SIST EN ISO 12872:2023
(Main)Olive oils and olive-pomace oils - Determination of the 2-glyceryl monopalmitate content (ISO 12872:2022)
Olive oils and olive-pomace oils - Determination of the 2-glyceryl monopalmitate content (ISO 12872:2022)
ISO 12872:2010 specifies a procedure for the determination of the content, as a percentage mass fraction, of 2-glyceryl monopalmitate content in olive oils and olive-pomace oils that are liquid at ambient temperature (20 °C).
Olivenöle und Oliventresteröle - Bestimmung des Gehalts an 2‑Glycerylmonopalmitat (ISO 12872:2022)
Dieses Dokument legt ein Verfahren fest zur Bestimmung des Gehalts, als Massenanteil in Prozent, an 2 Glycerinmonopalmitat in Olivenölen und Oliventresterölen, die bei Umgebungstemperatur (20 °C) flüssig sind.
ANMERKUNG Dieses Dokument basiert auf COI/T.20/Doc. No 23/Rev.1:2017 [7].
Huiles d'olive et huiles de grignons d'olive - Détermination de la teneur en 2-glycéryl monopalmitate (ISO 12872:2022)
L'ISO 12872:2010 spécifie une méthode pour la détermination de la teneur, sous forme de fraction massique en pourcentage, en 2-glycéryl monopalmitate des huiles d'olive et des huiles de grignons d'olive liquides à température ambiante (20 °C).
Oljčna olja in olja iz oljčnih tropin - Določevanje vsebnosti 2-gliceril monopalmitata (ISO 12872:2022)
Standard ISO 12872:2010 navaja postopek za določevanje vsebnosti 2-gliceril monopalmitata kot odstotek masnega deleža v oljčnih oljih in oljih iz oljčnih tropin, ki so pri temperaturi okolja (20° C) v tekočem stanju.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 12872:2023
01-januar-2023
Nadomešča:
SIST EN ISO 12872:2014
Oljčna olja in olja iz oljčnih tropin - Določevanje vsebnosti 2-gliceril monopalmitata
(ISO 12872:2022)
Olive oils and olive-pomace oils - Determination of the 2-glyceryl monopalmitate content
(ISO 12872:2022)
Olivenöle und Oliventresteröle - Bestimmung des Gehalts an 2‑Glycerylmonopalmitat
(ISO 12872:2022)
Huiles d'olive et huiles de grignons d'olive - Détermination de la teneur en 2-glycéryl
monopalmitate (ISO 12872:2022)
Ta slovenski standard je istoveten z: EN ISO 12872:2022
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
SIST EN ISO 12872:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 12872:2023
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SIST EN ISO 12872:2023
EN ISO 12872
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2022
EUROPÄISCHE NORM
ICS 67.200.10 Supersedes EN ISO 12872:2014
English Version
Olive oils and olive-pomace oils - Determination of the 2-
glyceryl monopalmitate content (ISO 12872:2022)
Huiles d'olive et huiles de grignons d'olive - Olivenöle und Oliventresteröle - Bestimmung des
Détermination de la teneur en 2-glycéryl Gehalts an 2-Glycerylmonopalmitat (ISO 12872:2022)
monopalmitate (ISO 12872:2022)
This European Standard was approved by CEN on 20 September 2022.
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, Türkiye 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
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 12872:2022 E
worldwide for CEN national Members.
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SIST EN ISO 12872:2023
EN ISO 12872:2022 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 12872:2023
EN ISO 12872:2022 (E)
European foreword
This document (EN ISO 12872:2022) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with 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 March 2023, and conflicting national standards shall
be withdrawn at the latest by March 2023.
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 ISO 12872:2014.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations 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, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 12872:2022 has been approved by CEN as EN ISO 12872:2022 without any modification.
3
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SIST EN ISO 12872:2023
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SIST EN ISO 12872:2023
INTERNATIONAL ISO
STANDARD 12872
Second edition
2022-08
Olive oils and olive-pomace oils —
Determination of the 2-glyceryl
monopalmitate content
Huiles d'olive et huiles de grignons d'olive — Détermination de la
teneur en 2-glycéryl monopalmitate
Reference number
ISO 12872:2022(E)
© ISO 2022
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SIST EN ISO 12872:2023
ISO 12872:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2022 – All rights reserved
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SIST EN ISO 12872:2023
ISO 12872:2022(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 1
6 Apparatus . 3
7 Sampling . 4
8 Preparation of the test sample. 4
9 Procedure .4
9.1 Preparatory steps . 4
9.2 Column chromatography . 5
9.2.1 Conventional procedure . 5
9.2.2 Procedure when ready-to-use silica SPE cartridges are employed . 5
9.3 Hydrolysis with pancreatic lipase . 5
9.4 Preparation of the silylated derivatives and gas chromatography . 5
9.5 Gas chromatography . 5
9.5.1 Operating conditions . 5
9.5.2 Identification of peaks . 6
9.5.3 Quantitative evaluation . 6
10 Expression of results . 6
11 Precision . 6
11.1 Interlaboratory test . . 6
11.2 Repeatability . 6
11.3 Reproducibility . 6
12 Test report . 7
Annex A (informative) Chromatograms . 8
Annex B (informative) Results of interlaboratory test and comparison study .11
Annex C (informative) Preparation and activity of the lipase .13
Bibliography .15
iii
© ISO 2022 – All rights reserved
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SIST EN ISO 12872:2023
ISO 12872:2022(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11,
Animal and vegetable fats and oils, in collaboration with the European Committee for Standardization
(CEN) Technical Committee CEN/TC 307, Oilseeds, vegetable and animal fats and oils and their by-products
— Methods of sampling and analysis, in accordance with the Agreement on technical cooperation
between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 12872:2010), which has been technically
revised.
The main changes are as follows:
— the use of iso-octane as an alternative to hexane has been added;
— precision data of the method using iso-octane compared with hexane have been added.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
© ISO 2022 – All rights reserved
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SIST EN ISO 12872:2023
ISO 12872:2022(E)
Introduction
As part of the Trade standard applying to olive oils and olive-pomace oils, the International Olive Council
[6]
(IOC) published COI/T.20/Doc. No 23:2006 .
COI/T.20/Doc. No 23 was applicable to olive and olive-pomace oils and was used to distinguish between
lampante virgin olive oils and crude olive-pomace oils. Olive pomace is the residual paste which still
contains a variable amount of water and oil after pressing or centrifuging.
In 2008, the IOC submitted the document to ISO/TC 34/SC 11 for adoption as an International Standard.
[7]
In 2017, the IOC published a revision of COI/T.20/Doc. No 23/Rev.1 , and this revised document is an
adoption of the IOC revised method.
v
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SIST EN ISO 12872:2023
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SIST EN ISO 12872:2023
INTERNATIONAL STANDARD ISO 12872:2022(E)
Olive oils and olive-pomace oils — Determination of the
2-glyceryl monopalmitate content
1 Scope
This document specifies a procedure for the determination of the content, as a percentage mass fraction,
of 2-glyceryl monopalmitate in olive oils and olive-pomace oils that are liquid at ambient temperature
(20 °C).
[7]
NOTE This document is based on COI/T.20/Doc. No 23/Rev.1:2017 .
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.
ISO 661, Animal and vegetable fats and oils — Preparation of test sample
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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/
3.1
2-glyceryl monopalmitate content
mass fraction of 2-glyceryl monopalmitate in the monoacylglycerol fraction
Note 1 to entry: This is determined according to the method specified in this document.
Note 2 to entry: The 2-glyceryl monopalmitate content is expressed as a percentage.
4 Principle
The oil, after suitable preparation, is subjected to the action of pancreatic lipase. A partial
hydrolysis takes place that is specific for positions 1 and 3 of the triacylglycerol molecule so that
2-monoacylglycerols are obtained as reaction products. The percentage of 2-glyceryl monopalmitate in
the monoacylglycerol fraction is determined, after silylation, by capillary gas chromatography.
5 Reagents
WARNING — Technical, organizational and personal safety measures shall be followed.
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade, and
distilled or demineralized water or water of equivalent purity.
1
© ISO 2022 – All rights reserved
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SIST EN ISO 12872:2023
ISO 12872:2022(E)
5.1 Silica gel, with a particle size of 0,063 mm to 0,200 mm (70/280 mesh), prepared as follows:
— put the silica gel into a porcelain cup, dry in an oven at 160 °C for 4 h, then cool at ambient temperature
in a desiccator;
— add a volume of water equivalent to 5 % of the mass of the silica gel as follows:
— weigh 152 g of silica gel into a 500 ml Erlenmeyer flask, add 8 g of water, stopper and homogenize
carefully;
— leave to settle for at least 12 h before using.
5.2 n-Hexane, chromatography grade.
Hexane may be replaced by iso-octane (2,2,4-trimethylpentane of chromatography grade), provided
that comparable precision values are achieved (see the precision values of the method with the use of
iso-octane in Annex B).
5.3 Isopropanol.
5.4 Isopropanol-water mixture, volume fractions 50 ml/100 ml.
5.5 Pancreatic lipase, activity between 2,0 and 10 lipase units per milligram (see Annex C).
NOTE Pancreatic lipase with an activity between 2 and 10 units per mg of enzyme is available commercially.
5.6 Buffer solution of tris(hydroxymethyl)aminomethane: prepare an aqueous solution (1 mol/l)
with pH 8 and mix with concentrated HCl, volume fractions 50 ml/100 ml.
5.7 Sodium cholate, special enzyme grade, aqueous solution, mass fraction 0,1 g/100 g.
Use this solution within 15 days of preparation.
5.8 Calcium chloride, aqueous solution, mass fraction 22 g/100 g.
5.9 Diethyl ether, chromatography grade.
5.10 Elution solvent 1: mixture of n-hexane-diethyl ether, volume fraction of n-hexane 87 ml/100 ml
and of diethyl ether 13 ml/100 ml.
5.11 Sodium hydroxide, aque
...
SLOVENSKI STANDARD
oSIST prEN ISO 12872:2022
01-maj-2022
Oljčna olja in olja iz oljčnih tropin - Določevanje vsebnosti 2-gliceril monopalmitata
(ISO/DIS 12872:2022)
Olive oils and olive-pomace oils - Determination of the 2-glyceryl monopalmitate content
(ISO/DIS 12872:2022)
Olivenöle und Oliventresteröle – Bestimmung des Gehalts an 2-Glycerylmonopalmitat
(ISO/DIS 12872:2022)
Huiles d'olive et huiles de grignons d'olive - Détermination de la teneur en 2-glycéryl
monopalmitate (ISO/DIS 12872:2022)
Ta slovenski standard je istoveten z: prEN ISO 12872
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
oSIST prEN ISO 12872:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 12872:2022
---------------------- Page: 2 ----------------------
oSIST prEN ISO 12872:2022
DRAFT INTERNATIONAL STANDARD
ISO/DIS 12872
ISO/TC 34/SC 11 Secretariat: BSI
Voting begins on: Voting terminates on:
2022-03-16 2022-06-08
Olive oils and olive-pomace oils — Determination of the
2-glyceryl monopalmitate content
Huiles d'olive et huiles de grignons d'olive — Détermination de la teneur en 2-glycéryl monopalmitate
ICS: 67.200.10
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 12872:2022(E)
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. © ISO 2022
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oSIST prEN ISO 12872:2022
ISO/DIS 12872:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2022 – All rights reserved
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oSIST prEN ISO 12872:2022
ISO/DIS 12872:2022(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 1
6 Apparatus . 2
7 Sampling . 3
8 Preparation of the test sample. 4
9 Procedure .4
9.1 Preparatory steps . 4
9.2 Column chromatography . 4
9.2.1 Conventional procedure . 4
9.2.2 Procedure when ready-to-use silica SPE cartridges are employed . 5
9.3 Hydrolysis with pancreatic lipase . 5
9.4 Preparation of the silylated derivatives and gas chromatography . 5
9.5 Gas chromatography . 5
9.5.1 Operating conditions . 5
9.5.2 Identification of peaks . 5
9.5.3 Quantitative evaluation . 6
10 Expression of results . 6
11 Precision . 6
11.1 Interlaboratory test . . 6
11.2 Repeatability . 6
11.3 Reproducibility . 6
12 Test report . 6
Annex A (informative) Chromatograms . 7
Annex B (informative) Results of an interlaboratory test .10
Annex C (informative) Preparation and activity of the lipase .12
Bibliography .14
iii
© ISO 2022 – All rights reserved
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oSIST prEN ISO 12872:2022
ISO/DIS 12872:2022(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International
Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies
casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 12872 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11,
Animal and vegetable fats and oils.
iv
© ISO 2022 – All rights reserved
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oSIST prEN ISO 12872:2022
ISO/DIS 12872:2022(E)
Introduction
As part of the Trade standard applying to olive oils and olive-pomace oils, the International Olive Council
[6]
(IOC) published COI/T.20/Doc. 23:2006 . COI/T.20/Doc. 23 was applicable to olive and olive-pomace
oils and was used to distinguish between lampante virgin olive oils and crude olive-pomace oils. Olive
pomace is the residual paste which still contains a variable amount of water and oil after pressing or
centrifuging.
In 2008, the IOC submitted the document to ISO/TC 34/SC 11 for adoption as an International Standard.
In 2017, the IOC published a revision of the COI/T.20/Doc. n°23 (Rev.1), and this revised ISO document
is an adoption of the IOC revised method.
v
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oSIST prEN ISO 12872:2022
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oSIST prEN ISO 12872:2022
DRAFT INTERNATIONAL STANDARD ISO/DIS 12872:2022(E)
Olive oils and olive-pomace oils — Determination of the
2-glyceryl monopalmitate content
1 Scope
This International Standard specifies a procedure for the determination of the content, as a percentage
mass fraction, of 2-glyceryl monopalmitate in olive oils and olive-pomace oils that are liquid at ambient
temperature (20 °C).
[6]
NOTE This International Standard is based on COI/T.20/Doc. n°23/Rev.1:2017 .
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 661, Animal and vegetable fats and oils — Preparation of test sample
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
2-glyceryl monopalmitate content
mass fraction of 2-glyceryl monopalmitate in the monoacylglycerol fraction, determined according to
the method specified in this International Standard
Note 1 to entry: The 2-glyceryl monopalmitate content is expressed as a percentage.
4 Principle
The oil, after suitable preparation, is subjected to the action of pancreatic lipase. A partial
hydrolysis takes place that is specific for positions 1 and 3 of the triacylglycerol molecule so that
2-monoacylglycerols are obtained as reaction products. The percentage of 2-glyceryl monopalmitate in
the monoacylglycerol fraction is determined, after silylation, by capillary gas chromatography.
5 Reagents
WARNING — Comply with any local regulations which specify the handling of hazardous
substances. Technical, organizational and personal safety measures shall be followed.
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade, and
distilled or demineralized water or water of equivalent purity.
5.1 Silica gel, with a particle size of 0,063 mm to 0,200 mm (70/280 mesh), prepared as follows: put
the silica gel into a porcelain cup, dry in an oven at 160 °C for 4 h, then cool at ambient temperature in a
desiccator. Add a volume of water equivalent to 5 % of the mass of the silica gel as follows: weigh 152 g
of silica gel into a 500 ml Erlenmeyer flask, add 8 g of water, stopper and homogenize carefully. Leave to
settle for at least 12 h before using.
5.2 n-Hexane, chromatography grade.
1
© ISO 2022 – All rights reserved
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oSIST prEN ISO 12872:2022
ISO/DIS 12872:2022(E)
NOTE Hexane may be replaced by iso-octane (2,2,4- trimethylpentane in chromatography grade), provided
that comparable precision values are achieved (see Precision values of the method with the used of isooctane in
Annex B).
5.3 Isopropanol.
5.4 Isopropanol-water mixture, volume fractions 50 ml/100 ml.
5.5 Pancreatic lipase, activity between 2,0 and 10 lipase units per milligram (see Annex C).
NOTE Pancreatic lipase with an activity between 2 and 10 units per mg of enzyme is available commercially.
5.6 Buffer solution of tris(hydroxymethyl)aminomethane: prepare an aqueous solution (1 mol/l)
with pH 8 and mix with concentrated HCl, volume fractions 50 ml/100 ml.
5.7 Sodium cholate, special enzyme grade, aqueous solution, mass fraction 0,1 g/100 g.
Use this solution within 15 days of preparation.
5.8 Calcium chloride, aqueous solution, mass fraction 22 g/100 g.
5.9 Diethyl ether, chromatography grade.
5.10 Elution solvent 1: mixture of n-hexane-diethyl ether, volume fraction of n-hexane 87 ml/100 ml
and of diethyl ether 13 ml/100 ml.
5.11 Sodium hydroxide, aqueous solution, mass fraction 12 g/100 g.
5.12 Phenolphthalein, ethanolic solution, mass concentration 1 g/100 ml.
5.13 Carrier gas: hydrogen or helium, gas chromatography grade.
5.14 Auxiliary gases: hydrogen, free from moisture and organic substances, and synthetic air, gas
chromatography grade.
5.15 Silylation reagent: mixture of pyridine, hexamethyldisilazane (HMDS) and trimethylchlorosilane
(TMCS); volume fractions: 9 ml/13 ml, 3 ml/13 ml, and 1 ml/13 ml, respectively.
NOTE Ready-to-use solutions are available commercially. Other silylation reagents can be used, e.g. bis
trimethylsilyl trifluoracetamide + 1 % trimethylchlorosilane, diluted with an identical volume of anhydrous
pyridine.
5.16 Reference samples: pure monoacylglycerols and mixtures of monoacylglycerols with a known
composition similar to that of the sample.
5.17 Elution solvent 2: mixture of n-hexane-diethyl ether, volume fraction of n-hexane 90 ml/100 ml
and of diethyl ether 10 ml/100 ml.
6 Apparatus
Usual laboratory equipment and in particular the following.
6.1 Erlenmeyer flasks, of capacity 25 ml.
2
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oSIST prEN ISO 12872:2022
ISO/DIS 12872:2022(E)
6.2 Beakers, of capacities 100 ml, 250 ml, and 300 ml.
6.3 Glass chromatography column, 21 mm to 23 mm internal diameter, 400 mm in length, with
septum and stopcock.
[2]
6.4 Measuring cylinders, of capacities 10 ml, 50 ml, 100 ml, and 200 ml, ISO 4788 class A.
6.5 Round-bottomed flasks, of capacities 100 ml and 250 ml.
6.6 Rotary evaporator.
6.7 Centrifuge tubes, conical bottom, of capacity 10 ml, with ground-glass stopper.
6.8 Centrifuge, suitable for 10 ml and 100 ml tubes.
6.9 Water bath, capable of maintaining a temperature of (40 ± 0,5) °C.
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6.10 Graduated pipettes, of capacities 1 ml and 2 ml, ISO 835 class A.
6.11 Hypodermic syringe, 1 ml.
6.12 Microsyringe, 100 µl.
6.13 Separating funnel, 1 000 ml.
6.14 Gas chromatograph, suitable for use with capillary columns, equipped with the components
specified in 6.14.1 to 6.14.5.
6.14.1
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