ISO 21362
(Main)Nanotechnologies — Analysis of nano-objects using asymmetrical flow and centrifugal field-flow fractionation
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
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Standards Content (Sample)
FINAL DRAFT
International
Standard
ISO/FDIS 21362
ISO/TC 229
Nanotechnologies — Analysis of
Secretariat: BSI
nano-objects using asymmetrical
Voting begins on:
flow and centrifugal field-flow
2025-10-15
fractionation
Voting terminates on:
2025-12-10
Nanotechnologies — Analyse des nano-objets par fractionnement
flux asymétrique et flux force centrifuge
Member bodies are requested to consult relevant national interests in IEC/TC
113 before casting their ballot to the e-Balloting application.
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Reference number
ISO/FDIS 21362:2025(en) © ISO 2025
FINAL DRAFT
ISO/FDIS 21362:2025(en)
International
Standard
ISO/FDIS 21362
ISO/TC 229
Nanotechnologies — Analysis of
Secretariat: BSI
nano-objects using asymmetrical
Voting begins on:
flow and centrifugal field-flow
fractionation
Voting terminates on:
Nanotechnologies — Analyse des nano-objets par fractionnement
flux asymétrique et flux force centrifuge
Member bodies are requested to consult relevant national interests in IEC/TC
113 before casting their ballot to the e-Balloting application.
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2025
IN ADDITION TO THEIR EVALUATION AS
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BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
ISO/CEN PARALLEL PROCESSING
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
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Published in Switzerland Reference number
ISO/FDIS 21362:2025(en) © ISO 2025
ii
ISO/FDIS 21362:2025(en)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms. 8
5 Principles of operation .10
5.1 Field-flow fractionation (FFF) — General .10
5.2 Specific applications by applied field.11
5.2.1 Flow field .11
5.2.2 Centrifugal field . 13
6 Method development for asymmetrical flow field-flow fractionation (AF4) .15
6.1 General . 15
6.2 Sample specifications . 15
6.3 Mobile phase specifications .16
6.4 Fractionation .17
6.4.1 Channel and membrane selection .17
6.4.2 Injection and relaxation .19
6.4.3 Optimizing flow conditions . 20
6.4.4 Elution programme . 20
6.4.5 Using field-flow fractionation (FFF) theory to select initial flow settings .21
7 Method development for centrifugal field-flow fractionation (CF3) .21
7.1 General .21
7.2 Choice of mobile phase .21
7.3 Field strength selection . 22
7.4 Field decay programme . 22
7.5 Channel flow rate selection . 22
7.6 Calculation of the relaxation time . 23
7.7 Calculation of sample injection delay . 23
7.8 Using field-flow fractionation (FFF) theory to select initial settings . 23
8 Analysis of nano-objects .23
8.1 General . 23
8.2 Online size analysis . 23
8.3 Online concentration analysis . 25
8.3.1 General . 25
8.3.2 Mass-based methods . 25
8.3.3 Number-based methods . . 26
8.4 Online material identification or composition .27
8.5 Off-line analysis (fraction collection) .27
8.6 Alternative and emerging methods . 28
9 Qualification, performance criteria and measurement uncertainty .29
9.1 System qualification and quality control . 29
9.1.1 Basic system qualification . 29
9.1.2 Focusing performance . 30
9.1.3 Flow rate of the carrier liquid. 30
9.1.4 Separation field . 30
9.2 Method performance criteria .31
9.2.1 Recovery .31
9.2.2 Selectivity .32
9.2.3 Retention ratio .32
9.2.4 Resolution .32
iii
ISO/FDIS 21362:2025(en)
9.3 Method precision and measurement uncertainty .32
10 General procedures for measurement of samples .33
10.1 Introduction . 33
10.2 Calibration of retention time for online size analysis . 33
10.2.1 Calibration of the asymmetrical flow field-flow fractionation (AF4) channel. 33
10.2.2 Calibration of asymmetrical flow field-flow fractionation (AF4) retention time
for online size measurements . . 34
10.3 Asymmetrical flow field-flow fractionation (AF4) general measurement procedure . 34
10.4 Centrifugal field-flow fractionation (CF3) general measurement procedure . 35
11 Test report .36
11.1 General . 36
11.2 Apparatus and measurement parameters . 36
11.2.1 Asymmetrical flow field-flow fractionation (AF4) recording and reporting
specifications . . 36
11.2.2 Centrifugal field-flow fractionation (CF3) recording and reporting specifications .37
11.3 Test report . 38
Annex A (informative) Summary of interlaboratory comparison .39
Bibliography .54
iv
ISO/FDIS 21362:2025(en)
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
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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).
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This document was prepared jointly by Technical Committee ISO/TC 229, Nanotechnologies and Technical
Committee IEC/TC 113, Nanotechnology standardization for electrical and electronic products and systems, in
collaboration with the European Committee for Standardization (CEN) Technical Committee CEN/TC 352,
Nanotechnologies, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna
Agreement). The draft was circulated for voting to the national bodies of both ISO and IEC.
This first edition cancels and replaces ISO/TS 21362:2018, which has been technically revised.
The main changes are as follows:
— addition of subclause 8.6 addressing alternative and emerging methods;
— revision of technical content to reflect the current state of the art;
— addition of Annex A summarizing an interlaboratory comparison conducted through VAMAS.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
ISO/FDIS 21362:2025(en)
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 proposed in Reference [1] 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 associated with the
abbreviation 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), the original “flow-based” technique
[2] [3]
as first described in 1976, has been supplanted commercially by 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
[4]
applied was described in 1974, although a centrifugal field-based FFF system was first developed and
[5]
tested independently in 1967. Other FFF field variants, such as thermal, electrical and magnetic, provide
unique capabilities, but are 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 estimate 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 micrometres, this document focuses primarily on materials
in the nanoscale regime and their associative structures. However, the basic underlying principles,
experimental approach, and hardware described here can be more broadly applied.
For general references and further reading for FFF theory and practise, as well as AF4 and CF3 applications
to nanotechnology, see References [6] to [18]. Annex A summarizes a Versailles Project on Advanced
Materials and Standards (VAMAS) interlaboratory comparison conducted to evaluate the capacity of AF4
and CF3 techniques to separate and characterize components of a complex multimodal mixture of analytes
reproducibly and with acceptable recovery and resolution across laboratories using different commercial
instrument platforms and instrument configurations.
vi
FINAL DRAFT International Standard ISO/FDIS 21362:2025(en)
Nanotechnologies — Analysis of nano-objects using
asymmetrical flow and centrifugal field-flow fractionation
1 Scope
This document describes the general principles of field-flow fractionation and specifies parameters,
conditions and minimal reporting requirements, as part of an integrated measurement system, required
to develop and validate methods for the application of asymmetrical flow and centrifugal field-flow
fractionation in the analysis of nano-objects and their aggregates and agglomerates in aqueous media.
General guidelines and procedures are provided to aid the user.
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 80004-1:2023, Nanotechnologies – Vocabulary — Part 1: Core vocabulary
ISO/TS 80004-6:2021, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 80004-1 and 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 https:// www .electropedia .org/
— ISO Online browsing platform: available at https:// www .iso .org/ obp
3.1
nanoscale
length range approximately from 1 nm to 100 nm
[SOURCE: ISO 80004-1:2023, 3.1.1]
3.2
nano-object
discrete piece of material with one, two, or three external dimensions in the nanoscale (3.1)
[SOURCE: ISO 80004-1:2023, 3.1.5]
3.3
nanoparticle
nano-object (3.2) with all external dimensions in the nanoscale (3.1)
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms such as nanofibre or
nanoplate are preferred to the term nanoparticle.
[SOURCE: ISO 80004-1:2023, 3.3.4]
ISO/FDIS 21362:2025(en)
3.4
field-flow fractionation
FFF
separation technique where a field is applied to a liquid suspension passing along a narrow channel (3.7) 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, liquid flow, electrical or magnetic.
Note 2 to entry: Using a suitable detector after or during separation allows determination of the mean size and size
distribution of nano-object (3.2) populations.
3.5
asymmetrical flow field-flow fractionation
AF4
separation technique that uses a cross flow (3.19) field applied perpendicular to the channel flow (3.21) 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 (3.2) generally fractionate by the “normal” mode, where diffusion dominates and the
smallest species elute first. In the micrometre size range, the “steric-lift hyperlayer” mode of fractionation is generally
dominant, with the largest species eluting first. The transition from normal to steric-lift hyperlayer mode (3.33) 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-lift hyperlayer modes, the technique has the capacity to separate
particles ranging in size from approximately 1 nm to about 50 µm.
3.6
centrifugal field-flow fractionation
CF3
separation technique that uses a centrifugal field applied perpendicular to a circular channel (3.7) 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.
3.7
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 (3.21)
Note 1 to entry: Channel thickness (3.9) can vary and is nominally determined by a spacer (3.8) insert, while fixed-
height channels have a predefined thickness and do not use inserts.
Note 2 to entry: In asymmetrical flow field-flow fractionation (3.5), 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 (3.12)] is solid (impermeable) and the
opposing surface [accumulation wall (3.11)] consists of a semipermeable membrane on a porous frit.
Note 4 to entry: In centrifugal field-flow fractionation (3.6), both the inner and outer walls of the 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.
ISO/FDIS 21362:2025(en)
3.8
spacer
thin plastic film with a cut-out that defines the thickness and lateral dimensions
of the channel (3.7)
Note 1 to entry: Trapezoidal or rectangular cut-outs are most commonly used in asymmetrical flowfield-flow
fractionation (3.5).
Note 2 to entry: Typical spacer thickness used for separation of nano-objects (3.2) ranges from 190 µm to 500 µm.
Note 3 to entry: Fixed-height channels do not use a spacer; in this case the channel shape and thickness are predefined.
3.9
channel thickness
w
nominal thickness as defined by the spacer (3.8) or predefined in a fixed-height
channel (3.7)
3.10
effective channel thickness
w
eff
varying from the nominal value due to compressibility or swelling of the
semipermeable membrane at the accumulation wall (3.11)
Note 1 to entry: The value of the effective thickness can differ from the nominal value for a given spacer (3.8) and may
be determined using a well-defined analyte of known diffusivity under the test conditions.
Note 2 to entry: The measured effective channel thickness can depend on other factors, such as interactions between
the analyte and the membrane and variability in spacer manufacturing.
3.11
accumulation wall
surface of a field-flow fractionation (3.4) channel toward which sample components are forced by the applied
field acting perpendicular to the channel flow (3.21)
Note 1 to entry: In asymmetrical flow field-flow fractionation (3.5), 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 (3.12). In the rare case that the particles have a
lower density than the aqueous medium, the depletion and accumulation walls are reversed.
3.12
depletion wall
surface of a field-flow fractionation (3.4) channel opposite the accumulation wall (3.11), 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 (3.6), 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.
3.13
mobile phase
carrier liquid
eluent
liquid phase used to achieve separation and transport of analytes
Note 1 to entry: The eluent or mobile phase can contain one or more salts, surfactants, and other chemical constituents
that are required for optimized separation and recovery (3.35) of an analyte.
ISO/FDIS 21362:2025(en)
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.
3.14
elution
process by which analytes in the mobile phase (3.13), or eluent, are transported
through, and exit from, the fractionation channel (3.7)
Note 1 to entry: Elution begins after injection, focusing (3.16) and other pre-elution steps have completed.
Note 2 to entry: Elution can occur with or without an applied field.
3.15
elution time
elapsed time after initiation of elution (3.14) and excluding preliminary steps such
as injection, focusing (3.16) or other transitions
Note 1 to entry: Elution and retention share the same timeline and can be used interchangeably.
Note 2 to entry: The horizontal (time) axis of a fractogram (3.34) is generally expressed as elution time.
3.16
focusing
application of counter-balanced flow from opposite ends
(inlet and outlet) of the channel (3.7) to focus sample components into a thin band near the inlet port at the
accumulation wall (3.11)
Note 1 to entry: This step is required to minimize band broadening (3.30) and to allow components to achieve an
equilibrium localization [relaxation (3.17)] within the channel.
Note 2 to entry: Focusing does not occur during frit-inlet injection.
3.17
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 (3.14) is initiated
Note 1 to entry: In flow field-flow fractionation (3.4) there are two means to achieve relaxation: normal focusing (3.16),
relaxation and frit inlet or hydrodynamic relaxation.
Note 2 to entry: In centrifugal field-flow fractionation (3.6), stop-flow is used to achieve relaxation.
3.18
injection flow
flow that drives the sample out of the injection loop and into the fractionation
channel (3.7)
Note 1 to entry: Depending on instrument design, injection can occur via a separate injection port or through the
channel inlet port.
3.19
cross flow
flow field applied perpendicular to the channel flow (3.21) to achieve
separation of analytes
Note 1 to entry: In asymmetrical flowfield-flow fractionation (3.5), cross flow is created by the pressure differential
across a permeable membrane at the accumulation wall (3.11), which results in a force directed toward the
accumulation wall 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.
ISO/FDIS 21362:2025(en)
3.20
inlet flow
mobile phase (3.13), or eluent, that enters the channel (3.7) at the front end
(upstream)
Note 1 to entry: In asymmetrical flow field-flow fractionation (3.5), inlet flow is split between cross flow (3.19) and
channel flow during elution (3.14).
3.21
channel flow
parabolic laminar flow through the channel (3.7) and parallel to the accumulation
wall (3.11)
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 flowfield-flow fractionation (3.5), 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.
3.22
void volume
V
fluid volume defined by the channel (3.7) dimensions plus the volume between the
channel exit and the first detector
3.23
void peak
peak appearing in the fractogram (3.34) that corresponds to unretained material
not in equilibrium with the separation field
Note 1 to entry: The void peak travels at the average carrier fluid 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.
Note 3 to entry: A void peak is generated by the mechanical disruption or change in flow conditions when elution
(3.14) is initiated; in this context the void peak should ideally contain only mobile phase (3.13) and small molecular
species unaffected by the applied field.
3.24
void time
t
time between initiation of elution (3.14) and detection of the void peak (3.23) defined at its maximum signal
intensity
3.25
retention time
t
R
time between initiation of elution (3.14) 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.
Note 2 to entry: Retention time and elution time (3.15) represent equivalent timelines. The latter is generic, while the
former is typically used in the context of an analyte peak.
Note 3 to entry: The net retention time can be obtained by subtracting the void time (3.24) from the measured peak
retention time. This equates to normalizing retention time to the elution of unretained material traveling at the mean
velocity of the channel parabolic flow.
ISO/FDIS 21362:2025(en)
3.26
retention parameter
λ
dimensionless parameter equal to the ratio of the analyte zone centre-of-mass
distance (from the accumulation wall (3.11)) to the channel (3.7) thickness
Note 1 to entry: A measure of the strength of interaction between the applied field and the analyte.
3.27
retention ratio
R
ratio of the mean velocity of the analyte zone to the mean velocity of the mobile
phase (3.13) in the channel (3.7) during elution (3.14)
Note 1 to entry: This can be calculated theoretically or determined empirically from the ratio of the elution times
(3.15) associated with the void and analyte peaks and is directly related to the retention parameter (3.26).
3.28
selectivity
measure of the ability of a method to separate analytes of different diffusion
coefficient or size
Note 1 to entry: Empirically, selectivity is calculated from the slope of a double logarithmic plot of diffusion coefficient
versus retention ratio (3.27) or retention time (3.25) versus analyte diameter.
Note 2 to entry: A high selectivity reflects a large change in retention time with a small variation in analyte size.
Note 3 to entry: In centrifugal field-flow fractionation (3.6), selectivity is also dependent on effective mass, but the
empirical relationship is defined in the same manner as asymmetrical flow field-flow fractionation (3.5).
3.29
resolution factor
fractionation power
R
s
ratio of the difference in retention time (3.25) 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.
3.30
band broadening
overall dispersion or widening of an analyte band as the analyte passes through a separation system
3.31
zone broadening
broadening of the width of the sample zone during separation in the channel (3.7)
3.32
normal mode
Brownian mode
mode of elution (3.14) in which diffusion is the dominant opposing force to the
applied orthogonal force (e.g., cross flow (3.19) or centrifugal), resulting in an elution (3.14) sequence where
smaller particles elute before larger particles
Note 1 to entry: All nanoparticles (3.3) are subject to normal or Brownian mode elution, which is dominant for particle
diameters smaller than approximately 0,5 µm; nano-objects (3.2) with at least one dimension greater than 0,5 µm can
be subject to steric-lift hyperlayer mode (3.33) 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 centrifugalfield-flow fractionation (3.6), 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.
ISO/FDIS 21362:2025(en)
3.33
steric-lift hyperlayer mode
mode of elution (3.14) in which diffusion forces are negligible, and motion of
particles due to the applied orthogonal force (e.g., cross flow (3.19) or centrifugal) is essentially impeded by
resistance of the accumulation wall (3.11) itself, resulting in an elution sequence that is reversed compared
to normal mode (3.32)
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-lift hyperlayer mode elution is not well defined and can depend on both
material and measurement factors such as the channel thickness (3.9) and flow rate or the applied field strength.
Generally, particles with an effective diameter greater than about 1 µm are subject to steric-lift hyperlayer elution, but
the onset of steric-lift hyperlayer effects can occur over a range from about 0,5 µm to about 2 µm. The inversion point
can be determined experimentally for a given set of conditions using an appropriate detection method like MALS.
3.34
fractogram
two-dimensional graphic representation of data derived from an experiment,
typically with one or more detector signals on the ordinate and retention time (3.25) on the abscissa
Note 1 to entry: This is analogous to a chromatogram in traditional chromatography.
3.35
recovery
R%
ratio of the analyte quantity eluted to the analyte quantity entering the
fractionation channel (3.7) expressed as a percentage
Note 1 to entry: Determined experimentally using a mass-sensitive detector, either off-line (directly measured in the
sample before injection and after collection of eluting peak) or online [by comparison of peak areas obtained with and
without the applied force field and focusing (3.16)]. The off-line approach assesses recovery for the entire instrumental
system, whereas the on-line approach assesses recovery principally in the fractionation channel.
Note 2 to entry: Recovery can be estimated based on the signal from non-mass-sensitive detectors, such as light
scattering, subject to the influence of changes in detector response due to changes in the size distribution or other
experimental conditions.
...
ISO/FDIS 21362:2025(en)
ISO/TC 229/ JWG2
Secretariat: BSI
Date: 2025-06-03
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
FDIS stage
ISO/DISFDIS 21362:20242025(en)
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
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Phone: + 41 22 749 01 11
E-mail: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO/DISFDIS 21362:20242025(en)
Contents
Foreword . v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms . 8
5 Principles of operation . 11
5.1 Field-flow fractionation (FFF) — General . 11
5.2 Specific applications by applied field . 12
6 Method development for asymmetrical flow field-flow fractionation (AF4) . 16
6.1 General . 16
6.2 Sample specifications . 16
6.3 Mobile phase specifications . 17
6.4 Fractionation . 18
7 Method development for centrifugal field-flow fractionation (CF3) . 23
7.1 General . 23
7.2 Choice of mobile phase . 23
7.3 Field strength selection . 23
7.4 Field decay programme . 24
7.5 Channel flow rate selection . 24
7.6 Calculation of the relaxation time . 24
7.7 Calculation of sample injection delay . 25
7.8 Using field-flow fractionation (FFF) theory to select initial settings . 25
8 Analysis of nano-objects . 25
8.1 General . 25
8.2 Online size analysis . 25
8.3 Online concentration analysis . 27
8.4 Online material identification or composition . 29
8.5 Off-line analysis (fraction collection) . 30
8.6 Alternative and emerging methods . 31
9 Qualification, performance criteria and measurement uncertainty . 31
9.1 System qualification and quality control . 31
9.2 Method performance criteria . 33
9.3 Method precision and measurement uncertainty . 35
10 General procedures for measurement of samples . 36
10.1 Introduction . 36
10.2 Calibration of retention time for online size analysis . 36
10.3 Asymmetrical flow field-flow fractionation (AF4) general measurement procedure . 37
10.4 Centrifugal field-flow fractionation (CF3) general measurement procedure . 38
11 Test report . 39
11.1 General . 39
11.2 Apparatus and measurement parameters . 39
11.3 Test report . 41
Annex A (informative) Summary of interlaboratory comparison. 43
Bibliography . 58
iii
ISO/DISFDIS 21362:20242025(en)
iv
ISO/DISFDIS 21362:20242025(en)
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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO [had/had not] received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that this
may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared jointly by Technical Committee ISO/TC 229, Nanotechnologies and Technical
Committee IEC/TC 113, Nanotechnology standardization for electrical and electronic products and systems, and
in collaboration with the European Committee for Standardization (CEN) Technical Committee CEN/TC 352,
Nanotechnologies, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna
Agreement). The draft was circulated for voting to the national bodies of both ISO and IEC.
This first edition cancels and replaces ISO/TS 21362:2018, which has been technically revised. The main
changes compared to the previous edition are as follows:
— Additional clause (8.6)The main changes are as follows:
— addition of subclause 8.6 addressing alternative and emerging methods;
— — Technical revisionsrevision of technical content to update information toreflect the current state of
the art.;
— — Annex Aaddition of Annex A summarizing an interlaboratory comparison conducted through VAMAS.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
ISO/DISFDIS 21362:20242025(en)
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-
[1]
flow fractionation (FFF), conceptually proposed by J. Calvin Giddings in Reference [11966 ,] 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 associated with the
abbreviation 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), the original “flow-based” technique as first
[2[2] ] [3[3] ]
described in 1976, , has been supplanted commercially by 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
[4[4] ]
described by Giddings and coworkers in 1974, , although a centrifugal field-based FFF system was first
[5[5] ]
developed and tested independently by Berg and Purcell in 1967. . Other FFF field variants, such as thermal,
electrical and magnetic, provide unique capabilities, but are 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 estimate 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 micrometres, this document focuses primarily on materials in
the nanoscale regime and their associative structures. However, the basic underlying principles, experimental
approach, and hardware described here can be more broadly applied.
GeneralFor general references and further reading for FFF theory and practise, as well as AF4 and CF3
applications to nanotechnology, see References [6] to [18are listed in the Bibliography [6]-[18]. Annex A].
Annex A summarizes a Versailles Project on Advanced Materials and Standards (VAMAS) interlaboratory
comparison conducted to evaluate the capacity of AF4 and CF3 techniques to separate and characterize
vi
ISO/DISFDIS 21362:20242025(en)
components of a complex multimodal mixture of analytes reproducibly and with acceptable recovery and
resolution across laboratories using different commercial instrument platforms and instrument
configurations.
vii
DRAFT International Standard ISO/DIS 21362:2024(en)
Nanotechnologies — Analysis of nano-objects using asymmetrical
flow and centrifugal field-flow fractionation
1 Scope
This document describes the general principles of field-flow fractionation and specifies parameters,
conditions and minimal reporting requirements, as part of an integrated measurement system,
necessaryrequired to develop and validate methods for the application of asymmetrical flow and centrifugal
field-flow fractionation in the analysis of nano-objects and their aggregates and agglomerates in aqueous
media. General guidelines and procedures are provided to aid the user.
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 80004--1:2023, Nanotechnologies – Vocabulary — Part 1: Core vocabulary
ISO/TS 80004--6:2021, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 80004-1 and 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 https://www.electropedia.org/
— — ISO Online browsing platform: available at https://www.iso.org/obp
3.1 3.1
nanoscale
length range approximately from 1 nm to 100 nm
[SOURCE: ISO 80004-1:2023, 3.1.1]
3.2 3.2
nano-object
discrete piece of material with one, two, or three external dimensions in the nanoscale (3.1(3.1))
[SOURCE: ISO 80004-1:2023, 3.1.5]
3.3 3.3
nanoparticle
nano-object (3.2(3.2)) with all external dimensions in the nanoscale (3.1(3.1))
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms such as nanofibre or
nanoplate are preferred to the term nanoparticle.
ISO/DISFDIS 21362:20242025(en)
[SOURCE: ISO 80004-1:2023, 3.3.4]
3.4 3.4
field-flow fractionation
FFF
separation technique where a field is applied to a liquid suspension passing along a narrow channel (3.7(3.7))
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, liquid flow, electrical or magnetic.
Note 2 to entry: Using a suitable detector after or during separation allows determination of the mean size and size
distribution of nano-object (3.2) populations.
3.5
3.5 Asymmetrical
asymmetrical flow field-flow fractionation
AF4
separation technique that uses a cross flow (3.19(3.19)) field applied perpendicular to the channel flow
(3.21(3.21)) 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 (3.2) generally fractionate by the “normal” mode, where diffusion dominates and the
smallest species elute first. In the micrometre size range, the “steric-lift hyperlayer” mode of fractionation is generally
dominant, with the largest species eluting first. The transition from normal to steric-lift hyperlayer mode (3.33) 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-lift hyperlayer modes, the technique has the capacity to separate
particles ranging in size from approximately 1 nm to about 50 µm.
3.6 3.6
centrifugal field-flow fractionation
CF3
separation technique that uses a centrifugal field applied perpendicular to a circular channel (3.7(3.7)) 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.
3.7 3.7
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 (3.21(3.21))
Note 1 to entry: Channel thickness (3.9) can vary and is nominally determined by a spacer (3.8) insert, while fixed-height
channels have a predefined thickness and do not use inserts.
Note 2 to entry: In asymmetrical flow field-flow fractionation (3.5,), 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.
ISO/DISFDIS 21362:2025(en)
Note 3 to entry: In asymmetrical flow, one channel surface ([depletion wall (3.12))] is solid (impermeable) and the
opposing surface ([accumulation wall (3.11))] consists of a semipermeable membrane on a porous frit.
Note 4 to entry: In centrifugal field-flow fractionation (3.6,), both the inner and outer walls of the 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.
3.8 3.8
spacer
thin plastic film with a cut-out that defines the thickness and lateral dimensions of
the channel (3.7(3.7))
Note 1 to entry: Trapezoidal or rectangular cut-outs are most commonly used in asymmetrical flow fieldflowfield-flow
fractionation (3.5.).
Note 2 to entry: Typical spacer thickness used for separation of nano-objects (3.2) ranges from 190 µm to 500 µm.
Note 3 to entry: Fixed-height channels do not use a spacer; in this case the channel shape and thickness are predefined.
3.9 3.9
channel thickness
w
nominal thickness as defined by the spacer (3.8(3.8)) or predefined in a fixed-height
channel (3.7(3.7))
3.10 3.10
effective channel thickness
w
eff
varying from the nominal value due to compressibility or swelling of the
(3.11))
semipermeable membrane at the accumulation wall (3.11
Note 1 to entry: The value of the effective thickness can differ from the nominal value for a given spacer (3.8) and may be
determined using a well-defined analyte of known diffusivity under the test conditions.
Note 2 to entry: The measured effective channel thickness can depend on other factors, such as interactions between the
analyte and the membrane and variability in spacer manufacturing.
3.11 3.11
accumulation wall
surface of a field-flow fractionation (3.4(3.4)) channel toward which sample components are forced by the
applied field acting perpendicular to the channel flow (3.21(3.21))
Note 1 to entry: In asymmetrical flow field-flow fractionation (3.5,), 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 (3.12.). In the rare case that the particles have a lower
density than the aqueous medium, the depletion and accumulation walls are reversed.
3.12 3.12
depletion wall
surface of a field-flow fractionation (3.4(3.4)) channel opposite the accumulation wall (3.11(3.11),), 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.
ISO/DISFDIS 21362:20242025(en)
Note 2 to entry: In centrifugal field-flow fractionation (3.6,), 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.
3.13
mobile phase
3.13
carrier liquid
eluent
mobile phaseliquid phase used to achieve separation and transport of analytes
Note 1 to entry: The eluent or mobile phase can contain one or more salts, surfactants, and other chemical constituents
that are required for optimized separation and recovery (3.35) 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.
3.14 3.14
elution
process by which analytes in the mobile phase (3.13(3.13),), or eluent, are
transported through, and exit from, the fractionation channel (3.7(3.7))
Note 1 to entry: Elution begins after injection, focusing (3.16) and other pre-elution steps have completed.
Note 2 to entry: Elution can occur with or without an applied field.
3.15 3.15
elution time
elapsed time after initiation of elution (3.14(3.14)) and excluding preliminary steps
such as injection, focusing (3.16(3.16)) or other transitions
Note 1 to entry: Elution and retention share the same timeline and can be used interchangeably.
Note 2 to entry: The horizontal (time) axis of a fractogram (3.34) is generally expressed as elution time.
3.16 3.16
focusing
application of counter-balanced flow from opposite ends (inlet
and outlet) of the channel (3.7(3.7)) to focus sample components into a thin band near the inlet port at the
accumulation wall (3.11(3.11))
Note 1 to entry: This step is necessaryrequired to minimize band broadening (3.30) and to allow components to achieve
an equilibrium localization ([relaxation (3.17))] within the channel.
Note 2 to entry: Focusing does not occur during frit-inlet injection.
3.17 3.17
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 (3.14(3.14)) is initiated
Note 1 to entry: In flow field-flow fractionation (3.4) there are two means to achieve relaxation: normal focusing (3.16),
relaxation and frit inlet or hydrodynamic relaxation.
Note 2 to entry: In centrifugal field-flow fractionation (3.6,), stop-flow is used to achieve relaxation.
ISO/DISFDIS 21362:2025(en)
3.18 3.18
injection flow
flow that drives the sample out of the injection loop and into the fractionation
channel (3.7(3.7))
Note 1 to entry: Depending on instrument design, injection can occur via a separate injection port or through the channel
inlet port.
3.19 3.19
cross flow
flow field applied perpendicular to the channel flow (3.21(3.21)) to achieve
separation of analytes
Note 1 to entry: In asymmetrical flow fieldflowfield-flow fractionation (3.5,), cross flow is created by the pressure
differential across a permeable membrane at the accumulation wall (3.11,), which results in a force directed toward the
accumulation wall 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.
3.20 3.20
inlet flow
mobile phase (3.13(3.13),), or eluent, that enters the channel (3.7(3.7)) at the front
end (upstream)
Note 1 to entry: In asymmetrical flow field-flow fractionation (3.5,), inlet flow is split between cross flow (3.19) and
channel flow during elution (3.14.).
3.21 3.21
channel flow
parabolic laminar flow through the channel (3.7(3.7)) and parallel to the
accumulation wall (3.11(3.11))
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 fieldflowfield-flow fractionation (3.5,), 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.
3.22 3.22
void volume
V
fluid volume defined by the channel (3.7(3.7)) dimensions plus the volume between
the channel exit and the first detector
3.23 3.23
void peak
peak appearing in the fractogram (3.34(3.34)) that corresponds to unretained
material not in equilibrium with the separation field
Note 1 to entry: The void peak travels at the average carrier fluid 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.
ISO/DISFDIS 21362:20242025(en)
Note 3 to entry: A void peak is generated by the mechanical disruption or change in flow conditions when elution (3.14)
is initiated; in this context the void peak should ideally contain only mobile phase (3.13) and small molecular species
unaffected by the applied field.
3.24 3.24
void time
t
time between initiation of elution (3.14(3.14)) and detection of the void peak (3.23(3.23)) defined at its
maximum signal intensity
3.25 3.25
retention time
t
R
time between initiation of elution (3.14(3.14)) 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.
Note 2 to entry: Retention time and elution time (3.15) represent equivalent timelines. The latter is generic, while the
former is typically used in the context of an analyte peak.
Note 3 to entry: The net retention time can be obtained by subtracting the void time (3.24) from the measured peak
retention time. This equates to normalizing retention time to the elution of unretained material traveling at the mean
velocity of the channel parabolic flow.
3.26 3.26
retention parameter
λ
dimensionless parameter equal to the ratio of the analyte zone centre-of-mass
distance (from the accumulation wall (3.11(3.11)))) to the channel (3.7(3.7)) thickness
Note 1 to entry: A measure of the strength of interaction between the applied field and the analyte.
3.27 3.27
retention ratio
R
ratio of the mean velocity of the analyte zone to the mean velocity of the mobile
phase (3.13(3.13)) in the channel (3.7(3.7)) during elution (3.14(3.14))
Note 1 to entry: This can be calculated theoretically or determined empirically from the ratio of the elution times (3.15)
associated with the void and analyte peaks and is directly related to the retention parameter (3.26.).
3.28 3.28
selectivity
measure of the ability of a method to separate analytes of different diffusion
coefficient or size
Note 1 to entry: Empirically, selectivity is calculated from the slope of a double logarithmic plot of diffusion coefficient
versus retention ratio (3.27) or retention time (3.25) versus analyte diameter.
Note 2 to entry: A high selectivity reflects a large change in retention time with a small variation in analyte size.
Note 3 to entry: In centrifugal field-flow fractionation (3.6,), selectivity is also dependent on effective mass, but the
empirical relationship is defined in the same manner as asymmetrical flow field-flow fractionation (3.5.).
ISO/DISFDIS 21362:2025(en)
3.29 3.29
resolution factor
fractionation power
R
s
ratio of the difference in retention time (3.25(3.25)) 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.
3.30 3.30
band broadening
overall dispersion or widening of an analyte band as the analyte passes through a separation system
3.31 3.31
zone broadening
broadening of the width of the sample zone during separation in the channel
(3.7(3.7))
3.32 3.32
normal mode
Brownian mode
mode of elution (3.14(3.14)) in which diffusion is the dominant opposing force to
the applied orthogonal force (e.g., cross flow (3.19(3.19)) or centrifugal), resulting in an elution (3.14(3.14))
sequence where smaller particles elute before larger particles
Note 1 to entry: All nanoparticles (3.3) are subject to normal or Brownian mode elution, which is dominant for particle
diameters smaller than approximately 0,5 µm; nano-objects (3.2) with at least one dimension greater than 0,5 µm can be
subject to steric-lift hyperlayer mode (3.33) 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 fieldcentrifugalfield-flow fractionation (3.6,), 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.
3.33 3.33
steric-lift hyperlayer mode
mode of elution (3.14(3.14)) in which diffusion forces are negligible, and motion of
particles due to the applied orthogonal force (e.g., cross flow (3.19(3.19)) or centrifugal) is essentially impeded
by resistance of the accumulation wall (3.11(3.11)) itself, resulting in an elution sequence that is reversed
compared to normal mode (3.32(3.32))
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-lift hyperlayer mode elution is not well defined and can depend on both
material and measurement factors such as the channel thickness (3.9) and flow rate or the applied field strength.
Generally, particles with an effective diameter greater than about 1 µm are subject to steric-lift hyperlayer elution, but
the onset of steric-lift hyperlayer effects can occur over a range from about 0,5 µm to about 2 µm. The inversion point
can be determined experimentally for a given set of conditions using an appropriate detection method like MALS.
ISO/DISFDIS 21362:20242025(en)
3.34 3.34
fractogram
two-dimensional graphic representation of data derived from an experiment,
typically with one or more detector signals on the ordinate and retention time (3.25(3.25)) on the abscissa
Note 1 to entry: This is analogous to a chromatogram in traditional chromatography.
3.35 3.35
recovery
R%
ratio of the analyte quantity eluted to the analyte quantity entering the fractionation
channel (3.7(3.7)) expressed as a percentage
Note 1 to entry: Determined experimentally using a mass-sensitive detector, either off-line (directly measured in the
sample before injection and after collection of eluting peak) or online ([by comparison of peak areas obtained with and
without the applied force field and focusing (3.16).)]. The off-line approach assesses recovery for the entire instrumental
system, whereas the on-line approach assesses recovery principally in the fractionation channel.
Note 2 to entry: Recovery can be estimated based on the signal from non-mass-sensitive detectors, such as light
scattering, subject to the influence of changes in detector response due to changes in the size distribution or other
experimental conditions.
3.36 3.36
residual
referring to material or a peak that elutes from the fractionation channel (3.7(3.7))
after removal of the applied field
Note 1 to entry: Residual peaks contain unfractionated material that, due to its physicochemical interaction with the
accumulation wall (3.11,), is retained during application of the field ([e.g. cross flow (3.19) or centrifugal)] and released
once this field decays to zero or is otherwise removed. Frequently this peak contains agglomerates or other relatively
large objects that have insufficient diffusional motion to counteract the applied field. The specific origins of residual
material isare not well defined and can involve multiple factors related to the nature of the analyte, accumulation wall
interactions or the experimental configuration (e.g. flow rates and applied field strength) .).
3.37 3.37
field-on
referring to a state in which the applied field is active during an elution (3.14(3.14))
program
3.38 3.38
field-off
referring to a state in which the applied field is inactive during an elution
(3.14(3.14)) program
4 Symbols and abbreviated terms
AF4 asymmetrical flow field-flow fractionation
BSA bovine serum albumin
CF3 centrifugal field-flow fractionation
BSA
bovine serum albumincertified reference materialcellulose triacetate
CRM
CTA
ISO/DISFDIS 21362:2025(en)
CTA cellulose triacetate
CTAB cetyltrimethylammonium bromide, (C H )N(CH ) Br
16 33 3 3
DAD diode array detector
DLS dynamic light scattering
F4 flow field-flow fractionation
AF4 asymmetrical flow field-flow fractionation
CF3 centrifugal field-flow fractionation
EAF4 electrical asymmetrical flow field-flow fractionation
F4 flow field-flow fractionation
FFF field-flow fractionation
FL-70 commercial formulation containing nonionic and ionic surfactants
FWHM full width at half maximum
HF5 hollow fibre flow field-flow fractionation
HPLC high performance liquid chromatography
ICP-MS inductively coupled plasma mass spectrometry
ICP-OES inductively coupled plasma optical emission spectroscopy
TOC total organic carbon
MALS multi-angle light scattering (static light scattering)
MWCO molecular weight cut-off
NOAA nano-objects and their aggregates and agglomerates
NIR near infrared
PEG
polyethylene glycolpolyethersulfone
PES
PES polyethersulfone
PSL polystyrene latex
PTA particle tracking analysis
RC regenerated cellulose
RM Raman microspectroscopy
RSD relative standard deviation
SDS sodium dodecyl sulfate, CH (CH ) SO Na
3 2 11 4
SPP sodium pyrophosphate
FL-70 commercial formulation containing nonionic and ionic surfactants
SEC size exclusion chromatography
sp single particle (as with inductively coupled plasma mass spectrometry)
SPR surface plasmon resonance
ISO/DISFDIS 21362:20242025(en)
UV-Vis ultraviolet-visible (wavelength range)
VAMAS Versailles Project on Advanced Materials and Standards
λ retention parameter
R retention ratio
t void time
t retention time (of analyte peak)
R
Δt retention time difference for two eluting peaks, centre-to-centre
R
t injection delay time
D
average width of two adjacent eluting peaks, typically defined as the full width at half
w
avg
maximum
w channel thickness (nominal, as defined by a spacer in AF4)
w effective channel thickness (as determined empirically in AF4)
eff
l centre of gravity distance from accumulation wall
D translational diffusion coefficient
˙
𝑉𝑉̇𝑉𝑉 volumetric cross flow rate in AF4
𝑐𝑐 𝑐𝑐
˙
volumetric channel inlet flow rate
𝑉𝑉̇𝑉𝑉
𝑖𝑖𝑖𝑖 𝑖𝑖𝑖𝑖
˙
𝑉𝑉̇𝑉𝑉 volumetric channel outlet flow rate
˙
𝑉𝑉̇𝑉𝑉 volumetric injection flow rate
𝑖𝑖 𝑖𝑖
𝑣𝑣 migration velocity of the analyte zone in the channel
⟨ ⟩
𝑣𝑣〈𝑣𝑣〉 average longitudinal carrier (mobile phase) velocity in the channel
V void volume
V volume between the injection valve and the channel injection port
i
V sample injection loop volume
l
S selectivity
R resolution factor
s
R% recovery
A peak area of eluted sample
S
A peak area of sample injected directly into detector or eluted without cross flow
D
˙
t
1 time over which 𝑉𝑉̇𝑉𝑉 or centrifugal field is held constant during an elution programme
𝑐𝑐 𝑐𝑐
r radius of gyration (mass-averaged root mean square radius)
g
d hydrodynamic diameter (sphere-equivalent)
H
d volume-based diameter (sphere-equivalent)
V
RPS rotational speed in revolutions per second
RPS rotational speed in revolutions per second at the beginning of the decay period
G centripedal acceleration (also referred to as centrifugal field strength)
ISO/DISFDIS 21362:2025(en)
5 Principles of operation
5.1 Field-flow fractionation (FFF) — General)
Field-flow fractionationFFF is a flow-based separation methodology. Typical FFF operation predicates the
release of a narrow sample band into a flowing mobile phase contained within a thin, well-defined channel.
By comparison, traditional liquid chromatography utilizes a packed column containing a solid stationary
phase that interacts with the analyte to achieve separation. In FFF, the mobile phase and analyte flow
longitudinally through the channel. The channel is designed to separate the sample components along its
length, resulting in the elution of constituents at different times. The channel and its large aspect ratio is
designed to promote parabolic or near-parabolic laminar flow between two infinite planes under normal
operational conditions. Though generally ignored, viscous drag at the edges of the channel can be accounted
[19[19] ]
for using a correction factor. . Under parabolic flow, longitudinal fluid velocity at the walls of the channel
approaches zero due to frictional drag, and increases with distance from the walls. Maximum velocity occurs
at the channel centre.
To induce separation between different constituents of a sample, a field, such as cross flow or centrifugal, is
applied in a direction perpendicular to the flow axis (see Figure 1Figure 1).). The force of the applied field
moves constituents toward the accumulation wall (depicted at bottom in Figure 1Figure 1)) and is opposed
by the diffusional force inherent to the affected species. Con
...
PROJET FINAL
Norme
internationale
ISO/FDIS 21362
ISO/TC 229
Nanotechnologies — Analyse des
Secrétariat: BSI
nano-objets par fractionnement flux
Début de vote:
asymétrique et flux force centrifuge
2025-10-15
Nanotechnologies — Analysis of nano-objects using asymmetrical
Vote clos le:
flow and centrifugal field-flow fractionation
2025-12-10
LES DESTINATAIRES DU PRÉSENT PROJET SONT
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ISO/FDIS 21362:2025(fr) © ISO 2025
PROJET FINAL
ISO/FDIS 21362:2025(fr)
Norme
internationale
ISO/FDIS 21362
ISO/TC 229
Nanotechnologies — Analyse des
Secrétariat: BSI
nano-objets par fractionnement flux
Début de vote:
asymétrique et flux force centrifuge
2025-10-15
Nanotechnologies — Analysis of nano-objects using asymmetrical
Vote clos le:
flow and centrifugal field-flow fractionation
2025-12-10
LES DESTINATAIRES DU PRÉSENT PROJET SONT
INVITÉS À PRÉSENTER, AVEC LEURS OBSERVATIONS,
Il est demandé aux comités membres de consulter les intérêts nationaux
NOTIFICATION DES DROITS DE PROPRIÉTÉ DONT ILS
AURAIENT ÉVENTUELLEMENT CONNAISSANCE ET À
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FOURNIR UNE DOCUMENTATION EXPLICATIVE.
plateforme de e-Balloting.
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ii
ISO/FDIS 21362:2025(fr)
Sommaire Page
Avant-propos .v
Introduction .vi
1 Domaine d'application . 1
2 Références normatives . 1
3 Termes et définitions . 1
4 Symboles et abréviations . 8
5 Principes de fonctionnement.11
5.1 Fractionnement par couplage flux-force (FFF) — Généralités .11
5.2 Applications spécifiques selon le type de champ appliqué . 12
5.2.1 Champ d'écoulement . 12
5.2.2 Champ centrifuge .14
6 Développement de la méthode de fractionnement par couplage flux-force asymétrique
(AF4) .16
6.1 Généralités .16
6.2 Spécifications de l'échantillon .16
6.3 Spécifications de la phase mobile .17
6.4 Fractionnement .18
6.4.1 Choix du canal et de la membrane .18
6.4.2 Injection et relaxation .21
6.4.3 Optimisation des conditions d'écoulement .21
6.4.4 Programme d'élution . 22
6.4.5 Utilisation de la théorie du fractionnement par couplage flux-force (FFF) pour
le choix des paramètres d'écoulement initiaux . 23
7 Développement de la méthode de fractionnement par couplage flux-force à force
centrifuge (CF3) .23
7.1 Généralités . 23
7.2 Choix de la phase mobile . 23
7.3 Choix de l'intensité de champ .24
7.4 Programme de diminution du champ .24
7.5 Choix du débit du canal . 25
7.6 Calcul du temps de relaxation . 25
7.7 Calcul du retard à l'injection de l'échantillon . 25
7.8 Utilisation de la théorie du fractionnement par couplage flux-force (FFF) pour le choix
des paramètres initiaux . 25
8 Analyse des nano-objets .26
8.1 Généralités . 26
8.2 Analyse granulométrique en ligne . 26
8.3 Analyse de concentration en ligne . 28
8.3.1 Généralités . 28
8.3.2 Méthodes basées sur la masse . 28
8.3.3 Méthodes basées sur le nombre . . 29
8.4 Identification des matériaux ou composition des échantillons en ligne . 30
8.5 Analyse en mode «hors ligne» (collecte de fractions) .31
8.6 Méthodes alternatives et émergentes .31
9 Qualification, critères de performance et incertitude de mesure .32
9.1 Qualification du système et contrôle de la qualité .32
9.1.1 Qualification du système de base .32
9.1.2 Performances de focalisation . 33
9.1.3 Débit de l'éluant . 34
9.1.4 Champ de séparation . 34
9.2 Critères de performance de la méthode . 34
iii
ISO/FDIS 21362:2025(fr)
9.2.1 Recouvrement . 34
9.2.2 Sélectivité . 35
9.2.3 Rapport de rétention . 35
9.2.4 Résolution . 36
9.3 Précision de la méthode et incertitude de mesure . 36
10 Procédures générales de mesure des échantillons .37
10.1 Introduction .37
10.2 Étalonnage du temps de rétention pour une analyse granulométrique en ligne .37
10.2.1 Étalonnage du canal de fractionnement par couplage flux-force asymétrique
(AF4) .37
10.2.2 Étalonnage du temps de rétention de fractionnement par couplage flux-force
asymétrique (AF4) pour les mesurages granulométriques en ligne. 38
10.3 Procédure générale de mesure par fractionnement par couplage flux-force asymétrique
(AF4) .38
10.4 Procédure générale de mesure par fractionnement par couplage flux-force à force
centrifuge (CF3) . 39
11 Rapport d'essai .40
11.1 Généralités . 40
11.2 Appareillage et paramètres de mesure . 40
11.2.1 Spécifications d'enregistrement et de rapport pour le fractionnement par
couplage flux-force asymétrique (AF4) . 40
11.2.2 Spécifications d'enregistrement et de rapport pour le fractionnement par
couplage flux-force à force centrifuge (CF3).41
11.3 Rapport d'essai .42
Annexe A (informative) Résumé d'une comparaison interlaboratoires .44
Bibliographie . 61
iv
ISO/FDIS 21362:2025(fr)
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes nationaux
de normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est en général
confiée aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude a le droit de faire
partie du comité technique créé à cet effet. Les organisations internationales, gouvernementales et non
gouvernementales, en liaison avec l'ISO participent également aux travaux. L'ISO collabore étroitement avec
la Commission électrotechnique internationale (IEC) en ce qui concerne la normalisation électrotechnique.
Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont
décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier, de prendre note des différents
critères d'approbation requis pour les différents types de documents ISO. Le présent document
a été rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2
(voir www.iso.org/directives).
L'ISO attire l'attention sur le fait que la mise en application du présent document peut entraîner l'utilisation
d'un ou de plusieurs brevets. L'ISO ne prend pas position quant à la preuve, à la validité et à l'applicabilité
de tout droit de propriété revendiqué à cet égard. À la date de publication du présent document, l'ISO [avait/
n'avait pas] reçu notification qu'un ou plusieurs brevets pouvaient être nécessaires à sa mise en application.
Toutefois, il y a lieu d'avertir les responsables de la mise en application du présent document que des
informations plus récentes sont susceptibles de figurer dans la base de données de brevets, disponible à
l'adresse www.iso.org/brevets. L'ISO ne saurait être tenue pour responsable de ne pas avoir identifié tout ou
partie de tels droits de brevet.
Les appellations commerciales éventuellement mentionnées dans le présent document sont données pour
information, par souci de commodité, à l'intention des utilisateurs et ne sauraient constituer un engagement.
Pour une explication de la nature volontaire des normes, la signification des termes et expressions
spécifiques de l'ISO liés à l'évaluation de la conformité, ou pour toute information au sujet de l'adhésion de
l'ISO aux principes de l'Organisation mondiale du commerce (OMC) concernant les obstacles techniques au
commerce (OTC), voir www.iso.org/avant-propos.
Le présent document a été élaboré conjointement par le comité technique ISO/TC 229, Nanotechnologies et le
comité technique IEC/TC 113, Nanotechnologies relatives aux appareils et systèmes électrotechnologiques, en
collaboration avec le comité technique CEN/TC 352, Nanotechnologie, du Comité européen de normalisation
(CEN) conformément à l'Accord de coopération technique entre l'ISO et le CEN (Accord de Vienne). Le présent
projet de document a été mis en circulation auprès des organismes nationaux de l'ISO et de l'IEC en vue d'un vote.
Cette première édition annule et remplace l'ISO/TS 21362:2018, qui a fait l'objet d'une révision technique.
Les principales modifications sont les suivantes:
— ajout de paragraphe 8.6 traitant de méthodes alternatives et émergentes;
— révision du contenu technique pour refléter l'état actuel de la technique;
— ajout de l'Annexe A résumant une comparaison interlaboratoires effectuée dans le cadre de VAMAS.
Il convient que l'utilisateur adresse tout retour d'information ou toute question concernant le présent
document à l'organisme national de normalisation de son pays. Une liste exhaustive desdits organismes se
trouve à l'adresse www.iso.org/fr/members.html.
v
ISO/FDIS 21362:2025(fr)
Introduction
La capacité à isoler et analyser des populations diverses de nano-objets et leurs agglomérats ou agrégats,
généralement en suspension dans des matrices complexes ou qui en sont extraits, est essentielle pour de
nombreuses applications, de la mise au point de nouveaux matériaux et de la nano-fabrication à la surveillance
réglementaire et à l'évaluation des risques environnementaux. De plus, la possibilité de caractériser ces
analytes en réduisant autant que possible les impacts sur leur état naturel ou natif est indispensable. Les
techniques disponibles pour atteindre de tels objectifs sont limitées, et bien que toutes les techniques
présentent à la fois des avantages et des inconvénients, aucune en particulier n'est en soi adéquate ou
adaptée à toutes les applications et à tous les matériaux possibles. Un groupe de techniques de séparation
connexes, collectivement désignées sous le nom de fractionnement par couplage flux-force (FFF) et qui ont
été proposées conceptuellement dans la Référence [1], se distingue par les nombreux avantages qu'il offre
pour leurs utilisations avec les nanotechnologies. Dans les techniques de FFF, l'analyte, en suspension dans
un milieu liquide, est fractionné par l'application d'un champ (par exemple un flux, un champ centrifuge, un
champ électrique, un gradient thermique, un champ magnétique) perpendiculairement au sens d'écoulement
de l'analyte et de la phase mobile qui est éluée à travers un mince canal. La séparation se produit lorsque
l'analyte réagit au champ appliqué, de sorte que les populations ayant différentes sensibilités de réponse
atteignent des positions d'équilibre (autrement dit, qu'elles se trouvent en équilibre avec les forces de
diffusion) plus ou moins hautes ou basses dans l'épaisseur du canal ou avec des profils de vitesse plus ou
moins élevés de l'écoulement laminaire du canal, de manière à être éluées différemment.
Parmi les variantes de la technique FFF, le système asymétrique de fractionnement par couplage flux-force
(désigné dans la littérature par les abréviations AF4, A4F, AFFFF, AfFFF ou AsFlFFF) et le fractionnement
par couplage flux-force à force centrifuge (abrégé en CF3, également appelé FFF de sédimentation associé
à l'abréviation SdFFF), sont disponibles dans le commerce et ont été largement adoptés dans le domaine
de la nanotechnologie (pour des raisons de commodité et de simplicité, les abréviations AF4 et CF3 sont
utilisées dans l'ensemble du présent document). La technique AF4 est incontestablement la plus polyvalente
compte tenu de la grande diversité d'applications, de matériaux et de granulométries pour lesquels elle a été
[2]
appliquée. Le FFF symétrique (fFFF), technique initiale «de flux» décrite pour la première fois en 1976, a
[3]
été remplacé commercialement par la technique AF4, introduite en 1987, en raison de plusieurs avantages,
notamment une conception de canal simplifiée, la possibilité de visualiser l'échantillon à travers une
paroi supérieure de canal transparente, et une réduction de la largeur de bande des analytes. La théorie
[4]
et l'application actuelles du CF3 ont été décrites en 1974 , bien qu'un système de FFF à base de champ
[5]
centrifuge ait été développé et soumis à essai pour la première fois indépendamment en 1967 . D'autres
variantes de champ de FFF, telles que thermiques, électriques et magnétiques, fournissent des capacités
uniques, mais sont limitées dans le domaine de leurs applications vis-à-vis de la nanotechnologie ou de la
disponibilité commerciale.
Alors que le FFF était, à ses débuts, essentiellement réservé aux spécialistes, ces instruments se sont
démocratisés pour la caractérisation des nano-objets et sont de plus en plus utilisés dans les laboratoires
institutionnels, industriels et universitaires. Deux facteurs sous-tendent ce recours croissant à la
nanotechnologie: la maturation de l'instrumentation commerciale et la polyvalence qui permet d'associer
une grande diversité de détecteurs aux systèmes de FFF. Dans ce dernier cas, les récents développements ont
conduit à l'utilisation de détecteurs élémentaires extrêmement sensibles (par exemple des spectromètres
de masse à plasma à couplage inductif ou ICP-MS), qui offrent de meilleures capacités de caractérisation
et de quantification pour de nombreux matériaux. En outre, les détecteurs classiques de concentration
ou de taille, notamment la spectroscopie d'absorption UV-visible (UV-Vis), la fluorescence, la diffusion de
la lumière multi-angles (MALS) et la diffusion dynamique de la lumière (DLS), produisent des données
en ligne pour les populations à élution et fournissent en théorie des informations plus précises que celles
pouvant être obtenues par des mesurages hors ligne de systèmes polydisperses non fractionnés. Le temps
de rétention mesuré d'un pic d'élution peut également être utilisé pour estimer la taille hydrodynamique par
la technique AF4 à partir de relations théoriques ou d'un étalonnage reposant sur un étalon de taille connue.
La technique CF3 a la particularité de séparer rapidement les espèces de même taille mais de différentes
densités.
Bien que les techniques FFF aient la capacité de séparer et caractériser des analytes sur une très vaste
plage granulométrique, de 1 nm environ jusqu'à quelques dizaines de micromètres, le présent document
se concentre principalement sur les matériaux à l'échelle nanométrique ainsi que sur leurs structures
vi
ISO/FDIS 21362:2025(fr)
associées. Cependant, les principes fondamentaux sous-jacents, l'approche expérimentale et le matériel
décrits ici peuvent être appliqués dans un domaine plus large.
Pour les références générales ainsi que d'autres publications consacrées aux descriptions théoriques et
pratiques du FFF et aux applications d'AF4 et de CF3 aux nanotechnologies, voir les Références [6] à [18].
L'Annexe A résume une comparaison interlaboratoires menée dans le cadre d'un Programme de Versailles
sur les matériaux de pointe et les normes (VAMAS) effectuée pour évaluer la capacité des techniques AF4
et CF3 à séparer et caractériser les composants d'un mélange multimodal complexe d'analytes de manière
reproductible et avec un recouvrement et une résolution acceptables dans les laboratoires utilisant
différentes plates-formes d'instruments commerciales et configurations d'instrument.
vii
PROJET FINAL Norme internationale ISO/FDIS 21362:2025(fr)
Nanotechnologies — Analyse des nano-objets par
fractionnement flux asymétrique et flux force centrifuge
1 Domaine d'application
Le présent document décrit les principes généraux du fractionnement par couplage flux-force et spécifie les
paramètres, les conditions et les exigences minimales en matière de rapports, dans le cadre d'un système de
mesures intégrées, requis 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
leurs agrégats et agglomérats en dispersion dans des milieux aqueux. Afin d'aider l'utilisateur, des lignes
directrices et des procédures générales sont fournies.
2 Références normatives
Les documents suivants sont cités dans le texte de sorte qu'ils constituent, pour tout ou partie de leur
contenu, des exigences du présent document. Pour les références datées, seule l'édition citée s'applique. Pour
les références non datées, la dernière édition du document de référence s'applique (y compris les éventuels
amendements).
ISO 80004-1:2023, Nanotechnologies — Vocabulaire — Partie 1: Vocabulaire "cœur"
ISO/TS 80004-6:2021, Nanotechnologies — Vocabulaire — Partie 6: Caractérisation des nano-objets
3 Termes et définitions
Pour les besoins du présent document, les termes et les définitions de l’ISO 80004-1 et l’ISO 80004-1 et
l'ISO/TS 80004-6, ainsi que les suivants s'appliquent.
L'ISO et l'IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en normalisation,
consultables aux adresses suivantes:
— IEC Electropedia: disponible à l'adresse https:// www .electropedia .org/
— ISO Online browsing platform: disponible à l'adresse https:// www .iso .org/ obp
3.1
échelle nanométrique
échelle de longueur s'étendant approximativement de 1 nm à 100 nm
[SOURCE: ISO 80004-1:2023, 3.1.1]
3.2
nano-objet
portion discrète de matériau dont une, deux ou les trois dimensions externes sont à l'échelle nanométrique (3.1)
[SOURCE: ISO 80004-1:2023, 3.1.5]
3.3
nanoparticule
nano-objet (3.2) dont toutes les dimensions externes sont à l'échelle nanométrique (3.1)
Note 1 à l'article: Si les dimensions diffèrent de façon significative (généralement d'un facteur supérieur à trois), des
termes tels que nanofibre ou nanoplaque sont préférés au terme «nanoparticule».
ISO/FDIS 21362:2025(fr)
[SOURCE: ISO 80004-1:2023, 3.3.4]
3.4
fractionnement par couplage flux-force
FFF
technique de séparation dans laquelle un champ est appliqué à une suspension liquide passant dans un canal
(3.7) étroit de manière à provoquer la séparation des particules présentes dans le liquide en fonction de leur
différence de mobilité sous l'effet de la force exercée par le champ
Note 1 à l'article: Le champ peut être, par exemple, gravitationnel, centrifuge, un écoulement de liquide, un champ
électrique ou magnétique.
Note 2 à l'article: L'utilisation d'un détecteur approprié, après ou pendant la séparation, permet de déterminer la taille
moyenne des populations de nano-objets (3.2) ainsi que leur distribution granulométrique.
3.5
fractionnement par couplage flux-force asymétrique
AF4
technique de séparation qui utilise un champ d'écoulement transversal (3.19) appliqué perpendiculairement
au flux du canal (3.21) pour obtenir une séparation en fonction du coefficient de diffusion ou de la taille des
analytes
Note 1 à l'article: L'écoulement transversal se produit au moyen d'une paroi semi-perméable dans le canal (paroi
d'accumulation), alors que l'écoulement transversal est nul sur une paroi opposée non perméable (déplétion).
Note 2 à l'article: Par comparaison, dans un écoulement symétrique, l'écoulement transversal pénètre à travers une
paroi perméable (fritté) et sort à travers une paroi opposée semi-perméable, et est généré séparément du flux du canal.
Note 3 à l'article: Les nano-objets (3.2) se fractionnent généralement selon le mode «normal», où la diffusion domine
et où la plus petite espèce est éluée en premier. À l'échelle micrométrique, le mode de fractionnement dit «stérique
lift hyperlayer» est généralement dominant, avec l'espèce la plus grande qui est éluée en premier. Le passage du
mode normal au mode stérique lift hyperlayer (3.33) peut être influencé par les propriétés des matériaux ou par les
paramètres de mesure et, par conséquent, il n'est pas définitivement identifié; cependant, cette transition peut être
définie de façon explicite pour un ensemble donné de conditions expérimentales; en règle générale, celle-ci intervient
sur une plage granulométrique approximativement comprise entre 0,5 µm et 2 µm.
Note 4 à l'article: En incluant à la fois le mode normal et le mode stérique lift hyperlayer, la technique permet de séparer
des particules couvrant une plage granulométrique d'environ 1 nm à 50 µm.
3.6
fractionnement par couplage flux-force à force centrifuge
CF3
technique de séparation qui utilise un champ centrifuge appliqué perpendiculairement à un canal (3.7)
circulaire qui tourne autour de son axe pour obtenir une séparation des particules d'une granulométrie
approximativement comprise entre 10 nm et 50 µm
Note 1 à l'article: La séparation est régie à la fois par la granulométrie et par la densité effective des particules.
Note 2 à l'article: La plage granulométrique applicable dépend de et est limitée par la densité effective des particules.
3.7
canal
système semblable à un mince ruban, ayant un profil
d'écoulement parabolique nécessaire pour permettre la séparation de particules grâce à un champ appliqué
perpendiculairement au flux du canal (3.21)
Note 1 à l'article: L'épaisseur du canal (3.9) peut varier et est nominalement déterminée par l'insertion d'une entretoise
(3.8), tandis que les canaux à hauteur fixe ont une épaisseur prédéfinie et n'utilisent pas d'inserts.
Note 2 à l'article: Dans un système de fractionnement par couplage flux-force asymétrique (3.5), un canal trapézoïdal,
généralement d'une largeur maximale d'environ 20 mm à 25 mm et d'une longueur d'environ 100 mm à 300 mm, est
habituellement utilisé.
ISO/FDIS 21362:2025(fr)
Note 3 à l'article: Dans un système asymétrique, une des surfaces du canal [paroi de déplétion (3.12)] est solide
(imperméable) et la surface opposée [paroi d'accumulation (3.11)] se compose d'une membrane semi-perméable
déposée sur un fritté poreux.
Note 4 à l'article: Dans un système de fractionnement par couplage flux-force à force centrifuge (3.6), les parois interne et
externe du canal sont toutes les deux solides (non poreuses) et le canal est incurvé. Un canal trapézoïdal, généralement
d'une largeur maximale d'environ 10 mm à 20 mm et d'une longueur d'environ 300 mm à 550 mm, est habituellement
utilisé.
3.8
entretoise
mince film plastique présentant une découpe qui définit l'épaisseur
et les dimensions latérales du canal (3.7)
Note 1 à l'article: Des découpes trapézoïdales et rectangulaires sont généralement utilisées dans la technique de
fractionnement par couplage flux-force asymétrique (3.5).
Note 2 à l'article: L'épaisseur typique des entretoises utilisées pour la séparation de nano-objets (3.2) est comprise
entre 190 µm et 500 µm.
Note 3 à l'article: Les canaux à hauteur fixe n'utilisent pas d'entretoise; dans ce cas, la forme et l'épaisseur du canal
sont prédéfinies.
3.9
épaisseur du canal
w
épaisseur nominale telle que définie par l'entretoise (3.8) ou
prédéfinie dans un canal (3.7) à hauteur fixe
3.10
épaisseur effective du canal
w
eff
variant de la valeur nominale en raison de la compressibilité ou du
gonflement de la membrane semi-perméable au niveau de la paroi d'accumulation (3.11)
Note 1 à l'article: La valeur de l'épaisseur effective peut différer de la valeur nominale pour une entretoise (3.8) donnée
et peut être déterminée à l'aide d'un analyte bien défini de la diffusivité connue dans les conditions d'essai.
Note 2 à l'article: L'épaisseur effective du canal mesurée peut dépendre d'autres facteurs, tels que les interactions
entre l'analyte et la membrane ou la variabilité des méthodes de fabrication de l'entretoise.
3.11
paroi d'accumulation
surface d'un canal de fractionnement par couplage flux-force (3.4) vers laquelle est forcé l'acheminement des
composants d'échantillon sous l'effet du champ appliqué perpendiculairement au flux du canal (3.21)
Note 1 à l'article: Dans un système de fractionnement par couplage flux-force asymétrique (3.5), la paroi d'accumulation
est plane et se compose d'une membrane semi-perméable déposée sur un fritté poreux.
Note 2 à l'article: Dans un système de fractionnement par couplage flux-force à force centrifuge, la paroi d'accumulation
est imperméable et est située à une plus grande distance de l'axe de rotation par rapport à la paroi de déplétion (3.12).
Dans le cas rare où les particules présentent une densité inférieure à celle du milieu aqueux, les parois de déplétion et
d'accumulation sont inversées.
3.12
paroi de déplétion
surface d'un canal de fractionnement par couplage flux-force (3.4) opposée à la paroi d'accumulation (3.11),
dont la concentration de l'analyte diminue sous l'effet du mouvement de l'analyte en direction de la paroi
d'accumulation grâce au champ appliqué
Note 1 à l'article: Dans un système asymétrique de fractionnement par couplage flux-force, la paroi de déplétion est
plane et imperméable.
ISO/FDIS 21362:2025(fr)
Note 2 à l'article: Dans un système de fractionnement par couplage flux-force à force centrifuge (3.6), la paroi de déplétion
est imperméable et incurvée, et se trouve plus près de l'axe de rotation par rapport à la paroi d'accumulation. Lorsque
la densité effective des particules est inférieure à la densité du milieu, les parois de déplétion et d'accumulation sont
inversées.
3.13
phase mobile
liquide porteur
éluant
phase liquide utilisée pour obtenir la séparation et le transport des analytes
Note 1 à l'article: L'éluant ou la phase mobile peut contenir un ou plusieurs sels, des tensioactifs et d'autres constituants
chimiques nécessaires pour optimiser la séparation et le recouvrement (3.35) d'un analyte.
Note 2 à l'article: Dans le présent document, seules les phases aqueuses sont pertinentes, mais des solvants organiques
peuvent également être utilisés sous réserve de compatibilité de l'équipement et du canal.
3.14
élution
procédé au cours duquel les analytes contenus dans la phase
mobile (3.13) ou l'éluant sont transportés à travers le canal (3.7) de fractionnement jusqu'à leur sortie
Note 1 à l'article: Une élution commence après que l'injection, la focalisation (3.16) et les autres étapes de pré-élution
se sont achevées.
Note 2 à l'article: Une élution peut se produire avec ou sans un champ appliqué.
3.15
temps d'élution
temps écoulé après le début de l'élution (3.14) et à l'exclusion des
étapes préliminaires telles que l'injection, la focalisation (3.16) ou d'autres transitions
Note 1 à l'article: L'élution et la rétention partagent la même durée et peuvent être utilisées de manière interchangeable.
Note 2 à l'article: L'axe horizontal (temps) d'un fractogramme (3.34) est généralement exprimé en temps d'élution.
3.16
focalisation
application d'un flux contre-équilibré des extrémités
opposées (entrée et sortie) du canal (3.7) pour concentrer les composants de l'échantillon dans une fine
bande près de l'orifice d'entrée au niveau de la paroi d'accumulation (3.11)
Note 1 à l'article: Cette étape est requise pour réduire au minimum l'élargissement de la bande (3.30) et pour permettre
aux composants d'atteindre une position d'équilibre [relaxation (3.17)] à l'intérieur du canal.
Note 2 à l'article: La focalisation ne se produit pas pendant l'injection d'un fritté d'entrée.
3.17
relaxation
procédé au cours duquel les composants d'un échantillon
atteignent leur état d'équilibre par rapport aux forces opposées de diffusion et au cours duquel le champ
appliqué est initié avant l'élution (3.14)
Note 1 à l'article: Dans un système de fractionnement par couplage flux-force (3.4) à flux, il existe deux façons
d'atteindre la relaxation: la relaxation normale après la focalisation (3.16) et la technique dite du frit inlet ou la
relaxation hydrodynamique.
Note 2 à l'article: Dans un système de fractionnement par couplage flux-force à force centrifuge (3.6), la relaxation est
obtenue par la méthode d'interruption de flux («stop-flow»).
ISO/FDIS 21362:2025(fr)
3.18
flux d'injection
flux qui conduit l'échantillon hors de la boucle d'injection et
l'achemine dans le canal (3.7) de fractionnement
Note 1 à l'article: Selon la conception de l'instrument, l'injection peut se produire par un port d'injection distinct ou à
travers le port d'entrée du canal.
3.19
écoulement transversal
champ d'écoulement appliqué perpendiculairement au flux
du canal (3.21) pour obtenir une séparation des analytes
Note 1 à l'article: Dans un système asymétrique de fractionnement par couplage flux-force (3.5), l'écoulement transversal
est créé par une pression différentielle à travers une membrane perméable sur la paroi d'accumulation (3.11), ce qui
entraîne une force dirigée vers la paroi d'accumulation qui diminue à mesure que la distance par rapport à celle-ci.
Note 2 à l'article: L'écoulement transversal est obtenu au moyen d'un régulateur de débit associé à une pompe unique
ou par l'utilisation d'une deuxième pompe dédiée.
3.20
flux à l'entrée
phase mobile (3.13), ou éluant, qui pénètre dans le canal (3.7) par
l'avant (en amont)
Note 1 à l'article: Dans un système de fractionnement par couplage flux-force asymétrique (3.5), le flux à l'entrée est
réparti entre l'écoulement transversal (3.19) et le flux du canal pendant l'élution (3.14).
3.21
flux du canal
flux laminaire parabolique à travers le canal (3.7) et parallèlement
à la paroi d'accumulation (3.11)
Note 1 à l'article: Le flux dans le canal équivaut généralement au flux de l'éluant qui sort du canal et qui pénètre dans les
détecteurs dans des conditions expérimentales données, mais il peut différer en cas de division du flux en sortie du canal.
Note 2 à l'article: Dans un système asymétrique de fractionnement par couplage flux-force (3.5), la perte de fluide à
travers la paroi d'accumulation perméable entraîne une diminution linéaire de la vitesse d'écoulement dans le canal.
Ce gradient peut être compensé au moyen d'une conception de canal trapézoïdale dans laquelle la largeur du canal
diminue vers la sortie.
3.22
volume mort
V
volume de fluide défini par les dimensions du canal (3.7)
auxquelles s'ajoute le volume entre la sortie du canal et le premier détecteur
3.23
pic au volume mort
pic observé sur le fractogramme (3.34), qui correspond à un
matériau non retenu qui n'a pas atteint l'état d'équilibre avec le champ de séparation
Note 1 à l'article: Le pic au volume mort suit la vitesse moyenne du liquide porteur et est élué avant les composants
retenus.
Note 2 à l'article: Dans ce contexte, «non retenu» renvoie aux composants qui ne sont pas séparés par le champ et qui sont
élués avec le pic au volume mort. Cette notion a une signification différente dans les séparations chromatographiques
traditionnelles basées sur l'enthalpie.
Note 3 à l'article: Un pic au volume mort est généré par la perturbation mécanique ou le changement des conditions
d'écoulement lorsque l'élution (3.14) est initiée; dans ce contexte, il convient que le pic au volume mort contienne
idéalement uniquement la phase mobile (3.13) et de petites espèces moléculaires qui ne sont pas affectées par le champ
appliqué.
ISO/FDIS 21362:2025(fr)
3.24
temps mort
t
temps s'écoulant entre le début de l'élution (3.14) et la détection du pic au volume mort (3.23) défini par son
maximum d'intensité du signal de détection
3.25
temps de rétention
t
R
temps s'écoulant entre le début de l'élution (3.14) et la détection d'un pic d'analyte défini à son maximum
d'intensité du signal de détection
Note 1 à l'article: Dans le cas d'un pic gaussien, la valeur maximale équivaut au centre du pic.
Note 2 à l'article: Le temps de rétention et le temps d'élution (3.15) représentent des durées équivalentes. Ce dernier
terme est générique, tandis que le premier est habituellement utilisé dans le contexte d'un pic d'analyte.
Note 3 à l'article: Le temps de r
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