SIST EN ISO 13885-3:2021

SLOVENSKI STANDARD
oSIST prEN ISO 13885-3:2021
01-maj-2021
Gelska permeacijska kromatografija (GPC) - 3. del: Voda kot izpiralna tekočina
(eluent) (ISO 13885-3:2020)
Gel permeation chromatography (GPC) - Part 3: Water as eluent (ISO 13885-3:2020)
Gelpermeationschromatographie (GPC) - Teil 3: Wasser als Elutionsmittel (ISO 13885-
3:2020)
Chromatographie par perméation de gel (GPC) - Partie 3: Eluant à l'eau (ISO 13885-
3:2020)
Ta slovenski standard je istoveten z: prEN ISO 13885-3
ICS:
87.060.20 Veziva Binders
oSIST prEN ISO 13885-3:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 13885-3:2021

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oSIST prEN ISO 13885-3:2021
INTERNATIONAL ISO
STANDARD 13885-3
First edition
2020-07
Gel permeation chromatography
(GPC) —
Part 3:
Water as eluent
Chromatographie par perméation de gel (GPC) —
Partie 3: Eluant à l'eau
Reference number
ISO 13885-3:2020(E)
©
ISO 2020

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oSIST prEN ISO 13885-3:2021
ISO 13885-3:2020(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
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Email: copyright@iso.org
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Published in Switzerland
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oSIST prEN ISO 13885-3:2021
ISO 13885-3:2020(E)

Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Apparatus . 2
5.1 Eluent supply . 3
5.2 Pump . 3
5.3 Injection system . 3
5.4 Separation columns . 3
5.5 Column temperature control . 5
5.6 Detector . 6
6 Reagents . 6
7 Calibration of the apparatus . 6
7.1 General . 6
7.2 Specification for the calibration standard . 6
7.3 Preparation of the calibration solutions for injection . 7
7.4 Conditions for calibration runs . 7
7.5 Measurement of elution volume . 7
7.6 Plotting the calibration curve . 7
8 Sampling . 8
9 Preparation for the test . 8
9.1 Preparation of the injection solution . 8
9.2 Preparation of the apparatus . 9
10 Analytical parameters . 9
11 Data acquisition and evaluation . 9
11.1 General . 9
11.2 Calculation of the net chromatogram from the raw data .10
11.2.1 Determination of the baseline .10
11.2.2 Correction of the measured values and of the net chromatogram .10
11.2.3 Evaluation limits .10
11.3 Calculation of the average values .10
11.4 Calculation of the distribution curves .11
12 Precision .12
12.1 General .12
12.2 Repeatability .12
12.3 Reproducibility .12
13 Test report .13
13.1 General .13
13.2 General data on the equipment and settings .13
13.2.1 Data on the equipment used .13
13.2.2 Calibration .13
13.2.3 Evaluation .14
13.3 Special data on the sample .14
Annex A (informative) Conversion of experimental parameters for variant column sizes .15
Annex B (informative) Example of a data sheet for a polymer standard .16
Annex C (informative) Explanations .18
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Bibliography .23
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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
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.
A list of all parts in the ISO 13885 series can be found on the ISO website.
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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oSIST prEN ISO 13885-3:2021
INTERNATIONAL STANDARD ISO 13885-3:2020(E)
Gel permeation chromatography (GPC) —
Part 3:
Water as eluent
WARNING — This document can involve hazardous materials, operations or equipment. It
does not purport to address all of the safety problems, if any, associated with its use. It is the
responsibility of the user to establish appropriate safety and health practices and to ensure
compliance with any national regulatory conditions.
1 Scope
This document specifies the determination of the molar-mass distribution and the average molar
mass values M (number average) and M (weight average) of polymers that are soluble in water by gel
n w
permeation chromatography (GPC).
NOTE Also known as size exclusion chromatography (SEC).
This method is applicable to neutral polymers and polyanions (e.g. polycarboxylates,
polysaccharides, fully hydrolyzed polyvinyl alcohols and high-molecular polyethylene oxides).
It is not applicable to polycations [e.g. polyvinylpyrrolidone, polyvinylpyridine, salts of
poly(diallyl-N,N-dimethyl-azacyclopentane), chitosan].
Despite good solubility in the mobile phase and even though the chromatograms obtained show good
repeatability, it is possible that this method cannot be used with certain polymer types because of
specific interactions (e.g. adsorption) within the sample/eluent/column system (see also Clause 12).
The conditions specified in this document are not applicable to the GPC analysis of polymer samples
6
with M values greater than 10 g/mol and/or polymers with elution limits outside the calibration
w
range (see 7.6 and Annex C).
This document includes no correction methods (e.g. for the elimination of peak broadening). If absolute
molar mass values are required, an absolute method (e.g. membrane osmometry for M or light
n
scattering for M ) can be used.
w
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 1513, Paints and varnishes — Examination and preparation of test samples
ISO 4618, Paints and varnishes — Terms and definitions
ISO 15528, Paints, varnishes and raw materials for paints and varnishes — Sampling
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 4618 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
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oSIST prEN ISO 13885-3:2021
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— IEC Electropedia: available at http:// www .electropedia .org/
3.1
gel permeation chromatography
GPC
separation of molecules, mainly based on exclusion effects such as differences in size and/or shape of
molecules (size exclusion chromatography) or in charge (ion exclusion chromatography)
[SOURCE: ISO 13885-1:2020, 3.1]
3.2
system peak
signal peculiar to the gel permeation chromatography (3.1) using a refractive index detector
Note 1 to entry: These signals appear at the total penetration limit of the columns and are not part of the sample,
but of the overall system.
[SOURCE: ISO 13885-1:2020, 3.2]
3.3
polycation
water-soluble polymer that carries permanent positive charges or forms temporary cationic structures
due to its pK value under the measurement conditions
B
3.4
polyanion
water-soluble polymer that carries permanent negative charges or forms temporary anionic structures
due to its pK value under the measurement conditions
S
3.5
neutral polymer
water-soluble polymer that carries no charges and forms o-charged groups or structures under the
measurement conditions
4 Principle
The dissolved (molecularly disperse) molecules of a polymer sample are fractionated on a porous
column material, with separation taking place according to the size of the molecule (or, more precisely,
the polymer coil size which forms in this eluent). Small molecules diffuse into the pores of the column
material more frequently and are therefore retarded more than large molecules. Thus, large molecules
are eluted earlier, small molecules later. Under the test conditions given, the elution volume is solely a
function of the coil size of the molecule.
The polymer content of a sample is determined, the sample is then diluted with eluent to give a
concentration of less than 5 g/l and an aliquot of the diluted sample is injected into the GPC system. The
concentration of the molecules eluted from the column is measured in order of decreasing coil size with
a concentration-sensitive detector (typically a differential refractometer). With the aid of a calibration
curve that has been determined for the particular GPC system, the relative molar-mass distribution, the
relative quantities M and M and the heterogeneity or polydispersity M /M are calculated from the
n w w n
chromatogram obtained.
5 Apparatus
The apparatus shall consist of the components shown in Figure 1, which are described below.
All components which come into contact with the eluent or the sample solution shall not exhibit any
adsorption and memory effects and shall not exhibit any expansion effects at the increased temperature.
The eluent described in Clause 6 can cause corrosion in the case of long-term use. For this reason, it is
necessary that high-quality steel, titanium, polyetherketones or polytetrafluoroethylene are used for
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all components and that the individual modules are connected to one another by means of capillary
tubes made of high-quality steel or titanium.
Figure 1 — Block diagram of a GPC apparatus
5.1 Eluent supply
The eluent reservoir shall adequately protect the eluent against external influences such as the
atmosphere, 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.
5.2 Pump
The pump shall ensure that the eluent flow through the separation column is as smooth and pulse-free
as possible. The flow rate shall be 1 ml/min (see Annex A). To fulfil these requirements, the pump shall
operate at optimum efficiency at this flow rate.
The flow rate of the pump used shall have a variation of max. 0,1 %.
It shall be ensured that the specifications required in 5.6 for detector noise can be adhered to by means
of an appropriate pump structure or a downstream pulsation damper.
5.3 Injection system
The injection system serves to introduce a given amount of the sample solution into the eluent stream
in a rapid and smooth fashion. This introduction can be carried out either manually or automatically.
If the introduction is carried out manually, ensure that the sample loop is filled completely with solvent
before loading with the sample.
Memory effects from the previous sample solution in the injection system shall be avoided by design
measures or by adequate flushing.
5.4 Separation columns
The apparatus shall have one or more columns connected in series and packed with spherical porous
material, the diameter of the pores corresponding to the size of the polymer molecules being analysed.
The packing material typically consists of an acrylate copolymer, produced by a special polymerization
process, with OH functionality and without basic groups. The packing material shall swell only slightly
in the eluent and therefore cannot deform under the pressure developed at the set flow rate.
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To keep adsorptive interactions between the packing material surface and the polymer molecules
to be investigated as low as possible, silica phases and modified silica phases shall not be used.
Furthermore, the sample being analysed shall not be changed, either chemically or structurally, within
the chromatographic system.
Certain polymers interact with the surface of the packing material (e.g. by adsorption), and other
effects can sometimes interfere with the GPC separation mechanism. Details of such effects and notes
on possible remedies are discussed in Annex C. If it is intended to compare analyses of such polymers by
different laboratories, the laboratories shall agree on details of the test conditions that are not covered
by this document.
For good repeatability of test results, it is necessary to adhere to the minimum requirements specified
below with regards to peak broadening (expressed in terms of a number of theoretical plates) and
separation efficiency.
a) 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 (see Figure 2). Inject up to 20 µg of ethylene
glycol on to each column under the same test conditions as are used for analysing polymers; the
injection volume shall not exceed 20 µl (see Annex A). Evaluate the chromatogram obtained using
the Formula (1):
2
 
V
100
e
N =×55, 4   × (1)
 
W L
1/2
 
where
V is the elution volume measured at the peak maximum;
e
W is the peak width at half height (see Figure 2); the same units shall be used for V and W ;
1/2 e 1/2
L is the length of the column (column combination), in centimetres.
Express as the result the number of theoretical plates per metre of column length. To conform to the
requirements of this document, a column combination shall have at least 10 000 theoretical plates
per metre.
NOTE See Annex C for tailing and fronting (asymmetry) of the peak used to calculate the plate count.
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Key
X elution volume
Y peak intensity
1 injection
V elution volume measured at the peak maximum
e
W peak width at half height of the peak
1/2
h maximum peak height
σ standard deviation
Figure 2 — Determination of the number of theoretical plates by the half-height method
b) Separation efficiency
To ensure adequate resolution, the log M versus elution volume, V , calibration curve for the
10 e
column combination used shall not exceed a specified gradient. For the purposes of this document,
the relation given in Formula (2) shall apply in the area of the peak maximum for the polymer
sample under investigation:
VV−
e,M
e,()10×M
x
x
>60, (2)
A
c
where
V is the elution volume for pullulan of molar mass, M , in cubic centimetres;
e,M x
x
V is the elution volume for 10 times that molar mass, in cubic centimetres;
e,1()0×M
x
A is the cross-sectional area of the column, in square centimetres;
c
M is the molar mass of pullulan; it shall be selected such that the peak maximum for the
x
polymer sample under investigation lies approximately halfway between these two
elution volumes.
5.5 Column temperature control
Carry out the test at room temperature (15 °C to 35 °C) or at a higher temperature of max. 40 °C. The
temperature of the column shall not change by more than 1 °C during the analysis (see Annex C).
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5.6 Detector
Use a differential refractometer detector. The cell volume shall not exceed 0,010 ml (see Annex A).
NOTE For the restriction to a single detector type, see Annex C.
The detector response obtained using the injection amounts specified in this document shall, at the
lowest setting for electronic damping, exhibit a noise level of less than 1 % of the maximum height of
the polymer peak.
The signals from the detector are recorded by means of an electronic data system (see Clause 11 for
details).
6 Reagents
A solution of sodium chloride (analytical quality), c(NaCl) = 0,1 mol/l, in water of grade 1 according to
ISO 3696 (conductivity: <0,01 mS/m, extinction at 254 nm and 1 cm optical path length: 0,001) is used
as the eluent. The pH value is adjusted to 7,5 to 8,5 using an aqueous sodium hydroxide solution. Other
components shall not be added.
Discard the eluent used to condition the columns or to perform the analyses, and do not return it to the
eluent reservoir.
7 Calibration of the apparatus
7.1 General
The method is not an absolute one and requires calibration with commercially available unbranched
pullulan standards that have been characterized by independent absolute methods. The results for
samples of polymers with different chemical structures are therefore only comparable within groups of
samples of the same type.
Calibrate the GPC apparatus with a series of unbranched pullulan samples of narrow molar mass
distribution (see Annex C) and whose molar masses have been determined by independent, absolute
methods. Glucose, maltose and maltotriose are used for calibration in the low-molar-mass range. The
result is a calibration curve for the evaluation of GPC analyses of unbranched polymethyl methacrylate
samples. If this calibration curve is used to analyse samples of other compositions, containing molecules
with other structures, the results shall be expressed as the “pullulan molar mass equivalents”.
7.2 Specification for the calibration standard
The molar-mass distribution of the standards shall be narrower than the limits given below as a
function of the molar mass at the peak maximum, M :
p
M < 2 000 g/mol M /M ≤ 1,3
p w n
2 000 g/mol ≤ M < 400 000 g/mol M /M ≤ 1,2
p w n
400 000 g/mol ≤ M M /M ≤ 1,5
p w n
The following minimum requirements shall be fulfilled in the characterization of each individual
pullulan standard used for calibration:
a) at least one average molar mass value, M , M or M , shall be determined by an absolute method;
n w z
b) at least one method shall be used to determine the molar-mass distribution;
c) all the parameters involved in the method used shall be indicated;
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d) the results and data for each batch analyzed shall be presented in a comprehensible form for the user.
NOTE An example of a data sheet is given in Annex B.
If the calibration standards give a shoulder on either side of the peak, pre-peaks or a tailing peak, the
area represented by these anomalies shall be less than 2,0 % of the peak area, otherwise the calibration
standard shall be rejected.
7.3 Preparation of the calibration solutions for injection
Dissolve the calibration standards in the eluent at ambient temperature without shaking or stirring.
The minimum dissolving time is 12 h. Then homogenize the solutions carefully. Store the solutions at
ambient temperature.
The soaking periods are relatively long during dissolving of the calibration standards. The solutions
shall not be shaken during the soaking phase so that the polymer chains do not tear.
Filter the solutions manually through a 0,2 µm to 0,45 µm membrane filter. If the filter shows signs of
blocking, the solution is unsuitable for calibration purposes.
The solutions shall be used within 48 h.
Several calibration standards may be
...