ISO 21846:2018

INTERNATIONAL ISO
STANDARD 21846
First edition
2018-08
Vegetable fats and oils —
Determination of composition of
triacylglycerols and composition and
content of diacylglycerols by capillary
gas chromatography
Corps gras d'origine végétale — Détermination de la composition
des triacylglycérols et de la teneur en diacylglycérols par
chromatographie en phase gazeuse sur colonne capillaire, dans les
huiles végétales
Reference number
©
ISO 2018
© ISO 2018
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Published in Switzerland
ii © ISO 2018 – All rights reserved

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Apparatus . 2
6 Reagents . 2
7 Procedure. 3
7.1 Gas chromatographic apparatus and capillary column condition . 3
7.2 Choice of operating conditions . 3
7.3 Performance of the analysis . 3
7.4 Peak identification . 3
7.5 Determination of percentage content of each triacylglycerol class . 4
7.6 Determination of percentage content of each 1,2 diacylglycerol . 4
7.7 Determination of weight percentage total content of diacylglycerols . 4
8 Expression of results . 5
9 Precision of the method . 5
9.1 Repeatability, r .5
9.2 Reproducibility, R .5
10 Test report . 5
Annex A (informative) Examples of a typical chromatograms . 6
Annex B (informative) Results of an interlaboratory test .11
Bibliography .13
Foreword
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This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11,
Animal and vegetable fats and oils.
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iv © ISO 2018 – All rights reserved

INTERNATIONAL STANDARD ISO 21846:2018(E)
Vegetable fats and oils — Determination of composition
of triacylglycerols and composition and content of
diacylglycerols by capillary gas chromatography
1 Scope
This document specifies the determination of composition of triacylglycerols and the determination of
the composition and content of diacylglycerols by capillary gas chromatography in vegetable oils with
a lauric acid content below 1 %.
Applying certain technological processing 1,2-diacylglycerols (1,2-DAGs) are transformed to the more
stable isomeric 1,3-diacylglycerols (1,3-DAGs) due to acidic catalysed reaction. During storage, the
speed and amount of this rearrangement depends on the acidity of the oil. The transformation normally
reaches an equilibrium between the two isomeric forms. The relative amount of 1,2-DAGs is related to
oil freshness or to a possible technological treatment. Therefore, it is possible to use the ratio of 1,2-
DAGs to 1,3-DAGs as a quality criterion for vegetable fats and oils.
The triacylglycerols profile is of potential interest for the fingerprint of each vegetable oil and may help
the detection of certain types of adulteration, such as the addition of high oleic sunflower oil or palm
olein in olive oil.
NOTE This document is based on Reference [3].
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
4 Principle
After the addition of an internal standard the oil sample is silylated, dissolved in a suitable reagent and
directly injected in the gas chromatographic apparatus. Triacyglycerols are separated into classes on
the basis of their carbon atom number, while diacylglycerols are separated in function of their carbon
atom number and structure, as 1,2 structures show a lower retention time than 1,3 ones.
Unsaturated diacylglycerol structures do not affect retention time. Therefore, saturated and
unsaturated diacylglycerols elute together, so 1,2 and 1,3-diacylglycerol structures are identified by
their peak retention time. The percentage content of 1,2 structure is determined through the ratio of
1,2-diacylglycerol areas to the sum of areas of all the diacylglycerol peaks.
The diacylglycerol total content is calculated by means of an internal standard.
The percentage content for each triacylglycerol class is calculated after normalization to 100 % of all
the triacylglycerol peaks.
5 Apparatus
5.1 Analytical balance suitable to perform weighing to an accuracy of within +/−0,1 mg.
5.2 Gas chromatograph for use with a capillary column, equipped with a system for direct on-column
for cold injection or a programmed temperature vaporizer.
5.3 Thermostat-controlled oven with temperature programming.
5.4 Cold injector for on-column injection or programmed temperature vaporizer.
5.5 Flame-ionization detector and converter-amplifier.
5.6 Recorder-integrator for use with the converter-amplifier (5.5), with a rate of response below 1 s
and variable paper speed, or any suitable device for data capture and handling.
5.7 Capillary column, fused silica, 6 m to 8 m length, 0,25 mm to 0,32 mm internal diameter,
internally coated with SE 52, SE 54 liquid phase to a uniform thickness of 0,10 μm to 0,15 μm.
5.8 Microsyringe, 10 μl, with a hardened needle for on-column injector.
5.9 Microsyringe, 100 μl, with a hardened needle.
5.10 Usual laboratory glassware.
6 Reagents
WARNING — Attention is drawn to the regulations which specify the handling of hazardous
substances. Technical, organizational and personal safety measures shall be followed.
Unless otherwise stated analytically pure reagents shall be used.
6.1 Carrier gas: hydrogen or helium, pure, for gas chromatography.
6.2 Auxiliary gases:
— hydrogen, pure, for gas chromatography;
— air, pure, for gas chromatography.
6.3 Silylating reagent, mix equal volumes of
— pyridine, and
— bistrimethylsilylfluoroacetamide (BSTFA)-trimethylchlorosilane (TMCS), 99:1, volume fraction.
6.4 n-Heptane.
6.5 Reference samples: pure diacylglycerols and triacylglycerols and their mixtures, with known
composition.
6.6 Methyl tert-butyl ether.
2 © ISO 2018 – All rights reserved

6.7 Dinonadecanoin sample solution (internal standard), 0,1 % mass/ volume in methyl tert-
butyl ether.
7 Procedure
7.1 Gas chromatographic apparatus and capillary column condition
Fit the column to the gas chromatograph (5.2), connecting the inlet port to the on-column system
and the outlet port to the detector. Check the gas chromatography apparatus (operation of gas loops,
detector and recorder efficiency, etc.).
Run a light flow of gas through the column, then switch on the gas chromatography apparatus. Gradually
heat until a temperature of 350 °C is reached after approximately 4 h.
Maintain this temperature for at least 2 h, then regulate the apparatus to the operating conditions
(regulate gas flow, light flame, connect to electronic recorder, regulate oven temperature for column,
regulate detector, etc.). Record the signal at a sensitivity at least twice as high as that required for the
analysis. The base line should be linear, with no peaks of any kind, and shall not have any drift.
Negative straight-line drift indicates that the column connections are not correct. Positive drift
indicates that the column has not been properly conditioned.
7.2 Choice of operating conditions
The operating conditions are generally as follows.
— The injector temperature shall be at least 10 °C below the vaporization temperature (99 °C) of the
employed solvent (n-Heptane).
— Detector temperature: 350 °C.
— Column temperature: 80 °C at first (1 min), ramp at 20 °C/min to 220 °C, ramp at 5 °C/min to 340 °C
(10 min).
— Carrier gas: hydrogen or helium at the optimal linear speed for the chosen gas.
— Amount of injected substance: 0,5 μl to 1 μl of solution prepared as in 7.3.
7.3 Performance of the analysis
Weigh (5.1) exactly 100 mg of oil in a glass bottom conical tube and add 1 ml of internal standard
solution (6.7). Shake the sample up to a complete solution, take up with a microsyringe (5.8) 20 μl to
30 μl of solution, put it inside a new glass tube (with a stopper) and dry by nitrogen gentle stream. Add
200 μl of silylation reagent. Af
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