CEN ISO/TS 21362:2021

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
• Publication Date: 06-Apr-2021
• ICS: 07.120 - Nanotechnologies
• Technical Committee: CEN/TC 352 - Nanotechnologies
• Drafting Committee: CEN/TC 352 - Nanotechnologies
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 07-Apr-2021
• Completion Date: 07-Apr-2021
• Ref Project: SIST-TS CEN ISO/TS 21362:2021 - Nanotechnologies - Analysis of nano-objects using asymmetrical-flow and centrifugal field-flow fractionation (ISO/TS 21362:2018)

Relations

• Revised: FprEN ISO 21362 - Nanotechnologies - Analysis of nano-objects using asymmetrical flow and centrifugal field-flow fractionation (ISO/FDIS 21362:2025)
• Effective Date: 19-Jan-2023

Overview

CEN ISO/TS 21362:2021 (identical to ISO/TS 21362:2018) defines measurement parameters and procedures for the analysis of nano-objects and their aggregates/agglomerates dispersed in aqueous media using field‑flow fractionation techniques - specifically asymmetrical‑flow field‑flow fractionation (AF4) and centrifugal field‑flow fractionation (CF3). The technical specification supports integrated measurement systems that combine fractionation with detectors for size, size distribution, concentration, and material identification, and sets out general guidelines, method development steps and minimal reporting requirements needed to reproduce and validate methods.

Key topics and requirements

• Scope and principles: Explanation of FFF operation and specific principles for AF4 and CF3 separation modes.
• Method development: Structured guidance including sample specifications, mobile phase selection, channel and membrane choice, injection/relaxation procedures, flow/field optimization and elution programming.
• Online and off‑line analysis: Procedures for online size and concentration determination during fractionation and for collecting fractions for off‑line analysis.
• Detectors and outputs: Use of one or more suitable detectors to obtain size, concentration and material identification information as part of an integrated system.
• Qualification and performance: System qualification, quality control checks (focusing, flow and field performance), method performance criteria (recovery, selectivity, retention, resolution), and measurement uncertainty considerations.
• Calibration and procedures: Calibration of retention time for online size analysis, AF4/CF3 general measurement procedures and recommended measurement workflows.
• Reporting requirements: Minimal recording and reporting elements to ensure reproducibility - apparatus, measurement parameters, flow/field settings, calibration data and results.

Applications

This Technical Specification is practical for laboratories and organizations that need validated, reproducible fractionation-based characterization of nanoparticles:

• Characterizing size distributions, particle concentration and aggregation state in aqueous dispersions.
• Coupling fractionation with spectroscopic or mass‑based detectors for material identification or composition analysis.
• Quality control and R&D in nanomaterials, pharmaceuticals, environmental monitoring, and advanced materials manufacturing.
• Method development, inter‑laboratory studies and validation where traceable reporting and uncertainty assessment are required.

Who should use this standard

• Analytical and characterization laboratories working with nanomaterials
• Method developers and instrument manufacturers for AF4/CF3 systems
• Quality assurance teams in industries using nanoparticle dispersions
• Regulatory bodies and researchers performing standardized nanoparticle measurements

Related standards

Refer to ISO/CEN publications from ISO/TC 229 (Nanotechnologies) and national CEN members for complementary guidance on terminology, sampling and broader nanotechnology measurement standards.

Standards Content (Sample)

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

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.

Contents Page
European foreword . 3

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.
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
ISO/TS 21362:2018(E)
© 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: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved

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/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

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/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

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 .
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/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”.]
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.
2 © ISO 2018 – All rights reserved

ISO/TS 21362:2018(E)
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 thickness depends on other factors, such as interactions between
the analyte and the membrane and variability in spacer manufacturing.
3.10
accumulation wall
surface of a field-flow fractionation channel toward which sample components are forced by the applied
field acting perpendicular to the channel flow
Note 1 to entry: In asymmetrical-flow field-flow fractionation, the accumulation wall is flat and consists of a
semipermeable membrane on a porous frit substrate.
Note 2 to entry: In centrifugal field-flow fractionation, the accumulation wall is impermeable and curved, and is
located farther from the axis of rotation relative to the depletion wall. In the rare case that the particles have a
lower density than the aqueous medium, the depletion and accumulation walls are reversed.
3.11
depletion wall
surface of a field-flow fractionation channel opposite the accumulation wall, which is depleted in
analyte due to the movement of analyte toward the accumulation wall in the applied field
Note 1 to entry: In asymmetrical-flow field-flow fractionation, the depletion wall is flat and impermeable.
Note 2 to entry: In centrifugal field-flow fractionation, the depletion wall is impermeable and curved, and located
closer to the axis of rotation relative to the accumulation wall. When the effective particle density is lower than
the density of the medium, the depletion and accumulation walls are reversed.
3.12
carrier liquid
eluent
mobile phase
liquid phase used to achieve separation and transport of analytes
Note 1 to entry: The eluent or mobile phase may contain salts, surfactants, and/or other chemical constituents
that are required for optimized separation and recovery of an analyte.
Note 2 to entry: In this document, only aqueous phases are relevant, but organic solvents can also be used if
equipment and channel are compatible.
3.13
elution
process by which analytes in the mobile phase, or eluent, are transported
through, and exit from, the fractionation channel
ISO/TS 21362:2018(E)
3.14
focusing
process by which, during and after sample injection a
counter-balanced flow entering from opposite ends of the channel (inlet and outlet) is applied to focus
the sample components into a thin band close to the inlet port and near the accumulation wall
Note 1 to entry: This step is necessary to minimize band broadening and to allow components to achieve an
equilibrium localization (relaxation) within the channel.
Note 2 to entry: During focusing outward flow occurs only through the permeable membrane at the
accumulation wall.
3.15
relaxation
process by which the sample components assume their equilibrium state
with respect to the opposing forces of diffusion and the applied field before elution is initiated
Note 1 to entry: In flow field-flow fractionation there are two means to achieve relaxation: normal focusing
relaxation and frit inlet or hydrodynamic relaxation.
Note 2 to entry: In centrifugal field-flow fractionation, stop-flow is used to achieve relaxation.
3.16
injection flow
flow that drives the sample out of the injection loop and into the
fractionation channel
Note 1 to entry: Depending on instrument design, injection can occur via a separate injection port or through the
channel inlet port.
3.17
cross flow
flow field applied perpendicular to the channel flow to achieve
separation of analytes
Note 1 to entry: In asymmetrical-flow field-flow fractionation, cross flow is created by the pressure differential
across a permeable membrane at the accumulation wall, which results in a downward force that decreases with
increasing distance from the accumulation wall.
Note 2 to entry: Cross flow is generated by using a flow controller combined with a single pump or by use of a
second dedicated pump.
3.18
channel inlet flow
eluent that enters the channel at the front end (upstream)
Note 1 to entry: In asymmetrical-flow field-flow fractionation, inlet flow is split between cross flow and channel
flow during elution.
3.19
channel flow
eluent flow through the channel
Note 1 to entry: Channel flow is generally equivalent to the flow exiting the channel and entering the detectors
under typical experimental conditions, but can differ if flow exiting the channel is split.
Note 2 to entry: In asymmetrical-flow field-flow fractionation, fluid loss through the permeable accumulation
wall leads to a linearly decreasing channel-flow velocity. This gradient can be compensated using a trapezoidal
channel design with decreasing channel breadth toward the outlet.
4 © ISO 2018 – All rights reserved

ISO/TS 21362:2018(E)
3.20
void volume
fluid volume defined by the channel dimensions plus the volume between the
channel exit and the first detector
3.21
void peak
a peak appearing in the fractogram that corresponds to unretained, typically
small sample components that are not in equilibrium with the separation field
Note 1 to entry: The void peak travels at the average carrier velocity and elutes before retained components.
Note 2 to entry: In this context, unretained means components that are not separated by the field and elute with
the void peak. Unretained has a different meaning in traditional enthalpic-based chromatographic separations.
3.22
void time
time between initiation of elution and detection of the void peak defined at its maximum signal intensity
3.23
retention time
time between initiation of elution and detection of an analyte peak defined at its maximum signal
intensity
Note 1 to entry: For a Gaussian peak, the maximum and peak centre are equivalent.
3.24
retention parameter
dimensionless parameter equal to the ratio of the analyte zone centre-of-
mass distance (from the accumulation wall) to the channel thickness
Note 1 to entry: A measure of the strength of interaction between the applied field and the analyte.
3.25
retention ratio
ratio of the mean velocity of the analyte zone to the mean velocity of the
mobile phase in the channel during elution
Note 1 to entry: This can be calculated theoretically or determined empirically from the ratio of the retention
times associated with the void and analyte peaks, and is directly related to the retention parameter.
3.26
selectivity
measure of the ability of a method to separate analytes of different diffusion
coefficient or size; empirically, the slope of a double logarithmic plot of diffusion coefficient versus
retention ratio for analytes of known size, where a high value reflects a large change in retention time
with a small variation in analyte size
Note 1 to entry: In centrifugal field-flow fractionation, selectivity is also dependent on effective mass, but the
empirical relationship is defined in the same manner as asymmetrical-flow field-flow fractionation.
3.27
resolution factor
fractionation power
ratio of the difference in retention time to the average of the peak widths measured as the full width at
half maximum for two adjacent eluting analytes
Note 1 to entry: Measure of the degree of separation between neighbouring or overlapping peaks.
3.28
band broadening
overall dispersion or widening of an analyte band as the analyte passes through a separation system
ISO/TS 21362:2018(E)
3.29
zone broadening
broadening of the width of the sample zone during separation in the channel
3.30
normal mode (of elution)
Brownian mode
mode of elution in which diffusion is the dominant opposing force to the
applied orthogonal force (e.g., cross flow or centrifugal), resulting in relatively faster migration of
smaller particles through the channel due to their higher location within the parabolic flow profile and
an elution sequence where smaller particles elute before larger particles
Note 1 to entry: All nanoparticles are subject to normal or Brownian mode elution, which is dominant for particle
diameters smaller than approximately 0,5 µm; nano-objects with at least one dimension greater than 0,5 µm
might be subject to steric-hyperlayer mode elution. The upper limit for normal mode elution is not well defined
and depends on both material and measurement factors.
Note 2 to entry: For centrifugal field-flow fractionation, the stated elution sequence assumes all particles have
the same density; for particles that differ in both size and density, it is possible for the elution sequence to be
reversed.
3.31
steric-lift hyperlayer mode (of elution)
elution in which diffusion forces are negligible, and motion of particles due
to the applied orthogonal force (e.g., cross flow or centrifugal) is essentially impeded by resistance of
the accumulation wall itself, resulting in an elution sequence that is reversed compared to normal mode
Note 1 to entry: Steric effects occur when larger particles form layers at the accumulation wall that, on average,
project higher into the parabolic flow profile of the channel. As a result, larger particles will migrate faster than
smaller particles. Hyperlayer or lift-hyperlayer occurs when the particles form thin layers above (extended from)
the accumulation wall due to hydrodynamic effects, with larger particles more elevated than smaller particles
resulting in their faster migration. Because steric and lift-hyperlayer are closely related, forming a continuum,
and produce similar elution behaviour, they are commonly merged together.
Note 2 to entry: The lower limit for steric-hyperlayer mode elution is not well defined and can depend on both
material and measurement factors such as the channel thickness and flow rate or the applied field strength.
Generally, particles with an effective diameter greater than about 1 µm are subject to steric-hyperlayer elution,
but the onset of steric-hyperlayer effects can occur over a range from about 0,5 µm to about 2 µm. The transition
can be determined experimentally for a given set of conditions.
3.32
fractogram
two-dimensional graphic representation of data derived from an experiment,
typically with one or more detector signals on the ordinate and retention time on the abscissa
Note 1 to entry: This is analogous to a chromatogram in traditional chromatography.
3.33
recovery
ratio of the mass eluted during fractionation to the initial injected mass
expressed as a percentage
Note 1 to entry: Determined experimentally using an appropriate mass sensitive detector, either off-line (directly
measured in sample before injection and after collection of elu
...