Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free and total glycerol and mono-, di-, triglyceride contents

The purpose of this European Standard is to determine the free glycerol and residual mono-, di- and triglyceride contents in fatty acid methyl esters (FAME) intended for addition to mineral oils. 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  For the purposes of this European Standard, the term "% (m/m)" is used to represent respectively the mass fraction.
WARNING - The use of this method may involve hazardous equipment, materials and operations. This method does not purport to address to all of the safety problems associated with its use. It is the responsibility of the user of this standard 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 freies Glycerin und Reste an Mono-, Di- und Triglyceriden in Fettsäure-Methylester (FAME) zu bestimmen. Der Gesamtglyceringehalt wird anschließend aus den erhaltenen Messwerten berechnet.
Die Bestimmungsgrenzen sind für die festgelegten analytischen Bedingungen 0,001 % (m/m) für freies Glycerin und 0,10 % (m/m) für alle Glyceride (Mono, Di- und Tri-). Das Verfahren ist einsetzbar für FAME aus Rapsöl, Sonnenblumenöl und Sojaöl, Palmöl, für tierische Fette und Öle und für deren Mischungen. Es kann nicht eingesetzt werden für FAME aus Kokosnuss- und Palmkernölen, da hier überlappende Peaks auftreten.
ANMERKUNG 1 Für die Zwecke dieser Europäischen Norm 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 ein Vorhandensein von Squalen vermutet wird, kann EN 17057 zur Unterscheidung zwischen Squalen und Glycerin-Monostearat angewendet werden.
WARNUNG — Die Anwendung dieser Norm kann die Anwendung gefährlicher Stoffe, Arbeitsgänge und Geräte mit sich bringen. Diese Norm beansprucht nicht, alle damit verbundenen Sicherheitsprobleme zu behandeln. Es liegt in der Verantwortung des Anwenders dieser Norm, vor ihrer 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, ou contenant des dérivés d’huiles de coprah ou de palmiste en raison du chevauchement de différents pics de glycérides.
NOTE 1 Pour les besoins du présent document, le terme « % (m/m) » est employé 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 — La présente norme peut impliquer l’utilisation de produits et la mise en oeuvre de modes opératoires et d’appareillages à caractère dangereux. La présente norme n’est pas destinée à traiter de tous les problèmes de sécurité liés à son utilisation. Il incombe aux utilisateurs de la présente norme 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 prostega in celotnega glicerola ter mono-, di- in trigliceridov

General Information

Status
Published
Public Enquiry End Date
19-Feb-2020
Publication Date
16-Dec-2020
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
11-Dec-2020
Due Date
15-Feb-2021
Completion Date
17-Dec-2020

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SLOVENSKI STANDARD
SIST EN 14105:2021
01-januar-2021
Nadomešča:
SIST EN 14105:2011

Derivati maščob in olj - Metil estri maščobnih kislin (FAME) - Določevanje 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: EN 14105:2020
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
SIST EN 14105:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 14105:2021
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SIST EN 14105:2021
EN 14105
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2020
EUROPÄISCHE NORM
ICS 67.200.10 Supersedes EN 14105:2011
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 European Standard was approved by CEN on 2 November 2020.

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, 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, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14105:2020 E

worldwide for CEN national Members.
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SIST EN 14105:2021
EN 14105:2020 (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 ..................................................................................................................................... 6

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 .................................................................. 7

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 ..................................................................................................................................................... 9

10.4 Glycerides .......................................................................................................................................................... 9

10.5 Total glycerol .................................................................................................................................................. 10

11 Expression of results ................................................................................................................................... 10

12 Precision .......................................................................................................................................................... 10

12.1 Interlaboratory study (ILS) ....................................................................................................................... 10

12.2 Repeatability, r .............................................................................................................................................. 11

12.3 Reproducibility, R ......................................................................................................................................... 11

13 Test report ....................................................................................................................................................... 11

Annex A (informative) Sample chromatogram ............................................................................................... 12

Annex B (informative) Calibration function calculation ............................................................................. 20

Annex C (informative) Worked example ........................................................................................................... 22

Annex D (informative) Results of the interlaboratory trial........................................................................ 24

Bibliography ................................................................................................................................................................. 27

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SIST EN 14105:2021
EN 14105:2020 (E)
European foreword

This document (EN 14105:2020) 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 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 June 2021, and conflicting national standards shall be

withdrawn at the latest by June 2021.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document supersedes EN 14105:2011.

In comparison with the previous edition, the following technical modifications have been made:

— document revised editorially;
— improvement of the quality of the figures;
— addition of figures in Annex A for clarification.

According to the CEN-CENELEC Internal Regulations, the national standards organisations 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, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
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SIST EN 14105:2021
EN 14105:2020 (E)
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 terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
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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SIST EN 14105:2021
EN 14105:2020 (E)

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.
6.5 Volumetric flasks, 10 ml capacity, Grade A.
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SIST EN 14105:2021
EN 14105:2020 (E)
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 (50,0 ± 0,1) mg 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 (50,0 ± 0,1) mg 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 (50,0 ± 0,1) 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.
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.
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SIST EN 14105:2021
EN 14105:2020 (E)
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 s
...

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