ISO/TS 17306:2016

Petroleum products — Biodiesel — Determination of free and total glycerin and mono-, di- and tracylglycerols by gas chromatography

ISO/TS 17306:2016 establishes a methodology for quantitative determination of free glycerol, mono-, di-, triacylglycerols and total glycerol by gas chromatography in biodiesel produced from any raw material including coconut or palm oil and animal fat. It is not applicable for biodiesel from castor oil. In most actual cases, biodiesel is based on fatty acid methyl esters (FAME). These have also been used during the precision study for this test method. There is no indication that the methodology does not apply to other ester types, but the precision has not been determined nor compared. NOTE For the purposes of this Technical Specification, the term "% (m/m)" is used to represent the mass fraction, µ.

Produits pétroliers — Biogazole — Détermination de glycerine libre et totale et des mono-, di- et tracylglycerols avec chromatographie gazeuse

General Information

• Status: Published
• Publication Date: 18-Jan-2016
• ICS: 75.160.20 - Liquid fuels
• ICS: 75.160.40 - Biofuels
• Technical Committee: ISO/TC 28/SC 7 - Liquid Biofuels
• Drafting Committee: ISO/TC 28/SC 7 - Liquid Biofuels
• Current Stage: 9060 - Close of review
• Start Date: 02-Dec-2025

Standards Content (Sample)

TECHNICAL ISO/TS
SPECIFICATION 17306
First edition
2016-02-01
Petroleum products — Biodiesel
— Determination of free and
total glycerin and mono-, di- and
tracylglycerols by gas chromatography
Produits pétroliers — Biogazole — Détermination de glycerine
libre et totale et des mono-, di- et tracylglycerols avec
chromatographie gazeuse
Reference number
ISO/TS 17306:2016(E)
©
ISO 2016

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ISO/TS 17306:2016(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
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
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www.iso.org
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ISO/TS 17306:2016(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Apparatus . 2
6 Reagents and materials . 3
7 Preparation of the apparatus . 3
8 Sampling . 4
9 Preparation of the standard solutions . 4
9.1 Preparation of the stock solutions . 4
9.2 Preparation of the calibration curve . 5
9.3 Preparation of the sample . 6
9.4 Procedure . 7
10 Calculation and expression of results . 8
11 Precision .10
11.1 General .10
11.2 Repeatability, r .10
11.3 Reproducibility, R .10
12 Test report .11
Annex A (normative) Check chromatograms .12
Annex B (normative) Calculation of the coefficients for determination of the combined glycerol .16
Annex C (informative) Information on interlaboratory study .20
Bibliography .21
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ISO/TS 17306:2016(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 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 28, Petroleum products and related products of
synthetic or biological origin, Subcommittee SC 7, Liquid biofuels.
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ISO/TS 17306:2016(E)

Introduction
This Technical Specification establishes a method for quantitative determination of free glycerol,
mono-, di-, triacylglycerols and total glycerol in fatty acid methyl esters (biodiesel) by gas
chromatography. High concentrations of these components can contribute to formation of deposits on
the pistons and valves of diesel cycle engines. Additionally, they can cause problems during storage and
in the engine’s fuel injection system.
[2] [3]
Alternative methods for similar determinations exist in ASTM D6584 and EN 14105, which
are tailor made to regional quality specification needs. This Technical Specification describes an
alternative technique using more easily available internal standards, instrumentation that can also
measure esters and a procedure applicable to short chain fatty acid esters, such as those from palm
kernel and coconut oil. This Technical Specification thus provides a wider usage with similar or worse
precision as other techniques.
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TECHNICAL SPECIFICATION ISO/TS 17306:2016(E)
Petroleum products — Biodiesel — Determination of free
and total glycerin and mono-, di- and tracylglycerols by gas
chromatography
WARNING — The use of this Technical Specification may involve the usage of dangerous
materials and equipment. It is the responsibility of the user to establish the appropriate
security, health and environmental practices, and to determine the applicability of regulatory
limitations before their use.
1 Scope
This Technical Specification establishes a methodology for quantitative determination of free glycerol,
mono-, di-, triacylglycerols and total glycerol by gas chromatography in biodiesel produced from any raw
material including coconut or palm oil and animal fat. It is not applicable for biodiesel from castor oil.
In most actual cases, biodiesel is based on fatty acid methyl esters (FAME). These have also been used
during the precision study for this test method. There is no indication that the methodology does not
apply to other ester types, but the precision has not been determined nor compared.
NOTE For the purposes of this Technical Specification, the term “% (m/m)” is used to represent the mass
fraction, µ.
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 3170, Petroleum liquids — Manual sampling
ISO 3171, Petroleum liquids — Automatic pipeline sampling
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
biodiesel
fuel comprised of mono-alkyl esters of fatty acids, derived from vegetable oils or animal fats
3.2
bonded glycerol
glycerol portion of the mono-, di-, and triacylglycerols molecules
3.3
total glycerol
sum of free glycerol and bonded glycerol
3.4
monoacylglycerols
sum of monostearin, monopalmitin, monoolein, monolinolein, concentrations and/or other
monoacylglycerols present in the biodiesel
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3.5
diacylglycerols
sum of diolein, dilinolein concentrations and/or other diacylglycerols present in the biodiesel
3.6
triacylglycerols
sum of triolein, trilinolein concentrations and/or other triacylglycerols present in the biodiesel
3.7
silylation
reaction to substitute the active hydrogen present in the mono- and diacylglycerol molecules to obtain
more volatile and stable compounds
4 Principle
A sample is injected into a gas chromatograph after silylation with N-methyl-N-trimethylsilyltrifluoro-
acetamide (MSTFA). The identification of the components in the sample is done by comparing the
retention times of four reference materials (glycerol, monoolein, diolein and triolein). The quantification
is done using calibration curves with internal standardization. For quantifying the glycerin and the
acylglycerols, ethylene glycol and tricaprin are used as internal standard, respectively. The total
glycerol is obtained from the sum of free and bonded glycerol concentrations.
5 Apparatus
5.1 Gas chromatograph equipped with flame ionization detector (FID), an on-column (or equivalent)
injector and oven with temperature programming.
5.2 Data acquisition system, an electronic instrument to obtain and record the peak area in the
chromatograms.
5.3 Column, fused silica capillary column, 30 m × 0,32 mm × 0,1 µm, with stationary phase 95 %
dimethylpolysiloxane and 5 % phenyl-methylpolysiloxane for high temperature (up to 400 °C).
NOTE 1 Any column with better or equivalent efficiency and selectivity can be used. Their usefulness can be
observed by comparing the chromatogram obtained with chromatograms presented in Annex A.
NOTE 2 A retention gap of 0,53 mm of internal diameter can be used.
5.4 Automatic sampler.
5.5 Balance, with resolution of 0,1 mg.
5.6 Volumetric flasks of 50 ml, 25 ml and 10 ml
5.7 Appropriate vials for automatic sampler, screw top vials can lead to sample evaporation.
5.8 Flask, with a capacity of 10 ml, with polytetrafluoroethylene (PTFE) faced septa.
5.9 Microlitre syringes, with a capacity of 5 µl for sample injection.
5.10 Microlitre syringes or micropipette, with a capacity of 100 µl and 250 µl for the preparation of
the solutions.
5.11 Pasteur pipettes.
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ISO/TS 17306:2016(E)

5.12 Volumetric pipettes, graduated, with a capacity of 10 ml and 20 ml.
6 Reagents and materials
6.1 n-heptane, 99,0 % minimum purity.
6.2 Pyridine (dried), 99,0 % minimum purity, with a maximum water content of 0,1 %.
It is recommended that the pyridine be stored with a molecular sieve 5A, 4/8 mesh. Its conditioning
should be undertaken in a lab oven, at 350 °C throughout the night. Allow cooling down in a desiccant,
without silica.
1)
6.3 1-Glycerolmonooctadecenoate (glycerol monooleate or monoolein) (CAS No. 111-03-5),
99,0 % minimum purity.
6.4 1,3 Glycerol dioctadecenoate (glycerol dioleate or diolein) (CAS No. 2465-32-9), 99,0 %
minimum purity.
6.5 Glycerol trioctadecenoate (glycerol trioleate or triolein) (CAS No. 122-32-7), 99,0 %
minimum purity.
6.6 Glycerol (CAS Nº 56-81-5), 99,5 % minimum purity.
6.7 Ethylene glycol (CAS No. 107-21-1), 99,0 % minimum purity.
6.8 Tricaprin (CAS Nº 621-71-6), 99,0 % minimum purity.
6.9 N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), reagent grade.
IMPORTANT — For silylation purposes, interaction with water shall be prevented.
6.10 Carrier gas, hydrogen or helium, 99,999 % minimum purity, as carrier gas (for detector gas a
FID suitable grade is allowed).
6.11 Nitrogen, grade suitable for FID.
6.12 Synthetic air, grade suitable for FID.
7 Preparation of the apparatus
7.1 Install the column according to the instructions of the manufacturer.
7.2 Establish a carrier gas flow of around 3,0 ml/min in the column (pressure of 180 kPa and average
linear velocity of around 0,54 m/s if helium is used or 105 kPa and 0,70 m/s to hydrogen).
7.3 Adjust the following typical operating conditions on the gas chromatograph:
1) Represents the register of chemical substances catalogued in the CAS system. CAS numbers have no chemical
meaning, these are numbers designated in sequential order for each substance added to the system.
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a) oven temperature programming:
Oven program rate Temperature Holding time
°C/min °C min
— 50 1
15 180 0
7 230 0
20 380 10
b) carrier gas: helium or hydrogen;
c) detector temperature: 380 °C;
d) injector temperature: oven tracking;
e) nitrogen flow (make-up gas): 30 ml/min;
f) hydrogen flow to the detector: 35 ml/min;
g) synthetic air flow to the detector: 350 ml/min;
NOTE The detector flows recommended by the manufacturer can also be used.
h) volume injected: 1,0 µl;
i) run time: 35 min.
7.4 Evaluate the stability of the baseline running a blank.
7.5 After system stabilization, baseline subtraction or electronic compensation following the
procedures described in the equipment’s manual can be applied to eliminate the deviation of the
baseline due to the temperature programming of the oven. Care should be taken not to lose the raw data
if required for good laboratory practices.
8 Sampling
Unless otherwise specified, obtain representative samples for analysis in accordance with the
procedures given in ISO 3170, ISO 3171 or an equivalent National Standard.
9 Preparation of the standard solutions
9.1 Preparation of the stock solutions
In a flask with adequate capacity, prepare stock solutions for each substance according to Table 1,
recording the respective masses.
Store and keep the solutions in the refrigerator when not in use.
NOTE Under these conditions, the stock solutions can be stored for one month.
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Table 1 — Preparation of the stock solutions
Approximate mass Pyridine mass
Compound
mg G
Glycerin 25 49,0
Monoolein 50 9,80
Diolein 50 9,80
Triolein 50 9,80
Ethylene glycol (internal standard 1) 25 24,5
Tricaprin (internal standard 2) 80 9,80
Calculate the concentrations of the stock solutions using Formula (1):
m
ss
C =×100 (1)
ss
m
T
where
C is the concentrations of the compounds in the stock solutions in % (m/m);
SS
m is the mass of the compounds used in the preparation of the stock solutions, with a precision
SS
of 0,1 mg;
m is the total mass (m + m ), with a precision of 0,1 mg.
T compound pyridine
9.2 Preparation of the calibration curve
9.2.1 In a flask with a capacity of 10 ml (5.8), prepare the standard solution one by adding the stock
solutions according to the indications on Table 2. Record the masses of each stock solution added.
9.2.2 Repeat the same procedure for the other standard solutions 2, 3, 4 and 5 from Table 2.
9.2.3 Add to each of the five standard solutions 100 µl of MSTFA. Close the flask and shake vigorously.
Let it react for at least 20 min at room temperature.
9.2.4 After the reaction time, add approximately 8 ml of n-heptane and shake, transfer a portion to an
automatic sampler vial and seal.
9.2.5 Inject 1,0 µl of each solution, at least twice. Identify the peaks according to their retention times;
an elution order is presented in Figure A.1.
Table 2 — Preparation of the standard solutions
Standard solution 1 2 3 4 5
Stock solution of glycerin (µl) 10 30 50 70 100
Stock solution of monoolein (µl) 20 50 100 150 200
Stock solution of diolein (µl) 10 20 40 70 100
Stock solution of triolein (µl) 10 20 40 70 100
Stock solution of ethylene glycol (µl) 100 100 100 100 100
Stock solution of tricaprin (µl) 100 100 100 100 100
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9.2.6 Use Formula (2) to calculate the mass of all the compounds present in the standard solutions
which will be used in calibration curves.
C
S
m =×m (2
...