Materials Sciences and Applications, 2011, 2, 346-354
doi:10.4236/msa.2011.25045 Published Online May 2011 (http://www.SciRP.org/journal/msa)
Crosslinking as an Efficient Tool for Decreasing
Moisture Sensitivity of Biobased Nanocomposite
Films
Jari Vartiainen, Ali Harlin
VTT Technical Research Centre of Finland, Espoo, Finland.
Received March 17th, 2011; revised March 25th, 2011; accepted March 29th, 2011.
ABSTRACT
Chitosan-nanoclay bio-hybrid films were successfully crosslinked with glutaraldehyde, genipin and glyoxal. Moisture
sensitivity of films decreased as a result of crosslinking which led to improved barrier properties against water vapor
and oxygen. Films containing chitosan (6.6 g/m2) with genipin (3.3 g/m2) and nanoclay (6.6 g/m2) had water vapor
transmission rate of 72 g × 100 µm/(m2 × 24 h) which was 34% lower as compared to pure chitosan and 30% lower as
compared to chitosan/nanoclay without crosslinkers. Glyoxal induced crosslinking resulted in 92% reduction in oxygen
transmission rate at 80% relative humidity as compared to pure chitosan films. Oxygen transmission through glyoxal
(3.3 g/m2) treated chitosan/nanoclay film was 2.8 cm3 × 100 µm/( m2 × 24 h) which was 53% lower as compared to chi-
tosan/nanoclay without crosslinkers. In addition, nanoclay and especially glyoxal crosslinking prevented the water va-
por sorption of chitosan considerably. Crosslinking may be used as an efficient tool for enhancing the exploitability of
naturally hydrophilic biopolymers towards new high-value applications, such as food packaging.
Keywords: Chitosan, Nanoclay, Crosslinking, Barrier, Packaging, Glutaraldehyde, Genipin, Glyoxal
1. Introduction
Green economy, also referred to as biobased economy,
utilizes biomass derived raw materials for high-volume
applications, such as packaging. Barrier properties are
extremely important for biobased food packaging mate-
rials as both gas and water vapor transmission through
packaging reduce the quality of food resulting shorter
shelf-lives, increased costs and eventually more waste.
Nanoclays (or nanolayered silicates) such as hectorite,
saponite and montmorillonite are promising additives
with high aspect ratio and surface area [1-3]. Due to their
unique platelet-like structure nanoclays have been widely
studied as regards the barrier properties. Chitosan is a
polysaccharide prepared by the N-deacetylation of chitin,
the second most abundant natural biopolymer after cel-
lulose. As chitosan is both hydrophilic and cationic in
acidic conditions, it has usually good miscibility with
negatively charged nanoclays. Chitosan chains may eas-
ily intercalate into the clay interlayer by means of catio-
nic exchange [4]. Chitosan/layered silicate nanocompo-
sites have been used to improve the barrier properties
against oxygen, water vapor, grease and UV-light trans-
mission as well as the mechanical, thermal and antimi-
crobial properties [5-9]. Studies on the barrier properties
of polymer/layered silicate nanocomposites have tradi-
tionally focused on systems with low organoclay con-
tents. Recently, it has been reported that nanoclays can
produce substantial improvements in barrier properties
over the full range of compositions even up to organoc-
lay content of 80 vol% withou t loosing th e flexibility an d
transparency [10]. Correspondingly, we have recently
demonstrated an 88% improvement in the oxygen barrier
properties of chitosan films in high humidity conditions
with 67 wt% of nanoclay. The main goal of this work
was to verify the effects of crosslinking on the barrier
properties of chitosan in high humidity. Glutaraldehyde,
genipin and glyoxal were selected as crosslinking agents
due to their well-recognised efficiency in cross- linking
chitosan [11-15].
2. Experimental
2.1. Materials
Chitosan was obtained from Fluka BioChemika (low-vis-

Crosslinking as an Efficient Tool for Decreasing Moisture Sensitivity of Biobased Nanocomposite Films 347
cous with a molecular weight of 150 kDa) and hydro-
philic bentonite nanoclay (Nanomer PGV) from Aldrich.
According to manufacturer, nanoclay was untreated (no
organic modification) hydrophilic clay (> 98% montmo-
rillonite) with aspect ratio of 150 - 200. Glutaraldehyde,
25% solution, was obtained from Merck-Schuchardt, ge-
nipin powder from Challenge Bioproducts and glyoxal,
40% solution, from Sigma-Aldrich.
2.2. Preparation of Nanocomposite Films
1% nanoclay was swelled in 50 mL of distilled water and
dispersed using ultrasonification tip (Branson Digital
Sonifier) for 10 min. The dispersion was added into 50
mL of 1% chitosan in 1% acetic acid, followed by soni-
cation for 10 min. Finally, 0.25% or 0.025% crosslinker
(glutaraldeh yde, g enip in or glyox al) w as d isso lv ed in 100
mL of ethanol and added under rigorous mixing. 15 mL
of each solution was cast onto polystyrene Petri dish (Ø
8.5 cm) and dried at room temperature. The obtained
films were peeled off from the Petri dishes and stored at
room temperature and 50% relative hu midity before tests.
The composition of the films can be found from Table 1.
2.3. Viscosity
Viscosity increment of chitosan solutions after adding
crosslinkers was measured using Brookfield Model DVIII
Rheometer at 23˚C with spindel DIN87, model LV and
rotation speed of 25 rpm.
Table 1. Composition of films expressed as grammages of
each component in film.
Crosslinker Crosslinker/nanoclay/chitosan [g/m2]
- 0/0/6.6
- 0/6.6/6.6
Glutaraldehyde 3.3/6.6/6.6
Glutaraldehyde 0.3/6.6/6.6
Glutaraldehyde 3.3/0/6.6
Glutaraldehyde 0.3/0/6.6
Genipin 3.3/6.6/6.6
Genipin 0.3/6.6/6.6
Genipin 3.3/0/6.6
Genipin 0.3/0/6.6
Glyoxal 3.3/6.6/6.6
Glyoxal 0.3/6.6/6.6
Glyoxal 3.3/0/6.6
Glyoxal 0.3/0/6.6
2.4. Scanning Electron Microscopy (SEM)
Structures of pure nanoclay in sonicated dispersions were
analyzed using scanning electron microscopy (SEM,
LEO DSM 982 Gemini FEG-SEM). SEM samples of
aqueous dispersions of pure nanoclay were prepared by
spreading dispersions on a polyvinyl amine premodified
silica surface using fast spinning (2800 rpm for 1 min).
Typically, no conductive coating was applied on the spe-
cimen prior SEM imaging. However, in some cases a
thin layer (~10 nm) of platinum was sputter coated on the
surface to improve conductivity and stability of the spe-
cimen. The SEM analyses of the aqueous dispersions
were conducted using electron energies of 1.0 kV and 2.0
kV [16].
2.5. Color
Hunter a- and b-values were measured using a colorime-
ter (CR200 Minolta Chroma Meter, Minolta Camera Co.,
Osaka, Japan). The values indicate the color directions:
+a (magenta), –a (green), +b (yellow) and –b (blue).
Color values were determined randomly at three different
positions on each film.
2.6. X-Ray Diffraction (XRD)
X-ray diffraction was used to determine the interlayer
distance of layered nanoclays and crosslinked chitosan
nanocomposites. Interlayer distances were calculated by
the Bragg’s equation: 2dsinθ = λ, where d is the interlay-
er distance, 2θ is the diffraction angle and λ is the wave-
length of the X-ray (λ = 1.542 Å). X-ray diffractograms
were run from the samples using Philips X’Pert MPD
diffractometer, powder method and Cu X-ray tube.
2.7. Water Contact Angle
Water contact angle of the film surface was measured
using CAM200 equipment (KSV Instruments, Finland)
in test conditions of 23˚C and 50% relative humidity af-
ter incubation for 2 s.
2.8. Water Vapor Transmission
Water vapor transmission rates of the films were deter-
mined gravimetrically using a modified ASTME-96 pro-
cedure. Samples with a test area of 25 cm2 were mounted
on a circular aluminium dish (H.A. Buchel V/H, A.v.d.
Korput, Baarn-Holland 45 M-141), which contained wa-
ter. Dishes were stored in test conditions of 23˚C and
50% relative humidity and weighed periodically until a
constant rate of weight reduction was attained .
2.9. Oxygen Transmission
Oxygen transmission measurements were performed with
Oxygen Permeation Analyser Model 8001 (Systech In-

Crosslinking as an Efficient Tool for Decreasing Moisture Sensitivity of Biobased Nanocomposite Films 348
struments Ltd. UK). The tests were carried out at 23˚C
and 80% relative humidity.
2.10. Water Vapor Sorption
Water vapor sorption isotherms were measured at 20˚C
with a dynamic water vapor sorption device (DVS-1,
Surface Measurement Systems, UK). The device con-
tained a microbalance and a humidity-regulated sample
module within a temperature controlled chamber. Hu-
midity was controlled by mixing dry and saturated (with
water vapor) nitrogen gases, which flowed through sam-
ple and reference (with an empty pan) cells. The weight
of the sample was recorded once a minute until the equi-
librium was reached.
3. Results and Discussion
Typically, the viscosity of the solutions increases as a
function of crosslinking and molecular weight. The vis-
cosity measurements indicated that after adding the cros-
slinkers the chitosan solutions finally converted into a
crosslinked gel [17]. Reaction between glutaraldehyde
and amino groups of chitosan took place immediately
whereas the crosslinking induced by glyoxal and espe-
cially genipin proceeded considerably slower. In each
case the final product formed gels with viscosity over the
range of measurement (Table 2).
Nanoclay was delivered as dry powder with particle
size < 25 µm. According to manufacturer, the nanoclay
was composed of high purity aluminosilicate minerals,
intended for use as additive to hydrophilic po lymers such
as polyvinylalcohols, polysaccharides and polyacrylic
acids. When fully dispersed, the nanoclay was supposed
to form nanocomposites with the host polymers. Hydro-
philic nanoclay was dispersed using ultrasonic energy in
aqueous suspension. Chitosan dissolved in 1% acetic
acid was then added to the mixture for adsorption to the
separated nanoclay sheets. After adding crosslinkers, the
nanocomposite films were obtained upon drying. Thick-
ness of the films varied between 8 and 15 µm. Acidic pH
was necessary for the protonation of amino groups of
Table 2. Viscosity (mPas) of 2% chitosan in 1% acetic acid
with 1% crosslinker measured 1, 150 and 500 min after
adding the crosslinker.
Crosslinker 1 min 150 min 500 min
 180 194 184
glutaraldehyde >10 000
(gelling) >10 000 (gelling) >10 000 (gelling)
genipin 160 257
>10 000
(gelling)
glyoxal 165
>10 000
(gelling) >10 000 (gelling)
chitosan. Adsorption process was mainly controlled by a
cationic exchange mechanism due to the coulombic inte-
ractions between the positive amino groups of chitosan
and the negative sites in the clay structure. Since chitosan
contains amino and hydroxyl groups, it can form strong
intermolecular hydrogen bonds with the silanol edges of
the nanoclays, which leads to the strong affinity b etween
the matrix and silicate layers [4].
As can be seen in Figure 1(a), dry nanoclay powder
consisted of round particles with coarse and platelety
surface. Diameters of nanoclay particles varied between
3 and 25 µm. By ultrasonic dispersing the nanoclay
platelets were effectively ripped off and uniformly dis-
tributed on the surface. Previous studies have demon-
strated that nanoclay platelets could easily orient parallel
to the surface of especially solution cast coatings [18,19].
The diameter of the intercalated nanoplatelets was < 400
nm (Figure 1( b)).
Chitosan dissolved in acetic acid formed completely
(a)
(b)
Figure 1. SEM images of (a) Typical nanoclay aggregates
prior dispergation and (b) Spincoated nanoclay platelets
after ultrasonic dispergation.

Crosslinking as an Efficient Tool for Decreasing Moisture Sensitivity of Biobased Nanocomposite Films 349
transparent and colorless films. Also the crosslinker so lu-
tions were colorless prior the reaction with chitosan.
Nanoclay addition made films slightly opaque and yel-
lowish but still transparent. Glutaraldehyde crosslinking
led to an intensive yellow color which was proportional
to the amount of crosslinker [11]. Genipin is a raw ma-
terial for gardenia blue pigment preparation, thus the
reaction between genipin and amino groups of chitosan
produced dark green-blue films [12]. The color formation
was attributed to double bonds in the genipin crosslink-
ing molecules [14]. Glyoxal reacted with chitosan did not
produce any measurable or visible color changes as
compared to equal films without crosslinker or nanoclay.
However, glyoxal slightly improved the color of nanoc-
lay containing films (Figure 2).
Crosslinking typically reduces the so lub ility and in this
case the films containing crosslinkers were completely
insoluble in 1% acetic acid. Chitosan films without na-
noclay and crosslinkers dissolved in 3 min under con-
stant mixing whereas the other films were intact after 60
days of immersion. Interestingly, also nanoclay contain-
ing chitosan film without crosslinkers kept its shape for 2
months in 1% acetic acid. Probably the clay sheets pre-
vented the solvent diffusion and the coulombic interac-
tions between the positive amino groups of chitosan and
the negative sites in the clay bound the components
tightly together.
Intercalated structures are formed when extended chi-
tosan chains are delaminated between the nanoclay layers.
The result is a well-ordered multilayer structure of alter-
nating biopolymeric and inorganic layers with a repeat
interlayer distance (d-value) between them. The d-values
were measured by XRD using the Braggs’s equation. The
pure powdery nanoclay had an interlayer distance of 1.2
nm. By sonication, the d-values were increased to 1.6 nm.
These results are consistent with our earlier studies [8].
As previously established, the thick ness of the individual
sheet of chitosan chain is 0.38 nm [20,21] In this case,
the diffractograms support the intercalation of chitosan in
a monolayer configuration. Monolayer adsorption was
mainly controlled by a cationic exchange mechanism due
to the coulombic interactions between the positive amino
groups of chitosan and the negative sites in the clay
structure [4,22]. Crosslinking did not have any notable
effects on delam i nat i on (Figure 3).
Surface wettability of chitosan was improved with na-
noclay. This is consisten t with our earlier findings where
water contact angles of chitosan-nanoclay films decrea-
sed as a function of nanoclay content [8]. Crosslinking
had positive effect on hydrophobicity and especially
Figure 2. Hunter a- and b-values indicate the color formation (green and yellow) of crosslinked films. Composition of films
was expressed as g/m 2 of each component in film (crosslinker/nanoclay/chitosan).

Crosslinking as an Efficient Tool for Decreasing Moisture Sensitivity of Biobased Nanocomposite Films 350
Figure 3. XRD-patterns of chitosan films containing nanoc-
lay and (a) 3.3 g/m2 glutaraldehyde, (b) 0.3 g/m2 glutaral-
dehyde, (c) no crosslinker s, (d) 3.3 g/m2 genipin, (e ) 0.3 g/m2
genipin, (f) 3.3 g/m2 glyoxal, (g) 0.3 g/m2 glyoxal, (h) Pure
powdery nanoclay, (i) Chitosan film without nanoclay and
crosslinkers, and (j) Silicon base-line.
the higher amount of crosslinkers increased the water
contact angles (Figure 4). As water vapor transmission
results indicated, nanoclay improved the barrier proper-
ties of chitosan films. Crosslinking, however, did not
provide any protection against water penetration, except
when used in combination with nanoclay. Higher amount
of crosslinkers clearly improved the barrier properties of
nanoclay containing films. Chitosan films with genipin
(3.3 g/m2) and nanoclay had water vapor transmission
rate of 72 g × 100 µm/(m2 × 24 h) which was 34% lower
as compared to pure chitosan and 30% lower as com-
pared to chitosan/nanoclay without crosslinkers. As was
observed earlier, the genipin-crosslinked chitosan net-
works prepared in acidic conditions typically consist of
short chains of cyclic crosslinking bridges [15]. This type
of bulky heterocyclic structure may hinder the relaxation
and diffusion more than the network crosslinked struc-
ture of the glutaraldehyde-crosslinked polymer [23,24].
Hydrophilic chitosan, however, is lacking the full capa-
bility of preventing the diffusion of water molecules, thus
total barrier effects were lower as compared to more hy-
drophobic PLA nanocomposites, where nanoclay incor-
poration decreased the water vapor transmission by about
40% - 50% [25].
In general, crosslinking reduces crystallinity of poly-
mers by interfering the formation of crystals. The net-
work junctions can not crystallize, and they may even
prevent the neighboring chains from entering into the
crystal phase [26]. This should eventually lead to reduced
barrier properties due to increased portion of amorphous
regions. On the other hand, crosslinking also prevents the
humidity induced swelling of chitosan which limits the
diffusion increase of water molecules (Figure 5). Marked
barrier improvements were achieved with the aid of in-
tercalated nanoclays. Due to intercalation, the effective
Figure 4. Water vapor transmission rates (columns) versus water contact angles (line) of glutaraldehyde, genipin and glyoxal
crosslinked chitosan-nanoclay films (measured at 23˚C, 50% RH). Composition of films was expressed as g/m2 of each com-
ponent in film (crosslinker/nanoclay/chitosan).

Crosslinking as an Efficient Tool for Decreasing Moisture Sensitivity of Biobased Nanocomposite Films 351
Figure 5. Effect of crosslinking: (a) Crystalline polymer, (b)
Swollen polymer, (c) Crosslinked polymer with reduced
crystallinity, (d) Swollen crosslinked polymer where cross-
links limit expansion.
path length for molecular diffusion increased and the
path became highly tortuous which decreased both oxy-
gen and moisture transmission through the film [27].
Chitosan and other biopolymers with crystalline struc-
ture and hydrogen bonds are typically very good oxygen
barriers, but only up to 50% relative humidity. In high
humidity conditions water molecules penetrate between
chitosan chains and destroy the hydrogen bonded struc-
ture and barrier properties. Nanoclay clearly improved
the oxygen barrier properties at 80% relative humidity
(Figure 6). Crosslinking improved barrier properties
only when applied together with nanoclay. Glyoxal in-
duced crosslinking of chitosan/nanoclay improved oxy-
gen barrier (92% reduction in oxygen transmission rate
as compared to pure chitosan films). Oxygen transmis-
sion through glyoxal (3.3 g/m2) treated chitosan/nanoclay
films was 2.8 cm3 × 100 µm/(m2 × 24 h) which was 53%
lower as compared to chitosan/nanoclay films without
crosslinkers. These results are slightly better than other
studies [8,28-31] where 15% - 88% reduction in oxygen
transmission rates has been attained with chitosan, PET
and PLA-based layered silicate nanocomposites.
Biopolymers, such as chitosan, have a high natural af-
finity for water, thus films without nanoclay or cross-
linkers absorbed almost 35% of water at 90% relative
humidity. Nanoclay and especially glyoxal crosslinking
prevented the sorption considerably (Figure 7). Nanoc-
lay without crosslinkers provided the lowest absorption
at lower humidities, whereas glyoxal linked chitosan
performed better at higher humidities. As was demon-
strated earlier, the glyoxal crosslinked chitosan is more
compact and hydrophobic and shows lower degree of
swelling as compared to pure chitosan and glutaralde-
hyde crosslinked chitosan [32]. These results are also
consistent with both water vapor and oxygen barrier im-
Figure 6. Oxygen transmission rate of glutaraldehyde, genipin and glyoxal crosslinked chitosan-nanoclay films (measured at
23˚C, 80% RH). Composition of films was expressed as g/m2 of each component in film (crosslinker/nanoclay/chitosan).

Crosslinking as an Efficient Tool for Decreasing Moisture Sensitivity of Biobased Nanocomposite Films 352
Figure 7. Sorption of glutaraldehyde, genipin and glyoxal crosslinked chitosan-nanoclay films. Composition of films was ex-
pressed as g/m2 of each component in film (crosslinker/nanoclay/chitosan).
provements. The absorbed water molecules weaken the
intermolecular interactions, such as hydrogen bonding
and crystallinity, leading to reduced barrier properties [6 ].
Nanoclay and crosslinking p revented the water solubility
and swelling of chitosan which increased the number of
silicate layers per unit volume eventually resulting in
lower permeability.
4. Conclusions
Chitosan-nanoclay bio-hybrid films were successfully
crosslinked with glutaraldehyde, genipin and glyoxal.
Moisture sensitivity of films decreased as a result of
crosslinking which led to improved barrier properties
against water vapor and oxygen. Crosslinking may be
used as an efficient tool for enhancing the exploitability
of naturally hydrophilic biopolymers towards new
high-value applications, such as food packaging.
5. Acknowledgements
The authors thank Heljä Heikkinen, Mari Leino, Juha
Hokkanen, Unto Tapper and Sirpa Vapaavuori for their
technical help. They also thank all project members as
well as VTT’s Industrial Biomaterials spearhead pro-
gramme for funding this study.
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