Role of the OH and NH vibrational groups in polysaccharide- nanocomposite interactions: A FTIR-ATR study on chitosan and chitosan/clay films

Polymer 99 (2016) 614e622
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Role of the OH and NH vibrational groups in polysaccharidenanocomposite interactions: A FTIR-ATR study on chitosan and
chitosan/clay films
C. Branca*, G. D'Angelo, C. Crupi, K. Khouzami, S. Rifici, G. Ruello, U. Wanderlingh
Department of Physics and Earth Sciences, University of Messina, Viale Stagno D'Alcontres 31, 98166 Messina, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 May 2016
Received in revised form
25 July 2016
Accepted 29 July 2016
Available online 30 July 2016
Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy was used to examine
the impact of hydrogen bonding on the reactive amine and hydroxyl groups of chitosan. Measurements
were performed on powder and film samples hydrated under different conditions. To resolve the individual OH and NH bands that overlap in the region between 3000 and 3800 cm1, the spectra were
deconvoluted using a Gaussian curve fitting method. By analyzing the changes recorded for each
Gaussian component along the dehydration process of the films at different pH values, a band assignment was proposed. This assignment was then used to analyze the ATR-FTIR spectra of the chitosan/
montmorillonite composites. The changes induced by the presence of the clay were ascribed to particular
mechanisms of interaction that involve the active sites of the clay and the amino or hydroxyl groups of
chitosan. The comparison between the FTIR and the XRD data, evidenced that these mechanisms are
intimately controlled by the type of nanostructures that are formed depending on the clay content.
© 2016 Elsevier Ltd. All rights reserved.
Keywords:
Chitosan
Montmorillonite
FTIR-ATR spectroscopy
1. Introduction
Naturally occurring polymers are extensively used as biocompatible and biodegradable materials and their importance will increase further in the future because of the growing urgency to
develop new bio based products. Most of the biopolymers are based
on biodegradable starch polymers, such as chitosan. Chitosan is the
only natural cationic polysaccharide derived from chitin through a
deacetylation process to obtain a linear structure of glucosamine
and N-acetylglucosamine linked in a b-1,4 manner. The chemically
active groups in the chitosan structure are the free amine groups,
located in the C2 position of the glucose residue in the polysaccharide chain, and the hydroxyl groups, that can be both
modified and involved in hydrogen or electrostatic bonding [1e3].
The amine groups can be easily protonated in acid environment
making chitosan soluble in aqueous acid solutions. Thanks to its
high biocompatibility, non toxicity, antifungal and antimicrobial
activities, chitosan is used for a broad range of applications
including drug carriers, food packaging films, bone substitutes and
so on [4e11]. However, its applications are limited because of the
* Corresponding author.
E-mail address: [email protected] (C. Branca).
http://dx.doi.org/10.1016/j.polymer.2016.07.086
0032-3861/© 2016 Elsevier Ltd. All rights reserved.
high moisture permeability and brittleness. The combination of
chitosan with clay nanofillers, such as montmorillonite, has proved
to be an effective method to overcome this limit creating new
nanocomposites showing improved mechanical, thermal and barrier properties usually even at very low concentrations [12e21].
Montmorillonite (hereafter MMT) is a hydrated alumina-silicate
layered clay made up of two silica tetrahedral sheets fused to an
edge-shared octahedral sheet of aluminum hydroxide. It is well
suitable for reinforcement purposes since it has a high surface area,
large aspect ratio (50e1000), and platelet thickness of 10 Å
[22e24]. Due to the relatively weak forces between the layers of
MMT, water and other polar molecules can enter between the unit
layers, causing the lattice to expand in the thickness direction.
Although several studies are reported in literature about
chitosan-clay composites, the overall understanding of the interactions underlying the formation of composite films still remains
elusive. It is generally reported that the incorporation of montmorillonite induces electrostatic interactions between the negatively charged groups of the clay and the positively charged eNHþ
3
groups of chitosan [12,13,15,17]. However, controversial results are
reported in literature [12,13,15,17,19,21] about the dependence on
the clay content shown by many properties of the polymer/clay
composites, such as tensile strength, elongation and swelling.
C. Branca et al. / Polymer 99 (2016) 614e622
615
Considering that interactions between different components are
governed by a variety of chemical bonds, the study of the vibrational properties can provide a valuable tool to understand the
nature of the interactions that strongly affects the properties of the
final products.
In the present work, by ATR-FTIR spectroscopy, we investigated
the role played by the OH and NH groups of chitosan in the formation of an hydrogen bonded network and how this is modified
by the presence of a nanocomposite structure. Measurements were
performed for chitosan powder, dry and hydrated films, and chitosan/montmorillonite nanocomposite films at different clay content. In particular, the time evolution of the absorption spectra of
the chitosan films, hydrated at different pH values, were carefully
monitored along the dehydration process. Infrared spectra were
recorded in the mid-ir region focusing the attention on the
stretching region that was deconvoluted using a Gaussian curve
fitting method.
The novelty of the present work lies in a comprehensive vibrational analysis of the spectral features of chitosan under different
environmental conditions that allowed a separation and identification of the intramolecular OH and NH stretching components.
The assignment of these modes, usually reported as overlapping
bands in the region 3000e3800 cm1, was crucial to get insights
into the existing correlation among mechanisms of interaction
between chitosan and clay, swelling properties and different architectures of nanostructures.
XRD mounts were obtained by pipetting a small amount of the
solutions onto glass plates. Silicon powder was added to the spread
solution, thus Silicon peak was used as a reference, in order to shift
spectra to the correct position and eliminate possible mistakes due
to incorrect vertical positioning of the sample. The solvent was then
removed by evaporation under nitrogen flux and the dry samples
were stored at 35 C. The drying of the XRD mounts was performed
at the same nitrogen flux and time duration for all the samples to
guarantee the comparability of XRD results.
2. Experimental
where We and Wd are the weights of the sample at equilibrium and
in the dry state, respectively.
2.4. Swelling
Dry chitosan films were immersed in water solutions at different
pH values, 3 and 11, and stored at 25 C. In order to avoid any
structural alteration of the phyllosilicate, all the chitosan-clay films
were swollen at pH 6.
After the removal of the excess surface water by filter paper, the
weights of the samples were measured at various time intervals.
The weighing was continued until equilibrium swelling was
attained. Each swelling experiment was repeated three times, and
the average values were reported.
The equilibrium degree of swelling (EDS) was calculated as
follows:
EDSð%Þ ¼
We Wd
*100
Wd
(1)
2.1. Materials
Low molecular weight chitosan (CS) (Cat. No. 448869,
190e310 kDa) and montmorillonite K10 were purchased by Sigma
Aldrich. MMT employed in this study has a CEC of 70e100 meq/
100 g. We used montmorillonite for its unique intercalation/exfoliation characteristics that can lead to platelets with very high
stiffness and strength when dispersed in a polymer matrix. All the
reagents were analytical grade and used without further
purification.
2.2. Sample preparation
Chitosan was dissolved in 1% acetic acid solution, magnetically
stirred for 24 h at 40 C. The pH of the polysaccharide solution was
adjusted to 5 with NaOH in order to avoid any structural alteration
of the phyllosilicate. We obtained a final concentration of 1.6% (w/
v). Prior to the addition of clay, an aliquot of the solution was cast on
a Teflon dish and left overnight for drying in an oven at 30 C. The
sample, labeled “as prepared film”, was kept in a desiccator before
characterization.
Two clay suspensions were prepared and slowly added to two
aliquots of the chitosan solution in order to obtain two physical
mixtures with a clay concentration of 2.5 wt% and 5 wt% based on
chitosan. For simplicity, these samples were labeled as CSM2.5 and
CSM5. The mixtures were stirred for 2 days, then poured onto a
Teflon surface and dried in an oven at 30 C. Finally, they were
peeled off, washed with bistilled water until free from acetate, and
stored in a desiccator until the tests were done.
2.5. ATR-FTIR
FTIR spectra were recorded at room temperature in the mid IR
range (400e4000 cm1) on a Bruker Vertex 80V FTIR spectrometer
equipped with a Bruker Platinum ATR accessory with a single
reflection diamond crystal.
Each spectrum was averaged over 128 scans with a resolution of
2 cm1. A background scan was recorded prior to the measurement
and subtracted from the sample spectra. The ATR correction to each
spectrum was applied using the OPUS software (Bruker optics). The
spectra were normalized to the same area and compared to each
other. Measurements were performed on powder, dry films and
hydrated films. In this latter case, the films were placed on the ATR
diamond and exposed to the dynamic vacuum of a diffusion pump
2.3. X-ray diffraction (XRD)
The diffractograms of the samples were recorded at room
temperature using a D8-ADVANCE Bruker diffractometer in BraggBrentano geometry using the Cu line l ¼ 1.542 Å. Each scan covered
2q from 2 to 12 with a step size of 0.02 , 1 s per step.
Fig. 1. A 3-D frequency-time-intensity plot for the dehydration process of a chitosan
film.
616
C. Branca et al. / Polymer 99 (2016) 614e622
at room temperature for fixed times to monitor the dehydrationinduced conformational changes. Spectra were recorded every
30 s initially and then every 2 min to 1 h. As an example, in Fig. 1 a
3D plot for the dehydration process of a chitosan film is reported.
In order to separate the OH and NH vibrational groups overlapping in the region between 3000 and 3800 cm1, a deconvolution of this spectral region into Gaussian components was
performed. According to the usual procedure, we started with two
components and then increased the number until the deconvolution was significantly improved. We used a curve-fitting procedure
using Peakfit software with all fit parameters having a 95% confidence interval. To avoid any ambiguity as to the number of components, we monitored both c2 and the residual sum of squares
(RSS) and selected the number of Gaussian components that
minimized both the parameters. No constraint was imposed on the
fitting parameters. To avoid any distortion of the band shape
resulting from an incorrect background subtraction, the whole region down to 1800 cm1 was deconvolved. It was observed that for
all the recorded spectra the same number of components were
required to satisfactorily reproduce the spectral profiles. After the
deconvolution was completed, the Gaussian components below
3000 cm1 were subtracted from each spectrum. Such a procedure
evidenced the presence of six components in the region between
3000 and 3800 cm1. This result is fully consistent with the previsions about the presumed minimum number of components
based on literature data according to which at least four components are expected (two for the primary amine group, one for the
secondary and at least one for the hydroxyl group).
3. Results and discussion
Fig. 2. (a) Equilibrium swelling degree of chitosan film at different pH values; b)
Equilibrium swelling degree of chitosan film and its composites at pH 6.
overlap in the same spectral region and, as a consequence, a clear
assignment of these modes is not trivial. The strategy followed in
this work to overcome this problem involved the use of a curve
fitting procedure and the analysis of chitosan under different forms.
To start with, the ATR-FTIR spectrum of the dry “as prepared”
chitosan film was compared to that of the chitosan powder. As one
can see from Fig. 3, both spectra present three main spectral regions: i) a broad asymmetric band between 3400 and 2500 cm1
encompassing the CH stretching modes at around 2927 and
2873 cm1 and, at higher wavenumbers, the overlapped OeH and
NeH stretching vibrations; ii) a region between 1700 and
1200 cm1 characteristic of the amide groups; iii) a strong absorption region between 800 and 1200 cm1 characteristic of the
(a)
(b)
C-O-C
Transmittance (a.u.)
Transmittance (a.u.)
As a preliminary phase, we investigated the swelling properties
of the prepared samples. They all swelled very rapidly and reached
equilibrium within half an hour. Confirming data from literature,
the chitosan films swelled to a larger degree at the lowest pH
values, see Fig. 2(a).
The increased swelling in acidic condition is generally ascribed
to the presence of more protonated amino groups that induce
electrostatic repulsions between the polymer segments. As pH increases, amino groups become deprotonated and repulsions recede
[25,26]. In the following, the relationship between swellability and
pH will be examined more closely with respect to changes in the
vibrational properties of some specific functional groups.
For what concerns the polymer-clay composites, the presence of
the clay induces a reduction in the swelling property with respect
to the chitosan film at the same pH of 6, see Fig. 2(b). This is
reasonable since it was proved that the dispersion of nanofillers
into a polymer matrix introduces breakable physical crosslinks
based on noncovalent interactions [18,27e31]. Consequently, a
denser structure with a reduced swelling capability has to be expected. However this effect is not linearly dependent on the clay
content; in fact, as shown in Fig. 2(b), the EDS (%) reached a minimum for a clay concentration of 2.5% and then increased by further
increasing the clay content. This result suggested that, depending
on the number of amino and hydroxyl groups involved in the
chitosan-clay interaction, peculiar nanostructures with different
hydrogen bonding network were likely to be formed.
In this frame, ATR-FTIR spectroscopy can give valuable information since it is particularly sensitive to the conformational
changes and reorganization of intra and intermolecular hydrogen
bonds involving the active groups of the compounds. In the present
work, we analyzed first the absorption spectra of chitosan and then
those of the chitosan/clay composites, focusing the attention on the
OH and NH stretching vibrations. This kind of study is rather
complex and challenging since the amino and hydroxyl groups
1555 1590
(a)
(b)
C-H
1400 1500 1600 -11700
Wavenumber (cm )
N-H, O-H
500 1000 1500 2000 2500 3000 3500 4000
-1
Wavenumber (cm )
Fig. 3. FTIR-ATR spectra of chitosan powder (a) and dry “as prepared” chitosan film (b).
The inset shows a magnification of the amide region.
C. Branca et al. / Polymer 99 (2016) 614e622
chitosan saccharide structure. Peaks at similar locations were
observed by several groups working on chitosan and its derivatives
[15,25,30e37]. The main differences between the film and the
powder spectra are visible in the amide region, see inset of Fig. 3,
where for the chitosan powder the characteristic peaks of amide I,
amide II and amide III are located at 1650 cm1, 1590 cm1 and
1317 cm1, respectively [32,33]. The peaks at 1375 cm1 and
1420 cm1 are assigned to the CH3 symmetrical deformation mode
[38,39]. For the “as prepared” film, the effects of protonation of the
amine functionalities in the acidic medium are evident. In fact,
protonation gives rise to two peaks both attributed to NHþ3 groups,
namely the antisymmetrical deformation that overlapped the
amide I band at 1646 cm1, and the symmetric deformation (dNH3)
at 1555 cm1 [13,36]. Significant differences in the spectral profiles
of the powder and the film appear also in the stretching region, see
Fig. 4(a) and (b). To better quantify these changes, a deconvolution
in Gaussian lineshapes, also displayed in the same figures, was
performed. In Table 1 the values of the central frequencies of the
Gaussian components together with the integrated areas are reported. Each of these components was numbered from A to F, as
reported in the table, and we will use this nomenclature in the
discussion that follows.
Notwithstanding previous studies, performed by using similar
approaches, often resulted in a controversial identification of the
spectral components in this stretching region, there is a general
agreement on the assignment of the high frequency side to the
vibrational stretching of the hydroxyl groups, and of the low frequency one to the amine stretching mode [40e44]. In order to
assign each of these components to a specific vibrational group, the
FTIR-ATR spectra of chitosan films at different hydration degree and
pH were also recorded and analyzed. In fact, it is reasonable to
expect that the OH and NH groups are involved to a different extent
in the protonation and hydration processes and that this is reflected
into some spectral changes.
For this reason, starting from an hydration level corresponding
to h z 1.1 (h ¼ g H2O/g dry chitosan) at pH 3 and 11, we monitored
the time evolution of the ATR-FTIR spectra along the dehydration
process by comparatively analyzing the changes exhibited in the
amide and stretching band.
In the following, for sake of clarity, only the spectra corresponding to three dehydration times are reported: tdehyd ¼ 0,
Absorbance (a.u.)
a)
b)
2800
3200
3600
-1
Wavenumber (cm )
Fig. 4. Spectral deconvolution for the chitosan powder (a) and “as prepared” dry film
(b) into Gaussian components. The experimental data are reported as squares; the best
fit is reported as a continuous line together with the single Gaussian contributions
(dashed lines).
617
Table 1
List of peak frequencies and integrated areas of the deconvoluted bands in the region
2800e3700 cm1 for the chitosan powder and the “as prepared” film.
Sample
Peak
Powder
A
B
C
D
E
F
3094
3223
3294
3358
3422
3550
Area%
Sample
Peak
19.0
26.5
11.0
5.4
31.7
6.4
As prepared film
A
B
C
D
E
F
Area%
3075
3225
3292
3355
3424
3564
26.4
25.9
6.3
4.8
31.2
5.4
hydrated sample, tdehyd ¼ 4 min, partially hydrated sample, and
tdehyd ¼ 24 h fully dried sample.
The ATR spectra reported in Figs. 5(a) and 6(a) show the dehydration evolution in the amide region for chitosan films at pH 11
and pH 3, respectively. For comparison, the spectrum of the dry “as
prepared” film is also reported as dashed line. The plots b, c and d of
Figs. 5 and 6 show the Gaussian deconvolution of the same spectra
in the high frequency region.
With reference to the amide region, what emerges at first sight
from these plots is that, whereas at pH 11 the effects of hydration
seem to be reversible, at pH 3 these effects modify permanently the
spectral profiles. This qualitative observation is confirmed by the
analysis of the frequency shifts observed for the two bands with
increasing dehydration degree at pH 3 and 11, see Fig. 7(a) and (b).
The frequency values corresponding to the dry “as prepared film”
are also reported as dashed lines.
As can be inferred from Fig. 7, at pH 11 (circle symbols), after a
slight frequency upshift determined by the initial hydration of the
film, both the bands moved back to the positions before the
swelling. On the contrary, at pH 3 (square symbols), the hydration
of the film induced a significant downshift in both peaks that
increased further in the first hour of dehydration and then levelled
off.
This behavior can be explained in terms of a protonation effect.
In fact, under acidic conditions, because of the excess of Hþ, we
expect a further protonation of both the amine and the carbonyl
groups at the oxygen with a consequent downshift of the corresponding bands. This is more evident as the dry state is approached
because of the antagonist “deprotonation” effect of water. However,
the larger downshift observed at pH 3 for the lower frequency
component, ~17 cm1, suggests an higher involvement of the amine
groups rather than the carbonyl ones in the hydration process. This
is in agreement with previous observations according to which the
higher swelling degree of chitosan film in acidic pH is attributed to
the presence of the ionic eNH3þ groups that cause the migration of
counter ions into the hydrogel favoring water uptake [26].
Starting from these observations, we have analyzed the effects
of the hydration/dehydration process on the peak positions and on
the fractional areas for the Gaussian components in which the
stretching region was deconvolved. Let's start with the component
labeled as E. Comparing Table 1 with Table 2, no change for this
component is observed between the powder and the film, whereas,
as a result of hydration, a significant downshift and an increase of
the percentage area is evident for any pH value.
These variations suggest that, in agreement with previous
studies [45e47], this component can be associated to the more
solvent exposed hydroxyl groups of chitosan that tend to form
hydrogen bonds with the water molecules rather than OHeOH or
NHeOH bonds within the polysaccharide itself.
Considering that the lower the pH, the higher the swelling and,
hence, the more the OH groups that are solvent exposed, it is not
surprising observing that the redshift for the E component is higher
under acidic conditions. Along the subsequent dehydration process,
tdehyd=0
tdehyd=24 h
1520
1560
1600
1640
-1
Absorbance (a.u.)
Wavenumber (cm )
1680
2800
3200
tdehyd=0
3200
3600
-1
Wavenumber (cm )
tdehyd=4 min
c)
b)
2800
Absorbance (a.u.)
a)
Absorbance (a.u.)
C. Branca et al. / Polymer 99 (2016) 614e622
Absorbance (a.u.)
618
3600
2800
-1
tdehyd=24 h
d)
Wavenumber (cm )
3200
3600
-1
Wavenumber (cm )
a)
tdehyd=0
tdehyd=24h
Absorbance (a.u.)
Absorbance (a.u.)
Fig. 5. FTIR-ATR spectra of chitosan film initially hydrated at pH 11 recorded for increasing times of dehydration. Plot a) shows the evolution of the spectra (solid lines) in the region
1500e1700 cm1 monitored at (from top to bottom) tdehyd ¼ 0, 4 min and 24 h. For comparison the spectrum of the “as prepared film” before swelling is also reported as dashed line.
Plots b), c) and d) show the Gaussian deconvolution of the same spectra in the region 2700e3800 cm1.
1480 1520 1560 1600 1640
1680
-1
2800
tdehyd=4 min
2800
3200
-1
Wavenumber (cm )
3600
Absorbance (a.u.)
Absorbance (a.u.)
Wavenumber (cm )
c)
b)
tdehyd=0
3200
tdehyd=24 h
d)
2800
3600
-1
Wavenumber (cm )
3200
3600
-1
Wavenumber (cm )
Fig. 6. FTIR-ATR spectra of chitosan film initially hydrated at pH 3 recorded for increasing times of dehydration. Plot a) shows the evolution of the spectra (solid lines) in the region
1500e1700 cm1 monitored at (from top to bottom) tdehyd ¼ 0, 4 min and 24 h. For comparison the spectrum of the “as prepared film” before swelling is also reported as dashed line.
Plots b), c) and d) show the Gaussian deconvolution of the same spectra in the region 2700e3800 cm1.
the position of this component shifts to higher frequencies suggesting the occurrence OH bond contraction related to the progressive decrease in the number of neighboring water molecules.
However, it is interesting observing that the extent of this bond
contraction is strongly dependent on pH; in fact, whereas for the
totally dehydrated sample at pH 3, the frequency of the E component is about 10 cm1 higher than that observed for the “as prepared film”, for the sample dehydrated at pH 11, it is practically the
same. Indeed, at pH 11, almost all the components are located at the
same frequencies of the “as prepared film”, thus confirming the
observation made for the amide region about the reversibility of
the hydration/dehydration process under basic conditions. Contrary, the differences observed in the dry state at pH 3 suggest the
occurrence of deep and permanent changes in the polymeric
network after swelling in acidic conditions. In particular, the hypothesized higher contraction of the OeH bond at pH 3, can be
1550
1540
1530
1300
tdehyd (min)
1400
-1
Peak frequency (cm )
0
b)
1648
1644
1640
1636
1632
1628
0
1300
tdehyd (min)
1400
Fig. 7. Peak frequency shifts for the deformation (a) and amide I (b) band as a function
of the dehydration time, tdehyd for chitosan films hydrated at pH 3 (squares) and pH 11
(circles). The values corresponding to the dry “as prepared film” are also reported as
dashed arrows for comparison.
Table 2
List of peak frequencies and integrated areas of bands in the region
2800e3700 cm1 for the chitosan film hydrated at pH 3 and pH 11 for different
dehydration times.
tdehydr ¼ 0 (h ¼ 1.1)
tdehydr ¼ 4 min
tdehydr ¼ 24 h (dry)
Peak (cm1)
Area (%)
Peak (cm1)
Area (%)
Peak (cm1)
Area (%)
25.9
27.8
4.1
1.2
39.2
1.8
3065
3217
3292
3354
3413
3578
28.0
27.4
4.0
2.1
37.1
1.4
3053
3222
3292
3355
3434
3581
39.2
25.2
3.1
4.2
27.1
1.2
24.0
26.1
4.8
2.7
37.1
5.3
3084
3222
3292
3356
3417
3567
23.9
26.2
4.9
3.8
36.0
5.2
3083
3223
3294
3354
3422
3568
24.2
26.2
6.3
5.1
31.8
6.4
pH3
A 3072
B 3215
C 3290
D 3354
E 3408
F
3577
pH11
A 3085
B 3222
C 3294
D 3356
E 3415
F
3568
ascribed to an excess of negative ions gathered around the NHþ
3
ions that, reducing the repellency among positive ions, favor the
folding of the polymer chains.
The behavior of the E component presents some analogies with
that observed for the B component. In particular, for the sample
hydrated at pH 3, a shift to a lower frequency and an increase of the
area for this component can be observed. Moreover, along the
dehydration, it moves again to higher frequencies tending to values
close to those measured for the “as prepared” chitosan film. This
similarity suggests the idea that also this component could be
assigned to stretching vibrations involving hydroxyl groups of
chitosan. However, the fact that at pH 11, both the hydration and
dehydration process does not induce any significant change in the
spectral characteristics of this component, suggests that the hydroxyl groups involved in this stretching vibration are those deeply
buried inside the polymer coil that are accessible to solvent only
after a strong swelling of the polysaccharide.
For what concerns the A component, it resulted to be the most
sensitive to pH changes. In particular, after the powder was treated
619
in an acidic solution to prepare the film, a strong downshift, of
about 20 cm1, and a decrease in the integrated area was observed
for this component, see Table 1. The subsequent hydration of the “as
prepared film” caused slight changes at pH 3 and a marked frequency Given these findings, we hypothesized to assign this
component to the stretching vibration of primary amine groups,
the most sensitive to pH changes, and to relate its frequency shifts
to the higher or lower upshift, with a reduction in the integrated
area, at pH 11, see Table 2. Along the dehydration process at pH 3, a
new strong peak downshift, associated with a consistent increment
in the integrated area, is observed for this component. Contrary, at
pH 11 the dehydration seems to have no effect.
Degree of protonation. More precisely, the positioning of this
component is the result of two competitive processes; the deprotonation from the water molecules, that is the dominant effect for
the most hydrated samples, especially for those under basic conditions, and the protonation of the amine groups at pH 3 that
obviously predominates in the dry state.
The F component deserves a particular discussion. Differently
from all the others components associated to functional groups of
chitosan, in our opinion it has to be attributed to water molecules
absorbed in the inner polymer and tightly bound to chitosan. The
presence of some residual water is widely reported in literature
[45e47]. The behavior observed for this component along the
initial film preparation and the following hydration/dehydration
process, is fully consistent with the hypothesis of a progressive
hydrogen bond weakening that involves the water molecules. The
fact that this effect results more marked under acidic conditions,
that is when the chitosan segments are loosened out, and that this
component is located, for the dried sample at pH 3, at the same
frequency observed for liquid water supports our hypothesis.
Finally, for what concerns the components C and D, whereas
their peak frequencies are practically the same at any experimental
condition, some changes are observed for the corresponding integrated areas. In particular, in the “as prepared” film, a strong
decrease in the area of the component C is observed in comparison
to the powder. Taking in mind that the strongest variation observed
along the film preparation involved the A component, we hypothesize that it could be assigned to primary amine groups not
involved in protonation. Finally, even if the recorded observations
are not sufficient to give a clear indication, we can assign the D
component to the secondary amine group by exclusion.
Given these findings, we proceeded, following the same criteria,
to analyze the spectra for the chitosan/clay nanocomposites.
In Fig. 8 the ATR spectrum of the “as prepared” chitosan film is
(a)
(b)
(c)
(d)
Transmittance (a.u.)
a)
1560
Transmittance (a.u.)
-1
Peak frequency (cm )
C. Branca et al. / Polymer 99 (2016) 614e622
1548 1555
1500
1600
(b)
(c)
(d)
1700
-1
Wavenumber (cm )
500
1000 1500 2000 2500 3000 3500 4000
-1
Wavenumber (cm )
Fig. 8. ATR-FTIR spectra of MMT (a), CSM5 (b), CSM2.5 (c) and “as prepared” (d) film.
An enlargement of the amide region for the chitosan film and its nanocomposites is
also reported.
C. Branca et al. / Polymer 99 (2016) 614e622
compared to the spectra of the polymer/clay composites at
different clay content prepared under the same conditions. The
spectrum of MMT is also reported. This latter is characterized by
typical bands responsible for the HeOeH stretching, 3426 cm1,
and the bending mode, 1636 cm1, of the adsorbed water. The peak
at 3626 cm1 is typical for AleAleOH stretching vibrations whereas
the sharp peak at 1038 cm1 is assigned to SieO stretching vibrations. Finally, the peak at 795 cm1 corresponds to quartz symmetrical stretching [13,17,48,49].
Because a relatively tiny amount of MMT was added, the ATRFTIR spectra of the chitosan/MMT composites present all the
vibrational peaks typical of chitosan. In the range between 950 and
1200 cm1 the characteristic bands of chitosan and MMT overlap,
giving rise to more intense bands for both the nanocomposite
samples. Major differences in peak position and intensity were
detected in the spectral regions between 1200e1750 and
3000e3700 cm1, and for this reason they were analyzed in more
details.
As shown in the inset of Fig. 8, for the film containing 5% MMT,
the frequency of the deformation vibration (dNH3), located at
1555 cm1 in the “as prepared film”, shifts towards a lower frequency, 1548 cm1. According to previous studies [13,15,30,48], this
shift is a signature of the intercalation of the biopolymer into the
silicate galleries, whose extent depends on the clay content. The
intercalation is favored, especially under acidic conditions, by the
electrostatic interactions between the negative charges on the clay
surface and the positive charges of chitosan.
In order to verify this hypothesis, the chitosan-clay films were
analyzed by XRD and the results are shown in Fig. 9. The XRD
patterns of MMT and of the “as prepared” chitosan film were also
recorded as reference. MMT presents a diffraction peak at
2q z 8.8 , corresponding to a d001 spacing of about 10 Å. In
agreement with previous investigations [12,17], the basal plane of
MMT disappears in the XRD pattern of the nanocomposite film at
the lowest clay content (2.5%), indicating the presence of a disordered structure. At a higher clay content, 5%, a broaden shoulder
was detected in the 2q range between 3.3 and 5.6 . This was much
lower than that of pristine MMT, suggesting that, consistently with
the infrared findings, intercalation occurred together with some
exfoliation [12,17,29].
It's reasonable expecting that the different nanostructures
modify the network of electrostatic interactions between chitosan
and MMT. Consequently, some changes on both the OH and NH
stretching modes are likely to happen. For this reason, following the
same criteria used for the chitosan films, a deconvolution of the
absorption spectra for the nanocomposite films was performed
between 2800 and 3700 cm1, see Fig. 10. The peak frequencies and
the corresponding areas resulting from deconvolution are presented in Table 3.
Intensity (a.u.)
a)
b)
Intercalated
nanocomposites
c)
d)
3
4
5
6
7
2θ (degree)
8
9
Exfoliated
nanocomposites
Fig. 9. XRD patterns of (a) MMT, (b) CSM5, (c) CSM2.5, and (d) chitosan film; a
schematic drawing of the different nanostructures formed is also reported.
a)
Absorbance (a.u.)
620
b)
2800
3200
3600
-1
Wavenumber (cm )
Fig. 10. Gaussian deconvolution of the ATR-FTIR spectra for the CSM2.5 (plot a) and the
CSM5 (plot b) nanocomposite film.
Table 3
List of peak frequencies and integrated areas of bands in the region
2800e3700 cm1 for the “as prepared” chitosan film, and the chitosan/clay films
with 2.5% and 5% of MMT.
A
B
C
D
E
F
As prepared CS film
CSM2.5
Peak (cm1)
Area (%)
Peak (cm1)
Area (%)
Peak (cm1)
Area (%)
3075
3225
3292
3355
3424
3564
26.4
25.9
6.3
4.8
31.2
5.4
3074
3223
3292
3354
3424
3562
27.1
27.2
5.1
5.0
30.1
5.5
3070
3228
3292
3352
3430
3568
29.7
25.8
6.6
6.1
28.8
3.0
CSM5
As for the chitosan, the deconvolution, performed leaving all the
fitting parameters free, evidenced the presence of six Gaussian
components that we labeled using the same nomenclature as
before. For what concerns the peak frequencies, it can be observed
that whereas the components B, E and F move to higher frequencies, the component A shifts to a lower one; moreover, also for
the nanocomposites, the components C and D result unchanged
with respect to the “as prepared” chitosan film. Consistently with
the observed frequency shifts, the incorporation of the clay determined a decrease of the integrated area for the components B, E and
F and in an increase for the A component.
Taking into mind that the component A was attributed to protonated amine groups, the observed downshift is fully consistent
with the formation of hydrogen bonds between the NHþ
3 groups of
chitosan and the negatively charged sites on the clay surface. Since,
as above reported, the swelling properties of the chitosan film are
ruled by to the presence and the mobility of protonated amine
groups, the involvement of these groups in hydrogen bonding with
the clay implies a reduction, in comparison to neat chitosan, in the
water sorption capability, as confirmed by our observations, see
Fig. 2. This behavior is in agreement with that reported by Abdollahi et al. [17] according to which the nanocomposites exhibit a
lower swelling capability. Moreover, they observed a minimum in
the water sorption for the nanocomposite containing 3% MMT; also
this result agrees very well with our experimental observations, see
Fig. 2.
This result is quite surprising since, if the reduced water sorption were just up to the reduced number of protonated amine
groups, we should expect a higher swelling at the lowest clay
C. Branca et al. / Polymer 99 (2016) 614e622
concentration, that is when the number of protonated amine
groups not involved in hydrogen bonding is higher. This contradiction can be solved if we take into account the different network
structures formed depending on the clay content. When intercalation occurs, the chitosan chains are forced to stretch along the
clay galleries; this arrangement increases the surface of interaction
favoring the electrostatic interactions between the NHþ
3 groups and
the clay platelet. At the same time, the elongation of the chain tends
to weaken and, eventually, to brake the bonds between the OH
groups of chitosan. This hypothesis is consistent with the FTIR results showing a blueshift, and a concomitant area decrease, for the
components B, and E both assigned to the hydroxyl groups of chitosan. These groups are thus available to bind with the water
molecules entering into the clay galleries.
Contrary, in presence of a complete exfoliation or homogeneous
dispersion of the clay platelets, the OH groups of chitosan are likely
involved in both intra and intermolecular bonds between adjacent
chitosan molecules. In such a configuration, the chains form of a
highly crosslinked network for which a reduced swelling is
expected.
4. Conclusions
We have presented a systematic investigation of the vibrational
spectra of chitosan under different forms and solvent conditions. By
applying a deconvolution analysis of the ATR-FTIR spectra in the
region 3000e3800 cm1, the OH and NH stretching vibrational
modes were differentiate and identified.
The assignment was based on the analysis of the changes in
peak position and integrated areas of the Gaussian components
upon variation of the hydration and pH conditions of the chitosan
film.
Starting from this assignment, the vibrational spectra of chitosan/montmorillonite nanocomposites at different clay content
were analyzed. The clay concentrations were accurately chosen in
order to realize, as evidenced by XRD data, exfoliated and intercalated nanostructures.
The observed changes in the spectral features of the OH and NH
stretching components revealed a strong influence of the architecture of the nanocomposites on the network of electrostatic interactions between chitosan and MMT. A direct consequence is that
the water sorption capability for the nanocomposites, cannot be
exclusively ascribed to the presence and mobility of the protonated
amino groups.
It was found that at the highest clay content investigated, the
intercalation of chitosan inside the clay galleries, reduces the intra
and intermolecular hydrogen bonding involving the hydroxyl
groups of the chitosan chains; the OH groups are thus available to
make hydrogen bonds to water molecules entering inside the
gallery.
Conversely, the minimum in the swelling properties, observed
for the nanocomposite with 2.5% MMT, is due to the existence of a
denser hydrogen bonded structure whose formation is favored by
the homogenous dispersion of the clay platelets within the
network of entangled chitosan chains.
The present study clarifies the main interaction mechanisms in
chitosan/clay composites and highlights the importance of a
controlled morphology synthesis for the design of highperformance bio-nanocomposites.
References
[1] R.A.A. Muzzarelli, Chitosan in Natural Chelating Polymers, Pergamon Press,
Oxford, 1973.
[2] R.A.A. Muzzarelli, C. Muzzarelli, Chitin and chitosan hydrogels, in:
621
G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, second ed.,
Woodhead Publishing Ltd., Cambridge, 2008, pp. 849e888.
[3] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci.
31 (2006) 603e632.
[4] M.N.V. Ravi Kumar, R.A.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A. Domb,
Chitosan chemistry and pharmaceutical perspectives, Chem. Rev. 104 (2004)
6017e6084.
[5] M.N.V. Ravi Kumar, A review of chitin and chitosan applications, React. Funct.
Polym. 46 (2000) 1e27.
[6] A. Di Martino, M. Sittinger, M.V. Risbud, Chitosan: a versatile biopolymer for
orthopaedic tissue-engineering, Biomaterials 26 (2005) 5983e5990.
ro
^me, Chitosan-based biomaterials for tissue engineering, Eur.
[7] F. Croisier, C. Je
Polym. J. 49 (2013) 780e792.
[8] M. Davidovich-Pinhas, Y. Danin-Poleg, Y. Kashi, H. Bianco-Peled, Modified
chitosan: a step toward improving the properties of antibacterial food packages, Food Packag. Shelf Life 1 (2014) 160e169.
[9] R. Hejazi, M. Amiji, Chitosan-based gastrointestinal delivery systems,
J. Control. Release 89 (2003) 151e165.
dio, M.T. Cerqueira, A.P. Marques, R.L. Reis, J.F. Mano, Cell selective
[10] C.A. Custo
chitosan microparticles as injectable cell carriers for tissue regeneration,
Biomaterials 43 (2015) 23e31.
[11] Y. Xiao, T. Gong, Y. Jiang, Y.P. Wang, Z.Z.T. Wen, S.B. Zhou, C.Y. Bao, X.M. Xu,
Fabrication and characterization of a glucose-sensitive antibacterial chitosanpolyethylene oxide hydrogel, Polymer 82 (2016) 1e10.
[12] Y. Xu, X. Ren, M.A. Hanna, Chitosan/clay nanocomposite film preparation and
characterization, J. Appl. Polym. Sci. 99 (2006) 1684e1691.
[13] M. Darder, M. Colilla, E. Ruiz-Hitzky, BiopolymerClay nanocomposites based
on chitosan intercalated in montmorillonite, Chem. Mat. 15 (2003)
3774e3780.
€ r, Synthesis and charac[14] E. Günister, D. Pestreli, C.H. Ünlü, O. Atici, N. Güngo
terization of chitosan-MMT biocomposite systems, Carbohyd. Polym. 67
(2007) 358e365.
[15] M. Lavorgna, F. Piscitelli, P. Mangiacapra, G.G. Buonocore, Study of the combined effect of both clay and glycerol plasticizer on the properties of chitosan
films, Carbohyd. Polym. 82 (2010) 291e298.
[16] J.W. Rhim, S.I. Hong, H.M. Park, P.K.W. Ng, Preparation and characterization of
chitosan-based nanocomposite films with antimicrobial activity, J. Agr. Food
Chem. 54 (2006) 5814e5822.
[17] M. Abdollahi, M. Rezaei, G. Farzi, A novel active bionanocomposite film
incorporating rosemary essential oil and nanoclay into chitosan, J. Food Eng.
111 (2012) 343e350.
[18] C. Branca, C. Crupi, G. D'Angelo, K. Khouzami, S. Rifici, A. Visco,
U. Wanderlingh, Effect of montmorillonite on the rheological properties of
dually crosslinked guar gum-based hydrogels, J. Appl. Polym. Sci. 132 (2014)
41373, http://dx.doi.org/10.1002/app.41373.
[19] B.R. dos Santos, F.B. Bacalhau, T.D. Pereira, C.F. Souza, R. Faez, Chitosanmontmorillonite microspheres: a sustainable fertilizer delivery system, Carbohyd. Polym. 127 (2015) 340e346.
[20] A.C.S. Alcantara, M. Darder, P. Aranda, A. Ayral, E. Ruiz-Hitzky, Bionanocomposites based on polysaccharides and fibrous clays for packaging
applications, J. Appl. Polym. Sci. 133 (2016) 42362.
[21] S.B. Ghelejlu, M. Esmaiili, H. Almasi, Characterization of chitosan-nanoclay
bionanocomposite active films containing milk thistle extract, Int. J. Biol.
Macromol. 86 (2016) 613e621.
[22] H. Uyama, M. Kuwabara, T. Tsujimoto, M. Nakano, A. Usuki, S. Kobayashi,
Green nanocomposites from renewable resources: plant OilClay hybrid
materials, Chem. Mat. 15 (2003) 2492e2494.
[23] Q. Wang, J.L. Mynar, M. Yoshida, E. Lee, M. Lee, K. Okuro, K. Kinbara, T. Aida,
High-water-content mouldable hydrogels by mixing clay and a dendritic
molecular binder, Nature 463 (2010) 339e343.
[24] M. Alexandre, P. Dubois, Polymer-layered silicate nano-composites: preparation, properties and uses of a new class of materials, Mat. Sci. Eng. 28 (2000)
1e63.
[25] K.M. Kim, J.H. Son, S.-K. Kim, C. Weller, M.A. Hanna, Properties of chitosan
films as a function of pH and solvent type, J. Food Sci. 71 (2006) E119eE124.
[26] T. Wang, M. Turhan, S. Gunasekaran, Selected properties of pH-sensitive,
biodegradable chitosanepoly(vinyl alcohol) hydrogel, Polym. Int. 53 (2004)
911e918.
[27] K. Haraguchi, T. Takehisa, Nanocomposite hydrogels: a unique organiceinorganic network structure with extraordinary mechanical, optical, and
swelling/deswelling properties, Adv. Mat. 14 (2002) 1120e1124.
[28] W.C. Lin, W. Fan, A. Marcellan, D. Hourdet, C. Creton, Large strain and fracture
properties of poly(dimethylacrylamide)/silica hybrid hydrogels, Macromolecules 43 (2010) 2554e2563.
[29] X. Wang, Y. Du, J. Yang, X. Wang, X. Shi, Y. Hu, Preparation, characterization
and antimicrobial activity of chitosan/layered silicate nanocomposites, Polymer 47 (2006) 6738e6744.
[30] S.F. Wang, L. Shen, Y.J. Tong, L. Chen, I.Y. Phang, P.Q. Lim, T.X. Liu, Biopolymer
chitosan/montmorillonite nanocomposites: preparation and characterization,
Polym. Degrad. Stabil. 90 (2005) 123e131.
[31] C. Paluszkiewicz, E. Stodolak, M. Hasik, M. Blazewicz, FT-IR study of montmorilloniteechitosan nanocomposite materials, Spectrochim. Acta A 79
(2011) 784e788.
[32] F.G. Pearson, R.H. Marchessault, C.Y. Liang, Infrared spectra of crystalline
polysaccharides. V. Chitin, J. Polym. Sci. 43 (1960) 101e116.
622
C. Branca et al. / Polymer 99 (2016) 614e622
[33] N.P.G. Roeges, A Guide to the Complete Interpretation of Infrared Spectra of
Organic Structures, Wiley, Chichester, 1994.
n, A.-C. Albertsson, Novel pH-sensitive chitosan hydrogels:
[34] X. Qu, A. Wirse
swelling behavior and states of water, Polymer 41 (2000) 4589e4598.
[35] I.A. Fadzallah, S.R. Majid, M.A. Careem, A.K. Arof, A study on ionic interactions
in chitosaneoxalic acid polymer electrolyte membranes, J. Membr. Sci. 463
(2014) 65e72.
[36] I.F. Amaral, P.L. Granja, M.A. Barbosa, Chemical modification of chitosan by
phosphorylation: an XPS, FT-IR and SEM study, J. Biomat. Sci.-Polym. E. 16
(2005) 1575e1593.
[37] T.T.M. Ho, K.E. Bremmell, M. Krasowska, S.V. MacWilliams, C.J.E. Richard,
D.N. Stringer, D.A. Beattie, In situ ATR FTIR spectroscopic study of the formation and hydration of a fucoidan/chitosan polyelectrolyte multilayer,
Langmuir 31 (2015) 11249e11259.
[38] T. Sannan, K. Kurita, Y. Iwakura, Studies on chitin, 2. Effect of deacetylation on
solubility, Makromol. Chem. 177 (1976) 3598e3600.
[39] T. Wang, M. Turhan, S. Gunasekaran, Selected properties of pH-sensitive,
biodegradable chitosanepoly(vinyl alcohol) hydrogel, Polym. Int. 53 (2004)
911e918.
[40] A.T. Paulino, J.I. Simionato, J.C. Garcia, J. Nozaki, Characterization of chitosan
and chitin produced from silkworm crysalides, Carbohyd. Polym. 64 (2006)
98e103.
[41] M.R. Kasaai, A review of several reported procedures to determine the degree
of N-acetylation for chitin and chitosan using infrared spectroscopy, Carbohyd. Polym. 71 (2008) 497e508.
[42] K.C. Gupta, F.H. Jabrail, Effects of degree of deacetylation and cross-linking on
physical characteristics, swelling and release behavior of chitosan microspheres, Carbohyd. Polym. 66 (2006) 43e54.
[43] K.V.H. Prashanth, F.S. Kittur, R.N. Tharanathan, Solid state structure of chitosan prepared under different N-decetylating conditions, Carbohyd. Polym. 50
(2002) 27e33.
~o, J. Rocha, An optimized method to
[44] M.L. Duarte, M.C. Ferreira, M.R. Marva
determine the degree of acetylation of chitin and chitosan by FTIR spectroscopy, Int. J. Biol. Macromol. 31 (2002) 1e8.
[45] A.G. Rusu, M.I. Popa, G. Lisa, L. Vereştiuc, Thermal behavior of hydrophobically
modified hydrogels using TGA/FTIR/MS analysis technique, Thermochim. Acta
613 (2015) 28e40.
[46] V. Georgieva, D. Zvezdova, L. Vlaev, Non-isothermal kinetics of thermal
degradation of chitosan, Chem. Cent. J. 6 (2012) 81e90.
[47] S.-K. Liao, C.-C. Hung, M.-F. Lin, A kinetic study of thermal degradations of
chitosan/polycaprolactam blends, Macromol. Res. 12 (2004) 466e473.
[48] J. Madejova, P. Komadel, Baseline studies of the clay minerals society source
clays: infrared methods, Clay Clay Min. 49 (2001) 410e432.
[49] P. Monvisade, P. Siriphannon, Chitosan intercalated montmorillonite: preparation characterization and cationic dye adsorption, Appl. Clay Sci. 42 (2009)
427e431.