Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 115–124

Contents lists available at ScienceDirect

Journal of Photochemistry & Photobiology, B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

Chitosan and gelatin based biodegradable packaging films with

UV-light protection
Shakeel Ahmed, Saiqa Ikram ⁎
Bio/Polymers Research Laboratory, Department of Chemistry, Jamia Millia Islamia (A Central University), New Delhi 110025, India

a r t i c l e i n f o a b s t r a c t

Article history: Biopolymers are polymers obtained from biological origins and used for various biological and industrial applica-
Received 20 July 2016 tions. A biopolymer should be non-toxic, non-antigenic, non-irritant, non-carcinogenic, sterilisable and ade-
Received in revised form 15 August 2016 quately available for their widespread applications. In this study, chitosan (CS) and gelatin (GL) based films
Accepted 16 August 2016
were prepared to be used as biodegradable packaging films. CS was blended with GL to improve various physi-
Available online 18 August 2016
cochemical properties. The blended CSGL films were crosslinked with boric acid (BA) to improve various proper-
Keywords:
ties viz. light barrier properties, Water Vapour Permeability (WVP), moisture content (%), Total Solubility Matter
Chitosan (TSM), most important to improve the strength. The studies of transparency, X-ray Diffraction (XRD), Scanning
Gelatin Electron Microscopy (SEM), and optical microscopy confirms that the synthesized films were found to be trans-
Crosslinker parent and homogenous indicating good compatibility among different components. The synthesized CS and GL
Plasticizer based films showed UV-light barrier properties as supported by data. The tensile strength of films increases, de-
Films creases water solubility, moisture content (%) and WVP on crosslinking. In order to make the crosslinked films
Packaging more flexible, Polyethylene glycol was used as plasticizer, making the films more flexible and transparent. This
study indicates that these biodegradable CS and GL based films are potent to be used as packing films.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Biopolymers are polymers obtained from biological origins and used
for various biological and industrial applications. A biopolymer should
be non-toxic, non-antigenic, non-irritant, non-carcinogenic, sterilisable
and adequately available for their widespread applications [1]. Chitosan
(CS) and gelatin (GL) both the natural polymers appear to offer most of
these features to serve in various applications. Both of these are non-
toxic, biodegradable and biocompatible polymers based on natural
and renewable resources, used as coatings and films in food and various
other applications [2].
CS is an unbranched polymer obtained from deacetylation of chitin,
with inherent antimicrobial character, carries a positive charge and is
soluble at pH below 6.5 [3–5]. Due to its cationic nature, it establish
the electrostatic interactions with other compounds; hence, CS has
been most widely used for fabrication of films with adequate barrier
to water vapour. However, it seems economically feasible to combine
it with other film forming biopolymers like GL/PVA, etc. considering
the cost of its preparation [6]. Blending of two different biopolymers
can intensely change the physico-chemical properties of film-forming
solutions (FFS), thus affecting the functionality of the resulting films.
Depending on their chemical structure, pH, molecular weight and
⁎ Corresponding author.
E-mail addresses: shakeelchem11@gmail.com (S. Ahmed), sikram@jmi.ac.in
(S. Ikram).
hydration behaviour among others, all these changes occur due to the
compatibility/incompatibility between both polymers [7].
GL is also one of biopolymers used in medical pharmaceutical appli-
cations with a good film forming ability used in production of biocom-
patible and biodegradable biomaterials, which can act as temporary
replacements [8]. GL can readily absorb UV-light due to presence of ar-
omatic amino acid in it. Several strategies have been employed to im-
prove the physical performance of CS films; including chemical [9–11]
or enzymatic treatment [12] and mixing with other polymers like poly-
ethylene glycol (PEG) [13,14], polyvinyl alcohol (PVA) [15,16], starch
[17], polyethylene glycol fumarate [8] and gelatin [18].
Furthermore, cross-linking has appeared as another important tech-
nique to improve the performance of CS/GL films. The cross-linkers like
borate, tripolyphosphate [19], glycerol [20], sodium trimetaphosphate
[21], and glutaraldehyde [22] can form cross-linked network structure
with CS and GL chain by non-covalent or covalent interactions. Howev-
er, an organic integrity of the merits of those developed cross-linking
techniques remains necessarily not only for designing CS/GL blends
with enhanced stability and strength, but also to expand their practical
applications in different areas. In order to improve flexibility and pro-
cessability of CS/GL films different types of plasticizers have been used
into a polymeric material. The improvement in the flexibility of the
polymer is there by increasing its intermolecular separation and lower-
ing its glass transition temperature [23].
However, traditional methods of crosslinking suffer from several
shortcomings such as lack of precise control over crosslinking, toxicity

http://dx.doi.org/10.1016/j.jphotobiol.2016.08.023
1011-1344/© 2016 Elsevier B.V. All rights reserved.
116 S. Ahmed, S. Ikram / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 115–124

Fig. 1. Digital images of the synthesized transparent crosslinked films.

of the cross linkers, etc. This applies particularly to formaldehyde which
in the presence of hydrochloric acid can form chloromethyl ether, the
use of which is highly restricted as it possess a significant hazards
[24]. Gluteraldehyde is considered as the most important crosslinker
in the sense as it increases the strength of polymers, but studies reveals
that it is carcinogenic in nature. Therefore, there is a necessity to use
eco-friendly crosslinker which inhibits the lethal effect. In the present
study, we tried to use a crosslinker which is biocompatible and safe to
use. Here, crosslinking of CS and GL blend film with boric acid (BA)
and the effect with its different concentration as crosslinker was inves-
tigated. BA is a non-volatile mineral and shows low-toxicity, fungicidal
effect and antimicrobial properties. BA and its salts, have been used in
medicine as a bactericide, a fungicide, and an antiseptic since the
1860s [25]. The objective of this study was to synthesized biodegradable
packaging films with good transparency and flexibility with enhanced
UV-light protection.
2. Materials and Methods
2.1. Materials
All the chemicals used were of analytical grade and were used as
purchased without further purification. CS from shrimp shells (Degree
of Deacetylation ≥ 75%) was purchased from HiMedia Laboratories,
Mumbai. GL powder and BA (extra pure) were purchased from
LobaChemie, Mumbai. PEG (mol. wt. 1400) was purchased from Thom-
as Baker Chemicals, Mumbai.
2.2. Preparation of Blend Films
Different ratios of solute and solvent were tried in order to obtain an
ideal film of the two individual polymers separately as well as of blend.
To prepare film forming solutions, GL was dissolved in distilled water
whereas the CS was dissolved in 2% acetic acid (AA) solution. These so-
lutions were kept on magnetic stirrer and stirred for 24 h at ambient
conditions. The 10%(w/v) of GL and 2%(w/v) of CS solution was found
to be suitable for synthesis of films and was optimized for fabrication
of films. The resulting solutions were filtered by using cheese cloth. In
order to form blend, both the solutions were mixed in different propor-
allowed to settle down. The films were fabricated by pouring these FFS
solutions with different ratio into petridishes further followed by evap-
oration of solvent at room temperature. The films were then peeled off
from petridishes and kept in a desiccator for further use. The ideal films
were then chosen for further investigations after examining their gener-
al morphology. The blend film having the concentration of
CS:GL(90:10) was optimized to be used for further studies.
2.3. Preparation of Crosslinked and Plasticized Blend Films
2.3.1. Optimization of Concentration of Crosslinker
Solutions of blend (2% CS (w/v) and 10% GL (w/v)) were mixed in
ratio of 90:10 (CS:GL). On stirring solutions, BA was added slowly to
blend solution. BA was used as crosslinker for the enhancement of phys-
ical and mechanical stabilities of the films. For this purpose a variation in
the concentration of BA (2–5%) as cross-linker was done. These FFS
were incubated at 30 °C for 24 h with gentle stirring using magnetic stir-
rer. These solutions were then filtered using cheese cloth, allowed to
settle down and were then poured into petridishes for casting. The
films so formed were peeled off from petridishes and kept in desiccator
for further studies.
2.3.2. Optimization of Concentration of Plasticizer
The crosslinked bio-polymeric films achieves a good mechanical
strength but lacks flexibility which is one of the important basic re-
quirement of any polymer to be modified as films for their respective
applications. Hence, PEG as plasticizer was subsequently added with
varying concentration (5%, 10%, and 20%) to achieve desired flexibil-
ities to the films. The solutions were then vigorously stirred for 24 h
followed by the film formation, with same procedure as mentioned
earlier.
CSGL
Transmittance(%)

tions, CS:GL (50:50, 60:40, 70:30, 80:20, 90:10 and 95:5), and was then

CS
Table 1
Film thickness and apparent density of synthesized films.

Films Film thickness(mm) Apparent density(gm/cm3)

CS 0.12 0.675
CSGL 0.15 1.744
CSGL2BA 0.13 1.459 3500 3000 2500 2000 1500 1000
CSGL3BA 0.12 1.554 wavenumber(cm-1)
CSGL4BA 0.13 1.582
CSGL5BA 0.14 1.56
Fig. 2. FTIR spectra of CS and CSGL films.
 S. Ahmed, S. Ikram / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 115–124 117

CSGL5BA
Transmittance(%)

CSGL4BA

CSGL3BA

CSGL2BA

Fig. 5. Total Soluble Matter (TSM) of films.

3500 3000 2500 2000 1500 1000 following equation:

wavenumber(cm )
-1
"
2 #
D
ρ ¼ W= π  H
Fig. 3. FTIR spectra of crosslinked CSGL films.
2

where, D, H and W are the diameter, height and weight of films

respectively.

2.4.3. Fourier Transform Infra-Red (FTIR) Spectroscopy

FTIR spectra were recorded from wave number 600–4000 cm− 1
CSGL5BA
(Bruker Tensor 37 spectrometer). The spectra were used to determine
Intensity (a.u.)

CSGL4BA
the presence of functional groups and interactions among various com-
ponents of the synthesized films.
CSGL3BA

CSGL2BA
2.4.4. X-Ray Diffraction (XRD)
X-Ray Diffractometer (Rigaku Ultima-IV) was used to determine
XRD patterns of the films equipped with a scintillation counter. The mo-
CSGL
lecular miscibility as well as crystallinity of films was determine by
CS using X-rays. XRD patterns of the films were recorded in the 2θ range
of 10–60°.

10 15 20 25 30 35 40 2.4.5. Light Transmission and Transparency

2θ
Film portions of 10 × 20 mm films were placed into the test cell of U-
3900UV Spectrophotometer Model 212-0013. An empty test cell was
Fig. 4. XRD patterns of CSGL and crosslinked films.
used as the reference. Transparency was determined by measuring the
absorbance at 600 nm by same method as reported earlier [26]. The
transparency of the films were calculated by the equation [27]:
2.4. Characterization Techniques


A600
2.4.1. Film Thickness Transparency ¼ −logT600=x
x
The thickness of films was measured by a handheld digital
micrometre with an accuracy of ±0.01 mm. The calliper was calibrated where, T600 is the transmittance at 600 nm, A is the absorbance at
every time before taking reading and six random locations around films 600 nm and x is the film thickness (mm). According to this equation;
were chosen for measuring their thickness. the high values of transparency indicates higher degree of opacity.

2.4.2. Apparent Density 2.4.6. Total Soluble Matter

To measure the apparent density of films, its portions were cut and Total Soluble Matter (TSM) was determined according to the meth-
selected. The apparent density (ρ) of films was measured using the od reported in literature [27,28]. The initial dry matter content of each

Table 2
Light transmittance (%T) at different wavelengths (nm) and transparency value at 600 nm.

Film Light transmittance(%) at different wavelength (nm) Transparency A600/x

200 250 300 350 400 450 500 550 600 650 700 750 800

CS 65.1 60.3 21.1 40 62.3 71.5 73.6 72.7 61 66.2 68.4 77.7 80.9 2.43
CSGL 73.9 68.7 50.2 60.4 77.1 83 81.9 73.3 71.6 75.2 81.0 86.0 87.1 1.59
CSGL2BA 50.3 40.9 5.6 18.2 37.8 48.0 50.6 48.0 48.4 50.3 53.6 57.6 59.2 1.18
CSGL3BA 60.2 49.2 11.3 26.7 56.6 70.4 76.7 77.8 78.9 79.7 80.4 81.0 80.9 1.21
CSGL4BA 69.1 57.2 22.7 38.1 61.4 70.1 74.2 74.8 75.8 76.8 77.7 78.5 78.6 1.27
CSGL5BA 69.7 64 35.6 49.6 70.2 77.5 77.5 69.9 68.7 72.1 77.6 82.4 83.4 1.81
118 S. Ahmed, S. Ikram / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 115–124

film was determined by drying to constant weight in an oven at 105 ±

2 °C. Films (2 × 2 cm diameter) were then cut, weighed and submerged
in 50 ml of distilled water in a beaker at 25 °C. After 24 h of immersion in
water, the pieces of film were taken out and dried to constant weight
again in an oven at 105 ± 2 °C to determine the weight of dry matter
which was not solubilized in water. The measurement of solubility of
the films was determined as follows:

TSM ¼ ðWi−Wf Þ=Wi  100

where, Wi and Wf are initial mass and final mass of the sample
respectively.
Fig. 6. Moisture content(%) of films.
2.4.7. Moisture Content (%)
Moisture Content (%) of the films was determined by measuring
weight loss of films upon drying in an oven at 105 ± 2 °C for 24 h.
The moisture content was calculated according to the following
equation:

Mw−Md
Moisture content ð%Þ ¼  100%
Mw

where, Mw is the weight of the films conditioned in 75% RH to moisture

equilibrium and Md is dry weight of the films.

2.4.8. Tensile Strength

The rectangular shaped films with dimensions (8.5 cm × 1 cm ×
0.15 mm) were fabricated and used to measure tensile strength. The
Fig. 7. Tensile strength of films.
two ends of films were installed vertically on two mechanical clamping

(a) SEM images of crosslinked films

(b) Optical images of crosslinked films

Fig. 8. (a) SEM images and (b) Optical images of crosslinked films.
 S. Ahmed, S. Ikram / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 115–124 119

CSGL4BA20PEG

Transmittance(%)
CSGL4BA10PEG

CSGL4BA5PEG

4000 3500 3000 2500 2000 1500 1000

Fig. 9. Water Vapour Permeability (WVP) of films. wavenumber(cm )
-1

Fig. 11. FTIR spectra of plasticized films.

units of tensile machine. The value of elongation and force were noted
and average of triplicate was taken to measure the tensile strength.
rate and placed at the maximum distance from the electron beam to
Force prevent damage to them. The images were obtained using an accelerat-
Tensilestrength ¼
t  w: ing voltage of 10–15 kV.

where, t and w are thickness and width of films respectively.

2.4.9. Water Vapour Permeability
Water Vapour Permeability (WVP) was determined by a gravimetric
method as reported by Pereda, et.al. [29]. Films were attached over the
openings of small bottles containing silica gel and were placed in a des-
iccator with distilled water at room temperature. The bottles were
weighed daily for 7 days and WVP of the films was calculated using
the equation:
w nents of films. If compatibility between films constituents was not
WVP ¼ x good, then due to reflection or dispersion of light at the phase in-
tAΔP
where w was weight gain (gm), x is the thickness of film (mm), t
elapsed time (hr) for the weight gain, A is the exposed area of film
(cm2) and ΔP the partial vapour pressure difference between the dry at-
mosphere and pure water (2642 Pa at 22 °C).
2.4.10. Surface Morphology
Morphology of fabricated films was determined by using a Lietz Op-
tical Microscope (Model Metallux-3). The surface morphology of the
films was also confirmed by using Field emission scanning electron mi-
croscopy (FESEM) (Nova Nano SEM 450, Fei). Before examination sam-
ples were sprayed with a fine layer of platinum using a low deposition
3. Results and Discussions
3.1. Effect of Crosslinker
3.1.1. Film Appearance, Thickness and Apparent Density
Homogeneous and flexible crosslinked films of CS and GL were
obtained and all films were transparent as observed by naked eye
observation (Fig. 1), indicating good compatibility among compo-
terface, the transparency would be low [30]. Given the fact that
there was no significant differences in thickness value and the re-
duction in opacity of CSGL films by increasing concentration of
crosslinker could indicate a good compatibility among different
components.
There was a variation in density of films in different concentration
and components (Table 1). The addition of GL to CS lead to increment
in density and further on addition of BA further increases the density
and results were found to be similar as already present in literature
[31]. This variation in apparent density can be attributed to the changes
of porosity and solid content density of films by adding different compo-
nents viz. GL or BA.

Fig. 10. Digital images of synthesized transparent plasticized films.

120 S. Ahmed, S. Ikram / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 115–124
Fig. 12. XRD pattern of plasticized membranes.
3.1.2. Fourier Transform Infra-Red (FTIR) Spectroscopy
The FTIR spectra of pure CS and CSGL blend film were carried out and
is shown in Fig. 2. The main characteristic absorption bands of the CS
film at 1645 cm−1 attributed to C_O stretching (amide I), 1570 cm−1
due to N\\H bending (amide II) and 1376 cm−1 assigned to\\OH bend-
ing, respectively. The absorption peak at 1050 cm− 1 is assigned to
C\\O\\C stretching of polysaccharide chain. Results obtained for CS
film are in good agreement with those reported earlier in literature
[29–30]. The physical blends vs chemical interactions are reflected by
changes in the characteristic spectral peaks, when two or more sub-
stances are mixed. It is predicted that if there are no interactions be-
tween CS and GL, all spectra must reproduce as of their virgin data;
however, the changes were observed when compared with individual
as well. Addition of GL into CS shifts the carbonyl and amino peaks
from 1645 to 1613 cm−1, 1570 to 1544 cm−1 shows the interaction be-
tween both the biopolymers in the blend film [34].
The main characteristic absorption bands (Fig. 3) with varying BA
(2–5%) as cross-linkers are in range of 2938 cm−1 and 2857 cm− 1
(\\CH2 stretching), 1632 cm−1 (C_O stretching amide I), 1529 cm−1
(amide II), 1449 cm− 1 (due to \\CH2 scissoring), 1376 cm−1 (\\OH
bending), and 1282 cm−1 (\\CN of amide-III of GL). On increasing the
concentration, there was decrease in intensity of peaks, indicating
crosslinking increases. It showed a broad band stretching frequency in
range of 3300–3500 cm−1 attributed to\\OH and\\NH2 stretching. Re-
sults obtained were in agreement with those reported earlier [35,36].
3.1.3. X-Ray Diffraction
The physical appearance in terms of crystallinity is one of the impor-
tant criterions of the films to design a particular application in the field
of packaging industries. The pure CS film showed four main diffraction
peaks at (2θ = 8.83°, 11.69°, 18.44° and 22.95°) present in its X-ray dif-
fraction pattern indicates its semi-crystalline state [29]. The peaks at
11.9° and 22.95° are the typical fingerprints of CS. If both the biopoly-
mers i.e. CS and GL have low compatibility, then each of the biopolymer
would have its own crystal region in the blend. But in XRD pattern of the
CSGL film, the diffraction peaks of CS almost disappear and becomes
broader, illustrating that the presence of protein (GL) reduces the
Table 3
Light transmittance (%T) at different wavelengths (nm) and transparency value at 600 nm.
Sample Light transmittance(%) at different wavelength (nm)
200 250 300 350 400 450
CSGL4BA5PEG 50.3 40.9 5.6 18.2 37.8 48.0
CSGL4BA10PEG 58.7 46.9 7.9 20.2 34.5 43.7
CSGL4BA20PEG 51.3 42.1 12.3 13.3 24.7 35.0
Fig. 13. Moisture content (%) of plasticized films.
crystallinity of CS. The CSGL blend film showed a broad diffraction
peaks around 2θ = 20° in the XRD pattern and the results are in line
with the literature [30,37,38]. Blending may result into an amorphous
matrix of the two biopolymers but the incorporation of crosslinker
may alter in the overall crystallinity of the fabricated films which may
be evaluated through XRD (Fig. 4). The crosslinked CSGL based films
showed broad diffraction peaks at (2θ = 18–22°) present in its X-ray
diffraction pattern showing an amorphous state, and the results were
in line with the literature [38]. These results suggested that there were
good compatibility and interaction among different components in the
films viz. crosslinker and biopolymers. X-ray diffraction results were
in good agreement with those of transparency and optical microscopy
as discussed further.
3.1.4. Light Transmission and Transparency
Light transmission and transparency of all films at selected wave-
lengths are shown in Table 2. The films were found to be transparent in-
dicating films to be homogenous i.e. there is no phase separation among
different components. If compatibility among different constituents of
films is not good, then the light transmittance is low or opacity is high
due to light reflection or dispersion at the two-phase interface [30].
Films have excellent barrier properties in the UV region, regardless
crosslinking content; therefore, CS/GL based films effectively prevented
the UV light, as supported by these results. This is attributed to the fact
that the presence of protein (GL); GL contains high content of aromatic
amino acids which absorb UV-light due to which it considers to have
high UV barrier properties [39]. At 300 nm all the films showed lowest
light transmittance which might be due to presence of protein based
composition of GL. All the films showed exceptional transparency that
did not change with BA content. The transmission of visible light was
N70% at 400–700 nm and N 80% at 800 nm for the films, while transpar-
ency value was b 2.2 at 600 nm.
3.1.5. Total Soluble Matter (TSM) and Moisture Content (%)
The plot of moisture content values obtained are shown in Fig. 6. It
was found that the moisture content of the film decreases with GL
blending. CS film shows higher moisture content (16.66%) due to avail-
ability of free \\OH and \\NH2 groups to interact with water and the
values were found to be similar as found in literature [40]. Blending of
CS with GL decreased the moisture absorption of films, due to interac-
tion of functional groups (\\OH, \\NH2, etc.) between CS and GL.
Crosslinking of CSGL films with BA underwent the reaction with
Transparency A600/x
500 550 600 650 700 750 800
50.6 48.0 48.4 50.3 53.6 57.6 69.2 1.18
49.9 54.2 57.6 60.3 62.3 64.1 65.6 0.48
44.9 51.5 56.4 62.3 66.2 69.1 71.3 0.74
 S. Ahmed, S. Ikram / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 115–124
Fig. 14. Total soluble matter of plasticized films.
functional groups forming the crosslinked network structure lead-
ing to decrease in mobility of polymer chains as well as decrease
in number of functional groups, thereby decreasing the value of
moisture content [41]. Higher the concentration of BA, higher was
the level of cross-linking and thus lower was their ability to absorb
moisture.
Total soluble matter (TSM) value is a measure of the resistance of
films against water [42] and is related to hydrophilicity of films. The
CS film is least solubility due to hydrophobic nature of chitosan. The
addition of hydrophilic gelatin lead to increase the TSM of blend film.
The blend film was slightly more soluble than pure CS film due to the
hydrophilic nature of gelatin or due to solubility of gelatin. Cross-
linked films were found to be less soluble than CSGL film (Fig. 5)
and increase in crosslinking further decrease their solubility in
water.
3.1.6. Tensile Strength
The mechanical properties are the favorable properties of the pack-
aging materials required to maintain film integrity and stability in
order to withstand the stress that occurs during storage, handling and
shipping. The loss of mechanical stability in packaging films is due to
their poor mechanical properties which reduces their effectiveness as
a barrier. It is of great importance to study mechanical properties of
films as these having influence on product performance and consumer
acceptance. The blending of CS with GL increases the strength of films
due to electrostatic interactions between different functional groups of
both the components (Fig. 7). When films were fabricated with the
crosslinker, the TS of crosslinked films increased due to the tight
crosslinks between two biopolymers. The strength of films increases
as the crosslinking between the polymer matrix increases, thus increas-
ing the polymer strength and stiffness up to 4% BA [43]. On further in-
creasing concentration of BA to 5%, the film becomes brittle and its
tensile strength decreases may be due to higher crosslinking due to
which flexibility of films decreases.
Fig. 15. Tensile strength of plasticized films.
121
3.1.7. Surface Morphology
To study the surface morphology of the fabricated films; the optical
microscopy (Fig. 8(b)) and scanning electron microscopy (Fig. 8(a))
were used. The crosslinked films were found to be of uniform, compact
and homogenous appearance and components were found to be well
dispersed. For cross-linked films, no phase separation detected, suggest-
ing a good compatibility among different components as already
discussed in transparency and XRD. It was found that the surface mor-
phology of the films were relatively smooth. These results indicated
that the GL, CS and BA were mixed well at the molecular levels indicat-
ing a good compatibility among the all components [32,33].
3.1.8. Water Vapour Permeability
Water vapour permeability (WVP) generally depends on both
diffusibility and solubility of water molecules in film matrix. WVP of
CSGL blend film decreases in comparison with that of pure CS film due
to interaction between two components. WVP further decreases with
addition of BA and further decreases with increasing concentration
(Fig. 9). On increasing the concentration of BA, the crosslinking between
the polymer matrix increases suggesting that polymeric chains accumu-
late compactness in chains which reduces interstitial spaces in polymer
matrix and thus allow for a decreased diffusion rate of the water
through polymeric chain. There are various factors that could affect
the water vapour permeability of films which include the molecular
weight of polymers, the type and amount of crosslinking and the pre-
paring conditions which would influence the network structure in poly-
meric films [44].
On comparing the various studies of the crosslinked films, it was
concluded that the films with 4% BA have good mechanical strength,
lowest water vapour permeability, and lower solubility; and hence op-
timized for further modifications.
3.2. Effect of Plasticizer
Although, the crosslinking of the two polymers provide the desired
mechanical stabilities to the films, but steals the flexibilities from
them, which is one of the most important requirement for their neces-
sary application as in packaging industries. The desired flexibility may
be introduced through the inclusion of a plasticizer without disturbing
the mechanical strengths. Polyethylene glycol (PEG) was used as the
plasticizer to achieve the required flexibility in the blend films. The in-
corporation of plasticizer into the crosslinked films increased their
transparency and the films become more transparent (Fig. 10).
3.2.1. Fourier Transform Infra-Red (FTIR) Spectroscopy
The changes in the intensities of FTIR spectra were observed as per
the variation in the content of plasticizer (5–20%) with a fixed concen-
tration of BA (4%). On the incorporation of plasticizer, the interaction
and proximity between chains of crosslinked films were reduced. The
FTIR spectra of films with optimized concentration of BA (4%) having
different concentration of PEG are in good agreement with those
which are already discussed above. The intensity and position of charac-
teristic peaks of\\OH group permit us to interpret rather easily the in-
fluence of interaction between PEG and functional group \\OH of
polymer films. From the spectra of films, (Fig. 11) it can be seen that hy-
droxyl band shifted to higher wavenumber decreasing intensity on in-
creasing the plasticizer content indicating the interactions between
the hydroxyl group of CS and GL with PEG. The well-known peaks of
CS/GL i.e. amide-I (1629 cm− 1), amide-II (1566 cm−1), amide-III
(1273 cm−1) and –C-O-C-(1067 cm−1) were found to be in same
range as described above [35,36]. On increasing the concentration of
plasticizer, there is increase in intensity of other peaks. There is not
any appreciable change in the FTIR spectra after addition of PEG, there-
fore PEG only increases the flexibility without much changing other
properties. Therefore, interactions between both components must be
considered giving a desired flexibilities to the films.
122 S. Ahmed, S. Ikram / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 115–124
Fig. 16. Surface morphology of plasticized films.
3.2.2. X-Ray Diffraction
A broad amorphous peak was present in range of 2θ = 20° in the
plasticized films, indicating that there was a molecular miscibility and
an interaction among these components in the films (Fig. 12). On in-
creasing the concentration of plasticizer, the intensity of the peaks de-
creases which showed spacing between polymer matrix increases
thereby increasing the flexibility of films. These results suggested that
there were good compatibility and interaction among different compo-
nents in the films [45,46]. X-ray diffraction results are in good agree-
ment with those of transparency and optical microscopy as discussed.
3.2.3. Light Transmission and Transparency
Plasticized films were found to be more transparent in comparison
to that of crosslinked one (Table 3). All the films fabricated were
found to be homogenous in nature i.e. there is no phase separation
found among different components. If compatibility among film constit-
uents is not good, then the light transmittance is low due to light disper-
sion or reflection at the two-phase interface [29,46]. The results are in
accordance as already reported [47,48] which states that films become
more transparent with addition of PEG. On increasing the concentration
of PEG, films become more transparent and similar results were found
in edible films of Jongjareonrak et al. [49].
3.2.4. Total Soluble Matter (TSM) and Moisture Content (%)
Plasticization of films tend to decrease the intermolecular interac-
tion between the polymer chains, thereby increasing the molecular
Fig. 17. Water Vapour Permeability (WVP) of plasticized films.
spaces in the films. Addition of PEG tends to increase the value of
total solubility matter (Fig. 14) due to free hydroxyl groups, as plas-
ticization decreases the intermolecular interaction (due to hydrogen
bonding). With the increment in concentration of PEG, the value of
moisture content increases because the plasticizer increases the in-
termolecular spaces between polymeric chains, therefore more
space is available for absorbing moisture (Fig. 13) in plasticized
films [29].
3.2.5. Tensile Strength
The studies showed that the tensile strength of polymeric films de-
crease on addition of plasticizer (Fig. 15). On increasing the concentra-
tion of PEG, the tensile strength decreases due to decrease in
intermolecular interaction among polymer matrix. Plasticizer interfere
with polymer chain by decreasing the intermolecular forces, soften
the rigidity of the film's structure and increase the polymer mobility,
thus decrease the tensile strength and making the films more flexible
and extensible [36].
3.2.6. Surface Morphology
For plasticized films, no phase separation detected, suggesting a
good compatibility among different components as discussed in trans-
parency data. It was found that the surface morphology of the films
were relatively smooth (Fig. 16). These results indicated that the differ-
ent components of plasticized films were well mixed at the molecular
levels. Plasticized films showed smooth and compact structures indicat-
ing a good compatibility among all the different components as already
discussed in transparency.
3.2.7. Water Vapour Permeability
Water Vapour Permeability (WVP) of the plasticized films was more
in comparison to the crosslinked one (Fig. 17). The plasticizer reduces
the intermolecular interaction, thereby increasing the molecular spaces
between the polymer matrixes thus, increasing the permeability of plas-
ticized films [48,50]. On increasing the concentration of plasticizer, the
molecular spaces between the polymer chains increases thereby de-
creasing the intermolecular interactions which tends to decrease the
mechanical strength thereby increases the flexibility of films.
 S. Ahmed, S. Ikram / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 115–124
4. Conclusions
In this study, different parameters of concentration of CS and GL to
obtain the homogenous, optimum and compact films were optimized.
The films were crosslinked using BA as crosslinker for the enhancement
of physical and mechanical stabilities of the films. All films were found
to be transparent by naked eye observation also confirmed by transpar-
ency data, indicating good compatibility among different components
of films. Films have excellent barrier properties in the UV region, regard-
less crosslinking content; therefore, CSGL based films effectively pre-
vent the UV-light. Crosslinking of CSGL films with BA underwent the
reaction with functional groups forming the crosslinked network struc-
ture leading to decrease in mobility of polymer chains as well as de-
crease in number of functional groups, thereby decreasing the value of
moisture content. WVP of films decreases and TS increases on increas-
ing crosslinking content. Although, the crosslinking of the two polymers
provide the desired mechanical stabilities to the films, but, steals the
flexibilities from them, which is one of the most important require-
ments for their necessary application as in packaging industries. PEG
was used as the plasticizer to achieve the required flexibility in poly-
meric films. On the incorporation of plasticizer, the interaction and
proximity between chains of crosslinked films were reduced. On in-
creasing the concentration of plasticizer, the intensity of the peaks de-
creases which showed spacing between polymer matrix increases
thereby increasing the flexibility of films. In addition, due to increase
in molecular spaces between the polymer matrixes, increasing the per-
meability of plasticized films. Plasticizer interfere with polymer chain by
decreasing the intermolecular forces, soften the rigidity of the film's
structure and increase the polymer mobility, thus decrease the tensile
strength and making the films more flexible and extensible. The plasti-
cizer reduces the intermolecular interaction, thereby increasing the mo-
lecular spaces. This is a preliminary investigation, further studies needs
to be carried out, so that films can be used as biodegradable packaging in
future.
Acknowledgements
Shakeel Ahmed gratefully acknowledges financial support from
University Grant Commission (UGC), New Delhi in the form of JRF
(NET) and SRF (NET).
References
[1] A. Silva-Weiss, V. Bifani, M. Ihl, P.J. a Sobral, M.C. Gómez-Guillén, Structural proper-
ties of films and rheology of film-forming solutions based on chitosan and chitosan-
starch blend enriched with murta leaf extract, Food Hydrocoll. 31 (2013) 458–466.
[2] M. Kolybaba, L.G. Tabil, S. Panigrahi, W.J. Crerar, T. Powell, B. Wang, et al., Biodegrad-
able Polymers : Past, Present , and Future, Paper Number: RRV03-0007 An ASAE
Meeting Presentation, 2003 1–15.
[3] S. Ahmed, M. Ahmad, S. Ikram, Chitosan : a natural antimicrobial agent-a review, J.
Applicable Chem. 3 (2014) 493–503.
[4] M. Ahmad, K. Manzoor, S. Ahmed, S. Ikram, Synthesis and characterization of
terepthalaldehyde–thiocarbohydrazide polymer doped with Cu(II) and Zn(II)
metal ions for solar cell applications, Opt. Int. J. Light Electron Opt. 127 (2016)
4329–4332.
[5] S. Ahmed, S. Ikram, Chitosan based scaffolds and their applications in wound
healing, Achiev. Life Sci. 10 (2016) 27–37.
[6] S. Ahmed, S. Ikram, Chitosan & its derivatives : a review in recent innovations, Int. J.
Pharmaceut. Sci. Res. 6 (2015) 14–30.
[7] P.B. Malafaya, G.A. Silva, R.L. Reis, Natural-origin polymers as carriers and scaffolds
for biomolecules and cell delivery in tissue engineering applications, Adv. Drug
Deliv. Rev. 59 (2007) 207–233.
[8] A. Hashemi Doulabi, H. Mirzadeh, M. Imani, N. Samadi, Chitosan/polyethylene glycol
fumarate blend film: physical and antibacterial properties, Carbohydr. Polym. 92
(2013) 48–56, http://dx.doi.org/10.1016/j.carbpol.2012.09.002.
[9] H. Sashiwa, N. Kawasaki, A. Nakayama, E. Muraki, N. Yamamoto, S. Aiba, Chemical
modification of chitosan. 14: synthesis of water-soluble chitosan derivatives by sim-
ple acetylation, Biomacromolecules 3 (2002) 1126–1128.
[10] G.E. Fredheim, B.E. Christensen, Polyelectrolyte complexes: interactions between
lignosulfonate and chitosan, Biomacromolecules 4 (2003) 232–239.
[11] J. Zhang, W. Xia, P. Liu, Q. Cheng, T. Tahirou, W. Gu, et al., Chitosan modification and
pharmaceutical/biomedical applications, Mar. Drugs. 8 (2010) 1962–1987.
123
[12] S. Kim, N. Rajapakse, Enzymatic production and biological activities of chitosan oli-
gosaccharides (COS): a review, Carbohydr. Polym. 62 (2005) 357–368.
[13] B. Gupta, S. Saxena, A. Arora, M.S. Alam, Chitosan-polyethylene glycol coated cotton
membranes for wound dressings, Indian J. Fibre Text. Res. 36 (2011) 227–280.
[14] C.-T. Chang, Y.-L. Lin, S.-W. Lu, C.-W. Huang, Y.-T. Wang, Y.-C. Chung, Characteriza-
tion of a Chitosanase from jelly fig (Ficus awkeotsang Makino) latex and its applica-
tion in the production of water-soluble low molecular weight Chitosans, PLoS One
11 (2016), e0150490.
[15] W.-Y. Chuang, T.-H. Young, C.-H. Yao, W.-Y. Chiu, Properties of the poly(vinyl alco-
hol)/chitosan blend and its effect on the culture of fibroblast in vitro, Biomaterials
20 (1999) 1479–1487.
[16] M. Kouchak, A. Ameri, B. Naseri, S.K. Boldaji, Chitosan and polyvinyl alcohol compos-
ite films containing nitrofurazone : preparation and evaluation, Iranian J. Basic Med.
Sci. 17 (2014) 14–20.
[17] T. Bourtoom, M.S. Chinnan, Preparation and properties of rice starch–chitosan blend
biodegradable film, LWT, Food Sci. Technol. 41 (2008) 1633–1641.
[18] A. Fernandes, I. Sousa, Biodegradable Fish Gelatin/Chitosan Composite Films: Homo-
geneous and Bi-Layer Structures, 2011 1–4.
[19] S. Rodrigues, A.M.R. da Costa, A. Grenha, Chitosan/carrageenan nanoparticles: effect
of cross-linking with tripolyphosphate and charge ratios, Carbohydr. Polym. 89
(2012) 282–289.
[20] J.F. Fundo, R. Fernandes, P.M. Almeida, A. Carvalho, G. Feio, C.L.M. Silva, et al., Molec-
ular mobility, composition and structure analysis in glycerol plasticised chitosan
films, Food Chem. 144 (2014) 2–8.
[21] V. Wintgens, C. Lorthioir, P. Dubot, B. Sébille, C. Amiel, Cyclodextrin/dextran based
hydrogels prepared by cross-linking with sodium trimetaphosphate, Carbohydr.
Polym. 132 (2015) 80–88.
[22] S. Ahmed, M. Ahmad, S. Ikram, Physicochemical characterization of gluteraldehyde
crosslinked chitosan-gelatin films, Mater. Focus. 5 (2016) 165–170.
[23] E. Snejdrova, M. Dittrich, Chapter 4, Pharmaceutically Used Plasticizers, Recent Adv.
Plast, Intech Publisher, 2012.
[24] NTP, Report on Carcinogens, 13th ed., 2014.
[25] R. Zan, I. Hubbezoglu, A.K. Ozdemir, T. Tunc, Z. Sumer, O. Alici, Antibacterial effect of
different concentration of boric acid against enterococcus Faecalis biofilms in root
canal, Marmara, Dent. J. 1 (2013) 76–80.
[26] L. Wang, Q. Wang, J. Tong, J. Zhou, Physicochemical properties of chitosan films in-
corporated with honeysuckle flower extract for active food packaging, J. Food Pro-
cess Eng. 1-8 (2015).
[27] Y. Peng, Y. Wu, Y. Li, Development of tea extracts and chitosan composite films for
active packaging materials, Int. J. Biol. Macromol. 59 (2013) 282–289.
[28] K.M. Kim, J.H. Son, S. Kim, C.L. Weller, M. Hanna, M.A. Hanna, Properties of chitosan
films as a function of pH and solvent type, J. Food Scince E Food Eng. Phys. Prop. 71
(2006) 119–124.
[29] M. Pereda, A.G. Ponce, N.E. Marcovich, R.A. Ruseckaite, J.F. Martucci, Chitosan-gelatin
composites and bi-layer films with potential antimicrobial activity, Food Hydrocoll.
25 (2011) 1372–1381.
[30] J.T. Martins, M.a. Cerqueira, A.a. Vicente, Influence of α-tocopherol on physicochem-
ical properties of chitosan-based films, Food Hydrocoll. 27 (2012) 220–227.
[31] M. Peter, N. Ganesh, N. Selvamurugan, S.V. Nair, T. Furuike, H. Tamura, et al., Prepa-
ration and characterization of chitosan–gelatin/nanohydroxyapatite composite scaf-
folds for tissue engineering applications, Carbohydr. Polym. 80 (2010) 687–694.
[32] S. Rivero, M.A. García, A. Pinotti, Crosslinking capacity of tannic acid in plasticized
chitosan films, Carbohydr. Polym. 82 (2010) 270–276.
[33] M. Cheng, J. Deng, F. Yang, Y. Gong, N. Zhao, X. Zhang, Study on physical properties
and nerve cell affinity of composite films from chitosan and gelatin solutions, Bio-
materials 24 (2003) 2871–2880.
[34] Z. Liu, X. Ge, Y. Lu, S. Dong, Y. Zhao, M. Zeng, Effects of chitosan molecular weight
and degree of deacetylation on the properties of gelatine-based films, Food
Hydrocoll. 26 (2012) 311–317.
[35] Q. Wang, Y. Du, L. Fan, Properties of chitosan/poly(vinyl alcohol) films for drug-con-
trolled release, J. Appl. Polym. Sci. 96 (2005) 808–813.
[36] L. Qi, Z. Xu, X. Jiang, C. Hu, X. Zou, Preparation and antibacterial activity of chitosan
nanoparticles, Carbohydr. Res. 339 (2004) 2693–2700.
[37] C.-H. Chen, F.-Y. Wang, C.-F. Mao, W.-T. Liao, C.-D. Hsieh, Studies of chitosan: II.
Preparation and characterization of chitosan/poly(vinyl alcohol)/gelatin ternary
blend films, Int. J. Biol. Macromol. 43 (2008) 37–42.
[38] H. Nagahama, H. Maeda, T. Kashiki, R. Jayakumar, T. Furuike, H. Tamura, Preparation
and characterization of novel chitosan/gelatin membranes using chitosan hydrogel,
Carbohydr. Polym. 76 (2009) 255–260.
[39] R.J.B. Pinto, S.C.M. Fernandes, C.S.R. Freire, P. Sadocco, J. Causio, C.P. Neto, et al., An-
tibacterial activity of optically transparent nanocomposite films based on chitosan
or its derivatives and silver nanoparticles, Carbohydr. Res. 348 (2012) 77–83.
[40] P.C. Srinivasa, M.N. Ramesh, K.R. Kumar, R.N. Tharanathan, Properties and sorption
studies of chitosan–polyvinyl alcohol blend films, Carbohydr. Polym. 53 (2003)
431–438.
[41] M.G.A. Vieira, M.A. da Silva, L.O. dos Santos, M.M. Beppu, Natural-based plasticizers
and biopolymer films: a review, Eur. Polym. J. 47 (2011) 254–263.
[42] L. Wang, Y. Dong, H. Men, J. Tong, J. Zhou, Preparation and characterization of active
films based on chitosan incorporated tea polyphenols, Food Hydrocoll. 32 (2013)
35–41.
[43] M.H. Jusof Khadidi, E.A. Hamid, A new flocculant-coagulant with potential use for in-
dustrial wastewater treatment, 2nd Int. Conf. Environ. Energy Biotechnol. 51 (2013)
139–142.
[44] L. Wang, Y. Dong, H. Men, J. Tong, J. Zhou, Preparation and characterization of active
films based on chitosan incorporated tea polyphenols, Food Hydrocoll. 32 (2013)
35–41.
124 S. Ahmed, S. Ikram / Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 115–124
[45] H. Zhuang, J.P. Zheng, H. Gao, K. Yao, In vitro biodegradation and biocompatibility of
gelatin/montmorillonite-chitosan intercalated nanocomposite, J. Mater. Sci. Mater.
Med. 18 (2007) 951–957.
[46] X. Meng, F. Tian, J. Yang, C.-N. He, N. Xing, F. Li, Chitosan and alginate polyelectrolyte
complex membranes and their properties for wound dressing application, J. Mater.
Sci. Mater. Med. 21 (2010) 1751–1759.
[47] M. Rodríguez, J. Osés, K. Ziani, J.I. Maté, Combined effect of plasticizers and surfac-
tants on the physical properties of starch based edible films, Food Res. Int. 39
(2006) 840–846.
[48] M. Tanaka, K. Iwata, R. Sanguandeekul, A. Handa, S. Ishizaki, Influence of plasticizers
on the properties of edible films prepared from fish water-soluble proteins, Fish. Sci.
67 (2001) 346–351.
[49] A. Jongjareonrak, S. Benjakul, W. Visessanguan, M. Tanaka, Effects of plasticizers on
the properties of edible films from skin gelatin of bigeye snapper and brownstripe
red snapper, Eur. Food Res. Technol. 222 (2006) 229–235.
[50] M.A. García, M.N. Martino, N.E. Zaritzky, Lipid addition to improve barrier properties
of edible starch-based films and coatings, J. Food Sci. 65 (2000) 941–944.