Liquid chromatography at critical conditions (LCCC) -- Chemical heterogeneity of polyethylene oxides

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.

Chromatographie liquide aux conditions critiques -- Hétérogénéité chimique des oxydes de polyéthylène

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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
---------------------- Page: 1 ----------------------
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
CP 401 • Ch. de Blandonnet 8
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
---------------------- Page: 2 ----------------------
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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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
---------------------- Page: 4 ----------------------
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).
© ISO 2020 – All rights reserved v
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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).
© ISO 2020 – All rights reserved 1
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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.
2 © ISO 2020 – All rights reserved
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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 %.
© ISO 2020 – All rights reserved 3
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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):
 
100
N=55, 4  (1)
 
 05, 
where

t is the retention time at the peak maximum (the same units shall be used, e.g. min);

w is the peak width at half height (see Figure 2);
0,5
L is the length of the column (column combination), in cm.
4 © ISO 2020 – All rights reserved
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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
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.

© ISO 2020 – All rights reserved 5
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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

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,

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
X2 ACN, as volume fraction in per cent
Y2 retention factor k
1 ACN / H O: 60 % / 40 %
2 ACN / H O: 55 % / 45 %
3 ACN / H O: 50 % / 50 %
4 ACN / H O: 49 % / 51 %
5 PEG 1460
6 PEG 3020
7 PEG 6600

Figure 3 — Schematic representation of the determination of the critical solvent composition

6 © ISO 2020 – All rights reserved
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ISO/TS 23973:2020(E)
The retention factor k is calculated using Formula (2):
tt−
k= (2)
where
t is the retention time of the standard in the relevant system;
t is the retention time of a non-delayed substance.
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;
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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ISO/TS 23973:2020(E)
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.
8 © ISO 2020 – All rights reserved
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ISO/TS 23973:2020(E)
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?
© ISO 2020 – All rights reserved 9
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ISO/TS 23973:2020(E)
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
S 2 PEG 3 000 Standard 3 000
S 3 PEG 6 000 Standard 6 000
A 2 PEO-A 3 000 Allyl
M 2 PEO-M 5 000 Methoxy
Mi 1 PEO-X Unknown Unknown
Mi 2 PEO-X Unknown Unknown

a) Polyethylene glycol standards in the molar mass range from 1 500 g/mol to 6 000 g/mol for

determination of the critical conditions of adsorption (S 1 to S 3).

b) Polyethylene oxides with different functionalities (A 2 and M 2), as well as their mixture for

separation under critical conditions and for quantification of the components (Mi 1 and Mi 2).

NOTE The sample solutions A 2, M 2 and mixture 1 needed to be manufactured by the participants (weighing,

dissolution). Mixture 2 was filled up with 2 ml of the eluent and was then available for measurement.

Columns: RP 18, <300 Å, <1 μm
Column temperature: (45 ± 1) °C
Eluent: acetonitrile / water (volume fraction or mass fraction)
Flow rate: 0,5 ml/min or 1 ml/min
Detection: ELSD, Corona, RI
Injection volume: 10 µl to 20 µl
Sample concentration: around 1 ml/ml to 2 mg/ml

The samples were dissolved in the relevant eluent (mixture 2 + 2 ml eluent). To rule out changes to the

polymers in solution as a function of the time as a possible source of error, the samples were analysed

10 © ISO 2020 – All rights reserved
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ISO/TS 23973:2020(E)

within 6 h of production of the solution. It was ensured that the temperature in the column remained

constant.
For the determination of the critical solvent com
...

TECHNICAL ISO/TS
SPECIFICATION 23973
First edition
Liquid chromatography at critical
conditions (LCCC) — Chemical
heterogenity of polyethylene oxides
Chromatographie liquide aux conditions critiques — Hétérogénéité
chimique des oxydes de polyéthylène
PROOF/ÉPREUVE
Reference number
ISO/TS 23973:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
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) ...........................................................................................18

Annex D (informative) Investigations of the long-term stability of the test mixture ......................................44

Bibliography .............................................................................................................................................................................................................................50

© ISO 2020 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
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 PROOF/ÉPREUVE © ISO 2020 – All rights reserved
---------------------- Page: 4 ----------------------
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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TECHNICAL SPECIFICATION ISO/TS 23973:2020(E)
Liquid chromatography at critical conditions (LCCC) —
Chemical heterogenity 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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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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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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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):
 
100
N=55, 4  (1)
 
 05, 
where

t is the retention time at the peak maximum (the same units shall be used, e.g. min);

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
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

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,

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
X2 ACN, as volume fraction in per cent
Y2 retention factor k
1 ACN / H O: 60 % / 40 %
2 ACN / H O: 55 % / 45 %
3 ACN / H O: 50 % / 50 %
4 ACN / H O: 49 % / 51 %
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−
k= (2)
where
t is the retention time of the standard in the relevant system;
t is the retention time of a non-delayed substance.
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;
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 23937:202X);

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 RAB, RAC and RBC in accordance

with [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
S 2 PEG 3 000 Standard 3 000
S 3 PEG 6 000 Standard 6 000
A 2 PEO-A 3 000 Allyl
M 2 PEO-M 5 000 Methoxy
Mi 1 PEO-X Unknown Unknown
Mi 2 PEO-X Unknown Unknown

a) Polyethylene glycol standards in the molar mass range from 1 500 g/mol to 6 000 g/mol for

determination of the critical conditions of adsorption (S 1 to S 3).

b) Polyethylene oxides with different functionalities (A 2 and M 2), as well as their mixture for

separation under critical conditions and for quantification of the components (Mi 1 and Mi 2).

NOTE The sample solutions A 2, M 2 and mixture 1 needed to be manufactured by the participants (weighing,

dissolution). Mixture 2 was filled up with 2 ml of the eluent and was then available for measurement.

Columns: RP 18, <300 Å, <1 μm
Column temperature: (45 ± 1) °C
Eluent: acetonitrile / water (volume fraction or mass fraction)
Flow rate: 0,5 ml/min or 1 ml/min
Detection: ELSD, Corona, RI
Injection volume: 10 µl to 20 µl
Sample concentration: around 1 ml/ml to 2 mg/ml

The samples were dissolved in the relevant eluent (mixture 2 + 2 ml eluent). To rule out changes to the

polymers in solution as a function of the time as a possible source of error, the samples were analysed

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