Crystallinities of Nanocrystalline and Nanofibrillated Celluloses by FTRaman Spectroscopy
Umesh P. Agarwala, Richard S. Reinera, Ilari Filpponenb, Akira Isogaic, Dimitris S.
Argyropoulosd
a
USDA Forest Service, Forest Products Laboratory,
Madison, WI
E-mail: [email protected]
b
TKK,
Espoo, Finland
c
Graduate School of Agricultural & Life Sciences
The University of Tokyo
Tokyo,Japan
d
Department of Forest Biomaterials
North Carolina State University
Raleigh, NC
ABSTRACT
Recently, a new method based on FT-Raman spectroscopy was proposed to determine cellulose I
crystallinity. It was reported that in the Raman spectrum of cellulosic materials the peak intensity
ratio of 380 and 1,096 cm-1 bands can be used to determine cellulose crystallinity. In that study,
Raman crystallinities of the calibration set Whatman CC31 samples were found to be greatly
correlated with the WAXS data (Segal-WAXS-21°; coefficient of determination R2 = 0.98).
Average standard error calculated from replicate Raman acquisitions indicated that the Raman
crystallinity model was highly reliable. In the present investigation, the Raman and WAXS
methods were applied to nanocrystalline and nanofibrillated celluloses. Calculated crystallinities
by the two methods were found to be significantly different.
INTRODUCTION
Cellulose nanocrystals (CNCs) and cellulose nanofibers (CNFs) are important new materials
in field of cellulose science and technology. While CNCs are elongated crystalline rod-like
nanoparticles NFCs consist of alternating crystalline and amorphous domains. These materials
have generated remarkable excitement in the scientific community. The materials have unique
physicochemical properties and have been used as reinforcing components in nanocomposites.
Additional benefits arise from the fact that they are derived from the biomass that is both
renewable and sustainable.
Among other things, crystallinity has an important effect on the physical, mechanical, and
chemical properties of cellulose based materials. For example, with increasing crystallinity,
tensile strength, dimensional stability, and density increase, while properties such as chemical
reactivity and swelling decrease. Cellulose crystallinity is defined as the mass fraction of
crystalline domains in cellulose materials.
1
Some of the frequently used techniques for estimating cellulose crystallinity are wide-angle Xray scattering (WAXS) [1-3], solid state 13C cross-polarization/magic-angle spinning (CP/MAS)
nuclear magnetic resonance (NMR) spectroscopy [4-6], and Fourier transform– infrared (FT-IR)
spectroscopy [7, 8]. Although WAXS is the most extensively used technique, the X-ray method
involves isolation of amorphous background from the diffraction pattern, which in the case of
cellulose crystallites is not always easy for reasons that have to do with cellulose crystallites
being small and, at lower crystallinities, the diffraction pattern being poorly defined (weak broad
features). Similarly, other techniques have limitations as well and these have been discussed in
the literature [e.g., 9 and references cited therein]. For the widely used simple Segal-WAXS
method [1], which involves measurement of 2 heights, the crystallinity values were significantly
higher than values from the other methods [10].
Raman spectroscopy has proven to be a useful technique in the cellulose and lignocellulose field
with numerous applications [11, 12]. It has become an important analytical technique for
nondestructive, qualitative, and quantitative analysis of cellulose-containing materials. In
particular, the FT–Raman technique has an added advantage due to its ability to successfully
analyze most materials that are fluorescent in conventional Raman (visible laser excitation).
Moreover, in the macro mode of sampling with 1,064-nm excitation, any anisotropy associated
with cellulose fiber materials is minimized because the scattering arises from the bulk of the
sample and is not limited to the surface. Crystallinity measurements using Raman on
semicrystalline polymers including cellulose [9, 13, 14, 15] have been performed. Of the two
Raman methods proposed for cellulose crystallinity estimation [9, 15], the method of Agarwal et
al. [9] is used here to calculate the crystallinities of CNCs and NFCs. The latter method produced
an improved correlation between Raman and WAXS crystallinities [9, 15].
EXPERIMENTAL
Materials
The nanocrystalline cellulose sample CNC-1 was prepared by acidic hydrolysis (similar to
the procedure used by Araki et al. [16]). A typical procedure was as follows: 2.0 g of cellulose
pulp obtained from Whatman #1 filter paper was blended by an Osterizer Blender. Resulting
pulp was hydrolyzed with 100 mL of 2.5 M HBr at 100°C for 3 hours. The ultrasonication was
applied during (every 60 minutes) the reaction (Omni-Ruptor 250W ultrasonic homogenizer,
50% power, 5 min). The resulting mixture was diluted with de-ionized (D.I) water followed by
five cycles of centrifugation at 1500g for 10 min. (IEC Centra-CL3 Series) to remove excess acid
and water soluble fragments. The fine cellulose particles became dispersed in the aqueous
solution approximately at pH 4. The turbid supernatant containing the polydisperse cellulose
particles was then collected for further centrifugation at 15000 g for 45 min (Automatic Servall
Superspeed Centrifuge) to remove ultra-fine particles. Ultra-fine particles with small aspect ratio
were removed from the upper layer, and the precipitation (after the high-speed centrifugation)
was dried using a lyophilizing system (Labconco, Kansas City, MU). CNC-2 sample was
produced at Forest Products Laboratory by sulfuric acid hydrolysis of wood pulp [17] using 64%
sulfuric acid (acid:cellulose = 8:1).
For NFCs, bleached softwood kraft pulp sample was TEMPO oxidized (1.3 mmol/g of COONa)
2
at the Department of Biomaterial Sciences (The University of Tokyo). The method used was
similar to that of Saito and Isogai [18]. The TEMPO treated sample was disintegrated in water to
obtain NFCs.
FT–Raman and crystallinity
CNCs, NFCs, and other cellulose samples were analyzed with a Bruker RFS 100 and
MultiRam spectrometers (Bruker Instruments Inc., Billerica, Massachusetts). Both the Raman
systems are equipped with a 1,064-nm 1,000-mW continuous wave (CW) diode pumped
Nd:YAG laser. About 20-30 mg of dry CNCs and NFCs were sampled in “Aluminum well” (a
Raman sampling accessory) or a pellet was made with ~ 100 mg of the sample. For rest of the
cellulose samples, approximately 250 mg of each sample was pressed into a pellet with the help
of a hydraulic press. To make a pellet, 276 9 106 dyn/cm2 compressive pressure was applied.
The laser power used for sample excitation was 600 mW, and 1,024 scans were accumulated.
Bruker’s OPUS software program was used to find peak positions and process the spectral data.
From the Raman spectra, amorphous contributions in the frequency region 250–700 cm-1 were
removed by first normalizing (making band intensity equal) the spectra on 897 cm-1 band and
then subtracting the corresponding spectrum of 120-min milled sample [9]. This was necessary
because Raman spectroscopy is a semi-quantitative technique. 897 cm-1 band in the spectrum
was chosen because its peak height was minimally impacted by change in crystallinity. For
plotting purposes, data were converted to ASCII format and then exported to Excel (Microsoft
Corp., Redmond, Washington). Raman crystallinity was determined using the univariate method
of Agarwal et al. [9] and consisted of calculating the peak height ratio I380/I1096 from the spectra
of samples from which spectrum of amorphous cellulose was subtracted. Cellulose crystallinities
in the samples were estimated by using the following correlation equation
X Raman =
(I
380
I 1, 096 ) − 0.0286
0.0065
X-ray and crystallinity
With the exception of CNCs-1 (see Table 1 later), wide-angle X-ray diffraction profiles were
recorded on a Bruker X-ray diffractometer with a Hi-Star 2-D area detector at the Materials
Research Science and Engineering Center, University of Wisconsin, Madison. Diffractograms
were obtained on the same sample pellets (or powder in the case of CNCs-2) that were analyzed
in FT–Raman. For CNCs-1, Omni Instruments Wide Angle XRD was used and the diffractogram
was obtained on the powdered sample. For calculating the crystallinity index, the ratio of the
crystallinity part of the 002 peak to the total absolute peak height was used. The peak present at
about 22.5° (2θ) corresponds to the [002] crystal planes. The crystalline portion of the total
contribution at 22.5° was calculated by Segal method (Segal et al. 1959) and involved
subtracting out the amorphous contribution at 21° (2θ). In the following, this crystallinity is
designated as Segal-WAXS-21°. The latter was the peak position in the diffractrogram of 120min milled amorphous sample. The method is same as used in reference [9]. WAXS crystallinity
was also calculated in the conventional manner by subtracting out the amorphous contribution at
18° (2θ). Here, the latter is specified as Segal-WAXS-18°.
3
RESULTS AND DISCUSSION
As reported previously [9] and shown in Fig.1, for the calibration set cellulose samples, the
Raman ratio plot for 380/1,096 (Fig. 1, Raman-Univariate) generated excellent regression (R2 =
0.992) and showed good sensitivity to change in crystallinity. Correlation of the calibration set
crystallinities with Segal-WAXS-21° is also shown in Fig. 1 (WAXS) and evidently, compared
to Raman-Univariate is somewhat inferior. The calibration set consisted of control (80.5%
crystalline) and 120-min milled (0% crystalline) Whatman CC31 and six cellulose mixtures
produced with crystallinities in the range 10.9–64% [9].
Fig. 1 Univariate analysis of the calibration set samples showing the correlation between
calibration set crystallinity versus 380/1,096 Raman intensity ratio or WAXS crystallinity [9].
Univariate-Raman, Segal-WAXS-18°, and Segal-WAXS-21° crystallinities of CNCs, NFCs,
and selected cellulose samples [9] are reported in Table 1. Clearly, the Raman crystallinities of
the CNCs-1 and CNCs-2 were identical (Table 1). However, although obtained on two different
X-ray instruments, that was not the case for either sets of Segal-WAXS crystallinity data. The
reasons for different Segal-WAXS-18° (or Segal-WAXS-21°) crystallinity values are not clear. In
any case, Segal-WAXS-18° crystallinity for CNC-1 in Table 1 was similar to that reported in
literature for other nanocelluloses [19].
4
Table 1: Crystallinities of CNCs, CNFs, and other cellulose products
Sample
Univariate-Raman Segal-WAXS (18°) Segal-WAXS (21°)
a
CNCs-1
66.0
91.0 b
80.0 b
Control-1
70.7
80.0 b
75.0b
CNCs-2
66.0
51.3 (67.3b)
43.7 (57.6b)
Control-2
69.6
77.0b
NFCs-1c
40.4
NAd
TEMPO-NFCs-1
40.5
NAd
NFCs-2e
33.0
NAd
TEMPO-NFCs-2
54.4
NAd
Starting Pulp-NFCs-2
62.3
NAd
Other cellulose materials
Whatman CC31
78.0f
90.3
80.5f
Whatman CC41
77.9f
87.8
74.6f
CF-11
72.0f
84.0
74.4f
Avicel PH-101
59.5f
75.6
60.9f
Whaman 541 paper
56.2f
57.1
50.0f
a
From Whatman # 1 paper
b
After a baseline is drawn under the diffractogram
c
From bleached softwood kraft pulp
d
NA = not yet available
e
From eucalyptus pulp
f
Ref. [9]
For CNCs-1, the difference of 11% between the two Segal-WAXS crystallinities (Table 1) is
understandable because it is well known that compared to other methods Segal-WAXS-18° gives
higher values [10, 20]. Actually, WAXS itself gives different values of crystallinity depending
upon how the crystallinity is calculated from the X-ray intensity data [10, 20]. The issue of
cellulose crystallinity index vis-à-vis measurement techniques was discussed by Park et al. [20]
where four different techniques incorporating X-ray diffraction and solid-state 13C nuclear
magnetic resonance (NMR) were compared. It was concluded that the Segal-WAXS-18°
produced significantly higher crystallinity values and did not provide an accurate measure of the
crystallinity of cellulose. Additionally, earlier Park et al. [10] used three X-ray methods (peak
height, peak deconvolution, and amorphous contribution subtraction) to calculate the crystallinity
index of 11 cellulose samples. The three methods produced significantly different crystallinity
indexes. Compared to the deconvolution approach, peak height method gave values that were
significantly higher – 20 to 30 points higher. Moreover, worth noting was also that the
differences depended upon the nature of the samples. For the 11 samples analyzed, the average
crystallinity indexes for the peak height, peak deconvolution, and amorphous contribution
subtraction methods were 86, 60, 68, respectively. This indicated that the index varied
significantly and depended upon how the analysis was performed. Agarwal et al. [9] have also
discussed this issue in their Raman crystallinity publication [9] and suggested that instead of
5
measuring the amorphous contribution at 18°, as is done in the traditional Segal method, the
measurement be carried out at 21° (called here Segal-WAXS-21°). Indeed, these researchers
have done that [9] for the cellulose materials investigated in that study and correlated thus
calculated WAXS crystallinities (Segal-WAXS-21°) to the ones obtained by Raman methods
(e.g., Fig. 1, univariate-Raman). For a number of cellulose products –Whatman CC31, CF-11,
Avicel PH-101, Whaman 541 paper, and Whatman CC41, both the Segal-WAXS-18° and SegalWAXS-21° crystallinities are listed in Table 1. For these samples, Segal-WAXS-21°
crystallinities were significantly lower compared to the Segal-WAXS-18° data but were similar
to univariate-Raman values (Table 1).
Notwithstanding the discrepancy between Segal-WAXS-21° and Raman methods, comparing
exclusively the Raman crystallinity data for nanocellulose samples (both CNCs and CNFs), it
can be noted that not only both the CNC samples gave same crystallinity (66%) the value was
significantly higher (≥26%) compared to the NFCs (Table 1). Although CNCs are supposedly
100% crystalline, it is not clear why a reduced value of 66% is generated. Perhaps, it has
something to do with either the small size of the CNCs or the increased number of molecules on
the crystallites’ surface. The reasons for lower CNCs crystallinity presently remain under
investigation. Similarly, Raman crystallinity data on NFCs (Table 1) indicated that the
crystallinity was lower, compared to CNCs, significantly (≥26) and for NFCs-1 remained same
as that of the control. As of now, the reasons behind the crystallinity drop for the other NFCs
sample (NFCs-2 in Table 1) remain to be investigated.
CONCLUSION
For the two CNC samples the Raman crystallinity of 66% was obtained. On the other hand,
values of 40 and 33% were generated for the two NFCs which were more than ≥26% lower
compared to the crystallinity of the CNCs. Between the NFCs, compared to control the
crystallinity of NFCs-2 dropped by about 21 points. However, the NFCs-1 sample showed no
such reduction. In contrast to Raman, the WAXS methods gave mixed results – higher
crystallinity for CNCs-1 and similar or lower crystallinity for CNCs-2.
ACKNOWLEDGEMENTS: The authors would like to thank Sally Ralph for the help received
during freeze drying and Raman spectra acquisitions.
REFERENCES
1. Segal L, Creely JJ, Martin AE, Conrad CM (1959) An empirical method for estimating the
degree of crystallinity of native cellulose using the X-ray diffractometer. Textile Res J 29:786–
794.
2. Jayme G, Knolle H (1964) The empirical X-ray determination of the degree of crystallinity of
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Structure of cellulose and microcrystalline cellulose from various wood species, cotton and
flax studied by X-ray scattering. Cellulose 16:999–1015.
4. Horii F, Hirai A, Kitamaru R (1987) CP/MAS 13C NMR spectra of the crystalline
components of native celluloses. Macromolecules 20:2117–2120.
5. Newman RH, Hemmingson JA (1990) Determination of the degree of cellulose crystallinity in
wood by carbon-13 nuclear magnetic resonance spectroscopy. Holzforschung 44:351–355
6
6. Newman RH (1999) Estimation of the lateral dimensions of cellulose crystallites using 13C
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7. Richter U, Krause T, Schempp W (1991) Untersuchungen zur Alkalibehandlung von
Cellulosefasern. Teil 1. Infrarotspektroskopische und Ro¨ntgenographische Beurteilung der
Anderung des Ordnungszustandes. Angew Makromol. Chem. 185(186):155–167.
8. Hulleman SHD, Van Hazendonk JM, Van Dam JEG (1994) Determination of crystallinity in
native cellulose from higher plants with diffuse reflectance Fourier transform infrared
spectroscopy. Carbohydrate. Res. 261:163–172.
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index of cellulose by solid state, 13C nuclear magnetic resonance. Cellulose 16:641–647.
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functional versatility, 2nd edn. Marcel Dekker, New York, pp 123–157.
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(Ed.) Characterization of lignocellulose materials. Blackwell, Oxford, pp 17–35.
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Biotechnology for Biofuels 3:10.
7
Crystallinities of Nanocrystalline and
Nanofibrillated Celluloses by FTRaman Spectroscopy
Presented by: Umesh Agarwal
Institution: USDA FS FPL
Outline
• Various methods for cellulose I crystallinity
determination
• Crystallinity by FT-Raman
• Crystallinity of CNCs and CNFs
How did we get into this?
Sour Orange: Control -1, 2, 7, 8; elevated CO2 – 3-6
Characterization of Lignocellulosic Materials – Ed.,Hu (2008), pp. 30
Various Methods
Park et al. Biotechnology for Biofuels, 3, 10(2010)
CI of Avicel-PH-101
Park et al. Biotechnology for Biofuels, 3, 10(2010)
Crystallinity from a Diffraction Pattern
Andersson et al.,Trees 18, 346(2004)
Cellulose I crystallinity determination
by FT-Raman spectroscopy
Agarwal et al., Cellulose 17, 721(2010)
Calibration Set Raman spectra
Univariate-Raman and WAXS
Correlations
Correlation between M-Segal-21°
WAXS and Raman
Raman Crystallinity
X Raman =
(I
380
I 1, 096 ) − 0.0286
0.0065
Table 1: Raman vs. WAXS
Sample
UnivariateRaman
Segal-WAXSa
(18°)
M-Segal-WAXS
(21°)
Whatman CC31
78.0
95.3
80.5
Whatman CC41
77.9
94.7
74.6
CF-11
72.0
89.6
74.4
Avicel PH-101
Whatman 541
paper
59.5
82.4
60.9
56.2
67.5
50.0
Segal et al., Textile Res. J. 29, 786(1959)
Comparison: CNCs-1 vs. CNCs-2
CNCs-1
• Source – Whatman FP #1
• Preparation – HBr
• Appearance – rod like; 78x100-400 nm
CNCs-2
• Source – Whatman CF1
• Preparation – H2SO4
• Appearance – rod like;
Sample
UnivariateRaman
Segal-WAXSa
(18°)
M-Segal-WAXS
(21°)
CNCs-1b
66.0
92.3
68.4
Control-1*
70.7
94.7
77.3
Sample
UnivariateRaman
Segal-WAXSa
(18°)
M-Segal-WAXS
(21°)
CNCs-2
66.0
92.6
70.8
Control-2
69.6
94.0
75.4
Comparison: CNFs-1 vs. CNFs-2
•
•
•
•
•
CNFs-1
Bleached softwood kraft
Aqueous media, pH 10
Regioselective oxidation
@ C6-OH
Disintegration – blender
Carboxylate content – 1.3
mmol/g
•
•
•
•
•
CNFs-2
Bleached Eucalyptus
kraft
Aqueous media, pH 10
Regioselective oxidation
@ C6-OH
Disintegration –
Centrifugal pump and
microfluidizer
Carboxylate content – 1.3
mmol/g
Sample
UnivariateRaman
Segal-WAXSa
(18°)
M-Segal-WAXS
(21°)
CNFs-1d
40.4
66.0
31.8
TEMPO-CNFs-1
58.5
91.4
69.2
Further support for CNFs-1 being more amorphous
Sample
UnivariateRaman
Segal-WAXSa
(18°)
M-Segal-WAXS
(21°)
CNFs-2e
34.5
73.1
39.1
TEMPO-CNFs-2
Starting PulpCNFs-2
54.4
83.2
52.2
60.5
79.6
50.0
Table 2: CNCs and CNFs
Sample
UnivariateRaman
Segal-WAXSa
(18°)
M-Segal-WAXS
(21°)
CNCs-1b
66.0
92.8 c
68.4
Control-1*
70.7
94.7
77.3
CNCs-2
66.0
92.6
70.8
Control-2
69.6
94.0
75.4
CNFs-1d
40.4
66.0
31.8
TEMPO-CNFs-1
58.5
91.4
69.2
CNFs-2e
34.5
73.1
39.1
TEMPO-CNFs-2
Starting PulpCNFs-2
54.4
83.2
52.2
60.5
79.6
50.0
Conclusions
• For CNCs, compared to M-Segal-WAXS-21 Raman gave
some what lower crystallinities (4-7% lower)
• For CNFs:
(a) In both Raman and WAXS the
crystallinity was lower compared to TEMPO
tr. control
(b) The avg. values were similar between
the methods (37.5 in Raman to 35.5 in
WAXS)
• Raman method can reliably estimate the crystallinity of
cellulose nanomaterials
Collaborators
• Rick Reiner – FPL
• Sally Ralph – FPL
• Ilari Filpponen – TKK, Espoo
• Akira Isogai – Univ. Tokyo
• Dimitris Argyropoulos – NCSU