ISO/TS 17764-2:2002

TECHNICAL ISO/TS
SPECIFICATION 17764-2
First edition
2002-11-01

Animal feeding stuffs — Determination of
the content of fatty acids —
Part 2:
Gas chromatographic method
Aliments des animaux — Détermination de la teneur en acides gras —
Partie 2: Méthode par chromatographie en phase gazeuse




Reference number
ISO/TS 17764-2:2002(E)
©
ISO 2002

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ISO/TS 17764-2:2002(E)
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ISO/TS 17764-2:2002(E)
Foreword
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technical committee may decide to publish other types of normative document:
 an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in
an ISO working group and is accepted for publication if it is approved by more than 50 % of the members
of the parent committee casting a vote;
 an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical
committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting
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ISO/TS 17764-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10,
Animal feeding stuffs.
ISO/TS 17764 consists of the following parts, under the general title Animal feeding stuffs — Determination of
the content of fatty acids:
 Part 1: Preparation of methyl esters
 Part 2: Gas chromatographic method

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TECHNICAL SPECIFICATION ISO/TS 17764-2:2002(E)

Animal feeding stuffs — Determination of the content of fatty
acids —
Part 2:
Gas chromatographic method
1 Scope
ISO/TS 17764 specifies methods for the quantitative determination of individual fatty acids and of the sum of
the fatty acids (elutable fatty acids).
This part of ISO/TS 17764 specifies the application of gas chromatography with capillary columns and flame
ionization detection for the determination of the quantitative content of fatty acids in a fat by making use of the
methyl esters of the fatty acids obtained in accordance with the method specified in ISO/TS 17764-1.
This part of ISO/TS 17764 is applicable to the investigation of animal and vegetable fats, oils and fatty acid
mixtures for incorporation in animal feeding stuffs and fat extracts of animal feeding stuffs and raw materials
for compound animal feeds, including fats and fatty acid mixtures containing butyric acid.
This method is not applicable to polymerized fatty acids.
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 3696:1987, Water for analytical laboratory use  Specification and test methods
ISO/TS 17764-1, Animal feeding stuffs — Determination of the content of fatty acids — Part 1: Preparation of
methyl esters
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
fatty acid content
mass fraction of the fatty acids in the test portion of oil, fat, fat extract, free fatty acids or soaps
NOTE The fatty acid content is expressed in grams per kilogram.
3.2
content of elutable material
mass fraction of the sum of all the fatty acids elutable using a gas chromatographic column as described in
this part of ISO/TS 17764
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ISO/TS 17764-2:2002(E)
4 Principle
The methyl esters prepared from fatty acids in accordance with ISO/TS 17764-1 are separated with gas-liquid
chromatography, making use of a capillary column. The peaks in the chromatogram are defined with the help
of a reference sample of known composition and are quantified by means of an internal standard.
5 Reagents
Use only reagents and solvents of recognized analytical grade.
5.1 Water, complying with at least grade 3 in accordance with ISO 3696:1087.
5.2 n-Hexane or n-heptane.
5.3 n-Pentane.
5.4 Reference sample: an oil or fat sample with exactly known fatty acid pattern, or a mixture of reference
fatty acid methyl ester materials or reference fatty acids materials.
NOTE If the BF method for esterification is used, a mixture of reference fatty acid methyl esters cannot be used for
3
calibration or correction factors for fatty acids with a chain length of less than 10 carbon atoms, because of possible
solubility of the methyl esters in the water phase.
6 Apparatus
Usual laboratory equipment and, in particular, the following.
6.1 Gas chromatograph, comprising a capillary column and an injection system specially designed for use
with such columns.
It may be of the split or the split-less type or a cold on-column injector. However, a warm split-less injector is
not suitable for the analysis of milk fats due to overlap of the solvent peak with the butyric acid peak.
6.2 Column, constructed of inert material (fused silica or glass) with a stationary phase preferable
chemically bonded to the wall of the column.
Column dimensions and film thickness are important factors in determining the separation efficiency and
capacity of the column. A resolution of at least 1,25 for the fatty acids C16:0 and C16:1, and C18:0 and C18:1
should be accomplished.
NOTE In most cases a moderately polar phase will suit. In special cases, for instance for the separation of cis-trans-
isomers and/or positional isomers, or if one must be sure that no peaks coincide, a more polar phase is warranted. The
desirable effectiveness and capacity of the column should also be considered for column dimensions and film thickness.
Moderately polar phases are, for instance, various esters of poly(ethylene glycol). More polar phases are often of the
cyano-propyl-polysiloxane type.
6.3 Injection system, for manual injection, with a capacity of at most 10 µl, and graduated in 0,1 µl
divisions, suited for the injector (6.2), or an automatic injection system.
NOTE The use of an automatic system is preferable and can improve repeatability and reproducibility.
6.4 Signal evaluation apparatus: an electronic system fitted with a recorder to transform the detector
signal to a chromatogram (an integrator or a data station).
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ISO/TS 17764-2:2002(E)
7 Procedure
7.1 Preparation of methyl esters
Prepare the methyl esters of the fatty acids of the test portion and the reference sample (5.4) in accordance
with ISO/TS 17764-1.
7.2 Selection of optimum operating conditions
Optimize the equipment in accordance with the instructions given by the manufacturer.
Optimize the flow of carrier gas in accordance with the recommendations of the column manufacturer for the
chosen column and the carrier gas.
Maintain a detector temperature of 20 °C to 50 °C above the highest temperature of the column in a
programmed heating, but at least at 150 °C.
The injector temperature depends on the type of the injector; follow the instructions given in the equipment
manual.
When using a split injector, set the split ratio between 1:30 and 1:100.
7.3 Analysis
7.3.1 Dissolve the fatty acid methyl esters of the test portion and the test portion with added internal
standard in n-hexane (5.2) to a content of 1 % (mass fraction) when using a split injector, or 0,05 % (mass
fraction) in the case of a split-less injector or a cold on-column injector.
Prepare a solution of the fatty acid methyl esters of the reference sample (7.1) in n-hexane with a comparable
concentration.
Inject separately 0,1 µl to 1 µl of the test sample, the test sample with internal standard, and when necessary
the reference sample.
When using cold on-column injection, the use of n-pentane as solvent is necessary for a good separation of
the fatty acid methyl esters with a chain length of less than 10 carbon atoms. Dissolve the methyl esters of the
fatty acids in the test portions and the reference sample in the same solvent.
7.3.2 Select a temperature programme depending on the fatty acid composition, allowing an effective
resolution in the shortest possible time. Take into account the criteria mentioned in 6.3.
Programme the oven temperature starting from 60 °C if the sample contains fatty acids with a chain length
shorter than 12 carbon atoms.
If necessary, progress isothermally after the highest temperature in the programme has been reached until all
components have been eluted.
When using a cold on-column injector, start with an oven temperature of not more than 10 °C higher than the
boiling point of the solvent at the prevailing pressure (50 °C for n-pentane).
Start the temperature programme immediately after the injection. Follow the manufacturer's instructions.
8 Peak identification
Identify the methyl ester peaks of the test portion according to the retention times in comparison with the
retention times of the peaks of known fatty acid methyl esters in the reference sample. Peaks in the
chromatogram of the test portion with the same retention time as peaks in the reference sample are
considered to represent the same fatty acids.
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ISO/TS 17764-2:2002(E)
9 Calculation
9.1 Correction for heptadecanoic acid in the test portion
Correct the peak area of heptadecanoic acid in the test portion with the added internal standard for
heptadecanoic acid originating from the test sample, using the equation:

A AAA++
( )
s17:0 sr16:0 sr18:0 sr18:1
...