SIST EN ISO 14673-1:2004

SLOVENSKI STANDARD
SIST EN ISO 14673-1:2004
01-maj-2004
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Milk and milk products - Determination of nitrate and nitrite contents - Part 1: Method
using cadmium reduction and spectrometry (ISO 14673-1:2004)
Milch und Milchprodukte - Bestimmung des Nitrat- und Nitritgehaltes - Teil 1: Verfahren
mit Cadmiumreduktion und Spektrometrie (ISO 14673-1:2004)
Lait et produits laitiers - Détermination des teneurs en nitrates et en nitrites - Partie 1:
Méthode par réduction au cadmium et spectrométrie (ISO 14673-1:2004)
Ta slovenski standard je istoveten z: EN ISO 14673-1:2004
ICS:
67.100.01 0OHNRLQPOHþQLSURL]YRGLQD Milk and milk products in
VSORãQR general
SIST EN ISO 14673-1:2004 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 14673-1:2004

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SIST EN ISO 14673-1:2004
EUROPEAN STANDARD
EN ISO 14673-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2004
ICS 67.100.10 Supersedes EN ISO 14673-1: 2001
English version
Milk and milk products - Determination of nitrate and nitrite
contents - Part 1: Method using cadmium reduction and
spectrometry (ISO 14673-1:2004)
Lait et produits laitiers - Détermination des teneurs en Milch und Milchprodukte - Bestimmung des Nitrat- und
nitrates et en nitrites - Partie 1: Méthode par réduction au Nitritgehaltes - Teil 1: Verfahren mit Cadmiumreduktion
cadmium et spectrométrie (ISO 14673-1:2004) und Spektrometrie (ISO 14673-1:2004)
This European Standard was approved by CEN on 2 February 2004.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, 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
© 2004 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14673-1:2004: E
worldwide for CEN national Members.

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SIST EN ISO 14673-1:2004
EN ISO 14673-1:2004 (E)
CORRECTED  2004-02-25
Foreword
This document (EN ISO 14673-1:2004) 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 August 2004, and conflicting national
standards shall be withdrawn at the latest by August 2004.
This document supersedes EN ISO 14673-1:2001.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Endorsement notice
The text of ISO 14673-1:2004 has been approved by CEN as EN ISO 14673-1:2004 without any
modifications.
NOTE Normative references to International Standards are listed in Annex ZA (normative).
2

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SIST EN ISO 14673-1:2004
EN ISO 14673-1:2004 (E)
Annex ZA
(normative)
Normative references to international publications
with their relevant European publications
This European Standard incorporates by dated or undated reference, provisions from other
publications. These normative references are cited at the appropriate places in the text and the
publications are listed hereafter. For dated references, subsequent amendments to or revisions of
any of these publications apply to this European Standard only when incorporated in it by
amendment or revision. For undated references the latest edition of the publication referred to
applies (including amendments).
NOTE Where an International Publication has been modified by common modifications, indicated
by (mod.), the relevant EN/HD applies.
Publication Year Title EN Year
ISO 1042 1998 Laboratory glassware - One- EN ISO 1042 1999
mark volumetric flasks
3

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SIST EN ISO 14673-1:2004

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SIST EN ISO 14673-1:2004



INTERNATIONAL ISO
STANDARD 14673-1
IDF
189-1
Second edition
2004-02-01


Milk and milk products — Determination
of nitrate and nitrite contents —
Part 1:
Method using cadmium reduction and
spectrometry
Lait et produits laitiers — Détermination des teneurs en nitrates et en
nitrites —
Partie 1: Méthode par réduction au cadmium et spectrométrie




Reference numbers
ISO 14673-1:2004(E)
IDF 189-1:2004(E)
©
ISO and IDF 2004

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SIST EN ISO 14673-1:2004
ISO 14673-1:2004(E)
IDF 189-1:2004(E)
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©  ISO and IDF 2004
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 or IDF at the respective
address below.
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Published in Switzerland

ii © ISO and IDF 2004 – All rights reserved

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SIST EN ISO 14673-1:2004
ISO 14673-1:2004(E)
IDF 189-1:2004(E)
Contents 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 . 5
8 Preparation of test sample. 5
8.1 Dried milk and dried whey. 5
8.2 Caseins and caseinates. 5
8.3 Cheese. 6
8.4 Whey cheese . 6
9 Procedure. 6
9.1 Preparation of the copperized cadmium column . 6
9.2 Checking the reducing capacity of the column . 6
9.3 Regeneration of the column. 7
9.4 Preparation of test portion. 7
9.5 Extraction and deproteination . 8
9.6 Reduction of nitrate to nitrite. 8
9.7 Blank test . 9
9.8 Determination . 9
9.9 Preparation of calibration graph . 9
10 Calculation and expression of results . 9
10.1 Nitrite content. 9
10.2 Nitrate content. 10
11 Precision . 10
11.1 General. 10
11.2 Repeatability. 10
12 Test report. 11
Bibliography . 13

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SIST EN ISO 14673-1:2004
ISO 14673-1:2004(E)
IDF 189-1:2004(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 14673-1IDF 189-1 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.
This second edition cancels and replaces the first edition (ISO 14673-1IDF 189-1:2001), of which it
constitutes a minor revision.
ISO 14673IDF 189 consists of the following parts, under the general title Milk and milk products —
Determination of nitrate and nitrite contents:
— Part 1: Method using cadmium reduction and spectrometry
— Part 2: Method using segmented flow analysis (Routine method)
— Part 3: Method using cadmium reduction and flow injection analysis with in-line dialysis (Routine method)
iv © ISO and IDF 2004 – All rights reserved

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SIST EN ISO 14673-1:2004
ISO 14673-1:2004(E)
IDF 189-1:2004(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
the National Committees casting a vote.
ISO 14673-1IDF 189-1 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 Minerals and minor compounds, of the
Standing Committee on Minor components characterization of physical properties, under the aegis of its
project leader, Mr G. Bråthen (NO).
This second edition, together with ISO 14673-2IDF 189-2 and ISO 14673-3IDF 189-3, cancels and replaces
IDF 84A:1984, IDF 95A:1982, IDF 96A:1987, IDF 97A:1985 and IDF 120:1984, which have been technically
revised.

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SIST EN ISO 14673-1:2004

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SIST EN ISO 14673-1:2004
ISO 14673-1:2004(E)
INTERNATIONAL STANDARD
IDF 189-1:2004(E)

Milk and milk products — Determination of nitrate and nitrite
contents —
Part 1:
Method using cadmium reduction and spectrometry
WARNING — The use of this International Standard may involve hazardous materials, operations and
equipment. This standard does not purport to address all the safety problems associated with its use.
It is the responsibility of the user of this standard to establish safety and health practices and
determine the applicability of regulatory limitations prior to use.
1 Scope
This part of ISO 14673IDF 189 specifies a method for the determination of the nitrate and nitrite contents of
milk and milk products by cadmium reduction and spectrometry. The method is applicable to
 whole and partly skimmed and skimmed dried milk;
 hard, semi-hard and soft cheeses;
 processed cheese;
 whey cheese, caseins and caseinates, and dried whey.
The method may be performed using automatic equipment, in particular by segmented flow analysis (SFA) or
flow injection analysis (FIA), thus reducing cadmium contamination in laboratory work places and waste water.
NOTE These methods are described in ISO 14673-2IDF 189-2 and ISO 14673-3IDF 189-3, respectively.
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 565, Test sieves — Metal wire cloth, perforated metal plate and electroformed sheet — Nominal sizes of
openings
ISO 648, Laboratory glassware — One-mark pipettes
ISO 835-1, Laboratory glassware — Graduated pipettes — Part 1: General requirements
ISO 1042, Laboratory glassware — One-mark volumetric flasks
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SIST EN ISO 14673-1:2004
ISO 14673-1:2004(E)
IDF 189-1:2004(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
nitrate content
mass fraction of nitrate determined by the procedure specified in this part of ISO 14673IDF 189
–
NOTE The nitrate content is expressed as the mass in milligrams of nitrate ions (NO ) per kilogram of product.
3
3.2
nitrite content
mass fraction of nitrite determined by the procedure specified in this part of ISO 14673IDF 189
–
NOTE The nitrite content is expressed as the mass in milligrams of nitrite ions (NO ) per kilogram of product.
2
4 Principle
A test portion is dispersed in warm water, with precipitation of the fat and proteins, then filtration. The nitrate
ions are reduced to nitrite ions in a portion of the filtrate by means of copperized cadmium.
A red colour is developed in portions of both unreduced filtrate and the reduced solution, by addition of
sulfanilamide and N-1-naphthyl ethylenediamine dihydrochloride. Spectrometric measurements are carried out
at a wavelength of 538 nm.
The nitrite content of the sample and the total nitrite content after reduction of nitrate ions are calculated by
comparing the measured absorbances with those of a set of sodium nitrite calibration solutions. The nitrate
content is calculated from the difference between these two contents.
5 Reagents
Use only reagents of recognized analytical grade unless otherwise specified.
5.1 Water, distilled or deionized, or water of equivalent purity, free from nitrate and nitrite ions.
To avoid the possible inclusion of small gas bubbles in the copperized cadmium column (9.1.6), freshly boil
the distilled or deionized water and cool to room temperature. Use the thus-prepared water for the preparation
of the column (9.1), to check the reducing capacity of the column (9.2), and to regenerate the column (9.3).
5.2 Cadmium granules, of diameter 0,3 mm to 0,8 mm.
Prepare cadmium granules, if not available commercially, as follows.
Place a suitable number of zinc rods in a beaker. Cover the rods with cadmium sulfate solution (5.3). Scrape
the cadmium sponge from the rods from time to time over a period of 24 h. Remove the zinc rods and decant
the liquid until only sufficient remains to cover the cadmium sponge. Wash the sponge two or three times with
water. Transfer the cadmium sponge to a laboratory blender together with 400 ml of the hydrochloric acid
working solution (5.7) and blend for a few seconds to obtain granules of the required size. Return the contents
of the blender to the beaker and leave to stand for several hours, while stirring occasionally to remove bubbles.
Decant most of the liquid and immediately copperize the granules as described in 9.1.
WARNING — Because of its toxicity, the used cadmium should be delivered as chemical waste to the
relevant authorities.
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SIST EN ISO 14673-1:2004
ISO 14673-1:2004(E)
IDF 189-1:2004(E)
5.3 Cadmium sulfate solution, c(CdSO ⋅8H O) = 40 g/l.
4 2
Dissolve 40 g of cadmium sulfate solution in water in a 1 000 ml volumetric flask (6.4). Dilute to the mark with
water and mix.
5.4 Copper(II) sulfate solution, c(CuSO ⋅5H O) = 20 g/l.
4 2
Dissolve 20 g of copper(II) sulfate in water in a 1 000 ml volumetric flask (6.4). Dilute to the mark with water
and mix.
5.5 Hydrochloric acid (HCl), (ρ = 1,19 g/ml).
20
5.6 Dilute hydrochloric acid, c(HCl) ≈ 2 mol/l.
Carefully add 160 ml of hydrochloric acid (5.5) to about 700 ml of water in a 1 000 ml volumetric flask (6.4)
while regularly swirling the contents. Cool the contents to room temperature. Dilute to the mark with water and
mix carefully.
5.7 Hydrochloric acid working solution, c(HCl) ≈ 0,1 mol/l.
Add 50 ml of dilute hydrochloric acid (5.6) to a 1 000 ml volumetric flask (6.4). Dilute to the mark with water
and mix.
5.8 Zinc sulfate solution, c(ZnSO ⋅7H O) = 535 g/l.
4 2
Dissolve 53,5 g of zinc sulfate in water in a 100 ml volumetric flask (6.4). Dilute to the mark with water and mix.
5.9 Potassium hexacyanoferrate(II) solution, c(K [Fe(CN) ]⋅3H O) = 172 g/l.
4 6 2
Dissolve 17,2 g of potassium hexacyanoferrate(II) trihydrate in water in a 100 ml volumetric flask (6.4). Dilute
to the mark with water and mix.
5.10 Disodium ethylenediaminetetraacetate dihydrate (EDTA) solution (Na C H N O ⋅2H O).
2 10 14 2 8 2
Dissolve 33,5 g of EDTA in about 900 ml water in a 1 000 ml volumetric flask (6.4). Dilute to the mark with
water and mix.
5.11 Solution I.
Carefully add 450 ml of hydrochloric acid (5.5) to a 1 000 ml volumetric flask (6.4). Dilute to the mark with
water and mix.
5.12 Solution II.
Dissolve by heating on a water bath (6.15) 0,5 g of sulfanilamide (NH C H SO NH ) in a mixture of 75 ml of
2 6 4 2 2
water and 5 ml of hydrochloric acid (5.5) in a 100 ml volumetric flask (6.4). Cool to room temperature. Dilute to
the mark with water and mix. Filter the obtained solution, if necessary.
5.13 Solution III.
Dissolve 0,1 g of N-1-naphthyl ethylenediamine dihydrochloride (C H NHCH CH NH ·2HCl) in water in a
10 7 2 2 2
100 ml volumetric flask (6.4). Dilute to the mark with water and mix. Filter the obtained solution, if necessary.
The solution may be stored for up to 1 week in a well-stoppered brown bottle in a refrigerator.
5.14 Sodium nitrite stock solution (NaNO ).
2
Dry a few grams of sodium nitrite in an oven (6.16) at 110 °C to 120 °C to constant mass (i.e. until the
difference between two successive weighings does not exceed 1 mg). Dissolve 0,150 g of the sodium nitrite in
water in a 1 000 ml volumetric flask (6.4). Dilute to the mark with water and mix.
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SIST EN ISO 14673-1:2004
ISO 14673-1:2004(E)
IDF 189-1:2004(E)
5.15 Sodium nitrite working solution.
Prepare the sodium nitrite working solution on the day of use. Transfer with a pipette (6.5), 10 ml of the stock
solution (5.14) and 20 ml of the buffer solution (5.19) to a 1 000 ml volumetric flask (6.4). Dilute to the mark
with water and mix. The nitrite content of the sodium nitrite working solution is 1 µg/ml.
5.16 Potassium nitrate stock solution (KNO ).
3
Dry a few grams of potassium nitrate in an oven (6.16) at 110 °C to 120 °C to constant mass (i.e. until the
difference between two successive weighings does not exceed 1 mg). Dissolve 1,468 g of the potassium
nitrate in water in a 1 000 ml volumetric flask (6.4). Dilute to the mark with water and mix.
5.17 Potassium nitrate working solution.
Prepare the potassium nitrate working solution on the day of use. Transfer with a pipette (6.5), 5 ml of the
potassium nitrate stock solution (5.16) and 20 ml of the buffer solution (5.19) to a 1 000 ml volumetric flask
(6.4). Dilute to the mark with water and mix. The nitrate content of the potassium nitrate working solution is
4,50 µg/ml.
5.18 Ammonia solution (NH ), (ρ = 0,91 g/ml).
3 20
If an ammonia solution of above-mentioned concentration is not available, an equivalent amount of a more
concentrated ammonia solution may be used in 5.19 [e.g. 103 ml of a 35 % (mass fraction) ammonia solution
(ρ = 1,19 g/ml)].
20
5.19 Buffer solution, pH 9,6 to 9,7.
Dilute 50 ml of hydrochloric acid (5.5) with 600 ml of water in a conical flask (6.3) and mix. Add 135 ml of the
ammonia solution (5.18) and dilute to 1 000 ml with 215 ml of water and mix. Adjust the pH, if necessary, to
between 9,6 and 9,7.
6 Apparatus
Clean all glassware thoroughly and rinse with distilled water to ensure that it is free from nitrate and nitrite ions.
Usual laboratory equipment and, in particular, the following.
6.1 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg.
6.2 Sample container, provided with an airtight lid.
6.3 Conical flasks, of capacity 250 ml, 500 ml and 1 000 ml.
6.4 Volumetric flasks, of nominal capacity 100 ml, 500 ml and 1 000 ml, complying with the requirements
of ISO 1042, class B.
6.5 Pipettes, capable of delivering 2 ml, 4 ml, 5 ml, 6 ml, 8 ml, 10 ml, 12 ml, 20 ml and 25 ml, complying
with the requirements of ISO 648, class A, or ISO 835-1. Where appropriate, burettes may be used instead of
pipettes.
6.6 Measuring cylinders, of capacities 5 ml, 10 ml, 25 ml, 100 ml, 250 ml, 500 ml and 1 000 ml.
6.7 Glass funnels, of diameter 7 cm, with short stem.
6.8 Filter paper, medium grade, of diameter about 15 cm, free from nitrate and nitrite ions.
6.9 Reduction column, made of glass, an example of which is given in Figure 1.
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SIST EN ISO 14673-1:2004
ISO 14673-1:2004(E)
IDF 189-1:2004(E)
6.10 Spectrometer, suitable for measuring absorbance at a wavelength of 538 nm, with cells of optical path
length 1 cm to 2 cm.
6.11 Grinding device, appropriate for grinding the test sample, if necessary. To avoid loss of moisture, the
device should not produce undue heat. A hammer shall not be used.
6.12 Laboratory mixer or homogenizer, with glass containers of capacity 250 ml or 400 ml, suitable for
suspending test portions of cheese and whey cheese.
6.13 Test sieve, of woven wire cloth, of diameter 200 mm, nominal size of openings 500 µm, and a receiver
complying with the requirements of ISO 565.
6.14 Magnetic stirrer.
6.15 Water bath, capable of boiling water.
6.16 Oven, capable of maintaining a temperature of between 110 °C and 120 °C.
7 Sampling
Sampling is not part of the method specified in this part of ISO 14673IDF 189. 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.
Store the test sample in such a way that deterioration and change in composition are prevented.
8 Preparation of test sample
8.1 Dried milk and dried whey
Transfer the test sample to a sample container (6.2) of capacity about twice the volume of the test sample.
Close the container immediately. Mix the test sample thoroughly by repeatedly shaking and inverting the
container until a homogeneous sample is obtained.
8.2 Caseins and caseinates
8.2.1 Thoroughly mix the test sample, if necessary after transferring all of it to a sample container (6.2) of
suitable capacity, by repeatedly shaking and inverting the container.
8.2.2 Transfer 50 g of the test sample to the test sieve (6.13). If the 50 g portion passes directly through the
sieve, or almost completely, pass the whole mixed test sample (8.2.1) through the sieve.
8.2.3 If the test sample does not pass completely through the sieve, use the grinding device (6.11) to
achieve that condition. Immediately transfer all the sieved test sample to the sample container (6.2) and mix
thoroughly in the closed container. During these operations, take precautions to avoid any change in the water
content of the product.
8.2.4 After the test sample has been prepared, proceed with the preparation of the test portion (9.4) as
soon as pos
...