ISO/TS 23973:2020

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 2020
© ISO 2020
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ii © ISO 2020 – All rights reserved

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
Foreword
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iv © ISO 2020 – All rights reserved

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).
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).
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.
2 © ISO 2020 – All rights reserved

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 ge
...