ISO/TR 19716:2016
(Main)Nanotechnologies — Characterization of cellulose nanocrystals
Nanotechnologies — Characterization of cellulose nanocrystals
ISO/TR 19716:2016 reviews commonly used methods for the characterization of cellulose nanocrystals (CNCs), including sample preparation, measurement methods and data analysis. Selected measurands for characterization of CNCs for commercial production and applications are covered. These include CNC composition, morphology and surface characteristics.
Nanotechnologies — Caractérisation des nanocristaux de cellulose
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TECHNICAL ISO/TR
REPORT 19716
First edition
2016-05-01
Nanotechnologies — Characterization
of cellulose nanocrystals
Nanotechnologies — Caractérisation des nanocristaux de cellulose
Reference number
ISO/TR 19716:2016(E)
©
ISO 2016
---------------------- Page: 1 ----------------------
ISO/TR 19716:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TR 19716:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Terms and definitions . 1
3 Symbols and abbreviated terms . 2
4 Production of cellulose nanocrystals (CNCs) . 3
5 Composition . 6
5.1 Chemical composition . 6
5.2 Surface functional groups . 7
5.2.1 Determination of sulfate half-esters . 7
5.2.2 Determination of carboxylic acids .11
5.3 Degree of polymerization .12
5.4 Crystallinity . .13
5.4.1 General.13
5.4.2 X-ray diffraction .14
5.4.3 Nuclear magnetic resonance .16
5.4.4 Vibrational spectroscopy .18
5.4.5 Crystallinity measurements for CNCs .18
5.5 Moisture content .20
5.6 Contaminants .20
5.6.1 General.20
5.6.2 Residual impurities derived from cellulosic biomass .21
5.6.3 Metal ions .21
5.6.4 Detection of contaminants by X-ray photoelectron spectroscopy .21
6 CNC Morphology .22
6.1 Distributions of length and cross-section from microscopy .22
6.1.1 General.22
6.1.2 Electron microscopy .23
6.1.3 Atomic force microscopy .25
6.1.4 Image analysis considerations .27
6.1.5 Microscopy size distributions for CNCs .27
6.2 Size measurement by dynamic light scattering (DLS) .31
7 CNC Surface characteristics .33
7.1 Specific surface area .33
7.2 Surface charge .34
8 Miscellaneous .35
8.1 Thermal properties .35
8.2 Viscosity .38
9 Concluding comments .38
Bibliography .40
© ISO 2016 – All rights reserved iii
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ISO/TR 19716:2016(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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 229, Nanotechnologies.
iv © ISO 2016 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TR 19716:2016(E)
Introduction
Cellulose nanomaterials, including cellulose nanocrystals (CNCs) and cellulose nanofibrils, are
anticipated to have significant commercial impact. Cellulose nanocrystals are extracted from naturally
occurring cellulose, primarily from wood and annual plants, by acid hydrolysis, or chemical or
[1][2][3]
enzymatic oxidation. Their production from cellulose sources, such as wood pulps makes them a
candidate for use as a potentially non-toxic, biodegradable and sustainable nanomaterial. Furthermore,
the recent demonstration of the feasibility of CNC production on a large scale 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
[4][5]
tons annually, depending on the predicted applications and the estimated market penetration.
Standards for characterization of CNCs are required for material certification to allow sustained
commercial development and applications.
Cellulose nanocrystals are nanorods that have high aspect ratio, surface area and mechanical strength
and assemble to give a chiral nematic phase with unique optical properties. They are smaller than
cellulose nanofibrils and have a higher crystalline content. These properties, plus the ability to control
CNC surface charge and chemistry for dispersion in a variety of matrices, lead to potential applications
in many areas including nanocomposite materials, paints and adhesives, optical films and devices,
rheology modifiers, catalysts and biomedical products. There are currently no International Standards
for this emerging commercial nanomaterial, although an ISO/TC 229 project on terminology is in
progress, a Canadian National Standard (CSA Z5100) was published in 2014 and two CNC reference
materials were released in 2013. This Technical Report reviews information on sample preparation,
data collection and data analysis/interpretation for the measurands that are predicted to be important
for the development of commercial products containing CNCs. Information for the following CNC
properties is included: composition (crystallinity, surface functional groups, degree of polymerization
and contaminants), morphology as assessed by microscopy and light scattering methods, surface
charge and specific surface area, viscosity and thermal stability. The Technical Report reviews various
approaches that have been used for specific properties, but does not recommend standard methods or
provide detailed information on the techniques. The coverage is restricted to CNCs as produced and
does not extend to post-production modified CNCs or CNC-enhanced materials or products.
© ISO 2016 – All rights reserved v
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TECHNICAL REPORT ISO/TR 19716:2016(E)
Nanotechnologies — Characterization of cellulose
nanocrystals
1 Scope
This Technical Report reviews commonly used methods for the characterization of cellulose
nanocrystals (CNCs), including sample preparation, measurement methods and data analysis. Selected
measurands for characterization of CNCs for commercial production and applications are covered.
These include CNC composition, morphology and surface characteristics.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
agglomerate
collection of weakly or medium strongly bound particles where the resulting external surface area is
similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example, van der Waals forces or
simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.3]
2.2
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area is
significantly smaller than the sum of surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example, covalent bonds, or those
resulting from sintering or complex physical entanglement, or otherwise combined former primary particles.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.4]
2.3
nanocrystal
nano-object with a crystalline structure
[SOURCE: ISO/TS 80004-2:2015, 4.15]
2.4
nanofibre
nano-object with two external dimensions in the nanoscale and the third dimension significantly larger
Note 1 to entry: The largest external dimension is not necessarily in the nanoscale.
Note 2 to entry: The terms nanofibril and nanofilament can also be used.
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ISO/TR 19716:2016(E)
2.5
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each other.
[SOURCE: ISO/TS 80004-2:2015, 2.2]
2.6
nanorod
solid nanofibre
[SOURCE: ISO/TS 80004-2:2015, 4.7]
2.7
nanoscale
size range from approximately 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from a larger size will typically, but not exclusively, be
exhibited in this size range. For such properties the size limits are considered approximate.
Note 2 to entry: The lower limit in this definition (approximately 1 nm) is introduced to avoid single and small
groups of atoms from being designated as nano-objects or elements of nanostructures, which might be implied
by the absence of a lower limit.
[SOURCE: ISO/TS 80004-2:2015, 2.1]
3 Symbols and abbreviated terms
For the purposes of this document, the following symbols and abbreviated terms apply.
AEC anion-exchange chromatography
AFM atomic force microscopy
BET Brunauer-Emmett-Teller (method for determination of specific surface area)
CrI crystallinity index (also CI)
CNC(s) cellulose nanocrystal(s)
CP-MAS cross polarization magic angle spinning
d hydrodynamic diameter
h
DP degree of polymerization
D translational diffusion coefficient
t
DSC differential scanning calorimetry
DLS dynamic light scattering
ε dielectric constant
EM electron microscopy
FE-SEM field emission-scanning electron microscopy
FTIR Fourier transform infrared spectroscopy
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ISO/TR 19716:2016(E)
GLC gas-liquid chromatography
ICP-MS inductively coupled plasma-mass spectrometry
ICP-OES inductively coupled plasma-optical emission spectroscopy
ID isotope dilution
IR infrared
k Boltzmann constant
PI polydispersity
ssNMR solid state nuclear magnetic resonance
SEC size exclusion chromatography
SEM scanning electron microscopy
TEM transmission electron microscopy
TEMPO 2,2,6,6-tetramethyl-1-piperidinyloxy free radical
TGA thermogravimetric analysis
U electrophoretic mobility
E
η viscosity
XPS X-ray photoelectron spectroscopy
XRD X-ray diffraction
4 Production of cellulose nanocrystals (CNCs)
Cellulose is a linear polysaccharide composed of anhydroglucose units linked by an oxygen atom between
the C1 and C4 carbons of adjacent glucose rings. In cellulose biosynthesis individual, polysaccharide
chains are assembled by an enzyme complex into an elementary fibril with stacked chains held together
by hydrogen bonding. The number and organization of polymer chains is specific to the organism. These
elementary fibrils are further assembled to give larger structures that contain ordered (crystalline), as
well as disordered cellulose and other components that depend on the organism.
Cellulose nanocrystals are formed from one or more elementary fibrils and contain primarily
crystalline and paracrystalline regions. CNCs have length and cross-sectional dimensions that depend
on the cellulose source with typical aspect ratios between 5 and 50 and do not exhibit branching or
network-like structures. The term nanocrystalline cellulose is synonomous with CNCs and the term
nanowhiskers has also been used frequently in the literature. Cellulose nanofibrils are typically larger
than CNCs and are branched, entangled and agglomerated structures. The nanofibrils have crystalline,
paracrystalline and amorphous regions and can contain non-cellulosic components. They have cross-
[6]
sections between 5 nm and 50 nm and aspect ratios that are greater than 50. An ISO/TC 229 project
aimed at standardizing the terminology for cellulose nanomaterials has recently been initiated.
Cellulose nanocrystals are produced from a variety of cellulose sources, primarily wood and other
[2][3][7][8][9][10][11][12][13]
plants, but also algae, bacteria and tunicates. Their extraction from cellulose-
containing biomass begins with mechanical and/or chemical pre-treatment to remove non-cellulose
components, reduce the particle size and increase the exposed surface area. This is followed by a
hydrolysis or oxidation step that digests the more reactive amorphous cellulose and liberates CNCs from
the larger cellulose fibrils (Figure 1). Acid hydrolysis with sulfuric acid is the most widely used method
for CNC production in both research laboratories and pilot scale commercial facilities, although other
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ISO/TR 19716:2016(E)
[2][3][7][8][9][14][15]
acids (e.g. hydrochloric, phosphoric, phosphotungstic) have also been employed. In
attempts to minimize the use of strong acids, a variety of other processes have also been examined
including ultrasonication-assisted hydrolysis (with or without an iron chloride catalyst), enzymatic
[16][17][18]
oxidation and ammonium persulfate oxidation. After the acid hydrolysis or oxidation step,
CNCs are purified by a combination of centrifugation or filtration and washing steps, followed by
dialysis to remove residual salt and/or acids. A typical sequence for CNC production by acid hydrolysis
is illustrated in Figure 2.
67
8
1
2
5
3
3
4
Key
1 micro-fibril
2 disordered
3 crystalline
4 elementary fibrils
5 hydrolysis or oxidation
6 cellulose
7 cellulose fibril
8 cellulose nanocrystals
Figure 1 — Cartoon description of the formation of CNCs from larger cellulose fibrils
4 © ISO 2016 – All rights reserved
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ISO/TR 19716:2016(E)
—
—
—
—
—
—
—
,
—
—
—
—
Figure 2 — Overview of a typical process for production of CNCs by acid hydrolysis
CNCs produced by sulfuric acid hydrolysis have negatively-charged sulfate half-esters on their surface
which result in stable aqueous colloidal suspensions. Negatively charged CNCs are also formed by
phosphoric acid hydrolysis, whereas hydrochloric acid gives uncharged CNCs with only surface
hydroxyl groups. Oxidation catalysed by TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy free radical)
can be used to convert surface hydroxyls to carboxylic acids for CNCs generated using either sulfuric or
[19][20] [16]
hydrochloric acid. Oxidation with ammonium persulfate also generates carboxylated CNCs.
The CNC dimensions vary with the source of the cellulose; CNCs derived from wood pulps typically have
average lengths of 100 nm to 200 nm and cross-sections of 4 nm to 9 nm, whereas those from bacterial
and tunicate sources can be considerably larger, with lengths of 1 μm to 2 μm and cross-sections up to
50 nm (as reviewed in Reference [2]). The preparation method, acid or oxidant concentration, reaction
time and temperature, and sonication steps during purification also affect the CNC dimensions and the
[21][22][23][24][25][26][27]
overall yield and kinetics.
The acidic CNC suspensions produced by acid hydrolysis can be used in never-dried form. However,
in most cases the proton can be replaced by other cations by neutralizing the CNC suspension with
aqueous bases, such as hydroxides (XOH) or carbonates (X CO ), to give a salt form of the CNCs (X-CNC,
2 3
where X is the counterion associated with the anionic group). The pH-neutral sodium form, Na-CNC,
is most typically produced commercially and at large scale by in-line neutralization of H-CNCs with
sodium hydroxide (NaOH) or sodium carbonate (Na CO ). Advantages, such as the water-dispersability
2 3
[28]
of the dried product, allowing spray-dried or freeze-dried CNCs to be stored and shipped in the dry
form at significantly lower cost and then re-suspended at the point of use, account for this preference.
Proton counterions are most often exchanged for others by neutralization of the acidic groups with
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ISO/TR 19716:2016(E)
[29]
aqueous hydroxide bases, but this can also be accomplished by treatment with the appropriate ion-
[30]
exchange resin.
Dry CNC samples are prepared from the initial aqueous suspensions by evaporation, oven-drying, freeze-
drying (lyophilization), or spray-drying. Some characterization methods require dry samples, whereas
others employ a dilute suspension of CNCs. If the CNCs are already available as an aqueous suspension,
the sample can be diluted to the required concentration using deionized water or dilute buffer or salt
(NaCl) solution. Dry samples can be redispersed in pure water; general guidelines for dispersion of
[31]
powders in liquids can be found in ISO 14887 . Although an ultrasonic treatment step is typically used
to break up aggregates and agglomerates, a lack of reproducibility might contribute to variability of
results, as summarized in a recent study aimed at standardizing procedures for ultrasonic dispersion of
[32]
nanoparticles. It is not trivial to obtain redispersed samples of CNCs that have size distributions and
levels of aggregates or agglomerates that are similar to those of a purified, but never-dried, sample. An
early study showed that films of CNCs with fully protonated sulfate half-esters could not be redispersed
after drying, whereas CNCs with monovalent cations, such as sodium were redispersed with mild
[29]
ultrasonic treatment to give stable colloidal suspensions that were similar to those prior to drying.
Detailed procedures for the redispersion of the neutral sodium-form of CNCs prepared by evaporation,
[28]
lyophilization or spray-drying have been reported. The counterion and moisture content of the dry
CNCs and the sonication conditions (energy, CNC concentration) were all shown to affect the CNC (re)
dispersibility. While the sodium-form CNCs were fully dispersible when completely dried, the protonated
CNCs were only fully dispersible above a threshold water content of 4 wt %.
In this Technical Report, emphasis is placed on CNCs manufactured using sulfuric acid, with sulfate
half-ester groups on the cellulose surface (cellulose sulfate); unless otherwise noted, all examples are
for this form of CNCs. This reflects the emphasis on this material, in both commercial and research
laboratories. Most of the characterization methods are also applicable, in some cases with appropriate
adjustments, to other chemical forms of CNCs or cellulose nanofibres. For example, the detection and
quantification of surface functional groups is specific to the specific CNC production method. The
nature of the CNC counterion is important for some measurands, notably determination of the surface
charge due to sulfate half-ester or carboxylate groups by conductometric titration (see 5.2.1 and 5.2.2)
and zeta potential (see 7.2). Unless otherwise mentioned, the particular counterion in the CNC sample
does not affect the characterization methods discussed in this Technical Report.
Cellulose nanocrystals have specific physico-chemical properties associated with both the underlying
cellulose particle and the surface chemistry imposed by its manufacturing process. At the point of
commercialization, it is necessary to clarify the several descriptive systems that have been used in this
field: the geometric forms in nanotechnology, the industrial production method, and the chemical form
used in national regulations. All three are found in the recent approval under Canada’s New Substances
[33]
Notification Regulations as it provides the following:
a) chemical description (cellulose, hydrogen sulfate, sodium salt with a total sulfur content greater
than or equal to 0,5 % and less than or equal to 1,0 % by weight);
b) production method description (obtained from sulfuric acid hydrolysis of bleached pulp);
c) geometric description of length (nominal length of 100 nm ± 50 nm) and cross-section (cross-
sectional dimensions of less than or equal to 10 nm). As suggested in ISO 12805, composition,
length, diameter and surface area are the critical parameters to be considered first in setting
[34]
specifications.
5 Composition
5.1 Chemical composition
The chemical identity of CNCs as cellulose can be assessed by a qualitative identification test employed
for microcrystalline cellulose; dispersion of dry CNCs in iodinated zinc chloride will result in a violet-
[35]
blue colour. Their composition can also be verified by elemental analysis, based on the formula
[(C H O ) ] and taking into account surface functional groups if their degree of substitution is known.
6 10 5 n
6 © ISO 2016 – All rights reserved
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ISO/TR 19716:2016(E)
Although elemental analysis provides some information on surface functionality (e.g. % S for sulfate
half-esters), more detailed tests are typically used to quantify surface functional groups (see 5.2). The
identity of inorganic metal counterions for CNCs with anionic surface groups can be determined by
inductively coupled plasma-optical emission spectroscopy (ICP-OES) using the procedure outlined
in 5.4.1 for sulfur. The density of CNCs has usually been assumed to be the same as other types of
[2] 3 3
cellulose, as confirmed by a recent determination of 1,56 g/cm and 1,63 g/cm for the densities of
[36]
sulfated and unsulfated CNCs.
5.2 Surface functional groups
5.2.1 Determination of sulfate half-esters
CNCs extracted by sulfuric acid hydrolysis have sulfate half-ester groups on their surface. The
concentration of these negatively charged groups determines the CNC surface charge density and
controls the colloidal stability of CNCs in aqueous suspension, along with the self-assembly behaviour
and rheological properties. Two approaches have been used to determine the sulfate half-ester content.
[26][27]
The first relies on measurement of total sulfur content by elemental analysis. In cases where
the sample has been purified to ensure removal of all residual unbound sulfate ions, the total sulfur
[37]
content can be converted directly to the CNC sulfate half-ester content. The second approach uses
conductometric titration of the acidic sulfate half-ester groups on the CNC surface using an aqueo
...
TECHNICAL ISO/TR
REPORT 19716
First edition
Nanotechnologies — Characterization
of cellulose nanocrystals
Nanotechnologies — Caractérisation des nanocristaux de cellulose
PROOF/ÉPREUVE
Reference number
ISO/TR 19716:2016(E)
©
ISO 2016
---------------------- Page: 1 ----------------------
ISO/TR 19716:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TR 19716:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Terms and definitions . 1
3 Symbols and abbreviated terms . 2
4 Production of cellulose nanocrystals (CNCs) . 3
5 Composition . 6
5.1 Chemical composition . 6
5.2 Surface functional groups . 7
5.2.1 Determination of sulfate half-esters . 7
5.2.2 Determination of carboxylic acids .11
5.3 Degree of polymerization .12
5.4 Crystallinity . .13
5.4.1 General.13
5.4.2 X-ray diffraction .14
5.4.3 Nuclear magnetic resonance .16
5.4.4 Vibrational spectroscopy .18
5.4.5 Crystallinity measurements for CNCs .18
5.5 Moisture content .20
5.6 Contaminants .20
5.6.1 General.20
5.6.2 Residual impurities derived from cellulosic biomass .21
5.6.3 Metal ions .21
5.6.4 Detection of contaminants by X-ray photoelectron spectroscopy .21
6 CNC Morphology .22
6.1 Distributions of length and cross-section from microscopy .22
6.1.1 General.22
6.1.2 Electron microscopy .23
6.1.3 Atomic force microscopy .25
6.1.4 Image analysis considerations .26
6.1.5 Microscopy size distributions for CNCs .27
6.2 Size measurement by dynamic light scattering (DLS) .31
7 CNC Surface characteristics .33
7.1 Specific surface area .33
7.2 Surface charge .34
8 Miscellaneous .35
8.1 Thermal properties .35
8.2 Viscosity .38
9 Concluding comments .38
Bibliography .40
© ISO 2016 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO/TR 19716:2016(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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 229, Nanotechnologies.
iv PROOF/ÉPREUVE © ISO 2016 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TR 19716:2016(E)
Introduction
Cellulose nanomaterials, including cellulose nanocrystals (CNCs) and cellulose nanofibrils, are
anticipated to have significant commercial impact. Cellulose nanocrystals are extracted from naturally
occurring cellulose, primarily from wood and annual plants, by acid hydrolysis, or chemical or
[1][2][3]
enzymatic oxidation. Their production from cellulose sources, such as wood pulps makes them a
candidate for use as a potentially non-toxic, biodegradable and sustainable nanomaterial. Furthermore,
the recent demonstration of the feasibility of CNC production on a large scale 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
[4][5]
tons annually, depending on the predicted applications and the estimated market penetration.
Standards for characterization of CNCs are required for material certification to allow sustained
commercial development and applications.
Cellulose nanocrystals are nanorods that have high aspect ratio, surface area and mechanical strength
and assemble to give a chiral nematic phase with unique optical properties. They are smaller than
cellulose nanofibrils and have a higher crystalline content. These properties, plus the ability to control
CNC surface charge and chemistry for dispersion in a variety of matrices, lead to potential applications
in many areas including nanocomposite materials, paints and adhesives, optical films and devices,
rheology modifiers, catalysts and biomedical products. There are currently no International Standards
for this emerging commercial nanomaterial, although an ISO/TC 229 project on terminology is in
progress, a Canadian National Standard (CSA Z5100) was published in 2014 and two CNC reference
materials were released in 2013. This Technical Report reviews information on sample preparation,
data collection and data analysis/interpretation for the measurands that are predicted to be important
for the development of commercial products containing CNCs. Information for the following CNC
properties is included: composition (crystallinity, surface functional groups, degree of polymerization
and contaminants), morphology as assessed by microscopy and light scattering methods, surface
charge and specific surface area, viscosity and thermal stability. The Technical Report reviews various
approaches that have been used for specific properties, but does not recommend standard methods or
provide detailed information on the techniques. The coverage is restricted to CNCs as produced and
does not extend to post-production modified CNCs or CNC-enhanced materials or products.
© ISO 2016 – All rights reserved PROOF/ÉPREUVE v
---------------------- Page: 5 ----------------------
TECHNICAL REPORT ISO/TR 19716:2016(E)
Nanotechnologies — Characterization of cellulose
nanocrystals
1 Scope
This Technical Report reviews commonly used methods for the characterization of cellulose
nanocrystals (CNCs), including sample preparation, measurement methods and data analysis. Selected
measurands for characterization of CNCs for commercial production and applications are covered.
These include CNC composition, morphology and surface characteristics.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
agglomerate
collection of weakly or medium strongly bound particles where the resulting external surface area is
similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example, van der Waals forces or
simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.3]
2.2
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area is
significantly smaller than the sum of surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example, covalent bonds, or those
resulting from sintering or complex physical entanglement, or otherwise combined former primary particles.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.4]
2.3
nanocrystal
nano-object with a crystalline structure
[SOURCE: ISO/TS 80004-2:2015, 4.15]
2.4
nanofibre
nano-object with two external dimensions in the nanoscale and the third dimension significantly larger
Note 1 to entry: The largest external dimension is not necessarily in the nanoscale.
Note 2 to entry: The terms nanofibril and nanofilament can also be used.
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2.5
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each other.
[SOURCE: ISO/TS 80004-2:2015, 2.2]
2.6
nanorod
solid nanofibre
[SOURCE: ISO/TS 80004-2:2015, 4.7]
2.7
nanoscale
size range from approximately 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from a larger size will typically, but not exclusively, be
exhibited in this size range. For such properties the size limits are considered approximate.
Note 2 to entry: The lower limit in this definition (approximately 1 nm) is introduced to avoid single and small
groups of atoms from being designated as nano-objects or elements of nanostructures, which might be implied
by the absence of a lower limit.
[SOURCE: ISO/TS 80004-2:2015, 2.1]
3 Symbols and abbreviated terms
For the purposes of this document, the following symbols and abbreviated terms apply.
AEC anion-exchange chromatography
AFM atomic force microscopy
BET Brunauer, Emmett and Teller (method for determination of specific surface area)
CrI crystallinity index (also CI)
CNC(s) cellulose nanocrystal(s)
CP-MAS cross polarization magic angle spinning
d hydrodynamic diameter
h
DP degree of polymerization
D translational diffusion coefficient
t
DSC differential scanning calorimetry
DLS dynamic light scattering
ε dielectric constant
EM electron microscopy
FE-SEM field emission-scanning electron microscopy
FTIR Fourier transform infrared spectroscopy
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GLC gas-liquid chromatography
ICP-MS inductively coupled plasma-mass spectrometry
ICP-OES inductively coupled plasma-optical emission spectroscopy
ID isotope dilution
IR infrared
k Boltzmann constant
PI polydispersity
ssNMR solid state nuclear magnetic resonance
SEC size exclusion chromatography
SEM scanning electron microscopy
TEM transmission electron microscopy
TEMPO 2,2,6,6-tetramethyl-1-piperidinyloxy free radical
TGA thermogravimetric analysis
U electrophoretic mobility
E
η viscosity
XPS X-ray photoelectron spectroscopy
XRD X-ray diffraction
4 Production of cellulose nanocrystals (CNCs)
Cellulose is a linear polysaccharide composed of anhydroglucose units linked by an oxygen atom
between the C1 and C4 carbons of adjacent glucose rings. In cellulose biosynthesis individual,
polysaccharide chains are assembled by an enzyme complex into an elementary fibril with stacked
chains held together by hydrogen bonding. The number and organization of polymer chains is specific
to the organism. These elementary fibrils are further assembled to give larger structures that contain
ordered (crystalline cellulose), as well as disordered cellulose and other components that depend on
the organism.
Cellulose nanocrystals are formed from one or more elementary fibrils and contain primarily
crystalline and paracrystalline regions. CNCs have length and cross-sectional dimensions that depend
on the cellulose source with typical aspect ratios between 5 and 50 and do not exhibit branching or
network-like structures. The term nanocrystalline cellulose is synonomous with CNCs and the term
nanowhiskers has also been used frequently in the literature. Cellulose nanofibrils are typically larger
than CNCs and are branched, entangled and agglomerated structures. The nanofibrils have crystalline,
paracrystalline and amorphous regions and can contain non-cellulosic components. They have cross-
[6]
sections between 5 nm and 50 nm and aspect ratios that are greater than 50. An ISO/TC 229 project
aimed at standardizing the terminology for cellulose nanomaterials has recently been initiated.
Cellulose nanocrystals are produced from a variety of cellulose sources, primarily wood and other
[2][3][7][8][9][10][11][12][13]
plants, but also algae, bacteria and tunicates. Their extraction from cellulose-
containing biomass begins with mechanical and/or chemical pre-treatment to remove non-cellulose
components, reduce the particle size and increase the exposed surface area. This is followed by a
hydrolysis or oxidation step that digests the more reactive amorphous cellulose and liberates CNCs from
the larger cellulose fibrils (Figure 1). Acid hydrolysis with sulfuric acid is the most widely used method
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for CNC production in both research laboratories and pilot scale commercial facilities, although other
[2][3][7][8][9][14][15]
acids (e.g. hydrochloric, phosphoric, phosphotungstic) have also been employed. In
attempts to minimize the use of strong acids, a variety of other processes have also been examined
including ultrasonication-assisted hydrolysis (with or without an iron chloride catalyst), enzymatic
[16][17][18]
oxidation and ammonium persulfate oxidation. After the acid hydrolysis or oxidation step,
CNCs are purified by a combination of centrifugation or filtration and washing steps, followed by
dialysis to remove residual salt and/or acids. A typical sequence for CNC production by acid hydrolysis
is illustrated in Figure 2.
67
8
1
2
5
3
3
4
Key
1 micro-fibril
2 disordered
3 crystalline
4 elementary fibrils
5 hydrolysis or oxidation
6 cellulose
7 cellulose fibril
8 cellulose nanocrystals
Figure 1 — Cartoon description of the formation of CNCs from larger cellulose fibrils
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—
—
—
—
—
—
—
,
—
—
—
—
Figure 2 — Overview of a typical process for production of CNCs by acid hydrolysis
CNCs produced by sulfuric acid hydrolysis have negatively-charged sulfate half-esters on their surface
which result in stable aqueous colloidal suspensions. Negatively charged CNCs are also formed by
phosphoric acid hydrolysis, whereas hydrochloric acid gives uncharged CNCs with only surface
hydroxyl groups. Oxidation catalysed by TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy free radical)
can be used to convert surface hydroxyls to carboxylic acids for CNCs generated using either sulfuric or
[19][20] [16]
hydrochloric acid. Oxidation with ammonium persulfate also generates carboxylated CNCs.
The CNC dimensions vary with the source of the cellulose, CNCs derived from wood pulps typically have
average lengths of 100 nm to 200 nm and cross-sections of 4 nm to 9 nm, whereas those from bacterial
and tunicate sources can be considerably larger, with lengths of 1 μm to 2 μm and cross-sections up to
50 nm (as reviewed in Reference [2]). The preparation method, acid or oxidant concentration, reaction
time and temperature, and sonication steps during purification also affect the CNC dimensions and the
[21][22][23][24][25][26][27]
overall yield and kinetics.
The acidic CNC suspensions produced by acid hydrolysis can be used in never-dried form. However,
in most cases the proton can be replaced by other cations by neutralizing the CNC suspension with
aqueous bases, such as hydroxides (XOH) or carbonates (X CO ), to give a salt form of the CNCs (X-CNC,
2 3
where X is the counterion associated with the anionic group). The pH-neutral sodium form, Na-CNC,
is most typically produced commercially and at large scale by in-line neutralization of H-CNCs with
sodium hydroxide (NaOH) or sodium carbonate (Na CO ). Advantages, such as the water-dispersability
2 3
[28]
of the dried product, allowing spray-dried or freeze-dried CNCs to be stored and shipped in the dry
form at significantly lower cost and then re-suspended at the point of use, account for this preference.
Proton counterions are most often exchanged for others by neutralization of the acidic groups with
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[29]
aqueous hydroxide bases, but this can also be accomplished by treatment with the appropriate ion-
[30]
exchange resin.
Dry CNC samples are prepared from the initial aqueous suspensions by evaporation, oven-drying, freeze-
drying (lyophilization), or spray-drying. Some characterization methods require dry samples, whereas
others employ a dilute suspension of CNCs. If the CNCs are already available as an aqueous suspension,
the sample can be diluted to the required concentration using deionized water or dilute buffer or salt
(NaCl) solution. Dry samples can be redispersed in pure water, general guidelines for dispersion of
powders in liquids can be found in ISO 14887. Although an ultrasonic treatment step is typically used
to break up aggregates and agglomerates, a lack of reproducibility might contribute to variability of
results, as summarized in a recent study aimed at standardizing procedures for ultrasonic dispersion of
[32]
nanoparticles. It is not trivial to obtain redispersed samples of CNCs that have size distributions and
levels of aggregates or agglomerates that are similar to those of a purified, but never-dried, sample. An
early study showed that films of CNCs with fully protonated sulfate half-esters could not be redispersed
after drying, whereas CNCs with monovalent cations, such as sodium were redispersed with mild
[29]
ultrasonic treatment to give stable colloidal suspensions that were similar to those prior to drying.
Detailed procedures for the redispersion of the neutral sodium-form of CNCs prepared by evaporation,
[28]
lyophilization or spray-drying have been reported. The counterion and moisture content of the dry
CNCs and the sonication conditions (energy, CNC concentration) were all shown to affect the CNC (re)
dispersibility. While the sodium-form CNCs were fully dispersible when completely dried, the protonated
CNCs were only fully dispersible above a threshold water content of 4 wt %.
In this Technical Report, emphasis is placed on CNCs manufactured using sulfuric acid, with sulfate
half-ester groups on the cellulose surface (cellulose sulfate), unless otherwise, noted all examples are
for this form of CNCs. This reflects the emphasis on this material, in both commercial and research
laboratories. Most of the characterization methods are also applicable, in some cases with appropriate
adjustments, to other chemical forms of CNCs or cellulose nanofibres. For example, the detection and
quantification of surface functional groups is specific to the specific CNC production method. The
nature of the CNC counterion is important for some measurands, notably determination of the surface
charge due to sulfate half-ester or carboxylate groups by conductometric titration (see 5.2.1 and 5.2.2)
and zeta potential (see 7.2). Unless otherwise mentioned, the particular counterion in the CNC sample
does not affect the characterization methods discussed in this Technical Report.
Cellulose nanocrystals have specific physico-chemical properties associated with both the underlying
cellulose particle and the surface chemistry imposed by its manufacturing process. At the point of
commercialization, it is necessary to clarify the several descriptive systems that have been used in this
field: the geometric forms in nanotechnology, the industrial production method, and the chemical form
used in national regulations. All three are found in the recent approval under Canada’s New Substances
[33]
Notification Regulations as it provides the following:
a) chemical description (cellulose, hydrogen sulfate, sodium salt with a total sulfur content greater
than or equal to 0,5 % and less than or equal to 1,0 % by weight);
b) production method description (obtained from sulfuric acid hydrolysis of bleached pulp);
c) geometric description of length (nominal length of 100 nm ± 50 nm) and cross-section (cross-
sectional dimensions of less than or equal to 10 nm). As suggested in ISO 12805, composition,
length, diameter and surface area are the critical parameters to be considered first in setting
[34]
specifications.
5 Composition
5.1 Chemical composition
The chemical identity of CNCs as cellulose can be assessed by a qualitative identification test employed
for microcrystalline cellulose, dispersion of dry CNCs in iodinated zinc chloride will result in a violet-
[35]
blue colour. Their composition can also be verified by elemental analysis, based on the formula
[(C H O ) ] and taking into account surface functional groups if their degree of substitution is known.
6 10 5 n
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Although, elemental analysis provides some information on surface functionality (e.g. % S for sulfate
half-esters) more detailed tests are typically used to quantify surface functional groups (see 5.2). The
identity of inorganic metal counterions for CNCs with anionic surface groups can be determined by
inductively coupled plasma-optical emission spectroscopy (ICP-OES) using the procedure outlined
in 5.4.1 for sulfur. The density of CNCs has usually been assumed to be the same as other types of
[2] 3 3
cellulose, as confirmed by a recent determination of 1,56 g/cm and 1,63 g/cm for the densities of
[36]
sulfated and unsulfated CNCs.
5.2 Surface functional groups
5.2.1 Determination of sulfate half-esters
CNCs extracted by sulfuric acid hydrolysis have sulfate half-ester groups on their surface. The
concentration of these negatively charged groups determines the CNC surface charge density and
controls the colloidal stability of CNCs in aqueous suspension, along with the self-assembly behaviour
and rheological properties. Two approaches have been used to determine the sulfate half-ester content.
[26][27]
The first relies on measurement of total sulfur content by elemental analysis. In cases where
the sample has been purified to ensure removal of all residual unbound sulfate ions, the total sulfur
[37]
content can be converted directly to the CNC sulfate half-ester cont
...
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