ISO/TS 21346:2021

TECHNICAL ISO/TS
SPECIFICATION 21346
First edition
2021-03
Nanotechnologies —
Characterization of individualized
cellulose nanofibril samples
Nanotechnologies — Caractérisation d'échantillons de nanofibrilles
individualisées de cellulose
Reference number
©
ISO 2021
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Characteristics to be measured of iCNF samples and their measurement methods .2
5.1 General . 2
5.2 Characteristics required to be measured or identified . 3
5.2.1 Morphology and size . 3
5.2.2 Total dry matter content . 4
5.2.3 Crystal structure . 4
5.2.4 Optical transmittance . 4
5.2.5 Surface functional groups: Types . 4
5.2.6 Surface functional groups: Content . 5
5.2.7 Viscosity . 5
5.3 Characteristics recommended to be measured or identified. 5
5.3.1 Width and height . 5
5.3.2 Length. 6
5.3.3 Molecular weight distribution . 6
5.3.4 Supernatant dry matter ratio . 6
5.3.5 Crystallinity . 7
5.3.6 Thermal stability. 7
5.3.7 Ash content . 7
5.3.8 Acid-soluble metal content . 8
5.3.9 Organic contaminant content . 8
5.3.10 Acetone-soluble matter content . 8
5.3.11 Constituent sugar content . 8
6 Reporting . 9
Annex A (informative) Protocols for sample preparation, measurement and data analysis .10
Annex B (informative) Description of individualized cellulose nanofibril (iCNF) .27
Bibliography .36
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation 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 by Technical Committee ISO/TC 229, Nanotechnologies.
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.
iv © ISO 2021 – All rights reserved

Introduction
Cellulose nanomaterials derived from naturally occurring cellulosic fibres are renewable advanced
materials with unprecedented properties. They are of wide variety in morphology, e.g. different shapes,
branching and networking. Basic research related to cellulosic nanomaterials has been increasingly
conducted worldwide. At the same time, manufacturing industries have already started to deliver
cellulose nanomaterials to the market. Application industries are also becoming more and more
interested in these new materials.
All native cellulosic fibres are composed of bundles in which the smallest fibril unit is an elementary
fibril originating from a cellulose terminal enzyme complex. An elementary fibril is made of a
certain number of cellulose molecules and contains crystalline regions predominantly. The size of an
elementary fibril is specific to the native cellulose source. In wood pulp, the cross-sectional dimension
of an elementary fibril is about 3 nm and its aspect ratio can reach more than 200. In native cellulose
fibres, elementary fibrils do not exist as single fibrils but adhere to each other through hydrogen
bonding and are densely packed to form a bundle of fibrils. Very recently, however, some novel methods
to extract and separate these elementary fibrils, through chemical modification of the outer surface
of the fibrils followed by mechanical treatment, were developed. The chemical modification methods
include TEMPO-mediated oxidation and phosphorylation. Using the above treatments, each native
elementary fibril can be converted to an individualized cellulose nanofibril (iCNF) with charges at its
surface. An iCNF has the functional groups on the outer surface of the fibril, and iCNFs can be separated
from each other, one by one, by the static repulsion due to the electrostatic charge of newly introduced
functional groups. Refer to Annex B for more explanations on iCNFs.
Several manufacturing companies have already begun producing iCNFs. iCNFs are now delivered
increasingly to the worldwide market for applications in the industrial fields of polymer composites,
adhesives, additives, gels, etc. Some examples of iCNF-containing commercial products are diapers with
deodorant performance and gel ink for ballpoint pens. In all applications, appropriate characterization
of the iCNF samples is necessary so that desired products can be manufactured.
This document provides a sound basis for the commercialization as well as the research and
development of iCNF materials.
TECHNICAL SPECIFICATION ISO/TS 21346:2021(E)
Nanotechnologies — Characterization of individualized
cellulose nanofibril samples
1 Scope
This document specifies characteristics to be measured of individualized cellulose nanofibril (iCNF)
samples in suspension and powder forms and their measurement methods. In addition, it provides
sample preparation, measurement and data analysis procedures.
This document does not apply to the characterization of iCNFs that have been modified after they are
manufactured.
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 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/TS 80004-2 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
elementary fibril
structure, originating from a single terminal enzyme complex, having a configuration of cellulose
chains specific to each cellulose-producing plant, animal, algal and bacteria species
[SOURCE: ISO/TS 20477:2017, 3.2.5]
3.2
cellulose nanofibril
CNF
cellulose nanofibre composed of at least one elementary  fibril (3.1), containing crystalline,
paracrystalline and amorphous regions, with aspect ratio usually greater than 10, which may contain
longitudinal splits, entanglement between particles, or network-like structures
[SOURCE: ISO/TS 20477:2017, 3.3.6, modified — The notes to entry have been deleted.]
3.3
individualized cellulose nanofibril
iCNF
discrete cellulose nanofibril (3.2) composed of one elementary fibril (3.1) with ionic functional groups on
its surface
4 Abbreviated terms
AFM atomic force microscopy
CNC cellulose nanocrystal
FT-IR Fourier transform infrared spectrometry
HPLC high performance liquid chromatography
IC ion chromatography
ICP-AES inductively coupled plasma - atomic emission spectrometry
ICP-MS inductively coupled plasma - mass spectrometry
ICP-OES inductively coupled plasma - optical emission spectrometry
NMR nuclear magnetic resonance
SEC-MALS size-exclusion chromatography - multi-angle laser light scattering
TEM transmission electron microscopy
TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl
TGA thermogravimetric analysis
UV-Vis ultraviolet-visible
XRD X-ray diffraction
5 Characteristics to be measured of iCNF samples and their measurement
methods
5.1 General
The characteristics of iCNF samples listed in Table 1 are required to be measured or identified. The
characteristics listed in Table 2 are recommended to be considered for measurement and identification
based on the agreement between a buyer and a seller of an iCNF material in the market. The iCNF
sample refers to a material taken from an iCNF product for characterization that contains iCNFs and
other substances as impurities as well as solvent in the case of suspension.
The measurement methods listed in Tables 1 and 2 are required and recommended, respectively, to be
adopted to determine the characteristics of an iCNF sample. Measurement protocols for characteristics
in Tables 1 and 2 are separately provided in Annex A for the individual characteristics.
Test specimens for measurements shall and should be prepared from the sample as specified in each
subclause in 5.2 and 5.3, respectively.
For each of the characteristics measured, the state of the test specimens, such as aqueous suspension
and air-dried, freeze-dried or oven-dried powders, shall be reported in accordance with Clause 6.
When the test specimen’s dispersibility is considered an important factor for measurement, such as
observation by electron microscopy, information about the dispersing methods used (e.g. sonication,
homogenization, the use of surfactants) shall also be reported.
2 © ISO 2021 – All rights reserved

Table 1 — Characteristics of iCNF samples that are required to be measured or identified
and their measurement methods
Characteristics Measurement methods
Morphology and size TEM or AFM
Total dry matter content Oven drying and weighing
Crystal structure X-ray diffractometry
Optical transmittance UV-Vis spectrophotometry
Surface functional groups: Types FT-IR
Surface functional groups: Content Conductometric titration
Viscosity Viscometry
NOTE  The characteristics are arranged from general to specific for identification of iCNFs. The
order indicates neither importance nor a flow of measurements.
Table 2 — Characteristics of iCNF samples that are recommended to be measured or identified
and their measurement methods
Characteristics Measurement methods
Width and height TEM or AFM
Length TEM
Molecular weight distribution SEC-MALS
Supernatant dry matter ratio Centrifugation, oven drying and weighing
Crystallinity Solid-state NMR
Thermal stability TGA
Ash content Combustion and weighing
Acid-soluble metal content Incineration, wet chemical analysis and ICP-
AES/OES or ICP-MS
Organic contaminant content Solid-state NMR
Acetone-soluble matter content Soxhlet extraction, oven drying and weighing
Constituent sugar content HPLC or IC, and chemical analysis
5.2 Characteristics required to be measured or identified
5.2.1 Morphology and size
An iCNF is distinctive and unique in shape and size. The morphology of an iCNF sample refers to the
shapes of iCNFs and other solid objects, such as bundle-formed cellulose nanomaterials that are not
individualized, contained in the sample. The size refers to the width, height and length of iCNFs and
other solid objects. The morphology and size are measured qualitatively to observe the presence of
iCNFs and other solid objects contained in an iCNF sample.
Microscopic images of solid objects in an iCNF sample shall be obtained by TEM or AFM. When the
sample is provided in powder form, a test specimen in aqueous suspension form is first prepared. The
iCNF suspension is diluted by adding deionized water at an adequate concentration for the TEM and
AFM measurements.
More than 10 images shall be provided at appropriate magnifications so that iCNFs can be clearly
observed. Each image accurately represents the solid objects contained in an iCNF sample. The scale
bar is shown on each image.
See examples of microscopic images of morphology and size in A.2.1.
Quantitative measurements of the size, width/height and length of iCNFs are separately described in
5.3.1 and 5.3.2, respectively.
5.2.2 Total dry matter content
An iCNF sample may contain solid components other than iCNFs as well as dissolved materials. The
total dry matter content of an iCNF sample in suspension or powder form is the ratio of the mass of the
iCNF sample after drying to that of the iCNF sample before drying.
The mass of total dry matter shall be measured by the oven drying method, which consists of drying
the sample to constant mass at a temperature of 105 °C ± 2 °C and weighing.
The results of total dry matter content measurement shall be expressed in the unit of kg/kg.
See measurement protocols for the total dry matter content in A.2.2.
5.2.3 Crystal structure
The crystal structure is distinctive and unique for a crystalline solid and enables its identification. An
iCNF derived from native cellulose has crystalline regions of cellulose I, which is a mixture of cellulose
Iα and Iβ. It is also important to confirm if there are any solid objects having crystalline structures
other than cellulose I in an iCNF sample.
The crystal structures shall be identified. Their amounts shall be qualitatively estimated by X-ray
diffractometry for a dried test specimen. When the sample is provided in suspension form, a dried test
specimen is prepared for the measurements.
The results shall be expressed as an X-ray spectral chart over the diffraction angles between 5° and 45°
(as 2θ) to cover the peaks of cellulose I and other crystal structures included in an iCNF sample.
See an example of an iCNF X-ray spectral chart in A.2.3.
5.2.4 Optical transmittance
As a native cellulose material is physically reduced to individual iCNFs, the optical transmittance of
the suspension increases due to a decrease of optical scattering by the cellulose fibrils. The optical
transmittance in the UV-Vis range can be an indication of dispersibility of an iCNF sample. The optical
transmittance is the ratio of the radiant flux of an optical beam that is transmitted through a test cell
containing an iCNF suspension to that through a blank test cell with pure dispersant.
The optical transmittance shall be measured over the wavelength range from 200 nm to 750 nm by UV-
Vis spectrophotometry. When the sample is provided in aqueous suspension form, a test specimen of a
1 % mass fraction total dry matter content is prepared by diluting or concentrating. When the sample
is provided in powder form, a test specimen of aqueous suspension of a 1 % mass fraction total dry
matter content is prepared. A cell with a 10 mm optical path length is used for the measurement.
The results of optical transmittance measurements shall be expressed as % or a dimensionless number
and illustrated as a spectral chart of the optical transmittance versus wavelength over the specified
wavelength range.
See examples of measurement results in A.2.4 and B.8.
5.2.5 Surface functional groups: Types
When iCNFs are manufactured from natural cellulose fibres, surfaces of the iCNF may be modified with
functional groups. The type of functional groups depends on the manufacturing methods used. A test
specimen in dried solid form shall be prepared and used for the measurement. The type of functional
groups which are newly introduced during manufacturing iCNFs shall be identified by FT-IR. The
−1 −1
spectral charts shall be shown in the range from 600 cm to 3 800 cm .
See examples of charts in A.2.5 and B.11.
4 © ISO 2021 – All rights reserved

5.2.6 Surface functional groups: Content
The surface functional group content of iCNF samples is the ratio of the amounts of the newly introduced
and charged functional groups (such as carboxylic acids) to the mass of the total dry matter in a sample.
The amounts of the negatively charged functional groups in CNF samples, such as carboxylic acids
and phosphoric acids, shall be measured by conductometric titration. When the sample is provided
in suspension form, a test specimen in powder form is prepared from the suspension sample for the
measurement.
The results of conductometric titration measurement for each negatively charged functional group
shall be expressed in the unit of mmol/g.
See an example of measurement results for carboxylic acids in A.2.6.1.
NOTE When dissolved salts are contained in a suspension sample, they are also detected by the
conductometric titration.
5.2.7 Viscosity
Viscosity is a rheological property of a fluid that expresses resistance to shearing flows. The viscosity
of an iCNF suspension is a significant fundamental characteristic for liquid applications.
When an iCNF sample is provided in suspension form, a test specimen of 1 % mass fraction total dry
matter content is prepared for the measurement by diluting or concentrating. When an iCNF sample
is provided in powder form, an aqueous suspension test specimen of 1 % mass fraction total dry
matter content is prepared for the measurement. The test specimen is stored sufficiently long prior to
measurement in order to avoid the influence of the thixotropy of iCNF suspensions.
The viscosity shall be measured by the rotational viscometer at one shear rate in the range between
−1 −1
0,2 s and 2 s and can be optionally measured at other shear rates.
The viscosity results at 25 °C shall be expressed in the unit of Pa·s. The shear rates at which the viscosity
measurements are taken, and the dispersion medium and viscometer type used (e.g. single cylinder,
concentric cylinder, cone and plate or others) shall be reported with the viscosity results.
See an example of measurement results in A.2.7.
5.3 Characteristics recommended to be measured or identified
5.3.1 Width and height
Width and height measurements of fibrous objects contained in an iCNF sample can clearly distinguish
iCNFs from other fibrous objects on a microscopic image. The cross-sectional dimensions of an iCNF are
approximately 3 nm and are uniform along the fibre axis while those of other fibrous objects, e.g. CNF
bundles, are much larger than those of iCNFs.
The width of a fibrous object is the distance on a two-dimensional image between the two edges on
a cross-sectional line orthogonal to the longitudinal direction. The height of a fibrous object is the
distance on a three-dimensional image between the top of the fibrous object and the substrate surface
when the fibrous object is deposited laterally on the substrate. Since the cross-section of an iCNF or
other fibrous object is not a perfect circle, the width and height measured on a microscopic image may
vary depending on the viewing or probing angles of TEM or AFM to the fibres. The average of width and
height can be obtained over randomly oriented viewing and probing angles.
One datapoint of width or height is obtained for each fibrous object. When the width or height varies
along the fibre axis on an image, the largest width or height should be measured and recorded. The
target fibrous objects to be measured should be representative of the fibrous solid objects contained in
an iCNF sample, i.e. all types of fibrous objects on an image should be equally selected. The number of
width or height data is more than 25.
Either the width or height may be measured for an iCNF sample. The width should be measured by
TEM and the height by AFM. When the sample is provided in aqueous suspension form, the target
concentration for a test specimen is obtained via dilution or concentration before testing, and then the
test specimen is used for the measurement. When the sample provided is in powder form, an aqueous
suspension test specimen is first prepared for the measurement.
The measurement results should be displayed as a histogram of the number of iCNFs and other fibrous
objects versus width or height at the interval of 0,5 nm. Also, the average (median) of width or height
data of iCNFs and other fibrous objects should be expressed in the unit of nm. It should be noted that
the measurement results can be qualitative with increased uncertainty when the observed microscopic
images are not representative of the sample.
See histogram examples in A.3.1.
5.3.2 Length
The length of a fibrous object not having branches is the longitudinal distance along the axis between
its two ends on a two-dimensional image. When there are kinks along the fibre, the length of the
fibrous object is the sum between adjacent kinks and a kink and an end. The target fibrous objects to
be measured should be representative of the iCNF sample, i.e. all types of fibrous objects on an image
should be equally selected. The number of length datapoints may be agreed between the buyer and
seller of an iCNF material.
The length of fibrous objects not having a branch should be measured by TEM with the aid of image
analysis techniques. When the sample is provided in powder form, a test specimen in aqueous
suspension form is first prepared for the measurement.
The measurement results should be displayed as a histogram exhibiting length distribution of iCNFs
and other fibrous objects. Also, the average (median) of fibrous object length data in an iCNF sample
should be expressed in the unit of nm or μm.
See examples of the histogram in A.3.2.
5.3.3 Molecular weight distribution
An iCNF is a longitudinal sequence of cellulosic structures having the same cross-sectional dimensions.
Although the length of an iCNF is not always equal to the lengths of the cellulose molecules composing
the iCNF due to variation of enzymic reactions during biosynthesis of the elementary fibrils, longer
fibrils can contain longer cellulose chains. Therefore, the molecular weight distribution of cellulose
molecules in an iCNF sample could be strongly related to the length distribution of the iCNFs themselves.
The molecular weight distribution of an iCNF sample should be measured by SEC-MALS measurement
after appropriate pre-treatment of the iCNF sample. A dried powder test specimen is prepared for the
measurement.
The results should be expressed as M (weight-average molecular weight) as well as two-dimensional
w
graphs obtained from SEC-MALS.
See an example of measurement results in A.3.3.
5.3.4 Supernatant dry matter ratio
Solid objects suspended in a fluid can be separated into lighter and heavier objects by centrifugation
where the separation depends on the mass and density of individual solid objects. When an appropriate
centrifugal separation is applied to an iCNF suspension sample, iCNFs and soluble matter remain in the
supernatant while other heavier solid objects deposit as sediments.
The supernatant dry matter ratio is the ratio of the dry matter content of a supernatant of an iCNF test
specimen after centrifugal separation to that of the iCNF test specimen before centrifugal separation.
6 © ISO 2021 – All rights reserved

When the sample is provided in suspension form, a test specimen prepared at a dry matter content
of 0,1 % mass fraction should be used for centrifugal separation. When an iCNF sample is provided in
powder form, a suspension test specimen is first prepared and then the same processes are followed as
for the suspension samples.
The centrifugal separation is usually performed at more than 12 000 g for longer than 20 min. The
mass of dry matter should be measured by the oven drying method as in 5.2.2.
The results of supernatant dry matter ratio should be expressed as % mass fraction or a
dimensionless ratio.
See measurement protocols for the total dry matter content in A.3.4.
5.3.5 Crystallinity
An iCNF contains regions of highly ordered (crystalline) cellulose and regions of disordered
(amorphous) cellulose. The fraction of crystalline cellulose depends on the cellulose source and the
iCNF manufacturing processes. The crystallinity of an iCNF sample is the ratio of the mass of crystalline
cellulose to that of the total (crystalline and amorphous) of cellulose.
The crystallinity of an iCNF sample should be measured by solid-state C NMR. A test specimen for
measurement is prepared in dried powder form from the powder or suspension form sample provided.
The C4 peak of the iCNF in the cross polarization - magic angle spinning (CP-MAS) spectrum is separated
1)
from the C2, C3 and C5 peaks. The spectral charts should be shown in the range from 20 ppm to
200 ppm. The results of crystallinity measurement are calculated by the ratio of the area of the peak
(87 ppm to 93 ppm region) assigned to C4 of crystalline cellulose to that of the peak (80 ppm to 93 ppm
region) assigned to all C4 of cellulose.
See examples of solid-state NMR spectral charts obtained with cellulosic samples in A.3.5 and B.10.
5.3.6 Thermal stability
The thermal stability indicates the quality and the ability of the substances present in an iCNF sample
to resist irreversible change in its chemical or physical structure by decomposition or depolymerization
when subjected to high temperatures. The measurement of thermal stability of an iCNF test specimen
refers to the mass loss of the specimen in dried powder form during heating to a sufficiently high
temperature.
The thermal stability should be measured by TGA. When the sample is provided in suspension form,
the dried test specimen is first prepared for the measurement. The mass loss of test specimen should be
measured for both dynamic and isothermal conditions.
The result should be expressed as a thermogravimetric curve showing the mass as a function of
temperature. Considering that the results depend on many experimental and instrumental variables,
relevant measurement conditions are also reported including the atmosphere (e.g. air, N , O ) and its
2 2
flow rate, the method of sample drying (e.g. freeze-dried or air-dried), and the temperature programme
used (e.g. heating ramp rate(s) and/or isothermal temperature(s)).
See examples of thermal stability measurement results in B.9.
5.3.7 Ash content
An iCNF sample is predominantly composed of cellulosic fibrils which can be eliminated by combustion.
However, the sample may contain metals and inorganic constituents which are left as ash after
combustion.
The ash content of an iCNF sample in suspension or powder form is the ratio of the mass of the residue
after complete combustion of the sample to that of the total dry matter of the sample.
1) Chemical shift values are in parts per million (ppm) relative to tetramethylsilane.
The ash content should be measured by weighing the residue after combustion at 900 °C ± 25 °C using
an electric furnace.
The results of ash content should be expressed as % mass fraction or in the unit of mg/kg.
See measurement protocols for the ash content in A.3.7.
5.3.8 Acid-soluble metal content
Metal constituents are likely introduced from the substrates used in manufacturing iCNFs.
The content of acid-soluble metals (magnesium, calcium, manganese, iron, copper, sodium and
potassium) is the ratio of the mass of metallic element contained in the residue from incineration at
900 °C of a dried iCNF test specimen to that of the dried test specimen before incineration.
The acid-soluble metal content should be measured by using incineration followed by wet chemical
digestion and ICP-AES/OES or ICP-MS. When the sample is provided in suspension form, a dried test
specimen is prepared from the suspension for the measurements.
The measurement results should be expressed as % mass fraction or in the unit of mg/kg for each
element present.
See measurement protocols for the acid-soluble metal content in A.3.8.
5.3.9 Organic contaminant content
An iCNF sample may contain organic compounds other than iCNFs, including lignin derived from wood
pulp, as well as polymers and other components. A solid-state NMR spectrum can provide qualitative
information about the presence of organic contaminants in an iCNF sample.
Organic contaminants should be identified by CP-MAS C NMR. When the sample is provided in
suspension form, a dried test specimen is first prepared for the measurement.
The measurement results should be expressed with a solid-state NMR spectral chart (0 ppm to
220 ppm) covering the peaks of iCNF and organic contaminants.
See measurement protocols for the organic contaminant content in A.3.9.
5.3.10 Acetone-soluble matter content
An iCNF sample may contain acetone-soluble matter including low molecular weight organic compounds
which are intentionally added into the sample or which are introduced as contaminants during the
manufacturing process. The acetone-soluble matter content is the ratio of the mass of low molecular
weight organic compounds extracted from an iCNF sample by acetone to that of the total dry matter of
the iCNF sample.
The mass of acetone-soluble matter in an air-dried iCNF powder sample should be measured by Soxhlet
extraction with boiling acetone followed by weighing of the extraction residue. When the sample is
provided in suspension form, a test specimen in air-dried powder form is first prepared and then the
same processes are followed as for the powder samples.
The results of acetone-soluble matter content should be expressed as % mass fraction or in the unit
of kg/kg.
See measurement protocols for the acetone-soluble matter content in A.3.10.
5.3.11 Constituent sugar content
When a wood pulp is the starting material for manufacturing iCNFs, an iCNF sample may include
hemicelluloses depending on the purity of the pulp. The amount of hemicelluloses in an iCNF sample can
8 © ISO 2021 – All rights reserved

be estimated from the quantities of their constituent sugars. The constituent sugar content is the ratio
of the mass of constituent sugar in an iCNF sample to that of the total dry matter of the iCNF sample.
The mass of constituent sugars in an iCNF air-dried powder sample should be measured by acid
hydrolysis followed by sugar determination using a quantitative analysis for each monosaccharide. The
separation and quantitative analysis of each sugar are conducted by using chromatography such as
HPLC or IC, and the calibration curve method. When the sample is provided in suspension form, a test
specimen in air-dried powder form is first prepared and then the same processes are followed as for
the powder samples.
The measurement results of individual constituent sugars contents should be expressed as % mass
fraction or in the unit of kg/kg.
See measurement protocols for the constituent sugar content in A.3.11.
6 Reporting
The characterization report shall include the following:
a) sample identification:
1) sample name;
2) manufacturer’s name;
3) lot number;
4) sample source, e.g. type of plant, marine organism or bacteria;
5) manufacturing method, e.g. TEMPO and others;
6) sample form; suspension or powder;
7) name of additives;
8) storage conditions prior to testing;
b) name of characteristic measured or identified that is listed in Tables 1 or 2;
c) measurement method used for the individual characteristic;
d) test specimen prepared for measurement:
1) suspension or powder;
2) dry matter content of suspension;
3) the solvent, such as deionized water, and dispersing method, if used, in the case that a
suspension test specimen is prepared from a powder sample;
e) dates of measurement and name of organization that made the measurements for the individual
characteristics;
f) quantitative and/or qualitative results of measurements with the sample state and dispersion
method, if appropriate, for the reported characteristics;
g) information on the uncertainty of results;
h) additional information, if any, supporting the measurement results;
i) deviations: if there are any deviations from this document, the name of, and detailed information
on, the measurement methods used and their justification.
Annex A
(informative)
Protocols for sample preparation, measurement and data analysis
A.1 General
The sample preparation and measurement procedures and data analysis methods that are generally
used are provided in this annex for individual characteristics.
Sampling and sample homogeneity should be carefully considered in accordance with ISO 14488 to
obtain accurate measurement results.
When the concentration of suspension is expressed as % mass fraction in this annex, it refers to the
percentage of dry matter content.
A.2 Protocols for measuring required characteristics
A.2.1 Morphology and size
TEM, AFM and image processing using software are used for the visualization of morphology and size.
For morphology and size, either TEM or AFM can be used. TEM is recommended to determine the object
width. AFM is used for measuring object height rather than width because of its tip broadening effect
on the width measurements.
For TEM, an aqueous suspension (0,01 % mass fraction or less) of iCNFs is prepared. The suspension
of an iCNF sample is dropped onto the surface of the elastic carbon film Cu microgrid pretreated by
hydrophilic treatment. The wet iCNF on the grid is subjected to negative staining, and the sample is
used for TEM observation after drying. The grid with dry iCNFs is set on the specimen mount holder of
the TEM, and TEM observation is performed under the conditions of appropriate accelerating voltage.
For AFM, an aqueous suspension (0,01 % mass fraction or less) of an iCNF sample is dropped onto a
freshly cleaved mica surface. The mica plate is used for AFM observation after drying. The grid with
dry iCNFs is set on the specimen mount holder of the AFM, and AFM observation is performed using
the tapping mode. Appropriate AFM images are acquired using magnification suitable for surveying the
height and length of iCNFs and other fibrous objects.
Representative data for morphology and size are shown in Figure A.1. See also B.2 and B.3.
10 © ISO 2021 – All rights reserved

a)  TEM b)  AFM
Figure A.1 — Images of iCNFs
NOTE 1 ISO/TR 19716 provides information on CNC characterization and includes reference to diameter
measurements by both TEM and AFM. ISO 13322-1 provides general guidance and a theoretical background
of size measurement from images including TEM and AFM. ISO 14487:1997 provides information on standard
water for physical testing (pulps).
NOTE 2 References [1] and [2] provide information on iCNF characterization and include reference to diameter
measurements by AFM. Reference [3] provides information on iCNF diameter measurements by both TEM and
AFM. Reference [4] provides information on the size evaluation of CNFs including iCNF by AFM.
NOTE 3 Reference [5] provides information on aspect ratios of iCNFs.
NOTE 4 Reference [6] provides information on sample preparation for measurement of CNC by AFM and TEM.
NOTE 5 References [7], [8] and [9] provide information on staining for TEM observation.
A.2.2 Total dry matter content
A glass container, oven, desiccator and precision balance are used for the measurement.
The measurement can be applied to an iCNF sample available in either aqueous suspension or powder
form. For samples in aqueous suspension form, an adequate amount of test specimen is used to ensure
that at a minimum 20 mg of solid material will remain after drying. For samples in powder form, a test
specimen of 1 g to 10 g is normally used.
Before the test specimen is placed in the container, the empty container (body and lid) shall be
previously heated to constant mass, cooled and weighed. The container with the test specimen is
weighed. The lid is open, and the lid and container with the test specimen is placed in the oven. They are
heated at 105 °C ± 2 °C for a sufficiently long period until constant mass is reached. After drying, the lid
is put on the container. The container with test specimen is cooled in a desiccator to room temperature.
The container is weighed after cooling to room temperature. Drying and weighing procedures are
repeated until the difference between two consecutive weighing results is less than 1 % mass fraction
of the test specimen mass after drying (i.e. constant mass is reached). At least two test specimens are
used for determination of the total dry matter content.
The total dry matter content is calculated by using Formula (A.1):
m −m
()
CT_after c
C = ×100 (A.1)
D
m −m
()
CT_before c
where
C is the total dry matter content (in % mass fraction);
D
m is the mass of the container (in g);
C
m is the mass of the container with the test specimen after drying (in g);
CT_after
m is the mass of the container with the test specimen before drying (in g).
CT_before
NOTE ISO 638-1 and ISO 638-2 specify protocols in more detail for the dry matter content measurement by
the oven-drying method for powder and suspension samples, respectively.
A.2.3 Crystal structure
An X-ray diffractometer and Cu Kα (radiation source) are used for the measurement.
A freeze-dried iCNF sample of 0,1 g or more is pelletized by pressing for 1 min at a pressure of 750 MPa.
A pellet sample having a thickness of about 1 mm is prepared. X-ray diffraction patterns of the pellet
sample are obtained under the condition of measuring range 2θ from 5° to 45°, from which the crystal
structures are identified.
Representative data for crystal
...