Preparation and Characterization

Of
Silk Films Coated With Chitosan
A Major Project Report submitted in partial fulfillment for the award
of the degree
Of

BACHELOR OF TECHNOLOGY
in

POLYMER SCIENCE AND CHEMICAL TECHNOLOGY

Submitted by
Himanshu Jain
Mansi Arora
Mohd Murtaza
Vinayak Sharma

Under the guidance of

Dr. Roli Purwar
Assistant Professor

DEPARTMENT OF APPLIED CHEMISTRY & POLYMER

TECHNOLOGY
DELHI TECHNOLOGICAL UNIVERSITY
MAIN BAWANA ROAD DELHI-11042

CERTIFICATE
This is to certify that the work incorporated in this major project entitled Insert Project
Name here submitted by Himanshu Jain, Mansi Arora, Mohd Murtaza, Vinayak Sharma
towards the requirement for the partial fulfilment for the award of the degree of B. Tech in
Polymer science and Technology of the Delhi Technological University was carried out
under my supervision and guidance during the session 2013-14. To the best of my knowledge
and belief, this work has not been submitted to any other university or institutions for the
award of any degree or diploma.

Dr. Roli Purwar

Prof. D. Kumar

Assistant Professor

Head of Department

Department of Applied

Department

Chemistry and

Chemistry and

Polymer Technology

Polymer Technology

Delhi Technological University

Delhi Technological University

Delhi

Delhi

of

Applied

ACKNOWLEDGEMENT
We wish to express our deep sense of gratitude and veneration to our project guide, Dr. Roli
Purwar, Assistant Professor, Department of Applied Chemistry and Polymer Technology,
Delhi Technological University, Delhi, for her perpetual encouragement, constant guidance,
valuable suggestions and continued motivation which has enabled us to complete this work.
We are deeply indebted to Prof. D. Kumar, Head, Dept. of Applied Chemistry and Polymer
Technology, for providing all the lab facilities and the opportunity to undertake this work.
Our special appreciation goes to Chandramohan Srivastava Sir for his invaluable support,
suggestions and guidance for timely completion of this project. We would also like to
acknowledge Prof. G. L. Verma, Prof. A.P.Gupta, Prof. R. C. Sharma, Dr. Archana Rani, Mr.
S. G. Warkar, Dr. RoliPurwar, Dr. RaminderKaur, Dr. Saurabh Mehta, Dr. D. Santhiya and
Dr. Anil Kumar for their support.
We are also thankful to all laboratory staff and colleagues for their co-operation.

Himanshu Jain
Mansi Arora
Mohd Murtaza
Vinayak Sharma

Index
S.No.

Topic

Abstract

Introduction

Silk and its properties

Chitosan and its properties

Wound dressing Application

Experimental Procedure

Results and Interpretation

Conclusion

References

Page No.

Abstract
In the present work Silk Films are produced from regenerative/degummed silk fibroins by
dissolving it in formic acid solution and chitosan coating of varied concentrations was done
on the silk films and their effect is studied. Change in mechanical properties and structural
properties is studied by using UTM machine and FTIR spectroscopic method respectively
with changing chitosan concentration. Chitosan is chosen as a coating material due to its
antibacterial properties and ability to provide strength to various natural fibres on coating.

Introduction
Silk from the silkworm Bombyx mori has been used as biomedical material for centuries.
This reflects the high biocompatibility of silk, despite silk being a foreign protein to
mammals. As a substrate, silk protein is good for mammalian cell adhesion and proliferation.
Recent studies reported the use of silk in oral administration. The excellent biocompatibility
and functionality of silk has led to the development of various biomedical devices.
Chitosan is the N-deacetylated derivative of chitin, a linear polysaccharide found in the
exoskeleton of crustaceans and other natural sources. Chemically, chitosan is (14)-2amino-2-deoxy--D-glucopyranose, which has a chemical structure similar to cellulose, but
with an amino or acetyl functional group substituting the hydroxyl group at carbon #2.
Among its many properties, chitosan is non-toxic, anti-microbial, soluble in dilute acids, nonsoluble in organic solvents, and capable of hydrogen and electrostatic bonding with organic
substrates. Importantly, chitosan is capable of coating and strengthening textile fibres.
Due to these properties of chitosan we have tried to investigate the effect of chitosan coating
on silk fibroin films dissolved in formic acid. Mainly emphasis is given to the changes in
mechanical properties.

SILK and its properties

Silk is a natural protein fiber, some forms of which can be woven into textiles. The protein fiber of
silk is composed mainly of fibroin and is produced by certain insect larvae to form cocoons. The bestknown type of silk is obtained from the cocoons of the larvae of the mulberry silkworm Bombyx
mori reared in captivity (sericulture). The shimmering appearance of silk is due to the
triangular prism-like structure of the silk fibre, which allows silk cloth to refract incoming light at
different angles, thus producing different colors.

Silks are produced by several other insects, but generally only the silk of moth caterpillars
has been used for textile manufacturing. There has been some research into other silks, which
differ at the molecular level. Many silks are mainly produced by the larvae of insects
undergoing complete metamorphosis, but some adult insects such as web spinners produce
silk, and some insects such as raspy crickets produce silk throughout their lives. Other types
of arthropod produce silk, most notably various arachnids such as spiders (see spider silk).
Production process
The entire production process of silk can be divided into several steps which are typically
handled by different entities. Extracting raw silk starts by cultivating the silkworms on
mulberry leaves. Once the worms start pupating in their cocoons, these are dissolved in
boiling water in order for individual long fibres to be extracted and fed into the spinning reel.
A special manufacturing process removes the outer irritant sericin coating of the silk, which
makes it suitable as non-absorbable surgical sutures. This process has also recently led to the
introduction of specialist silk underclothing for people with eczema where it can significantly
reduce it. New uses and manufacturing techniques have been found for silk for making
everything from disposable cups to drug delivery systems and holograms. To produce 1 kg of
silk, 104 kg of mulberry leaves must be eaten by 3000 silkworms. It takes about 5000
silkworms to make a pure silk kimono. The production of silk is called sericulture. The major
silk producers are China (54%) and India (14%).

Properties
Physical properties
Silk fibers from the Bombyx mori silkworm have a triangular cross section with rounded
corners, 510 m wide. The fibroin-heavy chain is composed mostly of beta-sheets, due to a
59-mer amino acid repeat sequence with some variations.[40] The flat surfaces of the fibrils
reflect light at many angles, giving silk a natural sheen. The cross-section from other
silkworms can vary in shape and diameter: crescent-like for Anaphe and elongated wedge
for tussah. Silkworm fibers are naturally extruded from two silkworm glands as a pair of
primary filaments (brin), which are stuck together, with sericin proteins that act like glue, to
form a bave.

Fig.1- SILK FIBROIN STRUCURE

Silk has a smooth, soft texture that is not slippery, unlike many synthetic fibers.
Silk is one of the strongest natural fibers, but it loses up to 20% of its strength when wet. It
has a good moisture regain of 11%. Its elasticity is moderate to poor: if elongated even a
small amount, it remains stretched. It can be weakened if exposed to too much sunlight. It
may also be attacked by insects, especially if left dirty.
Silk is a poor conductor of electricity and thus susceptible to static cling.
Unwashed silk chiffon may shrink up to 8% due to a relaxation of the fiber macrostructure,
so silk should either be washed prior to garment construction, or dry cleaned. Dry
cleaning may still shrink the chiffon up to 4%. Occasionally, this shrinkage can be reversed
by a gentle steaming with a press cloth. There is almost no gradual shrinkage nor shrinkage
due to molecular-level deformation.
Natural and synthetic silk is known to manifest piezoelectric properties in proteins, probably
due to its molecular structure.

Composition: The silk fibre is chiefly composed of 80% of fibroin, which is protein in nature
and 20% of sericin, which is otherwise called as silk gum.

Strength: Silk as a fibre, has good tensile strength, which allows it to withstand great pulling
pressure. Silk is the strongest natural fibre and has moderate abrasion resistance. The strength
of the thrown yarns is mainly due to the continuous length of the fibre. Spun silk yarn though
strong is weaker than thrown silk filament yarns.

Elasticity: Silk fibre is an elastic fibre and may be stretched from 1/7 to 1/5 of its original
length before breaking. It tends to return to its original size but gradually loses little of its
elasticity. This would mean that the fabric would be less sagging and less binding resulting in
the wearers comfort.

Resilience: Silk fabrics retain their shape and have moderate resistance to wrinkling. Fabrics
that are made from short staple spun silk have less resilience.

Drapability: Silk has a liability and suppleness that, aided by its elasticity and resilience,
gives it excellent drapability.

Heat Conductivity: Silk is a protein fibre and is a non-conductor of heat similar to that of
wool. This makes silk suitable for winter apparel.

Absorbency: Silk fabrics being protein in nature have good absorbency. The absorptive
capacity of the silk fabric makes comfortable apparel even for warmer atmosphere. Fabrics
made from silk are comfortable in the summer and warm in the winter. Silk fibre can
generally absorb about 11 percent of its weight in moisture, but the range varies from 10
percent to as much as 30 percent. This property is also a major factor in silks ability to be
printed and dyed easily.

Cleanliness and Washability: Silk fabric does not attract dirt because of its smooth surface.
The dirt, which gathers can be easily removed by washing or dry cleaning. It is often
recommended for the silk garments to be dry-cleaned. Silk fabrics should always be washed
with a mild soap and strong agitation in washing machine should be avoided. Silk water

spot easily, but subsequent washing or dry cleaning will restore the appearance of the fabric.

Reaction to Bleaches: Silk, like wool, is deteriorated with chlorine bleaches like sodium
hypochlorite. However, mild bleach of hydrogen peroxide or sodium per borate may be used
for silk.

Shrinkage: Silk fabrics are subjected only to normal shrinkage which can be restored by
ironing. Crepe effect fabrics shrink considerably in washing, but careful ironing with a
moderately hot iron will restore the fabric to its original size.

Effect of Heat: Silk is sensitive to heat and begins to decompose at 330 F (165 C). The silk
fabrics thus have to be ironed when damp.

Effect of Light: Silk fabric weakens on exposure to sun light. Raw silks are more resistant to
light than degummed silk.

Resistance to Mildew: Silks will not mildew unless left for sometime in a damp state or
under the extreme conditions of tropical dampness.

Resistance to Insects: Silk may be attacked by the larvae or clothe moths or carpet beetles.

Reaction to Alkalis: Silk is not as sensitive as wool to alkalis, but it can be damaged if the
concentration and the temperature are high. A mild soap or detergent in lukewarm water is
thus advisable.

Reaction to Acids: Concentrated mineral acids will dissolve silk faster than wool. Organic
acids do not harm silk.

Affinity for Dyes: Silk has good absorbency and thus has good affinity for dyes. Dyed silk is
colourfast under most conditions, but its resistance to light is unsatisfactory.

Resistance to Perspiration: Perspiration and sunlight weakens and yellows silk fabrics. The
silk itself deteriorates and the colour is affected causing staining. Garments worn next to the
skin should be washed or other wise cleaned after each wearing.

Tenacity - The silk filament is strong. This strength is due to its linear, beta configuration
polymers and very crystalline polymer system. These two factors permit many more
hydrogen bonds to be formed in a much more regular manner. Silk loses strength on wetting.
This is due to water molecules hydrolyzing a significant number of hydrogen bonds and in
the process weakening the silk polymer.

Specific gravity - Degummed silk is less dense than cotton, flax, rayon or wool. It has a
specific gravity of 1.25. Silk fibres are often weighted by allowing filaments to absorb heavy
metallic salts; this increases the density of the material and increases its draping property.
Elastic-plastic nature - Silk is considered to be more plastic than elastic because its very
crystalline polymer system does not permit the amount of polymer movement which could
occur in a more amorphous system. Hence, if the silk material is stretched excessively, the
silk polymers that are already in a stretched state (They have a betaconfiguration) will slide
past each other. The process of stretching ruptures a significant number of hydrogen bonds.

Elongation - Silk fibre has an elongation at break of 20-25% under normal condition. At
100%

R.H.

the

extension

at

break

is

33%.

Hygroscopic nature - Because silk has a very crystalline polymer system, it is less absorbent
than wool but it is more absorbent than cotton. The greater crystallinity of silk's polymer
system allows fewer water molecules to enter than do the amorphous polymer system of
wool.

It

absorbs

water

well

(M.R.11%),

but

it

dries

fairly

quickly.

Thermal properties - Silk is more sensitive to heat than wool. This is considered to be partly
due to the lack of any covalent cross links in the polymer system of silk, compared with the
disulphide bonds which occur in the polymer system of wool. The existing peptide bonds, salt
linkages and hydrogen bonds of the silk polymer system tend to break down once the
temperature

exceeds

1000C.

Electrical properties - Silk is a poor conductor of electricity and tends to form static charge
when it is handled. This causes difficulties during processing, particularly in dry atmosphere.

Hand feel - The handle of the silk is described as a medium and its very crystalline polymer
system imparts a certain amount of stiffness to the filaments. This is often misinterpreted, in
that

the

and

handle

is

regarded

regular

as

surface

soft,

because
of

of

the

smooth,

silk

even

filaments.

Drapes Property - Silk fibre is flexible enough and if silk fibre is used to make garments,
then the fabric drapes well and this is why it can be tailored well too.

Abrasion resistance - Silk fabric possess good abrasion resistance as well as resistance to
pilling.

Effect of sunlight - Silk is more sensitive light than any other natural fibre. Prolonged
exposure to sunlight can cause partially spotted color change. Yellowing of silk fibre is
generally occurred due to photo degradation by the action of UV radiation of sunlight. The
mechanism of degradation is due to the breaking of hydrogen bonds followed by the
oxidation

and

the

eventual

hydrolytic

fission

of

the

polypeptide

chains.

Chemical properties
Silk emitted by the silkworm consists of two main proteins, sericin and fibroin, fibroin being
the structural center of the silk, and serecin being the sticky material surrounding it. Fibroin is
made

up

of

the amino

acids Gly-Ser-Gly-Ala-Gly-Ala

and

forms beta

pleated

sheets. Hydrogen bonds form between chains, and side chains form above and below the
plane of the hydrogen bond network.
The high proportion (50%) of glycine, which is a small amino acid, allows tight packing and
the fibers are strong and resistant to breaking. The tensile strength is due to the many
interceded hydrogen bonds, and when stretched the force is applied to these numerous bonds
and they do not break.
Silk is resistant to most mineral acids, except for sulfuric acid, which dissolves it. It is
yellowed by perspiration. Chlorine bleach will also destroy silk fabrics.

Action of water - The absorption of water molecules takes place in the amorphous regions of
the fibre, where the water molecules compete with the free active side groups in the polymer

system to form cross links with the fibroin chains. As a result, loosening of the total
infrastructure takes place accompanied by a decrease in the force required to rupture the fibre
and increase extensibility. Treatment of silk in boiling water for a short period of time does
not cause any detrimental effect on the properties of silk fibre. But on prolonged boiling, silk
fibre tends to loss its strength to some degree, which thought to occur because of hydrolysis
action of water. Silk fibre withstands, however, the effect of boiling better than wool.

Effect of acids - Silk is degraded more readily by acids than wool. Concentrated sulfuric and
hydrochloric acids, especially when hot, cause hydrolysis of peptide linkages and readily
dissolve silk. Nitric acid turns the color of silk into yellow. Dilute organic acids show little
effect on silk fibre at room temperature, but when concentrated, the dissolution of fibroin
may take place. On treating of silk with formic acid of concentrated about 90% for a few
minutes, a swelling and contraction of silk fibre occur. Like wool, silk is also amphoteric
substance, which possesses the ability to appear as a function of the pH value either as an
acid or as a base.

Effect of alkalis - Alkaline solutions cause the silk filament to swell. This is due to partial
separation of the silk polymers by the molecules of alkali. Salt linkages, hydrogen bonds and
Van der Waals' forces hold the polymer system of silk together. Since these inter-polymer
forces of attraction are all hydrolyzed by the alkali, dissolution of the silk filament occurs
readily in the alkaline solution. Initially this dissolution means only a separation of the silk
polymers from each other. However, prolonged exposure would result in peptide bond
hydrolysis, resulting in a polymer degradation and complete destruction of the silk polymer.
Whatever, silk can be treated with a 16-18% solution of sodium hydroxide at low temperature
to produce crepe effects in mixed fabric containing cotton. Caustic soda, when it is hot and
strong, dissolves the silk fibre.

Action of oxidizing agent - Silk fibre is highly sensitive to oxidizing agents. The attack of
oxidizing agents may take place in three possible points of the protein 1. At the peptide bonds
of adjacent amino groups,
1. At the N-terminal residues and
2. At the side chains

Though fibroin is not severely affected by hydrogen peroxide solution, nevertheless may
suffer from the reduction of nitrogen and tyrosine content of silk indicate that hydrogen
peroxide may cause breakage of peptide bonds at the tyrosine residues resulting in the weight
loss of the fibre. The action of chlorine solution on the silk fibroin is more harmful than does
the solution of hypochlorite. These solutions, even at their lower concentration, cause damage
to fibroin.

Action of reducing agents - The action of reducing agents on silk fibre is still a little bit
obscure. It is, however, reported that the reducing agents that are commonly found in use in
textile processing such as hydrosulfite, sulfurous acids and their salts do not exercise any
destructive action on the silk fibre.

Chitosan
Chemistry and Applications
Chitosan is a de-acetylated derivative of chitin. Chitin is a natural linear chain polysaccharide
found in the exoskeletons of crustaceans and insects, the cell walls of fungi, and other natural
sources. Chitosan is produced in large amounts as a by-product of the food industry by
treating crustacean shells with acid. Chitosan is obtained by treating chitin with sodium
hydroxide. Chemically, chitin is - (14)-2-acetamido-2-deoxy--d-glucose, or N-acetylglucosamine. The structure and function of chitin is analogous to that of cellulose, but the
hydroxyl functional group at carbon #2 in cellulose is substituted by an acetamide group (
NHCOCH3) in chitin, and by an amino group (-NH2) in chitosan. Chitosan is (14)-2amino-2-deoxy--Dglucopyranose (or de-acetylated chitin, or poly(D-glucosamine)), but
since the degree of deacetylation varies, it may best be described as a copolymer of -(14)D-glucosamine and N-acetyl-D-glucosamine. Chitosan can be modified at its functional
groups (the amino and hydroxyl groups) to alter its properties and functionality, just as
cellulose can be modified to produce cellulose ethers, esters, and polymers. Unlike cellulose,
chitosan is hydrophobic. It is not soluble in most organic solvents, but is soluble in dilute
acidic aqueous solutions below pH 6. Due to its chemistry, chitosan is compatible with
organic substrates, including silk. Its amino and hydroxyl functional groups can form
hydrogen bonds with polar substrates. Chitosan is cationic, and can form stable electrostatic
bonds with negatively charged substrates.

Fig.2- CHITOSAN STRUCTURE

Additionally, chitosan is non-toxic and has anti-bacterial and anti-fungal effects. Chitosan is
used for various industrial purposes. Since chitosan is non-toxic, antimicrobial, biocompatible, bio-degradable, and non-allergenic, it is used in many biomedical applications,
including wound dressings, tissue engineering, and suturing material. It is also has uses in
food processing, agriculture, ophthalmology, photography, water treatment, textile
processing, and textile finishing.
Chitosan is also found in cosmetic products such as shampoos, as cationic chitosan can coat
negatively charged hair fibres, increasing their softness and mechanical strength. Chitosan is
used in textile processing to increase the wet strength and dye reception of cotton, and can be
used as a finishing agent for wool by coating wool scales to prevent felting. Chitosan is also
used as an antimicrobial finish for silk and cotton textiles. These various textile finishes
perform well and withstand multiple washings, demonstrating chitosans stability and
compatibility with natural fibre substrates. Chitosan has many attractive qualities, such as the
ability to bond to and coat silk fibres, impart them with strength, and provide them with
microbial resistance. Because of its polysaccharide structure, chitosan can form films on
organic substrates that are strong, durable, elastic, and gas and moisture-permeable. Chitosan
is also an abundant natural material, and is non-toxic to humans. The application method is
safe, as it does not require the use of organic solvents, only dissolution in dilute acids (acetic
acid is commonly used as it is readily available). Initial tests of chitosan consolidation in
conservation suggest chitosan imparts organic substrates with strength, without causing
colour change, but sometimes causes an increase in substrate stiffness. Tests of chitosan in
industrial settings (such as using chitosan to improve dye uptake and microbial resistance in
textiles) found that treated silks were stiffer than untreated silks.
Some studies have found that chitosan is not resistant to UV-induced photooxidation. It was
found that UV irradiation of chitosan caused chain scission, fibre weakening, and an increase
in the amount of carbonyl groups in the polymer due to oxidation, but UV exposure did not
cause cross-linking in the chitosan polymer; similar results were obtained by Bajer and
Kaszmarek (2010). These findings suggest chitosan may be vulnerable to yellowing due to
the formation of chromophoric carbonyl groups and embrittlement by chain scission when
exposed to UV light and photo-radicals. So, while chitosan may strengthen and consolidate
fragile silk textiles, it may also stiffen the fabrics and cause yellowing with age and light
exposure. These qualities would disqualify chitosan as a suitable treatment for silk textiles
and other artifacts. It is important to note, however, that these effects were observed in

experiments using chitosan for industrial applications, where testing conditions were
presumably more extreme than those encountered in museums or used in conservation
studies. Chitosan may be stable in the mild environmental conditions in museums, especially
if light is limited and filtered to remove UV radiation. When considering a new treatment
method for conservation, the question of reversibility arises. Chitosan is soluble in acidic
solutions below pH 6, so theoretically, it could be removed from an artifact, and the treatment
could be considered reversible.
If an artifact is so fragile that it requires consolidation, removal of the consolidant (reversal of
the treatment) would likely cause substantial damage to the object, if not complete
destruction. In some cases, invasive and essentially irreversible treatments like consolidation
and impregnation may be necessary to stabilize an artifact. Even adhesive impregnated
backings may not be easily removed from a textile, as some adhesive residue may remain on
textile fibres, but this may be acceptable if the treatment stabilizes the artifact. Future
treatments and examination of an artifact should be not hindered by conservation treatments,
so if not reversible, treatments should at least allow re-treatment of an artifact. It is
emphasized that the hypothetical re-treatability of silk textiles consolidated with chitosan.
Chitosan treatment would not interfere with other future treatments, as it is not soluble in
organic solvents or neutral pH water, solvents typically used in textile treatments.
Consolidated textiles could still be cleaned by chemical or mechanical means, and other
adhesive could still be used, without interfering with the chitosan consolidation.

Biomedical uses
Chitosan's properties allow it to rapidly clot blood, and has recently gained approval in the
United States and Europe for use in bandages and other hemostatic agents. Chitosan
hemostatic products have been shown in testing by the U.S. Marine Corps to quickly stop
bleeding and to reduce blood loss, and result in 100% survival of otherwise lethal arterial
wounds in swine. Chitosan hemostatic products reduce blood loss in comparison to gauze
dressings and increase patient survival. Chitosan hemostatic products have been sold to
the U.S. Army and are currently used by the UK military. Both the US and UK have already
used the bandages on the battlefields of Iraq and Afghanistan. Chitosan is hypoallergenic and
has natural antibacterial properties, which further support its use in field bandages.

Chitosan hemostatic agents are often chitosan salts made from mixing chitosan with an
organic acid (such as succinic or lactic acid). The hemostatic agent works by an interaction
between the cell membrane of erythrocytes (negative charge) and the protonated chitosan
(positive charge) leading to involvement of platelets and rapid thrombus formation. The
chitosan salts can be mixed with other materials to make them more absorbent (such as
mixing with alginate), or to vary the rate of solubility and bioabsorbability of the chitosan
salt. The chitosan salts are biocompatible and biodegradable making them useful as
absorbable haemostats. The protonated chitosan is broken down by lysozyme in the body to
glucosamine and the conjugate base of the acid (such as lactate or succinate) are substances
naturally found in the body. The chitosan salt may be placed on an absorbable backing. The
absorbable backing may be synthetic (for instance made from existing absorbable suture
materials e.g. Tephaflex polymer) or natural (e.g. cellulose or gelled/solidified honey).
Chitosan's properties also allow it to be used in transdermal drug delivery; it is mucoadhesive
in nature, reactive (so it can be produced in many different forms), and most importantly, has
a positive charge under acidic conditions. This positive charge comes from protonation of its
free amino groups. Lack of a positive charge means chitosan is insoluble in neutral and basic
environments. However, in acidic environments, protonation of the amino groups leads to an
increase in solubility. The implications of this are very important to biomedical applications.
This molecule will maintain its structure in a neutral environment, but will solubilize and
degrade in an acidic environment. This means chitosan can be used to transport a drug to an
acidic environment, where the chitosan packaging will then degrade, releasing the drug to the
desired environment. One example of this drug delivery has been the transport of insulin.

Applications Wound Dressing

The silk sutures are usually coated with wax or silicone to enhance the material properties
and reduce fraying. On the other hand, coated sutures may provoke inflammatory tissue
reactions if the pieces of the coating flake off and migrate into surrounding tissues. To reduce
this problem, coating should have an affinity for suture filaments. It has been reported that
the elimination of wax coating significantly diminished the high thrombotic response to silk.
Therefore, an innovative coating material is needed to improve the frictional characteristics
of silk sutures.
Furthermore, silk is a natural protein fiber and easily prone to microbial infection. The use of
antimicrobial sutures is expected to provide protection from wound infection. Several
approaches have been reported in developing antimicrobial sutures. Recently, the
incorporation of chitosan (Figure 1) on the surface of sutures is one of the approaches
adopted to impart antimicrobial activity to sutures. The application of chitosan coating to a
suture material has a benefit of smoothing suture surface, combating bacterial colonization
and enhancing certain other properties.
Chitosan is the deacetylated derivative of chitin, which is the main component of the shells of
crustaceans such as shrimps, crabs and lobsters. Chitosan has been found to inhibit the
growth of microbes in a large body of work that has been reported by several researchers.
The antimicrobial mechanism is not clear but is generally accepted that the primary amine
groups provide positive charges which interact with negatively charged residues on the
surface of microbes. Such interaction causes extensive changes in the cell surface and cell
permeability, leading to leakage of intracellular substances. The antimicrobial ability,
coupled with its nontoxicity, biodegradability and biocompatibility is facilitating chitosans
emerging applications in biomaterials engineering. In order to impart antimicrobial activity,
chitosan was successfully applied on to various suture materials, namely polylactic acid
sutures, polypropylene sutures, polyester sutures and cotton sutures. Even though several
attempts have been made to use chitosan polymer as antimicrobial agent, its use along with
silk suture has been limited. Frictional properties of textile materials are normally measured
over a range of applied normal loads and sliding speed. A large number of studies have been
carried out on the frictional properties of textile fiber assemblies. The effect of applied
normal loads and sliding speed on the frictional characteristics of woven and nonwoven

wipes was investigated by Das et al.. In another study, Das et al. have explored the frictional
characteristics of woven suiting and shirting fabrics. Ramkumar et al. have studied the effect
of applied normal loads and sliding speed on the frictional characteristics of needle punched
nonwoven fabrics. A very few literature is available with respect to the frictional properties
of chitosan-treated textiles. Bhuvana et al. investigated the frictional properties o chitosantreated wool fabrics. In another study, Jeong et al. characterized the frictional properties of
chitosan-treated wool fabrics using the Kawabata evaluation system.
To the authors best knowledge, there is no published data available on the frictional
properties of chitosan coated suture materials. In the present work, chitosan was applied on to
the braided silk suture (BSS) at three different concentrations. The effects of chitosan
treatment on the frictional, tensile and antimicrobial characteristics of BSS were studied.
Bacterial species are capable of colonizing different surfaces and proliferating on them,
forming adherent biofilms. This could represent a major problem for implantable suture
material. Experimental and clinical data indicate that most wound infections begin around
material left within the wound, and that the incidence of late suture complications are directly
related to the degree of contamination at the time of material placement. Measures for
reducing the risk of surgical site infections include surgical technique, appropriate
antimicrobial agent, adjunctive strategies for reducing wound contamination and promoting
wound healing.
In this study, chitosan was used as an antimicrobial agent to provide protection against
various microorganisms. Chitosan has been found to inhibit the growth of microbes in a large
body of work that has been reported by several researchers. Chitosan is a very attractive
material because it exhibits various promising biological activities such as antimicrobial
activity, biocompatibility, low toxicity, complete biodegradability hemostatic activity, scar
preventability and acceleration of wound healing. The antimicrobial efficiencies of both the
chitosan-treated and untreated BSS were assessed using agar diffusion method and shake
flask method. The gram positive S. aureus (ATCC 6538) and the gram-negative E. coli
(ATCC 11230) were used as test microorganisms because they are the major cause of cross
infection in hospitals. S. aureus is the most frequently evaluated species. It causes skin and
tissue infections, septicemia, endocarditis and meningitis. E. coli is a popular test organism
and is resistant to common antimicrobial agents. It causes urinary tract and wound infections,
common in gastrointestinal tract and accounts for 25% of hospital infections.

The zone of inhibition values for untreated and chitosan-treated BSS against both E. coli and
S. aureus are presented in Table 1. For the untreated BSS, no zone of inhibition was found
against both E. coli and S. aureus. For the treated BSS, antimicrobial activity is better against
both bacteria. The most accepted mechanism for microbial inhibition by chitosan is the
interaction of the positively charged chitosan with the negatively charged residues at the cell
surface of many fungi and bacteria, which causes extensive cell surface alterations and alters
cell permeability. This causes the leakage of intracellular substances such as electrolytes, UV
absorbing material, proteins, amino acids, glucose and lactate dehydrogenase. As a result,
chitosan inhibits the normal metabolism of microorganisms and finally leads to the death of
these cells. S. aureus being a gram-positive bacterium has a thicker cell wall, and hence, it is
more resistant to chitosan than E. coli. This fact is well documented by the previous studies
also. The microbial reduction percentage for untreated and chitosan -treated BSS against E.
coli and S. aureus determined by shake flask method is presented in the Table 1. It is
generally agreed that the bacterial reduction rates of at least 99% are needed to retard the
exponential growth of most microorganisms. The Table 1 shows that microbial reduction is
better at higher chitosan concentration against both bacteria. Furthermore, chitosan is more
effective against gram-negative bacterium than gram-positive bacterium. The reason for the
same has been explained earlier.

Experimental Procedure
Materials Used
The materials and apparatus used in this research were as listed below:

Mulberry Silk

Formic Acid

Chitosan

Deionized Water

Sodium Bicarbonate

Beakers (4-100ml, 2-500ml)

Measuring Cylinder

Heater And Oven

Weighing Balance

Petri Dishes (With Poly Styrene Sheet at the bottom)

Film Preparation

Mulberry Silk Fibers were extracted from silkworm cocoons manually.

As Silk Fiber protein cannot be directly soluble in Formic Acid, therefore they are
first degummed by boiling silk fibers in sodium bicarbonate for 1 hour.

This degummed fiber is thoroughly washed with deionized water twice to extract the
glue like sericin proteins.

Now this degummed fiber is dried in oven for 2 days at 40 degree Celsius.

The dried fiber is cut into very small pieces using a scissor for better dispersion in
solvent (Formic Acid).

Now 1.5 grams of fiber is weighed and dissolved in 10 ml of Formic acid Solution
slowly while stirring at 45 degree Celsius until fiber disperses in the solution.

This solution is cast on a petri dish with Poly Styrene sheets and dried for 12 hours at
50 degree Celsius.

5 such samples are prepared using the same process.

Now chitosan solution is prepared with varied concentrations (0.75%,1%.1.5%,2%

w/w) in 10ml of deionized water.

This solution is poured uniformly on silk fibroin films and the films are kept to dry for
12 hours at 45 degree Celsius.

Now these sheets are characterized by performing mechanical testing in an UTM machine
and FTIR spectra was also obtained using a FTIR spectrometer.

Formulations
Sample No. Silk Fibers (gm)

Formic Acid

Chitosan

(ml)

(% w/w)

S1.

1.5

10

0.1

S2.

1.7

10

0.2

S3.

1.5

10

0.1

S4.

1.5

10

1.0

S5.

1.5

10

0.8

Mechanical Properties
Tensile test was performed to evaluate the tensile strength and elongation at break. It was
tested using the UTM machine. The thickness of measured specimen was then placed in the
grips of the testing machine and the grips was tightened evenly and firmly to prevent
slippage. The tensile strength, and elongation at break was then calculated.

Tensile testing, also known as tension testing, is a fundamental materials science test in
which a sample is subjected to a controlled tension until failure. The results from the test are
commonly used to select a material for an application, for quality control, and to predict how
a material will react under other types of forces. Properties that are directly measured via a
tensile test are ultimate tensile strength, maximum elongation and reduction in area. From

these

measurements

the

following

properties

can

also

be

determined: Young's

modulus, Poisson's ratio, yield strength, and strain-hardening characteristics.

A tensile specimen is a standardized sample cross-section. It has two shoulders and a gauge
(section) in between. The shoulders are large so they can be readily gripped, whereas the
gauge section has a smaller cross-section so that the deformation and failure can occur in this
area.
The most common testing machine used in tensile testing is the universal testing machine.
This type of machine has two crossheads; one is adjusted for the length of the specimen and
the other is driven to apply tension to the test specimen. There are two types: hydraulic
powered and electromagnetically powered machines.
The machine must have the proper capabilities for the test specimen being tested. There are
four main parameters: force capacity, speed, and precision and accuracy. Force capacity
refers to the fact that the machine must be able to generate enough force to fracture the
specimen. The machine must be able to apply the force quickly or slowly enough to properly
mimic the actual application. Finally, the machine must be able to accurately and precisely
measure the gauge length and forces applied; for instance, a large machine that is designed to
measure long elongations may not work with a brittle material that experiences short
elongations prior to fracturing.
Alignment of the test specimen in the testing machine is critical, because if the specimen is
misaligned, either at an angle or offset to one side, the machine will exert a bending force on
the specimen. This is especially bad for brittle materials, because it will dramatically skew
the results. This situation can be minimized by using spherical seats or U-joints between the
grips and the test machine. If the initial portion of the stressstrain curve is curved and not
linear, it indicates the specimen is misaligned in the testing machine.
The strain measurements are most commonly measured with an extensometer, but strain
gauges are also frequently used on small test specimen or when Poisson's ratio is being
measured. Newer test machines have digital time, force, and elongation measurement systems
consisting of electronic sensors connected to a data collection device (often a computer) and
software to manipulate and output the data. However, analog machines continue to meet and
exceed ASTM, NIST, and ASM metal tensile testing accuracy requirements, continuing to be
used today.

The test process involves placing the test specimen in the testing machine and slowly
extending it until it fractures. During this process, the elongation of the gauge section is
recorded against the applied force. The data is manipulated so that it is not specific to the
geometry of the test sample. The elongation measurement is used to calculate
theengineering strain, , using the following equation:

where L is the change in gauge length, L0 is the initial gauge length, and L is the final
length. The force measurement is used to calculate the engineering stress, , using the
following equation:[4]

where F is the tensile force and A is the nominal cross-section of the specimen. The machine
does these calculations as the force increases, so that the data points can be graphed into
a stressstrain curve.

Chemical Interactions and Bonding

Fourier transform infrared spectroscopy (FTIR) was used to identify chemical features and
shifts due to interactions between functional groups via a Bruker FTIR with a resolution of 2
cm-1. The FTIR was used in reflectance mode using a Golden Gate attenuated total
reflectance attachment with a diamond objective.

FT-IR stands for Fourier Transform Infrared, the preferred method of infrared spectroscopy.
In infrared spectroscopy, IR radiation is passed through a sample. Some of the infrared
radiation is absorbed by the sample and some of it is passed through (transmitted). The
resulting spectrum represents the molecular absorption and transmission, creating a molecular
fingerprint of the sample. Like a fingerprint no two unique molecular structures produce the
same infrared spectrum. This makes infrared spectroscopy useful for several types of
analysis.
It can identify unknown materials
It can determine the quality or consistency of a sample

 It can determine the amount of components in a mixture

Infrared spectroscopy has been a workhorse technique for materials analysis in the laboratory
for over seventy years. An infrared spectrum represents a fingerprint of a sample with
absorption peaks which correspond to the frequencies of vibrations between the bonds of the
atoms making up the material.
Because each different material is a unique combination of atoms, no two compounds
produce the exact same infrared spectrum. Therefore, infrared spectroscopy can result in a
positive identification (qualitative analysis) of every different kind of material. In addition,
the size of the peaks in the spectrum is a direct indication of the amount of material present.
With modern software algorithms, infrared is an excellent tool for quantitative analysis.
Fourier Transform Infrared (FT-IR) spectrometry was developed in order to overcome the
limitations encountered with dispersive instruments. The main difficulty was the slow
scanning process. A method for measuring all of the infrared frequencies simultaneously,
rather than individually, was needed.
A solution was developed which employed a very simple optical device called an
interferometer. The interferometer produces a unique type of signal which has all of the
infrared frequencies encoded into it. The signal can be measured very quickly, usually on
the order of one second or so. Thus, the time element per sample is reduced to a matter of a
few seconds rather than several minutes.
Most interferometers employ a beam-splitter which takes the incoming infrared beam and
divides it into two optical beams. One beam reflects off of a flat mirror which is fixed in
place. The other beam reflects off of a flat mirror which is on a mechanism which allows this
mirror to move a very short distance (typically a few millimeters) away from the beamsplitter. The two beams reflect off of their respective mirrors and are recombined when they
meet back at the beam splitter. Because the path that one beam travels is a fixed length and
the other is constantly changing as its mirror moves, the signal which exits the interferometer
is the result of these two beams interfering with each other. The resulting signal is called an
interferogram which has the unique property that every data point (a function of the moving
mirror position) which makes up the signal has information about every infrared frequency
which comes from the source.

This means that as the interferogram is measured, all frequencies are being measured
simultaneously. Thus, the use of the interferometer results in extremely fast measurements.
Because the analyst requires a frequency spectrum (a plot of the intensity at each individual
frequency) in order to make an identification, the measured interferogram signal can not be
interpreted directly. A means of decoding the individual frequencies is required. This can
be accomplished via a well-known mathematical technique called the Fourier transformation.
This transformation is performed by the computer which then presents the user with the
desired spectral information for analysis

Results and Discussion

Mechanical Properties - Strength
SAMPLE
NO.

TENSILE STRENGTH
(In MPa)

ELONGATION AT
(in mm)

S1

0.10771

0.93109

S2

0.09914

1.10206

S3

0.14642

0.85787

S4

0.25584

1.04375

S5

0.55145

1.45619

BREAK

Tensile Strength(MPa)
0.6
0.55145
0.5
0.4
0.3
0.25584
0.2
0.14642
0.1

0.10771

0.09914

0
3

Graph.1- Tensile Strength

Elongation At Break(mm)
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0

Graph.2- Elongation at Break

From the above table it can be concluded that:

It is seen from the results that with increasing concentration of chitosan in coating on
silk films results in increasing tensile strength.
Also increasing the concentration of chitosan results in increasing elasticity and
higher elongation at break. This may be due to the better binding of fibers by the
chitosan thereby offering better resistance to the axial load.
Higher tensile strength along with higher elasticity by increasing chitosan
concentration in film coating is observed which can be advantageous in wound
dressing.

Structural Properties (FTIR Analysis)

IR Spectrum Of Silk Fibroin

IR Spectrum of Silk Coated With Chitosan

The above Infrared spectrum shows peaks at 1613 cm-1 and 1614 cm-1 corresponding to amide
I area and peaks at 1219 cm-1 and 1220 cm-1 appears in amide III area and c-c skeletal
stretching band appears at 1151 or 1158 cm-1. These bands are attributed to -helix and/or
random coil conformation indicating the molecular conformation of silk fibroin.
FTIR spectra of regenerated silk fibroin films show both random coil and/or -sheet structure
depends on the conformation of silk fibroin, which depends on types of regeneration solvent.
Hence addition of chitosan does not affect the -structure which is desirable.

Conclusion
The purpose of this experiment was to determine whether chitosan was an effective and
suitable consolidant for fragile silk films. The main areas of interest were the strengthening
and consolidating effects of chitosan and variation in structural properties of the films. Any
other visual, chemical, or physical changes in the substrate caused by treatment were also of
interest.
The addition of chitosan led to increase in mechanical properties such as tensile strength and
elongation at break. This may be attributed to the better binding of fibres by the chitosan
thereby offering better resistance to the axial load.
Also structural analysis shows that the -conformation of silk fibroins in the film is still intact
after addition of formic acid and chitosan. This is desirable for applications of silk films.
So due to increasing mechanical strength and stable structural properties further studies can
be done for wound dressing applications due to biocompatibility of silk films and anti
bacterial properties of chitosan blended together.

References

www.ijrat.org/downloads/jan-2014/paper%20id-212014103.pdf
http://en.wikipedia.org/wiki/Silk
http://en.wikipedia.org/wiki/Chitosan
http://cool.conservation-us.org/anagpic/2013pdf/anagpic2013_kata_paper.pdf
http://scholarsresearchlibrary.com/dpl-vol5-iss4/DPL-2013-5-4-88-97.pdf
www.jeffjournal.org/papers/Volume8/JEFF8-03-13.M.Montazer.pdf
http://teroauvinen3.files.wordpress.com/2013/07/journal-of-industrial-textiles-2013viju-256-68.pdf
http://www.sciencedirect.com/science/article/pii/S0928493113007042
www.researchgate.net/...silk.../0fcfd50a2344adb8e5000000