ISO TS 21356-1:2021

TECHNICAL ISO/TS
SPECIFICATION 21356-1
First edition
2021-03
Nanotechnologies — Structural
characterization of graphene —
Part 1:
Graphene from powders and
dispersions
Nanotechnologies — Caractérisation structurelle du graphène —
Partie 1: Graphène issu de poudres et de dispersions
Reference number
ISO/TS 21356-1:2021(E)
©
ISO 2021
ISO/TS 21356-1:2021(E)
© ISO 2021
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ii © ISO 2021 – All rights reserved

ISO/TS 21356-1:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 3
5 Sequence of measurement methods . 3
6 Rapid test for graphitic material using Raman spectroscopy . 5
7 Preparing a liquid dispersion . 7
7.1 General . 7
7.2 Preparing a dispersion of the correct concentration . 7
7.2.1 Powder samples . 7
7.2.2 Samples already in a dispersion . 8
8 Determination of methods . 8
9 Structural characterization using optical microscopy, SEM, AFM and Raman spectroscopy 8
10 Structural characterization using TEM . 9
11 Surface area determination using the BET method .10
12 Graphene lateral size and number fraction calculation .10
Annex A (informative) Rapid test for graphitic material using Raman spectroscopy .11
Annex B (informative) Structural characterization protocol using SEM, AFM and Raman
spectroscopy .14
Annex C (informative) Structural characterization using TEM .29
Annex D (informative) Lateral size and number fraction calculation .36
Annex E (informative) Brunauer–Emmett–Teller method .43
Annex F (informative) Additional sample preparation protocols — Silicon dioxide on
silicon wafer preparation and cleaning .47
Bibliography .48
ISO/TS 21356-1:2021(E)
Foreword
ISO (the International Organization for Standardization) and IEC (the International Electrotechnical
Commission) form the specialized system for worldwide standardization. National bodies that
are members of ISO or IEC participate in the development of International Standards through
technical committees established by the respective organization to deal with particular fields of
technical activity. ISO and IEC technical committees collaborate in fields of mutual interest. Other
international organizations, governmental and non-governmental, in liaison with ISO and IEC, also
take part in the work.
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 document 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 or www .iec .ch/ members
_experts/ refdocs).
Attention is drawn to the possibility that some of the elements of this document may be the subject
of patent rights. ISO and IEC shall not be held responsible for identifying any or all such patent
rights. Details of any patent rights identified during the development of the document will be in the
Introduction and/or on the ISO list of patent declarations received (see www .iso .org/ patents) or the IEC
list of patent declarations received (see patents.iec.ch).
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. In the IEC, see www .iec .ch/ understanding -standards.
This document was prepared jointly by Technical Committee ISO/TC 229, Nanotechnologies, and
Technical Committee IEC/TC 113, Nanotechnology for electrotechnical products and systems.
A list of all parts in the ISO/IEC 21356 series can be found on the ISO and IEC websites.
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 and www .iec .ch/ national
-committees.
iv © ISO 2021 – All rights reserved

ISO/TS 21356-1:2021(E)
Introduction
Due to the many superlative properties of graphene and related 2D materials, there are many
application areas where these nanomaterials could be disruptive, areas such as flexible electronics,
nanocomposites, sensing, filtration membranes and energy storage.
There are barriers to commercialisation that are impeding the progress of products containing
graphene, which need to be overcome. One of these crucial barriers is answering the question “What
is my material?”. End-users of the raw materials containing graphene should be able to rely on the
advertised properties of the commercial graphene on the global market, instilling trust and allowing
worldwide trade. Reliable and repeatable measurement protocols are required to address this challenge.
This document provides a set of flow-charts for analysts to follow in order to determine the structural
properties of graphene from powders and liquid dispersions (suspensions). Initially, a quick check
should be undertaken to determine if graphene and/or graphitic material is present. If it is, then further
detailed analysis is required to determine if the samples contain a mixture of single-layer graphene,
bilayer graphene, few-layer graphene, graphene nanoplatelets and graphite particles. Depending on
the methods used, the samples are typically analysed after deposition on a substrate. The document
describes how to assess what measurements are required depending on the type of sample and includes
decision trees and flow diagrams to aid the user. This document describes a selected set of measurands
that are needed, namely:
a) the number of layers/thickness of the flakes;
b) the lateral dimensions of flakes;
c) layer alignment;
d) the level of disorder;
e) the estimated number fraction of graphene or few-layer graphene;
f) the specific surface area of the powder containing graphene.
The above physical properties of the material can change during its processing and lifetime, for example,
the samples can become more agglomerated, obtain different surface functionalities. The above
measurand list for the initial material defines their inherent characteristics that, along with the chosen
manufacturing processes, will determine the performance of real-world products. Generally, different
material properties can be important in different application areas, depending on the functional role of
the material.
The document provides methods for structural characterization of individual flakes of graphene,
bilayer graphene, graphene nanoplatelets and graphite particles isolated from powders and/or liquid
dispersions. It does not provide methods for determination of whether the powders and/or dispersions
are composed solely of these materials. No recommendation is provided as to when or how often to
measure samples, although it is not expected this would be for every batch of the same material. It is up
to the user to determine when, how often and which characterization routes described in this document
to take. As with all microscopical investigations, care is needed in drawing statistical conclusions
dependant on representative sampling.
A set of annexes provide example protocols on how to prepare and analyse the samples, sources of
uncertainty and how to analyse the data. The methods used are Raman spectroscopy, scanning electron
microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) and the
BET (Brunauer–Emmett–Teller) method.
TECHNICAL SPECIFICATION ISO/TS 21356-1:2021(E)
Nanotechnologies — Structural characterization of
graphene —
Part 1:
Graphene from powders and dispersions
1 Scope
This document specifies the sequence of methods for characterizing the structural properties of
graphene, bilayer graphene and graphene nanoplatelets from powders and liquid dispersions using a
range of measurement techniques typically after the isolation of individual flakes on a substrate. The
properties covered are the number of layers/thickness, the lateral flake size, the level of disorder, layer
alignment and the specific surface area. Suggested measurement protocols, sample preparation routines
and data analysis for the characterization of graphene from powders and dispersions are given.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO/TS 80004-1:2015, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2:2015, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 80004-6:2021, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization
ISO/TS 80004-13:2017, Nanotechnologies — Vocabulary — Part 13: Graphene and related two-dimensional
(2D) materials
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/TS 80004-1:2015,
ISO/TS 80004-2:2015, ISO/TS 80004-6:2021, ISO/TS 80004-13:2017 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
graphene
graphene layer
single-layer graphene
monolayer graphene
single layer of carbon atoms with each atom bound to three neighbours in a honeycomb structure
Note 1 to entry: It is an important building block of many carbon nano-objects.
Note 2 to entry: As graphene is a single layer, it is also sometimes called monolayer graphene or single-layer
graphene and abbreviated as 1LG to distinguish it from bilayer graphene (2LG) (3.3) and few-layer graphene
(FLG) (3.4).
ISO/TS 21356-1:2021(E)
Note 3 to entry: Graphene has edges and can have defects and grain boundaries where the bonding is disrupted.
[SOURCE: ISO/TS 80004-13:2017, 3.1.2.1]
3.2
graphite
allotropic form of the element carbon, consisting of graphene layers (3.1) stacked parallel to each other
in a three-dimensional, crystalline, long-range order
Note 1 to entry: Adapted from the definition in the IUPAC Compendium of Chemical Terminology.
Note 2 to entry: There are two primary allotropic forms with different stacking arrangements: hexagonal and
rhombohedral.
[SOURCE: ISO/TS 80004-13:2017, 3.1.2.2]
3.3
bilayer graphene
2LG
two-dimensional material consisting of two well-defined stacked graphene layers (3.1)
Note 1 to entry: If the stacking registry is known, it can be specified separately, for example, as “Bernal stacked
bilayer graphene”.
[SOURCE: ISO/TS 80004-13:2017, 3.1.2.6]
3.4
few-layer graphene
FLG
two-dimensional material consisting of three to ten well-defined stacked graphene layers (3.1)
[SOURCE: ISO/TS 80004-13:2017, 3.1.2.10]
3.5
graphene nanoplate
graphene nanoplatelet
GNP
nanoplate consisting of graphene layers (3.1)
Note 1 to entry: GNPs typically have thickness of between 1 nm to 3 nm and lateral dimensions ranging from
approximately 100 nm to 100 μm.
[SOURCE: ISO/TS 80004-13:2017, 3.1.2.11]
3.6
lateral size
flake size
<2D material> lateral dimensions of a 2D material flake
Note 1 to entry: If the flake is approximately circular then this is typically measured using an equivalent circular
diameter or if not via x, y measurements along and perpendicular to the longest side.
[SOURCE: ISO/TS 80004-13:2017, 3.4.1.15]
3.7
graphene oxide
GO
chemically modified graphene (3.1) prepared by oxidation and exfoliation of graphite (3.2), causing
extensive oxidative modification of the basal plane
Note 1 to entry: Graphene oxide is a single-layer material with a high oxygen content, typically characterized by
C/O atomic ratios of approximately 2,0 depending on the method of synthesis.
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ISO/TS 21356-1:2021(E)
[SOURCE: ISO/TS 80004-13:2017, 3.1.2.13]
3.8
reduced graphene oxide
rGO
reduced oxygen content form of graphene oxide (3.7)
Note 1 to entry: This can be produced by chemical, thermal, microwave, photo-chemical, photo-thermal or
microbial/bacterial methods or by exfoliating reduced graphite oxide.
Note 2 to entry: If graphene oxide was fully reduced, then graphene (3.1) would be the product. However, in
3 2
practice, some oxygen containing functional groups will remain and not all sp bonds will return back to sp
configuration. Different reducing agents will lead to different carbon to oxygen ratios and different chemical
compositions in reduced graphene oxide.
Note 3 to entry: It can take the form of several morphological variations such as platelets and worm-like
structures.
[SOURCE: ISO/TS 80004-13:2017, 3.1.2.14]
4 Abbreviated terms
For the purposes of this document, the following abbreviated terms apply.
NOTE The final “M”, given as “microscopy”, can be taken equally as “microscope” depending on the context.
1LG single-layer graphene
2D two dimensional
2LG bilayer graphene
AFM atomic force microscopy
BET method Brunauer–Emmett–Teller method
CVD chemical vapour deposition
FLG few-layer graphene
FWHM full width at half maximum
GNP graphene nanoplate or graphene nanoplatelet
GO graphene oxide
NMP 1-methyl-2-pyrrolidinone also known as N-methylpyrrolidone
rGO reduced graphene oxide
SAED selected area electron diffraction
SEM scanning electron microscopy
TEM transmission electron microscopy
5 Sequence of measurement methods
This clause presents the sequence of measurement methods necessary to most efficiently characterize
graphene, bilayer graphene, few-layer graphene and graphene nanoplatelets from powders and liquid
dispersions (suspensions). In this document, graphene, bilayer graphene, few-layer graphene and
ISO/TS 21356-1:2021(E)
graphene nanoplatelets are in the form of flakes of limited lateral dimensions. However, samples also
typically contain significant amounts of flakes having thicknesses that exceed ten layers, which are
flakes of graphite by definition.
After an initial examination by Raman spectroscopy, and assuming the sample is graphene or graphitic
in nature, a more detailed characterization should follow. Various characterization routes are then
possible, as shown in Figure 1. The characterization method or methods to be used will depend on the
time and equipment available and the measurands that the user requires.
NOTE 1 As the flakes are from a powder or liquid dispersion, they will typically require deposition onto a
substrate before analysis.
NOTE The numbers in brackets refer to the clauses where the item is detailed.
Figure 1 — Overview of the sequence and process of the measurement methods used to
determine the structural properties of graphene from a powder or liquid dispersion sample
Firstly, determine if the sample contains graphene and/or graphitic material, that is bilayer graphene,
FLG, GNPs or graphite by undertaking a rapid analysis using Raman spectroscopy, as detailed in
Clause 6 and Annex A. The sample needs to be in powder form deposited as a thin layer on a substrate,
therefore if a liquid dispersion has been supplied, the material will first need to be removed from the
solvent, as detailed in A.2.
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ISO/TS 21356-1:2021(E)
Decide which methods to use as outlined in Clause 8. Either use TEM or a combination of SEM, AFM
and Raman spectroscopy to determine the distribution of lateral flake sizes and the relationship with
flake thickness. For this stage, clearly separated flakes on a substrate are required. To prepare these
samples by deposition, a liquid dispersion is initially required, therefore, if the material was provided
as a powder, it requires dispersing in a suitable solvent, as described in Clause 7, before subsequent
deposition onto a suitable substrate with example procedures outlined in Annexes B and C.
If TEM is used (see Clause 10), prepare the sample on a TEM support grid as outlined in C.2, otherwise
prepare the sample on a silicon dioxide on silicon substrate, B.2. Then use optical microscopy as a
quick quality check to determine if the sample is too agglomerated and therefore cannot be accurately
measured. Optimize the sample preparation until an even deposition of the material across the
substrate occurs. Then undertake a combination of SEM, AFM and Raman spectroscopy measurements
(see Clause 9 and Annex B) or TEM (see Clause 10, Annex C). SEM, AFM and Raman spectroscopy should
be used in combination and not in individual isolation in order to determine the measurands listed in
Figure 1.
If required, use BET to determine the specific surface area of the powder (see Clause 11 and Annex E).
Once all the necessary measurements have been undertaken, calculate the median lateral flake size,
the range of flake sizes, the graphene 1LG number fraction and FLG number fraction, as discussed in
Clause 12 and Annex D. Here, number fraction is the fraction by number of graphene or FLG over the
total number of flakes, this can also be expressed as a percentage.
NOTE 2 It is assumed that the sample contains graphene/2LG/FLG/graphite. If the sample has different
chemistries, for example contains graphene oxide or functionalised graphene, this will not produce the same
Raman spectroscopy results as those described in this document. However, optical microscopy, SEM and AFM
characterization of lateral dimensions and thicknesses (but not number of layers) can still be applied to these
materials.
NOTE 3 There is currently no quantitative or standardised method for determining the specific surface area
of the graphitic material when the sample is in or from a liquid dispersion form.
6 Rapid test for graphitic material using Raman spectroscopy
Firstly, test the sample, in powder form deposited on a substrate using Raman spectroscopy to
determine whether the sample supplied contains graphene and/or graphitic material. This test can
also provide qualitative information on the structural properties of the material, including the level of
disorder and the dimensions of the flakes. If the sample is supplied in a liquid dispersion, then remove
the liquid from the dispersion and analyse the sample in powder form.
A thin layer of powder is required for this rapid Raman spectroscopy analysis step. If a powder has
been provided, this should be analysed with a significant amount of the sample secured on adhesive
tape (see A.2) such that only the flakes rather than the substrate are analysed.
A measurement protocol and sample preparation method are detailed in Annex A.
−1
To confirm the presence of graphitic material, a sharp (< 30 cm full width at half maximum
−1
(FWHM)) G-peak at approximately 1 580 cm and a 2D-peak (sometimes referred to as the G’ peak)
−1
at approximately 2 700 cm should be consistently observed in the Raman spectra as shown in the
graphene spectrum in Figure 2. If an intense symmetric Lorentzian peak shape is found for the 2D-peak
with close to or greater intensity than the G-peak, this suggests the sample could contain single-layer
graphene. However, restacked few-layer graphene flakes can also show a single Raman 2D-peak. If the
2D-peak is not symmetric, this suggests flakes of multiple layers are present. A prominent shoulder in
the 2D-peak is indicative of layered material, with a thickness of over ten graphene layers (i.e. graphite).
If the G- and 2D-peaks are not present, further characterization is not required, as the sample does not
contain graphene or graphite, however, a sufficient ratio of the Raman peak signal to background noise
(S/N) ratio should be established before this conclusion can be made, see Annex A for example details.
To improve the S/N ratio, longer acquisition times can be used, or averaging multiple scans with short
acquisition times can be used.
ISO/TS 21356-1:2021(E)
If functionalised graphene or graphene oxide is present, Raman spectroscopy will show the D-
and G-peaks, but not necessarily a 2D-peak, and the D- and G-peaks will have much larger FWHM
−1
values (> 30 cm ) than expected for graphene. Here, additional chemical characterization should be
undertaken to determine the oxygen content and any other components, which if found to be high
means that the material is out of the scope of this document.
Key
−1
X Raman shift, cm
Y normalized intensity, arbitrary units
1 graphene
Figure 2 — Example Raman spectra of highly oriented pyrolytic graphite (HOPG), graphene,
reduced graphene oxide with lower oxygen content [rGO(L)], reduced graphene oxide with
higher oxygen content [rGO(H)] and graphene oxide (GO)
This step should not be confused with the processes used later for measurement of individual flakes
with AFM and Raman spectroscopy (detailed in Clause 9 and Annex B) after further sample preparation.
NOTE 1 Adhesive tape is specified to stop the powder moving for both health and safety reasons and to stop
possible electrostatic attraction and contamination of the lens.
NOTE 2 Chemical characterization of graphene including thermogravimetric analysis (TGA) and X-ray
photoelectron spectroscopy (XPS) will be detailed in a further ISO document in development at the time of
publication of this document.
NOTE 3 Other methods such as X-ray diffraction (XRD) can be used to determine the presence of graphitic
material. Raman spectroscopy is used here as a rapid confirmation step, as Raman spectroscopy is also required
for the detailed analysis of individual flakes (see Clause 9).
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ISO/TS 21356-1:2021(E)
7 Preparing a liquid dispersion
7.1 General
For further, more detailed characterization of the sample, the flakes should be prepared such that they
are isolated on a substrate. This allows the next characterization steps, as shown in Figure 1, to be
performed either using a combination of SEM, AFM and Raman spectroscopy with the sample on a
silicon dioxide on silicon substrate, or TEM with the sample on a TEM grid. For the preparation of the
flakes on a substrate, initially a liquid dispersion is required, therefore, if the material is provided as a
powder, it requires dispersing in a suitable solvent.
7.2 Preparing a dispersion of the correct concentration
7.2.1 Powder samples
Disperse the powder in a solvent such that a concentration of approximately 0,1 mg/ml is achieved. The
suitability of the solvent should be determined through the observation of how quick and how much, if
any, sedimentation of the material occurs. There are a number of different solvents that can be used.
Use the solvent that will disperse the powder and allow flakes to be characterized with the minimum of
unwanted residue on the surface. The order of preference of three solvents is given in Figure 3.
NOTE An order of preference of the solvent to be used is outlined.
Figure 3 — Flowchart for the creation of a dispersion
Firstly, try to disperse the powder with deionised water. Place the liquid and powder in a glass vial
or bottle and agitate. Sonicate the dispersion for up to a maximum of 10 min in a table-top ultrasonic
bath at 30 kHz to 40 kHz. Longer sonication times can cause changes to the structural properties,
including basal-plane scission (reducing the lateral size) and further exfoliation (reducing thickness/
layer number). Observe the dispersion over a period of several minutes. If a significant amount of
sedimentation occurs and occurs quickly then repeat the procedure using a different solvent.
If deionised water does not disperse the material, isopropanol should be used as the solvent using the
same method. If this does not work, then N-methylpyrrolidone (NMP) should be used as the solvent as
graphene disperses well in this. However, due to the high boiling point of NMP (203 °C), this can affect
the characterization results in the form of solvent residue.
The deposition of the material onto a substrate is detailed in Clauses 9 and 10 and in particular in
Annexes B and C.
Typically, graphene flakes will stay dispersed in deionised water only if a stabilizing agent, such as a
surfactant, is present as part of the manufacturing process. However, it should be noted that significant
use of surfactants can influence both the sample condition and the later measurement of the materials,
see examples in B.2.
ISO/TS 21356-1:2021(E)
Using a significant amount of ultrasonication to disperse the material can induce flake scission
and therefore affect the structural characterization results obtained for a sample. The amplitude
(commonly expressed as power) and duration of ultrasonication should therefore be kept to the
minimum required to disperse the flakes. A comparison of flake size measurement as a function of the
amplitude and duration of ultrasonication can be undertaken to check if flake scission occurs and to
optimize sonication conditions, if required.
1)
NOTE ISO/TS 22107 provides general guidance on the definition of dispersibility and deals with processing
and the achieved final dispersed state.
7.2.2 Samples already in a dispersion
If the sample is already provided as a dispersion, this should be diluted to approximately 0,1 mg/ml
using the same solvent. However, if the solvent is a water/surfactant mix, the dilution should be carried
out using deionised water, to reduce the level of surfactant.
NOTE In cases where the concentration of the dispersion provided is not known, the dilution needs to be
approximated. This concentration is chosen such to produce dispersed flakes in solution and individual flakes on
the substrate when cast.
8 Determination of methods
For detailed characterization, two characterization routes are possible, as shown in Figure 1. Determine
whether to use a combination of SEM, AFM and Raman spectroscopy measurements (see Clause 9 and
Annex B) or use TEM (see Clause 10 and Annex C). For powder samples, BET can be used to determine
the specific surface area, as described in Clause 11 and Annex E. Which method or methods are used
depends on the time and equipment available and the measurands that the user requires.
For either set of the microscopy methods, the samples shall be prepared firstly as a dispersion, as
detailed in Clause 7, and then deposited on the correct substrate as discussed in B.2 or C.2.
9 Structural characterization using optical microscopy, SEM, AFM and Raman
spectroscopy
This clause details the sequence of measurements to determine lateral flake dimensions, associated
flake thickness, level of disorder and number of graphene layers using a combination of SEM, AFM and
Raman spectroscopy. Use the methods as ordered in Figure 4.
1) Under preparation. Stage at the time of publication: ISO/DTS 22107:2021.
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ISO/TS 21356-1:2021(E)
Figure 4 — Flow diagram and decision-making process for determining the range of lateral
dimensions, thickness of flakes, number of layers and level of disorder
Firstly, the sample should be prepared from a liquid dispersion and placed on an appropriate substrate.
Use optical microscopy to check the sample preparation. Once an appropriate sample has been
produced, use SEM, AFM and Raman spectroscopy to characterize the sample and analyse the results to
extract the measurands as detailed in Figure 4.
Sample preparation methods, measurement protocols and data analysis protocols are outlined in
Annex B.
NOTE 1 The SEM measurements are performed on a different substrate and use different flakes to the AFM
and Raman spectroscopy measurements.
2)
NOTE 2 ISO 19749 provides guidance for measuring size and shape distribution of nanoparticles including
general principles, sample preparation, qualification of the SEM, image acquisition, particle and data analysis.
10 Structural characterization using TEM
In a transmission electron microscope (TEM) a high energy beam of electrons is passed through a thin
electron transparent sample in a high vacuum environment.
TEM can be used to determine the lateral size and number of layers in flakes, as well as layer alignment,
through diffraction contrast TEM imaging, lattice resolution imaging and selected area electron
diffraction (SAED), which are achievable with most modern TEM instruments. It should be noted that,
for liquid-phase exfoliated flakes, the presence of surfactants and common contaminants from the
environment (H, C, O, Si, Na and Cl) can cause difficulties in imaging.
Users should consult ISO 21363:2020 for useful information on instrument set up and particle analysis.
2) Under preparation. Stage at the time of publication: ISO/PRF 19749:2021.
ISO/TS 21356-1:2021(E)
Figure 5 — Flowchart for TEM to determine lateral size, number of layers and layer alignment
Follow the order of operations as detailed on Figure 5 to determine the lateral size, number of layers
and layer alignment of different flakes. The flakes should be deposited onto an appropriate TEM grid
from a dispersion. Optical microscopy should be used to check the sample preparation and positions of
flakes prior to analysis in the TEM. After TEM, the data should be analysed to determine the required
measurands.
A sample preparation method, measurement protocol and data analysis method are detailed in Annex C.
11 Surface area determination using the BET method
The Brunauer–Emmett–Teller (BET) method determines the total specific surface area of disperse
powders by measuring the amount of physically adsorbed gas. It utilizes the model developed by
Brunauer, Emmett and Teller for interpreting gas adsorption isotherms. Use the BET method to
determine the specific surface area of a powder sample.
A sample preparation method, measurement protocol and data analysis procedure are detailed in
Annex E.
12 Graphene lateral size and number fraction calculation
Analyse the data produced from the dimensional characterization. Calculate the median lateral flake
size, the range of flake sizes, the graphene 1LG and FLG number fraction and report which techniques
are used to do this. A method to calculate this data is given in Annex D.
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ISO/TS 21356-1:2021(E)
Annex A
(informative)
Rapid test for graphitic material using Raman spectroscopy
A.1 General
This annex details possible sample preparation steps and a measurement protocol for a rapid test to
confirm the presence of graphene, bilayer graphene, graphene nanoplatelets (GNPs) and/or graphite
using Raman spectroscopy.
A.2 Sample preparation
A.2.1 Sample preparation from a liquid dispersion
a) In a vacuum filtration kit, use a membrane with pore size of ≤ 0,2 µm to ensure that majority of the
smaller flakes are retained on the membrane.
1) The material of the membrane needs to be compatible with the solvent used to make the
dispersion.
2) Alumina or cellulose membranes should be used for common graphene solvents such as water,
isopropanol or NMP.
b) A pressure of ~100 mbar should be applied for the vacuum filtration step.
c) Collect the dried material on top of the filter at the end of the process as a supported or free-
standing graphene film.
The thickness of the film produced should be at least 1 μm to provide a strong Raman signal during
subsequent measurement and therefore a high enough concentration or large enough amount of
dispersion will be required to provide a film that can be handled.
NOTE There is no need to accurately measure the film thickness; if the signal from the material is not high
enough to perform the analysis in A.3, then the film is not thick enough.
A.2.2 Sample preparation from powder form
a) Before handling a powder sample of nano-objects, an appropriate risk assessment should be
performed and the required engineering controls, personal protective equipment and safety
processes employed.
b) Place double-sided adhesive tape on to a clean microscope slide.
c) Deposit a small amount of the powder on the adhesive tape, pressing down lightly with a spatula
to ensure adhesion. Adhesive tape is specified to stop the powder from moving for both health and
safety reasons and to stop possible electrostatic attraction of the powder to the microscope lens
and hence contamination of the lens.
To assess uniformity, material can be collected and prepared from more than one part of the batch
(e.g. top, middle and bottom of the container). However, as this step is for rapid analysis, a single
sample is sufficient.
d) Once the material is secured on the adhesive tape, excess and unsecured material should be
removed by tapping the microscope slide vertically. To prevent dust being raised, the material
ISO/TS 21356-1:2021(E)
should be collected onto a wet paper towel. An example is shown in Figure A.1. As described above,
sufficient material, such as shown in Figure A.1 should be deposited to provide a strong Raman
signal. If a signal is observed from the substrate, then more material should be deposited.
Figure A.1 — Photograph of a powder containing graphene deposited onto adhesive tape
NOTE An alternative sample preparation method would be to press the powder into a pellet.
A.3 Method
Raman spectroscopy should be undertaken in a backscattering geometry with preferably a 50 × or
100 × objective lens (NA ≥ 0,75). The system should be calibrated prior to measurements using the
user’s best practice. A red (typically 633 nm) or green (typically 532 nm or 514 nm) excitation laser
should be used. The positions of some of the peaks observed will be at different spectral positions,
depending on the wavelength of the excitation laser.
−1
The spectral range should be chosen such that the relevant Raman lines [D-band (~1 350 cm ), G-band
−1 −1
(~1 580 cm ), 2D-band (~2 700 cm )] and associated widths are included, so for example from
−1 −1
1 200 cm to 3 000 cm .
After locating a measurement area with the aid of optical microscopy, set the Z-focus position such
that the surface of the powder is in focus. Perform a single Raman spectroscopy measurement with
a laser power of less than 1 mW incident on the sample so as to minimize the damage to the sample,
with an exposure of 5 s to 10 s and two accumulations. This should provide a Raman peak intensity to
background noise (S/N) ratio of at least 10. If not, a longer measurement time can be used to increase
the S/N ratio.
Measurements should be performed from a minimum of three different areas of the sample to
understand the local variation across the sample as the material is generally in the form of aggregates.
−1 −1
To confirm the presence of graphene and/or graphite, a sharp (< 30 cm FWHM) G-peak at ~1 580 cm
−1
and a 2D-peak at ~2 700 cm should be consistently observed in the Raman spectra. If an intense
symmetric Lorentzian peak shape is found for the 2D-peak with close to or greater intensity than
the G-peak, this suggests the sample contains single-layer graphene. However, restacked few-layer
graphene flakes can also provide a single Raman 2D-peak. If the peak is not symmetric this suggests
multiple layers are present. A prominent shoulder in the 2D-peak is indicative of layered material, with
a thickness of over ten graphene layers (i.e. graphite). If the G- and 2D-peaks are not present, further
characterization is not required, as the sample does not contain graphene or graphite, however, a
sufficient S/N ratio should be established before this conclusion can be made.
Measurements of powders containing graphitic material also typically reveal a D-peak at approximately
−1
1 350 cm , as shown in Figure 2, due to flake edges activating the D-band as well as basal plane defects.
The intensity ratio of the D-peak relative to the G-peak (I /I ) is therefore correlated to the lateral size
D G
12 © ISO 2021 – All rights reserved

ISO/TS 21356-1:2021(E)
of the flakes, with a larger ratio typically indicating flakes with smaller lateral dimensions. Measure
the I /I ratio and compare to the results of the later characterization methods subsequently used,
D G
following the flowchart shown in Figure 1.
NOTE If functionalised graphene or graphene oxide is present, Raman spectroscopy shows the D- and
−1
G-peaks, but not necessarily a 2D-peak, and the D- and G-peaks have much larger FWHM values (>30 cm ) than
expected, for example, see References [5] and [6]. However, other carbon materials can also have these peaks, and
so it is recommended that chemical characterization is performed separately (details will be provided in an ISO
document on chemical characterization of graphene in development at the time of publication of this document).
ISO/TS 21356-1:2021(E)
Annex B
(informative)
Structural characterization protocol using SEM, AFM and Raman
spectroscopy
B.1 General
This annex details a set of measurement protocols to determine lateral flake size using SEM, lateral
flake size and thickness using AFM and the level of disorder and number of layers using Raman
spectroscopy. Sample preparation should be undertaken as detailed in B.2 prior to analysis as detailed
in B.3 through to B.5.
B.2 Sample preparation
B.2.1 Drop casting for SEM, AFM and Raman spectroscopy
To enable the measurement of flake dimensions for multiple flakes using optical microscopy, SEM, AFM
or Raman spectroscopy, the prepared dispersion should be deposited onto two types of substrate, one
substrate for SEM and one substrate for AFM and Raman spectroscopy.
For SEM, a silicon wafer with a thin native oxide should be used as a substrate. The oxide should be
thin enough to ensure good conductivity and prevent charging while imaging using SEM. For AFM and
Raman spectroscopy, a silicon wafer with a silicon dioxide layer of thickness of 300 ± 5 nm or 90 ± 5 nm
should be used in order to maximize the optical contrast between the flakes and the substrate.
For all three methods, the flakes should be deposited in such a way that a substantial fraction of them
are isolated from each other. For Raman spectroscopy, a flake should only be analysed if it is clearly
separated from another by a distance of approximately 1 μm. This is to avoid the analysis of more than
one flake at a time by the optical beam.
The procedure for deposition is as follows.
a) Prepare a stable dispersion as detailed in Clause 7.
b) The substrate should be cleaned as described in Annex F.
c) Place the cleaned substrate on a hot plate and set the temperature to be greater than the boiling
point of the solvent used for the dispersion.
d) Thoroughly mix the dispersion by shaking and then quickly extract a representative sample into
a pipette. Drop-cast a small volume of the dispersion onto the substrate (typically between 10 μl
and 100 μl is sufficient). A well-dispersed layer of flakes should then be left on the surface. Examine
under optical microscopy (see B.2.2) and determine whether the sample is suitable for analysis.
If it is not, then the sample preparation process should be repeated with different concentrations
and/or volumes of dispersion and/or different solvents. An example of a good sample is shown in
F
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