SIST EN ISO 17678:2010

Milk and milk products - Determination of milk fat purity by gas chromatographic analysis of triglycerides (Reference method) (ISO 17678:2010)

This International Standard specifies a reference method for the determination of milk fat purity using gas chromatographic analysis of triglycerides. Both vegetable fats and animal fats such as beef tallow and lard can be detected. By using defined triglyceride equations, the integrity of milk fat is determined. Basically, the method applies to bulk milk, or products made thereof, irrespective of feeding, breed or lactation conditions. In particular, the method is applicable to fat extracted from milk products purporting to contain pure milk fat with unchanged composition, such as butter, cream, milk, and milk powder. However, under the circumstances listed hereafter, a false positive result can be obtained. Hence, the method is not applicable to milk fat:
a) obtained from bovine milk other than cow's milk;
b) obtained from single cows;
c) obtained from cows which received an exceptionally high feeding of pure vegetable oils such as rapeseed oil;
d) obtained from colostrum;
e) subjected to technological treatment such as removal of cholesterol or fractionation;
f) obtained from skim milk or buttermilk;
g) extracted by using the Gerber, Weibull–Berntrop or Schmid–Bondzynski–Ratzlaff methods, or that has been isolated using detergents (e.g. the Bureau of Dairy Industries method). With the extraction methods specified in g), substantial quantities of partial glycerides or phospholipids can pass into the fat phase. Consequently, the scope of this International Standard excludes certain products and particularly cheese, whose ripening process can also affect the fat composition to such a degree that a false positive result is obtained. NOTE 1 In nature, butyric (n-butanoic) acid (C4) occurs exclusively in milk fat and enables quantitative estimations of low to moderate amounts of milk fat in vegetable and animal fats to be made. However, due to the large variation of C4, whose approximate content ranges from 3,1 % mass fraction to 3,8 % mass fraction, it is difficult to provide qualitative and quantitative information for foreign fat to pure milk fat ratios of up to 20 % mass fraction (see Reference [11]). NOTE 2 In practice, quantitative results cannot be derived from the sterol content of vegetable fats, because they depend on production and processing conditions. Furthermore, the qualitative determination of foreign fat using sterols is
ambiguous.

Milch und Milcherzeugnisse - Bestimmung der Reinheit des Milchfetts durch gaschromatographische Triglyceridanalyse (Referenzverfahren) (ISO 17678:2010)

Diese Internationale Norm legt ein Referenzverfahren zur Bestimmung der Reinheit des Milchfetts durch gaschromatographische Triglyceridanalyse fest. Es können sowohl pflanzliche als auch tierische Fette wie Rindertalg und Schweinefett nachgewiesen werden. Unter Verwendung festgelegter Triglyceridgleichungen wird die Integrität des Milchfetts bestimmt.
Hauptsächlich gilt das Verfahren für Sammelkuhmilch oder für Produkte, die daraus hergestellt werden, unabhängig von den Fütterungs , Zucht  oder Laktationsbedingungen. Das Verfahren ist besonders auf Fett anwendbar, das aus Milchprodukten extrahiert wurde, bei denen angegeben wird, dass sie reines Milchfett in unveränderter Zusammensetzung enthalten, wie z. B. Butter, Sahne, Milch und Milchpulver.
Jedoch kann unter den nachstehend erwähnten Fällen ein falsch positives Ergebnis ermittelt werden. Deshalb ist das Verfahren nicht anwendbar auf Milchfett
a)   das von einzelnen Kühen gewonnen wurde;
b)   das von Kühen gewonnen wurde, die eine außergewöhnlich hohe Fütterung von reinen Pflanzenölen wie Rapsöl erhalten haben;
c)   das aus dem Kolostrum (Vormilch) gewonnen wurde;
d)   das einer technischen Behandlung unterzogen wurde, wie z. B. das Entfernen von Cholesterol oder eine Fraktionierung;
e)   das aus Magermilch oder Buttermilch gewonnen wurde;
f)   das mit Verfahren nach Gerber, Weibull Berntrop oder Schmid Bondzynski Ratzlaff extrahiert wurde oder das mittels Detergenzien isoliert wurde (z. B. BDI Verfahren). Mit diesen Extraktionsverfahren können beträchtliche Mengen von Teilen der Glyceride oder Phospholipide in die Fettphase übertreten. Folglich werden bestimmte Produkte und besonders Käse (der Käsereifungsprozess kann die Fettzusammen¬setzung zusätzlich derartig beeinflussen, dass ein falsch-positives Ergebnis erhalten wird) aus dem Anwendungsbereich dieser Internationalen Norm ausgeschlossen.

Lait et produits laitiers - Détermination de la pureté des matières grasses du lait par analyse chromatographique en phase gazeuse des triglycérides (Méthode de référence) (ISO 17678:2010)

L'ISO 17678|FIL 202:2010 spécifie une méthode de référence pour la détermination de la pureté des matières grasses laitières par analyse chromatographique en phase gazeuse des triglycérides. La méthode permet de détecter des matières grasses d'origines végétale et animale telles que la graisse de bœuf et le saindoux. À l'aide de formules de triglycérides définies, l'intégrité de la matière grasse laitière est ainsi déterminée.
En principe, la méthode s'applique au lait en vrac, ou aux produits laitiers dérivés, indépendamment des conditions d'alimentation, d'élevage ou de lactation. Elle s'applique en particulier aux matières grasses extraites de produits laitiers supposés contenir des matières grasses laitières pures, présentant une composition non modifiée, comme le beurre, la crème, le lait et le lait en poudre.
Toutefois, dans les circonstances listées ci-après, un résultat faux positif peut être obtenu. Par conséquent, la méthode n'est pas applicable à la matière grasse laitière: a) issue de lait de bovin autre que le lait de vache; b) issue de vaches individuelles; c) issue de vaches ayant reçu une alimentation exceptionnellement riche en huiles végétales pures telles que l'huile de colza; d) issue du colostrum; e) soumise à un traitement technologique tel que l'élimination du cholestérol ou le fractionnement; f)issue du lait écrémé ou du lait battu (babeurre); g) extraite selon les méthodes Gerber, Weibull-Berntrop ou Schmid-Bondzynski-Ratzlaff, ou isolée au moyen de détergents [par exemple méthode BDI (Bureau of Dairy Industries)].
Avec les méthodes d'extraction spécifiées en g), d'importantes quantités de glycérides ou phospholipides partiaux peuvent passer dans la phase des matières grasses. Par conséquent, le domaine d'application de l'ISO 17678|FIL 202:2010 exclut certains produits et en particulier le fromage, dont le procédé d'affinage peut également affecter la composition de la matière grasse à tel point qu'un résultat faux positif est obtenu.

Mleko in mlečni proizvodi - Določevanje čistosti mlečne maščobe s plinsko kromatografsko analizo trigliceridov (referenčna metoda) (ISO 17678:2010)

Ta mednarodni standard določa referenčno metodo za določevanje čistosti mlečnih maščob s plinsko kromatografsko analizo trigliceridov. Tako rastlinske kot živalske maščobe kot goveji loj in svinjska mast so lahko zaznane. Z uporabo definiranih trigliceridnih enačb je določena celotna mlečna maščoba.  V bistvu metoda velja za večje količine mleka ali proizvode, izdelane iz njega, ne glede na krmljenje, pasmo ali pogoje izločanja mleka. Zlasti metoda velja za maščobe, ekstrahirane iz mlečnih proizvodov, ki domnevno vsebujejo čisto mlečno maščobo z nespremenjeno sestavo, kot so maslo, smetana, mleko in mleko v prahu. Vendar pod okoliščinami naštetimi v nadaljevanju, lahko dosežemo lažen pozitiven rezultat. Zato metoda ne velja za mlečne maščobe:
a) pridobljene iz govejega mleka razen kravjega mleka;
b) pridobljene iz posameznih krav;
c) pridobljene iz krav, ki so dobile izjemno veliko krme iz čistih rastlinskih olj, kot je na primer olje iz repičnih semen;
d) pridobljene iz kolostruma;
d) izpostavljene tehnološkim obdelavam, kot so odstranitev holesterola ali frakcioniranje;
f) pridobljene iz posnetega mleka ali sirotke;
g) ekstrahirane z uporabo Gerber, Weibull-Berntrop ali Schmid-Bondzynski-Ratzlaff metod, ali pa so bile izolirane z uporabo čistil (npr. metoda Urada za mlečno industrijo). Z metodami ekstrahiranja določenimi v g) lahko precejšne količine delnih gliceridov ali fosfolipidov preide v maščobno fazo. Posledično obseg tega mednarodnega standarda izključuje določene proizvode in posebne sire, katerih proces zorenja lahko vpliva na sestavo maščob do te mere, da lahko dobimo lažen pozitiven rezultat. OPOMBA 1: V naravi se butanojska (n-butanojska) kislina (C4) pojavlja samo v mlečni maščobi in omogoča izdelavo kvantitativne ocene nizkih do srednjih vrednostih mlečne maščobe v rastlinskih in živalskih maščobah. Vendar je zaradi velikih odstopanj C4, katerega približna vrednost je od 3.1 % masne frakcije do 3.8 masne frakcije, težko zagotoviti kvalitativne in kvantitativne informacije za razmerje tuje maščobe in čiste mlečne maščobe tudi do 20 % masne frakcije (glej Referenco [11]). OPOMBA: 2 V praksi se kvantitativni rezultati ne morejo izpeljati iz vsebnosti sterola v rastlinskih maščobah zaradi odvisnosti od pogojev pridelave in obdelave. Nadalje, kvalitativno določevanje tuje maščobe z uporabo sterolov je dvoumno.

General Information

• Status: Withdrawn
• Publication Date: 21-Jul-2010
• Withdrawal Date: 17-Jul-2019
• ICS: 67.100.10 - Milk and processed milk products
• ICS: 71.040.50 - Physicochemical methods of analysis
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 9900 - Withdrawal (Adopted Project)
• Start Date: 18-Jul-2019
• Due Date: 10-Aug-2019
• Completion Date: 18-Jul-2019
• Ref Project: EN ISO 17678:2010 - Milk and milk products - Determination of milk fat purity by gas chromatographic analysis of triglycerides (Reference method) (ISO 17678:2010)

Relations

• Replaced By: SIST EN ISO 17678:2019 - Milk and milk products - Determination of milk fat purity by gas chromatographic analysis of triglycerides (ISO 17678:2019)
• Effective Date: 01-Sep-2019

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 17678:2010
01-september-2010
0OHNRLQPOHþQLSURL]YRGL'RORþHYDQMHþLVWRVWLPOHþQHPDãþREHVSOLQVNR
NURPDWRJUDIVNRDQDOL]RWULJOLFHULGRYUHIHUHQþQDPHWRGD,62
Milk and milk products - Determination of milk fat purity by gas chromatographic analysis
of triglycerides (Reference method) (ISO 17678:2010)
Milch und Milcherzeugnisse - Bestimmung der Reinheit des Milchfetts durch
gaschromatographische Triglyceridanalyse (Referenzverfahren) (ISO 17678:2010)
Lait et produits laitiers - Détermination de la pureté des matières grasses du lait par
analyse chromatographique en phase gazeuse des triglycérides (Méthode de référence)
(ISO 17678:2010)
Ta slovenski standard je istoveten z: EN ISO 17678:2010
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
SIST EN ISO 17678:2010 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 17678:2010

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SIST EN ISO 17678:2010


EUROPEAN STANDARD
EN ISO 17678

NORME EUROPÉENNE

EUROPÄISCHE NORM
February 2010
ICS 67.100.99; 67.200.10
English Version
Milk and milk products - Determination of milk fat purity by gas
chromatographic analysis of triglycerides (Reference method)
(ISO 17678:2010)
Lait et produits laitiers - Détermination de la pureté des Milch und Milcherzeugnisse - Bestimmung der Reinheit des
matières grasses laitières par analyse chromatographique Milchfetts durch gaschromatographische Triglyceridanalyse
en phase gazeuse des triglycérides (Méthode de référence) (Referenzverfahren) (ISO 17678:2010)
(ISO 17678:2010)
This European Standard was approved by CEN on 13 February 2010.

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 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 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 and United Kingdom.






EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2010 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17678:2010: E
worldwide for CEN national Members.

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SIST EN ISO 17678:2010
EN ISO 17678:2010 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 17678:2010
EN ISO 17678:2010 (E)
Foreword
This document (EN ISO 17678:2010) has been prepared by Technical Committee ISO/TC 34 "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 2010, and conflicting national standards shall be withdrawn at
the latest by August 2010.
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 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 and the United Kingdom.
Endorsement notice
The text of ISO 17678:2010 has been approved by CEN as an EN ISO 17678:2010 without any modification.

3

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SIST EN ISO 17678:2010

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SIST EN ISO 17678:2010


INTERNATIONAL ISO
STANDARD 17678
IDF
202
First edition
2010-02-15


Milk and milk products — Determination
of milk fat purity by gas chromatographic
analysis of triglycerides (Reference
method)
Lait et produits laitiers — Détermination de la pureté des matières
grasses laitières par analyse chromatographique en phase gazeuse des
triglycérides (Méthode de référence)





Reference numbers
ISO 17678:2010(E)
IDF 202:2010(E)
©
ISO and IDF 2010

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SIST EN ISO 17678:2010
ISO 17678:2010(E)
IDF 202:2010(E)
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©  ISO and IDF 2010
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 2010 – All rights reserved

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SIST EN ISO 17678:2010
ISO 17678:2010(E)
IDF 202:2010(E)
Contents Page
Foreword .iv
1 Scope.1
2 Normative references.2
3 Terms and definitions .2
4 Principle .2
5 Reagents .2
6 Apparatus.3
7 Sampling .4
8 Procedure.4
8.1 Preparation of test samples .4
8.2 Preparation of fat sample solution .5
8.3 Chromatographic triglyceride determination .5
9 Integration, evaluation and control of the analytical performance .8
10 Calculation and expression of results .9
10.1 Triglyceride composition.9
10.2 S-values .10
10.3 Detection of foreign fat .10
11 Precision .11
11.1 Interlaboratory test.11
11.2 Repeatability .11
11.3 Reproducibility .12
12 Test report.12
Annex A (normative) Preparation of the packed column .13
Annex B (informative) Quantification of the foreign fat content.17
Annex C (informative) Uncertainty of measurement .19
Annex D (informative) Interlaboratory test.20
Bibliography.22

© ISO and IDF 2010 – All rights reserved iii

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SIST EN ISO 17678:2010
ISO 17678:2010(E)
IDF 202:2010(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 17678⎪IDF 202 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and
IDF.
iv © ISO and IDF 2010 – All rights reserved

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SIST EN ISO 17678:2010
ISO 17678:2010(E)
IDF 202:2010(E)
Foreword
IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide.
IDF membership comprises National Committees in every member country as well as regional dairy
associations having signed a formal agreement on cooperation with IDF. All members of IDF have the right to
be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in
the development of standard methods of analysis and sampling for milk and milk products.
The main task of Standing Committees is to prepare International Standards. Draft International Standards
adopted by the Standing Committees are circulated to the National Committees for endorsement prior to
publication as an International Standard. Publication as an International Standard requires approval by at least
50 % of the IDF National Committees casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. IDF shall not be held responsible for identifying any or all such patent rights.
ISO 17678⎪IDF 202 was prepared by the International Dairy Federation (IDF) and Technical Committee
ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF
and ISO.
All work was carried out by the Joint ISO-IDF Project Group on Foreign fats of the Standing Committee on
Analytical methods for composition under the aegis of its project leader, Dr J. Molkentin (DE).

© ISO and IDF 2010 – All rights reserved v

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SIST EN ISO 17678:2010

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SIST EN ISO 17678:2010
ISO 17678:2010(E)
INTERNATIONAL STANDARD
IDF 202:2010(E)

Milk and milk products — Determination of milk fat purity by
gas chromatographic analysis of triglycerides (Reference
method)
1 Scope
This International Standard specifies a reference method for the determination of milk fat purity using gas
chromatographic analysis of triglycerides. Both vegetable fats and animal fats such as beef tallow and lard
can be detected. By using defined triglyceride equations, the integrity of milk fat is determined.
Basically, the method applies to bulk milk, or products made thereof, irrespective of feeding, breed or lactation
conditions. In particular, the method is applicable to fat extracted from milk products purporting to contain pure
milk fat with unchanged composition, such as butter, cream, milk, and milk powder.
However, under the circumstances listed hereafter, a false positive result can be obtained. Hence, the method
is not applicable to milk fat:
a) obtained from bovine milk other than cow's milk;
b) obtained from single cows;
c) obtained from cows which received an exceptionally high feeding of pure vegetable oils such as rapeseed
oil;
d) obtained from colostrum;
e) subjected to technological treatment such as removal of cholesterol or fractionation;
f) obtained from skim milk or buttermilk;
g) extracted by using the Gerber, Weibull–Berntrop or Schmid–Bondzynski–Ratzlaff methods, or that has
been isolated using detergents (e.g. the Bureau of Dairy Industries method).
With the extraction methods specified in g), substantial quantities of partial glycerides or phospholipids can
pass into the fat phase. Consequently, the scope of this International Standard excludes certain products and
particularly cheese, whose ripening process can also affect the fat composition to such a degree that a false
positive result is obtained.
NOTE 1 In nature, butyric (n-butanoic) acid (C4) occurs exclusively in milk fat and enables quantitative estimations of
low to moderate amounts of milk fat in vegetable and animal fats to be made. However, due to the large variation of C4,
whose approximate content ranges from 3,1 % mass fraction to 3,8 % mass fraction, it is difficult to provide qualitative and
quantitative information for foreign fat to pure milk fat ratios of up to 20 % mass fraction (see Reference [11]).
NOTE 2 In practice, quantitative results cannot be derived from the sterol content of vegetable fats, because they
depend on production and processing conditions. Furthermore, the qualitative determination of foreign fat using sterols is
ambiguous.
© ISO and IDF 2010 – All rights reserved 1

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SIST EN ISO 17678:2010
ISO 17678:2010(E)
IDF 202:2010(E)
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 1211⎪IDF 1, Milk — Determination of fat content — Gravimetric method (Reference method)
ISO 2450⎪IDF 16, Cream — Determination of fat content — Gravimetric method (Reference method)
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 7328⎪IDF 116, Milk-based edible ices and ice mixes — Determination of fat content — Gravimetric
method (Reference method)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
milk fat purity
absence of vegetable and animal fats determined by the procedure specified in this International Standard
NOTE The purity is determined using S-values, which are calculated from the content of triglycerides. Triglyceride
mass fractions are expressed as percentages.
4 Principle
Fat extracted from milk or milk products is analysed by gas chromatography (GC) using a packed or a short
capillary column to determine triglycerides (TGs), separated by total carbon numbers. By inserting the mass
fraction, expressed as a percentage, of fat molecules of different sizes (C24 to C54, using even C numbers
only) into suitable TG equations, S-values are calculated. If the S-values exceed the limits established with
pure milk fat, the presence of foreign fat is detected.
NOTE 1 The suitability and equivalence of both packed and capillary columns have been demonstrated previously (see
References [8] to [10]).
NOTE 2 An S-value is the sum of weighted TG mass fractions.
5 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade.
5.1 Water complying with the requirements of ISO 3696, grade 2.
5.2 Carrier gas, nitrogen or, alternatively, helium or hydrogen, all with a purity of at least 99,995 % volume
fraction.
5.3 Fat standards, purity at least 99 % mass fraction, for standardizing the milk fat standard described in
8.3.3.
5.3.1 Triglyceride standards, saturated; suitable products are available commercially.
5.3.2 Cholesterol standard.
5.4 Methanol (CH OH), with a water content of not more than 0,05 % mass fraction.
3
2 © ISO and IDF 2010 – All rights reserved

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SIST EN ISO 17678:2010
ISO 17678:2010(E)
IDF 202:2010(E)
5.5 n-Hexane [CH (CH ) CH ].
3 2 4 3
5.6 n-Heptane [CH (CH ) CH ].
3 2 5 3
5.7 Other gases, hydrogen, purity at least 99,995 % volume fraction, free from organic impurities
(C H < 1 µl/l); synthetic air, free from organic impurities (C H < 1 µl/l).
n m n m
5.8 Anhydrous sodium sulfate (Na SO ).
2 4
6 Apparatus
Usual laboratory equipment and, in particular, the following.
6.1 High-temperature gas chromatograph, suitable for use at temperatures of at least 400 °C and
equipped with a flame ionization detector (FID). For capillary GC, an on-column or a programmed temperature
vaporization injector is indispensable while a split injector is unsuitable.
Septa used in the injector shall withstand high temperatures and exhibit a very low degree of “bleeding”.
Always use graphite seals to connect the column as well as injector and/or detector inserts (where applicable).
6.2 Chromatography column.
6.2.1 Packed column, glass, of internal diameter 2 mm and length 500 mm, packed with a stationary
1)
phase of 3 % OV-1 on 125 µm to 150 µm (100 mesh to 120 mesh) Gas ChromQ .
The preparation, silanization, packing and conditioning of the packed column is described in Annex A.
Alternatively, a capillary column (6.2.2) may be used.
6.2.2 Capillary column, short, e.g. of length 5 m, with a non-polar stationary phase that can withstand
2)
temperatures up to 400 °C or more .
Condition the column by performing 20 analyses of a milk fat solution (8.2) within no more than 2 days by
using the settings given in 8.3.4.2. After that, ensure that the response factors (8.3.3) are close to 1 and not
higher than 1,250 0.
Because of the variable overlap between C24 and cholesterol, a higher response factor may be accepted
for C24.
Columns with different dimensions and a different non-polar, highly temperature-resistant phase may be used
as long as their performance is consistent with this International Standard. However, the column length is
restricted by the indispensable limitation in resolution as shown in Figure 1. See also 8.3.4.2.
1)
6.3 Extrelut column , capacity 1 ml to 3 ml, filled with silica gel, for the extraction of milk fat in
accordance with 8.1.4 only.
6.4 Graphite seals, capable of withstanding temperatures of at least 400 °C; for the connection of the GC
column as well as for the injector and/or detector inserts.
6.5 Water bath, capable of being maintained at 50 °C ± 2 °C.
6.6 Oven, capable of operating at 50 °C ± 2 °C and 100 °C ± 2 °C.

1) Example of a suitable product available commercially. This information is given for the convenience of users of this
International Standard and does not constitute an endorsement by ISO or by IDF of this product.
2) CP-Ultimetal SimDist (5 m, 0,53 mm, 0,17 µm) is an example of a suitable product available commercially. This
information is given for the convenience of users of this International Standard and does not constitute an endorsement by
ISO or by IDF of this product.
© ISO and IDF 2010 – All rights reserved 3

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SIST EN ISO 17678:2010
ISO 17678:2010(E)
IDF 202:2010(E)
6.7 Micropipette.
[2]
6.8 Graduated pipette, capacity 5 ml, ISO 835 class A.
6.9 Round-bottomed flask, capacity 50 ml.
6.10 Erlenmeyer flask, nominal capacity 250 ml.
6.11 Funnel.
6.12 Fine-pored filter paper.
6.13 Rotary evaporator.
6.14 Ampoules, nominal capacity 1 ml, fitted with a polytetrafluoroethylene-lined aluminium crimp cap or
screw cap.
6.15 Injection syringe, with syringe plunger not reaching into the tip of the needle (packed column GC).
NOTE With these syringes, better repeatability of the results is obtained.
6.16 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg.
7 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling method
[1]
is given in ISO 707⎪IDF 50 .
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
8 Procedure
8.1 Preparation of test samples
8.1.1 General
For the preparation of test samples, use one of the milk fat isolation or extraction methods specified in 8.1.2 to
8.1.4.
8.1.2 Isolation from butter or butteroil
Melt 50 g to 100 g of test sample in the water bath (6.5) or the oven (6.6) at 50 °C.
Add 0,5 g to 1,0 g of sodium sulfate (5.8) to a folded filter paper (6.12). Preheat a 250 ml Erlenmeyer
flask (6.10) and a funnel (6.11) with the filter paper inserted, containing the sodium sulfate, in the oven (6.6) at
50 °C.
When a limited amount of test sample is available, use a smaller test sample and adapt the procedure
accordingly.
However, note that the handling of a smaller test portion involves a higher risk of obtaining a non-
representative sample.
While keeping the preheated flask, funnel, and inserted filter device in the oven, filter the fat layer of the
molten sample without transferring any serum.
NOTE 1 Butter can be obtained from cream by churning and thorough washing of the resulting butter grain.
NOTE 2 The milk fat obtained using the procedure in this subclause is almost free of phospholipids.
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SIST EN ISO 17678:2010
ISO 17678:2010(E)
IDF 202:2010(E)
8.1.3 Extraction according to the Röse–Gottlieb gravimetric method
Extract the fat fraction from the test sample by using the gravimetric method specified in one of:
ISO 1211⎪IDF 1, ISO 2450⎪IDF 16 or ISO 7328⎪IDF 116.
8.1.4 Extraction from milk using silica gel columns
Temper the milk to 20 °C. Using a micropipette (6.7), add 0,7 ml of the sample thus prepared into a 1 ml to
3 ml Extrelut column (6.3). Allow the sample to distribute uniformly on the silica gel for approximately 5 min.
To denature the protein–lipid complexes, using the graduated pipette (6.8), add 1,5 ml of methanol (5.4) to the
Extrelut column. Subsequently, extract the fat fraction from the test sample with 20 ml of n-hexane (5.5). Add
the n-hexane slowly in small amounts. Collect the solvent draining off in a 50 ml round-bottomed flask (6.9),
previously dried to a constant, known mass weighed to the nearest 1 mg and record the mass to 0,1 mg.
Allow the column to drain until empty after the extraction. Distil off the solvents from the eluate on a rotary
evaporator (6.13) with its water bath maintained at between 40 °C and 50 °C.
After distilling off the solvents, dry and subsequently weigh the round-bottomed flask and its contents to the
nearest 1 mg, recording the mass to 0,1 mg. Determine the fat mass yield by subtracting the mass of the dried
empty round-bottomed flask from the mass obtained.
Depending on the fat content of the milk and the required concentration of the sample solution, check whether
it is necessary to combine the yield of two or more extractions to obtain an adequate amount of fat.
8.2 Preparation of fat sample solution
For gas chromatography with a packed column, prepare a 5 % volume fraction solution of the fat obtained in
8.1.2, 8.1.3 or 8.1.4 in n-hexane (5.5) or n-heptane (5.6). Depending on the column dimensions, use a
concentration of 1 % [0,53 mm internal diameter (ID), wide-bore] or lower for on-column injection with a
capillary column.
When using the fat sample prepared in 8.1.4, calculate the amount of solvent (5.5 or 5.6) to be added to the
test sample in the flask based on the mass of fat obtained.
Completely dissolve the fat in the solvent used. Transfer approximately 0,5 ml to 1 ml of the fat sample
solution obtained into an ampoule (6.14).
8.3 Chromatographic triglyceride determination
8.3.1 Baseline drift
To minimize baseline rising, condition the column as specified in 6.2.2 (capillary column) or in Clause A.4
(packed column).
NOTE Because of the high column temperature, the analysis of TGs is particularly susceptible to a rise of the
baseline in the high carbon-number range.
8.3.2 Injection technique
8.3.2.1 Packed column
To avoid discrimination effects and to improve the quantification of the high-boiling TG components, apply the
hot-needle technique.
Fill the needle with air by drawing up the fat solution into the body of the syringe. Insert the needle into the
injector. Heat the needle prior to injection for about 3 s. Then, rapidly inject the syringe contents.
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SIST EN ISO 17678:2010
ISO 17678:2010(E)
IDF 202:2010(E)
8.3.2.2 Capillary column
When using cool on-column injection (8.3.4.2), insert the needle of the syringe and inject immediately. Choose
a suitable subsequent dwell time of the needle in the injector so as to avoid broad tailing of the solvent peak.
NOTE The optimum dwell time is typically about 3 s.
8.3.3 Calibration
8.3.3.1 General
For the calibration of test samples, perform two to three analyses of standardized milk fat at the beginning of
every working day. Use the last analysis of the standardized milk fat to determine the response factors, f
i
(mass fraction divided by area fraction), of the TGs and of cholesterol and apply these to the subsequent test
samples (see 10.1):
wA∑
ii
f = (1)
i
∑wA
ii
where
w is the mass fraction, expressed as
...