Download - CHITIN FROM JAMAICAN CRUSTACEANS

CHITIN:

ISOLATION AND

CHARACTERISATION

A Thesis

Submitted in Partial Fulfillment of the Requirement for the Degree of

Master of Philosophy in Chemistry

of

The University of the West Indies

by

Robert George Fowles

October 1999

Department of Chemistry

Faculty of Pure and Applied Sciences

Mona Campus

i

ABSTRACT

This thesis describes the isolation and characterisation of chitin obtained

from the exoskeleton of five Jamaican arthropods. These were the crustaceans

marine spiny lobster (Panulirus argus), the land crab (Gecarcinus ruricola), the

marine blue crab (Callinectes sapidus) and the giant Malaysian fresh water prawn

(Macrobracium rosenberg). The other arthropod investigated was the drummer

cockroach Blaberus discoidalis.

Isolation of chitin from crustacean shells involved acid digestion of

calcium salts, present in these shells followed by base hydrolysis of the shell

proteins. Instrumental Neutron Activation Analysis (INAA), weight loss

procedures, Atomic Absorption Spectroscopy (AAS) were the techniques

involved in the quantification of the isolated chitin.

INAA allowed for the elemental composition of the shell samples to be

determined. Shells were shown to contain calcium, sodium, potassium, bromine,

aluminium, manganese and chlorine. With the use of Gas Chromatography Mass

Spectrometry (GCMS) organic compounds like amines, high molecular weight

carboxylic acid and alkanes were also indicated. Complexation was shown to be a

workable alternative to acid digestion.

The percent content of calcium expressed as calcium carbonate of the

shells of the marine spiny lobster, land crab, blue crab and the giant Malaysian

fresh water prawn was determined to be 42, 70, 65 and 47%, respectively.

The digestion efficiency for extraction of calcium varied significantly with

species, as well as with the strength of the acid and the digestion time used.

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Standard acid hydrolysis was not effective in removing all calcium compounds

from the shells of some species of crustaceans.

The percentage by weight of chitin obtained from these crustacean shells

were found to be; Lobster 21%, land crab 18%, blue crab 19% and prawn 35%.

Characterisation involved the use of Thermogravimetric Analysis (TGA)

and Differential Scanning Calorimetry (DSC)), Scanning electron Microscopy

(SEM), carbon-13 NMR Spectroscopy and Infrared analysis. TGA and DSC show

that chitin is stable up to 394 °C. SEM showed by photographs the fibrous nature

of chitin. Carbon-13 NMR analysis showed chemical shift values that compared

well with literature values for glucose and IR analysis showed the characteristic

hydroxide band (3450 cm –1) and amide absorption band (1655 cm –1) associated

with chitin.

Characterisation of chitin also involved determination of the percentage

N-acetyl content (% N-Ac) by the use of two infrared analysis techniques where

(% N-Ac = A1655/A3450×115) and (% N-Ac = A1655/A3450×100/1.33). A typical

isolation process to produce chitin showed varying percent N-acetyl content,

which is affected by the alkaline conditions of the hydrolysis step as well as the

method of calculation.

The conversion of chitin to chitosan was also a method of characterisation

of chitin where chitosan was soluble in dilute acetic acid.

Key words: chitin, crustacean shells Instrumental Neutron Activation Analysis, weight loss, and calcium carbonate.

iii

ACKNOWLEDGEMENTS

I wish to acknowledge my supervisor, Dr. Keith Pascoe for his guidance

throughout the course of this project.

Special thanks to my co-supervisor, Dr. R. Rattray for his encouragement,

his unselfish help with the instrumental neutron activation analysis and atomic

absorption spectroscopy, and in completing this project.

Sincere thanks to the staff of The International Centre of Enviromental

and Nuclear Sciences UWI, Mona, for allowing me access to the SlOWPOKE 2

nuclear reactor and atomic absorption spectrophotometer and who from time to

time helped with information for this project; to Mr. Reid from the SEM unit for

his help with the scanning electron microscopy Studies; Mr. Aiken of the Life

Sciences Department UWI, Mona for identifying the crustaceans; Dr. Golden for

the gel electrophoresis analysis; Mr. Andrew Lewis for initial help with the

atomic absorption spectroscopy and Dr. Lancashire for some of the photographs.

Thanks to Dr. Paul Reese who was always ready to listen and make

suggestions for the various problems a graduate student faces.

I am indebted to Professor Dasgupta and the Chemistry Department for

the Departmental Award, the position as Tutorial Assistant and for the summer

jobs over the years.

I am thankful to all the kind staff members of the Chemistry Department

Miss Simon, Mrs. Chambers and Dr. Maragh to name a few. To my group

members Petrea, Fiona, Dionne, Susan and other past and present members of the

research laboratories – thanks for the love.

iv

DEDICATION

This work is dedicated to my Mother and Father, Almena and Alphonso;

to my brothers and sisters Chester, Clifton, Neville, Adrian, Kaye and Tonia,

“Oh how the years go by, oh how the love brings tear to my eye…we

laugh we cry as the years go by.” - Amy Grant

To my dear friend and wife Andrea truly beauty is your middle name.

v

TABLE OF CONTENTS

Pages

ABSTRACT i

ACKNOWLEDGEMENTS iii

DEDICATION iv

TABLE OF CONTENTS v

LIST OF COMPOUNDS ILLUSTRATED ix

LIST OF SCHEMATIC DIAGRAMS ix

LIST OF TABLES ix

LIST OF FIGURES xi

LIST OF PHOTOGRAPHS xii

CHAPTER ONE CHITIN

1.1 Introduction 2

1.2 History 3

1.3 Structure and Bonding 4

1.4 Biosynthesis 7

1.5 Polymorphic forms of chitin 10

1.6 Physical properties 12

1.7 Sources 14

1.8 The crustacean and exoskeleton 16

1.9 Techniques for extraction of chitin 20

1.10 Chitosan 24

1.11 Derivatives and uses 31

REFERENCES FOR CHAPTER ONE 38

vi

CHAPTER TWO ISOLATION OF CHITIN:

COMPOSITION AND CHARACTERISTIC OF

THE EXOSKELETON OF THE JAMAICAN

ARTHROPODS

2.1 Introduction 44

2.2 History, principles and instrumentation for instrumental neutron activation analysis (INAA) 45

2.3 Determination of percentage calcium in some Jamaican crustacean shells 54

2.3.1 Introduction 54

2.3.2 Digestion of lobster shells with different acids over varying times – optimising of digestion conditions by (a) weight loss percentages and (b) INAA 54

2.3.3 Calcium carbonate content of crustacean shells with optimised acid digestion conditions – as determined by weight loss 60

2.3.4 Calcium carbonate content of (a) crustacean shells and (b)chitin-protein residue - as determined by INAA 64

2.4 History principles and instrumentation for atomic absorption spectroscopy (AAS) 74

2.5 Calcium carbonate content - as determined by AAS 78


2.5.2 Results and discussion of AAS calcium carbonate determination 79

2.6 Chitin content of crustacean shells as determined by alkaline hydrolysis 82


vii

2.6.2 Percent unhydrolysed product (UHP%) after alkaline hydrolysis 83

2.6.3 Percent calcium carbonate impurities in unhydrolysed product 84

2.6.4 Composition of the exoskeleton 88

2.7 Removal of calcium from crustacean shell by complexation 92

2.7.1 Removal of calcium from crustacean shell by complexation with EDTA 92

2.7.2 Removal of calcium from crustacean shell by complexation with 18-Crown-6 ether 93

2.8 Chitin in cockroach 96

2.9 Summary 100

REFERENCES FOR CHAPTER TWO 100

CHAPTER THREE CHARACTERISATION OF CHITIN

3.1 Introduction 103

3.2 Thermal analysis 104

3.3 Scanning electron microscopy 109

3.4 Carbon-13 NMR analysis of chitin monomer 114

3.5 IR Spectral analysis – functional group analysis

and % N-acetylation determination. 117

3.5.1 Functional group analysis 117

3.5.2 Percentage N-acetylation (% N-Ac) 122

3.6 Chitosan from chitin 131

viii

REFERENCES FOR CHAPTER THREE 132

CHITIN AND ECONOMICS 133

APPENDIX ONE: EXPERIMENTAL DETAILS FOR

CHAPTER TWO 136

APPENDIX TWO: EXPERIMENTAL DETAILS FOR

CHAPTER THREE 145

ix

LIST OF COMPOUDS ILLUSTRATED

(1) Chitin 2

(2) Cellulose 4

(3) Hydrogen bonding in chitin 4

(4) Chitosan 5

(5) True chitin 5

(6) Chitin monomer 114

(7) glucose 114

(8) Biosynthetic (artificial) chitin 114

LIST OF SCHEMATIC DIAGRAMS

Scheme 1.1 Chitin hydrolysis 6

Scheme 1.2 Biosynthesis of chitin 9

Scheme 1.3 Formation of chitosan polycation 25

Scheme 1.4. Other derivatives of chitin 36

LIST OF TABLES

Table 2.1 Weight loss percentage on digestion of lobster shells with different acids over different digestion times 56

Table 2.2 Preliminary weight loss results of digestion of lobster shells

with 2M HCl 61 Table 2.3 Preliminary weight loss results of digestion of land crab shells

with 2M HCl 62

Table 2.4 Preliminary weight loss results of digestion of blue crab shells with 2M HCl 63

x

Table 2.5 Preliminary weight loss results of digestion of prawn shells

with 2M HCl 63

Table 2.6 Results of analysis of crustacean shells for calcium by INAA 65

Table 2.7 Comparison of percentage calcium (as calcium carbonate ) determined by INAA and average weight loss 66

Table 2.8 Results of analysis of chitin-protein residue obtained from 2M HCl digested shells for calcium by INAA 68

Table 2.9 New results of analysis of 2M HCl digested shells for

calcium (as calcium carbonate ) determined by INAA 69

Table 2.10 New weight loss percentages after 2M HCl digestion of crustacean shells 71

Table 2.11 Percentage calcium (as calcium carbonate) determined

by AAS and INAA experiments 80

Table 2.12 Alkaline hydrolysis of crustacean shells– percentage unhydrolysed product 84

Table 2.13 Calcium carbonate content of unhydrolysed product 85

Table 2.14 Elemental composition of shells 90

Table 2.15 Percentage calcium carbonate over different time periods using EDTA solution at roomtemperature 93

Table 2.16 Percentage weight loss by using 18 crown 6 – 1 ether 94

Table 2.17 Acid and alkaline hydrolysis of a Blaberus cockroach 96

Table 3.1 13 C data for hydrolysed chitin glucose and chitosan hydrochloride 115 Table 3.2 Percentage N-acetylation of chitin and chitosan samples 128

xi

LIST OF FIGURES

Figure 1.1 Cross section of the exoskeleton of a crustacean 17

Figure 2.1 Schematic diagram of sample flow from irradiation to counting stage 48

Figure 2.2 A typical INAA spectrum 49

Figure 2.3 INAA results after digestion of lobster shells with different acids over different times 59

Figure 2.4 Percent calcium present in crustacean shells 71

Figure 2.5 Percentage chitin calculated in (a) lobster and (b) prawn shells 98

Figure 3.1 TGA curves of prawn (cpwn2a) and lobster(clob2a) chitin 106

Figure 3.2 DSC curve of lobster chitin 106

Figure 3.3 DSC curve of prawn chitin 109

Figure 3.4 IR spectrum of unpurified crab chitin obtained from Sigma Co. 118

Figure 3.5 IR spectrum of sample chitin from lobster shells 119

Figure 3.6 IR spectrum of skin-like material obtained from the wing of an adult Blaberuscockroach after NaOH digestion 120

Figure 3.7 IR spectrum of powdered material obtained from the leg of an adult Blaberus cockroach after NaOH digestion 121

Figure 3.8 IR spectrum the wing ofan adult Blaberus Cockroach 121

Figure 3.9 IR spectrum of unpurified crab chitosan obtained from Sigma Co. 126

LIST OF PHOTOGRAPHS

Photograph 1.1 The Jamaican Marine Spiny Lobster 15

Photograph 2.1 Chitin and chitosan sample of prawn (left) and lobster (right) 87

xii

Photograph 3.1 SEM of lobster chitin (scale bar, 1mm) 110

Photograph 3.2 SEM of lobster chitin (higher magnification

scale bar, 10µm) 110 Photograph 3.3 SEM of chitin from Blaberus

cockroach leg( scale bar = 1mm) 111

Photograph 3.4 SEM of chitin from Blaberus cockroach leg

(higher magnification, scale bar = 10µm) 112

Photograph 3.5 SEM of chitin from Blaberus cockroach wings (scale bar = 1mm) 112

Photograph 3.6 SEM of chitin from Blaberus cockroach wings

(higher magnification, scale bar = 10µm) 113

Photograph 3.7 Chitin (left) and Chitosan (Right) of Sigma Co (Chitosan: 85% deacetylated) 129

1

CHAPTER ONE

CHITIN

2

1.1 INTRODUCTION

Chitin (1) is a sugar polymer, fibrous in nature and structurally similar to

cellulose. It is one of nature’s most common organic compounds second only to

cellulose 1, 2, 3. It has been known since the nineteenth century. Chitin is

commonly found in the exoskeleton of arthropods (particularly the crustaceans) or

fungi and green algae that utilize nitrogen containing sugars 4 and its biosynthesis

involves a series of enzymatic transformations from trehalose or glucose to the

formation of UDP-N-acetylglucosamine 5.

The proposed uses of chitin are very wide, from medical applications

(example, wound healing) 6 to waste water treatment 7. The derivatives used in

many commercial applications are made from chitosan, the deacetylated product

of chitin. Chitin is usually found present with other organic polymers and/or

inorganic salts 4 and its isolation usually involves hydrolysing and digesting these

molecular neighbours.

(1)

CHITIN

O

OH

n

NHCOCH3

HOH2C

NHCOCH3

NHCOCH3NHCOCH

3

HOH2C

HOH2C

HOH2C

O O

O

O

OHO

OH O

O

O

OH

3

1.2 HISTORY

Chitin was first described in 1811 by H. Braconnot 8, professor of natural

history, director of the botanical garden and a member of the Academy of

Sciences of Nancy, France. He isolated chitin from mushrooms by treatment with

warm alkali. Twelve years later A. Odier 8 again found chitin in insect cuticle and

some plant tissue. The silk worm was also discovered as a source of chitin when

in 1843 J. L. Lassaigne 8 isolated it from the Bombyx mori. In the same year, A.

Payen 8 initiated discussion about the differences between cellulose and chitin.

The monomeric unit of chitin (N-acetyl glucosamine) became known because of

the work of G. Ledderhose 8 in 1878 and E. Gilson 8 in 1894.

Rouget 9 discovered chitosan in 1859. He boiled chitin in potassium

hydroxide solution and found that the product chitosan dissolved in organic acid,

and was violet in diluted solutions of iodine and acid. In contrast, chitin is stained

brown in iodine-acid solution. Hoppe Seyler 9 coined the name chitosan in 1894

and in 1950, it was clearly described as a polymer of glucosamine 10.

In the first half of the twentieth century, research on chitin was mostly

directed toward the study of its occurrence in living organisms, its degradation by

bacteria, its uses in resin technology and its chemistry 9.

4

1.3 STRUCTURE AND BONDING

Chitin (poly-N-acetyl-D-glucosamine) (1) is a polysaccharide consisting

of beta (1-4) linkages. Therefore, it is sometimes referred to as beta (1-4)-2-

acetamido-2-deoxy-D-glucose. It is believed to be a derivative of natures most

common polysaccharide, cellulose (2) (beta (1-4) D-glucose) 11.

(2)

CELLULOSE

Glucose is the precursor of both molecules; both formed via primary

metabolism. The difference between chitin and cellulose occurs at position two

where in cellulose the hydroxy group replaces the acetamide group 13. Both chitin

and cellulose molecules are organised together in microfibrils consisting of

hydrogen bonds (3) 5.

(3)

HYDROGEN BONDING IN CHITIN

O

OH O

O

O

OHO

OH

O

O

OH

HOH2C

HOH2C

HOH2C

HOH2C

O

OH

OH

OH

OH

O

n

O NHCOCH3

HOH2C

NHCOCH3

NHCOCH3NHCOCH

3

HOH2C

HOH2C

HOH2C

O

H-O H-O

H-OH-O

n

O

O

O

O

O

O

O

hydrogen bond

5

Isolated chitin (true chitin) is not totally acetylated due to the partial

formation of the derivative chitosan (beta (1-4)-2-amino-2-deoxy-D-glucose) (4)

during isolation 13 and is best represented as structure (5). The result is that in a

few cases the carbon atom at position two will bear a NH2 group 6, 12 instead of

the acetamido group.

(4)

CHITOSAN

(5)

TRUE CHITIN

O

OH O

O

O

OHO

OH

O

O

OH

HOH2C

HOH2C

HOH2C

HOH2C

O

NH2

NH2

NH2

NH2

O

n

O

OH O

O

O

OHO

OH

O

O

OH

HOH2C

NHCOCH3NHCOCH

3

HOH2C

HOH2C

HOH2C

O

NH2

NH2

O

n

6

During isolation, chitin being a glucose polymer is also hydrolysed to its

monomeric units consisting of N-acetyl glucosamine (scheme 1.1). This

degradation is the result of the harsh conditions often associated with the isolation

procedures 4.

Scheme 1.1

Chitin Hydrolysis

O

O

O

O

C

H

HH 2

O

O

O

O

O

N C C

C

H

H

HH

3

2

O

OH

O

O

OH

O

NHCOCH3

HOH2C

NHCOCH3

HOH2C

O

n

H

H, OH

H, OH

NHCOCH3

7

1.4 BIOSYNTHESIS

The biosynthesis of chitin represents the first case in which substantial

evidence was presented for the formation of a polysaccharide from a sugar

molecule. The N-acetyl glucosamine monomer coupled with the appropriate

enzyme is the main ingredient for chitin biosynthesis. Glaser and Brown 14 in

1957 investigated an enzyme from the fungi Neurospora crassa. This enzyme

activated free N-acetyl glucosamine to produce chitin. In the laboratory chitin has

been biosynthesised by using a distorted glucosyl substrate monomer (chitobiose

oxazoline derivative), chitinase at pH 10.6 15.

Chitin synthesases have also been identified in S. cerevisiac, (a species of

yeast) which catalyses the transfer of N-acetyl-glucosamine from UDP-N-Acetyl

glucosamine to a growing chain of beta (1-4)-linked-N-acetylglucosamine

residues 16.

A detailed process of chitin formation has been outlined by E. Cohen 5.

Active catalytic units assembled in the cell membrane polymerise N- Acetyl

glucosamine into extracellular chitin chains. The substrate for polymerisation 5-

uridine diphospho-N-acetyl-D-glucosamine (UDP-N-acetylglucosamine) is an

end metabolite of a cascade of cytoplasmic biochemical transformation that starts

from the disaccharide trehalose or from glucose. The membrane bound chitin

sythesase (UDP-2-acetamido-2-deoxy-D-glucose: chitin 4-beta-actamidoglucosyl

transferase) is the essential enzyme in the chitin formation.

The pathway has also been outlined by Muzzarelli 17. Biosynthesis is

8

believed to occur in the hypodermis. First, it involves hydrolysis of trehalose

C12H22O11.2H2O a non-reducing disaccharide, with the enzyme trehalase to form

glucose. The glucose is phosphorylated by ATP in the presence of the enzyme

hexokinase to form glucose-6-phosphate, which is transformed to fructose-6-

phosphate in the presence of the enzyme glucose phosphate isomerase. Amination

occurs in the presence of glutamine aminotransferase and the amino acid

glutamine to form alpha-D-glucosamine-6-phosphate. Glutamic acid is the by-

product (Scheme 1.2).

Acetylation by acetylCoA in the presence of the enzyme glucosamine-6-

phosphate-N-acetyl transferase causes the formation of N-acetylglucosamine-6-

phosphate. The latter rearranges via the enzyme phosphoacetylglucosamine

mutase to form N-acetylglucosamine-1-phosphate, which is converted to

uridenediphosphate-N-acetyl glucosamine (UDP-N-acetyl glucosamine) via the

enzyme uridinediphosphate-N-acetylglucosamine pyrophosporylase, and UTP.

Pyrophosphate is the by-product. The final product chitin is produced via the

enzyme chitin synthesase by the loss of UDP. Chitin synthesase was responsible

for the polymerisation while the loss of UDP causes the absorption of free energy

for the glycoside formation 18.

9

Scheme 1.2

Biosynthesis of Chitin

O

OH

H

H

H

OH

OH

OH

HO

HO

O

OH

H

H

H

OH

HO

H

A D P

A T P

C H 2 O H

H

H

OH

H

H

OH

OH

HO

C H 2 O H

H

O

H

HOH

HO

H

O

OH

H

H

HOH

HO

C H 2 O H

H

OH

H

H

OH

OH

HO

H

O

H

H

OH

OH

HO

H

O

H

H

HOH

HO

C H 2 O H

H

HOH2C

O

OHO

H

HO

C H 2 O H- 2 O3 POCH2

OH

H

H

H

C H 2 O P O 3 2 -

H N C

O

C H 3

H N C

O

C H 3

H N C

O

C H 3

O PO32 -

- 2 O3 POCH2

HH

H

UDP

O

H

H

HOH

C H 2 O H

H

H

H N C

O

C H 3

O

O

O

alpha-D-glucosido-alpha-D-glycosidetrehalose ( )

glucose

glucose-6-phosphate

fructose-6-phosphate

alpha-D-glucosamine

N-acetyl glucosamine -6-phosphate

NH2

C H 2 O P O 3 2 -

-6-phosphate

UTP

pyrophosphate

UDP-N-acetylglucosamine

CHITIN

N-acetyl glucosamine-1-phosphate

trehalase

hexokinase

glucose phosphate isomerase

glutamine-fructose-6-phosphate amino

transferase

glutamine

glutamic acid

glucoseamine-6-phosphate-N-acetyl

transferase

acetyl-Co-A

CoA

phosphoacetylglucosamine mutase

UDP-N-acetylglucosaminepyrophoshorylase

chitin synthesase

UDP

10

1.5 POLYMORPHIC FORMS OF CHITIN

Chitin forms a dimer chitobiose C16 H 28 O 11 N 2 19 and chains classified

as alpha, beta or gamma 20. The alpha form is the most common with a tightly

packed structure and is the most crystalline form. Two antiparallel chains are

found in the alpha polymer, with intramolecular hydrogen bonds existing between

the CH2OH group of one residue and the carbonyl group of the next residue.

There is also intermolecular H-bonding, so that all hydroxyl groups are bonded.

Alpha chitin is found in the exoskeleton of arthropods and in some fungi.

Beta chitin chain forms sheets linked by C=O and H-N hydrogen bonds

and contains no hydrogen bonding between CH2OH groups. This crystalline

hydrate can be easily penetrated by water. Thus, beta chitin is less stable than

alpha chitin 20.

The gamma form has been found in the cocoons of the beetles Ptinus

tectus and Rhychaenus fage and has not been totally classified, however, an

arrangement of two parallel chains and one antiparallel has been suggested 19, 20.

Alpha and beta chitin can be differentiated by the fact that IR analysis

shows that alpha chitin has absorbances at 1655 and 1621 cm –1(referred to as a

doublet) whilst the beta chitin exhibits a singlet at 1631 cm-1 21.

Upon dissolution in 6M HCl, beta chitin converts into alpha chitin, the

more stable form. Once the alpha form has been reached, there is no reconversion

to the beta form. Thus, beta chitin is regarded as being a unique metastable entity

11

resulting from a specific biosynthetic mechanism different from that leading to

alpha chitin.

The three forms of chitin have been found in different parts of the squid

Loligo 20. The squid’s beak contains alpha chitin; its pen contains beta chitin and

its stomach lining gamma chitin. This fact indicates that the three forms are

relevant to functions and not to animal classification. In areas where extremes of

hardness are required alpha chitin is usually found frequently sclerotised and

encrusted with mineral deposits. Beta and gamma chitins are associated with

collagen type proteins providing toughness, flexibility and mobility, and may

have physiological functions such as support, control of electrolytes and transport

of ions 22.

12

1.6 PHYSICAL PROPERTIES

The physical properties of chitin investigated were molecular weight,

solubility, electrical properties, swelling and hydrophilicity.

(a) MOLECULAR WEIGHT

Chitin has an average molecular weight ranging from 1.036 million to 2.5

million Dalton (amu). The variation is a function of the extent of N-

acetylation 21, 23.

(b) SOLUBILITY

Chitin dissolves in concentrated solutions of lithium or calcium salts and

mineral acids, however extensive degradation occurs24. Precipitation from these

sources has been used as a means of purification.

Hexafluoroisopropanol and hexafluoro-acetone sesquihydrate are also

good solvents for chitin. Chloroalcohols for example, 2-chloroethanol, 1-chloro-

2-propanol and 3-chloro-1,2 propane diol, in conjunction with aqueous solutions

of mineral acids or with certain organic acids are also effective. These solvents

give relatively low viscosity solutions of chitin, dissolving it rapidly at room

temperature or mildly elevated temperatures. Degradation proceeds slowly 25.

(c) ELECTRICAL PROPERTIES

Alpha chitin has been reported to have electrical properties referred to as

piezoelectricity. This is electricity associated with anisotropic crystals when

13

subjected to pressure. Piezoelectricity then depends on the mechanical and

dielectric properties of chitin. The small values of dielectricity that have been

reported may be due to the many microvoids that exist in the polymer. The

dielectric constant increases where there is adsorbed water 26.

(d) CHITIN SWELLING AND HYDROPHILICITY

Repeatedly freezing and defreezing chitin in alkali solution causes it to

swell and dissolve, because the structure of the chitin becomes friable during

physical changes 27.

Water molecules are retained on the inner surface of chitin molecules. The

surface is less active and less permeable to water molecules than cellulose

fibres 27.

14

1.7 SOURCES

Chitin is found predominantly in the exoskeletons of members of the

phylum Arthropoda. This phylum includes the class Arachnida (spiders,

scorpions, ticks), class Insecta (cockroaches) and class Crustacea (lobsters, crabs

and shrimps). It is also found in some members of the phylum Annelida and

Mollusca.

The cell wall of members of the Fungi kingdom (yeast, mildews, rusts and

mushrooms); the divisions Chlorophyta (green algae), Phaeophyta (brown algae)

and Rhodophyta (red algae) are also noted sources. Photosynthetic plants utilize

nitrogen free sugars almost exclusively for their supporting structures and so lack

chitin 28, 29, 30.

Crustacean exoskeletons are probably the most readily available source of

chitin. The marine spiny lobster (Panulirus argus) - classified as a crayfish

(Photograph 1.1), the spotted spiny lobster (Panulirus guttatus), the long-armed

spiny lobster (Justitia longimanus), the copper lobster (Palinurellus gundlachi),

the spanish lobster (Scyllarides aequinoctialis), the slipper lobster (Parribacus

antarcticus) 31, the land crab (Gecarcinus ruricola), the blue crab (Callinectes

sapidus) and the giant Malaysian fresh water prawn (Macrobracium rosenberg)

are sources of chitin found in Jamaica.

15

Photograph 1.1

THE JAMAICAN MARINE SPINY LOBSTER

16

1.8 THE CRUSTACEAN AND EXOSKELETON

Crustaceans live in both aquatic and terrestrial environments 32 and their

bodies are designed to adapt to these environments. A tough heavily calcified

cuticle (the exoskeleton) covers their bodies, which protects the animals from

predators. This cuticle is resistant to changes in shape and the presence of joints

allows for the movement of the body.

The exoskeleton of crustaceans is composed of many layers. The

epicuticle is a thin light brown translucent waxy semipermeable outer layer of

lipoid material (3-6 µm thick), lacking chitin and lying on a protein layer. It is the

main waterproofing layer and gives protection against microorganisms. Because

of the tanning process the protein molecules are bound by oxidised phenolic

compounds which make the epicuticle very tough. The oxidised phenolic

compounds, are also responsible for the dark colouring of the exoskeleton. Being

lightly calcified and flexible, the epicuticle is ideal for resisting abrasion. It is

thicker in areas liable to wear and tear, such as in the tips of the walking legs or

between joints 33, 34.

Immediately underneath the epicuticle are the exocuticle and the

endocuticle, which make up the procuticle. The procuticle is a chitin-protein layer

of microfibrils. The microfibrils form monolayers or lamellae parallel to the

surface of the cuticle and within the lamellae all the microfibrils are parallel to

each other 35, 36 (Figure 1.1). The whole procuticle is strengthened by heavy

calcification within the chitin-protein matrix 35.

17

Figure 1.1

CROSS SECTION OF THE EXOSKELETON OF A CRUSTACEAN

The exocuticle can be clearly differentiated from the endocuticle. The

exocuticle is laid down in the form of hexagonal pillars oriented perpendicular to

the surface. Within the pillars, the chitin-protein lamellae are discontinuous and

irregular. In the inner exocuticle, the pillars coalesce and the lamellae become

continuous. The endocuticle forms lamellae running parallel to the surface of the

exoskeleton. In the exocuticle the lamellae are fine and tightly packed whereas in

the endocuticle they are larger and loosely stacked.

Tanned protein tails down from the epicuticle into the space between the

pillars of the exocuticle and the protein already present is also tanned. Tanned

18

proteins are absent from the endocuticle. Deposits of melanin occur throughout

the exocuticle, unlike the endocuticle, which is unpigmented. From a

development point of view, the epicuticle and the exocuticle are secreted before

moulting, while the endocuticle is produced after moulting.

Moulting or ecdysis is a process that allows the crustacean to grow. The

exoskeleton becomes loosened from the underlying hypodermis (lower layer of

the epidermis) as the epidermal layer secretes a new epicuticle. The hypodermis

then secretes chitinase and proteinase, which digest the old endocuticle 37. About

10% of the calcium compounds present are resorbed and stored and the rest lost to

the environment 38. The exoskeleton then softens at which point it is shed 36.

Protein and chitin are then synthesised in an effort to rebuild the exoskeleton. The

calcium compounds that were removed and stored are then returned to start the

hardening process. The rest of the calcium that is needed is absorbed from the

surrounding environment 38, 39. Glucose is used to provide carbon that is

incorporated into chitin during the early post molt period 40.

The chitin and protein in the exocuticle are believed to form a complex in

an approximate 55:45 ratio 41. A typical crustacean shell consists of about 25

percent complex (chitin-protein) and 75 percent calcium compounds 42. This ratio

is expected to change during growth and from species to species. There is no

apparent relationship between the proportion of chitin and the degree of

calcification.

Two types of protein are to be found in the shell. These are arthropodin

19

and resilin. Arthropodin forms a complex with chitin. Tanning increases its

degree of hardness and during this reaction, its molecular structure becomes much

firmer due to the formation of many additional crosslinkages at which point it

becomes known as sclerotonin. Resilin is an elastic protein made up of amino

acids running in all directions and randomly joined 35.

The innermost layer of the cuticle is a membranous layer lying on top of

the epidermis. This layer is similar to the endocuticle but is uncalcified. The

epidermal cells are capable of synthesising all precursors of chitin, from glucose

to uridine diphosphate-N-acetyl glucosamine 43, 44.

Below the epidermal layer are tegumentary glands, their ducts extending

through the exoskeleton to open on the surface. Tegumentary glands are most

common in areas prone to abrasion. They have been implicated in the repair to

damaged tissue by the secretion of epicuticlar like material. Running through the

cuticle are pore canals and the ducts of the tegumentary glands. The pore canals

probably assist in transport of material during exoskeleton growth. The pores

leading to bristles seem to have sensory functions.

The exoskeleton is arranged into plates called sclerites. At all movable

joints, the sclerites are fastened together by thin flexible articular membranes

made of chitin alone 44.

20

1.9 TECHNIQUES FOR EXTRACTION OF CHITIN

Several methods for the extraction of chitin from crustacean shells have

been reported in the literature. Some of the more widely used methods are

summarised below.

(a) METHOD OF HACKMAN 4, 45, 46

This is possibly the most popular method of isolation even if it is not

always referred to by name. Isolation of chitin results in a partly degraded product

and a mixture of chitin and chitosan (large deacetylation). Lobster shells were

dried in an oven at 100 °C. The shells were digested for 5 h with hydrochloric

acid (2 M) at room temperature, washed, dried and ground to a fine powder. The

powder was extracted for two days with hydrochloric acid (2 M) at 0 °C. The

resulting solid material was then collected by filtration, washed and extracted for

12 h with sodium hydroxide (1 M) at 100 °C. The alkali treatment was repeated

four more times. The resulting chitin was washed with water until neutral then

with ethanol and ether.

(b) METHOD OF WHISTLER AND BEMILLER 4, 45, 46

This method is milder than the method of Hackman because it does not

include boiling NaOH. Lobster shells were cleaned by washing and dried in an

oven at 50 °C. The shells, ground, were soaked for three days in 10% sodium

hydroxide solution previously deareated, at room temperature. Fresh hydroxide

solution was used each day. The deproteinised material was then washed until

21

free of alkali, then treated with ethanol (95%), to clean the product of pigments.

The protein free residue white in colour was washed with acetone, ethanol and

ether and then suspended in hydrochloric acid (37%) at –20 °C for 4 h. The

suspension was then filtered and the particles obtained washed with water, ethanol

and ether.

(c) METHOD OF HOROWITZ, ROSEMAN AND BLUMENTHAL 4, 45, 47

This method involved the use of shells partially digested with an organic

acid. The shells were digested for 5 h with HCl (2 M) at room temperature as

outlined by the method of Hackman 4, 46, 47. The decalcified lobster shells were

shaken for 18 h with concentrated formic acid (90%) at room temperature. After

filtration the residue was washed with water and treated for 2.5 h with sodium

hydroxide solution (10%) on a steam bath. The suspension was then filtered,

washed with water, ethanol and ether.

(d) METHOD OF FOSTER AND HACKMAN 45, 47

This method involves the use of the complexing agent ethylenediamine

acetic acid (EDTA) to remove calcium. Large cuticle fragments of the crab

Cancer parugus were attacked slowly (2 or 3 weeks) by EDTA at pH 9.0. The

residue was then further treated with EDTA at pH 3, and then extracted with

ethanol for pigment removal and with ether for the removal of lipids. The protein

was removed with formic acid (98-100%) followed by treatment with hot alkali.

Powdered shells having particle size 1-10 µm were decalcified more rapidly, in 15

minutes, under the same conditions.

22

(e) METHOD OF TAKEDA AND ABE AND TAKEDA AND KATSURA 48, 49

Most of the methods outlined before involved the use of drastic treatments

with concentrated acids and alkalis, sometimes at high temperatures. They

resulted in a decrease in the amount of chitin odtained since degradation occured.

The method of Takeda et. al is perhaps the mildest of the isolation techniques

reported in the literature and involves the use of the complexing agent EDTA for

calcium removal and the enzyme proteinase to digest the protein. King crab shells

were decalcified with EDTA at pH 10 and room temperature. Digestion followed

with a proteolytic enzyme such as tuna proteinase at pH 8.6 and 37.5 ºC, or

papain at pH 5.5-6.0 and 37.5 ºC or a bacterial proteinase at pH 7.0 and 60 ºC for

over 60 h. The protein still present in the chitin was about 5% which was removed

by treatment with sodium dodecylbenzensulfonate or dimethylformamide.

(f) METHOD OF BROUSSIGNAC 48, 49

This method is simple and perhaps suitable for the mass production of

chitin with little deacetylation. Decalcification was carried out by a simple

treatment with hydrochloric acid (1.4 M) at room temperature. This was done in a

plastic or wooden container. When treating large amounts of crab shell powder, a

series of containers were lined up and the acid solution from the most decalcified

chitin container is sent to the least decalcified in order to use the acid solution as

completely as possible. It was not necessary to cool the containers. This operation

took about 24 h and the carbon dioxide gas evolution in the beginning was

monitored, which stopped after one day. Before ending it was suggested to check

23

the ash content.

After completion of the decalcification treatment, proteins were removed

with papain, pepsin or trypsin, which allowed the chitin produced to be as little

deacetylated as possible.

(g) METHOD OF RIGBY 50

This method involves the use of hot sodium carbonate. Workability of this

method is questioned because sodium carbonate is a weak base. Crustacean shell

wastes were treated with hot 1% sodium carbonate solution followed by dilute

hydrochloric acid (1-5%) at room temperature, and then 0.4% sodium carbonate

solution.

(h) METHOD OF BLUMBERG 50

This method involves firstly the hydrolysis of protein present followed by

digestion of calcium carbonate an opposite procedure to the typical method of

Hackman). Lobster shells were treated with hot 5% sodium hydroxide solution,

cold sodium hypochlorite solution and warm 5% hydrochloric acid.

24

1.10 CHITOSAN

Chitosan (5) is the N-deacetylated derivative of chitin and perhaps the

most important derivative. The ratio of 2-acetamido-2-deoxy-D-glucopyranose to

2 amino-2-deoxy-D-glucopyranose determines the naming of a sample chitin or

chitosan 1. Therefore, if there are enough amino groups present to render the

polymer soluble in dilute aqueous acid (e.g. acetic acid), then the polymer is

called chitosan 51. This ratio is determined by H-NMR, IR and titration methods,

and is termed the degree of N-acetylation 1,2. The degree of N-acetylation

influences the physiological properties, chemical properties, the biodegradability

and immunological activity of chitosan 52.

Chitosan is soluble in organic acids because of the formation of a

polycation 53 (Scheme 1.3). The solubility in organic acids renders chitosan more

easily manipulated than chitin for industrial applications 54.

25

Scheme 1.3

FORMATION OF CHITOSAN POLYCATION

1.10.1 CONVERSION TECHNIQUES (PREPARATION OF CHITOSAN)

The following are some of the published methods used in the production

of chitosan.

(a) METHOD OF HOROWITZ 55, 56

This harsh method involves the use of solid potassium hydroxide and very

high temperatures. Chitin was converted to chitosan by fusion with solid

potassium hydroxide in a nickel crucible while stirring in a nitrogen atmosphere.

After 30 min. at 180 ºC, the melt was poured carefully into ethanol and the

O

OH O

O

O

OHO

OH

O

O

OH

O

OH O

O

O

OHO

OH

O

O

OH

+

+

+

+

H+

CHITOSAN POLYCATION

CHITOSAN

HOH2C

HOH2C

HOH2C

HOH2C

O

NH2

NH2

NH2

NH2

O

n

HOH2C

HOH2C

HOH2C

HOH2C

O

NH3

NH3

NH3

NH3

O

n

26

precipitate washed with water to neutrality.

(b) METHOD OF RIGBY, WOLROM, MAHER AND CHANEY AND WOLPHROM

AND SHEN-HAN 55, 57

This is one of the simpler methods but does not include a purification step.

Chitin was treated with aqueous solution of sodium hydroxide (40%) at 115 ºC for

6 h under nitrogen. After cooling, the mixture was filtered and washed with water

until neutral.

(c) METHOD OF FUGITA 57, 58

This method is simple and requires much less hydroxide than other

methods reported. Chitin was mixed with of sodium hydroxide, kneaded with

liquid paraffin in a 1: 1; 10 ratio, and stirred for 2 h at 120 °C. The mixture was

poured into cold water, filtered and thoroughly washed with water.

(d) METHOD OF BROUSSIGNAC 55, 57

This is another very harsh method and possibly results in extreme

degradation of the chitin sample. A solution containing KOH (50%), EtOH (96°,

25%) and monoethyleneglycol (25%) was prepared. The resulting mixture was

placed into a stainless steel reactor consisting of a steam heating system and a

stirrer along with chitin. The temperature of the system was 120 °C corresponding

to the boiling temperature of the mixture. The treatment was carried out for the

desired length of time and after filtration the chitosan was washed with water until

neutral, then dried at moderate temperatures.

27

(e) METHOD OF PENISTON AND JOHNSON 59

In this method chitosan is produced directly from the shellfish wastes

which permits recovery of proteins, sodium acetate and calcium carbonate as by-

products, providing nearly complete conversion of shellfish wastes into

marketable commodities. Shellfish waste ground to particle size of 3-6 mm, was

applied to a protein extraction apparatus where the shell was moved

countercurrently to the flow of dilute sodium hydroxide (0.5-2%). The amount of

extraction by alkali solution applied is controlled to maintain a residual of

alkalinity needed to form proteinate. The time of the extraction step was between

1-4 h, depending on the porosity of the shell, at temperatures in the range 50-

60 °C. Subsequent to removing the sodium proteinate solution, it was then

clarified by centrifugation or filtration. (The solution may also be treated with

refining agents to remove lipids or pigments). The clarified product was then

neutralised with hydrochloric acid to the pH of minimum solubility (4.5-3.4). This

depended upon the shellfish species and extraction conditions. The resulting

precipitated protein was collected, washed and dried by reslurrying and spray

drying.

Following protein removal, the shell was again extracted countercurrently

in a further series of extraction cells containing a concentrated sodium hydroxide

solution. The effluent from this operation contained excess sodium hydroxide,

sodium acetate and sodium carbonate. This was passed to a crystalliser to

precipitate sodium acetate and sodium carbonate as useful by-products which

28

were removed by filtration or centrifugation, washed and purified by conventional

means.

The mother liquor was diluted with water and treated with calcium

hydroxide in order to convert the remaining sodium carbonate back to sodium

hydroxide. The sodium carbonate crystallisation was also treated with calcium

hydroxide for sodium hydroxide recovery. The precipitated calcium carbonate

was then collected. The regenerated sodium hydroxide solution was combined

with added concentrated alkali and evaporated to the desired strength for use in

one of the early extraction processes.

The deacetylation and decarbonation process now completed, left behind

the residual shell consisting of chitosan and calcium hydroxide. This was washed

with carbonate-free water to remove residual sodium hydroxide.

The chitosan and calcium hydroxide mixture was then extracted with an

aqueous solution of sucrose. The calcium carbonate, which was dissolved as

calcium saccharate, was removed, leaving behind pure chitosan which was then

washed to neutrality and dried. The saccharate was then carbonated, precipitating

calcium carbonate, which was washed and passed to a calcium hydroxide kiln.

The sucrose solution was evaporated to the desired concentration and reused.

Other substances capable of chelating calcium, such as glycols, EDTA, sorbital or

gluconates may also be used instead of glucose.

29

(f) CHITOSAN BY FERMENTATION 60

Chitosan has also been prepared by fermentation. The fungal order

mucorales contains chitosan as a cell wall component. Absidia coerula a member

of this class was readily cultured on nutrients (example glucose or molasses) and

the cell wall material recovered by simple chemical procedures.

(g) AQUEOUS SODIUM HYDROXIDE METHOD 61

Probably the simplest of the procedures is the aqueous sodium hydroxide

method, easily carried out in a laboratory. In addition, a purification step is

present. NaOH (40%) was added to chitin and refluxed under N2 at 115 °C for

6 h. The cooled mixture was then filtered and washed with water until the

washings were neutral to phenolphthalein.

The crude chitosan was purified as follows. It was dispersed in acetic acid

(10%) and then centrifuged for 24 h, to obtain a clear supernatant liquid. The

latter was treated dropwise with aqueous sodium hydroxide (40%) solution and

the white flocculent precipitate formed at pH 7. The precipitate was then

recovered by centrifugation, washed repeatedly with water, ethanol and ether and

the solid collected and air-dried.

(h) HOMOGENOUS N-ACETYLATION OF CHITOSAN 2

Homogenous N-acetylation is geared towards making chitosan with a

required number of acetyl groups by adding a particular quantity of acetylating

agent.

30

Chitosan was dissolved in 1% aqueous acetic acid and the solution divided

into 5 equal portions. Ethanol was then added to each. Different volumes of

solutions of acetic anhydride in methanol (2 w%) were added to each solution..

After 1 h each solution was poured into a mixture of methanol and aqueous

ammonia (0.880 g / mL) (7/3 V/V). The precipitated polymer was then filtered,

washed well with methanol, then with ether and air-dried.

31

1.11 DERIVATIVES AND USES

There are various chitin derivatives, the main one being chitosan from

which many other derivatives are made. Many of the uses of chitin that are found

in the literature are also uses of chitosan, which demonstrates the importance of

chitosan to the chitin researcher. Some uses of chitin and chitosan are outlined

below.

(a) COMPLEXING AGENTS

Chitosan can absorb enzymes, anionic polysaccharides and is known to be

a good complexing agent that has been used to remove radioactive or toxic

elements, for example plutonium and arsenic, from various types of media 3, 62, 63.

Chitosan may be used to remove suspended particles from turbid

solutions. It helps to precipitate solids suspended in liquids by bonding to the

impurities. The impurities include alkali earth metals, vegetable matter and

proteins. Chitosan has been found to be as effective as seperan, a commercial

flocculating agent used in removing inorganic suspended solids in solutions 64. It

may be used along with coagulation aids like alum, ferric chloride or calcium

chloride in removing vegetable matter from tanks containing solutions 65.

(b) SHEET FORMING PROPERTIES

Chitin, chitosan and their derivatives have desirable sheet forming

properties. In solution, chitosan has been used in coatings or adhesives by the

paper industry, and has been reported as a filler or binder for cellulosic papers 51.

32

In solid form, chitin, chitosan and derivatives have demonstrated sheet-

forming properties. For example, Takai and co-workers 51 used chitin fibers to

make chitin papers by applying deproteinized, ground chitin particles from a

homogenised suspension to a bench-scale continuous papermaking machine.

Chitin acetate has also been used to make fibres.

(c) CHROMATOGRAPHY

Powdered chitin has been used as the stationary phase to separate mixtures

of phenols, amino acids, nucleic acid derivatives and inorganic ions by thin layer

chromatography. The results of separation equalled or surpassed those of

crystalline cellulose, silica gel or polyamide layers 66.

(d) WOUND HEALING

Chitin and some of its derivatives has been found to increase the rate at

which wounds heal. Chitosan for example when applied to a wound binds to fats

and help to initiate clotting of red blood cells 3, 67.

(e) DYE-SORPTION

Textile effluents usually contain very small amounts of dyes. They are

highly dispersible aesthetic pollutants that poison the aquatic environment. They

are difficult to treat because by design, they are highly stable molecules, made to

resist degradation by light, chemical, biological and other exposures. These dyes

are usually mixtures of large complexes and there is little certainty about their

molecular structure and properties. Other materials such as salts, surfactants, acids

33

and alkalis also accompany them.

Due to its unique molecular structure, chitosan has an extremely high

affinity for many classes of dyes so that it can be used to remove them from waste

products before they are released into the environment 7.

(f) GLASS FABRICS

It is difficult to use conventional dyes and techniques to dye glass fabrics,

because these dyes are deposited superficially and wash out simply by wetting.

Chitosan when applied to glass fibre forms a permanent coating with

many available sites thereby creating a product with physical characteristics

inherent to glass fibre and textiles, enhanced with chemical capacity to receive a

wide variety of dyes 68.

Other fibres, films, fabrics and yarns such as those made from olefins for

example polyethylene and polypropylene (plastic fibre) are also difficult to dye

with commercial dyes. Chitosan mixed with other compounds may be applied to

fabrics, which creates an electrostatic system to allow for the adsorption of these

dyes 69.

(g) BATIK DYEING

Chitosan salt solutions in a viscous and pastelike state react with all types of dyes

except cationic ones, producing water-insoluble precipitates. When they are

applied to a cloth and dried, a film with a strong resistance to peeling, suitable

34

plasticity, cuttable and scratchable, without causing its separation from the

material is formed. Thus, various designs can be cut or scratched in the cloth

without peeling 70.

(h) ANTISTATIC PROPERTIES

Substances with soil repellent and soil releasing properties are often added

to fabrics to reduce soiling. These substances may be strongly hydrophobic, for

example fluorinated polymers, or they may be hydrophilic polymers containing

carboxylic, phosphoric and or sulphonic acid groups. The hydrophobic polymeric

materials may become electrified readily when subjected to friction. Chemically

modified chitosan may be used to impart antistatic properties to these fabrics 71.

(i) PHOTOGRAPHIC FILMS

The photographic field is potentially very important for chitosan

applications. Chitosan is resistant to abrasion. Its film forming properties, its

optical characteristics and its behavior with silver complexes, make it important to

the photography industry. The chitosan film can be easily penetrated by solutions

carrying silver complexes 72.

(j) ADHESIVE PROPERTIES

Chitosan salt solutions are known for their adhesive properties. It is an

effective sealer and primer for wood, asbestos-cement board and paper, plasters,

brick and tile. Chitosan, when applied to these surfaces, decreases or prevents

35

penetration of contaminants (water, dirt, moisture, oils, grease, smoke and tar)

which cause deterioration of the surfaces due to the difficulties in cleaning 73.

(k) TOBACCO ADDITIVE

Chitosan solutions, when mixed with tobacco and other optional

ingredients may be formed into tobacco having good dry tensile properties and

good smoking characteristics 74, 75.

(l) LEATHER TANNING

Chitosan has been studied for its use in tanning, paste-drying and finishing

of leather, where it improves the quality of the material 76.

(m) BIOLOGICAL CARRIERS

Chitin is effective as an antigen when administered to animals attacked by

parasites such as ticks and mites and certain types of bacteria and fungi. Chitin

and chitosan derivatives have been used as enzymatically decomposable

pharmaceutical carriers. They are appealing as carriers because they are degraded

by lysozyme - an enzyme produced in the human body - and the degradation

products are not poisonous 77.

(n) ANTICOAGULANT

Heparin, one of the worlds most widely used blood anticoagulants was

isolated from liver cells in 1918 78. It is an expensive product and is in short

supply. Sulfated chitin has been investigated for its anticoagulant properties and

36

activity has been found in the fully amino group substituted polymer. Introduction

of uronic acid into chitin increases this activity.

(o) Other derivatives

Other derivatives that have been explored are summarized in Scheme 1.4.

Scheme 1.4.

OTHER DERIVATIVES OF CHITIN

O

O

O

O

O

O

O

O

OO

O

O

N

*

n

C C

C

H

H

HH

H

OO

OO

OO

OO

3

2

NHR2

ROH2C

H

1

n

( R1 = CH2CO-ARG-GLY-ASP-SER-OH

CH3COOH or R2 = H, AC)

RO

NHCOCH3

HOH2C

R = CO(CH2)mCH3

m - 2 = 8 ; n - 20 = 5000

chitin

NHCOCH2CH2CO2M

HOOCH2COH2C

n

M = group 1 or 2 metals

n - 10 = 5000

NHCOCH3

ROH2C

HO

R = (CH2)nCOOH or H

n > 1

*

n

chitin sulphate

79

8183

85

n

37

Cosmetics containing chitosan carboxy derivatives have been prepared. The

cosmetics showed excellent moisturising effect 79. Trimethylsilyl derivatives of

chitin have been prepared for possible industrial application 80.

Carboxymethylated derivatives of cell adhesion peptides have been prepared as

cancer metastasis inhibitors 81.

Chitin sulphates have been studied in order to prepare blood anti

coagulants 82. Nail polish containing chitin alkyl ester has been prepared which

served as a film-forming agent and or resin component 83. A substitute for eye

fluid containing O-carboxyalkyl chitin has been prepared 84.

Chitin has been used under the banner of a product “Fat Absorb” by diet

watchers. Capsules of chitin ingested after a meal are expected to bind with fats

and oils, preventing them from being digested by the body. They are therefore

easily egested 85.

Coating rice seeds with chitosan has been reported to cause higher yields.

A derivative of chitin developed by Harvard University 3 has been reported to

possibly halt the spread of AIDS. The compound slowed the synthesis of proteins

by the AIDS virus and prevented the virus from attaching to cell surfaces as well

as interfered with the activity of a key viral enzyme, reverse transcriptase 3.

38

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43

CHAPTER TWO

ISOLATION OF CHITIN:

COMPOSITION AND CHARACTERISTICS

OF THE EXOSKELETON OF SOME

JAMAICAN ARTHROPODS

44

2.1 INTRODUCTION

The original aim of this research was to find novel ways of isolating chitin

from the exokeleton of arthropods. The main concerns were the purity of the

isolated chitin and the long hours of acid and alkaline hydrolysis required by

published methods. Preliminary investigation of the percentage chitin present in

crustacean shells involved acid hydrolysis for 48 hours with 2M HCl, followed by

alkaline hydrolysis with 1M NaOH. These treatments were intended to remove

calcium carbonate and protein, respectively. The difference in weight before and

after the treatments was used to obtain the chitin content. The results suggested up

to 31% chitin in spiny lobster, 41% in the prawn and 57% in the land crab and

blue crab shells. These results however seemed to be high 1, 2 and it was suspected

that these inflated percentages were largely due to the presence of residual

calcium carbonate in the chitin samples thus obtained.

There was therefore an urgent need to assess the efficiency of the acid

digestion process. The use of INAA and AAS met this need and thus it was

possible to more accurately determine the percentages of chitin present in the

spiny lobster, land crab, blue crab and prawn shells. INAA allowed for

determination of calcium in the solid matrix before and after digestion with acid

whilst AAS allowed for quantification of calcium that went into solution after

acid digestion.

45

2.2 HISTORY, PRINCIPLES AND INSTRUMENTATION FOR

INSTRUMENTAL NEUTRON ACTIVATION ANALYSIS (INAA)

INAA was introduced by Von Hevesy and Levy 3 in 1936. It is a reliable

method for determining the elemental concentration of a sample. The method is

based upon the measurement of radioactivity induced in samples by irradiation

with neutrons of the appropriate energy 4.

Three sources of neutrons are employed in neutron activation methods.

These are radionuclides, accelerators and reactors.

Radioactive isotopes which produce neutrons in their decay schemes e.g.

californium-252, are convenient and relatively inexpensive sources. However,

neutron flux densities are relatively low, ranging from 10 5 to 10 8 n cm -2 s –1.

Detection limits are not as good as with other neutron sources such as nuclear

reactors 4. Accelerators produce highly energetic (MeV) neutrons that can be

moderated to reduce their energies. For example, the acceleration of deuterium

ions through a potential of about 150 kV to a target containing tritium absorbed

onto titanium or zirconium produces neutrons on impact that can be used for

INAA. 5.

Neutrons are produced in the fission of the uranium 235 fuel in nuclear

reactors. Reactors produce a neutron flux ranging from 10 11 to 10 14 n cm -2 s-1

and detection limits in the range 10 -3 to 10 µg 4, 6. The SLOWPOKE 2 nuclear

reactor 7 at the International Centre for Environmental and Nuclear Sciences

(ICENS), University of the West Indies, Mona was used for INAA in this work.

46

SLOWPOKE (Safe Low Power C(K)ritical Experiment) is a Canadian-made

reactor, light water cooled and moderated with a maximum neutron flux of

10 12 n cm–2 s–1.

When a sample is bombarded with neutrons a radioactive isotope of the

element of interest can be produced by a principle called neutron capture. Here

the nucleus of the sample is penetrated by a neutron to produce an isotope with a

mass number greater by one and the release of energy in the form of prompt

gamma rays. The atom is now in a highly excited state 5. For example, for the

calcium isotope 48Ca,

48Ca + 1n = 49Ca + γ…………………………………………Equation 2.1

If the radioactive isotope (e.g. Ca 49) decays with the emission of gamma rays,

they can be measured by the appropriate detector 8. The gamma energy is

characteristic of the isotope and hence it is used for element identification

(qualitative identification). The number of gamma rays emitted per unit time or

the intensity is dependent on the number of atoms present in the sample

(quantitative identification) 9, 10.

Samples may be solids, liquids or gases 11. Neither chemical treatment nor

addition of reagent is required to prepare samples for analysis 10. Standards should

approximate the sample closely, both physically and chemically. For most

reactors, a standard has to be irradiated with every sample, at the same time, in the

same container. However, the exceptional flux stability of the SLOWPOKE 3

allows standards to be done once for a batch of samples. Samples and standards

47

are placed in small polythene vials or heat-sealed quartz vials to carry out the

irradiation. They are usually exposed to the same neutron flux for the same length

of time, which can vary from several minutes to several hours. Usually an

exposure time, of three to five times the half-life of the analyte product is

employed.

After irradiation is terminated, the sample and standards are allowed to

decay or ‘cool’ for a period that varies from a few minutes to several weeks.

During this time potential interfering isotopes in the sample with shorter half lives

are allowed to decay. Cooling also reduces exposure of the laboratory personnel

to radiation 11.

After cooling, the sample is placed at a precise position on a detector for

counting. A multichannel analyser (MCA) connected to the detector displays the

range of energies and intensities of gamma rays (called the gamma spectrum)

emitted from the sample. A neutron activation analysis programme on a PC is

used to quantify the energy and intensity of the radiation in the gamma spectrum.

Figure 2.1 shows the basic steps and instrumentation involved in INAA. A

typical INAA gamma spectrum of peaks representing numbers of counts at

particular energies specific to an element is shown in Figure 2.2 7.

48

Figure 2.1

SCHEMATIC DIAGRAM OF SAMPLE FLOW

FROM IRRADIATION TO COUNTING STAGE 12

49

Figure 2.2

A TYPICAL INAA GAMMA SPECTRUM

The calculation of the elemental concentration in a sample by INAA is

based on the comparison of the radioactivity induced by neutron irradiation of that

element in the sample to that induced in a known standard treated under similar

conditions.

The activity A induced by neutron irradiation is determined by the

following equation

A = N ϕ σ (1 – e-λti) e-λtd 11………………………………Equation 2.2

Where

N = number of atoms of the element in the sample;

ϕ = neutron flux in neutrons cm-2 s-1;

σ = Cross section (related to probability of neutron capture by the

50

element) in barns (1 barn = 10-24 cm2);

λ = Radioactive decay constant;

ti = irradiation time;

td = decay time (time from end of irradiation to start of count);

Because the SLOWPOKE-2 reactor used for INAA at ICENS has

exceptional neutron flux stability. If the same irradiation times are used for both

standard and sample, it follows that

Asam / Astd = Nsam / Nstd ×e-λtdsam/ e-λtdstd…………Equation 2.3

Where,

Asam = activity induced in sample;

Astd = activity induced in standard;

Nsam = number of atoms of element in sample;

Nstd = number of atoms of element in sample;

However, the ratio of the concentration in the sample Csam to that in the standard

Cstd is

Csam / Cstd = Nsam / Nstd x Wstd / Wsam………………Equation 2.4

51

Where,

Wsam = weight of sample;

Wstd = weight of standard;

Therefore, the concentration in the sample is

Csam = Cstd x (Asam / Astd) x (Wstd/Wsam) x (e-λtdstd / e

-λtd sam)

………………………………………………………………………Equation 2.5

Experimentally, count rate (which is directly proportional to activity) is

usually measured instead of activity. A high count rate of decay is desirable to

minimise the duration of the counting period. However, high count rates can

cause ‘pulse pile up’ in the detector as the electronics can only process a certain

number of gamma rays per second. If counting rates exceed the resolving time of

the detector, a correction must be made to account for the difference between

elapsed time (clock) and live (available counting) time 13.

Good reproducibility is essential for all analytical techniques. Imprecise

results are not always due to the method but are often due to inhomogenous

distribution of the element of interest in the matrix being analysed. INAA results

are not usually affected by matrix effects. Because of this, it is often applied as an

independent check on a new analytical method to make sure no systematic error is

affecting the technique 14.

52

Accuracy of the INAA technique is excellent, depending mainly on the

accuracy of the standards being used for comparison with the sample. The

principal errors that arise during INAA analysis are due to self shielding, unequal

neutron flux for sample and standard, counting uncertainties and errors in

counting due to scattering, absorption and differences in counting or irradiation

geometry between sample and standard. The errors from these causes usually can

be reduced to less than 10% by acknowledging routine quality control methods.

Uncertainties in the range of 1 - 3% are frequently obtained.

Only a few milligrams of the samples are required and as little as 10 -5 µg

of several elements can be detected. The sensitivity of the method is limited by

the sensitivity of the detector, the decrease in activity at the time of counting, the

time available for counting and the magnitude of the background count rate

relative to the count rate of the analyte. Many authors overestimate the sensitivity

of a favoured technique, but sensitivity of a method can be dependent on the

sample matrix 15.

INAA has been used routinely to measure trace element concentrations in

complex matrices such as human and animal tissue, coal, fly ash, petroleum, river

sediments, urine, faeces, blood etc. More than 25 elements can be analysed at the

same time 10. Analysis can be performed without destroying the sample 9, 10 and it

is therefore popular in forensic science.

Other techniques used in elemental analysis include atomic emission,

absorption or fluorescence spectrometry and mass spectroscopy. No single

53

technique presents a general answer to the large variety of problems involved in

elemental analysis .

54

2.3 DETERMINATION OF PERCENTAGE CALCIUM IN SOME

JAMAICAN CRUSTACEAN SHELLS

2.3.1 INTRODUCTION

To determine the acid best suited for the digestion process, lobster shells

were digested with five different acids over varying times and the loss in weight

calculated. In addition, INAA was applied to the digested lobster shells for the

determination of percentage by weight of residual calcium present, expressed as

calcium carbonate. The results from the weight loss and INAA experiments were

compared, which allowed the efficiency of the acid digestion to be determined.

The best acid (most efficient) was then used to digest all the crustacean shells and

the percentage residual calcium (as calcium carbonate) determined by comparison

with the total percentage calcium (as calcium carbonate) also determined by

INAA. Experimental details are given in Appendix one.

2.3.2 DIGESTION OF LOBSTER SHELLS WITH DIFFERENT ACIDS OVER VARYING

TIMES – OPTIMISING OF DIGESTION CONDITIONS BY (a) WEIGHT LOSS

PERCENTAGES AND (b) INAA

(a) Weight loss

In an effort to assess the efficiency of calcium removal by acid digestion a

series of experiments was designed using lobster shells and different acids over

varying digestion times and the results compared. The best acid was expected to

55

be associated with the largest weight loss and percentage weight loss associated

with percentage calcium (present as calcium carbonate). Lobster shells were

chosen on the basis that they were most readily available and because their texture

was intermediate between the prawn shells (soft) and the crab shells (hard,

coarse).

Lobster shells obtained from fishermen at Port Henderson beach, St.

Catherine, Jamaica, were cleaned, oven dried, crushed, redried and weighed.

Accurately weighed samples of the shells were treated with aliquots of the

hydrochloric acid, nitric acid, trichloroacetic acid, acetic acid and sulphuric acid

(all 2 M) in round bottom flasks contained in ice baths of temperatures between 0

– 4 °C. Digestion times used were 1, 6 and 48 h.

The undigested portion of the shell, called the chitin-protein residue

(complex), was collected by filtration after the digestion period was complete,

washed with water until neutral (as indicated by filter paper), air-dried, weighed

and the percentage weight loss determined (Table 2.1).

56

Table 2.1

PERCENTAGE WEIGHT LOSS ON DIGESTION OF

LOBSTER SHELLS WITH DIFFERENT ACIDS OVER DIFFERENT DIGESTION TIMES

Weight loss

/ %

Time /h

Acids (2 M)

1 6 48

HCl 52 56 57

HNO3 54 57 56

CCl3COOH 53 55 60

CH3COOH 42 42 50

Progress of the digestion was evident by the frothing of the solution

associated with the production of carbon dioxide.

In a digestion time of 1 hour weight loss percentages obtained for HCl,

HNO3 and CCl3COOH, did not differ significantly. They were 52%, 54% and

53% respectively, all higher than the 42% obtained after using CH3COOH.

When the digestion time was increased to 6 hour the weight loss

percentages increased with the use of HCl, HNO3 and CCl3COOH; the values

were 56%, 57% and 55%, respectively. The weight loss percentage obtained from

using CH3COOH remained unchanged. (Table 2.1).

57

Increasing the digestion time to 48 hours generally increased the weight

loss percentages over those obtained for 6 hour period. There was a 1% increase

with HCl digestion, a 4% increase with CCl3COOH and an 8% increase with

CH3COOH. However, a decrease in weight loss percentage was obtained with the

HNO3 digestion where, the percentage changed from 57% (6 hour) to 56% (48

hour) (Table 2.1).

Weight loss percentages obtained for sulphuric acid could not be

determined conclusively because of the formation of calcium sulphate, which is

sparingly soluble in water. The weight loss percentages obtained were too small

for such a strong acid. Overall, HCl, HNO3 and CCl3COOH all appeared suitable

for digestion of shells over the 1, 6, or 48 hour periods.

The weight loss percentages obtained were expected to be related to the

amount of calcium salts that had gone into solution, which may be interpreted as

percentage calcium carbonate (calcium carbonate is the main calcium compound

found in crustacean shells) 2. However, there was the possibility of the digestion

of organic polymers that form a significant part of the shells. To determine

conclusively the percentage calcium carbonate hence the efficiency of the calcium

carbonate digestion process, INAA was used.

58

(b) INAA

INAA was used to confirm the best conditions required for digestion as

well as address the matter of efficiency of digestion. Approximately 0.25 g of

each of the chitin-protein residues obtained by digestion with the different acids

outlined in 2.3.1 a, was weighed out in polythene vials. INAA was used to

determine the percentage calcium using the OMNIGAM Neutron Activation

Analysis software package (EG&G Ortec, Oakridge Tennesse). To assess

analytical accuracy, the concentration of calcium in reference materials was also

determined in the same manner. The results obtained from the INAA experiments

are shown in Figure 2.3.

Digestion of lobster shells with 2 M acids over a 1 hour period left behind

a residue containing 14, 9, 8 and 25% calcium (as calcium carbonate) with use of

HCl, HNO3, CCl3COOH and CH3COOH respectively (Figure 2.3).

In the 6 hour digestion period HCl, HNO3, CCl3COOH and CH3COOH

were ineffective in removing 7, 3.9, 5 and 19% respectively calcium (as

carbonate) from the lobster shell samples.

With a 48 hour digestion period, only 1% calcium (as carbonate) remained

in the residue after applying HCl. In addition, 2, 3 and 16% calcium (as

carbonate) were left undigested when the acids HNO3, CCl3COOH, and

CH3COOH, respectively, were used (Figure 2.3).

Based on the findings of the optimisation experiments, 2M HCl was the

most effective acid for the digestion of lobster shells. By using a digestion time of

59

48 hour and keeping the reaction medium between 0 and 4 °C, a complex with

1% residual calcium carbonate was produced.The gypsum reference material

studied had percentage calcium expressed as carbonate 55% compared to 54 %

the value calculated from the manufacturer thus indicating the accuracy of the

results.

FIGURE 2.3

INAA RESULTS AFTER DIGESTION OF

LOBSTER SHELLS WITH DIFFERENT ACIDS OVER DIFFERENT TIMES

Gypsum reference material:

calcium expressed as calcium carbonate (manufacturer's value) 54%,

calcium expressed as calcium carbonate experimental value 55%

14

7

1

9

3.9

2

8

5

3

25

19

16

0

5

10

15

20

25

30

CA

LC

IUM

CA

RBO

NA

TE

IN R

ESID

UE / %

hydrochloric nitric trichloroacetic acetic

ACIDS

1 h

6 h

48 h

60

2.3.3 CALCIUM CARBONATE CONTENT OF CRUSTACEAN SHELLS WITH

OPTIMISED ACID DIGESTION CONDITIONS – AS DETERMINED BY WEIGHT

LOSS

The results obtained in the optimisation study indicated that digestion was

optimum when 2M HCl was used for 48 h. Consequently, it became the acid of

choice for digestion of calcium from lobster, land crab blue crab and prawn shells.

Lobster shells from the same batch used in the optimisation study, were

treated with 2M HCl for 48 hour in ice bath maintained at 0– 4 °C. Land crab

shells (obtained from Port Henderson beach, St. Catherine, Jamaica); blue crab

shells (obtained from Port Royal, Kingston, Jamaica) and prawn shells (obtained

from prawn bred by Best Dressed Chicken, Barton Isle, St. Elizabeth), were oven

dried, crushed and weighed. Samples of each of the shells were accurately

weighed and treated with 2M HCl for 48 h, the temperature of the reaction

medium kept between 0 and 4 °C.

The undigested portion of the shells (the chitin-protein residue) was

filtered from the solution, washed with wate, dried weighed and the weight loss

percentages calculated. Tables 2.2 - 2.5 show the weight loss percentages

obtained from the lobster, land crab, blue crab and prawn shells.

61

Table 2.2

PRELIMINARY WEIGHT LOSS RESULTS OF

DIGESTION OF LOBSTER SHELLS WITH 2M HCl

Shell sample name

Weight of shell / g

Weight of residue

/ g

Weight loss / %

RGf/1/25a 1.00 0.44 56

RGf/1/25b 1.03 0.46 56

RGf/1/25c 1.02 0.45 56

RGf/1/25d 1.06 0.46 57

RGf/1/25e 1.00 0.44 56

RGf/1/25f 1.01 0.44 57

RGf/1/25g 1.01 0.43 58

Average 0.45 57

62

Table 2.3


DIGESTION OF LAND CRAB SHELLS WITH 2M HCl

Shell sample name

Weight of shell / g

Weight of residue

/ g

Weight loss / %

RGf/1/28a 1.0140 0.5139 49

RGf/1/28b 1.0144 0.5396 47

RGf/128c 1.0198 0.5039 51

RGf/1/28d 1.0064 0.5102 49

RGf/1/28e 1.0146 0.4832 52

RGf/1/28f 1.0004 0.5100 49

RGf/1/28g 1.0136 0.5333 47

Average 0.5134 49

63

Table 2.4


DIGESTION OF BLUE CRAB SHELLS WITH 2M HCl

Shell sample name

Weight of shell / g

Weight of Residue

/ g

Weight loss / %

RGf/1/29a 1.0166 0.5163 49

RGf/1/29b 1.0107 0.4779 53

RGf/1/29c 0.9435 0.4329 54

RGf/1/29d 0.9487 0.4126 57

Average 0.4599 53

Table 2.5


DIGESTION OF PRAWN SHELLS WITH 2M HCl

Shell sample name

Weight of shell / g

Weight of Residue

/ g

Weight loss / %

RGf/1/30a 0.9888 0.4944 50

RGf/1/30b 1.0043 0.5544 45

RGf/1/30c 1.0072 0.5352 49

RGf/1/30d 0.9959 0.5440 45

Average 0.5320 47

64

Effervescence associated with the production of carbon dioxide

accompanied the digestion of the shells. The reaction involving the lobster shells

was the most vigorous followed by the prawn shells. The land crab shells were the

least active. The results show weight loss averaging 57, 49, 53, and 47% for

lobster, land crab, blue crab, and prawn, respectively (Tables 2.2, 2.3, 2.4

and 2.5).

Therefore, assuming that all except 1% of the calcium salts was dissolved

by acid digestion (based on optimisation/INAA results), lobster shells were

expected to have the most calcium present as calcium carbonate, followed by the

blue crab, land crab and prawn. The validity of the assumption was explored by

the use of INAA in the next section.

2.3.4 CALCIUM CARBONATE CONTENT OF (a) CRUSTACEAN SHELLS AND

(b AND c) CHITIN-PROTEIN RESIDUE - AS DETERMINED BY INAA

The weight loss percentages obtained in section 2.3.3 may not have been

equal to the total percentage of calcium salts that were present in the crustacean

shells. The aim of this investigation was to determine firstly the total percentage

calcium (as carbonate) that were present in the shells, by the use of INAA (a) and

secondly the percentage calcium (as carbonate) that remained in the chitin protein

residue after digestion with 2M HCl (b). Thus, the effectiveness of the acid

digestion process in the production of chitin from the lobster, land crab, blue crab

and prawn shells could be determined.

65

(a) CRUSTACEAN SHELLS

The total percentage of calcium expressed as calcium carbonate in lobster,

land crab, blue crab and prawn shells was determined by INAA. Samples of dried

and ground shells were weighed out in polythene vials and irradiated in the

nuclear reactor the gamma radiation emitted counted and the percent calcium

determined using the OMNIGAM neutron activation software package. The

results of the analysis are shown in Table 2.6.

Table 2.6

RESULTS OF ANALYSIS OF CRUSTACEAN SHELLS FOR CALCIUM BY INAA

Source Calcium / %

Calcium carbonate / %

Lobster 16.7 42

Land crab 27.8 70

Blue crab 25.8 65

Prawn (batch 1) 14.8 37

Prawn (batch 2) 18.8 47

Gypsum (experimental)

21.9 55

Gypsum (manufacturer's)

21.7 54

Empty ND ND

ND = Calcium not detected

The land crab shells contained the most calcium (as carbonate) (70%)

followed by blue crab shells (65%), lobster shells (42%) and prawn shells (37%).

66

A second batch of prawn shells obtained and irradiated had 47% calcium (as

carbonate) (Table 2.6). This difference in percentages between the two different

batches of prawn may have been due to their differing ages, as the calcium

carbonate content of the shell may vary with the stage of crustacean development.

The percentages of calcium (as carbonate) found by INAA in the shells of

the four crustacean species investigated varied significantly from the calcium

carbonate levels determined by weight loss. A comparison of the percentage

calcium carbonate determined in the shells (by INAA) and the average weight

loss percentage are shown in Table 2.7.

Table 2.7

COMPARISON OF PERCENTAGE CALCIUM

(AS CALCIUM CARBONATE) DETERMINED BY INAA AND AVERAGE WEIGHT LOSS

Source Average weight loss / %

Calcium carbonate in shells / %

Lobster 57 42

Land crab 49 70

Blue crab 53 65

Prawn 47 37

For lobster and prawn shells, weight loss percentages were higher than the

percentage calcium (as carbonate) present in these shells, as determined by INAA.

However, the weight loss percentages of the shells obtained for the two species of

crab were less than the percentage of calcium (as carbonate) determined by INAA

67

(Table 2.7). The lower percentage for the lobster and prawn (by INAA) compared

with weight loss suggested that all the calcium present as calcium carbonate was

dissolved from these shells, along with a small amount of the other main portion

of the shell 1, the organic portion (hydrolysis). The higher percentages for the

crabs (by INAA) compared with the weight loss percentage, indicated that the

calcium salts present as calcium carbonate were not totally digested from the

shells. There was also the possibility of hydrolysis of the organic polymers in the

crab shells although this was not indicated in these results.

The variation in the percentage calcium as calcium carbonate (by INAA)

and the weight loss percentages prompted a further investigation of the

effectiveness of 2M HCl digestion of all the crustacean shells studied. Thus, the

calcium contents of the chitin-protein residues were determined.

(b) (i) CHITIN-PROTEIN RESIDUE

A comparison of the weight loss percentages and percent calcium as calcium

carbonate determined by INAA suggested that there was incomplete removal of

the calcium salts from the land crab and blue crab shells. On the contrary, more

material than the calcium carbonate present in the lobster and prawn shells

appeared to be removed by this digestion. In addition, there could have been

incomplete calcium carbonate digestion in the lobster and prawn shells. The

effectiveness of the acid digestion was investigated in this section by determining

the percentage calcium as calcium carbonate that remained in all the chitin-

protein residues after digestion of the crustacean shells with HCl (2M).

68

A portion of chitin-protein residues produced (Section 2.3.3) was analysed

by INAA and the percentage calcium as calcium carbonate determined. The

possibility of calcium being present in the sample vials was investigated by

analysing an empty vial.The results of these experiments are summarised in

Table 2.8.

Table 2.8

RESULTS OF ANALYSIS OF CHITIN-PROTEIN RESIDUE

OBTAINED FROM 2M HCl DIGESTED SHELLS FOR CALCIUM BY INAA

Source Calcium / %

Calcium carbonate in residue

/ %

Percentage extraction

/ %

lobster 3.5 9 79

land crab 24.8 62 11

blue crab 21.8 55 15

prawn 0.59 2 96

gypsum (manufacturer's)

21.5 54 -

empty vial ND ND -

ND = Calcium not detected

The chitin-protein residues had varying levels of residual calcium

carbonate. The samples obtained from lobster and prawn shells had low levels of

residual calcium (as calcium carbonate), 9 and 2%, respectively, equivalent to 79

and 96% extraction efficiency as determined by Equation 2.6.

Extraction efficiency (%) = (CaS – CaR)/CaS × 100…………………Equation 2.6

69

Where,

CaS = calcium carbonate in shells by INAA (%);

CaR = calcium carbonate in residue by INAA (%);

The residues obtained from digestion of land crab and blue crab shells

however had high levels of residual calcium as calcium carbonate. The

percentages obtained were 62% (for land crab) and 55% (for blue crab)

corresponding to 11% and 15% extraction efficiency (Table 2.8). Therefore,

calcium carbonate was not being totally removed from the shells after digestion

with 2M HCl for 48 h. Calcium was not detected (ND) in the empty vial.

A comparison of percentage weight loss, the total percentage calcium as

calcium carbonate in the shells and the percent calcium as calcium carbonate

present in the chitin-protein residues (by INAA) were made. In the lobster, shells

the 57% weight loss compared with total 42% calcium carbonate in shells and 9%

calcium carbonate in chitin protein residue supported the suspicion that the

organic portion of the shell was hydrolysed during acid digestion. The 47%

weight loss, 47% calcium carbonate in shells and 2% calcium carbonate in chitin-

protein residue suggested that there was almost complete digestion of calcium

from the prawn shells. Land crab and blue crab shells showed 49% and 53%

weight loss respectively, compared with 70% and 65% total calcium carbonate in

the shells. This showed that, particularly in the land crab, the calcium was not

being effectively removed by acid digestion as the residue still contained 62% and

55% calcium carbonate.

70

(ii) CALCIUM CARBONATE CONTENT OF CHITIN-PROTEIN RESIDUE REDUCED

The percentage calcium carbonate measured for residues of lobster shells

after 2M HCl digestion during the optimisation studies, was 1%. When this was

repeated in section 2.3.4b, (Table 2.8), 9% calcium carbonate remained

undigested. This observation prompted a repeat of the experiment, vigorously

shaking the reaction vessel during the 48 hour digestion period and the residues

thoroughly washing in water before drying and weighing. The samples were then

analysed by INAA to determine the percentage calcium as calcium carbonate. The

weight loss percentages were also determined. These new results obtained by

INAA were recorded in Table 2.9. A graphical view of the improvements in

digestion made for each type of crustacean shell is shown in Figure 2.4. The

weight losses obtained after digestion with HCl (2M) are shown in Table 2.10.

Table 2.9

NEW RESULTS OF ANALYSIS OF 2M HCl DIGESTED SHELLS

FOR CALCIUM (AS CALCIUM CARBONATE) DETERMINED BY INAA

Source Average calcium carbonate / %

lobster < 1

land crab 52

blue crab 43

prawn < 1

gypsum standard 24.4 (24)

71

Figure 2.4

PERCENT CALCIUM PRESENT IN CRUSTACEAN SHELLS

BEFORE AND AFTER DIGESTION WITH 2M HCl AS DETERMINED BY INAA

Table 2.10

NEW WEIGHT LOSS PERCENTAGES AFTER

2M HCl DIGESTION OF CRUSTACEAN SHELLS

Source Average weight of shells

/ g

Average weight of chitin-protein

residue / g

Average weight loss / %

Lobster 4.9 2.1 57

Land Crab 4.8 2.0 58

Blue Crab 4.8 1.7 64

Prawn 4.9 2.1 58

The percentage calcium (as carbonate) that remained after digestion was

42

9

1

70

62

54

65

55

4347

2 1

0

10

20

30

40

50

60

70

80

90

100

CA

LC

IUM

CA

RBO

NA

TE / %

lobster land crab blue crab prawn

Source

before digestion after digestion after digestion (repeat)

72

less than 1% for lobster shells which was consistent with the results of the

optimisation study. The chitin-protein residue obtained from digesting the prawn

shells also contained less than 1% calcium (as carbonate) (Table 2.9). Thus, there

was improvement in the efficiency of digestion of these two types of crustacean

shells. Improvement in the level of calcium carbonate digestion was also evident

for the crab shells. The percentages went from 62 to 52% in the land crab and

from 55 to 43% in the blue crab shells (Figure 2.4). In the case of the lobster and

prawn shells, the calcium carbonate could be efficiently extracted by acid

digestion. In the case of the crab species the shells appeared to be very resistant to

acid digestion as the residues contained high calcium concentrations.

The weight loss results in Table 2.10 were in agreement with what was

previously observed. That is, hydrolysis of organic polymers occurred during acid

digestion. These effect was more pronounced for the blue crab shells. The shells

contained 65% calcium (as carbonate). After acid digestion the residue contained

43% calcium (as carbonate), yet weight loss percentage averaged 64% (Table

2.10).

A comparison of the average weight loss percentages obtained before and

the new average weight loss percentages were made. The weight loss percentages

generally increased with the improvement in the percentage calcium carbonate

removed, as expected. They went from 47% to 58% (2% to < 1% CaCO3) with

the prawn shells; 49% to 58% (62% to 54% CaCO3) with the land crabs and from

53% to 64% (55% to 43% CaCO3) in the blue crabs. The weight loss for the

lobster shells remained constant at 57% although the residual calcium carbonate

73

was brought to less than 1% by weight from 9%. Improving the efficiency of the

digestion of calcium carbonate may to some extent affect the quantity of chitin

produced because of the hydrolysis of chitin16, which can occur.

74

2.4 HISTORY, PRINCIPLES AND INSTRUMENTATION FOR

ATOMIC ABSORPTION SPECTROSCOPY (AAS)

AAS is an alternative technique to INAA that was used in this work for

analysing shells for their calcium content. INAA is the better technique since

AAS requires dissolution of the sample whereas INAA is a direct solid sample

analysis and is less prone to matrix interferences 14.

The foundation of atomic absorption dates back to 1802 when

Wollaston 17 discovered black lines in the spectrum of the sun, which were later

investigated by Fraunhofer 17. Brewster 17 postulated that absorption processes in

the atmosphere of the sun caused these lines. Kirchhoff and Bunsen 17 while

investigating the spectra of alkali and alkaline earth metals demonstrated that the

typical yellow line emitted by sodium salts in a flame is identical to the black line

in the spectrum of the sun.

When a gaseous atom in its ground state absorbs a specific quantum of

energy from an external source of radiation, it can attain an excited state in which

electrons surrounding the atom occupy higher energy levels than usual. This is an

unstable state and the atom quickly and spontaneously returns to its ground state

as the electrons return to their original orbital position. The exact amount of

energy that was absorbed during the excitation process is emitted during this

decay process. 18 The amount of the analyte element present is determined by

measuring a parameter called Absorbance 12 which is related to the reduction in

the intensity of the beam of radiation passing through the gaseous sample

75

(Equation 2.7) 20.

A = log (I0/I)……………………………………….Equation 2.7

Where,

A = Absorbance;

I0 = Intensity of radiation projected into sample;

I = Intensity of radiation passed through sample.

Quantitative measurements in atomic absorption are based on Beers’ law 21 which

states that concentration is proportional to Absorbance, where,

A = abc……………………………………………Equation 2.8

Where,

a = Absorption coefficient, a constant which is a characteristic of the

absorbing species at a particular wavelength;

b = Length of the radiation path intercepted by the absorption species in

the absorption cell;

c = Concentration of the absorbing species;

Absorbances of standard solutions containing known concentrations of analyte are

measured and the absorbance data are plotted against concentration. Ideally, this

should be a straight line as indicated by Beer’s law 21, and this is usually observed

76

at lower concentrations and absorbances. As concentration and absorbance

increase however non ideal behavior in the absorption process causes deviation

from linearity. The absorbance of the sample is measured and the concentration of

the analyte determined from the calibration curve. Modern atomic absorption

instruments have the ability to perform automatic curve correction, calibrate, and

compute concentrations using absorbance data from linear and non-linear

curves 22.

Initially the sample being analysed is atomised in a cell by a flame or an

electrically powered graphite furnace. Air - acetylene is the preferred flame for

the determination of many elements in atomic absorption, producing temperatures

of about 2300 °C 23.

The external radiation required for excitation is delivered by line sources,

for example, the hollow cathode lamp which are manufactured for individual

elements. Radiation passes from the source through the atomised sample to a

monochromater that disperses it and isolates a specific wavelength that is passed

directly to a detector, usually a photomultiplier tube (PMT). The PMT produces

an electrical current, the magnitude of which depends on the intensity of the

radiation falling on it. Comparison with known standards and the use of Beers

Law 21 enables the concentration of the analyte in the sample to be determined 24.

The above description is for a single beam spectrophotometer. In a double

beam spectrophotometer the light from its source is divided into a sample beam,

which is focused through the sample cell, and a reference beam, this is directed

77

around the sample cell. The actual readings obtained represent a ratio of the

sample and reference beams. The result is that fluctuations in source intensity are

not reflected in the read out obtained. No lamp warm up period is required in

contrast to the single beam spectrophotometer 25.

78

2.5 CALCIUM CARBONATE CONTENT - AS DETERMINED BY AAS

2.5.1 INTRODUCTION

INAA was used to determine the percentage calcium as calcium carbonate

present in both shells and chitin-protein residues and was conclusive. AAS is a

method which is cheaper, more readily available and was used to find the

percentage calcium present as calcium carbonate in the solution that is obtained

after acid digestion of the crustacean shells. Both the results of AAS experiments

and INAA experiments were expected to compliment each other in that the

following relationship was expected to hold:

(CaR × WR) + (CaF × WS) ÷ WS = TCa = CaS………………..Equation 2.9

where,

CaR = Calcium carbonate in chitin-protein residue by INAA (%);

WR = Weight of chitin-protein residue (g);

CaF = Calcium Carbonate in filtrate by AAS (%);

WS = Weight of shells (g);

TCa = Total percent calcium carbonate in shell calculated (%);

CaS = Calcium carbonate in shell by INAA (%).

AAS may therefore be used as a check, in conjunction with the information on

weight loss on acid digestion. To determine the percentage of the shell that was

79

not calcium salt (organic polymers) that had dissolved, Equation 2.10 was used.

Weight loss (%) = CaF + OP…………………………………Equation 2.10

where

CaF = Calcium carbonate in filtrate (%);

OP = Organic polymers (%).

2.5.2 RESULTS AND DISCUSSION OF CALCIUM CARBONATE

DETERMINATION BY AAS

Fresh samples of lobster and land crab shells were dried, weighed and

digested for 48 hour with 2M HCl. The digestion product obtained was filtered

and the residue washed with water, dried and collected for INAA to determine the

percentage calcium as calcium carbonate. The washings that were combined with

the filtrate were also collected and made up to 250 mL with distilled water.

Diluted portions of these solutions were analysed by AAS and the percentage

calcium, expressed as calcium carbonate determined. The results of both

experiments are shown in Table 2.11.

80

TABLE 2.11

PERCENTAGE CALCIUM (AS CALCIUM CARBONATE)

DETERMINED BY AAS AND INAA

Source WS

/ g

WR

/ g

Weight loss / %

CaS

/ %

CaR

/ %

CaF

/ %

TCa

/ %

lobster 3.002 1.235 59 42 0.125 42 42

land crab 1.004 0.3623 64 70 40 57 72

CaR = Calcium carbonate in chitin-protein residue by INAA (%), WR = Weight of chitin-protein

residue (g), CaF = Calcium Carbonate in filtrate by AAS (%), WS = Weight of shells (g), TCa =

Total percent calcium carbonate in shell calculated (%), CaS = Calcium carbonate in shell by

INAA (%)

For the lobster shell sample, the calculation showed that the total

percentage calcium as calcium carbonate calculated (TCa) was 42%

(Equation 2.9). This was equal to the total calcium carbonate in the shells (CaS).

The AAS determined percentage (CaF) was also equal to the latter, which

suggested that all the calcium carbonate was digested Table 2.11. In addition, for

the lobster sample 59% weight loss occurred to produce the chitin-protein residue.

With 42% calcium as calcium carbonate in the solution then, organic polymers

that were hydrolysed amounted to 17% of the shells (Equation 2.10).

In the land crab shell sample where calcium carbonate in residue (CaR)

was 40%, TCa was 72% (Equation 2.9). This was close to CaS Table 2.11. The

two differed by 2%. Digestion of the land crab shells resulted in 64% weight loss.

Therefore, organic polymer hydrolysed amounted to 7% (Equation 2.10). CaF

81

(57%) was less than CaS (70%) because the crab shell was incompletely digested.

The percentage organic polymers digested, 17% for the lobster shells and

7% for the land crab shells, confirmed that crab shells are more resistant to acid

than lobster shells.

Overall, it was shown that AAS was able to determine conclusively the

percentage calcium as calcium carbonate present in the shells of the more easily

digested crustacean shells for example, the lobster shells. This method however

was not sufficient for the harder, more acid resistant shells like the crab shells.

AAS is however suitable for routine check analysis on the samples as digestion

proceeds.

82

2.6 CHITIN CONTENT OF CRUSTACEAN SHELLS AS

DETERMINED BY ALKALINE HYDROLYSIS

2.6.1 INTRODUCTION

With the calcium present as calcium carbonate in crustacean shells

properly quantified, it became easier to determine their percentage of chitin. The

first step to obtaining the percentage chitin was to boil the chitin–protein residue

with sodium hydroxide and then weigh the unhydrolysed product (UHP)

(Equation 2.11).

Chitin-protein residue = UHP + Hydrolysed material…….Equation 2.11

Where,

UHP = Unhydrolysed product.

There may be reservations in calling the UHP, chitin, because of the

existing possibility of impurities mainly calcium. Therefore, the UHP was

analysed by INAA to determine if the hydrolysis process affected the percentage

of undigested calcium carbonate, particularly in the crab shells. By considering

the weight of UHP (WUHP) and the percentage calcium carbonate impurities

(CaUHP) the percentage pure chitin was determined (Equation 2.12).

83

Chitin% = [WUHP – (CaUHP × WUHP)] / WS × 100………….Equation 2.12

Where,

CaUHP = Calcium carbonate impurities in chitin (%);

Ws = Weight of shells.

An attempt was also made to determine the presence of and types of

amino acids and proteins that were present in the hydrolysed product. This

involved the use of Gas Chromatography – Mass Spectrometry (GC-MS), the

ninhydrin test and electrophoresis 26. GC-MS along with a total elemental analysis

aided the determination of the composition of the exoskeletons.

2.6.2 Percent unhydrolysed product (UHP%) after alkaline hydrolysis

The chitin-protein residues obtained after acid digestion were boiled with

1M NaOH for 48 hours. The unhydrolysed product (UHP) obtained was filtered

washed repeatedly with water until neutral, dried and then weighed. The

percentage UHP was calculated with Equation 2.13 and the results obtained after

duplicate experiments are shown in Table 2.12.

UHP% = WUHP / WS × 100…………………………….…..Equation 2.13

Where,

UHP% = unhydrolysed product (%);

WUHP = Weight of Unhydrolysed product;

WS = Weight of shells.

84

Table 2.12

ALKALINE HYDROLYSIS OF CRUSTACEAN

SHELLS – PERCENTAGE UNHYDROLYSED PRODUCT

Source Average weight of shells

/ g

Average weight of

Chitin-protein residue

/ g

Average weight of

unhydrolysed product

calculated / g

Average unhydrolysed

product / %

lobster 4.9 2.1 1.0 21

land crab 4.8 2.0 1.7 35

blue crab 4.8 1.8 1.7 36

prawn 4.9 2.1 1.7 35

Lobster shell samples had overall 21% unhydrolysed product after alkaline

hydrolysis. The land crab and prawn shell samples had on average 35%,

unhydrolysed product whilst the blue crab had 36%, after hydrolysis. These

results on their own suggested that the lobster shells would contain the least

amount of chitin, and the other three samples would contain about the same as

each other. This however did not take into account impurities in the UHP, which

will be discussed next.

2.6.3 PERCENT CALCIUM CARBONATE IMPURITIES

IN UNHYDROLYSED PRODUCT

In the land crab and blue crab shells, a large amount of calcium carbonate

was present after acid digestion and was expected to be present after the alkaline

hydrolysis process. It was therefore necessary to determine the percentage

85

calcium (as carbonate) in the unhydrolysed product in order to determine the

percent chitin present in the crustacean shells.

INAA was used to determine the percent calcium (as carbonate) present in

the UHP. A sample of practical grade crab chitin obtained from Sigma Co. was

also irradiated for comparison. The percentages are shown in Table 2.13.

Table 2.13

CALCIUM CARBONATE CONTENT OF UNHYDROLYSED PRODUCT

Source Average calcium

/ %

Average calcium carbonate

/ %

Lobster < 0.5 < 1

Land Crab 20 49

Blue Crab 19 49

Prawn < 0.5 < 1

Crab (Sigma Co.) 0.02 0.05

Gypsum 22.1 (22) -

Calcium std. 22.9 (22) -

In addition, GC-MS, the ninhydrin test and electrophoresis 26 were then

used to determine the presence of, and the type of amino acids and proteins that

were hydrolysed in the solution.

A portion of the solution obtained from alkaline hydrolysis of the chitin-

protein residues was filtered made more alkaline and extracted with a mixture of

dichloromethane. A diethyl ether extraction was also carried out after acidifying a

86

fresh portion of the solution. Extractions were done to obtain samples for GC-MS

the polypeptides and amino acids present. Another portion of the sample was

analysed using the ninhydrin and the electrophoresis 26 test.

UHP obtained from the prawn and lobster shells had less than 1% calcium

(as calcium carbonate) (Table 2.13). These were white compared to the brown

colour of the chitin-protein residue (Photograph 2.1). On average, 49% of the

UHP obtained from the land crab and blue crab shells was calcium carbonate

(CaUHP). The sample of practical grade crab chitin obtained from Sigma Co, when

analysed was shown to contain 0.05% calcium as calcium carbonate.

87

Photograph 2.1

CHITIN AND CHITOSAN SAMPLE OF PRAWN (LEFT) AND LOBSTER (RIGHT)

↓↓↓↓ CHITIN ↓↓↓↓ CHITOSAN ↓↓↓↓ CHITIN ↓↓↓↓ CHITOSAN

88

The CaUHP for the lobster and prawn were expected since the percentage

calcium (as calcium carbonate) present after acid digestion was very small (less

than 1%). For the land crab and blue crab samples, higher if not the same

percentages of calcium carbonate were expected after alkaline hydrolysis, since

the percentages were calculated with respect to the weight of the unhydrolysed

products (smaller weight compared with chitin-protein residue). The percentage

calcium carbonate in the land crab was 54% in the chitin-protein residue and 49%

in the UHP. In the blue crab it was 43% in the chitin-protein residue compared to

49% in the UHP, a reasonable change. A small increase in the percentage residual

calcium carbonate may be due to the small amount of protein that was present in

the chitin-protein residue of the crab shells.

The ninhydrin test, electrophoresis 26 and GC-MS suggested the absence of

any significant amount of protein in the solution obtained after alkaline

hydrolysis. The ninhydrin test indicated the presence of aminoacids, by the

characteristic blue colour obtained by heating ninhydrin and solution on filter

paper. However, the gel electrophoresis 26 that followed this test was negative.

The characteristic blue bands a positive sign for the presence of polypeptides and

amino acids were absent. The GC-MS indicated very few amino acids, for

example, glycine was present.

2.6.4 COMPOSITION OF THE EXOSKELETON

The exoskeleton is composed of chitin, calcium and other metals and non-

metals, proteins and other organic substances. Their final percentages are stated

89

below. The percentage chitin was determined using Equation 2.12. The

percentages of metals and nonmetals were determined by INAA and the organic

substances, excluding chitin were determined by GC-MS.

(a) Percentage chitin

The isolation of chitin involved two clear steps. These were digestion of

calcium present as calcium carbonate and hydrolysis of the chitin-protein residue

obtained. The percentage of UHp of all the crustacean shells were determined

based on the weight of the shells used. These were 21 and 35% in the lobster and

prawn shells. With less than 1% calcium as calcium carbonate present in these

UHP, it was concluded that the percentage chitin present in the lobster and prawn

shells were a minimum of 21 and 35%, respectively.

The percentage chitin in crab shells was calculated by considering the

percentage calcium carbonate in the UHP (Table 2.12 and Table 2.13), and

applying Equation 2.12. Therefore, for the land crab and blue crab shells,

percentage chitin was at least 18 and 19%, respectively.

(b) Elemental composition by INAA

The other elements apart from calcium that were present in the crustacean

shells, were determined.

The shells were found to contain small quantities of metals e.g. Na, K,

Mg, Al and Mn.; and non-metals e.g., Br and Cl (Tables 2.14).

90

Tables 2.14

ELEMENTAL COMPOSITION OF SHELLS

Shells Land Crab Blue Crab Lobster Prawn

/ %

Na 0.31 0.65 0.35 0.13

K 0.035 0.17 0.23 0.12

MgO 2.8 1.0 2.9 -

Shells Land Crab Blue Crab Lobster Prawn

/mg/kg

Br 31.0 105.0 390.0 221.0

Al2O3 ND 445.0 ND 276.0

Mn 70.0 137.0 11.0 42.0

Cl 140.0 776.0 476.0 293.0

ND = Not detected in shell

The quantities varied with species and may be an indication of variation in

the animals’ diets or habitats. For example, the land crab, which is not a marine

dweller, contained less of the halogens than the other species. The presence of

these elements coupled with the organic materials, make crustacean shells a

possible source of fertiliser. Many of these substances may not be eliminated

during acid and base hydrolysis and will remain as contaminants in chitin.

91

(c) OTHER ORGANIC SUBSTANCES

The other organic materials present in the crustacean shells were

determined using GC-MS.

The solutions obtained after alkaline hydrolysis of the chitin protein

residues were divided into two portions, one of which was made more alkaline

and the other acidic. The alkaline solution was extracted with dichloromethane

and the acidic solution with methylene chloride. The solvents containing the

components being analysed were then evaporated to dryness, derivatised with

bis(trimethylsilyl)trifluoroacetamide (BSTFA) and analysed by GC-MS and a

Pfleger/Maurer/Weber MS drug library used to determine its constituents.

The GC-MS and library revealed the presence of a variety of compounds:

aromatic as well as aliphatic amines, high molecular weight carboxylic acids and

alkanes.

92

2.7 REMOVAL OF CALCIUM FROM CRUSTACEAN SHELL

BY COMPLEXATION

The harsh conditions of acid digestion followed by base hydrolysis can

affect the isolation efficiency of chitin. Under these conditions chitin may be

deacetylated to chitosan or hydrolysed into its N-acetyl monomeric units 27.

Complexation is a mild alternative for the removal of calcium from lobster

shells 28. Any weight loss obtained from using complexing agents is expected to

be the result of removal of calcium without any effect on the organic polymers.

Thus the effectiveness of the complexation method was compared with the acid

digestion method on the basis of weight loss only.

2.7.1 REMOVAL OF CALCIUM FROM CRUSTACEAN SHELL BY COMPLEXATION

WITH EDTA

Ethylenediamine tetra acetic acid (EDTA) (tetrasodium salt)

[CH2.N(CH2.COONa)2] 2. 2H2O was the first complexing agent used to remove

calcium from lobster shells.

EDTA was dissolved in a a pH 9 solution. Dried and crushed lobster shells

were then added to the EDTA solution (0.03% w/v) (EDTA: shells, 1:2). The

mixture was then agitated for 15 minutes at room temperature and the solid

product collected by filtration, washed, dried and the weight loss percentage

determined. The experiment was repeated for 60 and 180 minutes. The weight

loss percentages are shown in Table 2.15.

93

TABLE 2.15

PERCENTAGE CALCIUM CARBONATE IN LOBSTER SHELLS OVER

DIFFERENT TIME PERIODS USING EDTA SOLUTION AT ROOM TEMPERATURE

Time for digestion / min.

Weight loss / %

15 23

60 40

180 48

The results in the table showed that the weight loss percentage increased

as the time of digestion increased. At room temperature and a digestion time of 60

and 180 minutes 40 and 48% respectively, weight losses were observed. This

compared well with the 42% calcium as calcium carbonate present in the lobster

shells, as determined by INAA. Weight loss percentage was about 57% when the

lobster shells were digested with HCl (Table 2.10), a higher value than that

obtained with the use of EDTA, probably because of the loss of weight from

hydrolysis of the organic polymers present.

2.7.2 REMOVAL OF CALCIUM FROM CRUSTACEAN SHELL BY COMPLEXATION

WITH 18-CROWN-6 ETHER

18-crown-6 ether was the second complexing agent used in the removal of

calcium from lobster shells.

18-crown-6 ether solutions were agitated with lobster shells for 1 hour and

the resulting solid collected by filtration, washed, dried and the weight loss

94

percentage determined. The solvents used were water and ethanol. The reaction

vessels were at room temperature (29 °C) and 80 – 85 °C. The pH of the solution

varied from pH 4.0 to pH 9.2. Table 2.16 shows the different reaction conditions

as well as the weight loss percentages obtained.

Table 2.16

PERCENTAGE WEIGHT LOSS BY USING 18 CROWN 6 ETHER

Lobster shell / g

18-Crown-6 / g

Chitin-protein residue

/ g

Weight loss / %

Reaction conditions

0.083 0.14 0.083 0 H2O, RT

0.17 0.20 0.14 20 H2O, 80-

85 °C

0.13 0.18 0.10 18 H2O, 80-

83°C

0.12 0.12 0.10 17 ETOH, RT

0.12 0.13 0.11 8 EtOH, pH 4, RT

0.12 0.13 0.11 8 EtOH, pH 9.2, RT.

RT = Room temperature; EtOH = Ethanol

The weight loss percentages obtained by using 18-crown-6 ether were less

than the percentages obtained by using EDTA. The highest percentage weight loss

obtained was 20% with the use of the H2O solvent and experimental temperature

95

of 80-85 °C. This was less than half the percentage CaCO3 present in the lobster

shells by INAA (42%). This was significantly less than the weight loss percentage

obtained by acid hydrolysis (57%).

On the basis of these weight loss experiments complexing agents are a

reasonable alternative to acids in removing Ca from lobster shells. Their

effectiveness will depend on the surface area of the shells being analysed, a higher

surface area will result in more sequestering.

96

2.8 CHITIN IN COCKROACH

Cockroaches are a nuisance to many homes and are found inhabiting many

drains and gutters. They are a source of chitin 1. They mature rapidly and are

readily available. Chitin was isolated from the cockroach by the same method

used for crustacean shells and the percentage present compared with those

obtained from crustacean shells.

The wings and legs of the cockroach Blaberus discoidalis obtained from

various sites in Mona, Kingston, Jamaica were agitated in 2M HCl for 48 h. The

resulting mixtures were then filtered and the undigested residue washed with

water and dried.

The chitin–protein residue thus obtained was boiled in 1M NaOH for 48 h,

and the product collected by filtration, dried, weighed, the percentage chitin

calculated and the IR spectra recorded (Chapter 3). The resulting percentages are

shown in Table 2.17.

Table 2.17

ACID DIGESTION AND ALKALINE HYDROLYSIS OF A BLABERUS COCKROACH

Source Weight loss after digestion

/ %

Chitin / %

wings 15 24

legs 17 28

Addition of acid to the exoskeleton of the Blaberus cockroach did not

97

produce the usual effervescence associated with the generation of carbon dioxide

as seen for the crustacean shells. This was perhaps due to the small amount or

absence of calcium carbonate in these arthropods 2. This was confirmed by the

small weight loss obtained after the acid treatment.

After alkaline hydrolysis, a skin-like material and a creamish white

powdered material were recovered. The IR spectra of both substances revealed

similarities to chitin obtained from crustaceans. Therefore with little or no

calcium carbonate to contend with as in the crustaceans it can be safely concluded

that the wings and legs contained 24 and 28% chitin respectively.

The relatively high percentages of chitin recorded suggested that the

cockroach was as good a source of chitin as the crustaceans.

98

2.9 SUMMARY

Weight loss analyses, Instrumental Neutron Activation Analysis (INAA)

and Atomic Absorption Spectroscopy (AAS) were used to determine the

percentage of calcium (expressed as calcium carbonate) in the shells of the

Jamaican marine spiny lobster (Panulirus argus), the land crab (Gecarcinus

ruricola), the blue crab (Callinectes sapidus), and the giant Malaysian fresh water

prawn (Macrobracium rosenberg). The percentage calcium aided determination

of the percentage of chitin present in these species.

Lobster shells contained at least 21% chitin by weight, 41% calcium as

calcium carbonate and 38% proteins and other types of materials (organic and

inorganic) (Figure 2.5).

Figure 2.5

PERCENTAGE CHITIN CALCULATED IN (A) LOBSTER AND (B) PRAWN SHELLS

The prawn shells contained no less than 35% chitin, 47% calcium as calcium

carbonate. Both lobster and prawn shells are soft and are easily digested with

acid.

(b) prawn shell

calc ium

carbonate

47%

protein

and other

materials

18%

chitin

35%

(a) lobster shell

calcium

carbonate

42%

protein

and other

materials

37%

chitin

21%

99

The land crab shell contained 18% chitin and 70% calcium as calcium

carbonate, whilst the blue crab shells contained about 19% chitin and 65%

calcium as calcium carbonate (Figure 2.4), the rest of the shells accounting for

the other organic and inorganic substances. The crab shells were tough and

difficult to digest with acid.

The merit of complexation with 18-crown-6 and EDTA as a method of

removing calcium ions was also briefly visited. On the basis of weight loss it was

a reasonable alternative to acid digestion.

In addition, weight loss experiments were applied to the wings and legs of

the Blaberus discoidalis cockroach in order to determine the amount of chitin they

contained. The wings and legs were shown to contain 24 and 28% chitin

respectively.

100

REFERENCES FOR CHAPTER TWO

1. P.W. Kent, and M.W. Whitehouse, “Biochemistry of the Amino Sugars,” Butterworths Scientific Publication, London, 1955, p 94.


3. De Soete, R. Gigbels and J. Hoste, “Neutron Activation Analysis,” John Wiley and Sons, London, 1972, Vol 34, p 1.

4. D.A. Skoog and J.J. Leary, “Principles of Instrumental Analysis,” A.

Harcourt Brace Janovich College Publishing, N. Y., 1992, p 410. 5, Reference 4, p 411. 6. J.C. Kotz and K.F. Purcell, “Chemistry and Chemical Reactivity,”

Saunders College Publishing, New York, 1987, p 1009.

7. G. C. Lalor, R. Rattray, H. Robotham, Jamaica Journal of Science and Technology, 1990, 1 (1), 65.

8. Reference 3, p 4. 9. Reference 4, p 413. 10. Nuclear Engineering Teaching Laboratory, Department of Mechanical

Engineering, University of Texas, Austin, 1995. 11. Reference 4, p 412.


13. Reference 3, p12

14. Reference 3, Vol 34, p 7.

15. Reference 3, Vol 34, p 8.


101

17. B. Welz, “Atomic Absorption Spectroscopy,” Verlag Chemie GmbH, D-6940 Weinheim, 1976, p 1.

18. R. D. Beaty, J. D. Kerber, “Concepts Instrumentation and Techniques in Atomic Absorption Spectrophotometry,” Perkin Elmer Co-orporation, Norwalk, 1993, p 1-1.

19. Reference 18, p 1-5. 20. Reference 18, p 1-6.

2.1 Perkin Elmer, “Analytical Methods for Atomic Absorption Spectrometry,” 1994, p 16.




25. Reference 21, p 6. 26. K. D. Golden, M Phil. Thesis, Beta galactosidase (beta-D-

galactohydrolase) (E. C. 3.2.1.23) from Coffea arabica, its possible role in fruit ripening and ethylene synthesis, Biochemistry Department, UWI, Mona, 1991, p 46.

27. R. A. A. Muzzarelli, Chitin, Pergamon Press N.Y., 1976, p 90.


102

CHAPTER THREE

CHARACTERISATION OF CHITIN

103

3.1 INTRODUCTION

Four techniques were used to characterise the isolated chitin. These were

Thermal Analysis, Scanning Electron Microscopy, Carbon-13 Nuclear Magnetic

Resonance Spectroscopy (13C NMR) and Infrared Spectroscopy (IR). IR was also

used in % N-acetylation determination.

Thermal analysis offered an insight into the physical changes of chitin as a

function of temperature. 13C NMR analysis performed on the monomer of the

chitin polymer allowed for comparison of spectral results with those of glucose

and a biosynthetic chitin.

Photography at the microscopic level is unique in that the sample is

observed in its original state and the result is not open to prejudice after a portion

of the sample has been selected for photography.

IR is the most common method of characterisation where the presence of

characteristic absorption peaks are investigated. The absorbance at 3450 cm -1

and 1655 cm -1, due to hydroxide and amide 1 groups respectively, were used in

the determination of % N-acetylation (% N-Ac) and the ratio of 2-acetamido-2-

deoxy-D-glucose to 2-amino-2-deoxy D-glucose monomeric units. If a chitosan

conversion method is applied to chitin, the % N-Ac is expected to decrease. A low

value of % N-Ac coupled with solubility in dilute acetic acid means that chitin has

been converted to chitosan, in which the majority of the monomers present are 2-

amino-2-deoxy D-glucose.

104

3.2 THERMAL ANALYSIS

Thermal analysis involves determining the physical parameters of a

system as a function of temperature. Two methods of thermal analysis were

employed, Thermal Gravimetric Analysis (TGA) and Differential Scanning

Calorimetry (DSC).

TGA gives the change in weight of the sample with increasing

temperature. If the molecular weight of the initial sample is known, the weight

loss obtained will aid in determination of the composition of the intermediate and

the final residue. Loss of weight is usually the result of evolution of a volatile

material physically or chemically bound to the sample. It can also be due to

decomposition of the sample 1.

The modern thermobalance used for TGA consist of a recording balance,

furnace, temperature programmer or controller and a recorder. The recording

balance records the weights as the temperature program controls the rate at which

the furnace heats the sample. The recorder produces the weight loss-temperature

curve, which provides information on the thermal stability of the sample 1.

In DSC, energy is applied to a sample and standard such that both

materials are isothermal to each other as they are heated or cooled at a linear

rate 2. The curve obtained is usually a recording of heat flow rate in mJ s-1 (mW)

as a function of temperature or time. Heat flow varies in a sample as a result of

the application of heat and these are due to endothermic and exothermic reactions.

The endothermic reactions include phase transitions, dehydration, reduction and

105

sometimes decompositions 3. Exothermic reactions are generally bond formation

reactions. On the curve of heat flow versus temperature, the modern convention is

that an endothermic peak is a minimum and an exothermic peak is a maximum.

The sample and reference are placed in sample holders of a furnace that is

sometimes electrically heated or by other means 4. The rate of temperature

increase of the furnace is controlled by a temperature programmer, which is

capable of linear temperature programming. To control the atmosphere within the

furnace and around the samples nitrogen or sometimes oxygen is used 5. The

temperature measurement system is very important. A thermocouple is used to

detect the temperature of the sample and reference holders. Electricity generated

by the thermocouple is proportional to the temperature required to maintain the

isothermal conditions 6. The thermocouple is attached to a recorder which

generates the curve of heat flow rate in mJ s-1 (mW) as a function of temperature

or time 2.

Chitin samples from lobster and prawn shells for TGA were heated under

nitrogen at a rate of 10 °C per minute from 25 °C to 1200 °C and the weight loss-

temperature curves plotted (Figure 3.1). Samples for DSC were heated at a rate of

10 °C per minute from 25 to 450 °C under nitrogen and the heat-flow rate –

temperature curves plotted (Figure 3.2 and 3.3).

106

Figure 3.1

TGA CURVES OF PRAWN (CPWN2a) AND LOBSTER(CLOB2a) CHITIN

Figure 3.2

DSC CURVE OF LOBSTER CHITIN

107

Figure 3.3

DSC CURVE OF PRAWN CHITIN

TGA curves are shown in Figure 3.1. The lobster and prawn chitin had

thermal stability up to 390 °C, after which the samples decomposed by about 80%

at 400 °C. There was an initial loss in weight between 80 and 250 °C, which may

have been due to loss of water trapped in the microvoids of the chitin structure. A

further loss in weight occurred after 390 °C, which was due to further

decomposition of the chitin and residue.

The DSC curves (Figures 3.2 and 3.3) exhibited broad endothermic

transitions at 80 – 200 °C, which was due to residual solvent. This confirmed that

the drop in weight between 80 – 250 °C in the TGA was due to water. The

exotherm at 307 or 302 °C in Figures 3.2 and 3.3 respectively was due to the

108

formation of crosslinkages in the molecule. At about 394 °C, decomposition of

the samples was confirmed by the small endotherm recorded. Therefore, chitin is

stable up to 394 °C. The presence of residual solvents in chitin suggests a

difficulty in drying chitin for weighing.

109

3.3. SCANNING ELECTRON MICROSCOPY (SEM)

Sir Charles W. Oatley 7 and his students developed the modern SEM at

Cambridge University in England from 1948 – 1961.

Microscopes magnify details that are invisible to the unaided eye. Objects

that are 0.1 mm apart can be differentiated. The optical microscope resolves

objects that are up to 0.2 µm apart. Scanning electron microscopes resolve objects

that are up to 3/10, 000 of a micron apart and magnify objects up to 800,000 times

their size. A finely focused electron beam irradiates the sample and secondary

electrons, backscattered electrons, X-rays and other types of radiation are

released. The secondary electrons are collected and amplified to produce an image

on a television screen 8.

Chitin samples obtained from lobster shells and the Blaberus cockroach

wings and legs were placed on a metal sample plate and observed by magnified

photographs taken by a Phillips 505 Scanning Electron Microscope. The

photographs were taken to give an overall view of the sample and a detailed view

of a selected portion.

Chitin (from lobster shells) observed by magnified photographs revealed

the fibrous nature 9 of the compound as shown by position s on the photograph

(Photograph 3.1).

110

Photograph 3.1

SEM OF LOBSTER CHITIN

(SCALE BAR, 1mm)

There were also white clumps of materials labeled c and an area sparsely covered

by more white materials. Higher magnification of the latter area revealed more of

fibres and clumps. These white clumps of materials appeared to be impurities

(Photograph 3.2).

Photograph 3.2

SEM OF LOBSTER CHITIN

(HIGHER MAGNIFICATION SCALE BAR, 10µµµµM)

111

In the photographs of chitin isolated from Blaberus cockroach legs

(Photograph 3.3), eggshell like materials es and white clumps c identical to those

present in Photograph 3.1 were observed.

Photograph 3.3

SEM OF CHITIN FROM BLABERUS

COCKROACH LEG( SCALE BAR = 1mm)

Photograph 3.4 shows the detail of one of the white clumps. Present

under these was the eggshell like material labeled es.

Photograph 3.5 shows the overall particle distribution of chitin obtained

from the wings of the Blaberus cockroach. Present were clumps of grey materials

g and the white clumps of materials c.

112

Photograph 3.4

SEM OF CHITIN FROM BLABERUS COCKROACH LEG

(HIGHER MAGNIFICATION, SCALE BAR = 10µµµµM)

Photograph 3.5

SEM OF CHITIN FROM BLABERUS

COCKROACH WINGS (scale bar = 1mm)

Photograph 3.6 was a higher magnification of g. Present on g were some

of the material labeled c. The grey material appeared to be a tightly woven

material. It seemed therefore that the typical chitin is riddled with various types of

impurities.

113

Photograph 3.6

SEM of chitin from Blaberus cockroach wings

(higher magnification, scale bar = 10µµµµm)

114

3.4 13 C NMR ANALYSIS OF CHITIN MONOMER

13 C NMR spectroscopy was used to determine the chemical shifts for each

carbon in the N-acetyl glucosamine monomer of chitin (6). These chemical shifts

were compared with those of the carbons of glucose (7) (in D2O) 10 and solid

biosynthetic chitin (called artificial chitin) (8) (cross polarisation magic angle

spinning - CP / MAS) 11.

O

OH

O

O

OH

O

NHCOCH3

NHCOCH3

HOH2C

O

n

51

23

4

HOH2C

O

OH

OH

OH

OH

CH2OH

O

O

O

N C CH

H

3H

H, OH

H, OH

CH2OH

1

3

4

6

122

3

4

55

6

6

CHITIN MONOMER

(6)(7)

GLUCOSE

(8)

BIOSYNTHETIC (ARTIFICIAL CHITIN)

7

8

Chitin obtained from lobster shells was hydrolysed in concentrated

hydrochloric acid. The unreacted residue was removed by filtration and the filtrate

collected. D2O and 3-(trimethylsilyl)-1-propane sulphonic acid salt was then

added to the solution and the 13 C NMR spectrum determined using a Bruker AC

115

200 instrument. The chemical shifts for each carbon were then determined and

compared with glucose and biosynthetic chitin from the literature (Table 3.1).

Table 3.1

13C DATA FOR HYDROLYSED CHITIN

GLUCOSE AND CHITOSAN HYDROCHLORIDE

Literature values

C Glucose (D2O/TMS)

/ δ ppm

Artificial chitin CP/MAS solid

/ δ ppm

Hydrolysed chitin (D2O/TMS)

/ δ ppm

1 93.6 105.0 99.9

2 73.2 56.2 61.7

3 74.5 74.3 76.9

4 71.4 84.4 96.4.

5 73.0 76.9 83.4

6 62.3 61.9 67.7

7 (C=O)

8 (CH3)

-

175.0

23.8

183.9

27.9

A value of δ 99.9 ppm was obtained for carbon 1, which was a little higher than

the sigma shift obtained for carbon 1 in glucose. This suggested that the ether

linkage was still present (incomplete hydrolysis). A high value of δ 105 ppm was

shown for the biosynthetic solid chitin where the entire C 1 – C 4 ether bonds

were intact, a highly deshielded environment. The chemical shift for carbon 2 was

δ 61.7 ppm a low value because of the shielding effect of the nitrogen atom. In

116

glucose where an OH was present, which was deshielding in effect, a value of

δ 73.2 ppm was obtained. The other carbons of the chitin monomer C 3, C 4, C 5

and C 6 had chemical shifts of δ 76.9,δ 96.4, δ 83.4and δ 67.7 ppm respectively.

Carbon 4 of the biosynthesised chitin had chemical shift δ 84.4 ppm. These high

values of δ 96.4 and 84.4 ppm may be due to the deshielding effect created by the

C 1 – C 4 linkages.

The chemical shift of the carbonyl group of the hydrolysed chitin was observed to

be δ 178 ppm. The methyl carbon resonated at δ 27.9 ppm. These values

compared favorably with those of the corresponding carbons of the biosynthetic

solid chitin, which suggested that the hydrolysis did not affect these group.

117

3.5 IR SPECTRAL ANALYSIS – FUNCTIONAL GROUP ANALYSIS

AND % N-ACETYLATION DETERMINATION.

3.5.1 FUNCTIONAL GROUP ANALYSIS

The characteristic absorptions of the main functional groups present in

chitin obtained from lobster were determined by IR spectroscopy and compared

with the spectrum of a sample of unpurified crab chitin obtained from Sigma Co.

The IR spectrum of the skin-like and powdered materials obtained from the

Blaberus cockroach exoskeleton was also determined.

Samples of chitin were ground with KBr and compressed into discs. The

chitin – KBr discs were placed into a Perkin Elmer FTIR Spectrophotometer

(previously standardised with polystyrene) and the absorbance or transmission

spectra determined. For comparison, the IR spectrum of the cockroach wing was

recorded. The wing was simply cut to fit the sample holder and placed into the

spectrophotometer.

Figure 3.4 and Figure 3.5 shows the IR spectra of the sample of

unpurified crab chitin obtained from Sigma Co and chitin from lobster shells.

118

Figure 3.4

IR SPECTRUM OF UNPURIFIED CRAB CHITIN OBTAINED FROM SIGMA CO.

25

35

45

55

65

75

500150025003500

Wavenumber cm -1

% T

ransm

itta

nce

119

Figure 3.5

IR SPECTRUM OF SAMPLE CHITIN FROM LOBSTER SHELLS

The IR spectra of residues (skin-like and powdered material) obtained

from the wings and legs of the Blaberus cockroach after alkaline hydrolysis are

shown in Figures 3.6 and 3.7. The IR spectrum of the wing of the cockroach is

shown in Figure 3.8.

40

45

50

55

60

65

70

75

550105015502050255030503550

Wavenumber / cm -1

% T

ransm

itta

nce

120

Figure 3.6

IR SPECTRUM OF SKIN-LIKE MATERIAL OBTAINED

FROM THE WING OF AN ADULT BLABERUS COCKROACH AFTER NAOH DIGESTION

Figure 3.7

0

10

20

30

40

50

60

70

80

90

100

450950145019502450295034503950

Wavenumber / cm -1

% T

ransm

itta

nce

121

IR SPECTRUM OF POWDERED MATERIAL OBTAINED

FROM THE LEG OF AN ADULT BLABERUS COCKROACH AFTER NAOH DIGESTION

Figure 3.8

IR SPECTRUM OF THE WING OF AN ADULT BLABERUS COCKROACH

0

20

40

60

80

100

450950145019502450295034503950

Wavenumber / cm -1

% T

ransm

itta

nce

0

20

40

60

80

100

4009001400190024002900340039004400

Wavenumber / cm -1

% T

ransm

itta

nce

122

The IR spectra of the chitin obtained from the lobster shell and crab shell (from

Sigma) confirmed bands at 3450 (OH), 2878 (C-H stretch), 1655 and 1630 (amide

1 or C=O stretch), 1560 (the amide 2 - NH bending), 1160 (bridge oxygen

stretching), 1070 and 1030 cm-1 (C-O stretches) as indicated by literature 12.

The IR spectrum of the skin-like material obtained from the wing of the

cockroach (Figure 3.6) showed the OH band at 3450 cm –1, with the doublet

characteristic. Also present was the C-H peak as well as the double at the C=O

stretch. The powdered material (Figure 3.7) obtained from the leg of the

cockroach varied from the spectrum of Figure 3.6, but the OH, C-H and C=O

were still evident. The spectrum of the wing of the cockroach (Figure 3.8) had the

characteristic hydroxide and amide peaks associated with chitin. This sugested

that a large portion of the cockroach wing may be chitin 13.

3.5.2 Percentage N-acetylation (% N-Ac)

The percentage N-acetylation of chitin is a long-standing method of

characterising chitin. The history concepts and principles involved in its

determination are outlined followed by the application of some of these concepts

to some of the chitin and chitosan samples studied. Specifically, two equations

have been applied to the determination of percentage N-acetylation of these

samples. These were proposed by Domzy and Roberts 14 and Baxter et. al 15.

123

(a) History, concepts and principles of percentage N-acetylation

determination

Many samples that are proposed to be chitin are a mixture of chitin and

chitosan. The value of the percentage N–acetylation tells how much of the

polymer is chitin, such that a 100% value indicates pure chitin 11.

An infrared spectroscopic technique for determining the degree of N-

acetylation of chitosan was proposed by G.K. Moore and G.A. Roberts (1955) 15

and later revisited by J. Domzy and G. A. Roberts (1985) 14. The method involves

the use of the amide band at 1655 cm-1 as a measure of the N-acetyl group content

and the hydroxyl band at 3450 cm -1 as an internal standard to correct for film

thickness or for differences in chitosan concentration if a KBr disc was used.

Domzy and Roberts 14 proposed that a fully N-acetylated compound should show

the ratio; of absorbance A 1655 cm-1 ÷ A 3450 cm

-1 to be 1.33, on the assumption that

the value of this ratio is zero for fully deacetylated chitosan, and that there is a

dependent relationship between the N-acetyl group content and the absorption of

the amide 1 band. The percentage of the acetamide groups was given as:

% N-acetyl = (A 1655 cm-1 ÷ A 3450 cm

-1) × 100 ÷ 1.33…………Equation 3.1

The absorbances were determined from designated baselines stretching across

these peaks.

Titration, NMR spectroscopy, mass spectrometry, circular dichroism,

HPLC, pyrolysis, gas chromatography and thermal analysis are also used to

124

determine degree of N-acetylation 15. The IR spectroscopic method proposed by

Moore and Roberts had a number of advantages; it is relatively quick and does not

require the purity of the sample to be determined separately. It is not sensitive to

the presence of moisture (standard drying techniques were applied to samples).

The method has been shown to have an acceptable level of precision, at least with

low acetylated (< 20%) samples, but the results were not good compared to other

methods (for example, when compared with the titration method): the values

obtained were too high. With % N-Ac greater than 20% however, the method

worked reasonably well 15.

Two additional absorption band ratios were proposed by Sannan 15 (1978)

and Miya et. al 15(1980) for percent N-acetylation determination:

A 1550 cm-1 ÷ A 2878 cm

-1 and A 1655 cm-1 ÷ A 2867 cm

-1, respectively. In both

cases, the C-H band is used as an internal standard.

These two ratios gave more accurate results at low % N-acetylation than the A1655

cm-1 / A3450 cm-1 ratio.

Miya et. al 15 found that the A1655 / A2867 ratio gave good agreement with

the colloidal titration method for samples having N-Ac. of less than 10%, whilst

samples having values of 10 - 25% N Ac were not in agreement. The use of the

A1550 / A2878 ratio is complicated by the considerable spectral changes that occur

in the 1595 - 1550 cm–1 region. In addition, for both ratios the use of the C-H band

as an internal reference was not good since this band decreases as the % N-Ac

decreases. The effect was small at low levels of % N-Ac but underestimates the

125

true values at higher levels; the comparison made with the titration method of

Broussignac 15.

Using A 1655 cm-1 / A3450 cm-1 (Domzy and Roberts 14) and a different

baseline proposed by Miya et. al 15, allowed for an accurate value of the percent

N-acetylation to be determined over a wider range of % N-Ac values than any

other absorption band ratio proposed (0 – 55%). However, two precautions must

be observed. The amount of sample in the beam must be small enough to ensure

that the 3450 cm-1 band has a transmission of at least 10% and if samples being

examined have been prepared by N-acetylation of chitosan any ester groups must

be removed by steeping in 0.5 M ethanolic KOH prior to recording the

spectrum 15. This formula that combined the ratio by Domzy and Roberts 14 and

the new baseline proposed by Miya et. al was put together by Baxter et. al (1992)

15 and is given as:

% N-acetyl = (A 1655 cm-1 / A 3450 cm-1) × 115 ……………..Equation 3.2

The value obtained will determine the proportion of chitin to chitosan that is

present in a sample which in effect will determine how a sample proposed to be

chitin will behave in dilute acetic acid. The baselines used by Domzy and Roberts

14 and Baxter et. al 15 are shown in Figure 3.9. The method of Domzy and

Roberts 14 required the use of Equation 3.1 and the baseline labeled (ΣΣΣΣ) and the

method of Baxter et. al 15 which required the use of Equation 3.2 the baselines

labeled (ΩΩΩΩ). The absorbances at 1655 cm-1 and 3450 cm-1 were determined from

the specified baselines.

126

Figure 3.9

IR SPECTRUM OF UNPURIFIED CRAB

CHITOSAN OBTAINED FROM SIGMA CO.

ΣΣΣΣ = the baselines involved in the method of Domzy and Roberts labeled ; ΩΩΩΩ = the baselines involved in the method of Baxter et. al. The absorbances at 1655 cm-1 and 3450 cm-1 were determined from the specified baselines.

(b) % N-ACETYLATION IN THE CHARACTERISATION OF CHITIN AND OF

CHITOSAN

Dried samples of chitin and chitosan were blended with KBr into discs.

The IR spectra of the samples were recorded using a Perkin Elmer FTIR

Spectrophotometer previously standardised using polystyrene. The % N-

acetylation was determined for the samples using the method of Domzy and

Roberts 14 and by the method of Baxter et. al 15. The percentages obtained are

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

550105015502050255030503550

Wave number / cm -1

Absorb

ance

127

shown in Table 3.2.

Samples analysed were crab chitin obtained from Sigma Co., crab chitosan

obtained from Sigma Co., lobster chitin and chitosan, prawn chitin and land crab

chitin. The chitin samples not obtained from sigma were prepared by acid

digestion followed by alkaline hydrolysis of crustacean shells. The chitosan

samples were prepared by refluxing chitin samples with concentrated sodium

hydroxide. The samples RGf/1/82a, RGf/1/82 c, RGf/1/82 d, and RGf/1/82 e were

prepared by homogenous N-acetylation of a chitosan sample RGf/1/81 (prepared

by refluxing lobster chitin). Homogenous N–acetylation involved acetylating with

different volumes of acetic anhydride, to effect conversion of the amine groups to

the corresponding acetamide.

When Equation 3.2 was used a wide variation of percentages were

recorded for the chitin and chitosan samples. The percentages obtained from using

Equation 3.1 showed a higher level of precision among the chitin samples where

higher % N-Ac values were expected.

128

Table 3.2

PERCENTAGE N-ACETYLATION OF CHITIN AND CHITOSAN SAMPLES

Sample N-acetyl

(A1655 cm-1/A3450 cm-1)

× (100/1.33)

/ %

N-acetyl

(A1655 cm-1/A3450 cm-1)

× 115

/ %

crab chitosan from Sigma Co., RGf/1/113a

8.7/16.3 × (100/1.33) = 40

2.5/16.3 × 115

= 18 (≤ 15)

crab chitin from Sigma Co., RGf/1/116a

69 51

lobster chitin, RGf/1/105b 63 54

lobster chitin RGf/1/21a-c 61 57

lobster chitin clob 61 42

lobster chitin, clob2c 66 67

prawn chitin, cpwn 60 61

prawn chitin, cpwn2b 60 42

land crab chitin, clc 48 60

lobster crude chitosan, RGf/1/80

40 30

N-Ac. chitosan, RGf/1/82a 49 31

N-Ac. chitosan, RGf/1/82c 55 37

N-Ac. chitosan, RGf/1/82d 59 45

N-Ac. chitosan, RGf/1/82e 61 57

lobster chitosan RGf/1/90 90 39

lobster chitosan RGf/1/97a 56 13

lobster chitosan RGf/1/114a 37 26

lobster chitosan, RGf/1/115b 40 21

lobster chitosan RGf/1/102 62. 18

129

Applying Equation 3.2 however gave better results where lower

percentages were expected. For example in the chitosan samples, a low value of

13% was obtained for RGf/1/ 97a, compared to 56% by using Equation 3.1.

The homogenous N-acetylated samples RGf/1/82 a, c, d and e showed the

effect of increasing the volume of the acetylating agent acetic anhydride. The

% N-Ac increased with increasing acetylating agent as expected.

The standard used in this experiment was crab chitosan obtained from the

Sigma Co. The manufacturers stated “minimum 85% deacetylated” (Photograph

3.7) which meant at least 15% N-Ac. When Equation 3.2 was applied 18% was

recorded whilst Equation 3.1 resulted in a percentage of 40% (Table 3.2).

Photograph 3.7

CHITIN (LEFT) AND CHITOSAN (RIGHT) FROM SIGMA CO.

(CHITOSAN: 85% DEACETYLATED)

130

Therefore Equation 3.2 was better for use with a wider variety of chitin

and chitosan samples even though it was less consistent when higher percentages

were expected as in the chitin samples. Equation 3.1 was better for use with the

chitin samples whilst Equation 3.2 was better for use with the chitosan samples.

Apart from the variation that results from using different equations in

calculation, % N-Ac varied because of inconsistencies in the reaction conditions

in the production of the various samples. For example, a sudden increase in

temperature may lead to an increase in the level of deacetylation.

131

3.6 CHITOSAN FROM CHITIN

If the chitin polymer the chitin polymer is converted fully to chitosan it is

expected to dissolve in 10% acetic acid. This is a simple test that aids in the

identification of chitin.

Chitosan was made from chitin by the aqueous sodium hydroxide method.

This method involves hydrolysis of chitin in NaOH (40 – 50%) under nitrogen for

6 h to obtain the crude chitosan. Purification was followed by adding the crude

chitosan to acetic acid (10%) and recovering the product obtained from the

solution at pH 7 by centrifugation, allowing it to dry and the yield calculated. The

dried product was then retested for its solubility in 10% acetic acid.

The purification process tended to be inefficient leading to a large loss of

product. For example, in a preparation deacetylation of the chitin resulted in a

70% yield of crude chitosan. Purification resulted in an overall yield of 10%.

As shown in section 3.5 b, the conversion method resulted in products

with various levels of % N-acetylation. Chitosan samples with low levels of % N-

Ac (13%, 18%) were soluble in 10% acetic acid and hence showed a successful

conversion of chitin to chitosan.

132

REFERENCES FOR CHAPTER 3

1. W.W.M. Wendhandt, “Thermal Methods of Analysis,” John Wiley and Sons, New York, 1974, Vol 19, p 6.






7. R. E. Lee, “Scanning Electron Microscopy and X-ray Analysis,” PTR Prentice-Hall Inc., New Jersey, 1993, p 9.

8. O. C. Wells, “Scanning Electron Microscopy,” McGraw-Hill Inc., New

York, 1974, p 2. 9. E. Cohen, Ann. Rev. Entomol, 1987, 32, 72.

10 T.E. Walker, R.E. London, T.W. Whaley, R. Barker and N.A. Matwiyoff, J. Am. Chem. Soc, 1976, 98:19,5808.

11. J. N. Bemiller, Meth. Carbohyd. Chem., 1965, 5, 103.

12. Y. Shigemasa, H. Matsurra and H.Saimoto, International Journal of Biological Molecules, 1966, 18, 237.

13. N. P. O. Green, G.W. Stout, D.J. Taylor and R. Soper, “Biological Science

Organisms, Energy and Environment,” Cambridge University Press, London, 1986, p 108.

14. G. Domszy, G. A. F. Roberts, Makromol. Chem., 1985, 186, 1671. 15. A. Baxter, M. Dillon, K.D.A. Taylor and G.A.F. Roberts, Int. J.

Macromol., 1992, 14, 166.

133

CHITIN AND ECONOMICS

134

The uses for chitin are many and constitute a multimillion-dollar industry.

these vary from medical applications to general industrial applications.

Lobsters are probably the most easily obtained shellfish in Jamaica.

Approximately 60,000 Kg are harvested each year (Fisheries division, Ministry of

Agriculture, Jamaica, 1996). This figure is obtained from over a dozen fishing

beaches around the island, where the crustacean supplies are very irregular.

A typical female spiny lobster of total weight 428 g, carapace length 8.5

cm consisted of 113.2 g (26%) shell and from this may be obtained 24 g of chitin

( assuming a chitin content of 21%).

A few of the types of chitin sold in Jamaica by Sigma Chemical Company

Distributor Industrial Technical Supplies Jamaica Limited gave an idea of the

earnings that were possible from chitin (figures for 1998).

EARNINGS FROM CHITIN

Description Price / $ Ja

Purified chitin powder from shrimp shell (5g) 11,550.70

Purified chitin powder from crab shell (5 g) 9,819.40

Unpurified chitin from crab shell (10 g) 525.05

If the lowest price is used, about $ Ja 1260 may be earned (before

production cost) from 24 g of chitin. Production costs include costs for acid,

135

alkali, fuel, equipment and labour. Hydrochloric acid costs 11.5 pounds per 500

mL and sodium hydroxide pellets cost 10.3 pounds per 500 g (prices of chemicals

from Sigma Co).

The feasibility of a chitin industry is often brought into question. The head

of the lobsters are discarded and whole crabs are sent to restaurants where they

are decorated and sold to the public. To have a vibrant chitin industry it would be

necessary to have a large collection drive. With such a small crustacean-eating

public the samples would degrade by the time enough had been collected.

Therefore, it is important to establish a reliable source of chitin, one of

which might be prawn. Prawn can be reared in ponds and their shells collected

after each moulting period. The adult prawn may also be uniquely stripped of its

exoskeleton before being sent to the supermarket or restaurant. The shrimp, which

is a smaller version of the prawn, may also be a viable alternative, where they

may be used whole, putting under one roof the production of proteins, chitosan

and chitin. Chitin may also be obtained from fungi grown on fermentation

systems to produce organic acids, antibiotics and enzymes.

136

APPENDIX ONE

EXPERIMENTAL DETAILS FOR CHAPTER TWO

137

PREPARATION OF SHELLS

Shells of the Jamaican crustaceans, the marine spiny lobster (Panulirus

argus), the land crab (Gecarcinus ruricola), the blue crab (Callinectes sapidus),

and the giant Malaysian fresh water prawn (Macrobracium rosenberg) were

scraped to remove all fleshy material washed and dried in an oven at 100 °C for

8 h. The dried shells were crushed and ground. (For each series of experiments

shells were redried at 100 °C for 1 h and cooled for 1 h in a dessicator before use).

INAA

Samples for Instrumental Neutron Activation Analysis (INAA) were

analysed using the SLOWPOKE-2 nuclear reactor at the International Centre for

Environmental and Nuclear Sciences, University of the West Indies, Mona. The

isotope Ca-49 (gamma energy 3084.4 keV, half-life 8.8 minutes) was used for

quantification.

Samples (0.25 g, undigested and digested shells), were accurately weighed

into acid-washed polyethylene vials for irradiation. A neutron flux of 2.5 x 1011 n

cm-2s -1 was used, with irradiation, decay and counting times of 300 seconds

each. Samples were counted 10 cm from the surface of a Canberra Reverse

Electrode Germanium gamma detector, which had a FWHM of 2.0 keV (at

1332.5 keV), and an efficiency of 15%. Conditions were chosen to avoid a

detector dead time of greater than 5% while providing adequate detection limits

and sample throughput.

138

Calcium carbonate (Aldrich) was used as a standard to calculate calcium

concentrations. To determine accuracy, a gypsum certified reference material

(GYP-C, Domtar, Quebec) was treated in the same manner as the samples. An

empty capsule was also analysed to provide a blank value.

Concentrations were calculated using version 3.5 of the OMNIGAM

Neutron Activation Analysis software package (EG&G Ortec, Oak Ridge,

Tennessee).

OPTIMISATION OF DIGESTION CONDITIONS

Dried lobster shells (five one gram portions) were accurately weighed into

containers (500 mL) and cooled in an ice bath (5° - 10°C) a low temperature

was used to prevent excessive hydrolysis of chitin.

Volumes of acids HCl, HNO3, CCl3COOH CH3COOH and H2SO4,

(all 2M) were measured out in separate containers (5.5 mL acid per gram sample)

and added simultaneously to the different containers of lobster shells (one acid per

container). Containers were made large enough to allow for the swelling of the

material as the carbon dioxide gas was given off. The mixtures were left in the ice

bath for 1 h with frequent agitation then filtered and the solid residues washed

with distilled water until free of acid as indicated by universal litmus paper. The

procedure was repeated for reaction times of 6 and 48 h. The products were dried

in an oven at 100 °C, cooled in a dessicator and weighed. The weight loss

percentages were then calculated (Table 2.1) and the percentage residual calcium

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as calcium carbonate determined by INAA. (Figure 2.3).

CALCIUM CARBONATE CONTENT OF CRUSTACEAN SHELLS - AS DETERMINED BY

WEIGHT LOSS

Fresh samples of the crustacean shells (lobster, land crab, blue crab and

prawn) (1 g), were accurately weighed into round bottom flasks (500 mL) and

cooled in an ice bath. The containers were made large enough to allow for the

swelling of the material as the carbon dioxide gas is given off). HCl (2 M, 5.5 mL

acid per gram of sample) was added slowly to the containers. The reactions were

left for 48 h during which the mixtures were agitated periodically and the

temperature maintained between 5 and 10 °C.

The mixtures were then filtered and the chitin-protein residue was washed

with distilled water until free of acid as indicated by universal litmus paper, dried

in an oven at 100 °C, cooled in a dessicator, then weighed. The weight loss

percentages were then calculated (Tables 2,2 - 2.5).

CALCIUM CARBONATE CONTENT OF CRUSTACEAN SHELLS AND CHITIN PROTEIN

RESIDUE WITH OPTIMISED ACID DIGESTION CONDITIONS – AS DETERMINED BY

INAA

Samples of the shells of the four crustacean species (0.25 g) were weighed

out and irradiated to determine their percentage calcium present as calcium

carbonate (Table 2.6).

Fresh samples of shells were again digested according to the weight loss

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procedures above and the percentage residual calcium as calcium carbonate

present, determined by INAA (Table 2.8). The digestion process was again

repeated in order to decrease the amount of residual calcium as calcium

carbonate. The new percentages obtained are shown in Table 2.9 and the

associated weigh tloss percentages presented in Table 2.10.

CALCIUM CARBONATE CONTENT - AS DETERMINED BY AAS

Shells (approximately 3 g) of two crustacean species (lobster and land

crab) were accurately weighed into round bottom flasks (500 mL) and cooled in

ice baths. HCl (5.5 mL acid per gram of sample) was then added slowly to the

containers. The reactions were left for 48 h during which the mixtures were

agitated periodically and the temperature maintained between 5 and 10 °C. The

mixtures were then filtered and the solid (chitin-protein residue) washed with

water (150 mL). The filtrate and washings were made up to zero with distilled

water (250 mL) in a volumetric flask. The residue was then analysed for its

percentage calcium by INAA (Table 2.11). The filtrates were diluted by a 1 / 50

dilution factor and the percentage calcium as calcium carbonate determined by a

Perkin Elmer 5100 PC Atomic Absorption Spectrophotometer (Table 2.10).

Calcium standards provided by the National Institute of Standards and

Technology Gathersburg, MD were also analysed. The samples were aspirated

into an air acetylene flame and the absorbance measured at wavelength 422.7 nm,

utilising a monochromator slit width of 0.7 nm.

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CHITIN CONTENT OF CRUSTACEAN SHELLS AS DETERMINED BY ALKALINE

HYDROLYSIS

The chitin-protein residue obtained from acid hydrolysis of the shell

samples was treated with NaOH (1 M, 5.5 mL per gram of solid). The mixtures

were refluxed at 100 °C for 12 h, cooled, filtered and the residues washed with

distilled water to remove hydrolysed protein. The residues were then returned to

the reaction vessels, and a fresh portion of NaOH added. The mixtures were then

refluxed for a further 12 h.

The process was repeated twice, after which the final residue was

thoroughly washed with water until free of base as indicated by universal litmus

paper, air-dried, weighed and the percentage unhydrolysed product determined

(Table 2.12). In addition, the percentage residual calcium carbonate present in the

unhydrolysed product was determined by INAA (Table 2.13). The weight of

unhydrolysed product and the percentage residual calcium carbonate were then

used to calculate the chitin composition of the different crustaceans under

investigation (Figure 2.5).

ANALYSES FOR THE PRESENCE OF AMINO ACIDS AND OTHER SUBSTANCES

PRESENT IN FRACTIONS OBTAINED FROM SODIUM HYDROXIDE HYDROLYSED

CHITIN-PROTEIN RESIDUE

Ninhydrin test

A drop of the filtrate obtained from lobster and prawn sample after

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alkaline hydrolysis was placed on a filter paper followed by ninhydrin. This was

allowed to dry and the paper heated for a minute and the colour of the paper

examined.

Gel electrophoresis

The filtrates obtained from lobster and prawn samples after alkaline

hydrolysis (60 µL) were added to 60 µL of sample buffer (0.01 M Tris-HCl,

0.001 M EDTA, SDS (1%), 2-mercaptoethanol (5%) (optional), pH 8.0). The

samples were heated for 3 minutes at 100 ºC in a water bath. Glycerol (40%, 30

µL) and tracking dye (5µL, bromothymol blue (1%)) were then added to the

sample. The sample (20 µL) each were then applied to gel - rods (polyacryl amide

(10%), containing SDS 0.53%) and subjected to electrophoresis at 100 V for

3.5 h. The electrophoresis tank contained electrophoresis buffer (EDTA (0.002

M), SDS (0.02%) at pH 7.4).

When the process was terminated the gels were treated with fixing agent

perchloric acid (3.5%), methanol (20%, v/v), stained with Coomassie Blue R

(250) (0.111g) in destaining solution (100 mL) and destained with ethanol (25%),

acetic acid (8%, v/v). The gels were then observed for the blue bands associated

with the presence of amino acids or polypeptides.

GC Mass Spectrometry

The filtrate (2 mL) obtained from NaOH (1M) treated lobster and prawn

chitin-protein residues were made more basic with concentrated ammonia

143

solution. The solutions were then extracted with two 5 mL portions of

dichloromethane. The dichloromethane fraction was then dried with sodium

sulphate.

A fresh portion of the filtrate (2 mL) was acidified with 6M HCland

heated for 15 minutes at 60 ºC, allowed to cool and at the end of the process

extracted with two 5 mL portions of diethyl ether

The acidic and basic fractions were evaporated to dryness, derivatised

with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and heated for 1 h at 40 ºC in

preparation for analysis by a Hewlett Packard 6890 Gas Chromatograph and Mass

Selective Detector, which produced their chromatograms. A Pfleger/ Maurer/

Weber MS Drug Library was used to determine the type of materials the samples

contained.

REMOVAL OF CALCIUM FROM CRUSTACEAN SHELL BY COMPLEXATION WITH

EDTA

A pH 9.2 tablet (tavollete tampone) was dissolved in water (100 mL).

Combined with ethylene diamine tetra-acetic acid disodium salt (EDTA) (3 g) and

added to some finely ground lobster shells (3 g).

The mixture was agitated for 15 minutes at room temperature and the solid

product collected by filtration, washed, dried and the weight loss percentage

determined. The experiment was repeated for 60 and 180 minutes (Table 2.15).

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REMOVAL OF CALCIUM FROM CRUSTACEAN SHELL BY COMPLEXATION WITH 18

CROWN-6 ETHER

8-crown-6 ether (0.1 g) was dissolved in water and agitated at room

temperature with lobster shells (0.1 g) for 1 h. The resulting solid was collected

by filtration, washed and dried and the weight loss percentage determined. The

experiment was repeated using ethanol instead of water at room temperature and

80 – 85 °C. In addition the pH of the solutions were varied from pH 4.0 - 9.2.

(Table 2.16)

CHITIN IN COCKROACH

The wings and legs of the cockroach Blaberus discoidalis obtained from

the gutters and drains of Mona (0.1 g) were accurately weighed and agitated in

HCl (2 M, 5.5 mL) for 48 h. The resulting mixtures were then filtered and their

undigested product washed with water and dried.

The product obtained after acid hydrolysis was then boiled in NaOH (1 M,

5.5 mL) for 48 h, and the product collected by filtration, dried, weighed and the

percentage chitin determined (Table 2.17). IR spectra were then recorded

(Chapter 3).

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APPENDIX TWO

EXPERIMENTAL DETAILS

FOR CHAPTER THREE

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THERMAL ANALYSIS OF CHITIN SAMPLES

Analyses were performed on a Universal V1 7 F T A Instrument. Chitin

samples of lobster (clob2a) and prawn (pwn2a) were heated in Nitrogen at 10 °C

per minute up to 1200 °C and the TGA curves determined. Fresh samples of the

shells and standard (Al pan) were also heated at the same rate up to 450 °C and

the DSC curves determined (Figures 3.1, 3.2 and 3.3).

SCANNING ELECTRON MICROSCOPY

Analyses were carried out on a Phillips Scanning Electron Microscope 505

at the Electron Microscopy Unit, U.W.I. Mona. Chitin samples from lobster shells

(RGf/1/21a-c) the Blaberus cockroach leg (RGf/1/31c, RGf/1/31d) and wing

(RGf/1/31e, RGf/1/31f) respectively were analysed by SEM. They were placed on

a metal sample plate and were illuminated by a beam of high-energy electron

beam and the image obtained from secondary electrons displayed on a screen

(Photographs 3.1 - 3.6).

13C NMR ANALYSIS OF CHITIN

Chitin obtained from lobster shells (RGf/1/21a-c) was boiled for 30

minutes in concentrated hydrochloric acid to hydrolyse it. The product obtained

was then filtered and the filtrate collected. D2O and 3(trimethylsilyl)-1-propane

sulphonic acid salt was then added to the solution and the 13C spectrum

determined using a Bruker AC 200 NMR spectrometer instrument (Table 3.1).

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PREPARATION OF CHITOSAN AND DETERMINATION

OF PERCENT N-ACETYL CONTENT OF CHITIN AND CHITOSAN

Preparation of chitosan from chitin samples

NaOH (40%, 490 mL) was added to chitin (RGf/1/21a-c, 10 g) and

refluxed under N2 at 110 °C for 6 h, cooled, filtered and the crude chitosan

residue (RGf/1/80) washed with water until the washings were neutral to

phenolphthalein then collected. This was then stirred for 24 h in a conical flask

with acetic acid (10% 177.5 mL).

The solution was then centrifuged to obtain a clear supernatant liquid. This

was treated dropwise with 40% aqueous sodium hydroxide solution where upon a

white flocculent precipitate formed at pH 7. The precipitate, recovered by

centrifugation, was washed repeatedly with water, ethanol and ether and the solid

collected and air-dried. The resulting purified chitosan (RGf/1/81) was then N-

acetylated to give N-acetylated chitosan samples RGf/1/82a, RGf/1/82c,

RGf/1/82d and RGf/1/82e. N-acetylation is covered in the next section.

The preparation from (RGf/1/21a-c) was repeated (without N-acetylation)

with the same ratio of samples to solvent to produce chitosan sample RGf/1/190.

NaOH (50%, 9.38 mL) was also used to carry out conversion of chitin

samples clob2b (0.1955 g) to chitosan sample RGf/1/97a. Chitin sample,

RGf/1/105b, when refluxed in two experiments (in similar ratio of sample to

alkaline in the preparation from RGf/1/21a-c) produced RGf/1/114a and

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RGf/1/115b.

Chitosan sample (RGf/1/90, 0.5207 g) was further deacetylated by

repeating the alkaline hydrolysis process with NaOH (40%, 24.5 mL) to produce

RGf/1/102.

Preparation of RGf/1/97a, RGf/1/114a, RGf/1/115b and RGf/1/102 did not

involve N-acetylation. All the chitosan samples were tested for their solubility in

10% acetic acid.

Homogenous N-acetylation of chitosan samples

Chitosan RGf/1/81 (5.27 g) was dissolved in acetic acid (1%, 523 mL )

solution for 24 h and the solution divided into five parts (~104 mL each).

Methanol (126 mL) was added to each part followed by volumes of acetic

anhydride (1.85%) in methanol solutions. The amounts of acetic acid/methanol

solutions were 3, 13, 17 and 25 mL. The solutions were left for 1 h after which the

precipitates developed were retrieved by centrifugation. These were then washed

thoroughly with water, methanol and ether and then air-dried. The products were

recorded as RGf/1/82a, RGf/1/82c, RGf/1/82d and RGf/1/82e.

Percent N-acetylation

Percent N-acetylation was determined for crab chitosan obtained from

Sigma Co. (RGf/1/113a), crab chitin from Sigma Co. (RGf/1/116a), lobster chitin

(RGf/1/105b), lobster chitosan (RGf/1/115b), lobster chitin (RGf/1/21a-c), lobster

chitin (clob), lobster chitin (clob2c), lobster chitin (clob2c), prawn chitin (cpwn),

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prawn chitin (cpwn2b), land crab chitin (clc), lobster crude chitosan (RGf/1/80),

homogenous N-Ac. Chitosan (RGf/1/82a), homogenous N-Ac. Chitosan

(RGf/1/82c), homogenous N-Ac. Chitosan (RGf/1/82d),homogenous N-Ac.

Chitosan (RGf/1/82e), lobster chitosan (RGf/1/90), lobster chitosan (RGf/1/97a),

lobster chitosan (RGf/1/102), lobster chitosan (RGf/1/101), and lobster chitosan

(RGf/1/114a). The chitin samples not obtained from sigma were prepared by acid

digestion followed by alkaline hydrolysis of crustacean shells.

Dried samples of the chitin and chitosan samples were blended with KBr

to form KBr discs. These were then placed into a Spectrum 1000 Perkin Elmer

FTIR Spectrometer, previously standardised with polystyrene, to determine the

absorbances of the functional groups present in the compounds. From the spectra,

the % N-acetylation were determined using the method of Domzy and Roberts 2

and Baxter et. al 1, the absorbances at 1655 and 3450 cm-1 and the baselines

labeled (Σ) and (Ω), shown in Figure 3.9 (crab chitosan sample, RGf/1/113a)

The method of Domzy and Roberts 2 and Baxter et. al 1 required the use of

the baseline labeled (Σ) and Equation 3.1, and baselines labeled (Ω) and

Equation 3.2 respectively.

% N-acetylation =(A1655 cm-1/A3450 cm-1) × (100/1.33)……………Equation 3.1

% N-acetylation = (A1650 ÷ A3450) × 115 ………………………….Equation 3.2)

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The absorbances at 1655 cm-1 and 3450 cm-1 were determined from these

specified baselines. The percentages obtained are shown in Table 3.2.

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