Report of UGC Minor Project
“Synthesis and Photophysical Properties of Fluorescent
Chitosan”
UGC Approval No. and Date: MRP(S)-1402/11-12/KLKE 046/UGCSWRO 28-09-12
Principal Investigator: Dr. Pearl Augustine
Co-Investigator: Dr. P. Manoj
Department of University/College: Department of Chemistry,
St. Michael’s College, Cherthala, Kerala.
Chapter I
INTRODUCTION AND LITERATURE REVIEW
1. General
Chitin is the second most abundant natural polymer in the world after
cellulose.1 Most commonly chitin means the skeletal material of invertebrates. It is found
in exoskeletons, peritrophic membranes and cocoons of insects. The shells of crabs,
prawns and lobsters coming from peeling machines in canning factories are used for the
industrial preparation of chitin. The isolated chitin is a highly ordered co-polymer of 2acetamido-2-deoxy--D glucose and 2-amino-2-deoxy -D-glucose. However, chitin has
poor solubility than cellulose because of the high crystallinity of chitin supported by
hydrogen bonds mainly through the acetamido group.2
1.1.Extraction of Chitin
Chitin can be extracted from crustacean shells by a treatment in a dilute solution
of NaOH (1-10%) at high temperature (85-100°C). Shells are then demineralised to
remove calcium carbonate by treating in a dilute solution of HCl (1-10%), at room
temperature. The degree of polymerization, acetylation and purity will be affected by the
severity of temperature, duration and concentration of chemicals.1
Chitosan is an important derivative of chitin. It is obtained by the deacetylation of
chitin. The removal of acetyl group is a harsh treatment usually performed with conc.
NaOH solution.
Chitin
Figure 1.1: Chemical structure of Chitosan
Chitosan is a biological product with cationic properties. It is of great interest,
than most of the polysaccharides of the same type as they are neutral or negatively
charged.1 Chitosan is a biocompatible, antibacterial and environmentally friendly
polyelectrolyte. Since it is biocompatible it does not elicit adverse reactions when in
contact with human cells. It is recognized by tumour cells and therefore it can bring the
drugs to their target selectively and hence has been widely studied as a drug carrier.
Chitosan is more versatile compared to chitin due to the presence of amino groups in C-2
position.2
1.2.Chitosan: Chemical Modifications
Chitosan is a linear polyamine having reactive amino groups and hydroxyl groups.
Researchers have achieved dramatic improvement in the properties of chitosan by
chemical manipulations of these functional groups on the chitosan backbone.
Modifications of chitin and chitosan often have to be conducted under heterogeneous
conditions with some exceptions in solution.
1.2.1. N-Deacetylation
Removal of the acetyl group from chitin is a harsh treatment with concentrated
NaOH solution; either aqueous or alcoholic .Protection from oxygen is necessary in order
to avoid depolymerisation and generation of reactive species. Deacetylation permits
bringing the polymer into solution by salt formation.2
1.2.2. Methylation
Trimethyl chitosan chloride (TMC) (80% degree of quaterenization),bearing
antennary galactose residue through a 6-0 linked carboxy methyl (CM) group served as
more suitable DNA carrier than plain chitosan .Synthesis of TMC is reported as treating
methyl iodide with low molecular weight chitosan.3
1.2.3. Tosylation
Introduction of bulky groups such as tosyl group destroys the tight crystalline
arrangement in chitosan and improves solubility.
1.2.4. Thiolation
Polymer bearing thiol groups provide much higher adhesive properties than
polymers generally considered to be mucoadhesive. Chitosan thioglycolic acid conjugates
were found to show ten-fold increase in adhesion property compared with unmodified
chitosan.4
1.2.5. Silylation
Silylation is a convenient modification for further reaction due to potential
solubility and reactivity .Both primary and secondary hydroxy groups get easily reacted
by silylation.
1.2.6. Azidated Chitosan
A new photo cross-linkable chitosan bearing p-azido benzoic acid has been
reported to have biomedical applications. This derivative can be crosslinked by UV
radiation and resulting hydrogel was found to have strong tissue-adhesion as well as
induction of wound contraction and healing.5
1.3.Chemically Modified Chitosan: Applications
Chitosan is a highly biocompatible, non-toxic, biodegradable polymer having anti
microbial activity as well as many biomedical applications. Chitosan is used as
biodegradable packing material for food wraps, in water treatment, chromatography, as
drug carrier, food additive, wound dressing materials, tissue engineering and skin
substitute.6
1.3.1. Industrial Applications
1.3.1.1. Water Engineering
Due to its polycationic nature chitosan can be used as a flocculating agent.
Chitosan is used in waste water treatment because it can absorb dyes and other heavy
metals from water. There are several reports on the metal chelating properties of
chemically modified chitosans.
1.3.1.2. Cosmetics
Since chitosan facilitates interaction of cosmetics with skin covers and hair it is
widely used in these applications. Chitosan is positive and hair is negative in charge .This
helps in the formation of a clear elastic film on hair increasing its softness and
smoothness. It can be used in shampoos, rinsers, permanent wave agents, dyes, as
moisturiser for skin, creams, lotions, nail enamel, etc. Chitosan is also used as dental filler
due its antimicrobial property.7
1.3.1.3. Photography
It is used as s fixing agent for the acid dyes in gelatine and improves diffusion in
developing films.
1.3.1.4. Chromatography
The presence of amino group, primary and secondary hydroxyl groups, makes it a
good support for chromatographic separation. Chlorophenols and phenols were separated
by HPLC using chitosan as a solid support.
1.3.1.5. Food Industry
Since it is non toxic, chitosan is used in manufacture of proteins fortified bread,
carrier for food ingredients like dyes and nutrients.8
1.3.2. Biomedical Applications
1.3.2.1. Drug Delivery
Chitosan is an alternative to synthetic drug carriers since it is non toxic and
biodegradable. The use of polymers susceptible to the hydrolytic bacterial enzymes seems
today to be the most convenient approach to the delivery of drugs. Chitosan is soluble at
acidic pH and becomes insoluble at approximately pH 6.5. Enteric coatings of chitosan
can protect chitosan from the acidity of stomach. In intestine enteric coating dissolves and
the chitosan core will be exposed to bacterial enzymes simultaneously releasing the
drugs.9
1.3.2.2. Gene Delivery
Chitosan has potential application as gene carrier. The low toxicity of chitosan
and its nature make it attractive for gene delivery purposes.11
1.3.2.3. Wound Healing
Chitosan based products provide improved healing of surgical wound. In all cases
the healing process is faster and smooth scars are retained.12
1.4. Scope and Objectives
Chemical modification of biopolymers, especially chitosan is an area of active
research. Due to its excellent properties like biocompatibility, biodegradability and
crystallinity, this polymer finds applications in a host of applications. However, there are
also a number of reports on the dramatic improvement of the properties of chitosan via
chemical modifications. For example, chitosan suffers from limited solubility in
physiological pH and causes presystemic metabolism of drugs in intestinal and gastric
fluids in the presence of proteolytic enzymes10. These inherent drawbacks of chitosan can
be overcome by chemical derivatisations like carboxylation, thiolation, etc. As a part of
an ongoing program to explore the chemical reactivity of chitosan as well as its potential
applications, in the present project we have prepared a chitosan derivative which would
be tried as a promising candidate in water treatment, antimicrobial applications as well as
drug delivery.
There are several reports on triazine linked chitosan as an intermediate for
conjugation with several bioactive molecules. Cyanuric chloride is the most versatile
linker employed for conjugation. The two labile chlorine atoms present on this
intermediate provides room for further conjugation with other nucleophiles. Several
derivatives of this intermediate prepared by reaction with corresponding amines have
been reported to complex cations from aqueous solutions13. We have explored the
possibility of forming simple Schiffs bases of chitosan by treatment with aromatic
aldehydes. There are several reports on the Schiff’s base derivatives of chitosan which
find applications in protein purifications, etc.14 Thus we have treated chitosan with
isovanillin to form the corresponding Schiffs base derivative of chitosan. Though the
synthesised Schiff base derivative of chitosan with isovanillin did not show any
fluorescent behaviour, efforts are being made to incorporate flurophores on to the
modified polymer. The chemically modified biopolymer was characterized by FT-IR
spectroscopy and later subjected to thermal decomposition studies by Differential
Scanning Calorimetry.
The main objectives of the study were:
1. Synthesis of Schiff’s bases of chitosan with isovanillin
2. Characterization of Schiff’s base of chitosan with isovanillin.
3. Thermal studies of the above said chitosan derivative by Differential Scanning
Calorimetry.
Chapter 2
MATERIALS AND METHODS
2.1. Materials
Chitosan and isovanillin used in this study were purchased from Sigma Aldrich.
All solvents were distilled and dried prior to use. FT-IR spectra were recorded on a
Shimadzu IR Affinity-1 spectrometer. Thermal studies were carried out on Mettler Toledo
DSC 822e.
2.2. Methods
2.2.1. Synthesis of Schiff’s Base derivative of Chitosan with Isovanillin
About 500 mg chitosan was dissolved in 25 mL of 2% acetic acid taken in a 100
mL RB flask. To this solution was added 2 g isovanillin in 5mL methanol and the
reaction mixture was stirred at room temperature for 24 hours. The solid product thus
obtained was filtered out and washed several times with methanol and diethyl ether. It
was then dried under vacuum and taken for characterization. (Scheme 1) The reaction of
chitosan with vanillin involves the condensation of a primary amino group of chitosan
with the carbonyl group of vanillin resulting in an unstable imino bond by the elimination
of a water molecule.
HO
HO
[O
HO
[O
OHC
O
O
NH2
NH2
O
OMe
O ]n
OH
HO
O
O
N
OH
OH
OMe
Scheme 1
]n
Chapter 3
RESULT AND DISCUSSION
Chemical modifications of biopolymers have been reported to impart significant
enhancement of their properties. For example, chitosan the versatile biopolymer has been
found to be an excellent platform for synthetic manipulations because of its reactive
functional groups which are amenable to chemical reactions. In the present work, we have
explored the chemical reactivity of chitosan with isovanillin. The prepared schiff’s base
derivative of chitosan was characterized by FT-IR spectroscopy and their thermal
behaviour was studied using differential Scanning Calorimetry.
3.1. Chitosan Derivative: FT-IR Studies
3.1.1. Chitosan-Isovanillin Schiffs base Derivative
The IR spectrum exhibits peaks at 1020 cm-1 corresponding to C-O-C and C-O
stretching of glucosamine unit. A new peak at 1640 cm-1 indicates the presence of C=N
(imine) bond. A peak at 3431 cm-1 corresponds to O-H stretching. Peaks at 1442 cm-1 and
1515cm-1 shows the presence of aromatic rings .Phenyl group is identified by the peaks at
750 cm-1 and 806 cm-1. A peak at 1280 cm-1 corresponds to para substituted -OCH3
stretching.
3.2. Chitosan Derivative: Thermal Studies
The DSC thermograms of deacetylated chitosan show a wide endothermic peak
at around 100 °C and an exothermic peak at around 273 °C. The endothermic peak could
be attributed the dehydration of the water contained in the chitosan whereas the
exothermic peak corresponds to the thermal decomposition of the polymer.
The thermogram of chitosan- isovanillin Schiff’s base derivative shows a shift in
the endothermic peak to a lower temperature when compared to chitosan [93.43°C].
During the chemical modification the amino group was replaced with a bulky
hydrophobic group which diminished the hydrogen bonding capacity of the modified
polymer. The exothermic peak appears at 283°C indicating that the modification does not
have influence on the thermal stability of the polymer.
55
2272.24
2124.68
2037.88
%T
452.33
671.26
569.99
895.01
1065.72
1020.39
3431.51
30
1280.79
35
1515.15
1442.82
2924.21
2858.63
1640.53
40
806.28
4695.90
45
4394.03
4291.80
50
25
4500
4000
3500
3000
2500
2000
1750
1500
1250
1000
Figure 3.1: FT-IR Spectrum of Chitosan Schiffs Base with Isovanillin
750
500
1/cm
Figure 3.2: DSC Thermogram of Chitosan Schiffs Base with Isovanillin
Chapter 5
CONCLUSION
Chitosan is a versatile biopolymer with a host of interesting properties that makes it
suited for a spectrum of applications including biomedical, pharmaceutical and engineering
applications. Chemical modifications of chitosan aimed at improvement or fine tuning of
properties is also an area of active pursuit. Thus in the present work, we have synthesized a
Schiff’s Base derivative of chitosan by treating chitosan with isovanillin. The chemically
modified chitosan polymer compound was characterized by FT-IR spectroscopy and later
subjected to thermal decomposition studies. Further attempts are being made to incorporate
fluorophores on to the modified polymer and explore the utility of these chemically modified
biopolymers in water treatment, protein chromatography and antimicrobial applications.
13
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14