SIST EN ISO 12228-2:2015

Determination of individual and total sterols contents - Gas chromatographic method - Part 2: Olive and olive pomace oils (ISO 12228:2014)

This part of ISO 12228 specifies a procedure for the gas chromatographic determination of the contents
and composition of sterols and triterpene dialcohols in olive and olive pomace oils. For the determination
of the contents and composition of sterols in all other animal and vegetable fats and oils, ISO 12228-1 is
to be used.
NOTE This part of ISO 12228 is technically identical to IOC Standard COI/T.20/Doc. No. 30 (November 2011).

Bestimmung der individuellen und der Gesamtsterine - Gaschromatographisches Verfahren - Teil 2: Oliven- und Oliventresteröle (ISO 12228:2014)

Diese Internationale Norm legt ein Verfahren zur gaschromatographischen Bestimmung der Zusammen-setzung und des Gehalts an Sterinen und Triterpen-Dialkoholen in Oliven  und Oliventresterölen fest. Für die Bestimmung der Gehalte und der Zusammensetzung der Sterine in allen anderen tierischen und pflanzlichen Fetten und Ölen muss der Teil 1 dieser Norm angewendet werden.
ANMERKUNG   Diese Internationale Norm ist technisch identisch mit dem IOC Standard COI/T.20/Doc. No. 30 (November 2011).

Détermination de la teneur en stérols individuels et totaux - Méthode par chromatographie en phase gazeuse - Partie 2: Huile d'olive et huile de grignons d'olive (ISO 12228:2014)

L'ISO 12228-2:2014 spécifie un mode opératoire de détermination de la teneur et de la composition des stérols et des dialcools triterpéniques contenus dans l'huile d'olive et l'huile de grignons d'olive par chromatographie en phase gazeuse. Pour déterminer la teneur et la composition des stérols dans tout autre corps gras d'origine animale ou végétale, l'ISO 12228-1 doit être utilisée.

Določevanje posameznih in celotnih sterolov - Plinska kromatografska metoda - 2. del: Oljčna olja in olja iz oljčnih tropin (ISO 12228-2:2014)

Ta del standarda ISO 12228 določa postopek za plinsko kromatografsko določevanje vsebnosti
in sestave sterolov in triterpen dialkoholov v oljčnih oljih in oljih iz oljčnih tropin. Za določevanje vsebnosti in sestave sterolov v vseh drugih živalskih in rastlinskih masteh in oljih se uporablja standard ISO 12228-1.
OPOMBA: Ta del standarda ISO 12228 je tehnično identičen kot standard IOC COI/T.20/št. dok. 30 (november 2011).

General Information

• Status: Published
• Public Enquiry End Date: 14-Jan-2013
• Publication Date: 16-Dec-2014
• ICS: 67.200.10 - Animal and vegetable fats and oils
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 06-Nov-2014
• Due Date: 11-Jan-2015
• Completion Date: 17-Dec-2014
• Ref Project: EN ISO 12228-2:2014 - Determination of individual and total sterols contents - Gas chromatographic method - Part 2: Olive and olive pomace oils (ISO 12228-2:2014)

Relations

• Replaces: SIST EN ISO 12228:2000 - Animal and vegetable fats and oils - Determination of individual and total sterols contents - Gas chromatographic method (ISO 12228:1999)
• Effective Date: 01-Jan-2015
• Replaces: SIST EN ISO 12228:2000 - Animal and vegetable fats and oils - Determination of individual and total sterols contents - Gas chromatographic method (ISO 12228:1999)
• Effective Date: 08-Jun-2022

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 12228-2:2015
01-januar-2015
1DGRPHãþD
SIST EN ISO 12228:2000
'RORþHYDQMHSRVDPH]QLKLQFHORWQLKVWHURORY3OLQVNDNURPDWRJUDIVNDPHWRGD
GHO2OMþQDROMDLQROMDL]ROMþQLKWURSLQ,62
Determination of individual and total sterols contents - Gas chromatographic method -
Part 2: Olive and olive pomace oils (ISO 12228:2014)
Bestimmung der individuellen und der Gesamtsterine - Gaschromatographisches
Verfahren - Teil 2: Oliven- und Oliventresteröle (ISO 12228:2014)
Détermination de la teneur en stérols individuels et totaux - Méthode par
chromatographie en phase gazeuse - Partie 2: Huile d'olive et huile de grignons d'olive
(ISO 12228:2014)
Ta slovenski standard je istoveten z: EN ISO 12228-2:2014
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
SIST EN ISO 12228-2:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 12228-2:2015

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SIST EN ISO 12228-2:2015

EUROPEAN STANDARD
EN ISO 12228-2

NORME EUROPÉENNE

EUROPÄISCHE NORM
October 2014
ICS 67.200.10 Supersedes EN ISO 12228:1999
English Version
Determination of individual and total sterols contents - Gas
chromatographic method - Part 2: Olive and olive pomace oils
(ISO 12228:2014)
Détermination de la teneur en stérols individuels et totaux - Bestimmung der individuellen und der Gesamtsterine -
Méthode par chromatographie en phase gazeuse - Partie 2: Gaschromatographisches Verfahren - Teil 2: Oliven- und
Huile d'olive et huile de grignons d'olive (ISO 12228-2:2014) Oliventresteröle (ISO 12228-2:2014)
This European Standard was approved by CEN on 6 September 2014.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 12228-2:2014 E
worldwide for CEN national Members.

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SIST EN ISO 12228-2:2015
EN ISO 12228-2:2014 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 12228-2:2015
EN ISO 12228-2:2014 (E)
Foreword
This document (EN ISO 12228-2:2014) 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 April 2015, and conflicting national standards shall be withdrawn at the
latest by April 2015.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 12228:1999.
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, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 12228-2:2014 has been approved by CEN as EN ISO 12228-2:2014 without any modification.
3

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SIST EN ISO 12228-2:2015

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SIST EN ISO 12228-2:2015
INTERNATIONAL ISO
STANDARD 12228-2
First edition
2014-10-01
Determination of individual
and total sterols contents — Gas
chromatographic method —
Part 2:
Olive oils and olive pomace oils
Détermination de la teneur en stérols individuels et totaux —
Méthode par chromatographie en phase gazeuse —
Partie 2: Huile d’olive et huile de grignons d’olive
Reference number
ISO 12228-2:2014(E)
©
ISO 2014

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SIST EN ISO 12228-2:2015
ISO 12228-2:2014(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2014
All rights reserved. Unless otherwise specified, 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
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved

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SIST EN ISO 12228-2:2015
ISO 12228-2:2014(E)

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 2
6 Apparatus . 3
7 Sample . 4
7.1 Sampling . 4
7.2 Preparation of the test sample . 4
8 Procedure. 4
8.1 Test portion . 4
8.2 Preparation of unsaponifiable matter. 4
8.3 Separation of the sterol and triterpene dialcohols (erythrodiol, uvaol) fractions by TLC . 5
8.4 Preparation of the trimethylsilyl ethers . 6
8.5 Gas chromatographic analysis . 6
9 Expression of results . 7
9.1 Quantitative evaluation . 7
9.2 Determination of the total sterol content . 8
9.3 Composition of sterols . 8
9.4 Composition of triterpene dialcohols . 8
10 Precision . 9
10.1 Interlaboratory test. 9
10.2 Repeatability limit, r . 9
10.3 Reproducibility limit, R . 9
11 Test report . 9
Annex A (informative) Figures .10
Annex B (informative) Interlaborative trial .13
Bibliography .16
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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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils.
This first edition of ISO 12228-2 cancels and replaces ISO 12228:1999, which has been technically
revised.
ISO 12228 consists of the following parts, under the general title Determination of individual and total
sterols contents — Gas chromatographic method:
— Part 1: Animal and vegetable fats and oils
— Part 2: Olive oils and olive pomace oils
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SIST EN ISO 12228-2:2015
INTERNATIONAL STANDARD ISO 12228-2:2014(E)
Determination of individual and total sterols contents —
Gas chromatographic method —
Part 2:
Olive oils and olive pomace oils
1 Scope
This part of ISO 12228 specifies a procedure for the gas chromatographic determination of the contents
and composition of sterols and triterpene dialcohols in olive and olive pomace oils. For the determination
of the contents and composition of sterols in all other animal and vegetable fats and oils, ISO 12228-1 is
to be used.
NOTE This part of ISO 12228 is technically identical to IOC Standard COI/T.20/Doc. No. 30 (November 2011).
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. 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
composition of sterols
composition of individual sterols in the sample, beginning with cholesterol and ending with ∆7-avenasterol
(see Table 1) under the conditions specified in this part of ISO 12228
Note 1 to entry: The composition is expressed as a percentage of all peak areas, normalized to 100 %.
3.2
total sterol content
mass fraction of the sum of all individual sterols, as determined in accordance with the method specified
in this part of ISO 12228, beginning with cholesterol and ending with ∆7-avenasterol (see Table 1),
divided by the mass of the test portion
Note 1 to entry: The content is expressed in milligrams per kilogram.
3.3
composition of triterpene dialcohols
composition of erythrodiol and uvaol in the sample under the conditions specified in this part of
ISO 12228
Note 1 to entry: The composition is expressed as a percentage of all peak areas, beginning with cholesterol and
ending with uvaol (see Table 1) under the conditions specified in this part of ISO 12228, normalized to 100 %.
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4 Principle
A test portion is saponified by boiling under reflux with an ethanolic potassium hydroxide solution. The
unsaponifiable matter is extracted with diethyl ether. The sterol and triterpene dialcohol fractions are
separated from the unsaponifiable matter by thin-layer chromatography on a basic silica gel plate. The
qualitative and quantitative compositions of the sterol and triterpene dialcohol fractions are determined
by gas chromatography of the trimethylsilyl ethers using cholestanol as internal standard.
5 Reagents
WARNING — Attention is drawn to the regulations which specify the handling of hazardous
substances. Technical, organizational and personal safety measures shall be followed.
Use only reagents of recognized analytical grade, unless otherwise stated, and water complying with
[1]
grade 3 of ISO 3696.
5.1 Potassium hydroxide, minimum mass fraction w = 85 g/100 g.
5.2 Potassium hydroxide, ethanolic solution, amount concentration, c, approximately 2 mol/l.
While cooling, dissolve 130 g of potassium hydroxide (5.1) in 200 ml of distilled water and then make up
to 1 l with ethanol (5.9). Keep the solution in well-stoppered dark glass bottles and store for a maximum
of 2 d.
5.3 Potassium hydroxide, ethanolic solution, amount concentration, c, approximately 0,2 mol/l.
Dissolve 13 g of potassium hydroxide (5.1) in 20 ml of distilled water and make up to 1 l with ethanol
(5.9).
5.4 Diethyl ether, for chromatography.
WARNING — Diethyl ether is highly flammable and can form explosive peroxides. Explosive
limits in air are 1,7 % to 48 % (volume fraction). Take special precautions when using it.
5.5 Anhydrous sodium sulfate.
5.6 Silica gel thin-layer chromatography (TLC) plates, commercially available, dimensions
20 cm × 20 cm, thickness of layer 0,25 mm, without fluorescence indicator.
5.7 Acetone, for chromatography.
5.8 n-Hexane, for chromatography.
5.9 Ethanol 96 %, minimum volume fraction φ = 95 %.
5.10 Ethyl acetate.
5.11 Reference solution for thin-layer chromatography, cholesterol or mixture of phytosterols, and
erythrodiol solution in ethyl acetate (5.10), mass concentration, ρ = 5 %.
5.12 2,7-dichlorofluorescein, ethanolic solution, mass concentration, ρ = 0,2 %.
Make slightly basic by adding a few drops of 2 mol/l alcoholic potassium hydroxide solution (5.2). Store
for a maximum of 1 year.
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5.13 α-cholestanol internal standard solution, mass concentration, ρ = 0,2 g/100 ml, in ethyl acetate
(5.10).
5.14 Phenolphthalein solution, mass concentration, ρ = 10 g/l, in ethanol (5.9).
5.15 Carrier gas for gas chromatography, helium or preferably hydrogen.
5.16 Auxiliary gases for gas chromatography, hydrogen, helium, nitrogen, and air.
5.17 Developing solvent, mixture of n-hexane and diethyl ether, volume concentrations are: σ(n-
hexane) = 65 ml/100 ml, σ(diethyl ether) = 35 ml/100 ml.
5.18 Hexamethyldisilazane.
5.19 Trimethylchlorosilane.
5.20 Silylation reagent, mixture of pyridine, hexamethyldisilazane, and trimethylchlorosilane.
Volume concentration σ(pyridine) = 9 ml/13 ml, σ(hexamethyl disilazane) = 3 ml/13 ml,
σ(trimethylchlorosilane) = 1 ml/13 ml. Prepare the mixture fresh daily.
NOTE Other silylation reagents can be used, e. g., mixture of N,O-bis-trimethylsilyl-trifluoroacetamide
(BSTFA) and trimethylchlorosilane (TMCS), σ(BSTFA) = 99 ml/100 ml and σ(TMSC) = 1 ml/100ml.
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 Round-bottomed flasks, of 250 ml, with ground neck.
6.2 Reflux condenser, with ground glass joint to fit the flask (6.1).
6.3 Separating funnel, of 500 ml capacity.
6.4 Developing tank, made of glass, with a ground glass lid, suitable for use with plates of dimensions
20 cm × 20 cm.
6.5 Ultraviolet lamp, wavelength of 366 nm or 254 nm.
6.6 Microsyringe, to deliver 100 µl, 500 µl, and 1000 µl.
6.7 Cylindrical filter funnel, with sintered-glass filter (G3, porosity 15 µm to 40 µm), diameter
approximately 2 cm, depth 5 cm, suitable for filtration under vacuum with male ground glass joint.
6.8 Conical flask, for operation under a vacuum, 50 ml with ground glass female joint to fit to the filter
funnel (6.7).
6.9 Test tube, 10 ml with a tapering bottom and a sealing glass stopper.
6.10 Gas chromatograph, for capillary columns, with split injector, consisting of:
— column oven, capable of maintaining the temperature with an accuracy of ±1°C;
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— temperature-controlled injection unit, with persilanised glass vaporising element and split
system;
— flame ionization detector (FID);
— data acquisition system.
6.11 Capillary column, made of fused silica, length 20 m to 30 m, internal diameter 0,25 mm to 0,32 mm,
coated with 5 % Diphenyl, 95 % Dimethyl polysiloxane (SE-52 or SE-54 or equivalent stationary phase),
with a temperature limit of at least 280 °C to 300 °C, film thickness between 0,1 µm and 0,30 µm.
6.12 Microsyringe for gas chromatography, of 1 µl and 10 µl capacity, for gas chromatography, with
cemented needle suitable for split injection.
6.13 Desiccator, containing an efficient desiccant, for storing the plates, e.g. calcium dichloride.
6.14 Oven, maintained at 103 °C ± 2 °C.
6.15 Rotary evaporator, attached to a vacuum pump and water bath maintained at 40 °C.
6.16 Analytical balance, capable of weighing to the nearest 0,001 g and displaying 0,000 1 g.
7 Sample
7.1 Sampling
Sampling is not part of the method specified in this part of ISO 12228. A recommended sampling method
[2]
is given in ISO 5555.
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or changed during transport or storage.
7.2 Preparation of the test sample
Prepare the test sample in accordance with ISO 661. Dry the samples if necessary by filtration.
8 Procedure
8.1 Test portion
By means of a micro syringe (6.6), fill 500 µl (for olive oils) or 1 500 µl (for olive pomace oils) of the
internal standard solution (5.13) in a 250 ml flask (6.1). Evaporate until dryness with a gentle current of
nitrogen in a warm water bath and cool the flask.
Weigh, to the nearest 10 mg, about 5 g of olive or olive pomace oil into the same flask and proceed with
8.2.
NOTE Olive (pomace) oils, containing appreciable quantities of cholesterol, might show a peak having a
retention time identical to cholestanol. In these cases, the sterol fraction shall be analysed in duplicate with and
without the addition of the internal standard.
8.2 Preparation of unsaponifiable matter
8.2.1 Add 50 ml of 2 mol/l ethanolic potassium hydroxide solution (5.2) and some pumice stones, fit
the reflux condenser, and heat to gentle boiling until the solution becomes clear (end of saponification).
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Continue heating for a further 20 min, add 50 ml of distilled water through the top of the condenser,
detach the condenser, and cool the flask to approximately 30 °C.
8.2.2 Transfer the contents of the flask quantitatively into a 500 ml separating funnel (6.3) using several
portions of distilled water (50 ml). Add approximately 80 ml of diethyl ether (5.4), shake vigorously for
approximately 60 s, periodically releasing the pressure by inverting the separating funnel and opening
the stopcock. Allow to stand until the separation of two phases is complete.
Draw off the soap solution as completely as possible into a second separating funnel. Perform two further
extractions on the water-alcohol phase in the same way using 60 ml to 70 ml of diethyl ether (5.4).
Emulsions shall be destroyed by adding small quantities of ethanol (5.9) and swirling gently.
8.2.3 Combine the three diethyl ether extracts in one separating funnel containing 50 ml of water.
Continue to wash with water (50 ml) until the wash water no longer turns pink on the addition of a drop
of phenolphthalein solution (5.14).
After the removal of the wash water, filter through anhydrous sodium sulfate (5.5) into a previously
weighed 250 ml flask, washing the funnel and filter with small quantities of diethyl ether (5.4).
8.2.4 Evaporate the solvent with a rotary evaporator at 30 °C under vacuum. Add 5 ml of acetone (5.7)
and remove the volatile solvent completely in a gentle current of air. Dry the residue in the oven (6.14) at
103 °C ± 2 °C for 15 min. Cool in a desiccator and weigh to the nearest 0,1 mg.
8.3 Separation of the sterol and triterpene dialcohols (erythrodiol, uvaol) fractions
by TLC
8.3.1 Immerse the silica gel plates (5.6) about 4 cm in the 0,2 mol/l ethanolic potassium hydroxide
solution (5.3) for 10 s, allow to dry in a fume cupboard for two hours, and place in an oven at 100 °C for
1 h.
Remove the plates from the oven and store in the desiccator (6.13) until use (plates shall be stored for
maximum of 15 d).
NOTE When basic silica gel plates are used to separate the sterol fraction, all compounds of an acidic nature
(fatty acids and others) are retained on the spotting line and the sterol band is clearly separated from the aliphatic
and triterpene alcohols band.
8.3.2 Fill the developing solvent (5.17) to a depth of approximately 1 cm into the development tank
(6.4). Close the glass lid and leave for at least half an hour in a cool place to get a liquid-vapour equilibrium.
Strips of filter paper dipping into the eluent should be placed on the internal surfaces of the chamber
to reduce the developing time by approximately one-third. In addition, it gives a better elution of the
components.
The developing solvent shall be replaced for every test, in order to achieve more reproducible elution
conditions. It is also possible to use a mixture of n-hexane and diethyl ether (volume concentrations
σ = 50 ml/100 ml) as an alternative developing solvent.
8.3.3 Prepare a 5 % solution of the unsaponifiable (8.2.4) in ethyl acetate (5.10). Using the 100 µl
microsyringe, apply 0,3 ml of the solution as a line at a distance of 2 cm from the lower edge onto a TLC
plate (5.6). Leave a gap of at least 3 cm from each side edge of the plate. Apply a spot of 5 µl of the TLC
reference solution (5.11) at 1,5 cm from the edges.
8.3.4 Place the plate into the tank, prepared according to 8.3.2, and develop it until the solvent reaches
approximately 1 cm to 2 cm from the upper edge of the TLC plate. The ambient temperature should be
kept constant. Remove the plate from the developing chamber and allow the solvent to evaporate in a
fume cupboard.
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8.3.5 Spray the plate lightly and uniformly with the 2,7-dichlorofluorescein solution (5.12) and
then leave to dry. Observe the plate in the ultraviolet light (6.5) and identify the sterols and triterpene
dialcohols bands with the aid of the spots from the reference solution (5.11). Mark the zones at the high
of the standard spots with a black pencil (shown in Figure A.1).
8.3.6 Scratch off the marked zone completely using a spatula and quantitatively collect the silica in the
filter funnel (6.7). Add 10 ml of hot ethyl acetate (5.10), mix carefully with the metal spatula, and filter
under vacuum, collecting the filtrate in a conical flask (6.8.) attached to the filter funnel. Wash the residue
in the flask three times with 10 ml of diethyl ether (5.4), and filter in the same flask attached to the funnel.
Evaporate the combined ether extracts to a volume of 4 ml to 5 ml, transfer the residual solution to the
previously weighed 10 ml test tube (6.9), and blow off the solvent with a gentle stream of nitrogen. Add a
few drops of acetone (5.7) and blow off the acetone to dryness. The residue in the test tube contains the
sterol and triterpene dialcohol fractions.
8.4 Preparation of the trimethylsilyl ethers
8.4.1 Add the silylation reagent (5.20) into the test tube. Use 50 µl reagent for 1 mg of sterols and
triterpene dialcohols.
NOTE Ready-to-use solutions are commercially available. Pyridine can be replaced by acetonitrile.
8.4.2 Stopper the test tube, shake carefully to dissolve completely. Leave to stand for at least 15 min
at ambient temperature and then centrifuge for a few minutes. The clear solution is ready for gas
chromatographic analys
...