Nanotechnologies - Analysis of nano-objects using asymmetrical-flow and centrifugal field-flow fractionation (ISO/TS 21362:2018)

This document identifies parameters and conditions, as part of an integrated measurement system, necessary to develop and validate methods for the application of asymmetrical-flow and centrifugal field-flow fractionation to the analysis of nano-objects and their aggregates and agglomerates dispersed in aqueous media. In addition to constituent fractionation, analysis can include size, size distribution, concentration and material identification using one or more suitable detectors. General guidelines and procedures are provided for application, and minimal reporting requirements necessary to reproduce a method and to convey critical aspects are specified.

Nanotechnologien - Analyse von Nanoobjekten mit Hilfe von Asymmetrischer-Fluss-Feldflussfraktionierung und zentrifugaler Feldflussfraktionierung (ISO/TS 21362:2018)

Dieses Dokument identifiziert als Teil eines integrierten Messsystems Parameter und Bedingungen, die zur Entwicklung und Validierung von Methoden für die Anwendung der Asymmetrischer-Fluss-Feldflussfraktionierung und zentrifugaler Feldflussfraktionierung zur Analyse von Nanoobjekten und ihren in wässrigen Medien dispergierten Aggregaten und Agglomeraten erforderlich sind. Zusätzlich zur Fraktionierung der Bestandteile kann die Analyse die Größe, Größenverteilung, Konzentration und Materialidentifizierung mit einem oder mehreren geeigneten Detektoren umfassen. Für die Anwendung werden allgemeine Anleitungen und Verfahren bereitgestellt, und es werden Mindestanforderungen an die Berichterstattung festgelegt, die zur Reproduzierung einer Methode und zur Vermittlung kritischer Aspekte erforderlich sind.

Nanotechnologies - Analyse des nano-objets par fractionnement par couplage flux-force asymétrique et à force centrifuge (ISO/TS 21362:2018)

Le présent document identifie, dans le cadre d'un système de mesures intégrées, les paramètres et conditions nécessaires au développement et à la validation de méthodes pour l'application des systèmes de fractionnement par couplage flux-force asymétrique et à force centrifuge à l'analyse de nano-objets et de leurs agrégats et agglomérats en dispersion dans des milieux aqueux. Outre le fractionnement des composants, l'analyse peut porter sur la taille, la distribution granulométrique, la concentration et l'identification des matériaux à l'aide d'un ou plusieurs détecteurs appropriés. Le présent document fournit des lignes directrices et procédures générales pour l'application et spécifie les exigences de rapport minimales afin de reproduire une méthode et de rendre compte des aspects critiques.

Nanotehnologije - Analiza nanoobjektov s frakcioniranjem asimetričnega in centrifugalnega poljskega pretoka (ISO/TS 21362:2018)

General Information

Status
Published
Public Enquiry End Date
17-Feb-2021
Publication Date
20-Apr-2021
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
15-Apr-2021
Due Date
20-Jun-2021
Completion Date
21-Apr-2021

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SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 21362:2021
01-junij-2021
Nanotehnologije - Analiza nanoobjektov s frakcioniranjem asimetričnega in
centrifugalnega poljskega pretoka (ISO/TS 21362:2018)

Nanotechnologies - Analysis of nano-objects using asymmetrical-flow and centrifugal

field-flow fractionation (ISO/TS 21362:2018)
Nanotechnologien - Analyse von Nanoobjekten mit Hilfe von Asymmetrischer-Fluss-

Feldflussfraktionierung und zentrifugaler Feldflussfraktionierung (ISO/TS 21362:2018)

Nanotechnologies - Analyse des nano-objets par fractionnement par couplage flux-force

asymétrique et à force centrifuge (ISO/TS 21362:2018)
Ta slovenski standard je istoveten z: CEN ISO/TS 21362:2021
ICS:
07.120 Nanotehnologije Nanotechnologies
SIST-TS CEN ISO/TS 21362:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST-TS CEN ISO/TS 21362:2021
---------------------- Page: 2 ----------------------
SIST-TS CEN ISO/TS 21362:2021
CEN ISO/TS 21362
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
April 2021
TECHNISCHE SPEZIFIKATION
ICS 07.120
English Version
Nanotechnologies - Analysis of nano-objects using
asymmetrical-flow and centrifugal field-flow fractionation
(ISO/TS 21362:2018)

Nanotechnologies - Analyse des nano-objets par Nanotechnologien - Analyse von Nanoobjekten mit

fractionnement par couplage flux-force asymétrique et Hilfe von Asymmetrischer-Fluss-

à force centrifuge (ISO/TS 21362:2018) Feldflussfraktionierung und zentrifugaler
Feldflussfraktionierung (ISO/TS 21362:2018)

This Technical Specification (CEN/TS) was approved by CEN on 19 March 2021 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to

submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS

available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in

parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 21362:2021 E

worldwide for CEN national Members.
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SIST-TS CEN ISO/TS 21362:2021
CEN ISO/TS 21362:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST-TS CEN ISO/TS 21362:2021
CEN ISO/TS 21362:2021 (E)
European foreword

The text of ISO/TS 21362:2018 has been prepared by Technical Committee ISO/TC 229

"Nanotechnologies” of the International Organization for Standardization (ISO) and has been taken over

as CEN ISO/TS 21362:2021 by Technical Committee CEN/TC 352 “Nanotechnologies” the secretariat of

which is held by AFNOR.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO/TS 21362:2018 has been approved by CEN as CEN ISO/TS 21362:2021 without any

modification.
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SIST-TS CEN ISO/TS 21362:2021
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SIST-TS CEN ISO/TS 21362:2021
TECHNICAL ISO/TS
SPECIFICATION 21362
First edition
2018-06
Nanotechnologies — Analysis of nano-
objects using asymmetrical-flow and
centrifugal field-flow fractionation
Nanotechnologies — Analyse des nano-objets par fractionnement flux
asymétrique et flux force centrifuge
Reference number
ISO/TS 21362:2018(E)
ISO 2018
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SIST-TS CEN ISO/TS 21362:2021
ISO/TS 21362:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

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 © ISO 2018 – All rights reserved
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SIST-TS CEN ISO/TS 21362:2021
ISO/TS 21362:2018(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Symbols and abbreviated terms ........................................................................................................................................................... 7

5 Principles of operation .................................................................................................................................................................................. 8

5.1 Field-flow fractionation (General) ......................................................................................................................................... 8

5.2 Specific applications by applied field ...............................................................................................................................10

5.2.1 Flow field .............................................................................................................................................................................10

5.2.2 Centrifugal field .............................................................................................................................................................11

6 Method development for AF4 ...............................................................................................................................................................13

6.1 General ........................................................................................................................................................................................................13

6.2 Sample specifications .....................................................................................................................................................................14

6.3 Mobile phase specifications ......................................................................................................................................................14

6.4 Fractionation .........................................................................................................................................................................................15

6.4.1 Channel and membrane selection ..................................................................................................................15

6.4.2 Injection and relaxation ..........................................................................................................................................17

6.4.3 Optimizing flow conditions .................................................................................................................................18

6.4.4 Elution programme ....................................................................................................................................................18

6.4.5 Using FFF theory to select initial flow settings ..................................................................................19

7 Method development for CF3 ...............................................................................................................................................................19

7.1 General ........................................................................................................................................................................................................19

7.2 Choice of mobile phase .................................................................................................................................................................20

7.3 Field strength selection ................................................................................................................................................................20

7.4 Field decay programme ................................................................................................................................................................20

7.5 Channel flow rate selection .......................................................................................................................................................21

7.6 Calculation of the relaxation time........................................................................................................................................21

7.7 Calculation of sample injection dela y ...............................................................................................................................21

8 Analysis of nano-objects ............................................................................................................................................................................21

8.1 General ........................................................................................................................................................................................................21

8.2 Online size analysis ..........................................................................................................................................................................22

8.3 Online concentration analysis.................................................................................................................................................23

8.3.1 General...................................................................................................................................................................................23

8.3.2 Mass-based methods .................................................................................................................................................23

8.3.3 Number-based methods .........................................................................................................................................24

8.4 Online material identification or composition .........................................................................................................25

8.5 Off-line analysis (Fraction collection) ..............................................................................................................................26

9 Qualification, performance criteria and measurement uncertainty ..........................................................26

9.1 System qualification and quality control .......................................................................................................................26

9.1.1 Basic system qualification ....................................................................................................................................26

9.1.2 Focusing performance ........................................................................................................................................... ...27

9.1.3 Flow rate of the carrier liquid ...........................................................................................................................28

9.1.4 Separation field ..............................................................................................................................................................28

9.2 Method performance criteria ..................................................................................................................................................28

9.2.1 Recovery ...............................................................................................................................................................................28

9.2.2 Selectivity ............................................................................................................................................................................29

9.2.3 Retention .............................................................................................................................................................................29

9.2.4 Resolution ...........................................................................................................................................................................29

9.3 Method precision and measurement uncertainty .................................................................................................29

© ISO 2018 – All rights reserved iii
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SIST-TS CEN ISO/TS 21362:2021
ISO/TS 21362:2018(E)

10 General procedures for measurement of samples .........................................................................................................30

10.1 General ........................................................................................................................................................................................................30

10.2 Calibration of retention time for online size analysis .........................................................................................30

10.2.1 Calibration of the AF4 channel .........................................................................................................................30

10.2.2 Calibration of AF4 retention time for online size measurements ......................................31

10.3 AF4 general measurement procedure .............................................................................................................................31

10.4 CF3 general measurement procedure..............................................................................................................................32

11 Test report ................................................................................................................................................................................................................33

11.1 General ........................................................................................................................................................................................................33

11.2 Apparatus and measurement parameters ...................................................................................................................33

11.2.1 AF4 recording/reporting specifications ...................................................................................................33

11.2.2 CF3 recording/reporting specifications ...................................................................................................34

11.3 Reporting test results .....................................................................................................................................................................34

Bibliography .............................................................................................................................................................................................................................35

iv © ISO 2018 – All rights reserved
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SIST-TS CEN ISO/TS 21362:2021
ISO/TS 21362:2018(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 on 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 the following

URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
© ISO 2018 – All rights reserved v
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SIST-TS CEN ISO/TS 21362:2021
ISO/TS 21362:2018(E)
Introduction

The capacity to isolate and analyse diverse populations of nano-objects and their agglomerates or

aggregates, often suspended in, or extracted from, complex matrices, is critical for applications ranging

from materials discovery and nanomanufacturing to regulatory oversight and environmental risk

assessment. Furthermore, the ability to characterize these analytes with minimal perturbation of

their natural or native state is highly desirable. The list of available techniques capable of achieving

such objectives is relatively short, and while all techniques have advantages and disadvantages, and no

single technique is solely adequate or appropriate for all possible applications and materials, a group

of related separation techniques known collectively as field-flow fractionation (FFF), conceptually

[1]

proposed by J. Calvin Giddings in 1966 , offers many advantages for nanotechnology applications. In

FFF, the analyte, suspended in a liquid medium, is fractionated by the application of a field (e.g. flow,

centrifugal, electric, thermal-gradient, magnetic) perpendicular to the direction of flow of the analyte

and mobile phase eluting through a thin defined channel. Separation occurs when the analyte responds

to the applied field, such that populations with different response sensitivities reach equilibrium

positions (i.e. in equilibrium with diffusional forces) higher or lower in the laminar flow streamlines

perpendicular to channel flow, thus eluting differentially.

Among the FFF variants, asymmetrical flow FFF (variously abbreviated in the literature as AF4,

A4F, AFFFF, AfFFF or AsFlFFF) and centrifugal FFF (abbreviated as CF3, also called sedimentation

FFF and abbreviated as SdFFF), are available commercially and have been most widely adopted in

the nanotechnology field (for convenience and simplicity, the abbreviations AF4 and CF3 are used

throughout this document). AF4 is arguably the most versatile technique with respect to the wide range

of applications, materials and particle sizes to which it has been applied. Symmetrical flow FFF (fFFF),

[2]

the original “flow-based” technique as first described in 1976 , has been supplanted commercially by

[3]

AF4, introduced in 1987 , due to several advantages, including a simpler channel design, the ability

to visualize the sample through a transparent top channel wall, and reduced analyte band width. The

theory and application of CF3 as it is presently applied was described by Giddings and coworkers in

[4]

1974 , although a centrifugal field-based FFF system was first developed and tested independently by

[5]

Berg and Purcell in 1967 . Other FFF field variants, such as thermal, electrical and magnetic, provide

unique capabilities, but have been limited in the scope of their applications vis-à-vis nanotechnology or

commercial availability.

Where FFF was once predominantly the domain of specialists, these instruments are now commonly

and increasingly utilized in government, industry and academic laboratories as part of the nano-

characterization toolbox. Two factors are driving this increase in nanotechnology utilization:

maturation of commercial instrumentation and versatility with respect to coupling a wide range

of detectors to FFF systems. In the latter case, recent developments have led to the use of highly

sensitive elemental detectors (e.g. inductively coupled plasma mass spectrometer or ICP-MS), which

offer enhanced characterization and quantification for many materials. Additionally, traditional

concentration or sizing detectors, such as ultraviolet-visible (UV-Vis) absorbance, fluorescence,

multi-angle light scattering (MALS) and dynamic light scattering (DLS), yield online data for eluting

populations, and theoretically provide more accurate information than obtainable using off-line

measurements of unfractionated polydisperse systems. The measured retention time of an eluting

peak can also be used to determine the hydrodynamic size by AF4 based on theoretical relationships or

calibration with a known size standard. CF3 has the unique capacity to rapidly separate species of the

same size but differing in density.

Although FFF based techniques have the capacity to separate and characterize analytes over an

extremely broad size range, from about 1 nm up to tens of micrometers, this document focuses primarily

on materials in the nanoscale regime and their associative structures. The basic underlying principles,

experimental approach, and hardware described here can be more broadly applied.

While this specification includes the most common online detection schemes for nano-object analysis,

other less common forms of detection have been utilized or reported in the literature, including

differential refractometry (primarily used for macromolecular analysis), particle tracking analysis,

graphite furnace atomic absorption spectrometry, single particle ICP-MS, and small-angle X-ray

vi © ISO 2018 – All rights reserved
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SIST-TS CEN ISO/TS 21362:2021
ISO/TS 21362:2018(E)

scattering. This number is likely to grow in the future, as new techniques emerge and existing ones are

modified and evaluated for coupling to FFF.

In order to develop and validate methods for application of FFF to the analysis of nano-objects and

their agglomerates or aggregates, and to properly report experimental results and conditions in order

to enable reproducibility across laboratories, it is critical to specify key parameters to be controlled

and reported. These parameters encompass all aspects of FFF methodology, including sample/analyte,

instrumentation, fractionation, calibration, qualification, performance specifications, measurement

uncertainty, and data analysis. This document identifies the key parameters and lays out a general

approach to method development for AF4 and CF3.

General references and further reading on FFF theory and practise, as well as AF4 and CF3 applications

[6]-[18]
to nanotechnology, can be found in the Bibliography .
© ISO 2018 – All rights reserved vii
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SIST-TS CEN ISO/TS 21362:2021
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SIST-TS CEN ISO/TS 21362:2021
TECHNICAL SPECIFICATION ISO/TS 21362:2018(E)
Nanotechnologies — Analysis of nano-objects using
asymmetrical-flow and centrifugal field-flow fractionation
1 Scope

This document identifies parameters and conditions, as part of an integrated measurement system,

necessary to develop and validate methods for the application of asymmetrical-flow and centrifugal

field-flow fractionation to the analysis of nano-objects and their aggregates and agglomerates dispersed

in aqueous media. In addition to constituent fractionation, analysis can include size, size distribution,

concentration and material identification using one or more suitable detectors. General guidelines and

procedures are provided for application, and minimal reporting requirements necessary to reproduce a

method and to convey critical aspects are specified.
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/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects

ISO/TS 80004-6, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization

3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO/TS 80004-1, ISO/TS 80004-2,

ISO/TS 80004-6 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 https: //www .iso .org/obp
3.1
nano-object

discrete piece of material with one, two or three external dimensions in the nanoscale (from

approximately 1 nm to 100 nm)
Note 1 to entry: Generic term for all discrete nanoscale objects.

[SOURCE: ISO/TS 80004-2:2015, 2.2, modified — In the definition, “(from approximately 1 nm to 100

nm)” has been added. Note 1 to entry has been changed.]
3.2
nanoparticle

nano-object with all external dimensions in the nanoscale where the lengths of the longest and the

shortest axes of the nano-object do not differ significantly

Note 1 to entry: If the dimensions differ significantly (typically by more than 3 times), terms such as nanofibre or

nanoplate may be preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
© ISO 2018 – All rights reserved 1
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SIST-TS CEN ISO/TS 21362:2021
ISO/TS 21362:2018(E)
3.3
field-flow fractionation
FFF

separation technique where a field is applied to a liquid suspension passing along a narrow channel in

order to induce separation of the particles present in the liquid, dependent on their differing mobility

under the force exerted by the field

Note 1 to entry: The field can be, for example, gravitational, centrifugal, a liquid flow, electrical or magnetic.

Note 2 to entry: Using a suitable detector after or during separation allows determination of the size and size

distribution of nano-objects.

[SOURCE: ISO/TS 80004-6:2013, 4.4, modified — The term “field flow” has been changed to “field-flow

...

SLOVENSKI STANDARD
kSIST-TS FprCEN ISO/TS 21362:2021
01-februar-2021
Nanotehnologije - Analiza nanoobjektov s frakcioniranjem asimetričnega in
centrifugalnega poljskega pretoka (ISO/TS 21362:2018)

Nanotechnologies - Analysis of nano-objects using asymmetrical-flow and centrifugal

field-flow fractionation (ISO/TS 21362:2018)
Nanotechnologien - Analyse von Nanoobjekten mit Hilfe von Asymmetrischer-Fluss-

Feldflussfraktionierung und zentrifugaler Feldflussfraktionierung (ISO/TS 21362:2018)

Nanotechnologies - Analyse des nano-objets par fractionnement par couplage flux-force

asymétrique et à force centrifuge (ISO/TS 21362:2018)
Ta slovenski standard je istoveten z: FprCEN ISO/TS 21362
ICS:
07.120 Nanotehnologije Nanotechnologies
kSIST-TS FprCEN ISO/TS 21362:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
kSIST-TS FprCEN ISO/TS 21362:2021
---------------------- Page: 2 ----------------------
kSIST-TS FprCEN ISO/TS 21362:2021
TECHNICAL ISO/TS
SPECIFICATION 21362
First edition
2018-06
Nanotechnologies — Analysis of nano-
objects using asymmetrical-flow and
centrifugal field-flow fractionation
Nanotechnologies — Analyse des nano-objets par fractionnement flux
asymétrique et flux force centrifuge
Reference number
ISO/TS 21362:2018(E)
ISO 2018
---------------------- Page: 3 ----------------------
kSIST-TS FprCEN ISO/TS 21362:2021
ISO/TS 21362:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

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 © ISO 2018 – All rights reserved
---------------------- Page: 4 ----------------------
kSIST-TS FprCEN ISO/TS 21362:2021
ISO/TS 21362:2018(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Symbols and abbreviated terms ........................................................................................................................................................... 7

5 Principles of operation .................................................................................................................................................................................. 8

5.1 Field-flow fractionation (General) ......................................................................................................................................... 8

5.2 Specific applications by applied field ...............................................................................................................................10

5.2.1 Flow field .............................................................................................................................................................................10

5.2.2 Centrifugal field .............................................................................................................................................................11

6 Method development for AF4 ...............................................................................................................................................................13

6.1 General ........................................................................................................................................................................................................13

6.2 Sample specifications .....................................................................................................................................................................14

6.3 Mobile phase specifications ......................................................................................................................................................14

6.4 Fractionation .........................................................................................................................................................................................15

6.4.1 Channel and membrane selection ..................................................................................................................15

6.4.2 Injection and relaxation ..........................................................................................................................................17

6.4.3 Optimizing flow conditions .................................................................................................................................18

6.4.4 Elution programme ....................................................................................................................................................18

6.4.5 Using FFF theory to select initial flow settings ..................................................................................19

7 Method development for CF3 ...............................................................................................................................................................19

7.1 General ........................................................................................................................................................................................................19

7.2 Choice of mobile phase .................................................................................................................................................................20

7.3 Field strength selection ................................................................................................................................................................20

7.4 Field decay programme ................................................................................................................................................................20

7.5 Channel flow rate selection .......................................................................................................................................................21

7.6 Calculation of the relaxation time........................................................................................................................................21

7.7 Calculation of sample injection dela y ...............................................................................................................................21

8 Analysis of nano-objects ............................................................................................................................................................................21

8.1 General ........................................................................................................................................................................................................21

8.2 Online size analysis ..........................................................................................................................................................................22

8.3 Online concentration analysis.................................................................................................................................................23

8.3.1 General...................................................................................................................................................................................23

8.3.2 Mass-based methods .................................................................................................................................................23

8.3.3 Number-based methods .........................................................................................................................................24

8.4 Online material identification or composition .........................................................................................................25

8.5 Off-line analysis (Fraction collection) ..............................................................................................................................26

9 Qualification, performance criteria and measurement uncertainty ..........................................................26

9.1 System qualification and quality control .......................................................................................................................26

9.1.1 Basic system qualification ....................................................................................................................................26

9.1.2 Focusing performance ........................................................................................................................................... ...27

9.1.3 Flow rate of the carrier liquid ...........................................................................................................................28

9.1.4 Separation field ..............................................................................................................................................................28

9.2 Method performance criteria ..................................................................................................................................................28

9.2.1 Recovery ...............................................................................................................................................................................28

9.2.2 Selectivity ............................................................................................................................................................................29

9.2.3 Retention .............................................................................................................................................................................29

9.2.4 Resolution ...........................................................................................................................................................................29

9.3 Method precision and measurement uncertainty .................................................................................................29

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10 General procedures for measurement of samples .........................................................................................................30

10.1 General ........................................................................................................................................................................................................30

10.2 Calibration of retention time for online size analysis .........................................................................................30

10.2.1 Calibration of the AF4 channel .........................................................................................................................30

10.2.2 Calibration of AF4 retention time for online size measurements ......................................31

10.3 AF4 general measurement procedure .............................................................................................................................31

10.4 CF3 general measurement procedure..............................................................................................................................32

11 Test report ................................................................................................................................................................................................................33

11.1 General ........................................................................................................................................................................................................33

11.2 Apparatus and measurement parameters ...................................................................................................................33

11.2.1 AF4 recording/reporting specifications ...................................................................................................33

11.2.2 CF3 recording/reporting specifications ...................................................................................................34

11.3 Reporting test results .....................................................................................................................................................................34

Bibliography .............................................................................................................................................................................................................................35

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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 on 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 the following

URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
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Introduction

The capacity to isolate and analyse diverse populations of nano-objects and their agglomerates or

aggregates, often suspended in, or extracted from, complex matrices, is critical for applications ranging

from materials discovery and nanomanufacturing to regulatory oversight and environmental risk

assessment. Furthermore, the ability to characterize these analytes with minimal perturbation of

their natural or native state is highly desirable. The list of available techniques capable of achieving

such objectives is relatively short, and while all techniques have advantages and disadvantages, and no

single technique is solely adequate or appropriate for all possible applications and materials, a group

of related separation techniques known collectively as field-flow fractionation (FFF), conceptually

[1]

proposed by J. Calvin Giddings in 1966 , offers many advantages for nanotechnology applications. In

FFF, the analyte, suspended in a liquid medium, is fractionated by the application of a field (e.g. flow,

centrifugal, electric, thermal-gradient, magnetic) perpendicular to the direction of flow of the analyte

and mobile phase eluting through a thin defined channel. Separation occurs when the analyte responds

to the applied field, such that populations with different response sensitivities reach equilibrium

positions (i.e. in equilibrium with diffusional forces) higher or lower in the laminar flow streamlines

perpendicular to channel flow, thus eluting differentially.

Among the FFF variants, asymmetrical flow FFF (variously abbreviated in the literature as AF4,

A4F, AFFFF, AfFFF or AsFlFFF) and centrifugal FFF (abbreviated as CF3, also called sedimentation

FFF and abbreviated as SdFFF), are available commercially and have been most widely adopted in

the nanotechnology field (for convenience and simplicity, the abbreviations AF4 and CF3 are used

throughout this document). AF4 is arguably the most versatile technique with respect to the wide range

of applications, materials and particle sizes to which it has been applied. Symmetrical flow FFF (fFFF),

[2]

the original “flow-based” technique as first described in 1976 , has been supplanted commercially by

[3]

AF4, introduced in 1987 , due to several advantages, including a simpler channel design, the ability

to visualize the sample through a transparent top channel wall, and reduced analyte band width. The

theory and application of CF3 as it is presently applied was described by Giddings and coworkers in

[4]

1974 , although a centrifugal field-based FFF system was first developed and tested independently by

[5]

Berg and Purcell in 1967 . Other FFF field variants, such as thermal, electrical and magnetic, provide

unique capabilities, but have been limited in the scope of their applications vis-à-vis nanotechnology or

commercial availability.

Where FFF was once predominantly the domain of specialists, these instruments are now commonly

and increasingly utilized in government, industry and academic laboratories as part of the nano-

characterization toolbox. Two factors are driving this increase in nanotechnology utilization:

maturation of commercial instrumentation and versatility with respect to coupling a wide range

of detectors to FFF systems. In the latter case, recent developments have led to the use of highly

sensitive elemental detectors (e.g. inductively coupled plasma mass spectrometer or ICP-MS), which

offer enhanced characterization and quantification for many materials. Additionally, traditional

concentration or sizing detectors, such as ultraviolet-visible (UV-Vis) absorbance, fluorescence,

multi-angle light scattering (MALS) and dynamic light scattering (DLS), yield online data for eluting

populations, and theoretically provide more accurate information than obtainable using off-line

measurements of unfractionated polydisperse systems. The measured retention time of an eluting

peak can also be used to determine the hydrodynamic size by AF4 based on theoretical relationships or

calibration with a known size standard. CF3 has the unique capacity to rapidly separate species of the

same size but differing in density.

Although FFF based techniques have the capacity to separate and characterize analytes over an

extremely broad size range, from about 1 nm up to tens of micrometers, this document focuses primarily

on materials in the nanoscale regime and their associative structures. The basic underlying principles,

experimental approach, and hardware described here can be more broadly applied.

While this specification includes the most common online detection schemes for nano-object analysis,

other less common forms of detection have been utilized or reported in the literature, including

differential refractometry (primarily used for macromolecular analysis), particle tracking analysis,

graphite furnace atomic absorption spectrometry, single particle ICP-MS, and small-angle X-ray

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scattering. This number is likely to grow in the future, as new techniques emerge and existing ones are

modified and evaluated for coupling to FFF.

In order to develop and validate methods for application of FFF to the analysis of nano-objects and

their agglomerates or aggregates, and to properly report experimental results and conditions in order

to enable reproducibility across laboratories, it is critical to specify key parameters to be controlled

and reported. These parameters encompass all aspects of FFF methodology, including sample/analyte,

instrumentation, fractionation, calibration, qualification, performance specifications, measurement

uncertainty, and data analysis. This document identifies the key parameters and lays out a general

approach to method development for AF4 and CF3.

General references and further reading on FFF theory and practise, as well as AF4 and CF3 applications

[6]-[18]
to nanotechnology, can be found in the Bibliography .
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TECHNICAL SPECIFICATION ISO/TS 21362:2018(E)
Nanotechnologies — Analysis of nano-objects using
asymmetrical-flow and centrifugal field-flow fractionation
1 Scope

This document identifies parameters and conditions, as part of an integrated measurement system,

necessary to develop and validate methods for the application of asymmetrical-flow and centrifugal

field-flow fractionation to the analysis of nano-objects and their aggregates and agglomerates dispersed

in aqueous media. In addition to constituent fractionation, analysis can include size, size distribution,

concentration and material identification using one or more suitable detectors. General guidelines and

procedures are provided for application, and minimal reporting requirements necessary to reproduce a

method and to convey critical aspects are specified.
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/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects

ISO/TS 80004-6, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization

3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO/TS 80004-1, ISO/TS 80004-2,

ISO/TS 80004-6 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 https: //www .iso .org/obp
3.1
nano-object

discrete piece of material with one, two or three external dimensions in the nanoscale (from

approximately 1 nm to 100 nm)
Note 1 to entry: Generic term for all discrete nanoscale objects.

[SOURCE: ISO/TS 80004-2:2015, 2.2, modified — In the definition, “(from approximately 1 nm to 100

nm)” has been added. Note 1 to entry has been changed.]
3.2
nanoparticle

nano-object with all external dimensions in the nanoscale where the lengths of the longest and the

shortest axes of the nano-object do not differ significantly

Note 1 to entry: If the dimensions differ significantly (typically by more than 3 times), terms such as nanofibre or

nanoplate may be preferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
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3.3
field-flow fractionation
FFF

separation technique where a field is applied to a liquid suspension passing along a narrow channel in

order to induce separation of the particles present in the liquid, dependent on their differing mobility

under the force exerted by the field

Note 1 to entry: The field can be, for example, gravitational, centrifugal, a liquid flow, electrical or magnetic.

Note 2 to entry: Using a suitable detector after or during separation allows determination of the size and size

distribution of nano-objects.

[SOURCE: ISO/TS 80004-6:2013, 4.4, modified — The term “field flow” has been changed to “field-flow”.]

3.4
asymmetrical-flow field-flow fractionation

separation technique that uses a cross flow field applied perpendicular to the channel flow to achieve

separation based on analyte diffusion coefficient or size

Note 1 to entry: Cross flow occurs by means of a semipermeable (accumulation) wall in the channel, while cross

flow is zero at an opposing nonpermeable (depletion) wall.

Note 2 to entry: By comparison, in symmetrical flow, the cross flow enters through a permeable wall (frit) and

exits through an opposing semipermeable wall and is generated separately from the channel flow.

Note 3 to entry: Nano-objects generally fractionate by the “normal” mode, where diffusion dominates and

the smallest species elute first. In the micrometre size range, the “steric-hyperlayer” mode of fractionation is

generally dominant, with the largest species eluting first. The transition from normal to steric-hyperlayer mode

can be affected by material properties or measurement parameters, and therefore is not definitively identified;

however, the transition can be defined explicitly for a given experimental set of conditions; typically, the

transition occurs over a particle size range from about 0,5 µm to 2 µm.

Note 4 to entry: Including both normal and steric-hyperlayer modes, the technique has the capacity to separate

particles ranging in size from approximately 1 nm to about 50 µm.
3.5
centrifugal field-flow fractionation

separation technique that uses a centrifugal field applied perpendicular to a circular channel that spins

around its axis to achieve size separation of particles from roughly 10 nm to roughly 50 µm.

Note 1 to entry: Separation is governed by a combination of size and effective particle density.

Note 2 to entry: Applicable size range is dependent on and limited by the effective particle density.

3.6
channel

thin ribbon-like chamber with a parabolic flow profile required for separation

under the influence of a field applied perpendicular to the channel flow
Note 1 to entry: Channel thickness can vary and is defined by a spacer insert.

Note 2 to entry: In asymmetrical-flow field-flow fractionation, a trapezoidal channel is commonly used, typically

with a maximum breadth of ca. 20 mm to 25 mm and length of ca. 100 mm to 300 mm.

Note 3 to entry: In asymmetrical-flow, one channel surface (depletion wall) is solid (impermeable) and the

opposing surface (accumulation wall) consists of a semipermeable membrane on a porous frit.

Note 4 to entry: In centrifugal flow field-flow fractionation, both the inner and outer walls of the circular channel

are solid (non-porous) and the channel is curved. A trapezoidal channel is commonly used, typically with a

breadth of 10 mm to 20 mm and length of 300 mm to 550 mm.
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3.7
spacer

thin plastic film with a cut-out that defines the thickness and lateral

dimensions of the channel

Note 1 to entry: Trapezoidal or rectangular cut-outs are most commonly used in asymmetrical-flow field-flow

fractionation.

Note 2 to entry: Typical spacer thickness used for separation of nano-objects ranges from 190 µm to 500 µm.

3.8
channel thickness
nominal thickness as defined by the spacer
3.9
effective channel thickness

thickness due to compressibility or swelling of the semipermeable membrane

at the accumulation wall, the effective value of which can differ from the nominal value for a given

spacer and is determined using a well-defined analyte of known diffusivity under the test conditions

Note 1 to entry: The measured effective channel
...

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