ISO 19929:2017

Plastics - Determination of average molecular mass and mixture ratio of poly(ethylene glycol) and its derivatives by MALDI-TOF-MS

ISO 19929:2017 specifies a general method for determining the average molecular mass and mixture ratio of poly(ethylene glycol) (PEG) and its derivatives with different end groups by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). It is applicable to PEG and its derivatives with molecular masses from 500 g mol−1 to 20 000 g mol−1. The composition is calculated by means of a calibration curve constructed using standard polymer mixtures, where the peak area ratio is plotted versus the mass ratio. This document can be applied to other polymers with monomeric unit similar to PEG.

Plastiques — Détermination de la masse moléculaire moyenne et du rapport de mélange du poly(éthylène glycol) et de ses dérivés par MALDI-TOF-MS

General Information

• Status: Published
• Publication Date: 22-Mar-2017
• ICS: 83.080.01 - Plastics in general
• Technical Committee: ISO/TC 61/SC 5 - Physical-chemical properties
• Drafting Committee: ISO/TC 61/SC 5/WG 11 - Analytical methods
• Current Stage: 9093 - International Standard confirmed
• Start Date: 15-Jun-2022
• Completion Date: 13-Dec-2025

Overview

ISO 19929:2017 specifies a standardized MALDI-TOF-MS method for determining the average molecular mass and mixture ratio of poly(ethylene glycol) (PEG) and its derivatives. The method covers PEG and derivatives with molecular masses from 500 g·mol−1 to 20 000 g·mol−1 and calculates composition using a calibration curve where peak area ratio is plotted against mass ratio. The standard can also be applied to other polymers having a monomeric unit similar to PEG.

Key Topics and Requirements

• Scope and applicability: Quantitative MALDI-TOF-MS analysis of PEG and related polymers (500–20 000 g·mol−1); applicable to mixtures of different end groups.
• Principle: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with calibration of time-of-flight to molecular mass.
• Sample preparation: Guidance on preparation of polymer standards, polymer/matrix/salt solutions, and deposition on the MALDI target.
• Recommended reagents:
  • Matrices: α-cyano-4-hydroxycinnamic acid (CHCA) and dithranol.
  • Salts: lithium, sodium, potassium salts (e.g., iodides, trifluoroacetates).
  • Solvents: methanol and tetrahydrofuran (THF).
• Calibration:
  • Mass-axis calibration using synthetic or biopolymer standards (see Annex A).
  • Intensity-axis calibration through standard polymer mixtures; calibration curve constructed from peak area ratio vs mass ratio.
• Data processing and results:
  • Calculation of molecular mass distribution (MMD), number-average molecular mass (Mn), peak areas, and mass ratios.
  • Software-supported integration of isotopic peaks or use of the most abundant isotopic peak where necessary.
• Quality and reporting: Requirements for precision, instrument settings, calibration records, and test reports.

Applications and Users

ISO 19929 is intended for practical use in:

• Polymer manufacturing quality control (PEG products and PEGylated materials)
• Analytical and research laboratories conducting polymer characterization
• Pharmaceutical and biotech labs analyzing PEG-based excipients or PEGylated drugs
• Instrument manufacturers and service labs validating MALDI-TOF-MS methods
• Interlaboratory studies and standardization projects to ensure reproducible quantitative MALDI data

Typical applications include composition analysis of polymer mixtures, monitoring polymer synthesis and end-group differentiation, and quantitative comparison of polymer batches.

Related Standards

• ISO 10927:2011 - Determination of molecular mass and molecular mass distribution by MALDI-TOF-MS
• ISO 472 - Plastics - Vocabulary

Keywords: ISO 19929, MALDI-TOF-MS, poly(ethylene glycol), PEG, molecular mass, mixture ratio, calibration curve, polymer analysis.

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 19929
First edition
2017-03
Plastics — Determination of average
molecular mass and mixture ratio
of poly(ethylene glycol) and its
derivatives by MALDI-TOF-MS
Plastiques — Détermination de la masse moléculaire moyenne et
du rapport de mélange du poly(éthylène glycol) et de ses dérivés par
MALDI-TOF-MS
Reference number
©
ISO 2017
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
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ii © ISO 2017 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principles . 3
5 Reagents . 3
6 Apparatus . 4
7 Procedure. 5
7.1 General . 5
7.2 Sample preparation . 5
7.2.1 General. 5
7.2.2 Preparation of polymer standard mixtures . 6
7.2.3 Preparation of polymer/matrix/salt solutions . 6
7.2.4 Deposition of the sample on the sample plate (target) . 6
7.2.5 Preparation of biopolymer/matrix solutions . 7
7.3 Instrument settings . 7
7.4 Spectra recording . 8
8 Data acquisition and processing . 8
8.1 General . 8
8.2 Calibration of mass axis . . 8
8.2.1 General. 8
8.2.2 Calibration of mass axis using synthetic polymer standards . 9
8.2.3 Calibration of mass axis using biopolymer standards . 9
8.2.4 Self-calibration method . 9
8.3 Generation of mass calibration curve . 9
8.4 Calibration of intensity axis . 9
9 Expression of results .10
9.1 Calculation of molecular mass distribution (MMD).10
9.2 Calculation of the number-average molecular mass .10
9.3 Calculation of peak area .10
9.4 Constructing calibration curve for intensity .10
9.5 Calculation of mass ratio .11
10 Precision .11
11 Test report .11
11.1 General .11
11.2 Apparatus .11
11.3 Calibration .11
11.4 Results .11
Annex A (normative) Calibrants .12
Bibliography .13
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
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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
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on the ISO list of patent declarations received (see www .iso .org/ patents).
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World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 5, Physical-
chemical properties.
iv © ISO 2017 – All rights reserved

Introduction
For quality control and research of polymeric materials, it is important to know the composition of
polymer mixtures with different terminal groups. In contrast to traditional methods such as liquid
chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS) is a rapid and effective method to characterize polymer mixtures because of its high
mass resolution. It can also be applied to quantitation of mixtures of different polymers. Interlaboratory
comparisons of quantitative MALDI-TOF-MS performed for mixtures of PEG and its derivatives can
ensure standardized conditions of measurement. Standardization of quantitative MALDI-TOF-MS may
promote increasing applications of this analytical technique.
INTERNATIONAL STANDARD ISO 19929:2017(E)
Plastics — Determination of average molecular mass and
mixture ratio of poly(ethylene glycol) and its derivatives by
MALDI-TOF-MS
1 Scope
This document specifies a general method for determining the average molecular mass and mixture
ratio of poly(ethylene glycol) (PEG) and its derivatives with different end groups by matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). It is applicable to PEG
−1 −1
and its derivatives with molecular masses from 500 g mol to 20 000 g mol . The composition is
calculated by means of a calibration curve constructed using standard polymer mixtures, where the
peak area ratio is plotted versus the mass ratio. This document can be applied to other polymers with
monomeric unit similar to PEG.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 472, Plastics — Vocabulary
ISO 10927:2011, Plastics — Determination of the molecular mass and molecular mass distribution of polymer
species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 472, ISO 10927 and the
following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
MALDI-TOF-MS
technique in which the separation is based on different flight times in a field free flight tube depending on
the mass of formed polymer ions after ionization by a laser, desorption and acceleration by high voltage
3.2
peak area
A
sum of peak areas, A , where A is an area under the curve of the mass, M , associated with the j-th
ij ij ij
species of polymer i
Note 1 to entry: As Figure 1 a) shows, integration for peak area, A , should be performed over all isotopes related
ij
to the j-th species. If the software is not able to integrate all isotopic peaks, the peak area of the most abundant
isotopic peak can be used instead [see Figure 1 b)]. For data handling, see ISO 10927:2011, 6.7.
a) Peak area of isotopic peaks b) Peak area or peak height of the most abundant
isotopic peak
Key
1 “peak area”, A
ij
+
2 “peak height” of the most abundant isotopic peak of [PEG(n = 43)+Na]
+
3 “peak area” of the most abundant isotopic peak of [PEG(n = 43)+Na]
X mass/charge
Figure 1 — Definitions of peak area and peak height
3.3
molecular mass
M
sum of the masses of atoms
Note 1 to entry: The molecular mass of the j-th species, M , is also calculated as the average mass of isotopes.
j
Note 2 to entry: The terms “molecular weight” and “molar mass” are also used instead of “molecular mass”.
3.4
number-average molecular mass
M
n
molecular mass defined as:
NM
jj
∑
j
M ≡
n
N
j
∑
j
Note 1 to entry: N is the number of molecules of species j of molecular mass M .
j j
3.5
weight-average molecular mass
M
w
molecular mass defined as:
NM×
()jj
∑
j
M ≡
w
NM×
()
∑ jj
j
2 © ISO 2017 – All rights reserved

3.6
z-average molecular mass
M
z
molecular mass defined as:
NM×
()
jj
∑
j
M ≡
z
NM×
()
jj
∑
j
4 Principles
The MALDI process involves the ablation and the ionization of an analyte dispersed in a small organic
molecule matrix. The matrix shall be able to absorb the laser energy. A metal salt may be added to
cationize the analyte. A polymer is co-crystallized or co-mixed with the matrix molecule and deposited
on the target. A short duration UV laser pulse is used to ablate the matrix and the analyte. The laser
energy is transferred to the matrix molecules, causing them to vaporize. Analyte and matrix molecules
leave the target surface in a plume. Due to the very short desorption time, polymer molecules do not
degrade. The polymer in the ablation plume gains a cation and is accelerated by a high voltage, drifts
down the field free flight tube and is detected at the end of the flight tube. The time-of-flight of the
species depends on their molecular masses, and needs to be calibrated with standards of known
molecular masses. Biopolymers such as proteins are often used to this end.
Ideally, the product of the ratio between the total sum of peak areas and that between number-average
molecular mass is proportional to the mass ratio, i.e.
M 
A  W
n,1
1 1
⋅ =k (1)
 
 
 
A M W
 0  n,0 0
 
where A , W and M are sum of peak areas in MALDI-TOF-MS spectra, mass and number-average
i i n,i
molecular mass of polymer i (i = 0 or 1), respectively. The proportionality constant, k , is experimental
and generally depends on the combination of polymers with different chemical structures. Ideally, if no
mass discrimination is observed, k = 1.
5 Reagents
5.1 Matrices.
α-Cyano-4-hydroxycinnamic acid (CHCA) and 1,8,9-trihydroxyanthracene (dithranol) are the
recommended matrices for this method. Other matrices can be used after examining sufficient
ionizing ability and solubility in solvents used. The procedure described in Clause 7 can be applied to
examination of the ability and solubility. All materials should be at least 97 % pure. They should be
stored in a freezer or refrigerator. They should be warmed up to room temperature right before use.
Regulated reagents shall be handled in accordance with regulations.
5.2 Salts.
Lithium, sodium and potassium salts, e.g. iodides or trifluoroacetates, are recommended.
5.3 Solvents.
Methanol and tetrahydrofuran (THF) are recommended since they are good solvents of PEG and its
derivatives. They also applied to polar polymers. The solvents should be at least 97 % pure. If the
solvents are regulated, they should be treated safely.
5.4 Molecular mass standards.
The calibration of the mass axis should be done using biopolymers and/or synthetic polymers with
known repeating units and defined end groups. The molecular mass of the standards shall be within
the range of the molecular mass of the investigated polymer. The software
...