Nanotechnologies — Particle size distribution for cellulose nanocrystals

This document describes methods for the measurement of particle size distributions for cellulose nanocrystals using atomic force microscopy and transmission electron microscopy. The document provides a protocol for the reproducible dispersion of the material using ultrasonication, as assessed using dynamic light scattering. Sample preparation for microscopy, image acquisition and data analysis are included.

Nanotechnologies — Distribution en taille des particules pour les nanocristaux de cellulose

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TECHNICAL ISO/TS
SPECIFICATION 23151
First edition
2021-09
Nanotechnologies — Particle size
distribution for cellulose nanocrystals
Nanotechnologies — Distribution en taille des particules pour les
nanocristaux de cellulose
Reference number
ISO/TS 23151:2021(E)
© ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 23151:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
© ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 23151:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction .................................................................................................................................................................................................................................v

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

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

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

4 Abbreviated terms ............................................................................................................................................................................................. 2

5 Dispersion of CNCs .............................................................................................................................................................................................2

5.1 General considerations.................................................................................................................................................................... 2

5.2 Dispersion of CNCs by sonication .......................................................................................................................................... 3

5.3 Dynamic light scattering assessment of dispersions ........................................................................................... 4

5.4 Determination of optimal sonication energy .............................................................................................................. 5

6 Sample preparation for microscopy ...............................................................................................................................................5

6.1 General considerations.................................................................................................................................................................... 5

6.2 AFM sample preparation ............................................................................................................................................................... 6

6.3 TEM sample preparation ............................................................................................................................................................... 6

7 Atomic force microscopy ............................................................................................................................................................................. 6

7.1 General ........................................................................................................................................................................................................... 6

7.2 Instrumentation and accessories .......................................................................................................................................... 7

7.3 Microscope calibration .................................................................................................................................................................... 7

7.4 Data acquisition ..................................................................................................................................................................................... 7

7.5 Image analysis......................................................................................................................................................................................... 8

8 Transmission electron microscopy ................................................................................................................................................. 8

8.1 General ........................................................................................................................................................................................................... 8

8.2 Instrumentation and accessories .......................................................................................................................................... 9

8.3 Microscope calibration .................................................................................................................................................................... 9

8.4 Data acquisition ..................................................................................................................................................................................... 9

8.5 Image analysis......................................................................................................................................................................................... 9

9 Data analysis .........................................................................................................................................................................................................10

9.1 General ........................................................................................................................................................................................................ 10

9.2 Assessment of data quality ....................................................................................................................................................... 10

9.3 Fitting distribution models to data ................................................................................................................................... 10

9.4 Measurement uncertainty ......................................................................................................................................................... 11

10 Test report ...............................................................................................................................................................................................................12

10.1 Atomic force microscopy ............................................................................................................................................................ 12

10.1.1 General information .....................................................................................................................................................12

10.1.2 Sample....................................................................................................................................................................................... 12

10.1.3 Data acquisition .................. ......................................................................................................................... .....................12

10.1.4 Image analysis ................................................................................................................................................................... 13

10.2 Transmission electron microscopy ................................................................................................................................... 13

10.2.1 General information .....................................................................................................................................................13

10.2.2 Sample....................................................................................................................................................................................... 13

10.2.3 Data acquisition .................. ......................................................................................................................... .....................13

10.2.4 Image analysis ................................................................................................................................................................... 14

Annex A (informative) Assessment of CNC dispersions ................................................................................................................15

Annex B (informative) Assessment of applied imaging force ................................................................................................16

Annex C (informative) Interlaboratory comparison results: AFM ...................................................................................18

Annex D (informative) Interlaboratory comparison results: TEM ..................................................................................25

Bibliography .............................................................................................................................................................................................................................34

iii
© ISO 2021 – All rights reserved
---------------------- Page: 3 ----------------------
ISO/TS 23151:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

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

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation 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 and www.iec.ch/national-

committees.
© ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 23151:2021(E)
Introduction

Cellulose nanomaterials, including cellulose nanocrystals (CNCs) and cellulose nanofibrils, are

anticipated to have significant commercial impact. Cellulose nanocrystals are produced from naturally

occurring cellulose, primarily from wood pulps and annual plants, by acid hydrolysis. Their production

from readily available cellulose sources makes them a candidate for use as a potentially non-toxic,

biodegradable and sustainable nanomaterial. The recent demonstration of the feasibility of large-scale

CNC production and the availability of infrastructure for harvesting raw materials will facilitate their

commercial development. CNCs and cellulose nanofibrils are produced in a number of countries on pilot,

pre-commercial or commercial scales. Estimates of the market potential for cellulosic nanomaterials

are as high as 35 million metric tons annually, depending on the predicted applications and the

[10],[11]

estimated market penetration . Standards for characterization of CNCs are required for material

certification to facilitate sustained commercial and applications development.

Cellulose nanocrystals have high crystallinity and are nanorods with high aspect ratio, surface area

and mechanical strength. They assemble to give a chiral nematic phase with unique optical properties

and their surface chemistry can be modified to ensure colloidal stability in water and to facilitate

dispersion in a variety of matrices. These properties, plus their biocompatibility, low cost and minimal

toxicity, enable many potential applications. Industrial producers are working with receptor industries

in various application areas, including nanocomposite materials, health and personal care products,

paints, adhesives and thin films, rheology modifiers and optical films and devices. Standardization

activities within ISO/TC 229 and ISO/TC 6 have focused on nomenclature and terminology,

characterization methods in general and specific methods for determining surface functional groups,

metal ion and dry ash content. Particle size distribution is also a key property for CNC characterization.

Particle morphology and size distribution control some properties of individual CNCs and contribute

in part to their organization in suspensions, dry films and matrices. These properties and chemical

characteristics determine CNC colloidal stability, viscosity and self-assembly, as well as performance

in applications (e.g. reinforcement of nanocomposites). Length distribution may also be used to

differentiate among cellulose nanocrystal grades or products.

This document describes a method for reproducibly dispersing dry CNCs for preparation of microscopy

samples, provides image acquisition protocols for atomic force and transmission electron microscopy

and summarizes image analysis procedures for determining particle size distributions. The methods

are compatible with analysis of CNCs as produced by several processes and can be extended to surface

modified CNCs with adjustment of dispersion and sample deposition methods. The two microscopy

methods provide complementary information, and both have been widely used for size analysis of CNCs.

© ISO 2021 – All rights reserved
---------------------- Page: 5 ----------------------
TECHNICAL SPECIFICATION ISO/TS 23151:2021(E)
Nanotechnologies — Particle size distribution for cellulose
nanocrystals
1 Scope

This document describes methods for the measurement of particle size distributions for cellulose

nanocrystals using atomic force microscopy and transmission electron microscopy. The document

provides a protocol for the reproducible dispersion of the material using ultrasonication, as assessed

using dynamic light scattering. Sample preparation for microscopy, image acquisition and data analysis

are included.
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

ISO 21363:2020, Nanotechnologies — Measurements of particle size and shape distributions by

transmission electron microscopy
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
cellulose nanocrystal

nanocrystal predominantly composed of cellulose with at least one elementary fibril (3.3), containing

predominantly crystalline and paracrystalline regions, with an aspect ratio of usually less than 50 but

usually greater than 5, not exhibiting longitudinal splits, inter-particle entanglement, or network-like

structures

Note 1 to entry: The dimensions are typically 3 nm to 50 nm in cross-section and 100 nm to several μm in length

depending on the source of the cellulose nanocrystal.

Note 2 to entry: The aspect ratio refers to the ratio of the longest to the shortest dimension.

Note 3 to entry: Historically cellulose nanocrystals have been called nanocrystalline cellulose (NCC), whiskers

such as cellulose nanowhiskers (CNW), and microfibrils such as cellulose microfibrils; they have also been called

spheres, needles or nanowires based on their shape, dimensions and morphology; other names have included

cellulose micelles, cellulose crystallites and cellulose microcrystals.
[SOURCE: ISO/TS 20477:2017]
© ISO 2021 – All rights reserved
---------------------- Page: 6 ----------------------
ISO/TS 23151:2021(E)
3.2
cellulose nanofibril

cellulose nanofibre composed of at least one elementary fibril (3.3), 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

Note 1 to entry: The dimensions are typically 3 nm to 100 nm in cross-section and typically up to 100 μm in

length.

Note 2 to entry: The aspect ratio refers to the ratio of the longest to the shortest dimensions.

Note 3 to entry: The terms “nanofibrillated cellulose”, “nanofibrillar cellulose”, “microfibrillated cellulose”,

“microfibrillar cellulose”, “cellulose microfibril” and “cellulose nanofibre” have been used to describe cellulose

nanofibrils produced by mechanical treatment of plant materials often combined with chemical or enzymatic

pre-treatment steps.

Note 4 to entry: Cellulose nanofibrils produced from plant sources by mechanical processes usually contain

hemicellulose and in some cases lignin.

Note 5 to entry: Some cellulose nanofibrils might have functional groups on their surface as a result of the

manufacturing process.
[SOURCE: ISO/TS 20477:2017, 3.3.6, modified — Note 6 to entry has been deleted.]
3.3
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]
4 Abbreviated terms
AFM atomic force microscopy
CNC(s) cellulose nanocrystal(s)
DLS dynamic light scattering
ILC interlaboratory comparison
PLL poly-L-lysine
PSD particle size distribution
PI polydispersity index
PVDF polyvinylidene difluoride
TEM transmission electron microscopy
VAMAS Versailles project on advanced materials and standards
5 Dispersion of CNCs
5.1 General considerations

Dry CNCs are aggregated and require energy input, typically by ultrasonication, for dispersion.

Previous studies have examined the sonication efficiency for CNCs derived from wood pulp by sulfuric

- +

acid hydrolysis and neutralization with sodium hydroxide which generates –SO Na groups on the

© ISO 2021 – All rights reserved
---------------------- Page: 7 ----------------------
ISO/TS 23151:2021(E)
[12]

surface . The average CNC size and size distribution varied with the sample concentration even when

the sonication energy divided by mass of CNC was kept constant; CNC suspensions with a mass fraction

of 2 % were shown to be optimal for efficient dispersion by sonication. The protocol below has been

developed using spray-dried sodium exchanged sulfated CNCs. The protocol may require optimization

[12],[13]

for freeze-dried CNCs , CNCs produced from other cellulose biomass sources and CNCs with a

different loading of sulfate half esters or other negatively charged surface groups.

A procedure for sample preparation and sonication (probe sonicator) to generate a well-dispersed

CNC suspension is provided in 5.2. Bath sonication has been shown to be inadequate for dispersion of

[12][14]

CNCs . A protocol for analysis of CNC suspensions by DLS is provided in 5.3; general details on the

[7]
use of DLS for particle size determination are available in ISO 22412 .

Representative results illustrating changes in size (Z-average) and PI as a function of sonication energy

are provided in Annex A. The Z-average is the intensity-weighted harmonic mean diameter derived

[7]
from a cumulants analysis of DLS data, as described in ISO 22412 .

The Z-average provides the equivalent hydrodynamic diameter, the diameter of a sphere that will

diffuse at the same rate as the acicular CNC particle.

Although the Z-average determined by DLS is not a direct measure of CNC particle size, it provides a

useful and rapid means of assessing changes in relative size for a large number of CNC suspensions.

Recent developments in the use of field flow fractionation coupled with multiple detection systems for

[15]

CNC analysis may provide an alternative to DLS analysis . The protocols for dispersion by sonication

[12]

and DLS assessment have been used by three laboratories with repeatable and reproducible results

[15][16]

Plots of Z-average and PI as a function of sonication energy can be used to select an appropriate

sonication energy for specific samples, see 5.4. This selection is a compromise between applying

sufficient sonication to disperse most aggregates while ensuring that the applied sonication energy

does not damage the sample.
5.2 Dispersion of CNCs by sonication

Remove dry CNC from low temperature storage and keep unopened until the sample reaches room

temperature (typically several hours).

Use an analytical balance to weigh the desired amount of CNC in a polypropylene centrifuge tube.

Amounts of CNC in the 50 mg to 300 mg range have been used with either 15 ml or 50 ml centrifuge

tubes in this protocol for preparation of 2 % mass fraction CNC suspensions. Glass tubes can be used,

although some optimization of the protocol may be required since the sonication efficiency is sensitive

to a number of factors, including the probe depth and placement and the container material and

[17]
geometry .

Add deionized water to the tube in the amount required to obtain a 2 % mass fraction suspension

of CNC, close the tube cap, and shake the tube vigorously by hand for a few seconds to promote CNC

dispersion. Freshly obtained deionized water (18,2 MΩ cm) filtered with a 0,22 μm filter (typically part

of the purification system) is used throughout.

The optimal concentration of CNC for dispersion by ultrasonication is 2 % mass fraction; disruption

of aggregates and agglomerates by sonication is less effective at lower concentrations. If suspensions

of lower concentration are required, dilute the sonicated 2 % mass fraction CNC suspension with

deionized water to the desired concentration.

Leave the mixture at room temperature for 24 h for the CNC to disperse. The mixture can be shaken by

hand periodically to accelerate dispersion; a tube shaker may also be used.

Check the condition of the ultrasonic probe (a 6-mm probe is recommended for the volumes used here)

and clean if pitting or roughness of the surface is observed.
© ISO 2021 – All rights reserved
---------------------- Page: 8 ----------------------
ISO/TS 23151:2021(E)

Sonication is most effective at low temperatures. Therefore, heating of the suspension during prolonged

sonication should be avoided. The temperature increase should not be more than 2 °C to 3 °C for the

amounts of dry CNC and processing energy recommended in this protocol, if the probe is in good working

condition, properly installed in the processor, and immersed in the suspension as recommended above.

During sonication, the tube may be placed in a room temperature water bath cooled when necessary

with a few ice cubes. Use of an ice bath is not recommended.

Immerse the ultrasonic probe in the suspension ensuring that the tip is centered in the tube and at least

1,3 cm both below the suspension surface and above the bottom of the tube.

Sonicate the suspension with the required energy (J/g dry CNC) at room temperature and an average

power of approximately 10 W. Ensure that the suspension surface remains as flat as possible and no

excessive aerosoling or bubbling is observed. If excessive aerosoling, bubbling, or suspension surface

fluctuation is observed, adjust the probe position immediately. Cover the tube to minimize loss of

suspension due to aerosoling.
[17]

The energy transfer efficiency may be measured calorimetrically to ensure that the applied

energy is reliable and does not change with time. Knowledge of the sonication energy is necessary for

comparisons between laboratories.

Remove the sample from the ultrasonic processor, and store for a short period of time at room

temperature (≈ 21 °C to 22 °C) or refrigerate (≈ 5 °C) for longer term storage.

NOTE This protocol has been tested with 50 mg to 300 mg dry CNC; preparation of suspensions with larger

amounts of CNC can require optimization of sonication conditions.
5.3 Dynamic light scattering assessment of dispersions
Set up the instrument as recommended in the manual.

Information on the importance of cell cleanliness and handling and proper technique for preparing and

[8]
transferring suspensions for DLS measurements is available in ISO/TR 22814 .

It is good laboratory practice to verify the operability of a DLS instrument by measuring a reference

nanomaterial (for which DLS data is available) to obtain Z-average and PI. Gold, silica and polystyrene

nanoparticles with diameter <100 nm are in the same size range as most CNC samples. For larger CNCs,

a reference material with diameter above 100 nm may be used. The use of a reference material from

[1]

a source qualified under ISO guidelines is recommended. The measured Z-average and PI should

be within the quoted uncertainty for the reference material. It is important to note that instrument

operability as verified using a reference material does not mean that a Z-average value obtained for

acicular CNCs is a quantitative or accurate measurement of diameter.

Dilute the 2 % mass fraction CNC suspensions to 0,1 % using deionized water, and then add 1 ml of

10 mmol/l NaCl solution to 1 ml of 0,1 % mass fraction CNC suspension to obtain 2 ml of 0,05 % mass

fraction suspension in 5 mmol/L NaCl. The 0,05 % suspension shall be analyzed within several hours of

preparation and shaken vigorously before transfer to the DLS cell. Filter the sample through a 0,45 μm

PVDF membrane syringe filter and discard the first several drops before adding the required volume to

the DLS cuvette. Ensure that there are no bubbles in the cell.

Place the cuvette in the instrument and equilibrate at the desired temperature. The time required for

equilibration will vary depending on the difference between the target temperature and the ambient

temperature. The equilibration time can be verified by measuring the temperature for an equivalent

volume of water under the same conditions. Adjust the scattering intensity using the instrument

software. Measure each sample three times with each measurement consisting of the average of a

number of runs (e.g. 10 runs of 10 s each).

Use the cumulants method to obtain the three-measurement average value and standard deviation for

Z-average and PI for the sample.

NOTE Different instrument optical configurations are available. The use of forward/backward scattering

and the scattering angle will affect the measured Z-average.
© ISO 2021 – All rights reserved
---------------------- Page: 9 ----------------------
ISO/TS 23151:2021(E)
5.4 Determination of optimal sonication energy

Sonicate CNC suspensions with varying energies and measure the DLS Z-average and PI for each sample

as described in 5.3. Plot Z-average and PI against sonication energy.

Select the optimal sonication energy for production of a well-dispersed suspension from a region of the

curve where the measured Z-average and PI change slowly with increasing energy. An example plot is

shown in Annex A.
To ensure reproducibility, measure a minimum of three replica
...

TECHNICAL ISO/TS
SPECIFICATION 23151
First edition
Nanotechnologies — Particle size
distribution for cellulose nanocrystals
Nanotechnologies — Distribution en taille des particules pour les
nanocristaux de cellulose
Member bodies are requested to consult relevant national interests in IEC/TC
113 before casting their ballot to the e-Balloting application.
PROOF/ÉPREUVE
Reference number
ISO/TS 23151:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 23151:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© 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 PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 23151:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

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

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

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

4 Abbreviated terms .............................................................................................................................................................................................. 2

5 Dispersion of CNCs .............................................................................................................................................................................................. 2

5.1 General considerations .................................................................................................................................................................... 2

5.2 Dispersion of CNCs by sonication ........................................................................................................................................... 3

5.3 Dynamic light scattering assessment of dispersions ............................................................................................. 4

5.4 Determination of optimal sonication energy ................................................................................................................ 5

6 Sample preparation for microscopy ................................................................................................................................................ 5

6.1 General considerations .................................................................................................................................................................... 5

6.2 AFM sample preparation ................................................................................................................................................................ 6

6.3 TEM sample preparation ................................................................................................................................................................ 6

7 Atomic force microscopy .............................................................................................................................................................................. 6

7.1 General ........................................................................................................................................................................................................... 6

7.2 Instrumentation and accessories ............................................................................................................................................ 7

7.3 Microscope calibration ..................................................................................................................................................................... 7

7.4 Data acquisition ..................................................................................................................................................................................... 7

7.5 Image analysis ......................................................................................................................................................................................... 8

8 Transmission electron microscopy ................................................................................................................................................... 8

8.1 General ........................................................................................................................................................................................................... 8

8.2 Instrumentation and accessories ............................................................................................................................................ 9

8.3 Microscope calibration ..................................................................................................................................................................... 9

8.4 Data acquisition ..................................................................................................................................................................................... 9

8.5 Image analysis ......................................................................................................................................................................................... 9

9 Data analysis ..........................................................................................................................................................................................................10

9.1 General ........................................................................................................................................................................................................10

9.2 Assessment of data quality ........................................................................................................................................................10

9.3 Fitting distribution models to data ....................................................................................................................................10

9.4 Measurement uncertainty ..........................................................................................................................................................11

10 Test report ................................................................................................................................................................................................................12

10.1 Atomic force microscopy .............................................................................................................................................................12

10.1.1 General information ...................................................................................................................................................12

10.1.2 Sample....................................................................................................................................................................................12

10.1.3 Data Acquisition ............................................................................................................................................................12

10.1.4 Image analysis .................................................................................................................................................................13

10.2 Transmission electron microscopy ....................................................................................................................................13

10.2.1 General information ...................................................................................................................................................13

10.2.2 Sample....................................................................................................................................................................................13

10.2.3 Data acquisition .............................................................................................................................................................13

10.2.4 Image analysis .................................................................................................................................................................14

Annex A (informative) Assessment of CNC dispersions .................................................................................................................15

Annex B (informative) Assessment of applied imaging force ..................................................................................................16

Annex C (informative) Interlaboratory comparison results: AFM ......................................................................................18

Annex D (informative) Interlaboratory comparison results: TEM .....................................................................................25

Bibliography .............................................................................................................................................................................................................................34

© ISO 2021 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO/TS 23151:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

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

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation 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 for electrotechnical products and systems.

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 a nd w w w . ie c . c h/ n at ion a l

-committees.
iv PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 23151:2021(E)
Introduction

Cellulose nanomaterials, including cellulose nanocrystals (CNCs) and cellulose nanofibrils, are

anticipated to have significant commercial impact. Cellulose nanocrystals are produced from naturally

occurring cellulose, primarily from wood pulps and annual plants, by acid hydrolysis. Their production

from readily available cellulose sources makes them a candidate for use as a potentially non-toxic,

biodegradable and sustainable nanomaterial. The recent demonstration of the feasibility of large-scale

CNC production and the availability of infrastructure for harvesting raw materials will facilitate their

commercial development. CNCs and cellulose nanofibrils are produced in a number of countries on pilot,

pre-commercial or commercial scales. See ISO/TC 6, Task Group 1 report: https:/ isotc .iso .org/ livelink/

livelink ?func = ll & objId = 8865729 & objAction = browse & viewType = 1). Estimates of the market potential

for cellulosic nanomaterials are as high as 35 million metric tons annually, depending on the predicted

[1][ 2]

applications and the estimated market penetration . Standards for characterization of CNCs are

required for material certification to facilitate sustained commercial and applications development.

Cellulose nanocrystals have high crystallinity and are nanorods with high aspect ratio, surface area

and mechanical strength. They assemble to give a chiral nematic phase with unique optical properties

and their surface chemistry can be modified to ensure colloidal stability in water and to facilitate

dispersion in a variety of matrices. These properties, plus their biocompatibility, low cost and minimal

toxicity, enable many potential applications. Industrial producers are working with receptor industries

in various application areas, including nanocomposite materials, health and personal care products,

paints, adhesives and thin films, rheology modifiers and optical films and devices. Standardization

activities within ISO/TC 229 and ISO/TC 6 have focused on nomenclature and terminology, an overview

of characterization methods and methods for determining surface functional groups, metal ion and

dry ash content. Particle size distribution is also a key property for CNC characterization. Particle

morphology and size distribution control some properties of individual CNCs and contribute in part to

their assemblies in suspensions, dry films and matrices. These properties and chemical characteristics

determine CNC colloidal stability, viscosity and self-assembly, as well as performance in applications

(e.g. reinforcement of nanocomposites). Length distribution may also be used to differentiate among

cellulose nanocrystal grades or products.

This document describes a method for reproducibly dispersing dry CNCs for preparation of microscopy

samples, provides image acquisition protocols for atomic force and transmission electron microscopy

and summarizes image analysis procedures for determining particle size distributions. The methods

are compatible with analysis of CNCs as produced by several processes and can be extended to surface

modified CNCs with adjustment of dispersion and sample deposition methods. The two microscopy

methods provide complementary information and both have been widely used for size analysis of CNCs.

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TECHNICAL SPECIFICATION ISO/TS 23151:2021(E)
Nanotechnologies — Particle size distribution for cellulose
nanocrystals
1 Scope

This document describes methods for the measurement of particle size distributions for cellulose

nanocrystals using atomic force microscopy and transmission electron microscopy. The document

provides a protocol for the reproducible dispersion of the material using ultrasonication, as assessed

using dynamic light scattering. Sample preparation for microscopy, image acquisition and data analysis

are included.
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 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects

ISO 21363:2020, Nanotechnologies — Measurements of particle size and shape distributions by

transmission electron microscopy
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 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
cellulose nanocrystal

nanocrystal predominantly composed of cellulose with at least one elementary fibril (3.3), containing

predominantly crystalline and paracrystalline regions, with an aspect ratio of usually less than 50 but

usually greater than 5, not exhibiting longitudinal splits, inter-particle entanglement, or network-like

structures

Note 1 to entry: The dimensions are typically 3 nm to 50 nm in cross-section and 100 nm to several μm in length

depending on the source of the cellulose nanocrystal.

Note 2 to entry: The aspect ratio refers to the ratio of the longest to the shortest dimension.

Note 3 to entry: Historically cellulose nanocrystals have been called nanocrystalline cellulose (NCC), whiskers

such as cellulose nanowhiskers (CNW), and microfibrils such as cellulose microfibrils; they have also been called

spheres, needles or nanowires based on their shape, dimensions and morphology; other names have included

cellulose micelles, cellulose crystallites and cellulose microcrystals.
[SOURCE: ISO/TS 20477:2017]
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ISO/TS 23151:2021(E)
3.2
cellulose nanofibril

cellulose nanofibre composed of at least one elementary fibril (3.3), 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

Note 1 to entry: The dimensions are typically 3 nm to 100 nm in cross-section and typically up to 100 μm in

length.

Note 2 to entry: The aspect ratio refers to the ratio of the longest to the shortest dimensions.

Note 3 to entry: The terms “nanofibrillated cellulose”, “nanofibrillar cellulose”, “microfibrillated cellulose”,

“microfibrillar cellulose”, “cellulose microfibril” and “cellulose nanofibre” have been used to describe cellulose

nanofibrils produced by mechanical treatment of plant materials often combined with chemical or enzymatic

pre-treatment steps.

Note 4 to entry: Cellulose nanofibrils produced from plant sources by mechanical processes usually contain

hemicellulose and in some cases lignin.

Note 5 to entry: Some cellulose nanofibrils might have functional groups on their surface as a result of the

manufacturing process.
[SOURCE: ISO/TS 20477:2017, 3.3.6, modified — Note 6 to entry has been deleted.]
3.3
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]
4 Abbreviated terms
AFM atomic force microscopy
CNC(s) cellulose nanocrystal(s)
DLS dynamic light scattering
ILC interlaboratory comparison
PLL poly-L-lysine
PSD particle size distribution
PI polydispersity index
PVDF polyvinylidene difluoride
TEM transmission electron microscopy
VAMAS Versailles project on advanced materials and standards
5 Dispersion of CNCs
5.1 General considerations

Dry CNCs are aggregated and require energy input, typically by ultrasonication, for dispersion. Previous

studies have examined the sonication efficiency for CNCs derived from wood pulp by sulfuric acid

- +

hydrolysis and neutralization with sodium hydroxide which generates –SO Na groups on the surface

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ISO/TS 23151:2021(E)
[12]

. The average CNC size and size distribution varied with the sample concentration even when the

sonication energy divided by mass of CNC was kept constant; 2 % mass fraction CNC suspensions were

shown to be optimal for efficient dispersion by sonication. The protocol below has been developed using

spray-dried sodium exchanged sulfated CNCs. The protocol may require optimization for freeze-dried

[12][13]

CNCs , CNCs produced from other cellulose biomass sources and CNCs with a different loading of

sulfate half esters or other negatively charged surface groups.

A procedure for sample preparation and sonication (probe sonicator) to generate a well-dispersed

CNC suspension is provided in 5.2. Bath sonication has been shown to be inadequate for dispersion of

[12][14]

CNCs . A protocol for analysis of CNC suspensions by DLS is provided in 5.3; general details on the

[7]
use of DLS for particle size determination are available in ISO 22412 .

Representative results illustrating changes in size (Z-average) and polydispersity index (PI) as a

function of sonication energy are provided in Annex A. The Z-average is the intensity-weighted

[7]

harmonic mean diameter derived from a cumulants analysis of DLS data, as described in ISO 22412 .

The Z-average provides the equivalent hydrodynamic diameter, the diameter of a sphere that will

diffuse at the same rate as the acicular CNC particle.

Although the Z-average determined by DLS is not a direct measure of CNC particle size, it provides a

useful and rapid means of assessing changes in relative size for a large number of CNC suspensions.

Recent developments in the use of field flow fractionation coupled with multiple detection systems for

[15]

CNC analysis may provide an alternative to DLS analysis . The protocols for dispersion by sonication

[12]

and DLS assessment have been used by three laboratories with repeatable and reproducible results

[15][16]

Plots of Z-average and PI as a function of sonication energy can be used to select an appropriate

sonication energy for specific samples, see 5.4. This selection is a compromise between applying

sufficient sonication to disperse most aggregates while ensuring that the applied sonication energy

does not damage the sample.
5.2 Dispersion of CNCs by sonication

Remove dry CNC from low temperature storage and keep unopened until the sample reaches room

temperature (typically several hours).

Use an analytical balance to weigh the desired amount of CNC in a polypropylene centrifuge tube.

Amounts of CNC in the 50 mg to 300 mg range have been used with either 15 ml or 50 ml centrifuge

tubes in this protocol for preparation of 2 % mass fraction CNC suspensions. Glass tubes can be used,

although some optimization of the protocol may be required since the sonication efficiency is sensitive

to a number of factors, including the probe depth and placement and the container material and

[17]
geometry .

Add deionized water to the tube in the amount required to obtain a 2 % mass fraction suspension

of CNC, close the tube cap, and shake the tube vigorously by hand for a few seconds to promote CNC

dispersion. Freshly obtained deionized water (18,2 MΩ cm) filtered with a 0,22 μm filter (typically part

of the purification system) is used throughout.

Leave the mixture at room temperature for 24 h for the CNC to disperse. The mixture can be shaken by

hand periodically to accelerate dispersion; a tube shaker may also be used.

Check the condition of the ultrasonic probe (a 6-mm probe is recommended for the volumes used here)

and clean if pitting or roughness of the surface is observed.

Immerse the ultrasonic probe in the suspension ensuring that the tip is centered in the tube and at least

1,3 cm both below the suspension surface and above the bottom of the tube.

Sonicate the suspension with the required energy (J/g dry CNC) at room temperature and an average

power of approximately 10 W. Ensure that the suspension surface remains as flat as possible and no

excessive aerosoling or bubbling is observed. If excessive aerosoling, bubbling, or suspension surface

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ISO/TS 23151:2021(E)

fluctuation is observed, adjust the probe position immediately. Cover the tube to minimize loss of

suspension due to aerosoling.

Remove the sample from the ultrasonic processor, and store for a short period of time at room

temperature (≈ 21 °C to 22 °C) or refrigerate (≈ 5 °C) for longer term storage.

NOTE 1 This protocol has been tested with 50 mg to 300 mg dry CNC; preparation of suspensions with larger

amounts of CNC may require optimization of sonication conditions.

NOTE 2 The optimal concentration of CNC for dispersion by ultrasonication is 2 % mass fraction; disruption

of aggregates and agglomerates by sonication is less effective at lower concentrations. If suspensions of lower

concentration are required, dilute the sonicated 2 % mass fraction CNC suspension with deionized water to the

desired concentration.

NOTE 3 Sonication is most effective at low temperatures. Therefore, heating of the suspension during

prolonged sonication should be avoided. The temperature increase should not be more than 2 °C to 3 °C for

the amounts of dry CNC and processing energy recommended in this protocol, if the probe is in good working

condition, properly installed in the processor, and immersed in the suspension as recommended above. During

sonication the tube may be placed in a room temperature water bath cooled when necessary with a few ice cubes.

Use of an ice bath is not recommended.
[17]

NOTE 4 The energy transfer efficiency may be measured calorimetrically to ensure that the applied energy

is reliable and does not change with time. Knowledge of the sonication energy is necessary for comparisons

between laboratories.
5.3 Dynamic light scattering assessment of dispersions
Set up the instrument as recommended in the manual.

Information on the importance of cell cleanliness and handling and proper technique for preparing and

[8]
transferring suspensions for DLS measurements is available in ISO/TR 22814 .

It is good laboratory practice to verify the operability of a DLS instrument by measuring a reference

nanomaterial (for which DLS data is available) to obtain Z-average and PI. Gold, silica and polystyrene

nanoparticles with diameter < 100 nm are in the same size range as most CNC samples. For larger CNCs

a reference material with diameter above 100 nm may be used. The use of a reference material from

[1]

a source qualified under ISO guidelines is recommended. The measured Z-average and PI should

be within the quoted uncertainty for the reference material. It is important to note that instrument

operability as verified using a reference material does not mean that a Z-average value obtained for

acicular CNCs is a quantitative or accurate measurement of diameter.

Dilute the 2 % mass fraction CNC suspensions to 0,1 % using deionized water, and then add 1 ml of

10 mmol/l NaCl solution to 1 ml of 0,1 % mass fraction CNC suspension to obtain 2 ml of 0,05 % mass

fraction suspension in 5 mmol/L NaCl. The 0,05 % suspension shall be analyzed within several hours of

preparation and shaken vigorously before transfer to the DLS cell. Filter the sample through a 0,45 μm

PVDF membrane syringe filter and discard the first several drops before adding the required volume to

the DLS cuvette. Ensure that there are no bubbles in the cell.

Place the cuvette in the instrument and equilibrate at the desired temperature. The time required for

equilibration will vary depending on the difference between the target temperature and the ambient

temperature. The equilibration time can be verified by measuring the temperature for an equivalent

volume of water under the same conditions. Adjust the scattering intensity using the instrument

software. Measure each sample three times with each measurement consisting of the average of a

number of runs (e.g. 10 runs of 10 s each).

Use the cumulants method to obtain the three-measurement average value and standard deviation for

Z-average and PI for the sample.

NOTE Different instrument optical configurations are available. The use of forward/backward scattering

and the scattering angle will affect the measured Z-average.
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