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Transcript of 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.
ii
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
2.5.1 Introduction 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
2.6.1 Introduction 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
xiii
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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41
Section of Medical Research, A division of the American Cyanamid Company, N. Y., 1983 (report).
61. D. Horton and D. R. Lineback, Meth. Carbohydr. Chem., 1995, 5, 405.
62. V. E. Tikhonov, L. A. Radigina and Y. A. Yamskov, Carbohydr. Res., 1996, 290, 33.
63. Reference 8, p 214.
64. Reference 8, p 248.
65. Reference 8, p 249.
66. Reference 8, p 183.
67. Reference 8, p 263.
68. Reference 8, p 231.
69. Reference 8, p 233.
70. Reference 8, p 235.
71. Reference 8, p 236.
72. Reference 8, p 238.
73. Reference 8, p 244.
74. Reference 8, p 246.
75. W. Schlotzhauer, O. Chortyk, P. Austin, J. Agric. Food Chem., 1976, 24 (1), 177.
76. Reference 8, p 247.
77. Reference 8, p 259.
78. Reference 8, p 260.
79. M. Kawakami, Jpn. Kokai Tokkyo Koho, JP06, 24, 934, 1994, CA 121: 65303n
80. R.E. Harmon, K.K. De and S.K. Gupta, Carbohyd. Res.,1973, 31, 408.
81. N. Nishikawa, Jpn Kokai Tokkyo Koho, JP05, 271, 094, 1995, CA 122, 32015n
42
82. K. R. Holme and A.S. Perlin, Carbohydr. Res, 1997, 302, 7.
83. E. Konrad, Ger Offen, DE 35, 537, 333, 1987, CA 107, 204935.
84. T. Miyata, Jpn Koho, JP 63, 220, 866, 1989, CA 111: 219319.
85. G. Rags Inc. 5000 Flat Creek Drive Ft. TX76179, 1999
http://www.fatabsorb.com/pinfo.htm
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
PRELIMINARY WEIGHT LOSS RESULTS OF
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
PRELIMINARY WEIGHT LOSS RESULTS OF
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
PRELIMINARY WEIGHT LOSS RESULTS OF
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.
2. Reference 1, p 92.
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.
12. Reference 4, p 414.
13. Reference 3, p12
14. Reference 3, Vol 34, p 7.
15. Reference 3, Vol 34, p 8.
16. Reference 1, p 92.
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.
22. Reference 21, p 17.
23. Reference 21, p 13.
24. Reference 21, p 4.
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.
28. Reference 27, p 91.
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.
2. Reference 1, p 193.
3. Reference 1, p 134.
4. Reference 1, p 212.
5. Reference 1, p 215.
6. Reference 1, p 242.
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
139
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
140
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.
141
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
142
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).
144
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).
145
APPENDIX TWO
EXPERIMENTAL DETAILS
FOR CHAPTER THREE
146
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).
147
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
148
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),
149
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)
150
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.