ISO/PRF TS 23151

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
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ISO/TS 23151:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

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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

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

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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

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

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.

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

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

For the purposes of this document, the terms and definitions given in ISO 80004-2 and the following

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

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

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

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 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

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

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

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

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

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

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

. 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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

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

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

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

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