MISCIBILITY STUDIES OF CHITOSANdigilib.library.usp.ac.fj/gsdl/collect/usplibr1/index/... · 2011....

Miscibility Studies of Chitosan with other polymers as a

function of Degree of Deacetylation of Chitosan

By

Parvish Nikesh KUMAR

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of

Master of Science in Chemistry

School of Biological, Chemical and Environmental Sciences, Faculty of Science,

Technology and Environment, The University of the South Pacific

2008

DECLARATION OF ORIGINALITY

Statement by Author

I hereby declare that the work contained in this thesis is my very own and where I have

used the thoughts and works of others I have clearly indicated this.

……………………………..

Parvish Nikesh Kumar

Student ID Number: S99007780

23rd of February 2009

Statement by Supervisor

I hereby confirm that the work contained in this thesis is the work of Parvish Nikesh

Kumar unless otherwise stated.

Dr. Jagjit Rai Khurma

Division Coordinator (Chemistry) and Senior Lecturer

23rd of February 2009

ii

ABSTRACT

Chitosan samples with 86, 91 and 97% degree of deacetylation (DD) values were

characterized using the titration and IR methodologies. The DD values were calculated

using graphs plotted from the second derivative of the titration data. It was found that the

titration method of DD analysis was more accurate than IR method.

Chitosan samples with different DD values were blended with PVB, PVF and PEO and

their blend properties were studied. The glass transition temperature of chitosan was

studied using Differential Scanning Calorimeter (DSC) and Dynamic Thermal

Mechanical Analyser (DMTA). Four thermal transitions between temperature ranges 90

to 2070C were identified for chitosan samples using DMTA.

It was also observed that solvent used to make the polymer films suppressed the thermal

transitions. An endothermic peak was observed at 69.440C in the DSC thermogram of

pure PEO powder while for PEO films made in formic acid showed this endothermic

peak at 64.740C. It was evident that the solvent had some effect on the glass transition

temperature.

Intermolecular interactions studied using Fourier Transform Infrared Spectrometer

(FTIR) showed the presence of hydrogen bonding in the Chs/PVB blend system.

Chs/PVF system showed possible loss of intermolecular interaction between the amide II

iii

proton of chitosan and the oxygen of the PVF glycosidic linkage. Chs/PEO systems

showed strong intermolecular interactions as well.

DSC, DMTA and Scanning Electron Microscope (SEM) analysis proved the Chs/PVB,

Chs/PVF and Chs/PEO systems to be immiscible. Despite the Chs/PVB, Chs/PVF and

Chs/PEO systems being immiscible, their mechanical properties were good. It was also

seen that with varying DD values the resultant blends had better tensile strength (TS) as

well as percentage elongations for the Chs/PVB blend system. Increasing the DD values

of chitosan increased the TS of Chs/PVF blend systems from 31.1N/mm2 (Chs80PVF

Chs1) to 33.1N/mm2 (Chs80PVF Chs3). Chs20PVF with the DD value of chitosan

sample being 97% had the highest tensile strength (38.0 N/mm2) in Chs/PVF blend

systems. PEO had the highest percentage elongation value (731.5%) in this research

work. Increasing DD values of chitosan samples brought about the loss of percentage

crystallinity of PEO in Chs/PEO blend system from 14.6% (Chs75PEO) in Chs/PEO

blend systems where DD of chitosan was 86% to 9.2% crytallinity (Chs75PEO) in

Chs/PEO blend film for chitosan having a DD value of 96%.

The antibacterial activity of chitosan and the blends showed that Chs/PVB and Chs/PVF

blend systems had almost the same trends in antibacterial activities. Chs/PEO blend

systems showed the best antibacterial activity. The antibacterial activity of chitosan

samples increased with increasing DD values. PVB and PVF had no antibacterial activity

while PEO showed some antibacterial activity.

iv

Chs90PEO with the DD of chitosan sample being 97% had the highest antibacterial

activity and produced the greatest inhibition zone in comparison to all other blend

systems. Chitosan sample with a DD value of 97% had maximum antibacterial activity in

comparison to all the antibacterial activities of pure polymers in this study.

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ACKNOWLEDGEMENTS

I extend my gratitude and thanks to the University of the South Pacific Research

Committee for providing partial financial support for my research.

My sincere thanks are also extended to my supervisor, Dr. Jagjit Rai Khurma for his

encouragement, assistance, guidance, wisdom and help which he provided throughout the

course of this research. This research couldn’t have been possible without Dr. Khurma’s

expert guidance.

I also thank Mr. David Rohindra for his discussions and assistance during my research.

My thanks are due to the technicians at the Department of Chemistry and the Department

of Biology of the University of the South Pacific. I am also grateful to the Medical

Laboratory Technology staff of Fiji School of Medicine and the staff of Microbiology

Department of Colonial War Memorial Hospital for their assistance during my

antimicrobial studies. The assistance provided by Prof. Kamal Kishore, Head of School

of Health Sciences, during the antimicrobial studies is also very much appreciated.

To Dr. Kevin Blake of James Cook University, Townsville, Australia for his expert

assistance provided to me during SEM studies and to Prof. David Yellowlees for

allowing me to use their facility at the campus, I extend my appreciation. I am grateful

and indebted to Professor Alan Easteal of Auckland University, New Zealand, for his

assistance during the DMTA analysis of my samples.

vi

My thanks are also due to Mr Ritesh Kumar, Mr Jagdishwar Chand, Mr Ashveen Nand,

Mr Thomas Tunidau, Mr Randhir Charan and Mr Ravin Deo for their company during

the long hours of research.

I also thank, Shalvin Kumar and Nilesh Kumar for their companionship during the

writing phase of my thesis, Dr. Vijay Singh and Dr. Pratim Choudhury (Fiji School of

Medicine) for their critical comments and discussion.

Finally, I convey my appreciation to my family, colleagues and all those who had helped

me during this research.

vii

Table of Contents

Page

i. Declaration of Originality ii

ii. Abstract iii

iii. Acknowledgement vi

iv. Table of Contents viii

v. List of Abbreviations xiii

1.0 Introduction 1

1.1 Polymers 1

1.1.1 Synthetic Polymers 3

1.1.1.1 Poly (vinyl Butyral) (PVB) 4

1.1.1.2 Poly (vinyl formal) (PVF) 4

1.1.1.3 Poly (ethylene oxide) (PEO) 5

1.1.2 Biopolymers 6

1.2 Polymer Miscibility 9

1.2.1 Thermodynamics of Polymer blends 9

1.2.2 Methods of Miscibility Study 12

2.0 Literature Review 14

2.1 Methods DD value determination 14

2.1.1 Titration 15

viii

2.1.2 Infrared (IR) Spectrometry 17

2.1.3 Other Methods 21

2.2 Effects of DD on physical and chemical properties of chitosan

polymer blends 23

2.2.1 Mechanical property evaluation studies 24

2.2.2 Thermal properties of chitosan 25

2.2.3 Anti-microbial studies 26

2.3 Blends of chitosan of different DD 30

2.3.1 Chitosan blends with Poly (�-caprolactone) (PCL) 31

2.3.2 Chitosan blends with Poly (vinyl pyrrolidone) PVP 32

2.3.3 Chitosan blends with Polyacrylamide 33

2.3.4 Chitosan blends with Poly (vinyl alcohol) PVA 33

2.3.5 Chitosan blends with Cellulose and Starch 35

2.3.6 Other polymers 36

3.0 Methodology 39

3.1 Materials 39

3.2 Determination of the Degree of Deacetylation (DD) of the samples 40

3.2.1 Titration 40

3.2.1.1 Standardization of sodium hydroxide and

hydrochloric acid solutions 40

3.2.1.2 Preparation of chitosan solution and titration 41

ix

3.3.2 FTIR Spectrometry 41

3.3 Miscibility Studies 42

3.3.1 FTIR Studies 43

3.3.2 DSC Studies 44

3.3.3 DMTA Studies 45

3.3.4 SEM Studies 45

3.4 Mechanical Property Studies 46

3.5 Antimicrobial Activity 46

3.5.1 Microbial suspension preparation 46

3.5.2 Conditions of incubation 47

3.5.3 Antimicrobial sensitivity disks 48

4.0 Results 49

4.1 Degree of Deacetylation Data 49

4.1.1 Titration 49

4.1.2 DD Determination using Infrared technique 54


4.2.1 Interaction studied using FTIR 56

4.2.1.1 Chitosan analysis 56

4.2.1.2 PVB analysis 58

4.2.1.3 PVF analysis 60

4.2.1.4 PEO analysis 62

4.2.1.5 Chs/PVB blend systems 64

x

4.2.1.6 Chs/PVF blend systems 68

4.2.1.7 Chs/PEO blend systems 72

4.2.2 Differential Scanning Calorimetry Data 76

4.2.2.1 Chs/PVB Systems 77

4.2.2.2 Chs/PVF Systems 78

4.2.2.3 Chs/PEO Systems 78

4.2.3 Dynamic Mechanical Thermal Analysis 82

4.2.4 Scanning Electron Microscopy (SEM) 83

4.3 Mechanical Property Studies 85

4.4 Antimicrobial Studies 92

4.4.1 Antimicrobial testing results 92

4.4.1.1 Antimicrobial testing results of pure polymers 93

4.4.1.2 Chs/PVB antimicrobial test results 94

4.4.1.3 Chs/PVF antimicrobial test results 95

4.4.1.4 Chs/PEO antimicrobial test results 96

5.0 Discussion 97

5.1 Determination of the Degree of Deacetylation 97

5.1.1 Comparison of DD values obtained using titration

and FTIR methodology 98


5.2.1 FTIR Studies 101

5.2.2 Thermal Studies 103

xi

5.2.3 Glass Transition temperature of Chs/PVB and

Chs/PVF blend films 104

5.2.4 Glass Transition temperature of Chs/PEO Systems 107

5.3 Mechanical property evaluations 109

5.4 Antimicrobial Studies 112

6.0 Conclusion 115

7.0 Bibliography 119

8.0 Appendix 133

8.1 KHP standard solution preparation 133

8.2 Standardization of Sodium Hydroxide Solution 133

8.3 Standardization of Hydrochloric Acid Solution 134

8.4 Determination of degree of deacetylation values of chitosan

samples using titration method 134

8.5 DD Determination using FTIR baseline (a) method 138

8.6 DSC Analysis of PVB and PVF 138

8.7 DSC Analysis of Chs/PVB System 140

8.8 DSC Analysis of Chs/PVF System 142

8.9 DSC Analysis of Chs/PEO System 143

8.10 DMTA Analysis 145

xii

LIST OF ABBREVIATIONS

C13 NMR Carbon-13 Nuclear Magnetic Resonance

CTFE Chlorotrifluorethylene

DA Degree of Acetylation

DD Degree of Deacetylation

DNA Deoxyribonucleic acid

DSC Differential Scanning Calorimetry

DMTA Dynamic Mechanical Thermal Analysis

ECTFE Ethylene-Chlorotrifluoroethylene

FEP Fluorinated Ethylene Propylene

FTIR Fourier Transform Infrared

GlcN Glucosamine

GlcNAc N-Acetyl Glucosamine

HBr Hydrobromic Acid

IR Infrared

PCL Poly (�-caprolactone)

PE Poly (ethylene)

PEG Poly (ethylene glycol)

PEO Poly (ethylene oxide)

PFA Perfluoroalkoxy

PHB Poly (3-hydroxybutyric acid)

PVA Poly (vinyl alcohol)

PVAc Poly (vinyl acetate)

xiii

xiv

PVB Poly (vinyl butyral)

PVF Poly (vinyl formal)

PVP Poly (vinyl pyrrolidone)

RNA Ribonucleic acid

SEM Scanning Electron Microscope

TEM Transmission Electron Microscope

TFE Tetrafluoroethylene

Tg Glass Transition Temperature

Tm Melting point temperature

UV Ultraviolet

UV/VIS Ultraviolet/Visible

v/v Volume/volume

WAXD Wide-Angle X-ray Diffraction

wt/v Weight/volume

wt/wt Weight/weight

XRD X-Ray Diffraction

ZCP Zero Crossing Point

Chapter 1.0 Introduction

CHAPTER 1.0

Introduction

1.1 Polymers

Polymers are long chain molecules which consist of small repeat units called monomers

chemically linked together. Classes of polymers known as plastics, which can be easily

molded into desired shapes, have rapidly become an integral part of our lives. Plastics

have replaced traditional materials such as metal where high strength to low mass ratios

is needed as in bulletproof jackets. Its stability under varying conditions has made its

application enormous and this high demand for polymers for specific applications has

created a global interest in obtaining new polymers with desired properties.

New materials with improved properties for specific purposes are being achieved to some

extent by polymer blending. Polymer blending is done by physically mixing different

polymers together. Solution blending, melt mixing and reactive blending are some of the

techniques used in blending polymers. Solution blending involves the physical mixing of

the component polymers using a common solvent to prepare a solution. The resulting

solution is poured on a clean surface like glass plate and the solvent is evaporated leaving

a thin layer of polymer blend film. Melt mixing is very similar to solution mixing. In melt

mixing, the component polymers are thoroughly heated till they melt and then the

component polymers are mixed in their molten states by mechanical mixing. An

advantage of melt mixing over solution mixing is that polymer blends could be prepared

for component polymers, which do not have a common solvent. Reactive blending is

mainly done for incompatible polymer systems by usually adding a multifunctional

1


copolymer (reactive ingredient) to the incompatible polymer pair. This reactive

ingredient binds both the component polymers together forming a blend.

Polymer blending has proved to be an excellent and inexpensive method of obtaining

novel materials with desired physical and chemical properties. Polymer blending and

evaluation of the resultant blends chemical and physical properties is now one of the

leading areas of research in polymer technology.

A lot of research in polymer technology is currently focused on the blending of synthetic

polymers with biopolymers. Synthetic polymers do not have a natural origin but are

rather made by chemical reactions. A very common example of a synthetic polymer is

poly (vinyl chloride) or PVC commonly used to make household water piping.

Biopolymers on the other hand are naturally occurring polymers. Biopolymers readily

biodegrade and for this reason a lot of polymer blending is done where synthetic

polymers are blended with biopolymers to produce a new noble material that has

properties of the synthetic polymer as well as the biodegradable characteristic of

biopolymers. Examples of biopolymers are many including even our DNA. For this

study, the biopolymer chitosan was used. Chitosan is a derivative of chitin which is

extracted from crustaceans’ shells. More notes on chitin and chitosan are under Section

1.1.2.

2


1.1.1 Synthetic Polymers

Synthetic polymers are produced commercially on large scales employing several

techniques and chemical processes depending on the type of polymer intended to be

produced. Different synthetic polymers have different properties and applications ranges

from its use in food-packaging (poly (ethylene), drug delivery devices (poly (ethylene

glycol), electrical appliances (polystyrene) to its use in body armor (Kevlar).

The first synthetic polymer dates to 1839 when Charles Goodyear heated a mixture of

sulfur and natural rubber on a stove, however science of synthetic polymers was

developed in the 1920s, and by the mid-1940s scientists had invented polystyrene,

polyvinyl chloride, low-density polyethylene, polyacrylates and glass-fiber reinforced

polyesters, to name a few.

Polymers can be divided into 3 classes; thermoplastics, thermosets and elastomers.

Thermoplastics soften with increasing temperature and return to their original hardness

when cooled as most are meltable, while the thermosets harden when heated and retain

hardness when cooled. Thermosets "set" into permanent shape by catalysis or when

heated under pressure. Elastomers have good elongation properties and are soft and

deformable. The softness characteristic of elastomers is due to segmental motions as

elastomers exist above their glass transition temperatures.

3


1.1.1.1 Poly (vinyl butyral) PVB

Figure 1.1 Structure of PVB

PVB, a thermoplastic is produced by condensation reaction between poly (vinyl alcohol)

(PVA) and butyraldehyde [CH3(CH2)2CHO], with an acid as catalyst. PVB has excellent

adhesion for glass, metal, ceramics, leather, and fibers. It also has good light-resistance,

cold-resistance and water-resistance. It is now being widely used in all departments of

industry such as middle film for safety glass, porcelain-use paper, protection primer of

metal, insulation paint, fiber processing agent, printing ink, dye, artificial sponge, etc.

PVB is resistant to inorganic acids and fats as well. Studying the mechanical properties of

PVB allows foreseeing its future use in fields where strength is also needed together with

its other properties as stated above.

1.1.1.2 Poly (vinyl formal) (PVF)

Figure 1.2 Structure of PVF

4


PVF is also a thermoplastic and used in the process industries. It is made by condensing

formaldehyde in the presence of PVA or by the simultaneous hydrolysis and acetylisation

of Poly (vinyl acetate) (PVAc). PVF can be molded into any shape as per the requirement

in the process industry. PVF resin is also used as an outdoor weather-resistant coating.

The chemical stability of PVF makes it an ideal polymer to be used as a drug delivery

tool and withstand the low pH levels of the stomach. A compatible chitosan/PVF system

would be ideal for use as a drug delivery tool as chitosan has been shown to be safe for

human body (Kumar et al., 2005). The compatibility of the chitosan/PVF system is

explored in this research.

1.1.1.3 Poly (ethylene oxide) (PEO)

Figure 1.3 Structure of PEO

PEO is a low-melting solid prepared by polymerization of ethylene oxide and derives its

importance from the fact that it is water-soluble. PEO has been blended with chitosan and

their miscibility studied by means of FTIR and SEM. Chitosan/PEO blend films have

good mechanical properties, which increase with increasing PEO proportion (Alexeev et

al., 2001). The good mechanical properties of Chitosan/PEO blend films together with

the water solubility characteristic of PEO and the biocompatibility of chitosan (Kumar et

al., 2005) makes Chitosan/PEO also an ideal candidate to be used as a tablet coating of

medicinal drugs. Hence the miscibility of the Chitosan/PEO blend system was

investigated in this research work.

5


1.1.2 Biopolymers

The increasing demand for biodegradable materials has created a need to blend synthetic

with those that are biodegradable. Biopolymers or biological polymers are renewable and

produced by biological systems such as plants and animals. These are macromolecules in

living organisms formed by the linking of several smaller molecules (monomers)

together. Deoxyribonucleic acid (DNA) is one of the most ancient biopolymer formed

from several nucleotides linked together. Some common examples of biopolymers

include starch, proteins and peptides, cellulose derivatives, gelatin, pectin, alginate,

polylactic acid, poly-3-hydroxybutyrate and chitin.

Chitin is the second most abundant natural polysaccharide (Knorr, 1982) and is found

mainly in the shells of crustaceans, such as crabs, shrimp and lobsters. Industrial

processing of exoskeletons of crustaceans yields between 20 to 40% of chitin (No and

Meyers, 1995). It is also found in the exoskeleton of marine plankton, including coral and

jellyfish. The wings of insects such as butterflies, ladybugs, cell walls of yeast,

mushrooms and other fungi also contain chitin (Riccardo et al., 1986).

Chemically, chitin is very similar to cellulose except that the 2-hydroxy group of the

cellulose has been replaced with an acetamide group.

6


Figure 1.4 Structural comparisons of Cellulose and Chitin

Although, there are many methods of extraction of chitin from the crustacean shells; the

underlying principles of extraction are relatively simple. The proteins are removed by

treatment in a dilute solution of sodium hydroxide (1-10%) at high temperature (85-

100°C). Shells are then demineralised to remove calcium carbonate. This is done by

treating the shells in a dilute solution of hydrochloric acid for 15 min at ambient

temperature in an excess of 0.25 M HCl with a solid-to-liquid ratio of about 1/40 (w/v)

(Percot et al., 2003).

The physico-chemical characteristics of the extracted chitin will vary depending on the

severity of these treatments such as temperature, duration, concentration of the chemicals,

concentration and size of the crushed shells. These treatments affect the three most

important characteristics of the chitin i.e., degree of polymerization, acetylation and

purity. Shells also contain lipids and pigments. A decolorising step is sometimes needed

to remove pigments and lipids for a white chitin sample. This is done either by soaking

the sample in organic solvents or in dilute solution of sodium hypochlorite. These

treatments however will have an influence on the characteristics of the chitin molecules

and its derivative chitosan.

7


Chitosan, a polycationic biopolymer, is the deacetylated derivative of chitin (Muzzarelli,

1983). The term chitosan is used when deacetylated chitin derivative can be dissolved in

weak acids. Chitosan is formed when chitin is heated in a strong solution of sodium

hydroxide (concentration >40% wt/v) at temperatures varying between 90-120°C. This

harsh treatment removes acetylic grouping on the amine radicals to a product (chitosan)

that could be dissolved in weak acids. At least 65% of the acetyl groups should be

removed on each monomeric chitin from the chitin polymeric chain to render it soluble

(Roberts, 1992).

Figure 1.5 Chitin and its derivative – Chitosan

The degree of deacetylation will vary according to the duration of treatment, the

temperature and the concentration of the sodium hydroxide solution. Many chemical

characteristics of chitosan (molecular weight, its polydispersity, the purity) are greatly

dependant on the choice of method, the equipment used and on the source of the shells. It

is necessary therefore, to control precisely the methods of production of chitosan to

obtain the desired characteristics needed for end use applications.

8


1.2 Polymer Miscibility

Miscibility of two or more polymers is an important criterion when using polymer

blending techniques to produce new polymer blends to suit specific needs. So the

complexity into the miscibility criteria of constituents in a polymer blend system is

important for investigations. It must be realized at this point that during solution blending

of two miscible polymers, two things happen; dissolution of component polymers with

the common solvent and miscibility of component polymers with each other.

The Flory-Huggins solution theory basically makes simplifying assumptions for the

Gibbs free energy change (�Gm) for the dissolution of polymer with solvent. This

research investigates miscibility between polymer and polymer. Despite the fact that

miscible polymer systems are usually preferred, immiscible polymer systems may also

have properties that are very much desired.

1.2.1 Thermodynamics of Polymer Blends

The enthalpy of a system with two or more components depends upon the interaction

between the molecules. Free energy of mixing (�Gmix), which includes both the entropic

and enthalpic terms, should be negative for a miscible polymer system.

�Gmix = �Hmix - T�Smix (1.1)

For complete miscibility, two conditions must be satisfied; �Gmix must be negative and

the second derivative of the free energy, �2�Gmix/��2, should be positive (Sergeant and

Koenig, 1993). The entropy of mixing is almost zero because of the large size of the

9


polymer chains. Therefore, the free energy is dependent only on the magnitude and the

sign of the enthalpy of mixing, �Hmix (Sergeant and Koenig, 1993).

The Flory-Huggins solution theory provides the extension to equating �Hmix and T�Smix

and has the following form:

�Hmix - T�Smix = RT[n1ln�1 + n2ln�2 + n1�2 12] (1.2)

Where R is the gas constant (8.314 J·K-1·mol-1) and T is the temperature. The n1 and �1

are the moles and volume fraction of solvent and n2 and �2 are moles and volume

fraction of polymer component. The parameter chi ( ) accounts for the energy of

interdispersing polymer and solvent molecules. Assuming that the polymer-solvent

system is occupying a common lattice structure, each site on the lattice is occupied by

one solvent molecule or a single monomer of polymer chain. Therefore the total number

of sites on the lattice structure is:

N = N1 + xN2 (1.3)

Where, N1 is the number of solvent molecules and N2 is the number of polymer molecules

with x number of monomers.

10


As the solvent and polymer chains are random variables, we define the entropy change

as:

�Sm = - k[N1ln(N1/N) + N2ln(xN2/N)] (1.4)

Where k is the Boltzmann’s constant (1.380 × 10�23JK-1). Equating N1/N and xN2/N as

volume fractions of solvent and polymer molecules respectively, we get:

Ø1 = N1/N

Ø2 = xN2/N

Where Ø1 is the volume fraction or probability that a given lattice site chosen at random

is occupied by the solvent molecule and Ø2 is the volume fraction or probability that a

given lattice site chosen at random is occupied by the polymer molecule. Substituting Ø1

and Ø2 in equation (1.4) we get:

�Sm = - k[N1ln Ø1+ N2ln Ø2] (1.5)

The solvent-solvent interaction parameter chi ( 12) equation (1.2) can be estimated from

the Hildebrand solubility parameters (�a and �b).

12 = Vseg (�a - �b)2/RT (1.6)

where Vseg is the actual volume of a polymer segment. �a and �b are the Hildebrand

solubility parameters of the solvent and polymer. The Hildebrand solubility parameter (�)

11


provides a numerical estimate of the degree of interaction between materials, and

materials with similar values of � are likely to be miscible.

1.2.2 Methods of Miscibility Study

There are a number of methods to study miscibility of polymer blends but it is commonly

studied by optical methods, cross-sectional examination using Scanning Electron

Microscope (SEM) and by studying glass transition temperatures (Tg) using Differential

Scanning Calorimeter (DSC) and Dynamic Thermal Mechanical Analyser (DMTA).

A miscible polymer system will show homogenous microstructures with small phase size

appearing uniform under a compound microscope on optical examination. Immiscible

blends appear opaque unless the refractive indices of the component polymers are

sufficiently close. SEM analysis of a miscible polymer blend system shows a single

continuous phase upon cross-sectional examination (Manisara et al., 2003).

DSC and DMTA are used to detect changes in the thermal transitions in polymer blends

during heating and cooling process. Tg is a characteristic transition from a glassy state to

a rubbery state. The absence of a single compositional dependent glass transition peak

indicates the absence of a single-phase, signifying the polymer blend system to be

immiscible (Geng et al., 2002). These transitions can be observed by use of DSC

however, DMTA has proven to be a very sensitive technique to study all kinds of

relaxation as well as the glass transition (Sakurai et al., 2000).

12


13

The presence of non-changing Tg in the DSC and DMTA scans of blend systems indicate

phase separation and immiscibility of the blend (Amiya et al., 2003).

Intermolecular interactions can give rise to miscibility. Infrared (IR) spectroscopy is a

very simple, reliable and sensitive technique of evaluating these interactions between

component polymers in a polymer blend. Shifts in band are the usual indications of

particular groups’ interactions with some other groups.

The aim of the present study was to study the miscibility of chitosan of different degrees

of deacetylation with other polymers. The degree of deacetylation (DD) values of the

three chitosan samples used for this study was to be determined using IR and titration

methodologies. The miscibility of the chitosan blends was to be studied using DSC and

DMTA. SEM analysis was also to be carried out to verify the DSC and DMTA findings.

Mechanical properties and antimicrobial properties of chitosan samples with three

different DD values and their blends with PVB, PVF and PEO were also studied.

Chapter 2.0 Literature Review

CHAPTER 2.0

Literature Review

This chapter is divided into 3 sections:

2.1 Methods for Degree of Deacetylation (DD) value determination

2.2 Effects of DD on chemical and physical properties of chitosan polymer

blends

2.3 Blends of chitosan of different DD

2.1 Methods of DD value determination

The extremity and time duration of the deacetylation process varies the degree of

deacetylation of chitin samples. The usual method of chitin deacetylation is the treatment

of the polymer with sodium hydroxide solution under nitrogen atmosphere at 1100C

(Khan et al., 2002).

Figure 2.1 Chitin deacetylation to Chitosan

14


The influences of alkaline concentration, temperature, time and the chitin/chitosan weight

to solution ratio has been investigated for their effect on the extent of deacetylation

(Methacanon et al., 2003). It was found that the extent of deacetylation was dependent

and increased mainly with increasing temperature, sodium hydroxide concentration and

length of the deacetylation process.

Research has also been done on the effects of processing sequence on the resultant

chitosan properties. It was found that process sequence did not alter the degree of

deacetylation, the intrinsic viscosity, or the molecular weight of chitosan. Temperature

and processing time had a significant impact on the molecular weight and the degree of

deacetylation (Rege and Block, 1999).

It takes 30 minutes processing of chitin samples at a temperature of 1000C with 60%

sodium hydroxide solution to obtain chitosan samples with a deacetylation value of 94%

(Castelli et al., 1996). The kinetics of homogeneous alkaline deacetylation of chitin has

been reported to be a pseudo-first-order reaction (Castelli et al., 1996). Given below are

number of techniques by which DD values can be determined.

2.1.1 Titration

The ability of the free primary amine groups of chitosan to form salts with acids have

been the basis for DD determination of chitosan samples with bases by acid-base titration

(Terayama, 1952). When hydrochloric acid is added to chitosan sample, the glucosamine

(GlcN) units immediately form a salt with HCl whereas the N-acetyl glucosamine

15


16

(GlcNAc) remain unreacted. Upon treatment of this chitosan salt with a base, an equal

amount of base molecules react with the total amount of chitosan salt in an acid-base

titration. By determining the amount of base used, the amount of GlcN repeat units in the

chitosan sample polymeric chain can be determined, hence the DD value (Dong, 2004).

In titrations, the base first reacts with the free (unbound) acids and then upon further

additions of base, second neutralisation reaction occurs between the base and the acid

bound to the chitosan moiety.

The difference in volume between the two inflexion points correlates with the amount of

chitosan molecules in the chitosan samples. The following reaction mechanism is

involved in getting the 1st and 2nd inflexion points:

Figure 2.2 Reaction mechanisms during the titration of acidified chitosan solution with

sodium hydroxide solution


Chitosan samples with unknown DD values have been analysed by titration with standard

0.1M sodium hydroxide solutions to determine the content of GlcN. Prior to titration, the

chitosan samples were treated with hydrobromic acid (HBr) to form chitosan

hydrobromide salts from the soluble fraction of chitosan samples (Ayer et al., 2000).

2.1.2 Infrared (IR) Spectrometry

IR spectroscopic method for the DD value determination of chitosan samples was

initially proposed during the late 70’s (Moore et al., 1978). The relative ease of analysis

using IR technique and the simple sample preparation steps have promoted the use of IR

spectrometric technique till date.

Figure 2.3 Baselines (a) and (b) used in determining DD values of chitosan samples

17


The Figure 2.3 indicates the baselines at (a) and (b) that are used for the DD value

determination. The peak at wavenumber 3450cm-1 is due to the hydroxyl groups of

chitosan samples, which is used in the equation as an internal standard to correct for film

thickness and the characteristic absorbance at 1655cm-1 of the amide-I band has been

used as a measure of the N-acetyl group content (Sabnis and Block, 1997).

The above-mentioned peaks have been used to develop an equation for the calculation of

the DD values. The equation for calculation of the DD value using the baseline (a)

(Domszy and Roberts, 1985) has been proposed as:

1655

3450

100100

1.33

AA

DD

� ��

� � � �

(2.1)

where the factor ‘1.33’ denotes the value of the ratio of A1655/A3450 for fully N-acetylated

chitosan. The assumption that went into the modelling of this baseline (a) equation was

that the ratio A1655/A3450 was taken as zero for fully deacetylated chitosan and there was a

rectilinear relationship between the N-acetyl group content and the absorbance of the

amide-I band.

A baseline (b) method, proposed by Baxter et al., (1992) is also used in determining the

DD values of chitosan samples.

18


Baseline (b) method is just a modification of baseline (a) method of computation for DD

value and has the following equation:

1655

3450

100 115ADDA

� ��

�� (2.2)

It has been reported that the DD values differ when computed using different baselines.

Baseline (b) method usually gave a higher than the actual DD value of samples (Khan et

al., 2002).

Chitosan samples prepared in the forms of potassium bromide (KBr) disk and films have

been studied (Khan et al., 2002). In this study, approximately 40-60mg of chitosan

powder and 120mg of KBr was triturated with agate mortar and pestle for 10 minutes.

40mg of the final mixture was compacted using an IR hydraulic press at a pressure of 8

tons for 60 seconds. Before analysis the chitosan/KBr mixture disk was conditioned in a

desiccator, which was placed in an oven for 16 hours. Chitosan films prepared by casting

0.5% wt/vv and 1.0% wt/v of chitosan solutions in 1% v/v acetic acid were also analysed

using IR spectrometer after drying the chitosan films in an oven at 600C for 16 hours.

Both baseline ‘a’ and ‘b’ methods were employed to determine the DD values of the

chitosan films. It was found that the DD values calculated were higher for films when

compared to the chitosan/KBr disk (Khan et al., 2002).

The higher calculated DD value for the chitosan films could be influenced by the

presence of residual acetic acid. The residual solvent is removed from the chitosan films

19


by lyophilising the films followed by rinsing with diethyl ether and finally drying the

films under reduced pressure at room temperature for 24 hours (Sakurai et al., 2000).

This method of subsequent drying of films was used in the research and drying process

was continued till the weight of films became constant which indicated removal of all

moisture. However, for films of chitosan prepared in acetic acid may not be pure chitosan

films at all but are films of chitosan acetate (Nunthanid et al., 2001) as the drying process

only removes moisture and the residual solvent which forms salt with the chitosan

polymer is not removed.

Chitosan/KBr disks and chitosan films have been prepared interchangeably for DD

determination using IR spectrometry technique and it has been observed that film forms

have usually a bit higher DD value than the disk form due to retained solvents which

exists as salts (Avadi et al., 2004; Dong et al., 2004; Kwon et al., 2003).

The relationship between the DD values of chitosan samples calculated and the technique

used has been investigated by Khan et al., (2002). The techniques used to evaluate the

DD values were first derivative UV spectrophotometry, titration and IR method using

both baseline ‘a’ and ‘b’ methods (Khan et al., 2002). It was revealed that the DD values

determined from the different techniques varied from one technique to another.

A similar study as done by Khan and his group (Khan et al., 2002) employing IR

spectrometry and titration method of DD determination also revealed that the DD values

determined were associated with the technique used (Avadi et al., 2004). Titration

20


method using sodium hydroxide solutions of concentration 0.1M was used to determine

the DD values of chitosan samples. The results were compared to the DD values as

determined using the baseline ‘a’ equation for IR spectrometry determination of the DD

value.

2.1.3 Other Methods

Various other documented methods exist for the determination of the DD value for

chitosan samples and has been well published (Khan et al., 2002). The choice of specific

method depends; on time frame of research, as some of the methods are too tedious, the

budget allocated for research, as nuclear magnetic resonance spectroscopy is costly for

routine analysis and other methods such as the “ninhydrin test” is destructive to the

sample. Many methods limit the range of degree of deacetylation to which they are

applicable as well (Domszy and Roberts, 1985).

A quantitative determination of the DD values of chitosan by reaction with ninhydrin

(Ninhydrin Test) was done using UV/VIS spectrophotometer (Curotto and Aros, 1993).

The ability of the 2-amino-2-deoxy-�-D-glucopyranose to form coloured product with

ninhydrin is the basic underlying principle behind this analytical technique in the

measurement of the amount of GlcN from the total amount of GlcNAc and GlcN present

in the given samples. Since the reaction of ninhydrin is specific to primary amino groups,

the reaction between GlcNAc and ninhydrin is not possible. Standards in the

concentration range of 10-120mgL-1 were used to plot a calibration curve before the

21


unknown samples were analysed at 570nm for the determination of the DD values. The

reaction of GlcN with ninhydrin was rapid, sensitive and reproducible.

The reaction between ninhydrin and primary groups of chitosan has been the basis of

several other DD values determination research’s as well. First derivative UV

spectrophotometry method for the determination of DD has been carried out using single

beam UV spectrophotometers (Tan et al., 1998). The DD values were calculated using

the concentration of the GlcNAc. The disadvantage of single beam UV

spectrophotometry is the search for the zero crossing point (ZCP). ZCP is the wavelength

(x-axis of UV spectrum) where the solute (GlcNAc) shows the maximum absorbance and

the solvent (either acetic or hydrochloric acid for chitosan systems) has the least

absorbance. Since the solvent affects the UV spectrum of the chitosan solution, it

becomes very important to remove this interference and hence to search for the ZCP.

Calibration graphs are then constructed by plotting a graph of ‘H’ values against the

corresponding GlcNAc concentrations. The ‘H’ values are the vertical distances from

ZCP to each GlcNAc solution spectrum (Khan et al., 2002). Apart from the UV

spectrophotometry, proton (H1) NMR spectrometry has also been used for DD value

determinations.

The hygroscopic nature of chitosan interferes with the computation of DD value by IR

methodologies as the absorbed moisture contributes to the hydroxyl peak intensity. This

hygroscopic nature of chitosan has been evaluated to be a non-interfering factor for DD

determination with H1NMR spectrometry as the chemical shift values of chitosan protons

22


23

used for the DD value determination appear at a different chemical shift value then

protons of water (Lavertu et al., 2003).

Elemental analysis has also been used to calculate DD of chitosan (Mima et al., 1983).

Initially the degree of acetylation (DA) was calculated using Perkin Elmer 2100 Auto

Analyser and then DD determined by the simple relationship:

100%DD DA� � (2.3)

Computations according to equation 2.3 have made characterisation of the degree of

deacetylation from the results of elemental analysis possible.

� �8.695 %(%) 100

1.799N

DA��

� ��

(2.4)

The degree of acetylation (DA) was calculated according to equation 2.4. The value

“8.695”, in equation 2.4 represents the percentage of N in fully deacetylated chitosan

while, “1.799”, is the percentage of N in fully acetylated chitin and % N is the percentage

of N calculated on the chitosan sample under investigation for its DD value (Taboada et

al., 2003).

2.2 Effects of DD on physical and chemical properties of chitosan polymer blends

Chitosan samples with different DD have been reported to differ in their chemical and

physical properties. This section focuses on studies involving chitosan samples with

different DD values and the associated results obtained.


2.2.1 Mechanical property evaluation studies

An important concept in the fields of material science, mechanical engineering and

structural engineering is Tensile Strength (TS). TS of a material is the maximum tensile

stress that it can be subjected to before it breaks. In the uniaxial manner of tension,

pulling forces across a specimen such as polymer films induces tensile stress. The units

for tensile strength is units of force per unit area (N/m2) or Pascal (Pa).

An equally important mechanical property of materials is its elongation. Elongation is of

two kinds, the ultimate elongation and the elastic elongation. It is the amount a sample

can be stretched before it breaks. Elastic elongation is the percent elongation that is

obtained without permanently deforming the sample. That is, how much the sample can

be stretched while still having the ability to snap back to its original length once the stress

on it is released. This is particularly important if the material is an elastomer.

Ability of a material to resist deformation is given by Young’s modulus (E) or modulus of

elasticity. It is stress to strain ratio on the loading plane along the loading direction and is

non-negative for all materials.

A study was conducted to understand the effect of DD on the mechanical properties of

chitosan films that were prepared in formic acid. Tensile strengths of the films were

measured and it was observed that increasing DD values resulted in an increase in tensile

strength of the chitosan films (Kwon et al., 2003). There was no effect on percentage

elongations of chitosan films with variations in DD values. Percentage elongation was

24


observed to increase with increasing molecular weight (Kwon et al., 2003; Chen and

Tsaih (1994); Chen et al., (1994); Blair et al., 1987).

Acetic acid has also been used as a solvent for chitosan films of varying DD values for

evaluation of mechanical properties in another study (Cervera et al., 2004). The resultant

films after drying were tested for their TS. It was observed that increasing DD values

increased the TS of the films accordingly. Again, no variation in percentage elongation

was seen with variations in DD values.

Both molecular weight and DD affects the pure chitosan film properties (Nunthanid et

al., 2001). It can be concluded that the increase in molecular weight of chitosan increases

the elongation as well as moisture absorption of the films, whereas the variations in the

deacetylation either increases or decreases the tensile strength of the films (Nunthanid et

al., 2001).

2.2.2 Thermal properties of chitosan

Not many studies have been conducted on the molecular and thermal relaxation

behaviour of chitosan. Although it is important to understand the physical and applied

properties of chitosan by investigating the glass transition temperature (Tg), studies on the

Tg of chitosan are difficult to pursue due to the difficulty in sample preparation and the

hygroscopic nature of samples. There is a small number of works concerning the Tg

measurement of chitosan. Ogura et al., (1980) reported the Tg of chitosan to around

1500C using the dynamic mechanical thermal analysis (DMTA) technique in the

25


temperature range from –150 to 1800C. It has also been reported that the Tg of chitosan is

around 300C (Ratto et al., 1995).

Ahn et al., (2001) observed a peak at 1610C in DMTA curves. Other researchers have

reported a much higher Tg values. Sakurai et al., (2000) estimated the Tg of chitosan to be

2030C from both the Differential Scanning Calorimetry (DSC) scans and tan � curves

based on DMTA.

The plethora of Tg values for chitosan that exists in the literature are due to the

differences in the degree of deacetylation values which changes the physical and

chemical properties of chitosan and thereby the Tg value (Nunthanid et al., 2001).

2.2.3 Anti-microbial studies

Due to chitosan’s well know anti-microbial properties against bacterial colonies such as

Staphylococcus aureus and Escherichia coli (Lim and Hudson, 2004), it has become a

candidate for medical research and applications and use of its blends in this field is

increasing rapidly.

The effects of the DD and preparation methods of chitosan have been evaluated for anti-

microbial activity (Tsai et al., 2002). Chitin was extracted from shrimp (Solenocera

Melontho) shell and then deacetylated using sodium hydroxide of varying concentrations

at different temperatures to obtain chitosan samples with DD values of 47, 74 and 95%.

Each chitosan sample was then used to make 50ml of chitosan solution in 0.1N

26


hydrochloric acid and the pH was adjusted to 6. To each chitosan solution, 100�L of test

bacterium was added and then shaken at 370C for 2 days. After thorough shaking for 2

days, 0.1mL of chitosan solution was withdrawn and spread on nutrition agar plates and

incubated for 2 days at 370C. After incubation, the colonies on the plates were counted.

It was observed that the anti-microbial activity of chitosan increased with increasing DD

value (Tsai et al., 2002).

Ikinci et al., (2002), undertook a study to determine the antimicrobial activity of chitosan

formulations either in gel or film form against a periodontal pathogen, Porphyromonas

gingialis. The viscosity, bioadhesive properties and antimicrobial activity of chitosan at

different molecular weight and DD were evaluated in the absence or presence of

chlorhexidine gluconate (Chx), incorporated into the formulations at concentrations of

0.1% and 0.2%. The flow properties of the gels were found to be suitable for topical

application on the oral mucosa and could be syringed into the periodontal pocket.

Chitosan was shown to have an antimicrobial activity against Porphyromonas gingialis

and this was higher with high molecular weight and higher deacetylated samples.

To study the relationship between anti-microbial activity and molecular structure of

chitosan, researchers used chitosan samples with different DD values and evaluated its

anti-microbial activity against gram-positive and gram-negative bacteria (Omura et al.,

2003). It was again concluded that anti-microbial activity of chitosan samples increased

with increasing DD and recommendation was made for its use in food preservation.

27


A year later, chitosan samples with varying DD values was studied for their anti-

microbial activity on food spoilage microorganisms in an attempt for use of chitosan in

seafood preservation (Tsai et al., 2004). Bacterial (Salmonella, Aeromonas and Vibrio)

counts in oyster stored at 50C decreased significantly on the addition of chitosan. In

general, chitosan with higher DD values showed better anti-microbial activity.

Chitosan (0.1g), assayed in a simple medium also reduced the viability of four lactic acid

bacteria isolated during the beer production process, whereas activity against seven

commercial brewing yeasts required up to 1g chitosan. Antimicrobial activity of chitosan

was shown to be inversely affected by the pH of the assay medium. In brewery wort, 0.1g

chitosan selectively inhibited bacterial growth without altering yeast viability or

fermenting performance (Gil et al., 2004).

The minimum antimicrobial growth inhibitory concentration (MIC) of chitosan against

methicillin-resistance Staphylococcus aureus (MRSA) has been investigated and the

concentration found was 100�g/ml in Mueller Hinton Broth medium (Seo et al., 1994).

An extensive review has shown that chitosan has strong anti-microbial effects and is safe

for human body due to its rapid excretion without any harmful side effects (Kumar et al.,

2005). Results have shown that the effects of chitosan are different for different kinds of

bacteria. The possible mechanism for anti-microbial activity on cultures of

Staphylococcus aureus have been shown to be from the formation of a polymer

membrane on the surface of cell, which results in the prevention of nutrients from

28


entering the cell. As for the inhibition of colony growth of Escherichia coli by chitosan,

the mechanism has been through cell pervasion. The mechanism and inhibition rate was

studied by spreading uniformly 0.25ml solution of chitosan solution and 0.25ml of

microbe suspension (cultured in peptone liquid) peptone culture plates, followed by

incubation and then counting number of colonies on the plates (Lian-Ying and Jiang-

Feng, 2003).

The inhibition rate was defined as:

1 2

1

= 100%N NN

� �� (2.5)

where N1 and N2 are the mean number of colony on the plates before and after inhibition,

respectively. N1 was obtained by smearing the bacterial solution on the peptone culture

plates and counting the number of colonies after incubation.

Antimicrobial properties of chitosan have been investigated in the solid and liquid culture

against bacteria associated with waterborne disease in order to assess the potential for

using chitosan as a natural disinfectant (Chen et al., 2002). Six strains which included

three gram-negative and three gram-positive bacteria were studied. The effects of the DD,

concentration, and molecular weight of chitosan on antibacterial activities were assessed.

Chitosan exhibited the highest antibacterial activity against the Pseudomonas aeruginosa

on the solid agar. Similar tendency was found when the bacteria were cultivated in liquid

broth. The higher DD and higher concentration of chitosan cause higher antibacterial

activities. The surface charge and persistence length illustrated the antibacterial

29


mechanism of chitosan. Results indicated that chitosan has potential as a natural

disinfectant (Chen et al., 2002). Colonies of Listeria monocytogenes has also been seen to

be affected by chitosan solutions (Zivanovic et al., 2004). The mechanism of chitosan’s

anti-microbial activity has been studied.

Since chitosan absorbs electronegative substance in the cell and flocculate them, it

disturbs the physiological activities of the bacteria and kills them. The anti-microbial

activity of chitosan appears to be mediated by the electrostatic forces between the

protonated NH2 group in chitosan and the negative residues at cell surfaces [Hadwiger et

al., 1981; Tsai and Su, 1999; Young et al., 1982).

The high-quality anti-microbial and mechanical properties of chitosan have made it an

ideal candidate of several research work focussing on polymer blending in pursuit of

finding better materials for use various facets of the modern world.

2.3 Blends of chitosan of different DD

The emerging lucrative markets of polymers have created much need in the laboratory-

based research of blending polymer systems. The concept of making new polymer

systems with desired properties or rather for the production of ‘tailor-made’ polymers for

specific purpose has resulted in numerous studies of chitin and its derivative chitosan

with other polymers.

30


Polymer miscibility is an important facet of polymer blending. Tools of miscibility

studies include from simple optical microscopes for surface studies in detection of

spherulites formation to the use of nuclear magnetic resonance spectrometry for

intermolecular interaction detections. Numerous studies have been done on blends of

chitosan with other polymers. A review of some of the studies is presented here. No

published article was found on miscibility studies of chitosan with PVB and minimal

irrelevant articles were found on chitosan and PVF studies despite exhaustive search.

2.3.1 Chitosan blends with Poly (�-caprolactone) (PCL)

Blends of chitosan/ PCL prepared by melt blending have been studied using DSC,

DMTA and FTIR spectrometer (Yang et al., 2001b). The study revealed that an increase

in the amount of chitosan did not show any change in melting temperature. However,

when chitosan butyrate was added to the PCL/chitin blends, both melting and

crystallization temperature of PCL was depressed implying an improvement in the

miscibility of the blend constituents.

FTIR studies suggested an interaction between the component polymers (Yang et al.,

2001b). To increase the miscibility between chitosan and PCL blends, butyralchitin was

blended in solution state to increase the intermolecular interactions between chitosan and

PCL in the blends. It was observed that the intermolecular interactions increased upon

increase in concentration of butyralchitin (Yang et al., 2001a).

31


Chitosan/PCL blends prepared by film casting were tested for water transmission rate

based on microcalorimetry and oxygen permeability. The miscibility was tested as well

using Scanning Electron Microscope (SEM). The SEM scans revealed the presence of a

single continuous phase indicating miscibility/compatibility of the blend film components

(Olabarrieta et al., 2001).

2.3.2 Chitosan Blends with Poly (vinyl pyrrolidone) (PVP)

Vinyl polymers with amide linkages in their side chains such as PVP, polyacrylamide and

poly (N-vinylacetamide) have been blended with water-soluble chitosan and

characterised by DSC and C13NMR spectroscopy for miscibility detection (Nishio et al.,

1999). It was seen that the Tg values of the blend films generally increased with

increasing chitosan content.

FTIR technique was used to investigate the intermolecular interactions in chitosan/PVP

blends. The amide peak shifts of chitosan with increasing concentration of PVP indicated

formation of intermolecular interactions. The chitosan/PVP films, which were optically

clear, were also analysed using DSC for the detection of the glass transition temperature

(Tg). The presence of a single Tg in the blend films throughout the chitosan concentration

range indicated the formation of a miscible phase, hence a miscible chitosan/PVP blend

film (Miyashita et al., 1995). This criterion has also been used in the current research in

determining the miscibility of chitosan with other polymers that were used in this

research.

32


Another similar research was undertaken as outlined above investigating thermal and

dynamic mechanical properties of chitosan/PVP blend films. The results of this

investigation also proved the chitosan/PVP blend system to be a miscible one (Sakurai et

al., 1996).

The compatibility of chitosan and PVP on a molecular level has been studied using IR

spectrometry (Cao et al., 1998). The clear blend films of chitosan and PVP prepared from

solution blending were investigated for the presence of carbonyl-hydroxyl hydrogen

bonding between the chains of the two component polymers. This interaction was also

investigated for the blend films at higher temperature as well and the results proved the

miscibility of the chitosan/PVP blend films. Miscibility of chitosan/PVP blend system

was also evident due to the presence of a single Tg in the blend films DSC thermograms.

2.3.3 Chitosan Blends with Polyacrylamide

Blend films of chitosan and polyacrylamide have been characterized using FTIR

spectroscopy and other techniques. The results showed the presence of intermolecular

interactions between the two polymers. Methods for improving the mechanical properties

of chitosan fibres have also been investigated and it was found that partially deacetylated

chitosan generally lowers the dry/wet strength properties of chitosan (Knaul et al., 1998).

2.3.4 Chitosan Blends with Poly (vinyl alcohol) (PVA)

Chitosan and poly (vinyl alcohol) (PVA) has been the focus of some investigations.

Blends of chitosan and PVA have been prepared using the solution blending technique

33


and the biodegradation and miscibility of the blends has been investigated (Lee et al.,

1996b). The compatibility of chitosan and PVA blends prepared by solution casting has

been investigated by DSC and the results showed that chitosan and PVA were partially

compatible (Wu et al., 1996).

A DSC study of chitosan/PVA blends was done for miscibility detection. Composition

dependent shift in the single Tg of PVA was observed for chitosan/PVA blend systems

(Miyashita et al., 1999) and melting point of PVA was depressed systematically with

increasing chitosan concentration indicating miscibility. Apart from DSC, FTIR

spectrometry has also been used in determining the miscibility of chitin/PVA miscibility.

The miscibility of chitin/PVA has been investigated using FTIR. The subtraction spectra

of the blends showed that intermolecular interactions between chitosan and PVA

disturbed the crystallisation of chitosan in the blends. The change in band shape and the

shift to lower frequencies of the band in the OH stretching region was due to hydrogen

bonding between the OH of PVA and the NH of chitosan (Miya et al., 1984; Kubota et

al., 1998).

It has been observed by IR spectroscopy that chitosan in the chitosan/PVA blends

improved light transmittance and mechanical properties, and decreased water absorption

of PVA (Yu et al., 2001b). Physical properties of PVA/chitosan blended films, cast from

different solvents, have been investigated and it was found that the solvent affected the

mechanical properties of the blends (Park et al., 2001).

34


The reduction of crystallinity of chitosan in the chitosan/PVA blend due to miscibility as

a result of intermolecular interaction has been studied by Wide-Angle X-ray Diffraction

(WAXD) (Lee et al., 1996a; Lee et al., 1996b). Miscibility was further investigated by

DSC and Transmission Electron Microscope (TEM) using ruthenium tetraoxide as a

staining agent.

C13NMR technique has also been utilised in the determination of the miscibility of

chitosan/PVA blend system (Kimura et al., 1997). It was observed that an increase in the

concentration of PVA in the chitosan/PVA blends systematically decreased the intensity

of the lower frequency of amide I doublet due to suppression of the intramolecular

hydrogen bonding of the amide C=O with OH and/or amine group of chitosan. FTIR

spectrometry was also used to supplement C13NMR data. Significant changes were seen

in the IR spectrum of PVA, indicating miscibility.

2.3.5 Chitosan blends with Cellulose and Starch

Structures of chitosan-cellulose mixtures in solution and solid state have been

investigated and it was found that cross-linking of the amino groups of chitosan with

hydroxyl groups of cellulose resulted in miscibility of the blends. The FTIR spectra

indicated the existence of hydrogen bonds between hydroxyl and amino groups,

indicating that miscibility occurred at the molecular level [Rogovina et al., 2001).

Characterisation of chitosan/cellulose blend films was done by XRD, Raman spectra and

by measurements of mechanical properties. Crystallinity of cellulose in the blend films

35


decreased with increasing chitosan content. Raman analysis indicated that chitosan and

cellulose molecules in the blend films seemed to have the same secondary structure as

those in 100% chitosan and cellulose films respectively. This indicated the presence of

interactions in the interfacial region between small domains of chitosan and cellulose

(Hasegawa et al., 1992).

Blend films of chitosan and starch were prepared and their structure and properties were

studied by FTIR, XRD, SEM and measurements of tensile strength. FTIR spectra and

SEM analysis showed that the two component polymers were miscible when the

composition of starch was 30% and less in the blend films. It was also observed that

crystallisation of starch was inhibited by chitosan (Du et al., 1997).

2.3.6 Other Polymers

Studies on the preparation, characterisation and properties of chitosan/nylon 1010

polymer blend films have been achieved using FTIR, XRD, DSC and SEM. The results

did not show the miscibility of the component polymers at the molecular level. The

blending of chitosan with nylon has shown to improve the mechanical properties and

biodegradability of nylon 1010 (Yu et al., 2001a).

Poly (ethylene oxide) (PEO) is a water-soluble polymer, which has been blended with

chitosan. The mechanical and structural properties of chitosan/PEO blend films were

studied and it was found that the mechanical properties of the blend films such as the

percent elongation, improved with increasing PEO composition (Alexeev et al., 2000). It

36


was shown that improvement in the mechanical properties correlated with a decrease in

the characteristic size of structural heterogeneities inherent in the two-phase system,

which was analysed by small-angle neutron scattering. The chitosan/PEO blend films had

the best mechanical properties at PEO concentration of 17-20%.

Miscibility of chitosan/ PEO blends has been studied by means of FTIR and SEM. The

results indicated that specific interactions between chitosan and PEO macromolecules

exist, which are mainly due to the formation of intermolecular hydrogen bonds between

bridge oxygens in the PEO main chains and amine groups on the chitosan chains. The

maximum interaction was observed at 50% PEO content in the blend films.

Chitosan blends with PEO were investigated as a candidate for oral gingival delivery

systems (Khoo et al., 2003). Results from DSC and DMTA, FTIR spectroscopy and

tensile testing, indicated that the chitosan/PEO blends showed some evidence of

miscibility. The study also indicated that chitosan blends were superior in properties

when compared to chitosan alone. These included improved film quality, improved

flexibility, and enhanced dissolution.

The phase behaviour of poly (ethylene glycol) (PEG)/chitosan blends has been

investigated with DSC and it was found that PEG did not melt when the chitosan content

was higher than 15% in the blend (Guo et al., 1999). The measurement of the combined

thermo gravimetric-differential thermal analyser (TG-DTA) indicated that PEG/chitosan

blend have good thermal stability.

37


38

Crystallization behaviour and environmental biodegradability of bacterial poly (3-

hydroxybutyric acid) (PHB) and chitosan blend has been reported (Ikejima and Inouse,

1999a). Blend films showed X-ray diffraction peaks that arose from the PHB crystalline

component in the blends and the films showed faster biodegradation than the pure-state

component polymers.

DSC was used to characterise PHB and chitosan blend films, which revealed that the

crystallisation of PHB in the chitosan/PHB blends was suppressed when the proportion of

chitosan was increased. This same tendency was evident from the FTIR band intensity of

the carbonyl stretching absorption from PHB indicating hydrogen bonds were formed

between the carbonyl groups of PHB and amide groups of chitosan (Ikejima et al.,

1999b).

Chitosan and silk fibrin have been blended using phosphoric acid as the solvent and the

resulting blends were investigated by FTIR (Park et al., 1999). The results indicated that

the carbonyl peak of silk fibrin shifted to longer wavelength, indicating interaction due to

hydrogen bond formation between the amino group of chitosan and the carbonyl group of

silk fibrin.

Chapter 3.0 Methodology

CHAPTER 3.0

Methodology

This section details the procedures used for the various studies carried out during this

research.

3.1 Materials

The following polymers were used in this study:

(i) Chitosan (medium molecular weight samples)

(ii) PVB (average molecular weight 50,000)

(iii) PVF (average molecular weight 11300)

(iv) PEO (average molecular weight 100,000)

Chitosan samples, with the following Degree of Deacetylation (DD) values as specified

by supplier (Fu Zhou Corona Science and Technology Development Co., Ltd), were used

in this study:

Chs1 - 85%

Chs2 - 87%

Chs3 - 94%

Formic acid (used as a common solvent), sodium hydroxide, hydrochloric acid and

potassium hydrogen phthalate were obtained from Aldrich Chemicals. All chemicals

were used without further purification except for formic acid, which was distilled twice

before use.

39


3.2 Determination of the Degree of Deacetylation (DD) of the samples

The values of DD for the chitosan samples were verified by titration and FTIR (using

baseline (a)) method.

3.2.1 Titration

The ability of the free primary amine group of chitosan to form salts with acids have been

the basis for DD determination of chitosan samples by acid-base titration (Terayama,

1952). The deacetylated chitosan sample and the commercial chitosan samples were all

analysed for their DD values before blending with other polymers. All titrations were

carried out in triplicates.

3.2.1.1 Standardisation of sodium hydroxide and hydrochloric acid solutions

A 1.00M solution of KHP (C8H5O4K - potassium hydrogen phthalate) was used to

standardise sodium hydroxide solution, which was prepared at an approximate

concentration of 0.1M.

The standardised sodium hydroxide solution (0.992M) was used to standardise

hydrochloric acid and also used as the titrant in the DD value determinations of chitosan

samples in the titrations. Please see Appendix Section 8.2 and 8.3 for calculations for the

standardisation results.

40


3.2.1.2 Preparation of chitosan solution and titration

Approximately one gram of the chitosan sample was completely dissolved in 100ml of

standardised hydrochloric acid by continuous stirring for 24 hours at ambient

temperature. 25ml from the resulting solution was titrated with a standard solution of

sodium hydroxide. The titrant volume was noted together with the chitosan solutions pH

values. The pH and volume data was later used to plot a graph of pH against titre volume.

A graph of �pH against �vol (titre) was also plotted. This derivative graph was used to

observe the inflexion points in the normal pH against titre volume graph (Tolaimate et

al., 2000).

The change in volume between the two inflection points on the titration curve was used

to calculate the DD by the method reported by Khan et al., (2002).

Sample calculations are given in the Appendix Section 8.4.

3.2.2 FTIR Spectrometry

FTIR analysis was done using the method described by Khan et al., (2002). The chitosan

films were prepared according to the method mentioned by Baxter et al., (1992), by

casting 1.5% (wt/v) chitosan in formic acid solutions on glass plates, followed by

thoroughly drying in an oven at 60° C for 1 week under reduced pressure. The drying

process were repeated and weight measurement taken till there was no further decrease in

weight of each films which indicated complete removal of moisture. The spectra of

41


chitosan film were obtained using a Perkin Elmer Infrared Spectrometer Spectrum 1000.

The films were scanned from 4000cm-1 to 400cm-1 at a resolution of 2cm-1. 64 scans were

taken for each sample. The DD of the chitosan samples was calculated using baseline (a)

method, as proposed by Domszy and Roberts, (1985).

3.3 Miscibility Studies

Miscibility studies of blends of chitosan with other polymers were carried out. FTIR

spectrometry was used to investigate the component polymer interactions, if any at the

molecular level. DSC was used to investigate the presence phase transitions occurring in

pure component polymers and their blends. The dependability of DSC data was

supplemented with DMTA in the case of Chs/PVB blends. DSC and DMTA were used to

observe the Tg, which indicates miscibility. A single compositional dependent Tg in a

polymer blend is an indication of miscibility whereas two separate Tg’s in a bi-polymer

blend system theoretically indicates immiscibility of the component polymers.

In this research work, the blend compositions for Chs/PVB and Chs/PVF blend systems

have been from chitosan 20% (wt/wt) (i.e. Chs20PVB and Chs20PVF) to chitosan 80%

(wt/wt) (i.e. Chs80PVB and Chs80PVF). This range of component compositions

produced clear films. For Chs/PEO systems, the chitosan composition in the blends

ranged from chitosan 75% (wt/wt) (i.e. Chs75PEO) to chitosan 90% (wt/wt) (i.e.

Chs90PEO). Composition of chitosan less than 75% (wt/wt) in Chs/PEO blend systems

produced films that were brittle and opaque.

42


Figure 3.1 Film clarity comparisons of Chs75PEO and Chs90PEO

A Chs20PVB (wt/wt) film constituted of 20% chitosan by weight and 80% of PVB by

weight. A Chs20PVF film constituted of 20% chitosan by weight and 80% of PVF by

weight. A Chs75PEO film constituted of 75% chitosan by weight and 25% of PEO by

weight.

3.3.1 FTIR Studies

All polymer films prepared from solution cast on flat glass plates were thoroughly dried

under reduced pressure at 60° C for 1 week to thoroughly remove all traces of residual

solvent. The dried polymer films were then analysed using FTIR. Spectra of all the

samples were signal averaged from sixty-four scans at a resolution of 2cm-1 from

wavenumber 4000-1 to 400cm-1. The spectra were corrected for baseline, normalised and

43


stored. Spectral analysis was carried out using the instrument’s spectra analysing

software.

Spectra subtractions were also carried out to study the behaviour of individual component

polymers in the blends. The peak shifts were noted and are reported in results section.

3.3.2 DSC Studies

Thermal characteristics of the pure polymers and blend films were measured using Perkin

Elmer Pyris 6 DSC. Blend and pure polymer films cast in glass petri dishes were

thoroughly dried under reduced pressure at 600C before DSC studies. Sample weight

ranged between 3-7 mg. The samples were scanned twice from 300C to 2500C at a

heating rate of 100C min-1 in an atmosphere of dry nitrogen at a flow rate of 30ml min-1.

The first heating cycle was carried out in order to remove any absorbed moisture from

chitosan since chitosan is apt to absorb moisture and the second heating cycle was done

to detect the temperature where the glass transition phase occurred. Scanning temperature

was not exceeded 2500C as thermal degradation of chitosan begins at about 2500C

(Sakurai et al., 2000). All DSC thermograms reported in this report are that as obtained

during the second heating run. Tg values were obtained directly from the thermograms

using the in-built software. The results were obtained at 2 decimal points.

44


3.3.3 DMTA Studies

DMTA was employed for Chs/PVB blends to supplement the DSC results as the signals

for the phase transitions in Chs/PVB blend system were weak in DSC scan in comparison

to other blends studied. DMT analysis of pure polymers (Chitosan and PVB) and blended

(Chs/PVB) films were done under nitrogen atmosphere by means of Rheometric

Scientific DMTA Mark IV instrument at a frequency of 1Hz. The temperature range for

analysis was from 250C to 2500C at a heating rate of 50Cmin-1. This part of the research

was done at the University of Auckland.

3.3.4 SEM Studies

Scanning Electron Microscopy was done for Chs/PVB and Chs/PVF blend systems at the

James Cook University, Townsville Campus, Australia. The presence of a single phase

upon cross-sectional examination of the blend system is an indication of a miscible

system (Manisara et al., 2003).

SEM analysis were done by mounting a section of the film vertically on an aluminium

stub and imaged using a JEOL JSM5410LV SEM operating at 10kV accelerating voltage.

The samples were given a platinum coat in order to provide an electrically conductive

surface necessary for examination by scanning electron microscopy. The

photomicrographs were captured digitally at an original magnification of 1500x.

45


3.4 Mechanical Property Studies

Blend films 10mm wide and 50mm long, were cut from film samples prepared on glass

plates. The film samples were measured for their thickness, length and width using a

micrometer and vernier calliper. The samples were thoroughly dried under reduced

pressure at 250C for 24 hours before tensile strength (TS) and percentage elongations

(%E) were measured using Shimadzu Table-Top Material Tester. There was no change in

film dimensions after drying. The samples were gripped in the machine and stretched to

rupture point. TS and %E were calculated using the machines software. All blend

composition films were run 5 times to get an average at a maximum load of 100N/mm2

and speed of 5mm/min for mechanical property evaluations. New blend films were used

for each run.

3.5 Antimicrobial Activity

The antimicrobial activity of chitosan of different degrees of deacetylation and its blends

were also studied. The bacteria used for this study was gram positive, Staphylococcus

Aureus as it is one of the safest bacteria to work with and the method followed was that

of Lian-Ying and Jiang-Feng (2003).

3.5.1 Microbial suspension preparation

The bacteria (Staphylococcus Aureus) was inoculated into 75ml of autoclaved peptone

liquid culture medium and incubated in air bath shaker (370C, 130rpm) for 12 hours. The

concentration of peptone liquid culture medium was 1% (wt/v). The bacterial population

was estimated using Haemocytometer.

46


A drop of peptone solution with bacteria was placed near the tip of the Haemocytometer

(covered by a coverslip) and through capillary action the solution was withdrawn in. The

bacterial count was taken by counting manually while viewing the Haemocytometer

under a microscope.

Figure 3.1 Haemocytometer

3.5.2 Condition of incubation

The peptone culture plates were prepared using 1% (wt/v) peptone and 1.5% (wt/v)

nutrient agar in distilled water. The culture media was autoclaved at 15 pounds of

pressure for 15 minutes at 1150C before preparation of culture plates. 0.25ml of bacterial

solution was added together with 0.25ml of polymer solutions on the culture plates and

spread uniformly. Each testing was done in duplicate. A blank (formic acid solution) was

also tested for comparison purpose as well as the growth of only the bacterial colony on

culture plate was also studied without the addition of polymer solution to obtain the

number of colonies on plate before inhibition. All the plates were incubated at 370C for

20 hours. The reported method of calculating the percentage inhibition (Lian-Ying and

Jiang-Feng, 2003) is defined as:

� = N1 – N2 X 100% (3.1)

N1Where:

N1 and N2 are the number of colony on the plates before and after inhibition, respectively.

All autoclaving was done at the Colonial War Memorial Hospital, Suva, Fiji.

47


48

3.5.3 Antimicrobial sensitivity disks

Ampicillin antimicrobial sensitivity disks was chosen as a means of detection of presence

of the bacterial cultures on the plate when only the bacterial colony was cultured without

any polymer solution to serve as a control over the antimicrobial testing.

The antimicrobial studies were carried out following the method published by Lian-Ying

and Jiang-Feng (2003).

Chapter 4.0 Results

CHAPTER 4.0

Results

4.1 Degree of Deacetylation Data

The DD values of samples were calculated using titration and FTIR. Data and

calculations are given under the respective techniques used.

4.1.1 Titration

Prior to titration of chitosan solution with sodium hydroxide solution, the sodium

hydroxide solution was standardised against KHP solution. The average concentration of

sodium hydroxide solution was 0.992M. Standardised sodium hydroxide solution was

used to standardise hydrochloric acid solution. The average concentration value of

hydrochloric acid solution was 0.302M. Calculations are given in Appendix Section 8.2

and 8.3.

The standardised sodium hydroxide solution was used as a titrant in the DD value

determination of the 3 chitosan samples using titration method. The changes in pH value

and accompanied titrant volume were carefully noted. Graphs were constructed using the

titration data. Each graph is also accompanied by its derivative graph to enable the

detection of titrant volumes causing the inflexion points.

49

Chapter 4.0 Results

50

0

2

4

6

8

10

12

0 2 4 6 8NaOH volume (ml)

pH

10

Figure 4.1 Graph of pH against NaOH volume (Chs1)

0

5

10

15

20

25


dpH

/dV

10

Figure 4.2 pH derivative graph for Chs1

Chapter 4.0 Results

51

02468

1012


pH

10


02468

10121416


dpH

/dV

10


Chapter 4.0 Results

52

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7 8 9NaOH volume (ml)

pH


0

2

4

6

8

10

12

14


dpH

/dV

10


Chapter 4.0 Results

Table 8.1, 8.2 and 8.3 in Appendix Section 8.4 contain the pH variations against titrant

volume data as well as the derivative data.

Sample calculation of DD value for Chitosan Sample # 1 (Chs1) is given in Appendix

Section 8.4.

The DD values of the three chitosan samples obtained by titration method are given in

Table4.1.

Table 4.1 DD values calculated using the titration method

Chitosan Sample DD Value (± 1)%

(Experimental Value)

DD Value (± 1)%

(As Specified by Supplier)

Chs1 86 85

Chs2 91 87

Chs3 97 94

The titration results of this research produced DD values that were close to that as

specified by the suppliers of the samples.

53

Chapter 4.0 Results

4.1.2 DD Determination using Infrared technique

For DD determination using IR technique the corrected area under the peaks at the

wavenumber 1655-1 and 3450-1cm were used. Areas under the respective peaks for the

three samples are given in Table 4.2.

Table 4.2 Areas of peak at wavenumber 1655cm-1 and 3450cm-1

Chitosan Sample

Area (corrected) (%T cm-1) obtained using baseline (a)

at wavenumber:

1655cm-1 3450 cm-1

Chs1 7351.06 54516.86

Chs2 3836.30 34073.39

Chs3 2618.11 69173.28

Using the corrected area for the peaks at wavenumber 1655cm-1 and 3450cm-1, obtained

using baseline (a), the following DD values of the three chitosan samples were calculated

using computation equation 2.1:

54

Chapter 4.0 Results

Table 4.3 DD values calculated using IR method

Chitosan

Sample

DD Value using baseline (a) (± 1)%


DD Value (± 1)%

(As Specified by Supplier)

Chs1 90 85

Chs2 92 87

Chs3 97 94

Sample calculation of DD value using baseline (a) method is given in Appendix Section

8.5.

The DD values obtained using titration method were more in agreement to that as

specified by the supplier in comparison to the DD values as determined using IR method.

DD calculations using IR method has some flaws as outlined in Discussions Section 5.1.

The DD values calculated using titration data is taken as the more correct DD values of

the supplied chitosan samples. Hence, the DD values of the chitosan samples used in this

research are determined to be as follows:

Table 4.4 DD values of the three chitosan samples used in this research

Chitosan DD Value (± 1 %)


Chs1 86

Chs2 91

Chs3 97

55

Chapter 4.0 Results

56


This section contains data obtained on intermolecular interactions and miscibility in the

polymer blends.

4.2.1 Interactions studied using FTIR

Pure polymer films as well as blend films were scanned using FTIR. Table 4.5 below

contains peak bands at respective wavenumbers and their peak assignments for chitosan

sample with a DD value of 86%. The scans are given in Figure 4.7. It can be seen that as

the DD values of the chitosan samples are increased from 86% to 97%, there is a general

shift in the carbonyl peak from 1666cm-1 to 1670cm-1 and this could be very much

attributed due to intramolecular hydrogen bonding between remaining carbonyl oxygen

and neighbouring protons on the amine groups.

4.2.1.1 Chitosan analysis

Table 4.5 Peak characteristics of chitosan

Wavenumber (cm-1) Peak Assignment

3372 OH stretching

1721 Amide II, N-H bending vibrations

1666 Amide I, C=O stretching vibrations

1586 N-H stretching vibrations of NH2 group

1378 CH3 in amide group

1317 OH vibrations of -CH2OH on the glucosamine ring

1154 -C-O-C- stretching

1073 -C-O-C bending modes

Cha

pter

4.0

R

esul

ts

Chs

1

Chs

2

Chs

3

Figu

re 4

.7 F

TIR

Spe

ctru

ms o

f Chi

tosa

n Sa

mpl

es

57

Chapter 4.0 Results

4.2.1.2 PVB analysis

Figure 4.8 shows the FTIR spectrum of PVB. PVB is synthesized from the hydrolysis of

poly (vinyl acetate) to poly (vinyl alcohol) and treated with n-butyral. The resulting PVB

contains some residual acetate, hydroxyl and n-butyl groups. The FTIR spectrum has

contributions from these groups. The peak data are given below in Table 4.6 with their

respective peak assignments.

Table 4.6 Peak characteristics of PVB

Wavenumber

(cm-1) Peak Assignment

3497 Intermolecular Hydrogen-bonding

2934 Asymmetric stretch of C-H groups

2873 Symmetric C-H group stretching

2733 C-H stretch of aldehyde

Carbonyl group vibrations of residual acetate and some remaining

formic acid which was used as solvent 1721

1432 Scissoring vibrations of CH2

1380 Asymmetric deformation of CH3 on ring

1180 Stretching and bending modes of C-O-C

1001

58

Cha

pter

4.0

R

esul

ts

99.9

PVB

1001

,64

1180

,14

1432

,77

2733

,94

2873

,71

1380

,71

1721

,28

2934

,54

3497

,72

95 90 85 80 75 70 65 60

%T

55 50 45 40 35 30 25 20

14

.4

40

00.0

3600

3200

2800

2400

2000

1800

1600

1400

1200

1000

800

600

400.

0cm

-1 Fi

gure

4.8

FTI

R S

pect

rum

of P

VB

59

Chapter 4.0 Results

4.2.1.3 PVF analysis

Figure 4.9 shows the FTIR spectrum of PVF. PVF is synthesised from the reaction

between PVA and formaldehyde under acidic conditions while PVA is the product of

hydrolysis of PVAc. Both the mentioned reactions do not go to completion; hence PVF

contains residual acetate and vinyl alcohol groups. The peak at 3518cm-1 in Figure 4.9, is

due to the stretching mode of O-H groups of vinyl alcohol while the peak at 1728cm-1 is

due to C=O group of the residual acetate.

The peak data are given below in Table 4.7 with their respective peak assignments.

Table 4.7 Peak characteristics of PVF


3518 Hydrogen-bonded hydroxyl groups

2919 Asymmetrical stretch of aliphatic C-H

2857 Symmetrical stretch of aliphatic C-H

2777 C-H stretch of aldehyde

1728 C=O stretching

1474 Bending of CH2 group attached to C=O group

1434 Bending of CH2 group attached away from C=O group

1372 and 1241 Bending and rocking modes of C-H group

1172, 1068 and 1020 Stretch, bending and rocking modes of C-O-C group

60

Cha

pter

4.0

R

esul

ts

4000

.0

3600

32

0028

00

2400

2000

1800

1600

1400

32

.1

35

40

45

50

55

60

65

70

75

80

86.1

%T

PVF

446,

69

3518

,61

2919

,42

2777

,59

2359

,72

1728

,34

1372

,45

791,

56

2857

,44

1474

,72

1434

,54

1241

,37

1172

,32

1068

,36

1020

,32

400.

060

080

010

0012

00cm

-1

Figu

re 4

.9 F

TIR

Spe

ctru

m o

f PV

F

61

Chapter 4.0 Results

4.2.1.4 PEO analysis

Figure 4.10 shows the FTIR spectrum of PEO. PEO is a low-melting solid prepared by

polymerization of ethylene oxide. Table 4.8 below contains the IR peaks and their

relative peak assignments.

Table 4.8 Peak characteristics of PEO


3510 Hydrogen-bonded O-H group

3260 CH2 asymmetric stretch

2880 Symmetric stretch of C-H

1457 CH2 symmetric wagging mode

1341 C2H5O stretch

1280 CH2 asymmetric twisting mode

1110 C-O-C asymmetric stretching mode

845 CH2 asymmetric rocking mode

62

Cha

pter

4.0

R

esul

ts

4000

36

00

3200

2800

2400

2000

1800

1600

1400

3.

2 10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

99.8

%T

PEO

521,

58

845,

11

959,

4

1280

,5

1110

,3

1341

,6

1457

,728

80,3

3510

,70

2167

,74

1967

,47

1639

,68

2240

,76

3260

,90

3736

,86

3848

,89

1241

,6

400

600

800

1000

1200

cm-1

Figu

re 4

.10

FTIR

Spe

ctru

m o

f PEO

63

Chapter 4.0 Results

4.2.1.5 Chs/PVB blend systems

Figures 4.11, 4.12 and 4.13 shows the FTIR spectrums of Chs/PVB blend systems with

varying DD values. No major trend was seen which could indicate intermolecular

interaction between chitosan and PVB in the Chs/PVB blend system.

64

Cha

pter

4.0

R

esul

ts

Figu

re 4

.11

FTIR

Spe

ctru

m o

f Chs

/PV

B (C

hs1)

65

Cha

pter

4.0

R

esul

ts

Figu

re 4

.12

FTIR

Spe

ctru

m o

f Chs

/PV

B (C

hs2)

66

Cha

pter

4.0

R

esul

ts

67

Figu

re 4

.13

FTIR

Spe

ctru

m o

f Chs

/PV

B (C

hs3)

Chapter 4.0 Results

4.2.1.6 Chs/PVF blend systems

Figures 4.14, 4.15 and 4.16 shows the FTIR spectrums of Chs/PVF blend systems with

varying DD values. A possible loss of intermolecular interaction between the amide II

proton and the oxygen of the PVF glycosidic linkage is seen in the spectrums.

68

Cha

pter

4.0

R

esul

ts

69

Figu

re 4

.14

FTIR

Spe

ctru

m o

f Chs

/PV

F (C

hs1)

4000

36

00

3200

28

00

2400

2000

1800

1600

1400

12

0010

0080

060

040

01

%T

Chs

20PV

F (C

hs1)

Chs

80PV

F (C

hs1)

3449

,5

2918

,2

1727

,1

1372

,3

1177

,110

24,1

791,

544

6,6

2779

,5

2853

,312

42,2

2918

,6

1718

,513

75,6

1176

,5

1577

,727

79,7

2853

,612

42,6

3421

,229

19,1

1723

,13

75,2

1026

,

790,

644

7,8

1665

,3

1585

,327

79,4

2853

,212

42,1

3377

,6

1715

,613

78,7

2779

,7

2853

,7

1242

,7

cm-1

C

hs40

PVF

(Chs

1)

Chs

60PV

F (C

hs1)

Cha

pter

4.0

R

esul

ts

Figu

re 4

.15

FTIR

Spe

ctru

m o

f Chs

/PV

F (C

hs2)

70

Cha

pter

4.0

R

esul

ts

4000

3000

20

00

1500

10

00

500

400

cm-1

Chs

20PV

F (C

hs3)

Chs

80PV

F (C

hs 3

)

3402

,5

2917

,9

1679

,813

78,1

4 12

42,1

310

36,4

787,

6161

6,62

899,

89

2771

,35

3289

,3

2921

,7

1663

,913

78,1

1 12

42,1

811

27,5

631,

46

899,

82

784,

58

2771

,29

1659

,10

1384

,19

1244

,39

1058

,463

1,53

2919

,17

3273

,5

1568

,15

899,

8478

7,71

3433

,10

2920

,9

1710

,11

1377

,16

1100

,3

790,

6544

7,75

899,

96

2778

,36

Chs

60PV

F (C

hs3)

Chs

40PV

F (C

hs3)

%

T

71

Figu

re 4

.16

FTIR

Spe

ctru

m o

f Chs

/PV

F (C

hs3)

Chapter 4.0 Results

4.2.1.7 Chs/PEO blend systems

Figures 4.17, 4.18 and 4.19 shows the FTIR spectrums of Chs/PEO blend systems with

varying DD values of chitosan. Chs/PEO system shows few strong trends in peak shifts

indicating intermolecular interactions between chitosan and PEO. These trends are also

seen to increase with increasing DD values.

72

Cha

pter

4.0

R

esul

ts

73

Figu

re 4

.17

FTIR

Spe

ctru

m o

f Chs

/PEO

(Chs

1)

4000

3000

20

0015

00

1000

500

400

cm-1

Chs

75PE

O (C

hs1)

Chs

90PE

O (C

hs1)

3363

,3

1588

,513

80,1

0 11

17,5

628,

38

2871

,5

900,

77

3254

,3

1603

,413

77,8

11

19,7

631,

30

2858

,6

900,

74

1604

,313

79,6

11

17,4

899,

7262

8,29

2852

,7

3247

,3

3456

,4

2889

,7

1654

,613

79,1

7 10

62,7

632,

4990

0,85

%T

Chs

80PE

O (C

hs1)

Chs

85PE

O (C

hs1)

Cha

pter

4.0

R

esul

ts

74

Figu

re 4

.18

FTIR

Spe

ctru

m o

f Chs

/PEO

(Chs

2)

4000

3000

20

0015

0010

0050

0 40

0 cm

-1

Chs

75PE

O (C

hs2)

Chs

90PE

O (C

hs2)

3395

,5

2883

,8

1662

,713

82,2

4

1087

,3

629,

56

1245

,51

3508

,3

1651

,311

50,6

899,

5584

2,45

630,

27

1245

,13

3355

,5

1598

,613

65,8

11

37,1

0

899,

74

635,

31

1245

,30

3417

,10

2881

,23

1662

,15

1585

,15

1382

,32

628,

6412

45,6

5

%T

Chs

80PE

O (C

hs2)

Chs

85PE

O (C

hs2)

Cha

pter

4.0

R

esul

ts

75

4000

30

00

2000

15

00

1000

500

400

cm-1

Chs

75PE

O (C

hs3)

Chs

90PE

O (C

hs3)

3378

,3

2880

,715

81,6

1382

,16

1084

,4

632,

5090

0,83

1245

,47

3376

,3

2882

,615

81,6

1381

,17

1087

,5

632,

5190

0,82

1245

,50

1597

,513

77,7

11

01,6

636,

28

900,

70

1245

,30

3369

,13

2884

,10

1666

,15

1585

,21

1380

,38

1106

,4

842,

7463

1,68

900,

8912

45,6

212

80,4

6

Figu

re 4

.19

FTIR

Spe

ctru

m o

f Chs

/PEO

(Chs

3)

Chs

80PE

O (C

hs3)

Chs

85PE

O (C

hs3)

%

T

Chapter 4.0 Results

4.2.2 Differential Scanning Calorimetry Data

The pure chitosan samples as well as its blends were analysed using DSC and the

thermograms showed no indications of phase transitions.

Figure 4.20 DSC thermograms of chitosan samples

Pure PVB, PVF and PEO samples were also analysed using DSC and produced good

results. It was observed that the solvent formic acid suppressed the Tg values of the pure

polymers. See Appendix Section 8.6 for the scans.

Table 4.9 contains the Tg data of the pure samples of PVB and PVF in film form made in

formic acid as well as Tg values of the polymer in its pure powder form.

76

Chapter 4.0 Results

Table 4.9 DSC data of pure PVB and PVF films made in formic acid

Sample Tg (Film in made in formic acid)

(±0.01)0C

Tg (Sample in pure powder form)

(±0.01)0C

PVB 47.54 75.16

PVF 93.35 110.94

Since all blend films were made in formic acid, for the purpose of this research the Tg

values of the pure polymers were taken as that which was obtained of the film form.

4.2.2.1 Chs/PVB Systems

All scans of Chs/PVB blend systems are given in Appendix Section 8.7. Below is Table

4.10 with all the data.

Table 4.10 DSC data of Chs/PVB blend films

Blend Composition Tg (±0.01)0C

Chs1

Tg (±0.01)0C

Chs2

Tg (±0.01)0C

Chs3

Chs20PVB 47.50 40.82 48.85

Chs40PVB 48.54 52.21 49.24

Chs60PVB 51.64 48.14 44.17

Chs80PVB 51.81 44.11 41.79

77

Chapter 4.0 Results

4.2.2.2 Chs/PVF Systems

All scans of Chs/PVF blend systems are given in Appendix Section 8.8. Below is Table

4.11 with all the data.

Table 4.11 DSC data of Chs/PVF blend films

Blend Composition Tg (±0.01) 0C

Chs1

Tg (±0.01)0C

Chs2

Tg (±0.01)0C

Chs3

Chs20PVF 95.38 95.43 89.05

Chs40PVF 97.41 100.02 100.38

Chs60PVF 97.55 99.09 97.09

Chs80PVF 101.39 95.17 -

4.2.2.3 Chs/PEO Systems

An endothermic peak was observed at 69.440C in the DSC thermogram of pure PEO

powder (Figure 4.21). PEO films made in formic acid showed this endothermic peak at

64.740C (Figure 4.22). No endothermic peak was found in the thermograms for pure

chitosan, PVB and PVF samples.

78

Chapter 4.0 Results

Figure 4.21 DSC thermogram of pure PEO powder

Figure 4.22 DSC thermogram of pure PEO film made in formic acid

79

Chapter 4.0 Results

Table 4.12 below has the Tm data on Chs/PEO systems.

Table 4.12 DSC data of Chs/PEO blend films

Blend Composition Tm (±0.01)0C

Chs1

Tm (±0.01)0C

Chs2

Tm (±0.01)0C

Chs3

Chs75PEO 61.01 61.11 61.39

Chs80PEO 61.74 60.45 61.13

Chs85PEO 61.44 60.47 61.48

Chs90PEO - - -

See Appendix Section 8.9 for the Chs/PEO blend DSC Thermograms

Table 4.13 Enthalpy of melting of PEO crystallites in Chs/PEO blend systems

Blend Composition �Hm (±0.001) Jg-1

Chs1

�Hm (±0.001) Jg-1

Chs2

�Hm (±0.001) Jg-1

Chs3

Chs75PEO 28.663 18.324 18.025

Chs80PEO 18.561 6.912 4.009

Chs85PEO 4.225 2.738 1.946

Chs90PEO - - -

�Hm is the melting enthalpy of PEO crystallites in the Chs/PEO blend films. The melting

enthalpy of 100% crystalline PEO is 196.4Jg-1 (Park and Kim, 2002). The �Hm of PEO

film in this study was 123.020Jg-1.

80

Chapter 4.0 Results

Table 4.14 below shows the percentage crystallinity in the Chs/PEO blend system. The

percentage crystallinity is calculated using the formula:

/

( )

% 100%m

m

T PEO Chs

T PEO Pure

HCrystallinity

H�

� ��

(4.1)

where:

�HTmChs/PEO is the enthalpy of melting crystalline PEO components in the Chs/PEO

blends and �HTmPEO is the melting enthalpy of pure 100% crystalline PEO. The value for

�HTmPEO is 196.400Jg-1

Table 4.14 Percentage crystallinity in Chs/PEO blend system

Blend

Composition

% Crystallinity

(±0.1)

Chs1

% Crystallinity

(±0.1)

Chs2

% Crystallinity

(±0.1)

Chs3

Chs75PEO 14.6 9.3 9.2

Chs80PEO 9.5 3.5 2.0

Chs85PEO 2.2 1.4 1.0

Chs90PEO - - -

Percentage crystallinity of Pure PEO film used in this study: 62.6%.

The percentage crystallinity was seen to decrease with increasing chitosan composition in

the Chs/PEO blend systems as shown in Figure 4.23 below. No Tm peak was detected for

Chs90PEO blend systems.

81

Chapter 4.0 Results

Figure 4.23 Graph of percentage crytallinity against blend compositions of Chs/PEO

blend systems

4.2.3 Dynamic Mechanical Thermal Analysis

DMTA is one of the most sensitive techniques to study all kinds of relaxation as well as

the glass transition of chitin/chitosan (Sakurai et al., 2000). Table 4.15 contains DMTA

data comparison of Chs1, Chs2 and Chs3.

Table 4.15 Thermal transition peaks of Chitosan samples as detected using DMTA

Chitosan

Sample

Transition 1

(0C)

Transition 2

(0C)

Transition 3

(0C)

Transition 4

(0C)

Chs1 90 120 183 207

Chs2 - 119 175 213

Chs3 - 118 190 212

82

Chapter 4.0 Results

The Tg of PVB from the tan delta against temperature graph of PVB from DMTA

analysis was 570C. Table 4.16 contains the DMTA data of Chs/PVB blend systems. See

Appendix Section 8.10.

Table 4.16 Thermal transition peaks of Chs/PVB blend system as detected using DMTA

Blend

Composition

Chs1 Chs2 Chs3

T1 T2 T3 T4 T5 T1 T2 T3 T4 T5 T1 T2 T3 T4 T5

Chs20PVB 51 - 105 181 215 42 - - - - 50 - - - -

Chs40PVB 51 - 122 181 211 45 95 - 186 - 50 - - 170 -

Chs60PVB 57 - 111 181 215 44 97 - - 215 50 - 118 190 215

Chs80PVB 50 - 111 - 215 39 98 - - 211 48 - 120 191 210

4.2.4 Scanning Electron Microscopy (SEM)

SEM was done for Chs/PVB and Chs/PVF blend systems to verify other miscibility

studies of Chs/PVB and Chs/PVF done in this research work. The photomicrographs

shown below were captured digitally at an original magnification of 1,500x.

Figure 4.24 PVB Figure 4.25 Chs10PVB

83

Chapter 4.0 Results

Figure 4.26 Chs30PVB Figure 4.27 Chs50PVB

Figure 4.28 Chs70PVB Figure 4.29 Chs90PVB

Figure 4.30 Chitosan Figure 4.31 Chs90PVF

84

Chapter 4.0 Results

Figure 4.32 Chs70PVF Figure 4.33 Chs50PVF

Figure 4.34 Chs30PVF Figure 4.35 Chs10PVF

Figure 4.36 PVF

4.3 Mechanical Property Studies

This Section 4.3 contains data on the blends tensile strength (strength parameter),

Young’s Modulus (YM) and percentage elongation (ductility) obtained using Instron

85

Chapter 4.0 Results

Tensile Testing Machine. Percentage elongations were calculated from the initial length

and elongation after subject to stress.

Table 4.17 TS, YM and %E of pure Chitosan, PVB, PVF and PEO films

Polymer Average TS

(± 0.1) N/mm2

Average YM

(± 1) N/mm2

Average % E

(± 0.2)%

Chs1 30.6 315 6.1

Chs2 30.9 311 6.0

Chs3 31.4 313 5.9

PVB 21.5 764 46.9

PVF 39.2 466 98.0

PEO 9.6 371 731.5

Table 4.18 contains the average tensile strength, young’s modulus and percentage

elongation data of the blend films.

86

Chapter 4.0 Results

Table 4.18 TS, YM and %E of Chs/PVB, Chs/PVF and Chs/PEO blend systems

Blend Composition

Chs1 Chs2 Chs3

TS* YM %E� TS* YM %E� TS* YM %E�

Chs20PVB 21.1 67 19.1 22.2 74 19.0 22.5 83 19.0

Chs40PVB 22.2 126 12.0 23.1 123 15.7 24.4 122 11.7

Chs60PVB 27.4 194 8.7 27.8 207 8.9 28.5 201 8.8

Chs80PVB 29.3 215 6.9 29.8 218 6.8 31.4 391 6.9

Chs20PVF 37.4 412 23.2 37.9 403 23.9 38.0 421 22.9

Chs40PVF 32.1 341 12.9 34.7 344 12.9 35.9 382 8.7

Chs60PVF 33.4 324 8.7 32.8 379 9.1 34.6 322 12.8

Chs80PVF 31.1 348 6.9 32.5 325 6.7 33.1 349 6.6

Chs75PEO 14.6 314 558.8 15.9 316 539.6 16.6 316 521.8

Chs80PEO 18.3 322 422.7 19.1 317 403.9 19.7 321 389.9

Chs85PEO 21.2 318 389.3 21.8 318 371.5 25.8 317 359.1

Chs90PEO 23.7 319 311.9 24.8 322 250.7 25.1 318 211.5

TS, YM and %E are average Tensile Strength, Average Young’s Modulus and Average

Percentage Elongation respectively.

* Error associated with Average Tensile Strength (± 0.1) N/mm2

Error associated with Average Young’s Modulus (± 1) N/mm2

� Error associated with Average Percentage Elongation (± 0.2)%

87

Chapter 4.0 Results

Figure 4.37 shows the average TS trend through blend composition in the three chitosan

samples while Figure 4.38 shows the trend of percentage elongation with blend

composition variations.

Figure 4.37 Graph of average TS variations against Chs/PVB blend compositions

Figure 4.38 Graph of average %E against Chs/PVB blend compositions

88

Chapter 4.0 Results

The maximum percentage elongation value calculated was 19.1 for Chs20PVB with the

chitosan sample having a DD value of 86%. It is apparent that increasing chitosan

concentration in the Chs/PVB blends increases the TS of the blend films and increasing

PVB components in the Chs/PVB blends increases the percentage elongation of the

resultant films.

Blending chitosan with PVF resulted in samples that had lower tensile strength as well as

percentage elongation values then the pure PVF. Figures 4.39 and 4.40 shows the

average TS and percentage elongation trends through blend composition in the three

chitosan samples.

Figure 4.39 Graph of average TS variations against Chs/PVF blend compositions

89

Chapter 4.0 Results

Figure 4.40 Graph of average %E against Chs/PVF blend compositions

PEO was evaluated for its mechanical properties as well. The average TS and %E values

of PEO was found to be 9.6N/mm2 and 731.5% respectively. PEO had the highest

analysed percentage elongation value in this research work. The trends in average TS and

percentage elongation properties with varying blend compositions are shown in Figures

4.41 and 4.42.

90

Chapter 4.0 Results

Figure 4.41 Graph of average TS variations against Chs/PEO blend compositions

Figure 4.42 Graph of average %E against Chs/PEO blend compositions

91

Chapter 4.0 Results

4.4 Antimicrobial Studies

Antimicrobial testing of chitosan samples and their blend films were done on the

bacterium staphylococcus aureus (S Aureus). After 12 hours of incubation of

staphylococcus aureus (S.Aureus) in 1% peptone liquid culture medium, the bacterial

count was done using a Haemocytometer and the concentration of the bacteria was found

to be about 2.714 x 105 cells ml-1.

4.4.1 Antimicrobial testing results

0.25 ml of bacterial solution placed on agar/peptone culture plate after 20 hours of

incubation produced immense patterns of bacterial colonies. A stage micrometer was

used to calibrate the microscope and determine the area observed through eyepiece and

ultimately the area over which the bacterial colonies grew. Despite higher magnification,

the number of bacterial colonies remained very high under each magnification and

existed as slime. Few colonies were seen separate from slime but ultimately counting the

colonies became impossible.

Inhibition rate couldn’t be determined as quantifying the results were impossible with the

high bacterial colonies, which formed slime on plates. Hence, in this study, the

antimicrobial results are treated with a qualitative approach and comparisons made

through visual inspection. The qualitative analysis is given in the Discussion Section 5.4.

92

Chapter 4.0 Results

4.4.1.1 Antimicrobial testing results of pure polymers

The peptone/agar nutrient culture plates are shown below with the bacterial colonies.

Figure 4.43 Figure 4.44 Figure 4.45



Figure 4.43 (S. Aureus) Figure 4.44 (S. Aureus with Ampicillin susceptibility disk) Figure 4.45 (S. Aureus & formic acid) Figure 4.46 (S. Aureus & PVB) Figure 4.47 (S. Aureus & PVF) Figure 4.48 (S. Aureus & PEO) Figure 4.49 (S. Aureus & Chs1) Figure 4.50 (S. Aureus & Chs2) Figure 4.51 (S. Aureus & Chs3)

93

Chapter 4.0 Results

4.4.1.2 Chs/PVB antimicrobial test results

Figure 4.52 Figure 4.53


Figure 4.56 Figure 4.57 Figure 4.52 Chs20PVB (Chs1) Figure 4.53 Chs80PVB (Chs1) Figure 4.54 Chs20PVB (Chs2) Figure 4.55 Chs80PVB (Chs2) Figure 4.56 Chs20PVB (Chs3) Figure 4.57 Chs80PVB (Chs3)

94

Chapter 4.0 Results

4.4.1.3 Chs/PVF antimicrobial test results



Figure 4.62 Figure 4.63 Figure 4.58 Chs20PVF (Chs1) Figure 4.59 Chs80PVF (Chs1) Figure 4.60 Chs20PVF (Chs2) Figure 4.61 Chs80PVF (Chs2) Figure 4.62 Chs20PVF (Chs3) Figure 4.63 Chs80PVF (Chs3)

95

Chapter 4.0 Results

4.4.1.4 Chs/PEO antimicrobial test results



Figure 4.68 Figure 4.69 Figure 4.64 Chs75PEO (Chs1) Figure 4.65 Chs90PEO (Chs1) Figure 4.66 Chs75PEO (Chs2) Figure 4.67 Chs90PEO (Chs2) Figure 4.68 Chs75PEO (Chs3) Figure 4.69 Chs90PEO (Chs3)

96

Chapter 5.0 Discussions

CHAPTER 5.0

Discussions

5.1 Determination of the Degree of Deacetylation

The DD values were determined using an acid base titration method (Avadi et al., 2004)

and IR method using baseline (a) equation (Khan et al., 2002). The titration graphs of pH

against titre volume showed two inflexion points. A derivative graph was plotted using

the pH and titre volumes as reported by (Tolaimate et al., 2000) (graphs in Results

Section 4.1.1) which showed two distinctive inflexion points.

The first inflexion point was achieved by the neutralisation reaction between NaOH and

free (unbound) HCl molecules to the chitosan moiety while the second inflexion point

was due to the neutralisation reaction between NaOH molecules and the bound HCl to

chitosan molecules (Terayama, 1952). The volume difference between the two inflexion

points was used in the DD value determinations. The DD values of the three samples

were found as 86, 91 and 97%.

Corrected areas under the peaks at wavenumber 1655cm-1 and 3450-1cm were used to

determine the DD values of chitosan samples using baseline (a) method as reported by

(Khan et al., 2002). Baseline (b) method of DD computation using IR method was not

used in this research as baseline (b) method usually gives a higher than the actual DD

value of samples (Khan et al., 2002).

97


The FTIR analysis of DD values using baseline (a) method produced DD values that

deviated more from the value as specified by supplier than the DD values as obtained

using titration methodology.

5.1.1 Comparison of DD values obtained using titration and FTIR methodology

It was observed that the DD values determined using the titration and IR method differed.

Table 5.1 below contains the data.

Table 5.1 DD values comparison as obtained using titration and IR method

Chitosan

Sample

DD value by titration

method (± 1)%

(Experimentally)

DD value by FTIR method

using baseline (a) (± 1)%

(Experimentally)

DD Value

(±1)%

(As specified

by supplier)

Chs1 86 90 85

Chs2 91 92 87

Chs3 97 97 94

The DD values obtained using titration method was more in agreement or closer to the

DD value as specified by the supplier. The titration method has the advantage that it

measures the protonated amine groups of chitosan directly and also there is no problem of

lack of accessibility of the amine groups during the protonation step as the salt was

formed during the dissolution of chitosan in excess HCl solution (Domszy and Roberts,

1985).

98


IR method of DD analysis has its advantages as well as disadvantages and major

advantage being the speed of analysis. Nonetheless, IR spectroscopy is primarily a solid-

state method, utilizing baseline for DD calculations and employment of different baseline

inevitably contributes to variations in DD values (Khan et al., 2002). The choice of

absorption band at wavenumber 3450cm-1 as an internal standard is questionable as

moisture absorbed by chitosan can readily enhance this peak (Blair et al., 1987). This

would in-turn increase the calculated DD value as is obvious from Table 5.1. The DD

values calculated using FTIR method was greater than those as calculated by titration

method.

On inspection of data in Table 4.2, it can be seen that the carbonyl peak area is

decreasing when moving from Chs1 to Chs3. This is understandable as the DD value of

Chs3 was greater than that of Chs1.

Figure 5.1 shows the carbonyl peak at wavenumber 1666cm-1 decreasing in size as the

DD values increase.

99


Figure 5.1 Variation in (C=O) and (NH2) peaks with DD variations

The peak at 1582cm-1, due to the NH2 was increasing in size as the carbonyl peak

decreased. This happened because as acetyl groups are removed from the acetamide

group, the (-NH) groups gain an extra proton to become (-NH2). The NH2 peak also shifts

from 1586cm-1 (Chs1) to 1580cm-1 (Chs3), indicating a possible decrease in

intramolecular interaction involving the protons on the NH2 groups and neighbouring

carbonyl oxygen (Cao et. Al., 1998).

There is an anomaly in the peak area of the hydroxyl groups at wavenumber 3450cm-1

though. This was due to either absorbed moisture or irregularities in the film thickness

(Brugnerotto et. al., 2001). The irregularity in film thickness is admissible as the

hydroxyl peak is itself an internal standard for irregularities in film thickness

100


(Brugnerotto et. al., 2001). On the other hand, absorbed moisture by the chitosan films is

a strong contender and an undeniable fact for these irregularities in the peak areas of the

hydroxyl band. Due to the hygroscopic nature of chitosan, (Khan et al., 2002) the

absorbed moisture would greatly increase this hydroxyl peak area at wavenumber

3450cm-1, resulting in a higher than actual DD value (Brugnerotto et. al., 2001). The

absorbed moisture decreases the overall value of the entire factor in square brackets as

shown in the equation below resulting in a higher DD value.

(5.1)


To study miscibility and intermolecular interactions between component polymers in the

blends in this study, FTIR, DSC, DMTA and SEM were used.

5.2.1 FTIR Studies

No major shift in peaks was observed for Chs/PVB blend systems for chitosan samples

with DD values of 86% and 91%. However, on inspection of Figure 4.13, it can be seen

that there is shift in the NH2 peak from 1589cm-1 (Chs20PVB) to 1561cm-1 (Chs80PVB),

indicating that the intramolecular hydrogen bonding of chitosan between NH2 groups and

the carbonyl carbon of the acetamide groups decreases as PVB content increases in the

Chs/PVB blend system for chitosan sample with a DD value of 97%. There was no

101


indication of intermolecular interaction between chitosan and PVB throughout the blend

composition in the three different chitosan samples.

It can be seen that as the content of PVF in the Chs/PVF system is decreased the amide II

peak of chitosan due to N-H bending, shifts to lower wavenumber. This indicates a

possible decrease in intermolecular interaction between the amide II proton and the

oxygen of the PVF glycosidic linkage. It is also seen that as the DD values are increased

the amide II peak shifts to a lower wavenumber from 1727cm-1 (Chs20PVF, Chs1) to

1710cm-1 (Chs20PVF, Chs3), indicating that as the DD values are increased there is a

probable decrease in intermolecular interaction between chitosan and PVF.

Chs/PEO systems showed strong intermolecular interactions as is clear from Figures

4.17, 4.18 and 4.19. There is a general shift in the peak due to the hydrogen bonded OH

stretching from 3254cm-1 (Chs85PEO, Chs1) to 3376cm-1 (Chs85PEO, Chs3), indicating

strong intermolecular interaction as the DD value of the chitosan sample is increased as

well as when the concentration of PEO is 15% and less (Nasir et al., 2005). This could be

a reason as to why percentage crystallinity was seen to decrease in the Chs/PEO system

when the concentration of PEO was increased (Table 4.13). The decrease in percentage

crystallinity and increasing intermolecular interaction is a strong indication of the

miscibility of the Chs/PEO system when the concentration of PEO less than 15% in

Chs/PEO system. This has also been reported in literature as well (Alexeev et al., 2000).

102


DSC analysis also did not show any Tm for Chs90/PEO systems while all other blend

compositions showed distinctive Tm. These are strong indications of Chs/PEO miscibility

when the concentration of PEO is less than 15%wt/wt in Chs/PEO systems. It was also

observed in Chs90/PEO compositions that higher DD values increased the intermolecular

interaction between chitosan and PEO where the NH2 peaks shifted from 1585cm-1

(Chs1) to 1597cm-1 (Chs3).

5.2.2 Thermal Studies

DSC is not sensitive enough to detect the phase transitions of chitosan samples in this

study as indicated in Figure 4.20. DMTA is one of the most sensitive techniques to study

all kinds of relaxation as well as the glass transition of chitin/chitosan (Sakurai et al.,

2000). Table 4.15 contains DMTA data comparison of Chs1, Chs2 and Chs3. Results

were obtained from first heating scan at a frequency of 1Hz. There were multiple peaks

observed in the tan delta against temperature curves of chitosan samples.

DMTA scans shows chitosan sample with a DD value of 86% having four transitions.

Chitosan is hygroscopic in nature. The transition at 900C is the water-induced relaxation

of chitosan sample and the transition at 1830C has been attributed due to the relaxation

(Dong et al., 2004). The transition at 1200C cannot be due to � relaxation, as the DD,

which relates to the amount of side group (i.e. acetamide group or amino group) would

affect this transition should it a � relaxation. It is noticeable from the DMTA analysis of

the three chitosan samples that all samples had a Tg at ~ 1200C. This does not indicate

103


that this transition is due to � relaxation. Ogura et al., (1980) reported the Tg of chitosan

to be around 1500C.

It has also been reported that the Tg value of chitosan does not vary with DD variations

(Dong et al., 2004). The transition at 1200C has been concluded as the glass transition

temperature of chitosan over DD values 86 to 97%.

The transition at 2070C could be either the thermal decomposition temperature of the

polysaccharide or attributed due to a liquid-liquid transition (Tl,l) (Dong et al., 2004).

Liquid-liquid transition was first found in PVC and PS in 1950 and suggests that the

liquid-liquid transition corresponds to the motion of very long segments or even the

whole chain from imaginary liquid to the real liquid above Tg (Dong et al., 2004).

The thermograms of pure polymers made in formic acid showed suppression in the Tg

values when Tg values of films made in formic acid were compared to the pure powdered

polymers DSC thermograms. This suppression in Tg values is due to the fact that

polymers such as PVB and PVF become plasticised in formic acid that causes the

polymers to become more pliable resulting in the lowering of the Tg value (Sakurai et al.,

2000). See Appendix Section 8.6 for the DSC thermograms of pure polymers.

5.2.3 Glass Transition temperature of Chs/PVB and Chs/PVF blend films

The Tg of polymer blends is a strong tool for the detection of miscibility. The DSC scans

of the Chs/PVB blends showed no Tg components of chitosan. The absence of the Tg

104


component of chitosan in the Chs/PVB blends DSC thermograms cannot be used as a

direct evidence for immiscibility as the DSC thermograms of pure chitosan also didn’t

have the Tg due to chitosan’s phase transitions. However, there was also no presence of a

single compositional dependent Tg in the scans, which could indicate the system to be a

miscible one (Geng et al., 2002). Contrary to a miscible system, Chs/PVB systems

showed weak single Tg in the DSC scans of the Chs/PVB blends in all three chitosan

sample blends, which remained affixed at the Tg value to that of PVB. This single non-

changing Tg in the Chs/PVB DSC scans proves the Chs/PVB system to be an immiscible

one (Geng et al., 2002).

Since the Tg values of DSC analysis of Chs/PVB systems were not very strong, therefore

DMT analysis of the system was done together with scanning electron microscopy in

determining the miscibility of the Chs/PVB system. Peak due to retained moisture (T2)

was not seen in the Chs/PVB blend systems where the DD values of chitosan were 86%

and 97% respectively. T2 was seen in Chs/PVB blend system through Chs40/PVB to

Chs80/PVB blend composition for chitosan sample with a DD value of 91%.

All blend films showed a Tg due to the PVB (T1) component, which remained constant

for all the different blend compositions, indicating immiscibility of the system. The Tg

peak of chitosan (T3) was also observed in the Chs/PVB blends. The presence of the

affixed component Tg’s (T1 and T3) in the blend systems indicate that PVB remains

phase separated regardless of compositional range proving the immiscibility of the

system (Amiya et al., 2003).

105


The relaxation temperature (T4) increased with increasing DD value of the chitosan

sample but had no trend within each chitosan sample through the blend composition. The

increase in the T4 values is most probably due to the fact that increasing DD values

produced more exposed –NH2 groups which formed more interactions with PVB

molecules.

The thermal decomposition temperature T5 remained the same in all the blend systems

over the entire compositional range. The values of the different transitions are given in

Table 4.16.

Chs/PVF blends also didn’t show Tg components of chitosan in the DSC scans. Chs/PVF

systems showed a single Tg in the DSC scans of the Chs/PVF blends in all three chitosan

sample blends, which remained affixed around the Tg value to that of pure PVF. This

single non-changing Tg in the Chs/PVF DSC scans proves the Chs/PVF system to be an

immiscible one, just like the Chs/PVB case.

SEM was done for Chs/PVB and Chs/PVF blend systems to verify other miscibility

studies of Chs/PVB and Chs/PVF done in this research work.

A miscible polymer blend system has a single continuous phase that can be seen upon

cross-sectional examination of a blend system using SEM (Manisara et al., 2003). The

scanning electron microscopy shows that the Chs/PVB and Chs/PVF blend systems do

not have a single continuous phase but rather the images are engrained with two different

layers. This further proved that the Chs/PVB and Chs/PVF blend systems are an

immiscible one (Manisara et al., 2003).

106


5.2.4 Glass Transition temperature of Chs/PEO Systems

The semi-crystalline nature of PEO produced an endothermic peak at 69.440C in the DSC

thermogram of pure PEO powder (Figure 4.21), which can be attributed to the melting

phase of PEO crystallites, in the DSC scan of pure PEO powder. The PEO films made in

formic acid had the above-mentioned endothermic peak suppressed to 64.740C (Figure

4.22). The Tm value of PEO as observed in this study has been very close to that of as

reported in literature at a value of 670C (Zoppi et al., 2001).

The Chs/PEO blend systems had single non-compositional dependent endothermic peaks

in their DSC scans due to the PEO crystalline component. The absence of a single

compositional dependent peak indicated absence of a single-phase as is the case in

miscible systems (Geng et al., 2002). Under immiscibility conditions, each of the

crystallisable components of the blend system exhibits the Tm of the corresponding pure

homopolymer (Zoppi et al., 2001) as is the case with Chs/PEO systems.

For Chs90/PEO systems, in all three chitosan samples, there was no phase transitions in

the DSC scans hence no Tm data was obtained. Chs/PEO blend films, where the

concentration of chitosan was greater than 90%, were much clearer than other Chs/PEO

blend compositions. See Appendix Section 8.9 for DSC thermograms.

The �Hm (melting enthalpy of PEO crystallites) of pure PEO film in this study was found

as 123.020Jg-1, which was in close agreement to that of literature value of 196.4Jg-1 for

perfect PEO crystals (Park and Kim, 2002).

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The percentage crystallinity of pure PEO was calculated to be 62.6%. This percentage

crystallinity of pure PEO was compared to the variations of percentage crystallinity of

PEO components in the Chs/PEO blend films. Data is given in Table 4.14. Figure 4.4

shows that upon increasing the concentration of chitosan component in Chs/PEO blend

system, the percentage crystallinity of PEO decreased. This decrease in percentage

crystallinity of Chs/PEO blend system is an indication of the formation of intermolecular

hydrogen bonds between bridge oxygens in the PEO main chains and amine groups on

the chitosan chains (Alexeev et al., 2000). If the interaction between two polymers is

strong in a blend system, crystallinity growth is retarded during the quenching period in

the DSC analysis and this brings about lowering of the melting point peaks in the DSC

thermograms, resulting in decreased percentage crystallisation (Park and Kim, 2002).

It can be seen from Figure 4.23, that the percentage crystallinity is decreasing rapidly as

the composition of chitosan is increased and this could be simply attributed to either

intermolecular interaction or dilution effect but at chitosan composition 90% (wt/wt) in

Chs/PEO blend systems in all three chitosan samples; it was observed that the melting

transition in the Chs/PEO blends disappears completely. Theoretically if the decrease in

percentage crystallinity is simple due to dilution effect there should be approximately

6.26% crystallinity observed in an immiscible binary mixture of chitosan and PEO and

melting transition detected as the composition of PEO in Chs90PEO is 10% and pure

PEO has percentage crystallinity value of 62.6%. This total loss of crystallinity could be

attributed to the fact that chitosan and PEO has strong intermolecular interactions and

when the composition of chitosan in the Chs/PEO is 90% and more, the immiscible

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Chs/PEO blend system attains miscibility and the crystallinity nature of PEO is

diminished. This has been reported as well by Khoo et al., (2003).

5.3 Mechanical property evaluations

The mechanical properties of pure polymer films as analysed in this research has been

very close to that reported in literature. For chitosan sample with a DD value of 86%, the

reported literature value of TS value is 26.8N/mm2 and the %E is reported as 4.6 (Cervera

et al., 2004) while in this study the TS and %E was 30.6 N/mm2 and 6.1% respectively.

The TS value of pure PEO has been reported as 9.2N/mm2 with a %E value of 800%

(Prodduturi et al., 2004). The TS of PEO in this study was determined as 9.6 N/mm2 with

average percentage elongation value of 731.5%. PVB had a TS value of 21.5 N/mm2,

which was also in good agreement to the literature value of 20 N/mm2 (Krüger, 1998).

PVB is used in safety glass, such as in vehicle windscreens and buildings, where it

provides resistance to breakage and holds broken glass fragments in cases of breakages.

The tensile strength of PVB was found to be 21.5 N/mm2. It was observed that upon

blending chitosan with PVB, the tensile strength of the resultant blends increased from

21.5N/mm2 to 29.3 N/mm2 (Chs80PVB Chs1). Chs/PVB blends with chitosan sample

with a DD value of 97% produced blends with the maximum TS values in comparison to

the other two chitosan samples with lower DD values. Chs80/PVB films had the

maximum average TS value of 31.4N/mm2 Figure 4.37. Figure 4.38 shows the

elongation trend for all blend compositions of the three chitosan samples.

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PVB has a higher elongation value then pure chitosan and decreasing the concentration of

PVB decreases the elongation property of the blend films. The maximum %E value

calculated was 19.1 for Chs20PVB with the chitosan sample having a DD value of 86%.

It is apparent that increasing chitosan concentration in the Chs/PVB blends increases the

TS of the blend films and increasing PVB components in the Chs/PVB blends increases

the percentage elongation of the resultant films. It is also noted that the TS values of the

Chs/PVB films increases as the DD values are increased of the component chitosan

samples. Percentage elongation values of the blend films decreased as the DD values are

increased of the chitosan samples.

Despite the immiscibility of the Chs/PVB blend system, it can be seen that the Chs/PVB

blend has good mechanical properties. Addition of chitosan to PVB improves the TS of

the PVB films while the elongation of the blend films improved due to the PVB

component.

The TS and %E of PVF in this research work was found to be 39.2N/mm2 and 98.0%

respectively. Chs/PVF blends apart from being immiscible also had lower tensile strength

as well as percentage elongation values then the pure PVF. Figure 4.39 and 4.40 shows

the average TS and %E trends through blend composition in the three chitosan samples.

Blending chitosan with PVF is useful though as pure chitosan has lower tensile strength

and percentage elongation values while the Chs/PVF blends had better mechanical

properties.

110


The decreases in average TS and %E values for the blend systems of the three chitosan

samples as the content of chitosan increase is due to the fact that the average TS and %E

values of chitosan is less then that of pure PVF. As the DD values of chitosan samples

were increased, the Chs/PVF blend systems average TS values increased as well. For

preparation of Chs/PVF film with the highest average TS value, the best blend

composition is Chs20PVF with the DD value of chitosan sample being 97%.

Chs/PVF system has been shown to be immiscible in this study. Chitosan on its own has

lower TS and %E properties in comparison to pure PVF films. Blending PVF with

chitosan improves chitosan’s mechanical properties as shown in Figures 4.39 and 4.40.

PEO was evaluated for its mechanical properties as well. The average TS and %E values

of PEO was found to be 9.6N/mm2 and 731.5% respectively which agreed with literature

value of 9.2N/mm2 with a %E value of 800 respectively (Prodduturi et al., 2004). The

average TS value of chitosan is more than that of PEO. Blending chitosan with PEO

improved the average TS values of Chs/PEO blend films as compared to the TS value of

PEO in pure state. PEO also complemented the properties of chitosan films as the

addition of PEO to chitosan resulted in Chs/PEO blend films with better elongation

properties. The trends in average TS and percentage elongation properties with varying

blend compositions are shown in Figures 4.41 and 4.42.

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5.4 Antimicrobial Studies

The concentration of Staphylococcus aureus cells in 75ml of 1% (wt/v) peptone was

2.714 x 105 cells/ml. About a quarter of 2.714 x 105 cells/ml of bacterial culture was

added to the agar/peptone culture plate.

The approximate number of bacterial cells added for culturing on the agar/peptone plate

was 67850 cells. Each bacterial cell has the capability of forming a colony. Since a large

number of bacterial cells were inoculated on the nutrient plates, this resulted in the

formation of a large number of colonies on the plates that existed very close to each other

resulting in the formation of slime.

A stage micrometer was used to calibrate the microscope in order to obtain the area under

view at high magnification. Despite several magnifications, the bacterial colonies in the

slime couldn’t be determined as they existed very close to each other and distinguishing

individual colonies were not possible. Hence the percentage inhibition rate could not be

calculated.

Since a quantitative analysis was impossible in this case, a qualitative approach was

taken. On inspection of the culture plates in Results Section 4.4.1.1, it can be seen that

formic acid did have some inhibition and the bacterial growth on formic acid plate was

same as to the plates. Pure PVB and PVF had almost the same size of colony growth as

that of formic acid plate. This probably indicates that PVB and PVF have no antibacterial

112


activity. On the other hand PEO showed an inhibition zone just like the ampicillin disk

indicating that PEO did have some antibacterial activity against Staphylococcus aureus.

Chitosan samples showed good antibacterial activity and it was observed that the

inhibition zones caused by chitosan samples increased as the DD values of the sample

increased indicating that as the DD values of chitosan increase the antibacterial activity

increases as well. This has been reported in literature as well (Tsai et al., 2002; Omura et

al., 2003; Tsai et al., 2004; Ikinci et al., (2002); Chen et al., 2002). Chitosan exposures

its antibacterial effect on Staphylococcus aureus by forming a polymer membrane on the

surface of the bacterial cell, resulting in the prevention of nutrients from entering the cell

(Lian-Ying and Jiang-Feng, 2003).

The trend in Chs/PVB and Chs/PVF blend systems was that as the chitosan content in the

blends increased, so did the inhibition of growth and colony formation. Chs80PVB and

Chs80PVF (Chs1) had more colony growths than Chs80PVB and Chs80PVF (Chs3).

Since both PVB and PVF didn’t show inhibition in pure states, the bacterial inhibition of

Chs/PVB and Chs/PVF blend systems is due to the chitosan component and using higher

DD samples increases the inhibition of the system.

Chs/PEO systems showed more inhibition than both Chs/PVB and Chs/PVF blend

systems and this is due to the finding that in this study both chitosan and PEO showed

antibacterial activity and PVB and PVF didn’t. The activity of Chs/PEO blend system

also increased with increasing chitosan content in the system.

113


114

All polymer blends’ antimicrobial activities increased with increasing chitosan content

and DD of chitosan samples had a directly proportional relation to the increase in

antimicrobial activity (Tsai et al., 2002; Omura et al., 2003; Tsai et al., 2004; Ikinci et

al., (2002); Chen et al., 2002).

Chapter 6.0 Conclusion

CHAPTER 6.0

Conclusion

Chitosan with three different DD values were blended with PVB, PVF and PEO. Prior to

blending, titration and IR means of DD analysis were employed following well-

documented and published methods. Titration method of DD analysis proved to be a

better and more sensitive method of DD analysis than IR method. The titration method

had the advantage that it measured the protonated amine groups of chitosan directly

(Domszy and Roberts, 1985) while DD analysis using IR method had flaws as utilizing

different baselines for DD calculations inevitably contributes to variations in DD values

(Khan et al., 2002). The absorption band at wavenumber 3450cm-1 also contributes to

errors in DD analysis using IR method as moisture absorbed by chitosan can readily

enhance this peak (Blair et al., 1987) which increases the calculated DD value as is

obvious from Table 5.1.

It was also seen that DSC method didn’t prove to be sensitive enough to detect the

transitions taking place in pure chitosan samples. DMTA was more sensitive and showed

four transitions happening in chitosan when it was thermally analysed for transitions.

Miscibility studies of chitosan polymer blends showed several results. There were no

major detectable intermolecular interactions taking place in Chs/PVB blend systems at

varying DD values of chitosan samples. However, there is shifting of the NH2 peak from

1589cm-1 (Chs20PVB) to 1561cm-1 (Chs80PVB) indicating that the intramolecular

hydrogen bonding on chitosan between protons on NH2 groups and possible the carbonyl

115


carbon of the acetamide groups decreases as PVB content increases in the Chs/PVB

blend system. Chs/PVF system showed loss of intermolecular interaction between the

amide II proton and the oxygen of the PVF glycosidic linkage (Miya et al., 1984). SEM

analysis also showed that Chs/PVB and Chs/PVF systems are immiscible.

Chs/PEO systems showed strong intermolecular interactions as there was a general shift

in the peak due to the hydrogen bonded OH stretching from 3254cm-1 (Chs85PEO, Chs1)

to 3376cm-1 (Chs85PEO, Chs3), indicating strong intermolecular interaction as the DD

value of the chitosan sample is increased as well as when the concentration of PEO is

15% and less (Nasir et al., 2005). The percentage crystallinity was also seen to decrease

in the Chs/PEO system when the concentration of PEO was increased (Table 4.13). The

decrease in percentage crystallinity, absence of Tm in Chs90PEO blend systems (while

Chs75PEO to Chs85PEO showed Tm) and increasing intermolecular interaction were

strong indications of the miscibility of the Chs/PEO system when the concentration of

PEO is 15% and less in Chs/PEO system (Alexeev et al., 2000). It was also observed in

Chs90/PEO compositions that higher DD values increased the intermolecular interaction

between chitosan and PEO where the NH2 peaks shifted from 1585cm-1 (Chs1) to

1597cm-1 (Chs3) (Nasir et al., 2005).

Despite the Chs/PVB, Chs/PVF and Chs/PEO (Chs/PEO from chitosan content 75% to

85% (wt/wt)) systems being immiscible, they had good mechanical properties. Blending

chitosan with PVB, increased the resultant blends TS as well as percentage elongations.

Chs/PVB blends with chitosan sample with a DD value of 97% produced blends with the

116


maximum TS values in comparison to the other two chitosan samples with lower DD

values. Hence higher DD values do improve Chs/PVB blend systems tensile strength.

Despite the immiscibility of the Chs/PVB blend system, chitosan improves the TS of the

PVB films while the elongation of the blend films improved due to the PVB component

in Chs/PVB blend systems. Blending chitosan with PVF is useful as pure chitosan has

lower TS and %E values while the Chs/PVF blends had better mechanical properties. As

the DD values of chitosan samples increased, the Chs/PVF blend systems’ average TS

values increased as well. For preparation of Chs/PVF film with the highest average TS

value, the best blend composition is Chs20PVF with the DD value of chitosan sample

being 97%. PEO had the highest %E value in this research work. Blending chitosan with

PEO improved the average TS values of Chs/PEO blend films as well as the elongation

properties of the blend system (Prodduturi et al., 2004).

Qualitative studies were done on the antimicrobial activity of chitosan, PVB, PVF, PEO

and chitosan blend systems with polymers mentioned. It was observed that PVB and PVF

had no antibacterial activity against Staphylococcus aureus. PEO had some antibacterial

activity. The antibacterial activity of chitosan samples increased with increasing DD

values of the chitosan samples. Chitosan sample with a DD value of 97% had no bacterial

growth. As the content of chitosan in the blend systems increased the antibacterial

activity increased as well. Chitosan/PEO had the greatest antibacterial activity at

Chs90PEO composition with the DD value of chitosan being 97%. Chs/PVB and

Chs/PVF systems had almost the same antibacterial activities.

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118

Further work is suggested for the evaluation of DD values of chitosan samples.

Techniques such as Proton Nuclear Magnetic Resonance (H1NMR) spectrometry, UV

and elemental analysis should be employed in DD analysis and comparison made to

titration method of analysis. Also the antimicrobial activity should be studied on other

bacteria as well, together with biodegradation of the chitosan blends. The thermal

transitions of chitosan as detected in this study should also be investigated further as

different published studies have reported different Tg values for chitosan with different

DD values.

The results of this research work have been presented by Dr. Khurma (my supervisor) at

The Indian Thermodynamics Society, Amritsar, National Conference on Thermodynamics

of Chemical and Biological Systems, Guru Nanak Dev University, Amritsar-143 005

(Pb.), INDIA (Dec. 28-30, 2005) and at the 9th European Symposium, September 9-12th

2007.

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Chapter 8.0 Appendix

CHAPTER 8.0

Appendix

This section contains data, calculations and figures that supplement discussions and

calculations in the thesis.

8. 1 KHP standard solution preparation

Moles of KHP used for preparing primary standard

Concentration of primary standard (KHP)

8.2 Standardisation of Sodium Hydroxide Solution

The primary standard of KHP solution was used to standardise NaOH solution. 25ml of

NaOH solution was used as aliquot in each titration. Three titrations were done for this

purpose. Below is the calculation using average titre volume of KHP.

133


8.3 Standardisation of Hydrochloric Acid Solution

A solution of 500ml of HCl was prepared. The standardised solution of NaOH was used

to standardise 25ml of HCl solution. Three titrations were done for this standardisation as

well. Below is the calculation using average titre volume of NaOH.

8.4 Determination of degree of deacetylation values of chitosan samples using titration

method

Chitosan samples were dissolved in 100ml of standardised HCl acid solution. These

solutions were stirred for 24 hours before being used for titrations. Of this 100ml

solution, 25 ml was withdrawn and titrated using NaOH solution.

The titration data was used to plot a graph of pH against titrant volume added. Two

inflexion points were weakly discernible. The derivative of data was taken and a second

graph plotted which showed two prominent peaks. The difference in volume between

these two peaks was used to determine the DD values of chitosan samples as shown in

the calculations below.

Sample 1

1.0029 gram of chitosan sample was dissolved in 100ml of 0.302M standardised HCl

acid solutions. From this 1.0029% (wt/vv) chitosan solution, 25ml was withdrawn and

then titrated using sodium hydroxide solution (0.992M).

134


The difference in titrant volume between the two peaks in the derivative graph was:

1.3ml with the first inflexion point being at titre volume 6.3ml. Hence 1.3ml of NaOH

was used to titrate the bond HCL to chitosan molecule.

Moles of Chitosan in 25ml chitosan solution (aliquot):

Moles of chitosan in 100ml of 1% (wt/vv) chitosan solution in HCl:

0.0012896(4) = 0.0051584 moles

Mass of chitosan component in 1.0029g of sample:

0.0051584 161 /0.8305

chitosann Mrg mol

g

��

Mass of chitin in 1.0029g of sample:

1.0029g – 0.8305g

0.1724g

Moles of chitin in 1.0029g of sample:

chitinn=m/Mr0.1724 203 /0.00084926

g molmoles

� ��

135


Degree of Deacetylation value for Chs1

#1

0.0051584 100%0.0051584 + 0.00084926

86%SampleDD

�

�

The same procedure as outlined above (including calculations) has been used to

determine the DD values of the other two chitosan samples. The DD values of the three

samples were: 86, 91 and 97 percent. The mass of chitosan sample used to prepare ~ 1%

(wt/vv) solutions varied. The masses of Chs2 and Chs3 were 1.0083 and 1.0089 grams

respectively.

136


137

Table 8.1 Table 8.2 Table 8.3

Chs1

Vol NaOH pH dpH/dV 0 0.79 1 0.82 0.03 2 0.91 0.09 3 1.01 0.14 1.15 0.14 5 1.31 0.16 6 1.51 0.2

6.1 1.55 0.46.2 1.61 0.66.3 1.91 36.4 2.11 26.5 2.22 1.16.6 2.31 0.96.7 2.41 16.8 2.55 1.46.9 2.66 1.17 2.77 1.1

7.1 3.01 2.47.2 3.31 37.3 3.71 47.4 4.1 3.97.5 4.8 77.6 6.9 21 7.7 8.1 12 7.8 9.1 10 7.9 10.05 9.58 11 9.5

Chs2

Vol NaOH pH dpH/dV0 0.83 1 1.1 0.27 2 1.5 0.43 1.8 0.34 2.1 0.35 2.5 0.46 3.1 0.6

6.1 3.2 16.2 3.5 36.3 3.7 26.4 3.8 16.5 3.9 16.6 4.05 1.56.7 4.2 1.56.8 4.4 26.9 4.5 17 4.6 1

7.1 4.7 17.2 4.8 17.3 4.9 17.4 5.1 27.5 5.4 37.6 6.9 15 7.7 7.8 97.8 8.6 87.9 9.3 78 9.9 6

8.1 10.5 6

Chs3

Vol NaOH pH dpH/dV 0 0.96 1 1.3 0.34 2 1.6 0.33 1.7 0.14 1.9 0.25 2.3 0.46 2.7 0.4

6.1 3.4 76.2 3.8 46.3 4.1 36.4 4.3 26.5 4.5 26.6 4.6 16.7 4.8 26.8 5 26.9 5.1 17 5.23 1.3

7.1 5.4 1.77.2 5.5 17.3 5.6 17.4 5.7 17.5 5.8 17.6 7.1 13 7.7 8.1 10 7.8 8.9 87.9 9.6 78 10.2 6

8.1 10.7 58.2 10.9 28.3 11.2 3


8.5 DD Determination using FTIR baseline (a) method

DD determination for Chs 1 using baseline (a) method:

1655

3450

100% 100

1.33

7351.06 10054516.86100

1.33

90%

AA

DD

� ��

��

�

8.6 DSC Analysis of PVB and PVF

Below are DSC thermograms for pure PVB and PVF

Figure 8.1 DSC thermogram of pure PVB in powder form

138


139

Figure 8.2 DSC thermogram of PVB film made in formic acid

Figure 8.3 DSC thermogram of pure PVF in powder form


140

Figure 8.4 DSC thermogram of PVF film made in formic acid

8.7 DSC Analysis of Chs/PVB System

Figure 8.5 DSC thermogram of Chs/PVB blend system (Chs1)




141


8.8 DSC Analysis of Chs/PVF System

Figure 8.8 DSC thermogram of Chs/PVF blend system (Chs1)


142



8.9 DSC Analysis of Chs/PEO System

Figure 8.11 DSC thermogram of Chs/PEO blend system (Chs1)

143


144




8.10 DMTA Analysis

DMTA analysis was done for only Chs/PVB blend system. Pure polymer spectra’s are

shown below and blend spectra data are shown in results sections.

Figure 8.14 DMTA Scan of Chs1


145



Figure 8.17 DMTA Scan of PVB

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