SVENSK STANDARD

SS-EN 14105:2020

Fett och oljederivater – Fettsyrametylester (FAME) – Bestämning

av fri och totalhalt glycerol och mono-, di, triglycerider

Fat and oil derivatives – Fatty Acid Methyl Esters (FAME) –

Determination of free and total glycerol and mono-, di-,
triglyceride contents

Språk: engelska / English
Utgåva: 3
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EUROPEAN STANDARD EN 14105

NORME EUROPÉENNE SS-EN 14105:2020 (E)

EUROPÄISCHE NORM December 2020

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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SS-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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SS-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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SS-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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SS-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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SS-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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SS-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 stable for one day.

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.

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SS-EN 14105:2020 (E)

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 (100,0 ± 0,1) mg of homogenized sample in a 10 ml vial (6.6).

Using precision pipettes or syringes (6.8 and 6.9), add 80 μl of 1,2,4-butanetriol stock solution (7.1),
200 μl of standard glycerides stock solution (7.3), 200 μl of pyridine (5.10) and 200 μl of MSTFA (5.1).
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.
9.5 Identification

The analysis of the calibration solutions under the same operating conditions as those used for the
analysis of the sample allows the identification of the peaks by comparison of the retention times. Due
to the overlapping of the elution zones of the methyl esters and of the monoglycerides, it is therefore
advised, in order to identify the monoglyceride peaks, to inject the commercial mixture composed of
monopalmitin, monosterarin and monoolein (7.4), the latter having been previously submitted to the
derivatisation reaction.
Chromatograms of a rapeseed oil and a palm oil methyl ester sample, obtained under the operating
conditions in 9.1 are presented in Annex A. In addition the chromatogram of a second palm oil methyl
ester sample containing fatty acid methyl ester dimers is presented. Internal glyceride standards may
be analysed under the above mentioned chromatographic conditions, after silyl derivatisation.
9.6 Calibration

For glycerol only, the study of the variation of weight ratio versus area ratio makes it possible to verify
the linearity of the response and to work out a calibration function.
For mono-, di- and triglycerides it is assumed that, within the considered concentration range, the
detector response is regarded as linear.
9.7 Column performance control

For each analysis, evaluate the relative response factor (RF) for glyceryl dinonadecanoate (Di C38)
versus glyceryl trinonadecanoate (Tri C57), by using Formula (1):

( ) (
RF = ADiC38 / M DiC38 / ATriC57 / M TriC57 ) (1)

where
ADi C38 is the peak area of internal standard Di C38;

M Di C38 is the weight of internal standard Di C38, in mg;

ATri C57 is the peak area of internal standard Tri C57;

M Tri C57 is the weight of internal standard Tri C57, in mg.

The results of the calculation of RF shall be lower than 1,8. For higher values, the gas chromatography
system is not suitable for analysis and shall be verified in order to improve triglyceride detection.

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SS-EN 14105:2020 (E)

10 Determination of results
10.1 Integration of the peaks

In each family of glycerides, there exist small peaks (see Annex A) which have to be integrated. This
method therefore calculates the percentage of mono-, di- and triglycerides (10.4) by summing the area
peaks for each family. It is advised to integrate jointly the two diglyceride peaks containing 36 atoms of
carbon, major compounds of this family, on account of an insufficient resolution which may induce
quantification errors if the two peaks are integrated separately. The presence of a double peak at the
level of the glycerol retention time shall lead to the verification of the silylation stage, which is probably
incomplete (presence of water in the samples).
10.2 Glycerol calibration function

The calibration function is given by Formula (2), obtained from the experimental data using the linear
regression method as in Annex B and according to:

(
M g / M ei a g Ag / Aei + bg
= ) (2)

where
M g is the weight of glycerol, in mg;

M ei is the weight of internal standard 1,2,4-butanetriol, in mg;

Ag is the peak area of glycerol;

Aei is the peak area of the internal standard 1,2,4-butanetriol;

a g , bg are the regression coefficients of the calibration function for glycerol.

The calibration function shall be regarded as correct only if the correlation coefficient, calculated
according to Annex B, is equal or greater than 0,9 (see Annex C for a worked example).
10.3 Free glycerol

Calculate the mass percentage of free glycerol (G) in % (m/m) in the sample using Formula (3):

= ( ) (
G a g Ag / Aei + bg  ⋅ M ei / m ⋅ 100
 
) (3)

where

Aei is the peak area of the internal standard 1,2,4-butanetriol;

M ei is the weight of internal standard 1,2,4-butanetriol, in mg;

m is the weight of the sample, in mg.

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SS-EN 14105:2020 (E)

10.4 Glycerides

Calculate the mass percentage of the mono-, di- and triglycerides in % (m/m) using Formulae (4), (5)
and (6):
M
= ( AMono / AMonoC19 ) ⋅ ( M MonoC19 / m) ⋅ 100 (4)

D= ( ADi / ADiC38 ) ⋅ ( M DiC38 / m) ⋅ 100 (5)

T= ( ATri / ATriC57 ) ⋅ ( M TriC57 / m) ⋅ 100 (6)

where
M , , 
DT are the mono-, di- and triglyceride concentration in the sample respectively;
AMono , , 
ADi ATri are the sums of the peak areas of the mono-, di- and triglycerides respectively;

AMonoC19 is the peak area of internal standard Mono C19;

M MonoC19 s the weight of internal standard Mono C19, in mg;

ADiC38 is the peak area of internal standard Di C38;

M DiC38 is the weight of internal standard Di C38, in mg;

ATriC57 is the peak area of internal standard Tri C57;

M TriC57 is the weight of internal standard Tri C57, in mg;

m is the weight of the sample, in mg.
10.5 Total glycerol

Calculate the percentage of total glycerol in the sample (Gtotal) in % (m/m) using Formula (7):
G total =
G + 0, 255  M + 0, 146  D + 0, 103  T (7)

11 Expression of results
Free and total glycerol content is expressed to the nearest 0,001 % (m/m).
All glyceride contents are each expressed to the nearest 0,01 % (m/m).

12 Precision
12.1 Interlaboratory study (ILS)

An ILS organized in 2008 at European level with the participation of 16 laboratories, each having
carried out two determinations on each sample, gave the statistical results indicated in Annex D.
The precision, as determined by statistical examination in accordance with EN ISO 4259:2006 is given
in 12.2 and 12.3.

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