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ISOLATION AND
CHARACTERISATION
A Thesis
of
by
October 1999
Department of Chemistry
Faculty of Pure and Applied Sciences
Mona Campus
i
ABSTRACT
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
calcium salts, present in these shells followed by base hydrolysis of the shell
aluminium, manganese and chlorine. With the use of Gas Chromatography Mass
carboxylic acid and alkanes were also indicated. Complexation was shown to be a
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.
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
were found to be; Lobster 21%, land crab 18%, blue crab 19% and prawn 35%.
(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.
N-acetyl content (% N-Ac) by the use of two infrared analysis techniques where
which is affected by the alkaline conditions of the hydrolysis step as well as the
method of calculation.
ACKNOWLEDGEMENTS
his unselfish help with the instrumental neutron activation analysis and atomic
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
the Departmental Award, the position as Tutorial Assistant and for the summer
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
DEDICATION
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
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
1.1 Introduction 2
1.2 History 3
1.4 Biosynthesis 7
1.7 Sources 14
1.10 Chitosan 24
2.1 Introduction 44
2.3.1 Introduction 54
2.5.1 Introduction 78
2.6.1 Introduction 82
vii
2.6.2 Percent unhydrolysed product (UHP%) after
alkaline hydrolysis 83
(1) Chitin 2
(2) Cellulose 4
(4) Chitosan 5
LIST OF TABLES
Table 2.3 Preliminary weight loss results of digestion of land crab shells
with 2M HCl 62
with 2M HCl 63
LIST OF FIGURES
Figure 3.1 TGA curves of prawn (cpwn2a) and lobster(clob2a) chitin 106
Figure 3.8 IR spectrum the wing ofan adult Blaberus Cockroach 121
LIST OF PHOTOGRAPHS
CHAPTER ONE
CHITIN
2
1.1 INTRODUCTION
fungi and green algae that utilize nitrogen containing sugars 4 and its biosynthesis
formation of UDP-N-acetylglucosamine 5.
The proposed uses of chitin are very wide, from medical applications
6
(example, wound healing) to waste water treatment 7. The derivatives used in
many commercial applications are made from chitosan, the deacetylated product
of chitin. Chitin is usually found present with other organic polymers and/or
inorganic salts 4 and its isolation usually involves hydrolysing and digesting these
molecular neighbours.
HOH2C HOH2C
NHCOCH3 NHCOCH3 O
O
O HO O HO O
O HO O HO
O O
NHCOCH3 NHCOCH3
HOH2C HOH2C
n
(1)
CHITIN
3
1.2 HISTORY
warm alkali. Twelve years later A. Odier 8 again found chitin in insect cuticle and
some plant tissue. The silk worm was also discovered as a source of chitin when
in 1843 J. L. Lassaigne 8 isolated it from the Bombyx mori. In the same year, A.
8
Payen initiated discussion about the differences between cellulose and chitin.
9
Rouget discovered chitosan in 1859. He boiled chitin in potassium
hydroxide solution and found that the product chitosan dissolved in organic acid,
and was violet in diluted solutions of iodine and acid. In contrast, chitin is stained
brown in iodine-acid solution. Hoppe Seyler 9 coined the name chitosan in 1894
In the first half of the twentieth century, research on chitin was mostly
directed toward the study of its occurrence in living organisms, its degradation by
HOH 2C HOH 2C
OH O OH O
O HO O HO O
O HO O HO
O O
OH OH
HOH 2C HOH2C
n
(2)
CELLULOSE
metabolism. The difference between chitin and cellulose occurs at position two
where in cellulose the hydroxy group replaces the acetamide group 13. Both chitin
HOH2C HOH2C
NHCOCH3 NHCOCH3 O
O
O H-O O H-O O
O H-O O H-O
O O
NHCOCH3 NHCOCH3
HOH2C HOH2C
hydrogen bond n
(3)
HOH2C HOH2C
NH2 O NH2 O
O HO O HO O
O HO O HO
O O
NH2 NH2
HOH2C HOH2C
n
(4)
CHITOSAN
HOH2C HOH2C
NHCOCH3 NHCOCH3 O
O
O HO O HO O
O HO O HO
O O NH2
NH2
HOH2C HOH2C
n
(5)
TRUE CHITIN
6
During isolation, chitin being a glucose polymer is also hydrolysed to its
degradation is the result of the harsh conditions often associated with the isolation
procedures 4.
Scheme 1.1
Chitin Hydrolysis
NHCOCH3
O HO
H, OH
O
HOH2C HOH2C
NHCOCH3 O
O HO O
O HO
O NHCOCH3
HOH2C
n HO H C
2
O
H, OH O
HO
NH C OC H3
7
1.4 BIOSYNTHESIS
1957 investigated an enzyme from the fungi Neurospora crassa. This enzyme
activated free N-acetyl glucosamine to produce chitin. In the laboratory chitin has
residues 16.
from the disaccharide trehalose or from glucose. The membrane bound chitin
17
The pathway has also been outlined by Muzzarelli . Biosynthesis is
8
believed to occur in the hypodermis. First, it involves hydrolysis of trehalose
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
Scheme 1.2
Biosynthesis of Chitin
CH2OH OH H
H H HO acetyl-Co-A glucoseamine-6-phosphate-N-acetyl
H
O
OH transferase
H HOH 2C CoA
OH
O O
H
HO
C H2 O P O 2-
H OH H 3
trehalose (alpha-D-glucosido-alpha-D-glycoside ) H OH
O
trehalase H
OH H
H
HO O
CH2OH
H HN C CH 3
H H
O
N-acetyl glucosamine -6-phosphate
H
OH phosphoacetylglucosamine mutase
OH
HO
H OH
glucose CH2OH
ATP H
O
O PO 2 -
3
hexokinase H
ADP H
OH
H
- 2 HO O
O3 POCH 2
H C
H H HN CH 3
O
N-acetyl glucosamine-1-phosphate
OH H
OH UTP
HO UDP-N-acetylglucosamine
OH pyrophoshorylase
H pyrophosphate
glucose-6-phosphate
CH2OH
CH2OH H
- 2O POCH2
3 OH H
O H
HO O
H HO
H HN C CH 3
HO HO UDP-N-acetylglucosamine
OH H
fructose-6-phosphate
chitin synthesase
glutamine UDP
glutamine-fructose-6-phosphate amino
transferase CH2OH
glutamic acid
H O
C H2O P O 3
2- O
H H H
O OH H
H O H
OH H O
OH H C
HO HN CH 3
H NH2
CHITIN
alpha-D-glucosamine -6-phosphate
10
19
Chitin forms a dimer chitobiose C16 H 28 O 11 N 2 and chains classified
20
as alpha, beta or gamma . 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.
Beta chitin chain forms sheets linked by C=O and H-N hydrogen bonds
hydrate can be easily penetrated by water. Thus, beta chitin is less stable than
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
–1
shows that alpha chitin has absorbances at 1655 and 1621 cm (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
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
of ions 22.
12
Chitin has an average molecular weight ranging from 1.036 million to 2.5
(b) SOLUBILITY
of mineral acids or with certain organic acids are also effective. These solvents
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
swell and dissolve, because the structure of the chitin becomes friable during
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
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
and Rhodophyta (red algae) are also noted sources. Photosynthetic plants utilize
nitrogen free sugars almost exclusively for their supporting structures and so lack
28, 29, 30
chitin .
(Photograph 1.1), the spotted spiny lobster (Panulirus guttatus), the long-armed
sapidus) and the giant Malaysian fresh water prawn (Macrobracium rosenberg)
Photograph 1.1
32
Crustaceans live in both aquatic and terrestrial environments and their
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
lipoid material (3-6 µm thick), lacking chitin and lying on a protein layer. It is the
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
surface of the cuticle and within the lamellae all the microfibrils are parallel to
35, 36
each other (Figure 1.1). The whole procuticle is strengthened by heavy
Figure 1.1
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
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
development point of view, the epicuticle and the exocuticle are secreted before
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
38 36
the environment . The exoskeleton then softens at which point it is shed .
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
38, 39
surrounding environment . 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
41
an approximate 55:45 ratio . A typical crustacean shell consists of about 25
percent complex (chitin-protein) and 75 percent calcium compounds 42. This ratio
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
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
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
joints, the sclerites are fastened together by thin flexible articular membranes
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.
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
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
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.
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
4, 46, 47
outlined by the method of Hackman . 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,
acetic acid (EDTA) to remove calcium. Large cuticle fragments of the crab
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
(e) METHOD OF TAKEDA AND ABE AND TAKEDA AND KATSURA 48, 49
Most of the methods outlined before involved the use of drastic treatments
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
with a proteolytic enzyme such as tuna proteinase at pH 8.6 and 37.5 ºC, or
over 60 h. The protein still present in the chitin was about 5% which was removed
This method is simple and perhaps suitable for the mass production of
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.
with papain, pepsin or trypsin, which allowed the chitin produced to be as little
deacetylated as possible.
This method involves the use of hot sodium carbonate. Workability of this
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.
Hackman). Lobster shells were treated with hot 5% sodium hydroxide solution,
1.10 CHITOSAN
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,
1,2
and is termed the degree of N-acetylation . The degree of N-acetylation
polycation 53 (Scheme 1.3). The solubility in organic acids renders chitosan more
Scheme 1.3
HOH2C HOH2C
NH2 O NH2 O
O HO O HO O
O HO O HO
O NH 2 O
NH2
HOH2C HOH2C
n
CHITOSAN
+
H
+ HOH2C + HOH 2C
NH 3 NH 3
O O
O HO O HO O
O HO O HO
O O
NH NH
+ 3 + 3
HOH2C HOH 2C
n
CHITOSAN POLYCATION
The following are some of the published methods used in the production
of chitosan.
This harsh method involves the use of solid potassium hydroxide and very
After 30 min. at 180 ºC, the melt was poured carefully into ethanol and the
26
precipitate washed with water to neutrality.
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.
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.
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
which permits recovery of proteins, sodium acetate and calcium carbonate as by-
marketable commodities. Shellfish waste ground to particle size of 3-6 mm, was
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-
refining agents to remove lipids or pigments). The clarified product was then
depended upon the shellfish species and extraction conditions. The resulting
precipitated protein was collected, washed and dried by reslurrying and spray
drying.
solution. The effluent from this operation contained excess sodium hydroxide,
means.
The mother liquor was diluted with water and treated with calcium
hydroxide. The sodium carbonate crystallisation was also treated with calcium
was then collected. The regenerated sodium hydroxide solution was combined
with added concentrated alkali and evaporated to the desired strength for use in
the residual shell consisting of chitosan and calcium hydroxide. This was washed
The chitosan and calcium hydroxide mixture was then extracted with an
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.
of this class was readily cultured on nutrients (example glucose or molasses) and
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
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
recovered by centrifugation, washed repeatedly with water, ethanol and ether and
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
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,
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 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.
impurities. The impurities include alkali earth metals, vegetable matter and
may be used along with coagulation aids like alum, ferric chloride or calcium
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-
51
forming properties. For example, Takai and co-workers used chitin fibers to
(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
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
(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.
affinity for many classes of dyes so that it can be used to remove them from waste
because these dyes are deposited superficially and wash out simply by wetting.
inherent to glass fibre and textiles, enhanced with chemical capacity to receive a
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.
Chitosan salt solutions in a viscous and pastelike state react with all types of dyes
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
Substances with soil repellent and soil releasing properties are often added
modified chitosan may be used to impart antistatic properties to these fabrics 71.
optical characteristics and its behavior with silver complexes, make it important to
the photography industry. The chitosan film can be easily penetrated by solutions
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.
ingredients may be formed into tobacco having good dry tensile properties and
Chitosan has been studied for its use in tanning, paste-drying and finishing
parasites such as ticks and mites and certain types of bacteria and fungi. Chitin
pharmaceutical carriers. They are appealing as carriers because they are degraded
(n) ANTICOAGULANT
Heparin, one of the worlds most widely used blood anticoagulants was
78
isolated from liver cells in 1918 . 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
Other derivatives that have been explored are summarized in Scheme 1.4.
Scheme 1.4.
1
ROH2C HOH2C
O O
O O
O HO O RO
NHR2 n NHCOCH3
n
( R1 = CH2CO-ARG-GLY-ASP-SER-OH R = CO(CH2)mCH3
chitin sulphate 83
81 m - 2 = 8 ; n - 20 = 5000
CH3COOH or R2 = H, AC)
H OH 2 C
O
* O
O HO
NC OC H3
n
chitin
HOOCH2COH2C ROH2C
O O
O * O
O HO
O HO
n
NHCOCH2CH2CO2M NHCOCH3
n
M = group 1 or 2 metals R = (CH2)nCOOH or H
n - 10 = 5000 79 n > 1 85
37
Cosmetics containing chitosan carboxy derivatives have been prepared. The
79
cosmetics showed excellent moisturising effect . Trimethylsilyl derivatives of
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
Coating rice seeds with chitosan has been reported to cause higher yields.
3
A derivative of chitin developed by Harvard University 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
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.
13. H. Blair, J. Guthrie, T. Lew and P. Turkington, J. Appl. Polymer Sc., 1987,
33, 641.
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.
39
19. Reference 8, p 45.
21. T.D. Rethke and S.M. Hudson, J. M. S – Rev. Macromol. chem. Phys,
1994, C 34, 378.
24. P.W. Kent, and M.W. Whitehouse, “Biochemistry of the Amino Sugars,”
M.W. Whitehouse, London, Butterworths Scientific Publication, 1955,
p 95.
29. Reference 8, p 6.
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.
34. A. E. Vines and N.Rees, “Plant and Animal Biology,” Pitman Publishing
Ltd., London, 1972, Vol. 1, p 647.
40. Reference 8, p 9.
52. K. Chang, G. Tsai, J. Lee, W. Fu, Carbohydr. Res., 1997, 303, 327.
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:
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
crustacean shells involved acid hydrolysis for 48 hours with 2M HCl, followed by
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
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
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
Detection limits are not as good as with other neutron sources such as nuclear
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
11 14 -2
reactors. Reactors produce a neutron flux ranging from 10 to 10 n cm s-1
(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.
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
48
Ca + 1n = 49Ca + γ…………………………………………Equation 2.1
49
If the radioactive isotope (e.g. Ca ) decays with the emission of gamma rays,
(qualitative identification). The number of gamma rays emitted per unit time or
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
3
same container. However, the exceptional flux stability of the SLOWPOKE
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.
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.
range of energies and intensities of gamma rays (called the gamma spectrum)
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
Figure 2.1
Figure 2.2
element in the sample to that induced in a known standard treated under similar
conditions.
following equation
11
A = N ϕ σ (1 – e-λti) e-λtd ………………………………Equation 2.2
Where
ti = irradiation time;
exceptional neutron flux stability. If the same irradiation times are used for both
Where,
However, the ratio of the concentration in the sample Csam to that in the standard
Cstd is
………………………………………………………………………Equation 2.5
minimise the duration of the counting period. However, high count rates can
cause ‘pulse pile up’ in the detector as the electronics can only process a certain
number of gamma rays per second. If counting rates exceed the resolving time of
the detector, a correction must be made to account for the difference between
results are not always due to the method but are often due to inhomogenous
distribution of the element of interest in the matrix being analysed. INAA results
are not usually affected by matrix effects. Because of this, it is often applied as an
accuracy of the standards being used for comparison with the sample. The
principal errors that arise during INAA analysis are due to self shielding, unequal
neutron flux for sample and standard, counting uncertainties and errors in
geometry between sample and standard. The errors from these causes usually can
the sensitivity of the detector, the decrease in activity at the time of counting, the
time available for counting and the magnitude of the background count rate
relative to the count rate of the analyte. Many authors overestimate the sensitivity
complex matrices such as human and animal tissue, coal, fly ash, petroleum, river
sediments, urine, faeces, blood etc. More than 25 elements can be analysed at the
same time 10. Analysis can be performed without destroying the sample 9, 10 and it
elemental analysis .
54
2.3.1 INTRODUCTION
To determine the acid best suited for the digestion process, lobster shells
were digested with five different acids over varying times and the loss in weight
calculated. In addition, INAA was applied to the digested lobster shells for the
calcium carbonate. The results from the weight loss and INAA experiments were
The best acid (most efficient) was then used to digest all the crustacean shells and
with the total percentage calcium (as calcium carbonate) also determined by
series of experiments was designed using lobster shells and different acids over
varying digestion times and the results compared. The best acid was expected to
55
be associated with the largest weight loss and percentage weight loss associated
chosen on the basis that they were most readily available and because their texture
was intermediate between the prawn shells (soft) and the crab shells (hard,
coarse).
Catherine, Jamaica, were cleaned, oven dried, crushed, redried and weighed.
Accurately weighed samples of the shells were treated with aliquots of the
hydrochloric acid, nitric acid, trichloroacetic acid, acetic acid and sulphuric acid
(complex), was collected by filtration after the digestion period was complete,
washed with water until neutral (as indicated by filter paper), air-dried, weighed
Table 2.1
Weight loss
/%
Time /h
1 6 48
Acids (2 M)
HCl 52 56 57
HNO3 54 57 56
CCl3COOH 53 55 60
CH3COOH 42 42 50
HNO3 and CCl3COOH, did not differ significantly. They were 52%, 54% and
53% respectively, all higher than the 42% obtained after using CH3COOH.
When the digestion time was increased to 6 hour the weight loss
percentages increased with the use of HCl, HNO3 and CCl3COOH; the values
were 56%, 57% and 55%, respectively. The weight loss percentage obtained from
loss percentages over those obtained for 6 hour period. There was a 1% increase
CH3COOH. However, a decrease in weight loss percentage was obtained with the
HNO3 digestion where, the percentage changed from 57% (6 hour) to 56% (48
sparingly soluble in water. The weight loss percentages obtained were too small
for such a strong acid. Overall, HCl, HNO3 and CCl3COOH all appeared suitable
amount of calcium salts that had gone into solution, which may be interpreted as
found in crustacean shells) 2. However, there was the possibility of the digestion
conclusively the percentage calcium carbonate hence the efficiency of the calcium
(b) INAA
INAA was used to confirm the best conditions required for digestion as
each of the chitin-protein residues obtained by digestion with the different acids
outlined in 2.3.1 a, was weighed out in polythene vials. INAA was used to
determined in the same manner. The results obtained from the INAA experiments
Digestion of lobster shells with 2 M acids over a 1 hour period left behind
a residue containing 14, 9, 8 and 25% calcium (as calcium carbonate) with use of
in the residue after applying HCl. In addition, 2, 3 and 16% calcium (as
carbonate) were left undigested when the acids HNO3, CCl3COOH, and
most effective acid for the digestion of lobster shells. By using a digestion time of
59
48 hour and keeping the reaction medium between 0 and 4 °C, a complex with
the value calculated from the manufacturer thus indicating the accuracy of the
results.
FIGURE 2.3
30
25
25
19
CALCIUM CARBONATE
20
IN RESIDUE / %
16
1h
14 6h
15
48 h
9
8
10 7
5
3.9
3
5 2
1
0
hydrochloric nitric trichloroacetic acetic
ACIDS
LOSS
The results obtained in the optimisation study indicated that digestion was
optimum when 2M HCl was used for 48 h. Consequently, it became the acid of
choice for digestion of calcium from lobster, land crab blue crab and prawn shells.
Lobster shells from the same batch used in the optimisation study, were
treated with 2M HCl for 48 hour in ice bath maintained at 0– 4 °C. Land crab
shells (obtained from Port Henderson beach, St. Catherine, Jamaica); blue crab
shells (obtained from Port Royal, Kingston, Jamaica) and prawn shells (obtained
from prawn bred by Best Dressed Chicken, Barton Isle, St. Elizabeth), were oven
dried, crushed and weighed. Samples of each of the shells were accurately
weighed and treated with 2M HCl for 48 h, the temperature of the reaction
filtered from the solution, washed with wate, dried weighed and the weight loss
percentages calculated. Tables 2.2 - 2.5 show the weight loss percentages
obtained from the lobster, land crab, blue crab and prawn shells.
61
Table 2.2
Average 0.45 57
62
Table 2.3
Average 0.5134 49
63
Table 2.4
Average 0.4599 53
Table 2.5
Average 0.5320 47
64
Effervescence associated with the production of carbon dioxide
accompanied the digestion of the shells. The reaction involving the lobster shells
was the most vigorous followed by the prawn shells. The land crab shells were the
least active. The results show weight loss averaging 57, 49, 53, and 47% for
lobster, land crab, blue crab, and prawn, respectively (Tables 2.2, 2.3, 2.4
and 2.5).
Therefore, assuming that all except 1% of the calcium salts was dissolved
expected to have the most calcium present as calcium carbonate, followed by the
blue crab, land crab and prawn. The validity of the assumption was explored by
The weight loss percentages obtained in section 2.3.3 may not have been
equal to the total percentage of calcium salts that were present in the crustacean
shells. The aim of this investigation was to determine firstly the total percentage
calcium (as carbonate) that were present in the shells, by the use of INAA (a) and
secondly the percentage calcium (as carbonate) that remained in the chitin protein
residue after digestion with 2M HCl (b). Thus, the effectiveness of the acid
digestion process in the production of chitin from the lobster, land crab, blue crab
land crab, blue crab and prawn shells was determined by INAA. Samples of dried
and ground shells were weighed out in polythene vials and irradiated in the
nuclear reactor the gamma radiation emitted counted and the percent calcium
Table 2.6
Lobster 16.7 42
Gypsum 21.9 55
(experimental)
Gypsum 21.7 54
(manufacturer's)
Empty ND ND
ND = Calcium not detected
The land crab shells contained the most calcium (as carbonate) (70%)
followed by blue crab shells (65%), lobster shells (42%) and prawn shells (37%).
66
A second batch of prawn shells obtained and irradiated had 47% calcium (as
carbonate) (Table 2.6). This difference in percentages between the two different
batches of prawn may have been due to their differing ages, as the calcium
carbonate content of the shell may vary with the stage of crustacean development.
the four crustacean species investigated varied significantly from the calcium
calcium carbonate determined in the shells (by INAA) and the average weight
Table 2.7
Lobster 57 42
Land crab 49 70
Blue crab 53 65
Prawn 47 37
For lobster and prawn shells, weight loss percentages were higher than the
However, the weight loss percentages of the shells obtained for the two species of
crab were less than the percentage of calcium (as carbonate) determined by INAA
67
(Table 2.7). The lower percentage for the lobster and prawn (by INAA) compared
with weight loss suggested that all the calcium present as calcium carbonate was
dissolved from these shells, along with a small amount of the other main portion
of the shell 1, the organic portion (hydrolysis). The higher percentages for the
crabs (by INAA) compared with the weight loss percentage, indicated that the
calcium salts present as calcium carbonate were not totally digested from the
shells. There was also the possibility of hydrolysis of the organic polymers in the
effectiveness of 2M HCl digestion of all the crustacean shells studied. Thus, the
the calcium salts from the land crab and blue crab shells. On the contrary, more
material than the calcium carbonate present in the lobster and prawn shells
incomplete calcium carbonate digestion in the lobster and prawn shells. The
the percentage calcium as calcium carbonate that remained in all the chitin-
protein residues after digestion of the crustacean shells with HCl (2M).
68
A portion of chitin-protein residues produced (Section 2.3.3) was analysed
Table 2.8.
Table 2.8
lobster 3.5 9 79
prawn 0.59 2 96
gypsum 21.5 54 -
(manufacturer's)
empty vial ND ND -
ND = Calcium not detected
carbonate. The samples obtained from lobster and prawn shells had low levels of
The residues obtained from digestion of land crab and blue crab shells
percentages obtained were 62% (for land crab) and 55% (for blue crab)
calcium carbonate was not being totally removed from the shells after digestion
with 2M HCl for 48 h. Calcium was not detected (ND) in the empty vial.
calcium carbonate in the shells and the percent calcium as calcium carbonate
present in the chitin-protein residues (by INAA) were made. In the lobster, shells
the 57% weight loss compared with total 42% calcium carbonate in shells and 9%
calcium carbonate in chitin protein residue supported the suspicion that the
organic portion of the shell was hydrolysed during acid digestion. The 47%
weight loss, 47% calcium carbonate in shells and 2% calcium carbonate in chitin-
protein residue suggested that there was almost complete digestion of calcium
from the prawn shells. Land crab and blue crab shells showed 49% and 53%
weight loss respectively, compared with 70% and 65% total calcium carbonate in
the shells. This showed that, particularly in the land crab, the calcium was not
being effectively removed by acid digestion as the residue still contained 62% and
after 2M HCl digestion during the optimisation studies, was 1%. When this was
shaking the reaction vessel during the 48 hour digestion period and the residues
thoroughly washing in water before drying and weighing. The samples were then
weight loss percentages were also determined. These new results obtained by
digestion made for each type of crustacean shell is shown in Figure 2.4. The
weight losses obtained after digestion with HCl (2M) are shown in Table 2.10.
Table 2.9
lobster <1
land crab 52
blue crab 43
prawn <1
Figure 2.4
100
CALCIUM CARBONATE / %
90
80 70
65
70 62
55
60 54
47
42 43
50
40
30
20 9
2 1
10 1
0
lobster land crab blue crab prawn
Source
before digestion after digestion after digestion (repeat)
Table 2.10
The percentage calcium (as carbonate) that remained after digestion was
72
less than 1% for lobster shells which was consistent with the results of the
optimisation study. The chitin-protein residue obtained from digesting the prawn
shells also contained less than 1% calcium (as carbonate) (Table 2.9). Thus, there
shells. Improvement in the level of calcium carbonate digestion was also evident
for the crab shells. The percentages went from 62 to 52% in the land crab and
from 55 to 43% in the blue crab shells (Figure 2.4). In the case of the lobster and
digestion. In the case of the crab species the shells appeared to be very resistant to
The weight loss results in Table 2.10 were in agreement with what was
previously observed. That is, hydrolysis of organic polymers occurred during acid
digestion. These effect was more pronounced for the blue crab shells. The shells
contained 65% calcium (as carbonate). After acid digestion the residue contained
43% calcium (as carbonate), yet weight loss percentage averaged 64% (Table
2.10).
the new average weight loss percentages were made. The weight loss percentages
removed, as expected. They went from 47% to 58% (2% to < 1% CaCO3) with
the prawn shells; 49% to 58% (62% to 54% CaCO3) with the land crabs and from
53% to 64% (55% to 43% CaCO3) in the blue crabs. The weight loss for the
lobster shells remained constant at 57% although the residual calcium carbonate
73
was brought to less than 1% by weight from 9%. Improving the efficiency of the
digestion of calcium carbonate may to some extent affect the quantity of chitin
AAS is an alternative technique to INAA that was used in this work for
analysing shells for their calcium content. INAA is the better technique since
AAS requires dissolution of the sample whereas INAA is a direct solid sample
Wollaston 17 discovered black lines in the spectrum of the sun, which were later
investigating the spectra of alkali and alkaline earth metals demonstrated that the
typical yellow line emitted by sodium salts in a flame is identical to the black line
energy from an external source of radiation, it can attain an excited state in which
electrons surrounding the atom occupy higher energy levels than usual. This is an
unstable state and the atom quickly and spontaneously returns to its ground state
as the electrons return to their original orbital position. The exact amount of
energy that was absorbed during the excitation process is emitted during this
18
decay process. The amount of the analyte element present is determined by
12
measuring a parameter called Absorbance which is related to the reduction in
the intensity of the beam of radiation passing through the gaseous sample
75
(Equation 2.7) 20.
Where,
A = Absorbance;
A = abc……………………………………………Equation 2.8
Where,
measured and the absorbance data are plotted against concentration. Ideally, this
should be a straight line as indicated by Beer’s law 21, and this is usually observed
76
at lower concentrations and absorbances. As concentration and absorbance
increase however non ideal behavior in the absorption process causes deviation
from linearity. The absorbance of the sample is measured and the concentration of
the analyte determined from the calibration curve. Modern atomic absorption
instruments have the ability to perform automatic curve correction, calibrate, and
curves 22.
electrically powered graphite furnace. Air - acetylene is the preferred flame for
for example, the hollow cathode lamp which are manufactured for individual
elements. Radiation passes from the source through the atomised sample to a
radiation falling on it. Comparison with known standards and the use of Beers
Law 21 enables the concentration of the analyte in the sample to be determined 24.
beam spectrophotometer the light from its source is divided into a sample beam,
which is focused through the sample cell, and a reference beam, this is directed
77
around the sample cell. The actual readings obtained represent a ratio of the
sample and reference beams. The result is that fluctuations in source intensity are
not reflected in the read out obtained. No lamp warm up period is required in
2.5.1 INTRODUCTION
present in both shells and chitin-protein residues and was conclusive. AAS is a
method which is cheaper, more readily available and was used to find the
after acid digestion of the crustacean shells. Both the results of AAS experiments
and INAA experiments were expected to compliment each other in that the
where,
weight loss on acid digestion. To determine the percentage of the shell that was
79
not calcium salt (organic polymers) that had dissolved, Equation 2.10 was used.
where
DETERMINATION BY AAS
Fresh samples of lobster and land crab shells were dried, weighed and
digested for 48 hour with 2M HCl. The digestion product obtained was filtered
and the residue washed with water, dried and collected for INAA to determine the
percentage calcium as calcium carbonate. The washings that were combined with
the filtrate were also collected and made up to 250 mL with distilled water.
Diluted portions of these solutions were analysed by AAS and the percentage
TABLE 2.11
residue (g), CaF = Calcium Carbonate in filtrate by AAS (%), WS = Weight of shells (g), TCa =
Total percent calcium carbonate in shell calculated (%), CaS = Calcium carbonate in shell by
INAA (%)
For the lobster shell sample, the calculation showed that the total
(Equation 2.9). This was equal to the total calcium carbonate in the shells (CaS).
The AAS determined percentage (CaF) was also equal to the latter, which
suggested that all the calcium carbonate was digested Table 2.11. In addition, for
the lobster sample 59% weight loss occurred to produce the chitin-protein residue.
With 42% calcium as calcium carbonate in the solution then, organic polymers
In the land crab shell sample where calcium carbonate in residue (CaR)
was 40%, TCa was 72% (Equation 2.9). This was close to CaS Table 2.11. The
two differed by 2%. Digestion of the land crab shells resulted in 64% weight loss.
The percentage organic polymers digested, 17% for the lobster shells and
7% for the land crab shells, confirmed that crab shells are more resistant to acid
Overall, it was shown that AAS was able to determine conclusively the
percentage calcium as calcium carbonate present in the shells of the more easily
digested crustacean shells for example, the lobster shells. This method however
was not sufficient for the harder, more acid resistant shells like the crab shells.
AAS is however suitable for routine check analysis on the samples as digestion
proceeds.
82
2.6.1 INTRODUCTION
first step to obtaining the percentage chitin was to boil the chitin–protein residue
with sodium hydroxide and then weigh the unhydrolysed product (UHP)
(Equation 2.11).
Where,
the weight of UHP (WUHP) and the percentage calcium carbonate impurities
Where,
CaUHP = Calcium carbonate impurities in chitin (%);
Ws = Weight of shells.
amino acids and proteins that were present in the hydrolysed product. This
ninhydrin test and electrophoresis 26. GC-MS along with a total elemental analysis
The chitin-protein residues obtained after acid digestion were boiled with
1M NaOH for 48 hours. The unhydrolysed product (UHP) obtained was filtered
washed repeatedly with water until neutral, dried and then weighed. The
percentage UHP was calculated with Equation 2.13 and the results obtained after
Where,
WS = Weight of shells.
84
Table 2.12
Lobster shell samples had overall 21% unhydrolysed product after alkaline
hydrolysis. The land crab and prawn shell samples had on average 35%,
unhydrolysed product whilst the blue crab had 36%, after hydrolysis. These
results on their own suggested that the lobster shells would contain the least
amount of chitin, and the other three samples would contain about the same as
each other. This however did not take into account impurities in the UHP, which
IN UNHYDROLYSED PRODUCT
In the land crab and blue crab shells, a large amount of calcium carbonate
was present after acid digestion and was expected to be present after the alkaline
INAA was used to determine the percent calcium (as carbonate) present in
the UHP. A sample of practical grade crab chitin obtained from Sigma Co. was
also irradiated for comparison. The percentages are shown in Table 2.13.
Table 2.13
Land Crab 20 49
Blue Crab 19 49
26
In addition, GC-MS, the ninhydrin test and electrophoresis were then
used to determine the presence of, and the type of amino acids and proteins that
protein residues was filtered made more alkaline and extracted with a mixture of
dichloromethane. A diethyl ether extraction was also carried out after acidifying a
86
fresh portion of the solution. Extractions were done to obtain samples for GC-MS
the polypeptides and amino acids present. Another portion of the sample was
UHP obtained from the prawn and lobster shells had less than 1% calcium
(as calcium carbonate) (Table 2.13). These were white compared to the brown
UHP obtained from the land crab and blue crab shells was calcium carbonate
(CaUHP). The sample of practical grade crab chitin obtained from Sigma Co, when
Photograph 2.1
calcium (as calcium carbonate) present after acid digestion was very small (less
than 1%). For the land crab and blue crab samples, higher if not the same
the percentages were calculated with respect to the weight of the unhydrolysed
calcium carbonate in the land crab was 54% in the chitin-protein residue and 49%
in the UHP. In the blue crab it was 43% in the chitin-protein residue compared to
49% in the UHP, a reasonable change. A small increase in the percentage residual
calcium carbonate may be due to the small amount of protein that was present in
The characteristic blue bands a positive sign for the presence of polypeptides and
amino acids were absent. The GC-MS indicated very few amino acids, for
The exoskeleton is composed of chitin, calcium and other metals and non-
metals, proteins and other organic substances. Their final percentages are stated
89
below. The percentage chitin was determined using Equation 2.12. The
percentages of metals and nonmetals were determined by INAA and the organic
The isolation of chitin involved two clear steps. These were digestion of
obtained. The percentage of UHp of all the crustacean shells were determined
based on the weight of the shells used. These were 21 and 35% in the lobster and
prawn shells. With less than 1% calcium as calcium carbonate present in these
UHP, it was concluded that the percentage chitin present in the lobster and prawn
percentage calcium carbonate in the UHP (Table 2.12 and Table 2.13), and
applying Equation 2.12. Therefore, for the land crab and blue crab shells,
The other elements apart from calcium that were present in the crustacean
The shells were found to contain small quantities of metals e.g. Na, K,
Tables 2.14
/mg/kg
the animals’ diets or habitats. For example, the land crab, which is not a marine
dweller, contained less of the halogens than the other species. The presence of
these elements coupled with the organic materials, make crustacean shells a
during acid and base hydrolysis and will remain as contaminants in chitin.
91
residues were divided into two portions, one of which was made more alkaline
and the other acidic. The alkaline solution was extracted with dichloromethane
and the acidic solution with methylene chloride. The solvents containing the
aromatic as well as aliphatic amines, high molecular weight carboxylic acids and
alkanes.
92
BY COMPLEXATION
affect the isolation efficiency of chitin. Under these conditions chitin may be
27
deacetylated to chitosan or hydrolysed into its N-acetyl monomeric units .
shells 28. Any weight loss obtained from using complexing agents is expected to
be the result of removal of calcium without any effect on the organic polymers.
Thus the effectiveness of the complexation method was compared with the acid
WITH EDTA
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
TABLE 2.15
15 23
60 40
180 48
The results in the table showed that the weight loss percentage increased
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
18-crown-6 ether was the second complexing agent used in the removal of
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
Table 2.16
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
effectiveness will depend on the surface area of the shells being analysed, a higher
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
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
and the product collected by filtration, dried, weighed, the percentage chitin
calculated and the IR spectra recorded (Chapter 3). The resulting percentages are
Table 2.17
wings 15 24
legs 17 28
as seen for the crustacean shells. This was perhaps due to the small amount or
that the wings and legs contained 24 and 28% chitin respectively.
2.9 SUMMARY
Jamaican marine spiny lobster (Panulirus argus), the land crab (Gecarcinus
ruricola), the blue crab (Callinectes sapidus), and the giant Malaysian fresh water
calcium carbonate and 38% proteins and other types of materials (organic and
Figure 2.5
chitin
21% c hitin
calcium c alc ium
35%
carbonate c arbonate
42% 47%
protein protein
and other and other
materials materials
37% 18%
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.
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
removing calcium ions was also briefly visited. On the basis of weight loss it was
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
2. Reference 1, p 92.
5, Reference 4, p 411.
8. Reference 3, p 4.
9. Reference 4, p 413.
CHAPTER THREE
CHARACTERISATION OF CHITIN
103
3.1 INTRODUCTION
Four techniques were used to characterise the isolated chitin. These were
Resonance Spectroscopy (13C NMR) and Infrared Spectroscopy (IR). IR was also
chitin polymer allowed for comparison of spectral results with those of glucose
observed in its original state and the result is not open to prejudice after a portion
and 1655 cm -1, due to hydroxide and amide 1 groups respectively, were used in
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
Calorimetry (DSC).
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
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
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)
the application of heat and these are due to endothermic and exothermic reactions.
reactions. On the curve of heat flow versus temperature, the modern convention is
The sample and reference are placed in sample holders of a furnace that is
furnace and around the samples nitrogen or sometimes oxygen is used 5. The
detect the temperature of the sample and reference holders. Electricity generated
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 –
Figure 3.1
Figure 3.2
Figure 3.3
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
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
the samples was confirmed by the small endotherm recorded. Therefore, chitin is
7
Sir Charles W. Oatley and his students developed the modern SEM at
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
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 were taken to give an overall view of the sample and a detailed view
of a selected portion.
(Photograph 3.1).
110
Photograph 3.1
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
(Photograph 3.2).
Photograph 3.2
µM)
(HIGHER MAGNIFICATION SCALE BAR, 10µ
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
Photograph 3.3
Photograph 3.4 shows the detail of one of the white clumps. Present
from the wings of the Blaberus cockroach. Present were clumps of grey materials
Photograph 3.4
µM)
(HIGHER MAGNIFICATION, SCALE BAR = 10µ
Photograph 3.5
material. It seemed therefore that the typical chitin is riddled with various types of
impurities.
113
Photograph 3.6
µm)
(higher magnification, scale bar = 10µ
114
13
3.4 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
10
were compared with those of the carbons of glucose (7) (in D2O) and solid
biosynthetic chitin (called artificial chitin) (8) (cross polarisation magic angle
6 6
CH2OH 4 CH2OH
4 O O
H, OH HO
5 1 5 1
HO H, OH HO 2 OH
2
3 NH C O C H3 3
OH
(7)
(6)
CHITIN MONOMER GLUCOSE
6
HOH2C
NHCOCH3 O
4 5
O HO O
1
O HO 2
O 8
3 NHCOCH3
HOH2C 7
n
(8)
BIOSYNTHETIC (ARTIFICIAL CHITIN)
hydrochloric acid. The unreacted residue was removed by filtration and the filtrate
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
13
C DATA FOR HYDROLYSED CHITIN
Literature values
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
solid chitin, which suggested that the hydrolysis did not affect these group.
117
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
Samples of chitin were ground with KBr and compressed into discs. The
chitin – KBr discs were placed into a Perkin Elmer FTIR Spectrophotometer
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.
75
65
% Transmittance
55
45
35
25
3500 2500 1500 500
Wavenumber cm -1
119
Figure 3.5
75
70
65
% Transmittance
60
55
50
45
40
3550 3050 2550 2050 1550 1050 550
Wavenumber / cm -1
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
Figure 3.6
100
90
80
70
% Transmittance
60
50
40
30
20
10
0
3950 3450 2950 2450 1950 1450 950 450
Wavenumber / cm -1
Figure 3.7
121
100
% Transmittance 80
60
40
20
0
4400 3900 3400 2900 2400 1900 1400 900 400
-1
Wavenumber / cm
Figure 3.8
100
% Transmittance
80
60
40
20
0
3950 3450 2950 2450 1950 1450 950 450
Wavenumber / cm -1
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
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
–1
cockroach (Figure 3.6) showed the OH band at 3450 cm , 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
to some of the chitin and chitosan samples studied. Specifically, two equations
samples. These were proposed by Domzy and Roberts 14 and Baxter et. al 15.
123
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.
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
-1
and the hydroxyl band at 3450 cm as an internal standard to correct for film
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:
these peaks.
HPLC, pyrolysis, gas chromatography and thermal analysis are also used to
124
15
determine degree of N-acetylation . The IR spectroscopic method proposed by
Moore and Roberts had a number of advantages; it is relatively quick and does not
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
-1 -1 -1 -1
A 1550 cm ÷A 2878 cm and A 1655 cm ÷A 2867 cm , respectively. In both
These two ratios gave more accurate results at low % N-acetylation than the A1655
15
Miya et. al 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.
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
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:
The value obtained will determine the proportion of chitin to chitosan that is
chitin will behave in dilute acetic acid. The baselines used by Domzy and Roberts
14 15
and Baxter et. al are shown in Figure 3.9. The method of Domzy and
Roberts 14 required the use of Equation 3.1 and the baseline labeled (Σ
Σ) and the
15
method of Baxter et. al which required the use of Equation 3.2 the baselines
Ω). The absorbances at 1655 cm-1 and 3450 cm-1 were determined from
labeled (Ω
Figure 3.9
0.5
0.45
0.4
Absorbance
0.35
0.3
0.25
0.2
0.15
3550 3050 2550 2050 1550 1050 550
-1
Wave number / cm
Σ = the baselines involved in the method of Domzy and Roberts labeled ; Ω = 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.
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
acetylation was determined for the samples using the method of Domzy and
14 15
Roberts and by the method of Baxter et. al . The percentages obtained are
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
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
Table 3.2
PERCENTAGE N-ACETYLATION OF CHITIN AND CHITOSAN SAMPLES
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.
effect of increasing the volume of the acetylating agent acetic anhydride. The
The standard used in this experiment was crab chitosan obtained from the
3.7) which meant at least 15% N-Ac. When Equation 3.2 was applied 18% was
Photograph 3.7
CHITIN (LEFT) AND CHITOSAN (RIGHT) FROM SIGMA CO.
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
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
dried product was then retested for its solubility in 10% acetic acid.
Ac (13%, 18%) were soluble in 10% acetic acid and hence showed a successful
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.
10 T.E. Walker, R.E. London, T.W. Whaley, R. Barker and N.A. Matwiyoff,
J. Am. Chem. Soc, 1976, 98:19,5808.
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.
The uses for chitin are many and constitute a multimillion-dollar industry.
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
Description Price / $ Ja
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
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
public the samples would degrade by the time enough had been collected.
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
APPENDIX ONE
PREPARATION OF SHELLS
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
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.
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
Electrode Germanium gamma detector, which had a FWHM of 2.0 keV (at
detector dead time of greater than 5% while providing adequate detection limits
(GYP-C, Domtar, Quebec) was treated in the same manner as the samples. An
Tennessee).
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
(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).
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
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
INAA
Samples of the shells of the four crustacean species (0.25 g) were weighed
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
carbonate. The new percentages obtained are shown in Table 2.9 and the
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
into an air acetylene flame and the absorbance measured at wavelength 422.7 nm,
HYDROLYSIS
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
The process was repeated twice, after which the final residue was
thoroughly washed with water until free of base as indicated by universal litmus
(Table 2.12). In addition, the percentage residual calcium carbonate present in the
unhydrolysed product and the percentage residual calcium carbonate were then
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
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
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
GC Mass Spectrometry
The filtrate (2 mL) obtained from NaOH (1M) treated lobster and prawn
sulphate.
heated for 15 minutes at 60 ºC, allowed to cool and at the end of the process
preparation for analysis by a Hewlett Packard 6890 Gas Chromatograph and Mass
Weber MS Drug Library was used to determine the type of materials the samples
contained.
EDTA
Combined with ethylene diamine tetra-acetic acid disodium salt (EDTA) (3 g) and
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
CROWN-6 ETHER
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
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
(Chapter 3).
145
APPENDIX TWO
EXPERIMENTAL DETAILS
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
at the Electron Microscopy Unit, U.W.I. Mona. Chitin samples from lobster shells
beam and the image obtained from secondary electrons displayed on a screen
13
C NMR ANALYSIS OF CHITIN
was then filtered and the filtrate collected. D2O and 3(trimethylsilyl)-1-propane
13
sulphonic acid salt was then added to the solution and the C spectrum
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
The solution was then centrifuged to obtain a clear supernatant liquid. This
was treated dropwise with 40% aqueous sodium hydroxide solution where upon a
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-
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
repeating the alkaline hydrolysis process with NaOH (40%, 24.5 mL) to produce
RGf/1/102.
involve N-acetylation. All the chitosan samples were tested for their solubility in
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
solutions were 3, 13, 17 and 25 mL. The solutions were left for 1 h after which the
thoroughly with water, methanol and ether and then air-dried. The products were
Percent N-acetylation
Sigma Co. (RGf/1/113a), crab chitin from Sigma Co. (RGf/1/116a), lobster chitin
chitin (clob), lobster chitin (clob2c), lobster chitin (clob2c), prawn chitin (cpwn),
149
prawn chitin (cpwn2b), land crab chitin (clc), lobster crude chitosan (RGf/1/80),
(RGf/1/114a). The chitin samples not obtained from sigma were prepared by acid
Dried samples of the chitin and chitosan samples were blended with KBr
to form KBr discs. These were then placed into a Spectrum 1000 Perkin Elmer
absorbances of the functional groups present in the compounds. From the spectra,
2
the % N-acetylation were determined using the method of Domzy and Roberts
and Baxter et. al 1, the absorbances at 1655 and 3450 cm-1 and the baselines
labeled (Σ) and (Ω), shown in Figure 3.9 (crab chitosan sample, RGf/1/113a)
The method of Domzy and Roberts 2 and Baxter et. al 1 required the use of
the baseline labeled (Σ) and Equation 3.1, and baselines labeled (Ω) and