TECHNICAL ISO/TS

SPECIFICATION 23647

First edition
2010-10-01

Vegetable fats and oils — Determination

of wax content by gas chromatography
Corps gras d'origine végétale — Détermination de la teneur en cires par
chromatographie en phase gazeuse

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Reference number
ISO/TS 23647:2010(E)

© ISO 2010
ISO/TS 23647:2010(E)

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 ISO/TS 23647:2010(E)

Contents Page

Foreword ............................................................................................................................................................iv
Introduction.........................................................................................................................................................v
1 Scope ......................................................................................................................................................1
2 Normative references............................................................................................................................1
3 Terms and definitions ...........................................................................................................................1
4 Principle..................................................................................................................................................1
5 Reagents and materials ........................................................................................................................1
6 Apparatus ...............................................................................................................................................2
7 Sampling.................................................................................................................................................3
8 Preparation of the test sample .............................................................................................................3
9 Procedure ...............................................................................................................................................3
9.1 Preparation of silver nitrate-impregnated silica gel 60 .....................................................................3
9.2 Column packing.....................................................................................................................................3
9.3 iTeh STANDARD PREVIEW
Isolation of waxes..................................................................................................................................3
9.4 Gas chromatographic determination of waxes ..................................................................................4
10
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Precision.................................................................................................................................................6
10.1 Results of interlaboratory test .............................................................................................................6
10.2 ISO/TS 23647:2010
Repeatability ..........................................................................................................................................6
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Reproducibility ......................................................................................................................................6
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11 Test report ..............................................................................................................................................7
Annex A (informative) Chromatograms ............................................................................................................8
Annex B (informative) Results of interlaboratory tests ................................................................................12
Annex C (informative) Preparation of crystalline sunflower wax standard ................................................13
Bibliography......................................................................................................................................................16

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ISO/TS 23647:2010(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.

In other circumstances, particularly when there is an urgent market requirement for such documents, a
technical committee may decide to publish other types of document:

⎯ an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in
an ISO working group and is accepted for publication if it is approved by more than 50 % of the members
of the parent committee casting a vote;
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⎯ (standards.iteh.ai)
an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical
committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting
a vote.
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An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a
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further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is
confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an
International Standard or be withdrawn.

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/TS 23647 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11,
Animal and vegetable fats and oils.

iv © ISO 2010 – All rights reserved

 ISO/TS 23647:2010(E)

Introduction
Vegetable fats and oils consist predominantly of triacylglycerols, but they also contain small amounts of
non-glyceridic substances often called minor components. The composition of these minor constituents (e.g.
sterols, steryl esters, triterpene dialcohols, and waxes) provides highly characteristic information about the
identity of the oils. Since the physical and chemical steps in the processing of vegetable fats and oils can
cause alteration in their composition and content, analysis of these minor components can be conveniently
applied to characterize the processing steps through which an oil was obtained.

Waxes are natural compounds occurring in various vegetable oils and can easily crystallize at low
temperatures resulting in cloudiness. Wax removal is very often a part of the vegetable oil-refining process
(e.g. sunflower, rice bran, corn oil) and its efficiency requires measurement.

This Technical Specification does not cover the analysis of waxes of olive oil.

With the exception of olive oil, there is currently no reliable official method to measure the wax content and
composition of vegetable fats and oils. Cold tests give neither qualitative nor quantitative results; methods
developed for olive oils are not applicable to seed oils and can cause a lot of problems in the interpretation of
results. There is an industrial and commercial need for an International Standard which is applicable to crude,
degummed, pre-dewaxed, winterized, and fully refined vegetable fats and oils.
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Waxes from different vegetable oils and fats are separated from triacylglycerols and from other non-glyceridic
compounds containing double bonds by column chromatography using a mixed column packing consisting of
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silica gel and silica gel impregnated with AgNO3.

The wax fraction is further analysed by capillary ISO/TS 23647:2010

gas chromatography. The method also gives information
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about the total content of waxes and their composition. The information given by the method can be easily
used to check oil quality and to track the f5efc8259f33/iso-ts-23647-2010
efficiency of the dewaxing process.

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TECHNICAL SPECIFICATION ISO/TS 23647:2010(E)

Vegetable fats and oils — Determination of wax content by gas

chromatography

1 Scope
This Technical Specification specifies a gas chromatographic method for determining the wax content of crude,
degummed, neutralized, winterized, and fully refined vegetable oils, such as sunflower, soybean, rapeseed,
corn, and rice bran oils. It is not applicable to olive oils or olive pomace oils.

Waxes are esters of long chain fatty acids and fatty alcohols (having C20 or longer saturated carbon chain).

The wax content is expressed in milligrams per kilogram of oil.

2 Normative references

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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
(standards.iteh.ai)
document (including any amendments) applies.

ISO 661, Animal and vegetable fats and oils — Preparation

ISO/TS 23647:2010of test sample
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3 Terms and definitions
For the purpose of this document, the following terms and definitions apply.

3.1
wax content
mass fraction of those substances in the sample, which are determined under the conditions specified in this
Technical Specification

NOTE The wax content is expressed in milligrams per kilogram of oil.

4 Principle
The waxes are separated by column chromatography using a mixed column packing consisting of silica gel
and silica gel impregnated with AgNO3. Determination of the waxes is carried out using capillary gas
chromatography (GC), applying an internal standard, previously added to the oil.

5 Reagents and materials

WARNING — Attention is drawn to the regulations which specify the handling of dangerous matter.
Technical, organizational and personal safety measures shall be followed.

During the analysis, unless otherwise stated, use only reagents of recognized analytical grade suitable for
HPLC column chromatography and distilled or demineralized water or water of equivalent purity.

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ISO/TS 23647:2010(E)

5.1 n-Hexane.

5.2 Dichloromethane.

5.3 n-Heptane.

5.4 Chloroform.

5.5 Silica gel 60, of particle size 0,063 mm to 0,200 mm (70 mesh to 230 mesh), such as Merck
No. 1077341).

5.6 Silver nitrate (AgNO3).

5.7 Hexatriacontane2) (C36 paraffin).

5.7.1 Internal standard solution, mass concentration 0,1 mg/ml in n-heptane.

5.7.2 Standard for determination of response factor, mass concentration 1 mg/ml in n-heptane.

5.8 Standards for peak identification in the gas chromatogram, pure waxes3), e.g. C40, C44.

5.8.1 Standard for determination of response factor, stearyl stearate or stearic acid stearyl ester
(C36 wax), mass concentration 1 mg/ml in chloroform.

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5.9 Crystallized sunflower wax, prepared from winter cake or from crude sunflower oil using crystallization.
For the preparation, see Annex C.
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5.10 Cotton wool, surgical quality, non-absorbent.
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6 Apparatus f5efc8259f33/iso-ts-23647-2010

6.1 Chromatographic column, made of glass, of internal diameter 30 mm and length 450 mm, equipped
with a polytetrafluoroethylene (PTFE) tap.

6.2 Glass rod, about 600 mm in length.

6.3 Pasteur pipette, ISO 7712[4].

6.4 Vacuum rotary evaporator.

6.5 Round-bottomed flask, capacity 250 ml (to collect wax fraction).

6.6 Pear-shaped flask, capacity 25 ml (to collect the concentrated wax fraction).

6.7 Gas chromatograph, equipped with flame ionization detector, split/splitless injector and integrator or
data acquisition system.

1) Merck No. 107734 is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of this product. Equivalent
products may be used if they can be shown to lead to the same results.
2) Hexatriacontane is available from Sigma-Aldrich with a purity of min. 99 % mass fraction. This information is given for
the convenience of users of this document and does not constitute an endorsement by ISO of this supplier.
3) Pure waxes e.g. behenic acid stearyl ester (C40), stearic acid stearyl ester (C36) and behenic acid behenyl ester (C44)
are available from Sigma-Aldrich, purity approximately 99 % mass fraction. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO of this supplier.

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6.8 Fused silica capillary column, length 25 m, internal diameter 0,32 mm or preferably 0,20 mm, coated
with dimethylpolysiloxane (e.g. HP-1 or OV-1 or equivalent), film thickness 0,11 µm.

7 Sampling
Sampling is not part of the method specified in this Technical Specification. A recommended sampling method
is given in ISO 5555[1].

It is important the laboratory receive a truly representative sample which has not been damaged or changed
during transport or storage.

8 Preparation of the test sample

Prepare the test sample in accordance with ISO 661.

Heat the laboratory sample up to 130 °C. Mix it so as to melt completely the crystallized waxes and remove
any moisture (a magnetic stirrer or microwave oven may be used).

9 Procedure

9.1 Preparation of silver nitrate-impregnated silica gel 60

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Place 100 g of silica gel 60 (5.5) into a ceramic bowl. Pour a silver nitrate solution, containing 5 g AgNO (5.6)
dissolved in 240 ml water on to (standards.iteh.ai)
3
the silica gel and, after thorough mixing, make its surface flat. Put this
suspension into a cold electric oven, heat to 170 °C and activate the gel overnight. Allow to cool slowly to
50 °C in the electric oven (in the dark). ISO/TS Keep this column packing in the dark in a well-sealed bottle. If
23647:2010
necessary, remove the dark particles from the impregnated silica gel surface.
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The gel contains 5 % mass fraction AgNO3.

9.2 Column packing

Using the glass rod (6.2) place a wad of the cotton wool (5.10) in the lower part of the column (6.1) and press
it down. Pour about 30 ml of n-hexane (5.1) into the column and remove air by pressing the cotton wool down
with the rod. Allow the solvent layer to rise approximately 2 mm to 3 mm above the cotton wool before putting
the silica gel into the column.

Introduce 3 g of impregnated silica gel (9.1) into the glass column (6.1), remove any entrapped air, and flatten
the surface. Add 12 g of silica gel 60 (5.5) on the top of the previous packing, remove any air, and protect the
packing against light. Wash the column packing with approximately 90 ml to 100 ml of n-hexane, removing
any air bubbles by slight tapping if necessary. The tap should be open when adding the solvent for washing.
Allow approximately 5 mm of solvent layer above the column packing before adding the sample solution.

9.3 Isolation of waxes

9.3.1 Sample preparation

Solvent mixture A: n-hexane (5.1) and dichloromethane (5.2), volume fraction of hexane,
ϕC H = 95 ml/100 ml and of dichloromethane, ϕCH Cl = 5 ml/100 ml.
6 14 2 2

Weigh the sample according to the expected wax content (sunflower oil: 3 g from crude oil and neutralized oil,
and 4 g to 4,5 g from winterized and fully refined oil, other oils: 4 g to 4,5 g). Add 3 ml of internal standard
solution (5.7.1) and add 7 ml of solvent mixture A to the sample. Mix thoroughly and transfer approximately
2 ml of this solution by means of a Pasteur pipette (6.3) into the column.

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ISO/TS 23647:2010(E)

Adjust the flow rate to 1,5 ml/min to 2 ml/min and wash the inner surface of the glass column three times with
approximately 3 ml of the solvent mixture A. Do not let the column run dry, allow approximately 5 mm of
solvent layer above the column packing before adding the next portion of the solvent.

9.3.2 Column chromatography

Solvent mixture B: n-hexane (5.1) and dichloromethane (5.2), volume fraction of hexane,
ϕC H = 80 ml/100 ml and of dichloromethane, ϕCH Cl = 20 ml/100 ml.
6 14 2 2

Elute the waxes with 190 ml of Solvent B. Adjust the flow rate to approximately 3 ml/min.

Collect all material eluting from the column in a round-bottomed flask (6.5), and evaporate the solvent by
means of a vacuum rotary evaporator (6.4). Dissolve the residue in a small quantity of chloroform (5.4)
transfer the solution into a pear-shaped flask (6.6), evaporate the solvent again and re-dissolve the waxes in
approximately 1 ml of chloroform. The GC analysis is carried out using this solution.

9.4 Gas chromatographic determination of waxes

9.4.1 Recommended gas chromatographic conditions

Column: HP-1 (25 m × 0,20 mm, 0,11 µm film)

Oven: 170 °C (0,1 min); 170 °C to 350 °C (6 °C/min); 350 °C isotherm (20 min)

Carrier gas: H2 iTeh STANDARD PREVIEW

Flow rate: 1,4 ml/min
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Injector: 355 °C, split ratio: 1:30 ISO/TS 23647:2010
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These conditions may be adjusted in accordance with the characteristics of the gas chromatograph apparatus
and the column to give good separation for the wax compounds.

9.4.2 Determination of response factor

Mix equal volumes of the standard solutions (5.7.2 and 5.8.1), and inject 1 µl of the mixture into the GC to
determine the response factor, Fr

AIS ρ
Fr =
A ρ IS

where

AIS is the area of the hexatriacontane (5.7.2) peak;

A is the area of the stearyl stearate peak;

ρ is the mass concentration of stearyl stearate (5.8.1);

ρIS is the mass concentration of hexatriacontane (5.7.2).

The value of Fr should be between 1,1 and 1,2. Keep this solution in a sealed vial in the dark and repeat the
determination as required, normally once a week.

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 ISO/TS 23647:2010(E)

9.4.3 Calculation of wax content

Inject 1,5 µl to 2 µl of solution of waxes (isolated according to 9.3) into the GC. The elution order is the
following: solvent, hydrocarbons, internal standard (hexatriacontane), waxes.

If the isolation (9.3) is done properly, the wax fraction (especially the sunflower wax) should show a typical
pattern: continuously decreasing peak area after that for C48 (see Figure A.1 and Table C.1). However,
overlapping peaks or big peaks (e.g. sterol esters and triglycerides) at the end of the wax region can make
data analysis impossible. In this case, repeat the column chromatography (see Figure A.4).

9.4.3.1 Sunflower oil

The main components of sunflower wax are: C44, C46, C48 waxes. The sunflower wax contains mainly waxes
with an even number of carbon atoms e.g. C44, C46, C48, C50, C52, C54. Among these major peaks, minor
peaks with an odd carbon number can also be found. All the peaks (minor and major peaks) are used in the
calculation.

The calculation is based on a series of previous measurements and the literature (References [5][6][7]). The
wax content, wt, expressed as a mass fraction in milligrams per kilogram oil, is given by:

Fr ⎡ A>C 44 + (1 + k ) AC 44 ⎤ m IS
⎣ ⎦
wt =
AIS m

where
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Fr is the response factor (9.4.2);
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A>C 44 is the total area of peaks due to all waxes having more than C44;
ISO/TS 23647:2010
AC 44 is the area of the C44 wax peak;
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AIS is the area of the hexatriacontane peak;

k is an empirical factor (k = 1,0 for refined oils and winterized oils; k = 0,5 for non-refined oils, see
Reference [5]);

mIS is the mass, in micrograms, of internal standard added to the oil;

m is the mass, in grams, of the oil.

9.4.3.2 Other oils

The main wax components of vegetable oils (soybean, rapeseed, and corn) are: C40, C41, C42, C44, C46
waxes. Their content is generally below 20 mg/kg in a well-refined deodorized oil. The exception is crude rice
bran oil, which can have 1 500 mg/kg to 2 000 mg/kg wax content with a similar composition to sunflower.

9.4.3.2.1 Rice bran oil

The wax content, wt, expressed as a mass fraction in milligrams per kilogram oil, is given by:

Fr ( AWC mIS )
42
wt =
AIS m

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