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
chitin: ISOLATION AND CHARACTERISATON. DSC, INAA, TGA, IR, NMR
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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.
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
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
REFERENCES FOR CHAPTER ONE
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2. J. Lehmann, "Carbohydrates Structure and Biology," Thieme Stuttgart, N.Y.,1998, p 81.
3. E. Pennisi, Science News, 1993, 144, 72.
4. J. N. Bemiller, Chitin, in "Methods in Carbohydrate Chemistry," Academic press N.Y., 1965, p103.
5. E. Cohen, Ann. Rev. Entomol, 1987, 32, 72.
6. Y. Shigemasa, H. Matsura and H. Saimoto, Int. J. Biol. Mol., 1966, 18, 237.
7. B. Smith, T. Koonce and S. Hudson, Polymer and Textile Chemistry, N.C.S.U., Raleigh, N.C., American Dyestuff Reporter, 1993, 20.
8. R.A.A. Muzzarelli, “Chitin,” Pergamon Press, N.Y., 1976, p 1.
9. Reference 8, p 2.
10. Reference 8, p 3.
11. R.A.A. Muzzarelli, “Natural Chelating Polymers: Alginic Acid, Chitin and Chitosan,” Pergamon Press, Oxford, 1973, p 83.
12. J. Mann, “Secondary Metabolism,” Oxford University Press, N.Y., 1987, p 8.
13. H. Blair, J. Guthrie, T. Lew and P. Turkington, J. Appl. Polymer Sc., 1987, 33, 641.
14. Reference 8, p 17.
15. S. Kobayashi, T. Kiyosada and S. Shoda, J. Am. Chem. Soc., 1996, 118, 13113.
16. E. Cabib, S.J. Silverman, J.A. Shaw, S. Dasgupta, H. Park, J.T. Mullings, P.C. Mol, B. Bowers, Pure and Appl. Chem., 1991, 63 (4), 485.
17. Reference 8, p 8.
18. D. Voet and J. G. Voel, “Biochemistry,” John Wiley and Sons Inc.N. Y., 1995, p 608.
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19. Reference 8, p 45.
20. Reference 8, p 46.
21. T.D. Rethke and S.M. Hudson, J. M. S – Rev. Macromol. chem. Phys, 1994, C 34, 378.
22. Reference 8, p 47.
23. Reference 8, p 79.
24. P.W. Kent, and M.W. Whitehouse, “Biochemistry of the Amino Sugars,” M.W. Whitehouse, London, Butterworths Scientific Publication, 1955, p 95.
25. Reference 8, p 58.
26. Reference 8, p 85.
27. Reference 8, p 67.
28. Reference 24, p 92.
29. Reference 8, p 6.
30. J. J. Skujins, H. J. Potgeiter and M. Alexander, Arch. Biochem. Biophys., 1965, 111, 358.
31. K. Aiken, Jamaica Journal, 1984, 17, 44.
32. 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.
33. G. F. Warner, “The Biology of Crabs,” Paul Eleck Scientific Ltd.,London, 1977, p 7.
34. A. E. Vines and N.Rees, “Plant and Animal Biology,” Pitman Publishing Ltd., London, 1972, Vol. 1, p 647.
35. Reference 32, p 109.
36. Reference 33, p 8.
37. R. D. Barnes, “Invertebrate Zoology,” Saunders College Publishing, Philadelphia, 1987, p 475.
38. R. S. Lowery, Growth Moulting and Production, in “Freshwater, Crayfish Biology, Management and Expoitation,” Eds. D.M Holdich and R.S.
40
Croom Helm Ltd, London, 1988, p 89.
39. Reference 38, p 83.
40. Reference 8, p 9.
41. Reference 24, p 94.
42. Reference 24, p 92.
43. Reference 8, p 10.
44. Reference 34, p 648.
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46. Reference 11, p 97.
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48. Reference 8, p 91.
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51. S. Salmon and S. M. Hudson, Journal of Polymer Science, Part B, Polymer Physics, 1995, 33, 1007.
52. K. Chang, G. Tsai, J. Lee, W. Fu, Carbohydr. Res., 1997, 303, 327.
53. Y. Chung Wei and S. Hudson, Macromolecules., 1993, 23, 4151.
54. B. Smith, T. Koonce and S. Hudson, Polymer and Textile Chemistry, N.C.S.U., Raleigh, N.C., American Dyestuff Reporter, 1993, 22.
55. Reference 11, p 145.
56. Reference 8, p 96.
57. Reference 8, p 97.
58. Reference 11, p 147.
59. Reference 8, p 98.
60. W. J. McGahren, G. A. Perkinson, J.A. Growich, R.A. Leese, G.A. Ellestad, ‘Chitosan by Fermentation,’ Process Research and Development
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.
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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.
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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
[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
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