment used should be appropriate to the risk
of contamination and oxidation during sample preparation.
6.1 Equipment for sample preparation in general.
6.1.1 Brushes for cleaning grinders, etc.
10 © ISO 2012 – All rights reserved

ISO 6498:2012(E)
6.1.2 Compressed air blower for cleaning.
6.1.3 Vacuum cleaner.
6.1.4 Systems for microbe reduction of mills, equipment for disinfection and flame treatment for
microbiological analysis.
6.2 Drying systems.
6.2.1 Lyophilization system, forced-air drying oven capable of being maintained at 55 °C ± 5 °C or
microwave oven, household type, or vacuum oven.
6.2.2 Moisture dish (pan) made of plastics, aluminium or glass, e.g. with ≥50 mm diameter, ≤40 mm deep.
6.3 Equipment for mass and particle size reduction of “wet” feeds (e.g. forages, silages).
6.3.1 Garden pruning clippers for cutting forages or a paper cutter for small sample volumes or a laboratory
forage chopper for large volumes and a ceramic cutter, especially when trace elements are of interest.
6.3.2 Cutting mill with 6 mm and 1 mm screens.
6.3.3 Shearing-type mill with forage head and 1 mm screen.
6.3.4 Riffle sample divider, the minimum chute width shall be at least 2d + 5 mm, where d is the diameter of
the largest particle.
6.3.5 Sterile cutter or disinfected mill when microbiological analysis (e.g. of probiotics) is of interest.
6.4 Equipment for mass and particle size reduction of “dry” feeds (e.g. cereals, mineral mixtures,
pelleted feeding stuffs).
6.4.1 Riffle divider.
6.4.2 Rotary divider with vibratory feeder.
6.4.3 Shearing grinding mill equipped with 1,0 mm, 0,5 mm and <0,5 mm sieves.
6.4.4 Cutting mill with 4 mm to 6 mm screens.
6.4.5 Shearing blending mill (e.g. household coffee mill).
6.5 Equipment for the storage of samples.
6.5.1 Sterile bottles with airtight lids (e.g. brown glass bottles for unstable parameters like vitamins) and
especially for microbiological purposes.
6.5.2 Wide-mouth bottles with screw cap, plastic.
6.5.3 Plastic bags with low microbe content, with an airtight closure or for setting to vacuum for
microbiological purposes.
6.5.4 Refrigerator.
ISO 6498:2012(E)
6.5.5 Freezer.
7 Procedure
7.1 General
After registration and a check, including temperature, of a laboratory sample (see 7.2), the homogenization
procedure consists of a mass reduction step (see 7.3).
In the second step, the particles in the test samples are reduced to adequate sizes to minimize the subsampling
error that arises when the test portion is taken from the test sample. Particle size reduction should be performed
without deteriorating the integrity of the substance to be analysed (see 7.4).
For feedstuffs with higher moisture content (dry mass fraction <85 %), partial drying below 55 °C to 60 °C can
be necessary before grinding a subsample in a mill, to a particle size of 1,0 mm, in order to analyse its stable
analytes (see 7.5).
For feedstuffs containing lumps or consisting of particle sizes >6 mm, grinding or chopping to particle sizes of
4 mm to 6 mm can be necessary before subsampling is possible (see 7.6).
For some fatty or sticky feedstuffs (e.g. oilseeds, pet foods, molasses block feed), special sample preparation
procedures are useful or necessary (see 7.7).
Finally, the samples are stored (see 7.8).
Samples taken for routine analysis by near-infrared reflectance (NIR) spectrometry should reflect the sample
preparation carried out to derive the calibration. By its very nature, NIR requires minimal or no samp
...