oSIST prEN 14105:2023
(Main)Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free and total glycerol and mono-, di-, triglyceride contents
Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free and total glycerol and mono-, di-, triglyceride contents
This document specifies a method to determine the free glycerol and residual mono-, di- and triglyceride contents in fatty acid methyl esters (FAME). The total glycerol content is then calculated from the obtained results.
Under the conditions described, the quantification limits are 0,001 % (m/m) for free glycerol, 0,10 % (m/m) for all glycerides (mono-, di- and tri-). This method is suitable for FAME prepared from rapeseed, sunflower, soybean, palm, animal oils and fats and mixture of them. It is not suitable for FAME produced from or containing coconut and palm kernel oils derivatives because of overlapping of different glyceride peaks.
NOTE 1 For the purposes of this document, the term "% (m/m)" is used to represent the mass fraction.
NOTE 2 Under the common EN 14105 GC conditions, squalene can coelute with alpha glycerol monostearate. If the presence of squalene is suspected, EN 17057 can be used to discriminate between squalene and glycerol monostearate.
WARNING - The use of this document can involve hazardous materials, operations and equipment. This document does not purport to address all of the safety problems associated with its use. It is the responsibility of users of this document to take appropriate measures to ensure the safety and health of personnel prior to application of the standard, and fulfil statutory and regulatory requirements for this purpose.
Erzeugnisse aus pflanzlichen und tierischen Fetten und Ölen - Fettsäure-Methylester (FAME) - Bestimmung des Gehaltes an freiem und Gesamtglycerin und Mono-, Di- und Triglyceriden
Dieses Dokument legt ein Verfahren fest, um den Gehalt an freiem Glycerin und restlichen Mono-, Di- und Triglyceriden in Fettsäure-Methylestern (FAME) zu bestimmen. Der Gesamtglyceringehalt wird anschließend aus den erhaltenen Ergebnissen berechnet.
Die Bestimmungsgrenzen sind unter den beschriebenen Bedingungen 0,001 % (m/m) für freies Glycerin und 0,10 % (m/m) für alle Glyceride (Mono-, Di- und Tri-). Das Verfahren ist geeignet für FAME aus Rapsöl, Sonnenblumenöl, Sojaöl, Palmöl, tierischen Ölen und Fetten und für deren Mischungen. Es ist nicht geeignet für FAME aus oder mit Kokosnuss- und Palmkernölderivaten, da hier verschiedene überlappende Glyceridpeaks auftreten.
ANMERKUNG 1 Für die Zwecke dieses Dokuments wird zur Angabe des Massenanteils einer Substanz der Ausdruck „% (m/m)“ verwendet.
ANMERKUNG 2 Unter den allgemeinen GC-Bedingungen nach EN 14105 kann Squalen mit Alpha-Glycerin-Monostearat koeluieren. Falls die Anwesenheit von Squalen vermutet wird, kann EN 17057 zur Unterscheidung zwischen Squalen und Glycerin-Monostearat angewendet werden.
WARNUNG — Die Anwendung dieses Dokuments kann die Anwendung gefährlicher Stoffe, Arbeitsgänge und Geräte mit sich bringen. Dieses Dokument beansprucht nicht, alle damit verbundenen Sicherheitsprobleme zu behandeln. Es liegt in der Verantwortung des Anwenders dieses Dokuments, vor der Anwendung geeignete Maßnahmen für die Sicherheit und den Gesundheitsschutz des Personals zu ergreifen, und dafür Sorge zu tragen, dass behördliche und gesetzliche Maßnahmen eingehalten werden.
Produits dérivés des corps gras - Esters méthyliques d'acides gras (EMAG) - Détermination de la teneur en glycérols libre et total et en mono-, di- et triglycérides
Le présent document spécifie une méthode de détermination des teneurs en glycérol libre et en mono-, di- et triglycérides résiduels des esters méthyliques d’acides gras (EMAG). La teneur en glycérol total est ensuite calculée à partir des résultats obtenus.
Dans les conditions décrites, les limites de quantification sont de 0,001 % (m/m) pour le glycérol libre et de 0,10 % (m/m) pour tous les glycérides (mono-, di- et tri-). Cette méthode convient pour les EMAG préparés à partir d’huiles de colza, de tournesol, de soja, de palme, d’huiles et de graisses d’origine animale et d’un mélange de celles-ci. Elle ne convient pas pour les EMAG produits à partir de dérivés d’huiles de coprah ou de palmiste, ou contenant ces dérivés, en raison du chevauchement de différents pics de glycérides.
NOTE 1 Pour les besoins du présent document, l’expression « % (m/m) » est utilisée pour représenter la fraction massique.
NOTE 2 Dans les conditions de CPG courantes indiquées dans l’EN 14105, le squalène peut coéluer avec le monostéarate de glycérol alpha. Si la présence de squalène est suspectée, l’EN 17057 peut être utilisée pour distinguer le squalène du monostéarate de glycérol.
AVERTISSEMENT - L’utilisation du présent document peut impliquer la mise en oeuvre de matériaux, d’opérations et de matériels dangereux. Ce document ne prétend pas couvrir tous les problèmes de sécurité liés à son utilisation. Il incombe aux utilisateurs du présent document de prendre, avant son application, des mesures appropriées de protection de la sécurité et de la santé du personnel et de s’assurer que les exigences statutaires et réglementaires sont satisfaites.
Derivati maščob in olj - Metil estri maščobnih kislin (FAME) - Določevanje vsebnosti prostega in celotnega glicerola ter mono-, di- in trigliceridov
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 14105:2023
01-maj-2023
Derivati maščob in olj - Metil estri maščobnih kislin (FAME) - Določevanje
vsebnosti prostega in celotnega glicerola ter mono-, di- in trigliceridov
Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free and
total glycerol and mono-, di-, triglyceride contents
Erzeugnisse aus pflanzlichen und tierischen Fetten und Ölen - Fettsäure-Methylester
(FAME) - Bestimmung des Gehaltes an freiem und Gesamtglycerin und Mono-, Di- und
Triglyceriden
Produits dérivés des corps gras - Esters méthyliques d'acides gras (EMAG) -
Détermination de la teneur en glycérols libre et total et en mono-, di- et triglycérides
Ta slovenski standard je istoveten z: prEN 14105
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
oSIST prEN 14105:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 14105:2023
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oSIST prEN 14105:2023
DRAFT
EUROPEAN STANDARD
prEN 14105
NORME EUROPÉENNE
EUROPÄISCHE NORM
March 2023
ICS 67.200.10 Will supersede EN 14105:2020
English Version
Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) -
Determination of free and total glycerol and mono-, di-,
triglyceride contents
Produits dérivés des corps gras - Esters méthyliques Erzeugnisse aus pflanzlichen und tierischen Fetten und
d'acides gras (EMAG) - Détermination de la teneur en Ölen - Fettsäure-Methylester (FAME) - Bestimmung
glycérols libre et total et en mono-, di- et triglycérides des Gehaltes an freiem und Gesamtglycerin und Mono-,
Di- und Triglyceriden
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 307.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 14105:2023 E
worldwide for CEN national Members.
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prEN 14105:2023 (E)
Contents Page
European foreword . 3
1 Scope . 4
2 Normative references . 4
3 Terms and definitions . 4
4 Principle . 4
5 Chemicals . 5
6 Apparatus . 5
7 Preparation of solutions . 6
7.1 1,2,4-Butanetriol stock solution, 1 mg/ml . 6
7.2 Glycerol stock solution, 0,5 mg/ml . 6
7.3 Standard glycerides stock solution, 2,5 mg/ml. 6
7.4 Commercial mixture of monoglycerides . 6
7.5 Calibration solutions . 7
8 Sampling . 7
9 Procedure . 7
9.1 Operating conditions . 7
9.2 Analysis of the calibration solutions. 7
9.3 Analysis of the commercial mixture of monoglycerides . 8
9.4 Preparation and analysis of the samples . 8
9.5 Identification . 8
9.6 Calibration . 8
9.7 Column performance control . 8
10 Determination of results . 9
10.1 Integration of the peaks . 9
10.2 Glycerol calibration function . 9
10.3 Free glycerol . 10
10.4 Glycerides . 10
10.5 Total glycerol . 10
11 Expression of results . 11
12 Precision . 11
12.1 Interlaboratory study (ILS) . 11
12.2 Repeatability, r . 11
12.3 Reproducibility, R . 11
13 Test report . 12
Annex A (informative) Sample chromatogram . 13
Annex B (informative) Calibration function calculation . 22
Annex C (informative) Worked example of calibration function calculation . 24
Annex D (informative) Results of the interlaboratory trial. 26
Bibliography . 28
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European foreword
This document (prEN 14105:2023) has been prepared by 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 document is currently submitted to the CEN Enquiry.
This document will supersede EN 14105:2020.
In comparison with the previous edition, the following technical modifications have been made:
— the weighing accuracy is specified.
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1 Scope
This document specifies a method to determine the free glycerol and residual mono-, di- and triglyceride
contents in fatty acid methyl esters (FAME). The total glycerol content is then calculated from the
obtained results.
Under the conditions described, the quantification limits are 0,001 % (m/m) for free glycerol,
0,10 % (m/m) for all glycerides (mono-, di- and tri-). This method is suitable for FAME prepared from
rapeseed, sunflower, soybean, palm, animal oils and fats and mixture of them. It is not suitable for FAME
produced from or containing coconut and palm kernel oils derivatives because of overlapping of different
glyceride peaks.
NOTE 1 For the purposes of this document, the term “% (m/m)” is used to represent the mass fraction.
NOTE 2 Under the common EN 14105 GC conditions, squalene can coelute with alpha glycerol monostearate. If
the presence of squalene is suspected, EN 17057 can be used to discriminate between squalene and glycerol
monostearate.
WARNING — The use of this document can involve hazardous materials, operations and equipment. This
document does not purport to address all of the safety problems associated with its use. It is the
responsibility of users of this document to take appropriate measures to ensure the safety and health of
personnel prior to application of the standard, and fulfil statutory and regulatory requirements for this
purpose.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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.
EN ISO 5555, Animal and vegetable fats and oils - Sampling (ISO 5555)
EN ISO 3170, Petroleum liquids - Manual sampling (ISO 3170)
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
The glycerol and the mono- and diglycerides are transformed into more volatile and stable silyl
derivatives in presence of pyridine and of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA).
The sample after silylation is analysed by gas chromatography on a short capillary column with thin film
thickness, with an on-column injector or equivalent device, and flame ionization detection.
After a calibration procedure, the quantification of glycerol is carried out in presence of the internal
standard 1,2,4-butanetriol.
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Mono-, di- and triglycerides are directly evaluated in presence of an internal standard for each glyceride
category:
— glyceryl monononadecanoate (Mono C19) for monoglycerides;
— glyceryl dinonadecanoate (Di C38) for diglycerides;
— glyceryl trinonadecanoate (Tri C57) for triglycerides.
5 Chemicals
Use only chemicals of recognized analytical grade, unless otherwise specified.
5.1 N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA).
5.2 Pyridine, max. 0,1 % water, stored on molecular sieve.
Pyridine silyl grade (5.10) can also be used.
5.3 Tetrahydrofurane (THF).
5.4 n-Heptane.
5.5 Glycerol.
5.6 1,2,4-Butanetriol.
5.7 1-Glyceryl monononadecanoate (Mono C19).
5.8 1-3 Glyceryl dinonadecanoate (Di C38).
5.9 Glyceryl trinonadecanoate (Tri C57).
5.10 Pyridine, silyl grade.
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 Gas chromatograph, equipped with an on-column injector or equivalent device, a temperature-
programmable oven and a flame ionization detector.
6.2 Capillary column, capable of being programmed up to 400 °C (“high temperature” type) for which
the following characteristics are advised:
— 100 % dimethylpolysiloxane or 95 % dimethyl-5 % diphenylpolysiloxane stationary phase;
— length 15 m;
— internal diameter 0,32 mm;
— film thickness 0,1 μm.
6.3 Volumetric flask, 50 ml capacity, Grade A.
6.4 Volumetric flasks, 20 ml capacity, Grade A.
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6.5 Volumetric flasks, 10 ml capacity, Grade A.
6.6 Screw-cap vials with PTFE-faced septa, 10 ml capacity.
6.7 Precision pipette, 1 ml capacity.
6.8 Precision pipette or syringe, 100 μl capacity.
6.9 Precision pipette or syringe, 500 μl capacity.
6.10 Graduated cylinder, 10 ml capacity.
6.11 Analytical balance, with a weighing accuracy of ± 1 mg or better and a readability of ± 0,1 mg
or better.
6.12 Carrier gas, hydrogen or helium.
6.13 Auxiliary gases, such as air, hydrogen and nitrogen.
7 Preparation of solutions
7.1 1,2,4-Butanetriol stock solution, 1 mg/ml
Weigh, to the nearest 0,1 mg, 40 mg to 60 mg of 1,2,4-butanetriol (5.6) in a 50 ml volumetric flask (6.3)
and fill up to the mark with pyridine (5.2 or 5.10).
7.2 Glycerol stock solution, 0,5 mg/ml
Weigh, to the nearest 0,1 mg, 40 mg to 60 mg of glycerol (5.5) in a 10 ml volumetric flask (6.5) and fill up
to the mark with pyridine (5.2 or 5.10). Transfer 1 ml of this solution into a 10 ml volumetric flask (6.5)
using a pipette (6.7) and fill up to the mark with pyridine (5.2 or 5.10).
7.3 Standard glycerides stock solution, 2,5 mg/ml
For each reference glyceride, monononadecanoate (5.7), dinonadecanoate (5.8) and trinonadecanoate
(5.9), weigh, to the nearest 0,1 mg, 40 mg to 60 mg in a unique 20 ml volumetric flask (6.4) and fill up to
the mark with tetrahydrofurane (5.3).
The solution shall be perfectly transparent at ambient temperature. After storage in a refrigerator at 4 °C
the solution can show a precipitate that shall re-dissolve spontaneously when restored at ambient
temperature, without any external heating.
NOTE If stored at 4 °C the solution is stable for almost 3 months.
7.4 Commercial mixture of monoglycerides
Made up of mono-palmitoylglycerol (monopalmitin), mono-stearoylglycerol (monostearin) and of mono
oleoylglycerol (monoolein), present in quantities having an identical mass.
Prepare a stock solution of this mixture by weighing approximately 100 mg in a 10 ml volumetric flask
(6.5) and fill up to the mark with pyridine (5.2 or 5.10).
This solution may be used to locate the relevant peaks in GC paths.
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7.5 Calibration solutions
Prepare four calibration solutions by transferring the volumes given in Table 1 of the glycerol stock
solutions (7.2) and of the 1,2,4-butanetriol (7.1) into a series of vials (6.6), using the 100 μl precision
pipette (6.8). If using a syringe, make sure that the needle and the body of the syringe are free from air
bubbles.
Table 1 — Preparation of calibration solutions
1 2 3 4 Syringe
Stock solution
(μl) (μl) (μl) (μl) (μl)
Glycerol solution (7.2) 10 40 70 100 100
Internal butanetriol solution (7.1) 80 80 80 80 100
8 Sampling
Samples shall be taken in accordance with EN ISO 5555 or EN ISO 3170.
9 Procedure
9.1 Operating conditions
The chromatographic analysis conditions shall be chosen taking into account the characteristics of the
column being used and the type of carrier gas (hydrogen or helium). It is however recommended to
observe an analysis time of about 30 min to 35 min to ensure triglycerides elution.
EXAMPLE By way of indication, an example of analysis conditions is described in Table 2:
Table 2 — Example of analysis conditions
Column temperature: 50 °C hold for 1 min, programmed at 15 °C/min up to 180 °C,
programmed at 7 °C/min up to 230 °C, programmed at 10 °C/min up
to 370 °C, final temperature hold for 15 min
Detector temperature: 380 °C
Carrier gas pressure 80 kPa or constant flow (about 2 ml/min)
(hydrogen):
Volume injected: 1 μl
9.2 Analysis of the calibration solutions
Using a precision pipette (6.9), add 150 μl of MSTFA (5.1) to each of the four calibration solutions (7.5),
close hermetically the vials and shake vigorously. Store 15 min at room temperature. Then open the vial
and add 8 ml of n-heptane (5.4) using a graduated cylinder (6.10). Close the vial hermetically again.
Analyse 1 μl of each reaction mixture by gas chromatography e.g. under the conditions indicated in 9.1,
using only the first part of the temperature programme, stopping the analysis when the temperature
of 230 °C has been reached. Samples are stable for some hours after derivatisation.
NOTE The silylated standard solutions are only stable for one day.
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9.3 Analysis of the commercial mixture of monoglycerides
Using precision pipette (6.9), transfer 200 μl of commercial mixture of monoglycerides dissolved in
pyridine (5.10) and 150 μl of MSTFA (5.1) into a 10 ml vial (6.6). Avoid contact with humidity.
Hermetically close the vial and shake vigorously.
Store 15 min at room temperature. Then open the vial and add 8 ml of n-heptane (5.4) using a graduated
cylinder (6.10). Close the vial hermetically again.
Analyse 1 μl of the reaction mixture by gas chromatography e.g. according to the conditions indicated
in 9.1.
9.4 Preparation and analysis of the samples
Weigh, to the nearest 0,1 mg, 80 mg to 125 mg of h
...
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