SIST EN ISO 12966-4:2015

Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 4: Determination by capillary gas chromatography (ISO 12966-4:2015)

This part of ISO 12966 specifies a method for the determination of fatty acid methyl esters (FAMEs) derived
by transesterification or esterification from fats, oils, and fatty acids by capillary gas chromatography
(GLC). Fatty acid methyl esters from C8 to C24 can be separated using this part of ISO 12966 including
saturated fatty acid methyl esters, cis- and trans-monounsaturated fatty acid methyl esters, and cis- and
trans-polyunsaturated fatty acid methyl esters.
The method is applicable to crude, refined, partially hydrogenated, or fully hydrogenated fats, oils, and
fatty acids derived from animal and vegetable sources.
This method is not suitable for the analysis of dairy, ruminant fats and oils, or products supplemented
with conjugated linoleic acid (CLA). Milk and milk products (or fat coming from milk and milk products)
are excluded from the scope of this part of ISO 12966.
This part of ISO 12966 is not applicable to di-, tri-, polymerized and oxidized fatty acids, and fats and oils.

Tierische und pflanzliche Fette und Öle - Gaschromatographie von Fettsäuremethylestern - Teil 4: Bestimmung von cis-, trans-, gesättigten, mono- und mehrfach ungesättigten Fettsäuren in pflanzlichen oder nicht-ruminanten Ölen und Fetten (ISO 12966-4:2015)

Dieser Teil der ISO 12966 legt ein Verfahren zur Bestimmung von Fettsäuremethylestern (FAME), die durch Umesterung oder Veresterung von Fetten, Ölen und Fettsäuren gewonnen wurden, mittels Kapillar-gaschromatographie (GLC) fest. Durch die Anwendung dieses Teiles von ISO 12966 können Fett-säuremethylester von C8 bis C24, einschließlich gesättigte Fettsäuremethylester, einfach ungesättigte cis  und trans Fettsäuremethylester sowie mehrfach ungesättigte cis  und trans-Fettsäure¬methylester abgetrennt werden.
Das Verfahren ist geeignet für rohe, raffinierte, teilweise oder vollständig hydrierte Fette, Öle und Fettsäuren, die aus tierischen und pflanzlichen Quellen gewonnen wurden.
Dieses Verfahren ist nicht geeignet für die Untersuchung von Milch, Fetten und Ölen von Wiederkäuern, oder Produkten ergänzt mit konjugierter Linolsäure (CLA). Milch und Milcherzeugnisse (oder Fett aus Milch und Milcherzeugnissen) sind vom Anwendungsbereich dieses Teiles der ISO 12966 ausgeschlossen.
Dieser Teil der ISO 12966 ist nicht geeignet für di-, tri-, polymerisierte und oxidierte Fettsäuren und Fette und Öle.

Corps gras d'origines animale et végétale - Chromatographie en phase gazeuse des esters méthyliques d'acides gras - Partie 4: Détermination des acides gras saturés, mono- et poly-insaturés, cis ou trans, dans les corps gras d'origines végétale ou animale (non ruminant)

ISO 12966-4:2015 spécifie une méthode de détermination des esters méthyliques d'acides gras (EMAG) obtenus par transestérification ou estérification de corps gras et d'acide gras par chromatographie en phase gazeuse (CPG) sur colonne capillaire. Les esters méthyliques d'acides gras de C8 à C24 peuvent être séparés en utilisant la présente partie de l'ISO 12966, y compris les esters méthyliques d'acides gras saturés, les esters méthyliques d'acides gras mono-insaturés cis et trans et les esters méthyliques d'acides gras poly-insaturés cis et trans.
La méthode s'applique aux corps gras et acides gras bruts, raffinés, partiellement hydrogénés ou totalement hydrogénés d'origines animale et végétale.
La présente méthode ne convient pas à l'analyse des matières grasses laitières provenant des ruminants, ou des produits supplémentés en acide linoléique conjugué (CLA). Le lait et les produits laitiers (ou corps gras dérivés du lait et des produits laitiers) ne relèvent pas du domaine d'application de la présente partie de l'ISO 12966.
ISO 12966-4:2015 ne s'applique pas aux acides gras et corps gras dimérisés, trimérisés, polymérisés et oxydés.

Živalske in rastlinske maščobe in olja - Plinska kromatografija metilnih estrov maščobnih kislin - 4. del: Določevanje s kapilarno plinsko kromatografsko metodo (ISO 12966-4:2015)

Ta del standarda ISO 12966 določa metodo za določanje metilnih estrov maščobnih kislin (FAME), pridobljenih s transesterifikacijo ali esterifikacijo iz maščob, olj in maščobnih kislin, s kapilarno plinsko kromatografsko metodo. Metilne estre maščobnih kislin C8 do C24 je mogoče ločiti z uporabo metode iz tega dela standarda ISO 12966, vključno z metilnimi estri nasičenih maščobnih kislin, metilnimi estri cis- in trans-enkratnenasičenih maščobnih kislin in metilnimi estri cis- in trans-večkratnenasičenih maščobnih kislin.
Ta metoda velja za surove, rafinirane in delno ali povsem hidrogenirane maščobe, olja in maščobne kisline, pridobljene iz živalskih in rastlinskih virov. Ta metoda ni primerna za analizo mlečnih izdelkov, maščob in olj prežvekovalcev ali izdelkov z dodano konjugirano linolno kislino (CLA). Mleko in mlečni izdelki (ali maščoba iz mleka in mlečnih izdelkov) niso zajeti v tem delu standarda ISO 12966. Ta del standarda ISO 12966 ne velja za dimerne, trimerne, polimerne in oksidirane maščobne kisline.

General Information

• Status: Published
• Public Enquiry End Date: 29-Apr-2014
• Publication Date: 04-Aug-2015
• ICS: 67.200.10 - Animal and vegetable fats and oils
• ICS: 71.040.50 - Physicochemical methods of analysis
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 08-Jul-2015
• Due Date: 12-Sep-2015
• Completion Date: 05-Aug-2015
• Ref Project: EN ISO 12966-4:2015 - Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 4: Determination by capillary gas chromatography (ISO 12966-4:2015)

Relations

• Replaces: SIST EN ISO 5508:1996 - Animal and vegetable fats and oils - Analysis by gas chromatography of methyl esters of fatty acids (ISO 5508:1990)
• Effective Date: 01-Sep-2015
• Replaces: SIST EN ISO 15304:2002 - Animal and vegetable fats and oils - Determination of the content of trans fatty acid isomers of vegetable fats and oils - Gas chromatographic method (ISO 15304:2002)
• Effective Date: 01-Sep-2015
• Replaces: SIST EN ISO 15304:2002/AC:2005 - Animal and vegetable fats and oils - Determination of the content of trans fatty acid isomers of vegetable fats and oils - Gas chromatographic method (ISO 15304:2002/Cor.1:2003)
• Effective Date: 01-Sep-2015
• Replaces: SIST EN ISO 5508:1996 - Animal and vegetable fats and oils - Analysis by gas chromatography of methyl esters of fatty acids (ISO 5508:1990)
• Effective Date: 08-Jun-2022

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 12966-4:2015
01-september-2015
1DGRPHãþD
SIST EN ISO 15304:2002
SIST EN ISO 15304:2002/AC:2005
SIST EN ISO 5508:1996
äLYDOVNHLQUDVWOLQVNHPDãþREHLQROMD3OLQVNDNURPDWRJUDILMDPHWLOQLKHVWURY
PDãþREQLKNLVOLQGHO'RORþHYDQMHVNDSLODUQRSOLQVNRNURPDWRJUDIVNRPHWRGR
,62
Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters -
Part 4: Determination by capillary gas chromatography (ISO 12966-4:2015)
Tierische und pflanzliche Fette und Öle - Gaschromatographie von
Fettsäuremethylestern - Teil 4: Bestimmung von cis-, trans-, gesättigten, mono- und
mehrfach ungesättigten Fettsäuren in pflanzlichen oder nicht-ruminanten Ölen und
Fetten (ISO 12966-4:2015)
Corps gras d'origines animale et végétale - Chromatographie en phase gazeuse des
esters méthyliques d'acides gras - Partie 4: Détermination des acides gras saturés,
mono- et poly-insaturés, cis ou trans, dans les corps gras d'origines végétale ou animale
(non ruminant)
Ta slovenski standard je istoveten z: EN ISO 12966-4:2015
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
SIST EN ISO 12966-4:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 12966-4:2015

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SIST EN ISO 12966-4:2015

EUROPEAN STANDARD
EN ISO 12966-4

NORME EUROPÉENNE

EUROPÄISCHE NORM
June 2015
ICS 67.200.10 Supersedes EN ISO 15304:2002, EN ISO 5508:1995
English Version
Animal and vegetable fats and oils - Gas chromatography of fatty
acid methyl esters - Part 4: Determination by capillary gas
chromatography (ISO 12966-4:2015)
Corps gras d'origines animale et végétale - Tierische und pflanzliche Fette und Öle -
Chromatographie en phase gazeuse des esters Gaschromatographie von Fettsäuremethylestern - Teil 4:
méthyliques d'acides gras - Partie 4: Détermination par Bestimmung mittels Kapillargaschromatographie (ISO
chromatographie capillaire en phase gazeuse (ISO 12966- 12966-4:2015)
4:2015)
This European Standard was approved by CEN on 16 April 2015.

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, Former Yugoslav Republic of Macedonia, 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

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 12966-4:2015 E
worldwide for CEN national Members.

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SIST EN ISO 12966-4:2015
EN ISO 12966-4:2015 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 12966-4:2015
EN ISO 12966-4:2015 (E)
Foreword
This document (EN ISO 12966-4:2015) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and
oils and their by-products - Methods of sampling and analysis” the secretariat of which is held by AFNOR.
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 2015, and conflicting national standards shall be withdrawn
at the latest by December 2015.
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.
This document supersedes EN ISO 5508:1995, EN ISO 15304:2002.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, 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.
Endorsement notice
The text of ISO 12966-4:2015 has been approved by CEN as EN ISO 12966-4:2015 without any modification.
3

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SIST EN ISO 12966-4:2015

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SIST EN ISO 12966-4:2015
INTERNATIONAL ISO
STANDARD 12966-4
First edition
2015-06-01
Animal and vegetable fats and oils —
Gas chromatography of fatty acid
methyl esters —
Part 4:
Determination by capillary gas
chromatography
Corps gras d’origines animale et végétale — Chromatographie en
phase gazeuse des esters méthyliques d’acides gras —
Partie 4: Détermination par chromatographie capillaire en phase
gazeuse
Reference number
ISO 12966-4:2015(E)
©
ISO 2015

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SIST EN ISO 12966-4:2015
ISO 12966-4:2015(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2015 – All rights reserved

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SIST EN ISO 12966-4:2015
ISO 12966-4:2015(E)

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Principle . 1
4 Reagents and materials . 1
4.1 Reference fatty acid methyl esters (FAMEs) . 2
4.2 Internal standards . 2
5 Apparatus . 3
6 Sampling . 4
7 Preparation of test sample . 4
8 Preparation of methyl esters from fats, oils, and fatty acids . 4
9 Procedure. 4
9.1 General . 4
9.2 GC conditions . 4
9.3 Performance check . 5
10 Calculations. 5
10.1 Qualitative analysis and peak identification . 5
10.2 Quantitative analysis . 5
10.2.1 Calculation of the composition of fatty acid methyl esters . 5
10.2.2 Calculation of the composition of fatty acid methyl esters using
correction factors . 6
10.2.3 Calculation of the composition of fatty acid methyl esters using an
internal standard . 6
11 Precision . 7
11.1 Results of interlaboratory test . 7
11.2 Repeatability . 7
11.3 Reproducibility . 7
12 Test report . 7
Annex A (informative) Theoretical flame ionization detector correction factor (TCF) for
fatty acid methyl esters (FAMEs) . 9
Annex B (informative) Examples of chromatograms .10
Annex C (informative) Comparison of FAME composition with two different GC columns.12
Annex D (informative) Results of an interlaboratory trial .14
Bibliography .20
© ISO 2015 – All rights reserved iii

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SIST EN ISO 12966-4:2015
ISO 12966-4:2015(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of any
patent rights identified during the development of the document will be in the Introduction and/or on
the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary Information
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils.
This first edition cancels and replaces ISO 5508:1990 and ISO 15304:2002, which have been
technically revised.
ISO 12966 consists of the following parts, under the general title Animal and vegetable fats and oils — Gas
chromatography of fatty acid methyl esters:
— Part 1: Guidelines on modern gas chromatography of fatty acid methyl esters
— Part 2: Preparation of methyl esters of fatty acids
— Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH)
— Part 4: Determination by capillary gas chromatography
iv © ISO 2015 – All rights reserved

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SIST EN ISO 12966-4:2015
INTERNATIONAL STANDARD ISO 12966-4:2015(E)
Animal and vegetable fats and oils — Gas chromatography
of fatty acid methyl esters —
Part 4:
Determination by capillary gas chromatography
1 Scope
This part of ISO 12966 specifies a method for the determination of fatty acid methyl esters (FAMEs) derived
by transesterification or esterification from fats, oils, and fatty acids by capillary gas chromatography
(GLC). Fatty acid methyl esters from C8 to C24 can be separated using this part of ISO 12966 including
saturated fatty acid methyl esters, cis- and trans-monounsaturated fatty acid methyl esters, and cis- and
trans-polyunsaturated fatty acid methyl esters.
The method is applicable to crude, refined, partially hydrogenated, or fully hydrogenated fats, oils, and
fatty acids derived from animal and vegetable sources.
This method is not suitable for the analysis of dairy, ruminant fats and oils, or products supplemented
with conjugated linoleic acid (CLA). Milk and milk products (or fat coming from milk and milk products)
are excluded from the scope of this part of ISO 12966.
This part of ISO 12966 is not applicable to di-, tri-, polymerized and oxidized fatty acids, and fats and oils.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 661, Animal and vegetable fats and oils — Preparation of test sample
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 6353, Reagents for chemical analysis
ISO 12966-2, Animal and vegetable fats and oils — Gas chromatography of fatty acid methyl esters — Part 2:
Preparation of methyl esters of fatty acids
ISO 12966-3, Animal and vegetable fats and oils — Gas chromatography of fatty acid methyl esters —
Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH)
3 Principle
Using capillary gas chromatography, FAMEs are separated on a highly polar stationary phase with
respect to their chain length, degree of (un)saturation, and geometry and position of the double bonds.
4 Reagents and materials
Unless otherwise stated, use only reagents as specified in ISO 6353-2 and ISO 6353-3 (if listed there). If
not, then use reagents of recognized analytical grade and water of at least grade 3, as defined in ISO 3696.
© ISO 2015 – All rights reserved 1

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SIST EN ISO 12966-4:2015
ISO 12966-4:2015(E)

WARNING — Attention is drawn to the regulations which specify the handling of dangerous
matter. Technical, organizational, and personal safety measures shall be followed.
4.1 Reference fatty acid methyl esters (FAMEs)
4.1.1 Reference mixtures of pure FAMEs and/or oils with known fatty acid composition should be used
for the identification of fatty acids analysed under the test conditions of this method.
4.1.2 Fats and oils with certified fatty acid composition, e.g. certified reference material BCR 162.
4.1.3 Reference fatty acid methyl esters (FAMEs) - Methyl esters of pure fatty acids, in particular, cis- and
trans-isomers of octadecenoic (oleic), trans-isomers of octadecadienoic (linoleic), and octadecatrienoic
(α-linolenic) acids. Wide ranges of cis- and trans-octadecenoic methyl ester isomers are available on the
market. Trans-geometrical isomers of linoleic and α-linolenic acids can be prepared in the laboratory
with the aid of p-toluenesulfonic acid. In addition to pure compounds, convenient mixtures of FAMEs are
also commercially available.
4.2 Internal standards
For the quantification of the fatty acids, in grams per 100 g, the use of a FAME as an internal standard
(IS) is necessary. An external calibration with mixtures of different fatty acids is also possible.
NOTE If it is necessary to check the recovery and the effectiveness of the derivatization method, then either
or both a TAG and a FAME internal standard should be used. While the TAG-IS is added to the sample prior to the
FAME preparation, the FAME-IS is added before or after the FAME preparation. The FAME-IS is used to calculate
the recovery of the FAME from the TAG-IS and therefore, the efficiency of the derivatisation procedure. In this
case, a different chain length of the standards is required.
Depending on the type of fat, different internal standards can be used (C11:0 FAME, C17:0 FAME, C19:0
FAME, C21:0 FAME, C23:0 FAME, etc.). An external calibration with mixtures of different fatty acids is
also possible. It is recommended to carry out further analysis of the sample without the addition of the
internal standard to check the natural content of the fatty acid which is used as the internal standard.
The content shall be considered in the calculation.
IMPORTANT — If the TAG-IS (4.2.2) is hard to dissolve in the cold, a hot methylation procedure,
as specified in ISO 12966‑2:2011, 4.3, 4.4, and 4.5, shall be used.
The internal standard solutions are stable if precautions are taken to eliminate the loss of solvent and
therefore, a change in the concentration of the IS. For example, store the solution in a refrigerator in
a well-sealed amber bottle when not in use. Pure standards are available on the market. Purity of the
IS shall be confirmed by thin-layer chromatography, high-performance liquid chromatography, gas
chromatography analysis, or by any other appropriate technique.
The following are examples of suitable standards (as FAME and TAG):
4.2.1 Fatty acid methyl ester (FAME) as internal standard (IS) solution:
C21:0 FAME – heneicosanoic acid methyl ester (purity >99 %), mass concentration 5,0 mg/ml in iso-
octane or MTBE should be used as the internal standard.
4.2.2 Triacylglycerol (TAG) internal standard (IS) solution:
C21:0 TAG - triheneicosanoin (purity >99 %), mass concentration 5,0 mg/ml in chloroform. The
TAG internal standard solution is stable if precautions are taken to eliminate the loss of solvent and
therefore, a change in the concentration of the IS. For example, store the solution in a refrigerator in a
well-sealed amber bottle when not in use. Pure triheneicosanoin is available on the market. Purity of
the IS shall be confirmed by thin-layer chromatography, high-performance liquid chromatography, gas
chromatography analysis, or by any other appropriate technique.
2 © ISO 2015 – All rights reserved

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SIST EN ISO 12966-4:2015
ISO 12966-4:2015(E)

Toluene can be used in place of chloroform with the following considerations. Triheneicosanoin is not as
soluble in toluene as it is in chloroform. A solution with a mass concentration of 2 mg/ml can be prepared
in toluene. It is necessary to warm the solution slightly to get it dissolved, but once in solution, it will stay
dissolved if kept at room temperature. If the solution is stored in a refrigerator, it will crystallize out,
but can be dissolved again by slight warming of the solution. Care has to be taken so none of the toluene
is evaporated during this warming procedure. Also, care has to be taken to prevent the loss of toluene
during storage. Solvents other than iso-octane (i.e. chloroform or toluene) have to be removed after the
addition of the TAG-IS as these solvents are not used in the derivatization according to ISO 12966-2.
4.3 Iso-octane (2,2,4-trimethyl pentane).
4.4 Methyl tert-Butyl ether (MTBE) (2-Methoxy-2-methylpropane).
4.5 Chloroform.
SAFETY PRECAUTIONS — Chloroform is classed as a carcinogenic solvent (Category 3).
4.6 n-Hexane.
4.7 n-Heptane.
SAFETY PRECAUTIONS — Prolonged exposure through inhalation and swallowing could cause
serious damage to health despite limited evidence on the carcinogenic effect (Category 3).
4.8 Toluene.
5 Apparatus
Usual laboratory equipment and, in particular, the following.
5.1 Gas chromatograph, equipped with flame ionization detector, split or splitless injector, and data
acquisition system.
NOTE The use of on-column and programmable temperature vaporizer (PTV) injectors are also possible.
1)
5.2 Capillary column, fused silica capillary 100 m and 0,25 mm i.d. coated with SP-2560 or CP-Sil 88 ,
100 % cyanopropylsilicone stationary phase to a thickness of 0,20 µm. Commercially prepared columns
are available from different suppliers.
NOTE The use of 100 m, 0,25 mm ID, 0,20 µm film thickness columns with SP-2560 or CP-Sil 88 as the
stationary phase are recommended as the separation capacity of these columns is sufficient to separate most
C18:1 trans- and cis-isomers. If this separation is not required, a 50 m or 60 m column can also be used. However,
some 50 m or 60 m long columns might also achieve this separation mostly for vegetable oils. Other types of
columns (BPX70, DB-23, HP-23, Rtx-2330, SP-2330, SP-2380, etc.) are also possible, but a shift in the elution order
is possible. For fast GC analysis, short columns are also possible (10 m to 15 m), but with limited information
which in certain cases, will not be a problem.
5.3 Micro syringe, for gas chromatography, 10 μl delivery with a hardened needle.
5.4 Carrier gas, hydrogen (recommended) or helium, 99,999 5 % pure or better, gas chromatography
quality, dried, oxygen removed by suitable filters (<0,1 mg/kg), free from organic impurities.
NOTE Nitrogen gas is not acceptable as a carrier gas for this method.
1) Examples of suitable products available commercially. This information is given for the convenience of users of
this document and does not constitute an endorsement by ISO of these products. Equivalent products may be used
if they can be shown to lead to the same results.
© ISO 2015 – All rights reserved 3

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SIST EN ISO 12966-4:2015
ISO 12966-4:2015(E)

WARNING — Hydrogen, which is used with capillary columns, can double the speed of the
analysis (in comparison with helium), but is hazardous. Hydrogen generators and safety devices
are available and it is essential that a suitable device be incorporated into the apparatus.
5.5 Flame gases, hydrogen and air, gas chromatography quality, free from organic impurities.
5.6 Make-up gas, nitrogen or helium, gas chromatography quality, free from organic impurities.
6 Sampling
A representative sample should be sent to the laboratory. It should not be damaged or changed during
transport or storage.
Sampling is not part of the method specified in this part of ISO 12966. A recommended sampling method
is given in ISO 5555.
7 Preparation of test sample
Prepare the test sample in accordance with ISO 661.
8 Preparation of methyl esters from fats, oils, and fatty acids
The fatty acid methyl esters shall be prepared in accordance with ISO 12966-2 or ISO 12966-3.
NOTE Prior to methylation, the internal standard solution, if required, is added to the reaction flask so that
after the oil or fat is added, the mass fraction is between 0,05 and 0,10 mg IS/mg oil or fat. Since a solvent is used
in the IS, it shall be evaporated from the flask prior to the methylation procedure.
Dissolve the prepared FAMEs in n-heptane, n-hexane, or iso-octane. The mass concentration should be
approximately 15 mg/ml to 20 mg/ml for split injection. For on-column injection, the mass concentration
should be adapted.
9 Procedure
WARNING — Due to the toxic character of some solvents, a ventilated hood shall be used.
9.1 General
The first sample in an analysis batch shall always be a blank FAME dissolution solvent. No peaks shall
be detected in this blank run.
9.2 GC conditions
Adapt the temperatures and GC conditions considering the type of fat, oil, or fatty acid analysed and the
apparatus used. The following conditions have been proven to be suitable for the separation of FAMEs
(C4 to C24) on 100 m columns. However, other conditions are also possible and can be used.
Injector temperature 250 °C
Detector temperature: 250 °C
Oven temperature: 120 °C to 240 °C with 4 °C/min, hold for further 7 min at 240 °C
Carrier gas hydrogen: column head pressure, 220 kPa
linear velocity; (30 to 40) cm/s, flow rate approx. 1,0 ml/min
4 © ISO 2015 – All rights reserved

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SIST EN ISO 12966-4:2015
ISO 12966-4:2015(E)

split ratio, 1:100
Injection volume: 1 µl (equivalent to 15 µg to 20 μg FAME)
Examples of chromatograms and alternative conditions are shown in Annex B and Annex C:
NOTE For the analysis of animal fats, the complete elution of all FAMEs can be checked with certified
reference standards.
9.3 Performance check
Column performance is checked using a suitable mixture of fatty acid methyl esters covering the range
of fatty acids under investigation. Since commercial GC designs are different and the separation obtained
is not identical to the example chromatograms, small changes in the sample size, sample concentration,
or oven temperature may be required. If so, adjust the sample size, sample concentration, or oven
temperature until the best separation results are obtained. If the column oven temperature needs to be
adjusted, it should be adjusted by small increments, preferably in steps of 1 °C.
NOTE On all cyanopropylsilicone capillary columns, the column temperature has a major effect on the elution
pattern of 13t- and 14t-C18:1, 16t-C18:1, 14c-C18:1, 9c,12c,15t-C18:3, 11c-C20:1 and 9c,12c,15c-C18:3.
10 Calculations
10.1 Qualitative analysis and peak identification
The individual FAMEs are identified by their retention times and in comparison with FAME
...