SIST-TS CEN ISO/TS 23973:2022
(Main)Liquid chromatography at critical conditions (LCCC) - Chemical heterogeneity of polyethylene oxides (ISO/TS 23973:2020)
Liquid chromatography at critical conditions (LCCC) - Chemical heterogeneity of polyethylene oxides (ISO/TS 23973:2020)
This document establishes a valid method for separation of chemically heterogeneous polyethylene
oxide (PEO) mixtures and for the determination the number and content of the chemically heterogeneous
species in the overall sample.
The method presented in this document serves as a technical guideline and enables laboratories to
learn the principle of “critical chromatography” on a validated system.
This method presented in this document with its stated system parameters is not applicable for other
polymer classes, due to the diversity of the interactions between the polymer/mobile phase/stationary
phase and the number of separation systems that are therefore available.
The evaluation of the interlaboratory testing has shown that many error sources relate to the technique
of liquid chromatography in general. Possible error sources are described in Annex A.
Details on the evaluation of the interlaboratory testing are given in Annex B.
Elugrams of the participants (excerpts) are given in Annex C.
Investigations of the long-term stability of the test mixture are given in Annex D
Flüssigchromatographie unter kritischen Bedingungen - Chemische Heterogenität von Polyethylenoxiden (ISO/TS 23973:2020)
Dieses Dokument beschreibt ein gültiges Verfahren für die Trennung von chemisch heterogenen Gemischen von Polyethylenoxid (PEO) und die Ermittlung der Anzahl sowie des Gehaltes der chemisch heterogenen Spezies in der Gesamtprobe.
Das in diesem Dokument dargestellte Verfahren dient als technischer Leitfaden und soll Laboratorien befähigen, das Prinzip der „kritischen Chromatographie“ an einem validierten System zu erlernen.
Auf Grund der Vielfalt der Wechselwirkungen Polymer/mobile Phase/stationäre Phase und der dadurch zur Verfügung stehenden Anzahl der Trennsysteme ist dieses Verfahren mit den angegebenen Systemparametern nicht allgemeingültig für andere Polymerklassen.
Die Auswertung der Ringversuche hat gezeigt, dass viele Fehlerquellen in der Technik der Flüssig-chromatographie allgemein zu suchen sind. Mögliche Fehlerquellen werden im Anhang A beschrieben.
Anhang B enthält Angaben zur Evaluierung des Ringversuchs.
Elugramme der Teilnehmer (Auszüge) sind im Anhang C enthalten.
Untersuchungen zur Langzeitstabilität des Prüfgemisches sind im Anhang D aufgeführt.
Chromatographie liquide aux conditions critiques - Hétérogénéité chimique des oxydes de polyéthylène (ISO/TS 23973:2020)
Tekočinska kromatografija pri kritičnih pogojih (LCCC) - Kemijska heterogenost polietilen oksidov (ISO/TS 23973:2020)
Ta dokument določa veljavno metodo za ločevanje kemijsko heterogenih zmesi polietilen oksida (PEO) ter za določevanje števila in vsebnosti kemijsko heterogenih vrst v celotnem vzorcu. Metoda, predstavljena v tem dokumentu, je podana kot tehnična smernica, ki laboratorijem omogoča, da uvedejo načelo »kritične kromatografije« v okviru potrjenega sistema. Metoda, predstavljena v tem dokumentu, in navedeni parametri sistema se zaradi raznolikosti interakcij med polimeri/mobilno fazo/stacionarno fazo in številom sistemov za ločevanje, ki so posledično na voljo, ne uporabljajo za druge razrede polimerov. Z vrednotenjem medlaboratorijskega preskušanja je bilo ugotovljeno, da se številni viri napak navezujejo na tehniko tekočinske kromatografije na splošno. Možni viri napak so opisani v dodatku A. Podrobnosti o vrednotenju medlaboratorijskega preskušanja so podane v dodatku B. Elugrami udeležencev (izvlečki) so podani v dodatku C. Raziskave dolgoročne stabilnosti preskusne zmesi so podane v dodatku D.
General Information
Buy Standard
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 23973:2022
01-marec-2022
Tekočinska kromatografija pri kritičnih pogojih (LCCC) - Kemijska heterogenost
polietilen oksidov (ISO/TS 23973:2020)
Liquid chromatography at critical conditions (LCCC) - Chemical heterogeneity of
polyethylene oxides (ISO/TS 23973:2020)
Chromatographie liquide aux conditions critiques - Hétérogénéité chimique des oxydes
de polyéthylène (ISO/TS 23973:2020)
Ta slovenski standard je istoveten z: CEN ISO/TS 23973:2021
ICS:
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
SIST-TS CEN ISO/TS 23973:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN ISO/TS 23973:2022
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SIST-TS CEN ISO/TS 23973:2022
CEN ISO/TS 23973
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
December 2021
TECHNISCHE SPEZIFIKATION
ICS 71.040.50
English Version
Liquid chromatography at critical conditions (LCCC) -
Chemical heterogeneity of polyethylene oxides (ISO/TS
23973:2020)
Chromatographie liquide aux conditions critiques -
Hétérogénéité chimique des oxydes de polyéthylène
(ISO/TS 23973:2020)
This Technical Specification (CEN/TS) was approved by CEN on 5 December 2021 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 23973:2021 E
worldwide for CEN national Members.
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SIST-TS CEN ISO/TS 23973:2022
CEN ISO/TS 23973:2021 (E)
Contents Page
European foreword . 3
2
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SIST-TS CEN ISO/TS 23973:2022
CEN ISO/TS 23973:2021 (E)
European foreword
The text of ISO/TS 23973:2020 has been prepared by Technical Committee ISO/TC 35 "Paints and
varnishes” of the International Organization for Standardization (ISO) and has been taken over as
CEN ISO/TS 23973:2021 by Technical Committee CEN/TC 139 “Paints and varnishes” the secretariat of
which is held by DIN.
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.
Any feedback and questions on this document should be directed to the users’ national standards body.
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 announce this Technical Specification: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO/TS 23973:2020 has been approved by CEN as CEN ISO/TS 23973:2021 without any
modification.
3
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SIST-TS CEN ISO/TS 23973:2022
TECHNICAL ISO/TS
SPECIFICATION 23973
First edition
2020-08
Liquid chromatography at critical
conditions (LCCC) — Chemical
heterogeneity of polyethylene oxides
Chromatographie liquide aux conditions critiques — Hétérogénéité
chimique des oxydes de polyéthylène
Reference number
ISO/TS 23973:2020(E)
©
ISO 2020
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SIST-TS CEN ISO/TS 23973:2022
ISO/TS 23973:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020
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
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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
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SIST-TS CEN ISO/TS 23973:2022
ISO/TS 23973:2020(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
5.1 General . 2
5.2 Eluent supply . 3
5.3 Pump . 3
5.4 Injection system . 4
5.5 Separation columns . 4
5.6 Column temperature control . 5
5.7 Detectors . 5
5.8 Eluent . 5
5.9 Data acquisition . 5
6 Sample preparation . 5
7 Performance of the measurements . 6
7.1 Determination of the critical conditions . 6
7.2 Analysis of the validation kit . 7
8 Test report . 7
Annex A (informative) Error sources . 9
Annex B (informative) Evaluation of the interlaboratory testing .10
Annex C (informative) Elugrams of the participants (excerpts) .17
Annex D (informative) Investigations of the long-term stability of the test mixture .43
Bibliography .49
© ISO 2020 – All rights reserved iii
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SIST-TS CEN ISO/TS 23973:2022
ISO/TS 23973:2020(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 35, Paints and varnishes.
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 2020 – All rights reserved
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SIST-TS CEN ISO/TS 23973:2022
ISO/TS 23973:2020(E)
Introduction
Since the first description of liquid chromatography at critical conditions (LCCC) in
1986 (see Reference [1]), the method has been continuously refined and has proved itself to be
indispensable for polymer characterisation. Separation is required not only for the quantitative
analysis of the individual species. It also offers the preconditions for qualitative characterisation of the
fractions by means of spectroscopic and spectrometric techniques. The key factor here is the reduction
of the polydispersity/chemical heterogeneity within a fraction, which represents a large problem for
mass-spectrometric investigations.
The method has been described extensively in professional circles over the last two decades for
different polymer systems, see References [2] to [9].
Within the framework of the Technical Committee, the extent that the method supplies consistent
results for a simple, chemically heterogeneous polymer mixture was clarified as part of interlaboratory
testing.
At this time, necessary experience relating to the selection of the system (interaction between the
polarities separation phase/eluent/sample) was not expected of any of the participating laboratories.
The interlaboratory testing has shown that, even with a well-characterized system and with
specification of all pertinent system parameters, it has to date not been possible to classify the process
as a routine method in laboratories with experience in polymer analytics.
The idea presents itself of offering a validation kit (polymer mixture with the expecting separation
result).
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SIST-TS CEN ISO/TS 23973:2022
TECHNICAL SPECIFICATION ISO/TS 23973:2020(E)
Liquid chromatography at critical conditions (LCCC) —
Chemical heterogeneity of polyethylene oxides
1 Scope
This document establishes a valid method for separation of chemically heterogeneous polyethylene
oxide (PEO) mixtures and for the determination the number and content of the chemically heterogeneous
species in the overall sample.
The method presented in this document serves as a technical guideline and enables laboratories to
learn the principle of “critical chromatography” on a validated system.
This method presented in this document with its stated system parameters is not applicable for other
polymer classes, due to the diversity of the interactions between the polymer/mobile phase/stationary
phase and the number of separation systems that are therefore available.
The evaluation of the interlaboratory testing has shown that many error sources relate to the technique
of liquid chromatography in general. Possible error sources are described in Annex A.
Details on the evaluation of the interlaboratory testing are given in Annex B.
Elugrams of the participants (excerpts) are given in Annex C.
Investigations of the long-term stability of the test mixture are given in Annex D.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
liquid chromatography at critical conditions
LCCC
special form of liquid chromatography of polymers at the point of adsorption, where chemically and
structurally identical polymers with a certain repeat unit elute independently of the molar mass at the
same retention time
Note 1 to entry: The individual monomer units do not contribute to the retention. Under these determined system
parameters (defined combination of separation column/eluent mixture/temperature), a separation of polymer
mixtures of the same repeat unit takes place based on chemical heterogeneity. Chemical heterogeneities can take
the form of different functional groups, end groups, differences in the microstructure (e.g. copolymers and their
composition) as well as topological differences (e.g. branching).
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SIST-TS CEN ISO/TS 23973:2022
ISO/TS 23973:2020(E)
4 Principle
In the following, the method for the separation of polyethylene oxides at critical conditions of adsorption
is described for the ethylene oxide repeat unit.
Three polyethylene glycols (PEG) with different molar masses (1 mg/ml to 2 mg/ml dissolved in the
relevant eluent mixture (see 7.1) are measured, starting with a high proportion of the thermodynamically
good solvent B (in this case acetonitrile). The eluent mixture is successively changed by increasing
the proportion of component A (in this case water). This is followed by measurements in different
compositions until all three standards elute independently of the molar mass at the same retention
time. The determined critical solvent composition (csc) corresponds to the critical conditions.
Afterwards, the unknown mixtures are dissolved in this eluent mixture and measured.
From the peaks of the resulting chromatograms, the number of species with different functionalities
and their relative content (taking into account the detection properties) can be determined.
Species contained in the mixtures:
(1) Polyethylene (2) Methylene polyethylene (3) Polyethylene glycol
glycol glycol monoallyl ether
5 Apparatus
5.1 General
The apparatus shall consist of the components shown in Figure 1, which are described in more
detail below.
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SIST-TS CEN ISO/TS 23973:2022
ISO/TS 23973:2020(E)
Key
1 LCCC mobile phase
2 pump, 0,1 ml/min to 2 ml/min
3 injection valve, autosampler
4 detectors: RI, ELSD, corona
5 data processing
6 waste
7 separation columns/column temperature control
Figure 1 — LCCC apparatus
All components shall come into contact with the eluent or the sample solution are resistant to them and
do not exhibit any adsorption or memory effects. The individual modules should be generally connected
with steel capillary tubes for polymer analytics.
5.2 Eluent supply
The eluent reservoir shall adequately protect the eluent against external influences such as the
atmosphere and light, if necessary by means of a blanket of inert gas above the liquid level.
The eluent reservoir shall contain a sufficient quantity of the eluent to bring the apparatus to
equilibrium and to carry out several repeat analyses.
The eluent shall be degassed either before it is introduced into the reservoir, or by use of a device fitted
between the reservoir and the pump, to prevent malfunctions of the pump or the formation of bubbles
in the detector. The method of degassing used (e.g. bubble trap, online purging with helium or vacuum
degassing) is open to choice, but shall be stated in the test report.
For polymer mixtures that contain chromophoric groups, other detectors may be used, e.g. UV or IR
detectors.
5.3 Pump
The pump ensures that the eluent flow through the separation columns is as smooth and pulse-free as
possible. The flow rate shall be 0,5 ml/min to 1 ml/min, depending on the dimensions of the columns
used. To fulfil these requirements, the pump shall operate at optimum efficiency at this flow rate and
at the counterpressure established in the process. The variation in the flow rate of the pump used may
have a variation of max. 0,1 %.
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SIST-TS CEN ISO/TS 23973:2022
ISO/TS 23973:2020(E)
5.4 Injection system
The injection system serves to introduce into the chromatographic separation system an exactly
specified amount of the sample solution (5 µl to 25 μl) in an eluent interval that is delimited as accurately
as possible. This introduction may be carried out either manually or automatically.
When filling the sample loop with sample solution and subsequently introducing the sample solution
into the eluent stream, the volume of liquid used shall be great enough to ensure that, even if laminar-
flow effects occur, the sample loop is completely filled with the sample solution and subsequently
completely flushed out.
Memory effects from the previous sample solution in the injection system shall be avoided by design or
by adequate flushing.
5.5 Separation columns
The separation system consists of one or more columns connected in series, which are packed with a
spherical (d < 10 μm) and porous separation material, the diameter of the pores corresponding to the
size of the polymer molecules being analysed (80 Å to 300 Å for this method).
Depending on the molar mass of the samples under investigation, the pore size of the separation
material may need to be adjusted accordingly.
At this point in the method, the separation material consists of a nonpolar/hydrophobic stationary
phase (octadecyl-modified silica gel, reversed phase, C18). The hydrophobic properties of the material
should have been further boosted by end-capping with trimethyl chlorosilane. The C-content thus
obtained is ≫10 %.
To meet the objective of this document of obtaining results that agree as well as possible in different
laboratories using different LC apparatus with the same sample, it is necessary to adhere to the
minimum requirements specified below with regard to peak broadening (expressed in terms of a
number of theoretical plates) and separation efficiency; the actually obtained values shall be stated in
the test report.
Number of theoretical plates
The number of theoretical plates N shall be determined, for the apparatus used per metre of column
used, from the peak width at half height. Inject up to 20 μl of a solution of the PEG 1 500 standard
(min. mass concentration 1 mg/ml) on to the column, and evaluate the chromatogram obtained under
the same conditions as are used for analysing polymers, using Formula (1):
2
t
100
R
N=55, 4 (1)
wL
05,
where
t is the retention time at the peak maximum (the same units shall be used, e.g. min);
R
w is the peak width at half height (see Figure 2);
0,5
L is the length of the column (column combination), in cm.
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ISO/TS 23973:2020(E)
Key
X retention time, in minutes
Y detection signal
t retention time at the peak maximum, in minutes
R
w peak width at half height of the peak
0,5
h maximum peak height
h/2 half peak height
Figure 2 — Determination of the number of theoretical plates by the half-height method
The result is expressed as the number of theoretical plates per metre of column length. A column
(column combination) should be used that achieves at least 5 000 theoretical plates per column (column
combination) used.
5.6 Column temperature control
The analyses can be performed in a temperature range from 20 °C to 45 °C. The temperature of the
column shall not change by more than 0,5 °C during the analysis. All analyses shall be performed at the
[1]
same temperature, as the LCCC is an enthalpic process and therefore temperature-dependent .
5.7 Detectors
The following can be used as detectors: RID (refractive index detector), ELSD (evaporative light
scattering detector) or Corona CAD (charged aerosol detector).
5.8 Eluent
All eluents that are used should have the purity “HPLC grade”. The eluents should not be returned to the
eluent reservoir.
5.9 Data acquisition
The signals from the detector are recorded by means of an electronic data system.
6 Sample preparation
Dissolve the samples (1 mg/ml to 2 mg/ml) in the relevant eluent. To rule out changes to the polymers
in solution as a function of the time as a possible source of error, the samples should be analysed within
6 h of production of the solution.
Fill up the validation kit (mixture 2) with 2 ml of the critical solvent composition.
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ISO/TS 23973:2020(E)
7 Performance of the measurements
7.1 Determination of the critical conditions
The critical conditions can be set up either by changing the temperature or by changing the composition
of the mobile phase.
In the following procedure, keep the temperature strictly constant, and successively change the
solvent composition with decreasing acetonitrile content. This can be done via separate mixing of the
components or with a binary pump.
Measure the three standards with different molar masses in the relevant chosen solvent composition.
At this point this this method, it is very important to make sure that the solvent in which the standard
is dissolved also complies with the eluent composition.
The critical solvent composition (ACN : H O) is attained when all molecules of the same chemical
2
structure (in this case PEG standards) elute at the same retention time. This means that, under these
conditions, the chromatographic behaviour is no longer determined by the size of the molecule, but by
chemical inhomogeneity.
To determine the critical solvent composition, plot lgM , in g/mol, over the retention time, in min, (Figure 3,
p
left), or the retention factor k, over the content of component B in the eluent, in % (Figure 3, right).
a) Logarithm of molar mass over retention time b) Retention factor over content of component
B in the eluent
Key
X1 retention time, in minutes
Y1 lg M , in grams per mole
p
X2 ACN, as volume fraction in per cent
Y2 retention factor k
1 ACN / H O: 60 % / 40 %
2
2 ACN / H O: 55 % / 45 %
2
3 ACN / H O: 50 % / 50 %
2
4 ACN / H O: 49 % / 51 %
2
5 PEG 1460
6 PEG 3020
7 PEG 6600
Figure 3 — Schematic representation of the determination of the critical solvent composition
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ISO/TS 23973:2020(E)
The retention factor k is calculated using Formula (2):
tt−
()
R0
k= (2)
t
0
where
t is the retention time of the standard in the relevant system;
R
t is the retention time of a non-delayed substance.
0
7.2 Analysis of the validation kit
Mix the sample with 2 ml of the critical solvent mixture and dissolve it at room temperature under
shaking.
To rule out changes to the polymers in solution as a function of the time as a possible source of error,
the samples should be analysed within 6 h of production of the solution. Ensure that the temperature in
the column remains constant.
The injection volume shall be matched to the set of columns used and can therefore be varied between
5 µl and 25 µl.
It is recommended to conduct a blind test (injection of the eluent/the pure sample solvent) before
measuring the sample in order to identify solvent signals or peaks that are caused by the injection.
The measurement of the sample ends after complete elution of the last system peak and after the signal
returns to the base line.
Conduct three repeat measurements and calculate the mean values of the retention time for the
separated peaks.
Calculate the chromatographic resolution of two peaks R as a quality criterion for the separation using
Formula (3):
()tt−
R2 R1
R=×11, 8 (3)
ww+
f,hm,f12,hm
where
t is the retention time;
R
w is the full width of the peak at half maximum.
f,hm
8 Test report
The test report shall contain at least the following information:
a) all details necessary for complete identification of the product tested;
b) a reference to this document (ISO/TS 23973:2020);
c) all information about the apparatus (pump, degasser, injection system, column oven, detectors,
evaluation software);
d) all information about the separation columns (manufacturer, separation material, pore size type,
grain size, number, dimensions, C-content);
e) the number of theoretical plates per column combination in the LC apparatus used;
© ISO 2020 – All rights reserved 7
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SIST-TS C
...
SLOVENSKI STANDARD
kSIST-TS FprCEN ISO/TS 23973:2021
01-september-2021
Tekočinska kromatografija pri kritičnih pogojih (LCCC) - Kemijska heterogenost
polietilen oksidov (ISO/TS 23973:2020)
Liquid chromatography at critical conditions (LCCC) - Chemical heterogeneity of
polyethylene oxides (ISO/TS 23973:2020)
Chromatographie liquide aux conditions critiques - Hétérogénéité chimique des oxydes
de polyéthylène (ISO/TS 23973:2020)
Ta slovenski standard je istoveten z: FprCEN ISO/TS 23973
ICS:
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
kSIST-TS FprCEN ISO/TS 23973:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST-TS FprCEN ISO/TS 23973:2021
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kSIST-TS FprCEN ISO/TS 23973:2021
TECHNICAL ISO/TS
SPECIFICATION 23973
First edition
2020-08
Liquid chromatography at critical
conditions (LCCC) — Chemical
heterogeneity of polyethylene oxides
Chromatographie liquide aux conditions critiques — Hétérogénéité
chimique des oxydes de polyéthylène
Reference number
ISO/TS 23973:2020(E)
©
ISO 2020
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kSIST-TS FprCEN ISO/TS 23973:2021
ISO/TS 23973:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020
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kSIST-TS FprCEN ISO/TS 23973:2021
ISO/TS 23973:2020(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
5.1 General . 2
5.2 Eluent supply . 3
5.3 Pump . 3
5.4 Injection system . 4
5.5 Separation columns . 4
5.6 Column temperature control . 5
5.7 Detectors . 5
5.8 Eluent . 5
5.9 Data acquisition . 5
6 Sample preparation . 5
7 Performance of the measurements . 6
7.1 Determination of the critical conditions . 6
7.2 Analysis of the validation kit . 7
8 Test report . 7
Annex A (informative) Error sources . 9
Annex B (informative) Evaluation of the interlaboratory testing .10
Annex C (informative) Elugrams of the participants (excerpts) .17
Annex D (informative) Investigations of the long-term stability of the test mixture .43
Bibliography .49
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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
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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).
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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 35, Paints and varnishes.
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.
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Introduction
Since the first description of liquid chromatography at critical conditions (LCCC) in
1986 (see Reference [1]), the method has been continuously refined and has proved itself to be
indispensable for polymer characterisation. Separation is required not only for the quantitative
analysis of the individual species. It also offers the preconditions for qualitative characterisation of the
fractions by means of spectroscopic and spectrometric techniques. The key factor here is the reduction
of the polydispersity/chemical heterogeneity within a fraction, which represents a large problem for
mass-spectrometric investigations.
The method has been described extensively in professional circles over the last two decades for
different polymer systems, see References [2] to [9].
Within the framework of the Technical Committee, the extent that the method supplies consistent
results for a simple, chemically heterogeneous polymer mixture was clarified as part of interlaboratory
testing.
At this time, necessary experience relating to the selection of the system (interaction between the
polarities separation phase/eluent/sample) was not expected of any of the participating laboratories.
The interlaboratory testing has shown that, even with a well-characterized system and with
specification of all pertinent system parameters, it has to date not been possible to classify the process
as a routine method in laboratories with experience in polymer analytics.
The idea presents itself of offering a validation kit (polymer mixture with the expecting separation
result).
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kSIST-TS FprCEN ISO/TS 23973:2021
TECHNICAL SPECIFICATION ISO/TS 23973:2020(E)
Liquid chromatography at critical conditions (LCCC) —
Chemical heterogeneity of polyethylene oxides
1 Scope
This document establishes a valid method for separation of chemically heterogeneous polyethylene
oxide (PEO) mixtures and for the determination the number and content of the chemically heterogeneous
species in the overall sample.
The method presented in this document serves as a technical guideline and enables laboratories to
learn the principle of “critical chromatography” on a validated system.
This method presented in this document with its stated system parameters is not applicable for other
polymer classes, due to the diversity of the interactions between the polymer/mobile phase/stationary
phase and the number of separation systems that are therefore available.
The evaluation of the interlaboratory testing has shown that many error sources relate to the technique
of liquid chromatography in general. Possible error sources are described in Annex A.
Details on the evaluation of the interlaboratory testing are given in Annex B.
Elugrams of the participants (excerpts) are given in Annex C.
Investigations of the long-term stability of the test mixture are given in Annex D.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
liquid chromatography at critical conditions
LCCC
special form of liquid chromatography of polymers at the point of adsorption, where chemically and
structurally identical polymers with a certain repeat unit elute independently of the molar mass at the
same retention time
Note 1 to entry: The individual monomer units do not contribute to the retention. Under these determined system
parameters (defined combination of separation column/eluent mixture/temperature), a separation of polymer
mixtures of the same repeat unit takes place based on chemical heterogeneity. Chemical heterogeneities can take
the form of different functional groups, end groups, differences in the microstructure (e.g. copolymers and their
composition) as well as topological differences (e.g. branching).
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4 Principle
In the following, the method for the separation of polyethylene oxides at critical conditions of adsorption
is described for the ethylene oxide repeat unit.
Three polyethylene glycols (PEG) with different molar masses (1 mg/ml to 2 mg/ml dissolved in the
relevant eluent mixture (see 7.1) are measured, starting with a high proportion of the thermodynamically
good solvent B (in this case acetonitrile). The eluent mixture is successively changed by increasing
the proportion of component A (in this case water). This is followed by measurements in different
compositions until all three standards elute independently of the molar mass at the same retention
time. The determined critical solvent composition (csc) corresponds to the critical conditions.
Afterwards, the unknown mixtures are dissolved in this eluent mixture and measured.
From the peaks of the resulting chromatograms, the number of species with different functionalities
and their relative content (taking into account the detection properties) can be determined.
Species contained in the mixtures:
(1) Polyethylene (2) Methylene polyethylene (3) Polyethylene glycol
glycol glycol monoallyl ether
5 Apparatus
5.1 General
The apparatus shall consist of the components shown in Figure 1, which are described in more
detail below.
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Key
1 LCCC mobile phase
2 pump, 0,1 ml/min to 2 ml/min
3 injection valve, autosampler
4 detectors: RI, ELSD, corona
5 data processing
6 waste
7 separation columns/column temperature control
Figure 1 — LCCC apparatus
All components shall come into contact with the eluent or the sample solution are resistant to them and
do not exhibit any adsorption or memory effects. The individual modules should be generally connected
with steel capillary tubes for polymer analytics.
5.2 Eluent supply
The eluent reservoir shall adequately protect the eluent against external influences such as the
atmosphere and light, if necessary by means of a blanket of inert gas above the liquid level.
The eluent reservoir shall contain a sufficient quantity of the eluent to bring the apparatus to
equilibrium and to carry out several repeat analyses.
The eluent shall be degassed either before it is introduced into the reservoir, or by use of a device fitted
between the reservoir and the pump, to prevent malfunctions of the pump or the formation of bubbles
in the detector. The method of degassing used (e.g. bubble trap, online purging with helium or vacuum
degassing) is open to choice, but shall be stated in the test report.
For polymer mixtures that contain chromophoric groups, other detectors may be used, e.g. UV or IR
detectors.
5.3 Pump
The pump ensures that the eluent flow through the separation columns is as smooth and pulse-free as
possible. The flow rate shall be 0,5 ml/min to 1 ml/min, depending on the dimensions of the columns
used. To fulfil these requirements, the pump shall operate at optimum efficiency at this flow rate and
at the counterpressure established in the process. The variation in the flow rate of the pump used may
have a variation of max. 0,1 %.
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5.4 Injection system
The injection system serves to introduce into the chromatographic separation system an exactly
specified amount of the sample solution (5 µl to 25 μl) in an eluent interval that is delimited as accurately
as possible. This introduction may be carried out either manually or automatically.
When filling the sample loop with sample solution and subsequently introducing the sample solution
into the eluent stream, the volume of liquid used shall be great enough to ensure that, even if laminar-
flow effects occur, the sample loop is completely filled with the sample solution and subsequently
completely flushed out.
Memory effects from the previous sample solution in the injection system shall be avoided by design or
by adequate flushing.
5.5 Separation columns
The separation system consists of one or more columns connected in series, which are packed with a
spherical (d < 10 μm) and porous separation material, the diameter of the pores corresponding to the
size of the polymer molecules being analysed (80 Å to 300 Å for this method).
Depending on the molar mass of the samples under investigation, the pore size of the separation
material may need to be adjusted accordingly.
At this point in the method, the separation material consists of a nonpolar/hydrophobic stationary
phase (octadecyl-modified silica gel, reversed phase, C18). The hydrophobic properties of the material
should have been further boosted by end-capping with trimethyl chlorosilane. The C-content thus
obtained is ≫10 %.
To meet the objective of this document of obtaining results that agree as well as possible in different
laboratories using different LC apparatus with the same sample, it is necessary to adhere to the
minimum requirements specified below with regard to peak broadening (expressed in terms of a
number of theoretical plates) and separation efficiency; the actually obtained values shall be stated in
the test report.
Number of theoretical plates
The number of theoretical plates N shall be determined, for the apparatus used per metre of column
used, from the peak width at half height. Inject up to 20 μl of a solution of the PEG 1 500 standard
(min. mass concentration 1 mg/ml) on to the column, and evaluate the chromatogram obtained under
the same conditions as are used for analysing polymers, using Formula (1):
2
t
100
R
N=55, 4 (1)
wL
05,
where
t is the retention time at the peak maximum (the same units shall be used, e.g. min);
R
w is the peak width at half height (see Figure 2);
0,5
L is the length of the column (column combination), in cm.
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Key
X retention time, in minutes
Y detection signal
t retention time at the peak maximum, in minutes
R
w peak width at half height of the peak
0,5
h maximum peak height
h/2 half peak height
Figure 2 — Determination of the number of theoretical plates by the half-height method
The result is expressed as the number of theoretical plates per metre of column length. A column
(column combination) should be used that achieves at least 5 000 theoretical plates per column (column
combination) used.
5.6 Column temperature control
The analyses can be performed in a temperature range from 20 °C to 45 °C. The temperature of the
column shall not change by more than 0,5 °C during the analysis. All analyses shall be performed at the
[1]
same temperature, as the LCCC is an enthalpic process and therefore temperature-dependent .
5.7 Detectors
The following can be used as detectors: RID (refractive index detector), ELSD (evaporative light
scattering detector) or Corona CAD (charged aerosol detector).
5.8 Eluent
All eluents that are used should have the purity “HPLC grade”. The eluents should not be returned to the
eluent reservoir.
5.9 Data acquisition
The signals from the detector are recorded by means of an electronic data system.
6 Sample preparation
Dissolve the samples (1 mg/ml to 2 mg/ml) in the relevant eluent. To rule out changes to the polymers
in solution as a function of the time as a possible source of error, the samples should be analysed within
6 h of production of the solution.
Fill up the validation kit (mixture 2) with 2 ml of the critical solvent composition.
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7 Performance of the measurements
7.1 Determination of the critical conditions
The critical conditions can be set up either by changing the temperature or by changing the composition
of the mobile phase.
In the following procedure, keep the temperature strictly constant, and successively change the
solvent composition with decreasing acetonitrile content. This can be done via separate mixing of the
components or with a binary pump.
Measure the three standards with different molar masses in the relevant chosen solvent composition.
At this point this this method, it is very important to make sure that the solvent in which the standard
is dissolved also complies with the eluent composition.
The critical solvent composition (ACN : H O) is attained when all molecules of the same chemical
2
structure (in this case PEG standards) elute at the same retention time. This means that, under these
conditions, the chromatographic behaviour is no longer determined by the size of the molecule, but by
chemical inhomogeneity.
To determine the critical solvent composition, plot lgM , in g/mol, over the retention time, in min, (Figure 3,
p
left), or the retention factor k, over the content of component B in the eluent, in % (Figure 3, right).
a) Logarithm of molar mass over retention time b) Retention factor over content of component
B in the eluent
Key
X1 retention time, in minutes
Y1 lg M , in grams per mole
p
X2 ACN, as volume fraction in per cent
Y2 retention factor k
1 ACN / H O: 60 % / 40 %
2
2 ACN / H O: 55 % / 45 %
2
3 ACN / H O: 50 % / 50 %
2
4 ACN / H O: 49 % / 51 %
2
5 PEG 1460
6 PEG 3020
7 PEG 6600
Figure 3 — Schematic representation of the determination of the critical solvent composition
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The retention factor k is calculated using Formula (2):
tt−
()
R0
k= (2)
t
0
where
t is the retention time of the standard in the relevant system;
R
t is the retention time of a non-delayed substance.
0
7.2 Analysis of the validation kit
Mix the sample with 2 ml of the critical solvent mixture and dissolve it at room temperature under
shaking.
To rule out changes to the polymers in solution as a function of the time as a possible source of error,
the samples should be analysed within 6 h of production of the solution. Ensure that the temperature in
the column remains constant.
The injection volume shall be matched to the set of columns used and can therefore be varied between
5 µl and 25 µl.
It is recommended to conduct a blind test (injection of the eluent/the pure sample solvent) before
measuring the sample in order to identify solvent signals or peaks that are caused by the injection.
The measurement of the sample ends after complete elution of the last system peak and after the signal
returns to the base line.
Conduct three repeat measurements and calculate the mean values of the retention time for the
separated peaks.
Calculate the chromatographic resolution of two peaks R as a quality criterion for the separation using
Formula (3):
()tt−
R2 R1
R=×11, 8 (3)
ww+
f,hm,f12,hm
where
t is the retention time;
R
w is the full width of the peak at half maximum.
f,hm
8 Test report
The test report shall contain at least the following information:
a) all details necessary for complete identification of the product tested;
b) a reference to this document (ISO/TS 23973:2020);
c) all information about the apparatus (pump, degasser, injection system, column oven, detectors,
evaluation software);
d) all information about the separation columns (manufacturer, separation material, pore size type,
grain size, number, dimensions, C-content);
e) the number of theoretical plates per column combination in the LC apparatus used;
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f) the column temperature;
g) the flow rate;
h) the critical solvent composition, in per cent, (mass fraction or volume fraction) with statement of
the method of mixing (gradient pump or separate production of the mixtures);
i) the concentration of the injected solution, in milligrams per millilitre (for samples to be weighed in
by yourself);
j) the injection volume, in microlitres;
k) the overlay of the elugrams of the three PEGs with different molar masses;
l) the validation kit surface percentage report, including chromatogram;
m) a statement of the mean values of the retention time;
n) a statement of the chromatographic resolution of the peaks R , R and R according to
AB AC BC
Reference [3];
o) the result of the test;
p) any deviation from the method specified;
q) any unusual features (anomalies) observed during the test;
r) the date of the test.
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Annex A
(informative)
Error sources
The evaluation of the interlaboratory testing has shown that many error sources can not only be
attributed specifically to a lack of experience in terms of the chromatography of polymers, but that they
relate to the technique of liquid chromatography in general.
As is well known, the resolution R depends on the separation factor α (for selectivity, measure of the
separation capability of the chromatographic system), the retention factor k (for capacity, measure of
the strength of the interactions) and on the number of plates N (for efficiency, measure of the band
broadening of the substance zone).
If the result does not correspond to the expectations, then the following aspects should be considered:
a) Have the column dimensions and packaging material been ideally chosen?
Improvement of the resolution via the number of plates:
— column as long as possible;
— particles as small as possible;
— optimal flow rates;
— determination of the adsorption range and exclusion rate for the selected column;
— determination of the number of plates (>25 000/m);
— adjustment of the pore size to the largest molar mass to be analysed (e.g. >100 Å for molar
masses above 6 000 g/mol)
b) Is the column contaminated/changed due to previous injections, is the specification no longer met?
This can alter the selectivity, and this influences the resolution more strongly than the number of
plates (separation factor α).
c) Are the solvent signals and system peaks overlaying the peaks of the analyte (particularly when
using RI [and UV] detectors)?
d) Have the rinsing times been complied with during the change in rinsing to new solvent compositions
(reference cell rinsing!)?
e) Do the internal diameters and lengths of the capillary tubes between the column and detector meet
the requirements (maximum internal diameter of 0,12 mm)?
f) Are there dead volumes (particularly in the case of detectors connected in series)?
g) Has the sample actually been analysed in the current eluent mixture?
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Annex B
(informative)
Evaluation of the interlaboratory testing
B.1 Participants in the interlaboratory testing
The 17 laboratories who have experience with HPLC and polymers as the substance class under
investigation took part in the interlaboratory testing.
B.2 System parameters for performance of the interlaboratory testing that were
made available to the participants
The samples are given in Table B.1.
Table B.1 — Samples
Designation Type Molar mass Composition
g/mol
S 1 PEG 1 500 Standard 1 500
...
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