ISO/TR 19716:2016

TECHNICAL ISO/TR
REPORT 19716
First edition
2016-05-01
Nanotechnologies — Characterization
of cellulose nanocrystals
Nanotechnologies — Caractérisation des nanocristaux de cellulose
Reference number
©
ISO 2016
© ISO 2016, Published in Switzerland
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ii © ISO 2016 – All rights reserved

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
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
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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).
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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

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.
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.
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
2 © ISO 2016 – All rights reserved

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
[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.
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

—
—
—
—
—
—
—
,
—
—
—
—
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
[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

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 aqueous
base. Both methods are described in this Clause, followed by a comparison of results for various CNCs.
Measurement of the total sulfur content can be accomplished by elemental analysis or by ICP-OES using
a spectrometer equipped with a concentric nebulizer, a cyclonic spray chamber and a quartz torch
with a quartz injector tube, optimized according to the manufacturer’s specifications. The sample is
completely solubilized by microwave assisted sample digestion using high purity nitric and hydrochloric
acids in high pressure closed vessels or, by wet ashing with strong acids, such as nitric and perchloric.
[38]
A block digestion system can be used to evaporate excess acids after the initial digestion. Analyses
are conducted by ICP-OES using the sulfur emission lines at 180,669 nm and 181,972 nm. Calibration is
accomplished by the method of additions (to compensate for any residual matrix interferences) in which
incremental spikes of sulfur as sulfate are prepared from a standard sulfur solution (e.g. a primary
standard, such as NIST SRM 3154). Samples can be diluted to ensure linearity of response. Samples are
gravimetrically spiked with at least two incremental levels of appropriate amounts of known calibration
standard. The levels should be chosen such that the spike results in a one to twofold increase in the total
sulfur concentration in the sample with each spike and the analytical response is linear. The calculation
of the concentration of sulfur requires a three point (minimum) standard additions calibration. Since the
slope of the standard additions calibration function for the sample and blank are likely not equivalent,
separate calibrations for C and C should be performed. Note that although total sulfur content can
s blk
also be obtained by inductively coupled plasma mass spectrometry (ICP-MS, see 5.6.3) ICP-OES is the
more reliable and straightforward method unless the sulfur concentration is very low.
The sulfate half-ester content can be determined by conductometric titration of the acidic sulfate half-
[30][39]
ester groups on the CNC surface using an aqueous base, such as sodium hydroxide. This is the
[40]
most commonly used method for this purpose. The sample should first be purified by extensive
dialysis to remove any residual ions and then treated with H-form strong acid cation-exchange resin to
ensure fully protonated sulfate half-esters. Commonly, CNC samples are diluted or re-dispersed with
deionized water (typically to ≤1,5 wt %) and dialyzed against pure water until the pH and conductivity
of the water surrounding the membranes no longer changes and approaches that of pure water. Hollow-
fibre membrane dialysis systems can also be used, they reduce dialysis time by drastically increasing
the exchange surface area and maintaining a large concentration gradient via counter-current sample
and dialysate flow. Prior to CNC protonation, the H-form strong acid cation-exchange resin should
be rinsed with a large excess of pure water until the filtrate is colourless and identical in pH and
conductivity to the wash water. A large excess of resin should be added to dialyzed CNC suspension
at a sufficiently low CNC concentration to ensure no coagulation, and the sample shaken to ensure
uniform mixing. The resin is then removed by filtration. Multiple successive treatments with fresh
resin might be required to achieve full protonation, particularly if the CNCs are in neutral salt form.
Alternatively, passing the diluted suspension through a column filled with such resin results in faster
[37]
treatment. Such treatment ensures complete protonation of a pure CNC suspension containing
no residual dissolved ions, yielding a 1:1 ratio of sulfate half-esters to protons and ensuring accurate
titration results. The concentration of the final protonated CNC sample is determined gravimetrically,
a sample of known volume is weighed and placed in a beaker with a dilute salt (NaCl) solution and
[30]
titrated against dilute sodium hydroxide using titration conditions optimized from the literature.
[41]
The sample conductivity is measured once the sample has equilibrated after the addition of each
aliquot of NaOH. Equivalence points are determined from the intersection of the regression lines fit to
the data points in the distinct regions of the titration curve (Figure 3).
8 © ISO 2016 – All rights reserved

a) H-CNCs containing strong acid sulfate half-ester groups and a small quantity of weak acid
carboxylic acid groups
b) TEMPO-oxidized CNCs generated with two different concentrations of oxidant
Key
X volume NaOH added (ml)
Y conductivity (μS/cm)
X′ volume NaOH (ml)
Y′ conductivity (mS/cm)
[37][42]
SOURCE Beck et al. 2014 and Habibi et al. 2006.
Figure 3 — Schematic illustration of conductometric titration curves
When interpreting conductometric titration data, it is important not to confuse the protons that are
actually detected (by neutralization with sodium hydroxide) with the sulfate half-ester content that is
calculated from the titration results. The calculations are based on the assumption that the protons in
the sample are in a 1:1 ratio with the sulfate half-ester groups. Despite this caveat, the titration method
does not require specialized and expensive equipment, and can be very useful for quality control during
CNC production.
Typical sulfate half-ester and sulfur content values for wood-based and other CNCs measured by
titration and elemental analysis are shown in Table 1. The differences between values obtained by
these two approaches have been discussed in the literature for CNCs of varying degrees of protonation.
[30][37][39][43][44] [40]
Titration values are often lower than those found by elemental analysis; this is
primarily due to insufficient sample preparation of the CNCs analysed by titration, notably failure to
ensure that the CNCs are fully protonated following purification by dialysis. Sodium-form CNCs are an
extreme example for which the sulfate half-ester content would not be measurable by conductometric
titration but would be by elemental analysis, such as ICP-OES. Treatment with mixed bed ion-exchange
resin (which contains hydroxide form anion exchange resin) has also been found to remove elemental
sulfur from CNC samples, and as such it is recommended to completely avoid the use of mixed bed ion-
[37]
exchange in CNC suspension purification; only dialysis should be used. Alternatively, the presence of
contamination in the form of sulfur-containing species, such as sulfate ions will yield erroneously high
elemental analysis (and conductometric titration, if they are protonated) results. This illustrates the
importance of dialysis for CNC suspension purification.
Differences between sulfate half-ester/sulfur contents measured by titration and elemental analysis
methods, respectively, might also be caused by the presence of sulfate half-ester groups that are
[39]
inaccessible to titrant or other forms of sulfur, introduced during biosynthesis of the source
[45]
cellulose. Owing to these discrepancies, if elemental analysis is used to estimate CNC surface charge
from sulfate half-esters, it is recommended to perform concurrent elemental analysis of the cellulose
source of the CNCs being studied to obtain an estimate of sulfur. It is important to understand that total
sulfur (measured by elemental analysis methods, such as ICP), titratable sulfur (protonated sulfate
half-ester groups that are accessible to titrant), sulfate half-ester content contributing to surface charge
(all surface sulfate half-ester groups) and total sulfate half-ester content (sulfate half-ester groups that
are titrant-accessible and -inaccessible, if any) are not necessarily equivalent values. However, a recent
study has shown that total sulfur and titratable sulfur are equivalent for softwood kraft pulp-derived
[37]
CNCs, indicating that all sulfate half-esters are at the surface. In general, elemental analysis of the
source cellulose and CNCs combined with conductometric titration of protonated CNCs will give the
most complete picture. Elemental analysis of CNCs extracted from the same source by HCl hydrolysis
might also be helpful in determining the “base sulfur content” of the CNCs. The above recommendations
are particularly important if knowledge of the precise quantitative surface charge or sulfur content
is required. Sulfur contents determined by the different methods typically vary by no more than
around 0,1 wt %, provided the CNCs contain no sulfur-containing impurities and are fully protonated if
[37]
required.
An additional complication in assessing sulfate half-ester content arises for samples that have
been extracted by sulfuric acid hydrolysis and then subjected to TEMPO-catalysed oxidation to
generate surface carboxylic acids. It is difficult to measure sulfate half-ester (strong acid) content by
conductometric titration if significant levels of weakly acidic carboxylic acids are present, but sulfate
half-esters can be determined in the presence of low quantities of carboxylic acid groups as shown in
[39][43]
Figure 3 a) for a non-oxidized CNC sample with a small number of weak carboxylic acid groups.
Finally, the surface sulfur content has also been measured by X-ray photoelectron spectroscopy (XPS).
[20][40][46][47][48][49]
Values typically range between 0,3 to 0,6 atomic % S, although in a few cases the
sulfur content was reported to be too low to be detected. In several examples, the surface sulfur content
has been compared to conductometric titration results for CNCs with sulfur content similar to the data in
[20][40][48]
Table 1. In one study, both methods showed lower sulfur content for desulfated CNCs than for the
[40]
initial CNCs obtained by sulfuric acid hydrolysis. However, quantitative agreement was poor, with the
sulfur content showing a sixfold change by titration but only a twofold change by XPS. Although the film
thickness was not reported, one should in principle obtain the same sulfur content for the two methods
when films of a single monolayer of CNCs are measured by XPS, since the depth penetration of ~10 nm is
greater than the particle cross-section (see 5.6.4 for more details on XPS measurements of CNCs).
10 © ISO 2016 – All rights reserved

Table 1 — Sulfate half-ester and sulfur contents (in mmol/kg CNC) for various CNC samples
Elemental
Titration
analysis
Cellulose source (standard Pre-treatment Reference
(standard
a
deviation)
a
deviation)
Dialysis, mixed bed
b
Cotton 205 (10) 220 (20) [44]
ion-exchange resin
Softwood (bleached kraft
c
84 240 Dialysis [39]
pulp)
Bacteria (Nata de coco) 5 — Dialysis [19]
Dialysis, mixed bed
Hardwood (eucalyptus) 250 — [21]
ion-exchange resin
Softwood (bleached sulfite Dialysis, mixed bed
290 (35) — [21]
pulp) ion-exchange resin
Softwood (dissolving-grade
d
293 0,57 atom % Dialysis [40]
sulfite pulp)
Dialysis, strong acid
e
Cotton 221 (6) 193 [30]
cation-exchange resin
Dialysis, mixed bed
e
Cotton 181 (6) 193 [30]
ion-exchange resin
Softwood (bleached kraft Dialysis, strong acid
f
225 (15) 225 (15) [37]
pulp) cation-exchange resin
a
Standard deviation is listed when it was provided in the literature reference.
b
Elemental analysis technique not specified.
c
Elemental analysis by X-ray fluorescence analysis.
d
Elemental analysis by XPS (based on C, O and S content).
e
Elemental analysis by quantitative conversion of sulfur to SO by combustion. The analyser uses IR or thermal
conductivity to detect sulfur in the combustion gases.
f
Elemental analysis by ICP-OES (total sulfur).
5.2.2 Determination of carboxylic acids
The carboxylate content of oxidized CNCs can be determined by conductometric titration with
sodium hydroxide using a similar approach to that described in 5.2.1. Typically, a known amount of
a strong acid, such as hydrochloric acid (HCl) is added prior to titration, ensuring full protonation of
[41][50][51]
the weak carboxylic acid groups. The carboxylate content is determined by extrapolating
and intersecting the three linear portions of the curve (strong acid, weak acid, excess titrant) to give
the two equivalence points (strong acid and total acid). Subtracting the strong acid content from the
total acid content gives the weak acid (carboxylate) content [Figure 3 b)]. As described in 5.4.1, the
presence of strong acid sulfate half-ester groups hinders the determination of carboxylic acid content
by conductometric titration in sulfated CNCs that have been highly oxidized (e.g. by TEMPO-mediated
oxidation).
Determination of surface carboxylic acids by conductometric titration has typically been reported as
[51]
degree of oxidation, which is defined as the mass fraction of carboxyl groups in the CNC sample. In
several cases, the degree of oxidation has been measured as a function of the oxidant/cellulose ratio
for TEMPO-catalysed oxidation; a plateau value is obtained that is hypothesized to represent complete
[19][42]
conversion of accessible surface hydroxyl groups to carboxylic acids. Reported degrees of
[16][42][50][52]
oxidation between 0,1 and 0,2 are typical; in two cases the degree of oxidation corresponds
[19][50]
to ~900 mmol/kg, which is considerably higher than the typical values of 200 mmol/kg for
sulfate half-esters (Table 1). Note that complete oxidation of surface hydroxyl groups will give different
degrees of oxidation for CNC particles with different surface area/mass ratios. In cases where the
fraction of surface, cellulose chains has been estimated based on the unit cell parameters for individual
[42][52]
crystallites, the predicted degree of oxidation is similar to that obtained experimentally.
Carboxylic acid groups have also been quantified by Fourier transform infrared spectroscopy (FTIR)
−1 [20]
using the strong absorption band due to the carbonyl stretch of the carboxylic acid at 1 634 cm .
[42][51][52] −1
The degree of oxidation is calculated as the ratio of the intensity of the 1 634 cm band to
−1
that of strongest cellulose backbone band at 1 050 cm . Note that use of the carbonyl stretch of the
−1
carboxylate anion at 1 608 cm should be avoided due to interference from adsorbed water in this
[51]
region. In several cases, the FTIR method was shown to be in reasonable agreement with the results
[20][42][52]
from conductometric titration, although it has been
...