Chitosan containing azo-based Schiff bases: Thermal, antibacterial and birefringence properties for bio-optical devices

Article abstract of DOI:10.1039/c5ra27210f

The present investigation describes the preparation of chitosan/2-hydroxy-5-(4-nitrophenylazo)-benzaldehyde (CHNAB) and chitosan/2-hydroxy-5-(4-tolylazo)-benzaldehyde (CHMAB) derivatives (in 4:5 molar ratio) under mild conditions. These derivatives were s

Full text of DOI:10.1039/c5ra27210f

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PAPER
Chitosan containing azo-based Schiff bases:
thermal, antibacterial and birefringence properties
Cite this: RSC Adv., 2016, 6, 5575
for bio-optical devices†
a
a
a
b
a
*
*
Nidhi Nigam, Santosh Kumar,‡ P. K. Dutta, S. Pei and Tamal Ghosh
The present investigation describes the preparation of chitosan/2-hydroxy-5-(4-nitrophenylazo)-
benzaldehyde (CHNAB) and chitosan/2-hydroxy-5-(4-tolylazo)-benzaldehyde (CHMAB) derivatives (in
4 : 5 molar ratio) under mild conditions. These derivatives were synthesized by 79% and #90%
deacetylated chitosan in an isopropyl alcohol/water mixture and dimethyl sulfoxide. The thermal
properties of the derivatives were examined by differential scanning calorimetry (DSC) and
thermogravimetric analysis (TGA). CHNAB shows an endothermic peak at 212 ꢀC and exothermic peak at
231 ꢀC due to phase change for cis–trans isomerisation, whereas, in CHMAB, only the endothermic peak
at 222 ꢀC is seen due to the absence of cis–trans isomerisation. Toxicity has been reduced in chitosan
based azo derivatives, as compared to the corresponding azo compounds, as seen by the antibacterial
results where S. aureus (Gram positive) shows ZOI 26 mm for CHNAB and 38 mm for CHMAB. The
transmitted signal intensity induced by the birefringence as shown by CHNAB as pdDn/l is much less
than 1. The transmitted signal is effectively proportional to (Dn)2 because of the nitro group behaving like
an auxochrome. The study on the optical birefringence properties of the azo-based chitosan derivatives
indicates that the prepared derivatives may be used as bio-optical devices for biomedical applications.
Received 19th December 2015
Accepted 2nd January 2016
DOI: 10.1039/c5ra27210f
www.rsc.org/advances
antibacterial nature.12 Chitosan in solution and gel forms
exhibit strong antimicrobial activity against a broad spectrum
Introduction
of both Gram-positive and Gram-negative medically relevant
microorganisms.13 The antimicrobial nature of chitosan is due
to surface–surface interactions between the biopolymer chains
and microbial cell walls. Thus, chitosan based materials have
the potential for antimicrobial action, the exploitation of which
is of particular interest to the development of medical tech-
nologies. Azo dye containing polymers are studied with
immense interest because of their unique photoinduced
effects.14,15 The photoisomerization process, which implies
mobility of the azo dye molecules, results in a facile change in
its orientation due to the polarized optical eld and has been
observed even in high glass transition polymer matrices.
In the present paper, we have described the preparation of
chitosan/2-hydroxy-5-(4-nitrophenylazo)-benzaldehyde (CHNAB)
and chitosan/2-hydroxy-5-(4-tolylazo)-benzaldehyde (CHMAB)
derivatives using 79% and #90% deacetylated chitosan in iso-
propyl alcohol/water mixture and dimethyl sulfoxide under mild
conditions. The thermal property of the derivatives has been
investigated by differential scanning calorimetry (DSC) and
thermogravimetric analysis (TGA). The methyl group containing
derivative CHMAB shows wide spectrum antimicrobial activity
against S. aureus which is much higher compared to free chito-
san. Chitosan derivatives, both having azobenzene chromophore
attached to chitosan polymer systems, display photoinduced
birefringence in the form of solid lms.
Polymer based azo materials have attracted huge attention in
the past few decades due to their enormous potential applica-
tions in optical information transmission, optical data storage,
optical switching, nonlinear optical (NLO) materials.1–6 Poly-
meric nonlinear optical (NLO) materials have been widely used
for lower switching voltage and higher band width electro-optic
devices and have become a subject area of intense research
interest in recent years. Properties such as high electro-optic
constant and low dielectric constant have made the NLO poly-
mers more advantageous which has been extensively demon-
strated by different research groups.7–11 For polymeric based
devices to effectively compete with inorganic materials, issues
such as optical insertion loss and thermal stability have to be
improved.
Chitosan possesses interesting characteristics, such as its
ability to induce a minimal foreign body reaction, an intrinsic
aDepartment of Chemistry, Motilal Nehru National Institute of Technology, Allahabad
– 211004, India. E-mail: pkd@mnnit.ac.in; tamalghosh@mnnit.ac.in; Fax: +91 532
2545341
bDepartment of Physics, Jilin University, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra27210f
‡ Present address: Department of Chemistry, University of Coimbra, Coimbra,
Portugal.
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(DMAc) and isopropyl alcohol solution and washed several
times with isopropyl alcohol/water mix-solvent. The nal
product was dried and kept in desiccators for further studies.
Experimental methods
Preparation of chitosan azo dye derivatives
2-Hydroxy-5-(4-nitrophenylazo)-benzaldehyde/2-hydroxy-5-(4-tolyl-
azo)-benzaldehyde was dissolved in 5 mL of isopropyl alcohol
and 1 mL of water was added to raise the dielectric constant of
the isopropyl alcohol more than 40% of the solution. 50 mg of
chitosan powder was immersed into the acetic acid solution at
pH 4 (maintaining 4 : 5 molar ratio for chitosan : azo dye) and
slowly stirred at room temperature for half an hour and the
swollen mixture was kept in 0 ꢀC for 3 days. The obtained
product was successively washed with an isopropyl alcohol and
water mixture (5 : 1, v/v) and dried in air (Scheme 1). The
product was re-crystallized from N,N-dimethylacetamide
Characterization of the azo based chitosan derivatives CHNAB
and CHMAB
The azo dyes 2-hydroxy-5-(4-nitrophenylazo)-benzaldehyde
(HNAB) and 2-hydroxy-5-(4-tolylazo)-benzaldehyde (HMAB)
were synthesized according to literature procedure.16 The
recrystallised azo dyes were nally added in chitosan matrix
resulting in the formation of the corresponding Schiff bases.
Chitosan derivative obtained from HNAB and HMAB is named
as CHNAB and CHMAB respectively. The azo based chitosan
derivatives were characterized by FTIR spectroscopy. The
Scheme 1 Preparation of chitosan derivatives CHNAB and CHMAB.
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infrared spectra were recorded on Perkin Elmer RX1 FTIR laser was 23 mW and the diameter of the beam was approxi-
spectrophotometer using KBr pallets. Thermogravimetric anal- mately 0.3 cm.17,18
ysis (TGA) with Perkin Elmer diamond and differential scanning
calorimetry (DSC) with Pyris-6 heating and cooling rates of 10 ꢀC
were used.
Results and discussion
Preparation
of
chitosan/2-hydroxy-5-(4-nitrophenylazo)-
benzaldehyde (CHNAB) and chitosan/2-hydroxy-5-(4-tolylazo)-
benzaldehyde (CHMAB) derivatives (in 4 : 5 molar ratio) under
mild conditions is shown in Scheme 1. We have synthesized two
different derivatives CHNAB and CHMAB to explore the effect of
electron-withdrawing (NO2) and electron-donating (CH3) group,
respectively in thermal, antibacterial and birefringence
properties.
Biological activity: antimicrobial assay
The antimicrobial activity of chitosan and chitosan containing
azo based Schiff bases were carried out using agar plate diffu-
sion method. In this method, the solution (1 mg mLꢁ1) of the
Schiff base was absorbed in sterilized discs (approximately 60
mL of solution) and the antimicrobial activity was evaluated
against two different test cultures viz. Gram negative bacteria
E. coli and Gram positive bacteria S. aureus. The sterilized discs
were placed on nutrient agar plates making lawns of the above
test cultures. The plates were then incubated at 37 ꢀC for 24 h.
The diameters of the inhibitory zone surrounding discs were
then measured.
Differential scanning calorimetry analysis
DSC curves of chitosan derivatives recorded in nitrogen from
ꢀ
ꢀ
10 C temperature to 400 C are shown in Fig. 2. The tempera-
tures for various thermal effects are represented for the deriv-
ꢀ
ative CHNAB, the endothermic peaks at 91 C due to the type
and length of the side chains. Endothermic peak at 212 ꢀC and
exothermic peak at 231 ꢀC is due to the phase change and
simultaneously decomposition of derivative, in most of the
Birefringence measurement
Birefringence in azo-polymer lms may be created by trans–cis–
trans photoisomerization processes produced with a linear
polarized light, with the induced orientation of the chromo-
phores being perpendicular to the polarization direction. The
anisotropic molecular orientation results in stable photoin-
duced birefringence or dichroism which can be erased by
randomization of the alignment with circularly polarized light
or heat. The experimental setup for the measurement of the
photoinduced birefringence is shown in Fig. 1. The 532 nm line
from a Nd:YAG laser was used as a pump (or writing) beam, and
the 632.8 nm line from a He–Ne laser was used as a probe (or
reading) beam. To carry out the experiments, a lm sample was
placed between a pair of crossed polarizers. To achieve
maximum signal, the polarization vector of the pump beam was
set to 45ꢀ with respect to the polarization vector of the probe
beam. When needed, a quarter-wave plate (not shown in the
gure) was inserted into the optical path of the pump beam
before the sample (the quarter-wave plate converted the linear
polarized beam to circular polarization which removed the
birefringence that had been induced). The power of the pump
Fig. 1 Experimental setup for the measurement of optically induced
birefringence.
Fig. 2 DSC thermogram of (a) CHNAB and (b) CHMAB.
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nitro group (–NO2) containing compounds such decomposition
trends is observed.19 In CHMAB, the endothermic peak at 73 ꢀC
is due to the presence of side chains, and peak at 222 ꢀC is due
to the phase change.
Thermogravimetric and differential thermal analysis
Fig. 3 shows the thermogravimetric analysis of chitosan deriv-
atives reveal the fact that the decomposition occurs in three
consecutive steps. In case of CHNAB degradation of rst step
4% loss by mass is in the temperature range of 60–94 ꢀC. Due to
the side chain groups present in derivative, second step 51%
loss by mass in the temperature range 94–273 ꢀC, due to acetyl
and various hydrogen bonding functional groups present in the
chitosan derivative. Third step 45% loss by mass in the
temperature range 273–480 ꢀC due to the loss of C]N and other
moieties. No residual is le lastly, so the nitro derivative is less
stable as compared to methyl one which can be justied by
ꢀ
decomposition temperature (Td) ¼ 227 C and where as Td for
methyl derivative is Td ¼ 250 ꢀC.
In case, of CHMAB the same rst step 20% loss by mass in
the temperature range of 60–260 ꢀC due to the groups present in
the chitosan derivative. Second step 27% loss by mass in
ꢀ
temperature range 260–370 C due to C]N and some groups.
Third step 67.6% loss by mass in the range 370–650 ꢀC and
residual of 6.4% is le. This shows that methyl derivative is
more stable as compared to nitro derivative which is also sup-
ported by DSC.
Fig. 4 DTA of (a) CHNAB and (b) CHMAB.
DTA of both derivatives shows the endothermic peaks
showing the phase change which can be easily observed in Fig. 4.
Antibacterial activity
Fig. 5 shows a typical antibacterial test result of chitosan as
control in DMSO (A) and chitosan based Schiff bases (B and C)
Fig. 5 Inhibitory effect of chitosan as control in DMSO (A), CHMAB (B)
and CHNAB (C) against E. coli (1) and S. aureus (2).
Fig. 3 TGA of (a) CHNAB and (b) CHMAB.
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Table 1 Diameter of inhibitory zone of chitosan in DMSO as control
and the Schiff bases against E. coli and S. aureus
polarization direction of the irradiating light nally leaves the
trans isomers predominantly oriented perpendicular to this
direction. Successive photoisomerization cycles result in a net
dichroism and birefringence induced in the material due to the
absorbance and refractive index difference in the parallel and
perpendicular directions (to the incident polarization
direction).
Fig. 6 shows the representative growth and decay traces of
the birefringence signal. As the azo compound molecules are
randomly oriented, no light is transmitted through the analyzer
in the beginning of the experiment. As the pump laser radiation
is introduced (point A in the gure), an anisotropic orientation
distribution is created as a result of the accumulation of cis
isomers and trans isomers that are oriented with the transition
dipole vertical to the polarization vector of the pump beam.
Light was, thus, transmitted through the analyzer because of
the onset of birefringence. When the excitation light is turned
off (point B in the gure), as shown in the gure, relaxation of
the anisotropic steady state occurs.
Diameter of inhibitory zone (mm)
Chitosan as control
Test culture
in DMSO (A)
CHMAB (B)
CHNAB (C)
E. coli
S. aureus
15
20
30
38
Nil
26
against S. aureus and E. coli as determined by the agar diffusion
plate method. As shown in the gure, antibacterial activity,
determined by the diameter of the growth inhibition zone, was
dependent on the test sample used. The inhibition zone
diameters (mm) of two samples were examined into two
microorganisms, and the results are shown in Table 1. The agar
diffusion test is a method commonly used to examine antimi-
crobial activity regarding the diffusion of the compound tested
through water-containing agar plate.
The appearance and size of the clear zone in the disk method
is mainly dependent on the ratio of disk area and size of inoc-
ulum, type of solid medium, and contact area. The surrounding
clearing zone of CHMAB showed a very clear inhibitory zone of
both Gram-positive and Gram-negative bacteria. It was gener-
ally caused by the maximum capability of chitosan polymer to
carry active agents beside the occurrence of functional groups
interaction phenomenon. The diffusion itself is dependent on
the size, shape and polarity of the diffusing material. The
antibacterial mechanism of chitosan is generally considered
due to its positively charged amino group at the C-2 position of
the glucosamine residue which interacts with negatively
charged microbial cell membranes, leading to the leakage of
proteinaceous and other intracellular constituents of the
microorganisms.20 The results revealed that chitosan azo
derivative with a methyl group shows a very clear and greater
inhibitory effect against S. aureus and E. coli in comparison to
the derivative with the nitro group. The effect of the polymer
complex on E. coli is not as effective as on S. aureus, which can
be attributed to their different cell wall structures, the major
constituent of its cell wall is peptidoglycan and there is very
little protein.
However, the relaxation is not complete; parts of the induced
birefringence are still held. To remove the remaining birefrin-
gence, circularly polarized light is introduced (point C in the
Birefringence studies
When illuminated with a polarized light of appropriate wave-
length, the azobenzene groups undergo a reversible trans–cis–
trans isomerisation process and an associated orientational
redistribution of the chromophores. Appropriate substitution
of the azobenzene chromophores may be used to tune the
chromatic features from UV through the visible spectrum. The
azo dye molecules are predominantly in the trans-state at room
temperature and are isotropically distributed. When excited by
a linearly polarized blue-green light (488 nm), preferential
molecular excitation and the associated photoisomerization
process results in an orientational hole burning. The orienta-
Fig. 6 Experimental birefringence results of CHNAB and CHMAB at
tional redistribution of the dye molecules with respect to the room temperature.
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bottom gure). The circularly polarized light effectively con-
verted the cis isomers to the trans form for all orientations. The
transmitted signal intensity induced by the birefringence is
proportional to sin2(pdDn/l), where d is the thickness of the
lm, Dn is the birefringence of the lm, and l is the wavelength
of the probe beam (632.8 nm).21,22 Because pdDn/l is much less
than 1, the transmitted signal is effectively proportional to
(Dn)2.23 Birefringence is observed more in case of CHNAB as
compared to the CHMAB because the derivative containing
nitro group (CHNAB) shows more transmittance as compared to
the methyl group derivative (CHMAB), as nitro group is acting
like an auxochrome.
Two competing processes dened the maximum achievable
birefringence: the orientation due to photoisomerization and
the thermal relaxation that tended to disorientate the azo-
benzene groups. As the temperature increases, the second
process becomes more important than the rst one. These two
competing processes are also likely to be relevant for the
kinetics of the build up and decay of birefringence. The curves
for the build up process can be well tted with a biexponential
function.23
Fig.
7
Polarized light induced photoisomerization of chitosan
attached azobenzene derivatives.
The curves of build up and decay processes can be well tted
by using
ꢁkbt
y ¼ A(1 ꢁ eꢁk t) + B(1 ꢁ e
)
(1)
a
mobility and relaxation of the polymer backbone. Recently
Kozanecka-Szmigiel et al. reported the photoinduced birefrin-
gence in azobenzene poly(esterimide) lm at blue excitation
wavelengths due to trans–cis–trans isomerization cycles associ-
ated with both the p–p* and n–p* transitions and the result is
in good agreement with our investigation.18
The tted dynamic parameters for both the derivatives are
listed in Table 2. In sample CHNAB the value of ka is less due to
the presence of intermolecular hydrogen bonding in cis
conformation, between nitro and hydroxyl group (Fig. 7) where
as in case of CHMAB hydrogen bonding is absent. Hence rate of
isomerisation is higher in CHMAB. Mobility rate kb is same in
both the derivative. Fast decay rate constant kc is higher in case
of CHNAB because cis–trans back-reaction starts more favour-
ably in case of CHNAB due to intramolecular hydrogen bonding
as compared to CHMAB. Slow rate constant kd is higher in case
of CHMAB as it needed more time to be in contact with the
circular polarized light.
where, A and B are pre exponential factors, t is the time, and ka
and kb are the rate constants for writing the fast and slow
processes, respectively. Actually, ka has been shown to depend
on the quantum yield of the isomerization reaction, the isom-
erization rate, and the local mobility of the azo dye, which are
affected by the size of the azo moiety, the free volume around it,
and the strength of the coupling interactions between the azo
dye and the polymer backbone. The magnitude of kb depends
mainly on the polymer mobility. The curves for the decay could
also be tted with another biexponential function:
y ¼ Ceꢁk t + Deꢁk t+ E
(2)
c
d
where C and D are the amplitudes associated with the processes
having rate constants kc and kd, respectively and E is the back-
ground intensity, which can be minimized if, as mentioned
above, the circular polarized light is introduced to erase the
residual birefringence. The fast decay is due to the fast
component of the thermal cis–trans isomerisation and dipole
reorientation, whereas the slow decay is associated with the
Long term stability of birefringence (En) is more in case of
CHNAB as compared with that of CHMAB. This is also shown in
the Fig. 6, when the pump laser turned off, the decreased values
of birefringence for sample CHNAB are much larger than that
for sample CHMAB. The rigid aromatic structure of CHMAB
limits the relaxation process of the oriented chromophore
groups.
Table 2 Fitted dynamic parameters at room temperature for CHNAB
and CHMAB
Parameters
ka (sꢁ1
Sample CHMAB
Sample CHNAB
Conclusions
)
0.034
0.004
0.0103
0.0025
0.76
0.016
0.004
0.034
0.023
0.64
Thermal study of azo derivatives established that CHMAB is
thermally more stable than CHNAB. The Schiff base containing
methyl group CHMAB, shows wide spectrum antimicrobial
activity against S. aureus which is much higher compared to free
chitosan. The results of the photoinduced birefringence showed
kb (sꢁ1
)
kc (sꢁ1
kd (sꢁ1
En
)
)
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