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
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.
Standard acid hydrolysis was not effective in removing all calcium compounds from the shells of some species of crustaceans. The percentage by weight of chitin obtained from these crustacean shells were found to be; Lobster 21%, land crab 18%, blue crab 19% and prawn 35%. Characterisation involved the use of Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC)), Scanning electron Microscopy (SEM), carbon-13 NMR Spectroscopy and Infrared analysis. TGA and DSC show that chitin is stable up to 394 °C. SEM showed by photographs the fibrous nature of chitin. Carbon-13 NMR analysis showed chemical shift values that compared well with literature values for glucose and IR analysis showed the characteristic hydroxide band (3450 cm –1) and amide absorption band (1655 cm –1) associated with chitin. Characterisation of chitin also involved determination of the percentage N-acetyl content (% N-Ac) by the use of two infrared analysis techniques where (% N-Ac = A1655/A3450×115) and (% N-Ac = A1655/A3450×100/1.33). A typical isolation process to produce chitin showed varying percent N-acetyl content, which is affected by the alkaline conditions of the hydrolysis step as well as the method of calculation. The conversion of chitin to chitosan was also a method of characterisation of chitin where chitosan was soluble in dilute acetic acid. Key words: chitin, crustacean shells Instrumental Neutron Activation Analysis, weight loss, and calcium carbonate.
ACKNOWLEDGEMENTS I wish to acknowledge my supervisor, Dr. Keith Pascoe for his guidance throughout the course of this project. Special thanks to my co-supervisor, Dr. R. Rattray for his encouragement, his unselfish help with the instrumental neutron activation analysis and atomic absorption spectroscopy, and in completing this project. Sincere thanks to the staff of The International Centre of Enviromental and Nuclear Sciences UWI, Mona, for allowing me access to the SlOWPOKE 2 nuclear reactor and atomic absorption spectrophotometer and who from time to time helped with information for this project; to Mr. Reid from the SEM unit for his help with the scanning electron microscopy Studies; Mr. Aiken of the Life Sciences Department UWI, Mona for identifying the crustaceans; Dr. Golden for the gel electrophoresis analysis; Mr. Andrew Lewis for initial help with the atomic absorption spectroscopy and Dr. Lancashire for some of the photographs. Thanks to Dr. Paul Reese who was always ready to listen and make suggestions for the various problems a graduate student faces. I am indebted to Professor Dasgupta and the Chemistry Department for the Departmental Award, the position as Tutorial Assistant and for the summer jobs over the years. I am thankful to all the kind staff members of the Chemistry Department Miss Simon, Mrs. Chambers and Dr. Maragh to name a few. To my group members Petrea, Fiona, Dionne, Susan and other past and present members of the research laboratories – thanks for the love.
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.
TABLE OF CONTENTS
Pages ABSTRACT ACKNOWLEDGEMENTS DEDICATION TABLE OF CONTENTS LIST OF COMPOUNDS ILLUSTRATED LIST OF SCHEMATIC DIAGRAMS LIST OF TABLES LIST OF FIGURES LIST OF PHOTOGRAPHS CHAPTER ONE 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 CHITIN Introduction History Structure and Bonding Biosynthesis Polymorphic forms of chitin Physical properties Sources The crustacean and exoskeleton Techniques for extraction of chitin Chitosan Derivatives and uses 2 3 4 7 10 12 14 16 20 24 31 38 i iii iv v ix ix ix xi xii
REFERENCES FOR CHAPTER ONE
ISOLATION OF CHITIN: COMPOSITION AND CHARACTERISTIC OF THE EXOSKELETON OF THE JAMAICAN
ARTHROPODS 2.1 2.2 Introduction History, principles and instrumentation for instrumental neutron activation analysis (INAA) Determination of percentage calcium in some Jamaican crustacean shells Introduction Digestion of lobster shells with different acids over varying times – optimising of digestion conditions by (a) weight loss percentages and (b) INAA Calcium carbonate content of crustacean shells with optimised acid digestion conditions – as determined by weight loss 44
Calcium carbonate content of (a) crustacean shells and (b)chitin-protein residue - as determined by INAA 64 History principles and instrumentation for atomic absorption spectroscopy (AAS) Calcium carbonate content - as determined by AAS Introduction Results and discussion of AAS calcium carbonate determination Chitin content of crustacean shells as determined by alkaline hydrolysis Introduction
Percent unhydrolysed product (UHP%) after alkaline hydrolysis Percent calcium carbonate impurities in unhydrolysed product Composition of the exoskeleton Removal of calcium from crustacean shell by complexation Removal of calcium from crustacean shell by complexation with EDTA Removal of calcium from crustacean shell by complexation with 18-Crown-6 ether 93 Chitin in cockroach Summary
96 100 100
REFERENCES FOR CHAPTER TWO CHAPTER THREE 3.1 3.2 3.3 3.4 3.5 CHARACTERISATION OF CHITIN Introduction Thermal analysis Scanning electron microscopy Carbon-13 NMR analysis of chitin monomer IR Spectral analysis – functional group analysis and % N-acetylation determination. 3.5.1 3.5.2 3.6 Functional group analysis Percentage N-acetylation (% N-Ac) Chitosan from chitin
103 104 109 114
117 117 122 131
REFERENCES FOR CHAPTER THREE CHITIN AND ECONOMICS APPENDIX ONE: EXPERIMENTAL DETAILS FOR CHAPTER TWO APPENDIX TWO: EXPERIMENTAL DETAILS FOR CHAPTER THREE
LIST OF COMPOUDS ILLUSTRATED
(1) Chitin (2) Cellulose (3) Hydrogen bonding in chitin (4) Chitosan (5) True chitin (6) Chitin monomer (7) glucose (8) Biosynthetic (artificial) chitin LIST OF SCHEMATIC DIAGRAMS Scheme 1.1 Chitin hydrolysis Scheme 1.2 Biosynthesis of chitin Scheme 1.3 Formation of chitosan polycation Scheme 1.4. Other derivatives of chitin LIST OF TABLES Table 2.1 Weight loss percentage on digestion of lobster shells with different acids over different digestion times Table 2.2 Preliminary weight loss results of digestion of lobster shells with 2M HCl Table 2.3 Preliminary weight loss results of digestion of land crab shells with 2M HCl Table 2.4 Preliminary weight loss results of digestion of blue crab shells with 2M HCl
2 4 4 5 5 114 114 114
6 9 25 36
Table 2.5 Preliminary weight loss results of digestion of prawn shells with 2M HCl Table 2.6 Results of analysis of crustacean shells for calcium by INAA Table 2.7 Comparison of percentage calcium (as calcium carbonate ) determined by INAA and average weight loss Table 2.8 Results of analysis of chitin-protein residue obtained from 2M HCl digested shells for calcium by INAA Table 2.9 New results of analysis of 2M HCl digested shells for calcium (as calcium carbonate ) determined by INAA Table 2.10 New weight loss percentages after 2M HCl digestion of crustacean shells 63 65
Table 2.11 Percentage calcium (as calcium carbonate) determined by AAS and INAA experiments Table 2.12 Alkaline hydrolysis of crustacean shells– percentage unhydrolysed product Table 2.13 Calcium carbonate content of unhydrolysed product Table 2.14 Elemental composition of shells Table 2.15 Percentage calcium carbonate over different time periods using EDTA solution at roomtemperature Table 2.16 Percentage weight loss by using 18 crown 6 – 1 ether Table 2.17 Acid and alkaline hydrolysis of a Blaberus cockroach Table 3.1 13 C data for hydrolysed chitin glucose and chitosan hydrochloride Table 3.2 Percentage N-acetylation of chitin and chitosan samples
84 85 90
93 94 96
LIST OF FIGURES Figure 1.1 Cross section of the exoskeleton of a crustacean Figure 2.1 Schematic diagram of sample flow from irradiation to counting stage Figure 2.2 A typical INAA spectrum Figure 2.3 INAA results after digestion of lobster shells with different acids over different times Figure 2.4 Percent calcium present in crustacean shells Figure 2.5 Percentage chitin calculated in (a) lobster and (b) prawn shells Figure 3.1 TGA curves of prawn (cpwn2a) and lobster(clob2a) chitin Figure 3.2 DSC curve of lobster chitin Figure 3.3 DSC curve of prawn chitin Figure 3.4 IR spectrum of unpurified crab chitin obtained from Sigma Co. Figure 3.5 IR spectrum of sample chitin from lobster shells Figure 3.6 IR spectrum of skin-like material obtained from the wing of an adult Blaberuscockroach after NaOH digestion Figure 3.7 IR spectrum of powdered material obtained from the leg of an adult Blaberus cockroach after NaOH digestion Figure 3.8 IR spectrum the wing ofan adult Blaberus Cockroach Figure 3.9 IR spectrum of unpurified crab chitosan obtained from Sigma Co. LIST OF PHOTOGRAPHS Photograph 1.1 The Jamaican Marine Spiny Lobster Photograph 2.1 Chitin and chitosan sample of prawn (left) and lobster (right) 15 17
98 106 106 109
120 121 121
Photograph 3.1 SEM of lobster chitin (scale bar, 1mm) Photograph 3.2 SEM of lobster chitin (higher magnification scale bar, 10µm) Photograph 3.3 SEM of chitin from Blaberus cockroach leg( scale bar = 1mm) Photograph 3.4 SEM of chitin from Blaberus cockroach leg (higher magnification, scale bar = 10µm) Photograph 3.5 SEM of chitin from Blaberus cockroach wings (scale bar = 1mm) Photograph 3.6 SEM of chitin from Blaberus cockroach wings (higher magnification, scale bar = 10µm) Photograph 3.7 Chitin (left) and Chitosan (Right) of Sigma Co (Chitosan: 85% deacetylated)
INTRODUCTION Chitin (1) is a sugar polymer, fibrous in nature and structurally similar to
cellulose. It is one of nature’s most common organic compounds second only to cellulose
1, 2, 3
. It has been known since the nineteenth century. Chitin is
commonly found in the exoskeleton of arthropods (particularly the crustaceans) or fungi and green algae that utilize nitrogen containing sugars 4 and its biosynthesis involves a series of enzymatic transformations from trehalose or glucose to the formation of UDP-N-acetylglucosamine 5. The proposed uses of chitin are very wide, from medical applications (example, wound healing)
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 NHCOCH3 O HO O O HOH2C HO
HOH2C NHCOCH3 HO O NHCOCH3 HOH2C O HO
O O NHCOCH3
Chitin was first described in 1811 by H. Braconnot 8, professor of natural history, director of the botanical garden and a member of the Academy of Sciences of Nancy, France. He isolated chitin from mushrooms by treatment with warm alkali. Twelve years later A. Odier 8 again found chitin in insect cuticle and some plant tissue. The silk worm was also discovered as a source of chitin when in 1843 J. L. Lassaigne 8 isolated it from the Bombyx mori. In the same year, A. Payen
initiated discussion about the differences between cellulose and chitin.
The monomeric unit of chitin (N-acetyl glucosamine) became known because of the work of G. Ledderhose 8 in 1878 and E. Gilson 8 in 1894. Rouget
discovered chitosan in 1859. He boiled chitin in potassium
hydroxide solution and found that the product chitosan dissolved in organic acid, and was violet in diluted solutions of iodine and acid. In contrast, chitin is stained brown in iodine-acid solution. Hoppe Seyler 9 coined the name chitosan in 1894 and in 1950, it was clearly described as a polymer of glucosamine 10. In the first half of the twentieth century, research on chitin was mostly directed toward the study of its occurrence in living organisms, its degradation by bacteria, its uses in resin technology and its chemistry 9.
STRUCTURE AND BONDING Chitin (poly-N-acetyl-D-glucosamine) (1) is a polysaccharide consisting
of beta (1-4) linkages. Therefore, it is sometimes referred to as beta (1-4)-2acetamido-2-deoxy-D-glucose. It is believed to be a derivative of natures most common polysaccharide, cellulose (2) (beta (1-4) D-glucose) 11.
HOH 2C OH O HO O O HOH 2C HO OH HOH2C O O OH HO O O
HOH 2C O O HO OH
Glucose is the precursor of both molecules; both formed via primary metabolism. The difference between chitin and cellulose occurs at position two where in cellulose the hydroxy group replaces the acetamide group 13. Both chitin and cellulose molecules are organised together in microfibrils consisting of hydrogen bonds (3) 5.
HOH2C NHCOCH3 O H-O O H-O O HOH2C hydrogen bond
HOH2C NHCOCH3 O H-O NHCOCH3 HOH2C O O H-O
O O NHCOCH3
(3) HYDROGEN BONDING IN CHITIN
Isolated chitin (true chitin) is not totally acetylated due to the partial formation of the derivative chitosan (beta (1-4)-2-amino-2-deoxy-D-glucose) (4) during isolation
and is best represented as structure (5). The result is that in a
few cases the carbon atom at position two will bear a NH2 group the acetamido group.
HOH2C NH2 O HO O O HOH2C HO NH2 HOH2C O NH2 O HO O O
HOH2C O O HO NH2
HOH2C NHCOCH3 O HO O O HOH2C HO
O O NH2
HOH2C NHCOCH3 HO O O HOH2C HO
O O NH2
(5) TRUE CHITIN
During isolation, chitin being a glucose polymer is also hydrolysed to its monomeric units consisting of N-acetyl glucosamine (scheme 1.1). This degradation is the result of the harsh conditions often associated with the isolation procedures 4. Scheme 1.1 Chitin Hydrolysis
NHCOCH3 O HO H, OH O HOH2C NHCOCH3 O HO O O HOH2C HO NHCOCH3 HOH2C O O
HO H C 2 O O HO NH C OC H3
BIOSYNTHESIS The biosynthesis of chitin represents the first case in which substantial
evidence was presented for the formation of a polysaccharide from a sugar molecule. The N-acetyl glucosamine monomer coupled with the appropriate enzyme is the main ingredient for chitin biosynthesis. Glaser and Brown
1957 investigated an enzyme from the fungi Neurospora crassa. This enzyme activated free N-acetyl glucosamine to produce chitin. In the laboratory chitin has been biosynthesised by using a distorted glucosyl substrate monomer (chitobiose oxazoline derivative), chitinase at pH 10.6 15. Chitin synthesases have also been identified in S. cerevisiac, (a species of yeast) which catalyses the transfer of N-acetyl-glucosamine from UDP-N-Acetyl glucosamine to a growing chain of beta (1-4)-linked-N-acetylglucosamine residues 16. A detailed process of chitin formation has been outlined by E. Cohen 5. Active catalytic units assembled in the cell membrane polymerise N- Acetyl glucosamine into extracellular chitin chains. The substrate for polymerisation 5uridine diphospho-N-acetyl-D-glucosamine (UDP-N-acetylglucosamine) is an end metabolite of a cascade of cytoplasmic biochemical transformation that starts from the disaccharide trehalose or from glucose. The membrane bound chitin sythesase (UDP-2-acetamido-2-deoxy-D-glucose: chitin 4-beta-actamidoglucosyl transferase) is the essential enzyme in the chitin formation. The pathway has also been outlined by Muzzarelli
. Biosynthesis is
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-6phosphate 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 byproduct (Scheme 1.2). Acetylation by acetylCoA in the presence of the enzyme glucosamine-6phosphate-N-acetyl transferase causes the formation of N-acetylglucosamine-6phosphate. 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.
Scheme 1.2 Biosynthesis of Chitin
CH2OH H H OH HO OH H O H H OH H HO OH HOH 2C O O H C H2 O P O H H OH HO CH2OH H H OH HO H glucose ATP hexokinase ADP HO O OH HO H OH UTP pyrophosphate CH2OH glucose phosphate isomerase
- 2O 3 2-
H trehalose (alpha-D-glucosido-alpha-D-glycoside ) trehalase
H O H HN C CH
N-acetyl glucosamine -6-phosphate phosphoacetylglucosamine mutase OH
OH CH2OH H H OH O H H O H HN C CH
PO 2 3
O3 POCH 2 H H
OH H glucose-6-phosphate
H CH2OH H OH HO H HO H
POCH2 H HO OH
H O C CH
fructose-6-phosphate glutamine glutamine-fructose-6-phosphate amino transferase C H2O P O H H OH HO H NH2 O H OH glutamic acid
chitin synthesase UDP CH2OH H H OH O H HN CHITIN O H H O C CH
POLYMORPHIC FORMS OF CHITIN Chitin forms a dimer chitobiose C16 H O 11 N 2
and chains classified
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. 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 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
(referred to as a
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.
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 Nacetylation 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-chloro2-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
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.
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)
, 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.
Photograph 1.1 THE JAMAICAN MARINE SPINY LOBSTER
THE CRUSTACEAN AND EXOSKELETON Crustaceans live in both aquatic and terrestrial environments
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 m 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
(Figure 1.1). The whole procuticle is strengthened by heavy
calcification within the chitin-protein matrix 35.
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
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
. 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 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 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 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
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.
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
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 m were decalcified more rapidly, in 15 minutes, under the same conditions.
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
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.
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
. 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.
Scheme 1.3 FORMATION OF CHITOSAN POLYCATION
HOH2C NH2 O HO O O HOH2C HO NH 2 HOH2C O O NH2 HO O O
HOH2C O O HO NH2
+ NH 3 O HO O O HOH2C
HOH2C O O HO NH + 3 HOH 2C HO
+ NH 3 O O
HOH 2C O O HO NH + 3
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
precipitate washed with water to neutrality. (b) METHOD RIGBY, WOLROM, MAHER
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.
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 byproducts, 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 5060 °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
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.
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.
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.
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.
In solid form, chitin, chitosan and derivatives have demonstrated sheetforming properties. For example, Takai and co-workers
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
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
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
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
. It is an expensive product and is in short
supply. Sulfated chitin has been investigated for its anticoagulant properties and
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
1 ROH2C O O O HO NHR2 ( R1 = CH2CO-ARG-GLY-ASP-SER-OH 81 CH3COOH or R2 = H, AC) n chitin sulphate O
HOH2C O O RO NHCOCH3 R = CO(CH2)mCH3 83 m - 2 = 8 ; n - 20 = 5000
H OH 2 C * O HO NC OC H3 chitin n O O
HOOCH2COH2C O O O HO n NHCOCH2CH2CO2M M = group 1 or 2 metals n - 10 = 5000 79 * O
ROH2C O O HO NHCOCH3 R = (CH2)nCOOH or H n > 1 85
Cosmetics containing chitosan carboxy derivatives have been prepared. The cosmetics showed excellent moisturising effect chitin have been prepared for
. Trimethylsilyl derivatives of 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
. Nail polish containing chitin alkyl ester has been prepared which
served as a film-forming agent and or resin component
. 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
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.
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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24.
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Croom Helm Ltd, London, 1988, p 89. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. Reference 38, p 83. Reference 8, p 9. Reference 24, p 94. Reference 24, p 92. Reference 8, p 10. Reference 34, p 648. Reference 8, p 90. Reference 11, p 97. Reference 33, p 98. Reference 8, p 91. Reference 11, p 100. Reference 8, p 92. S. Salmon and S. M. Hudson, Journal of Polymer Science, Part B, Polymer Physics, 1995, 33, 1007. K. Chang, G. Tsai, J. Lee, W. Fu, Carbohydr. Res., 1997, 303, 327. Y. Chung Wei and S. Hudson, Macromolecules., 1993, 23, 4151. B. Smith, T. Koonce and S. Hudson, Polymer and Textile Chemistry, N.C.S.U., Raleigh, N.C., American Dyestuff Reporter, 1993, 22. Reference 11, p 145. Reference 8, p 96. Reference 8, p 97. Reference 11, p 147. Reference 8, p 98. W. J. McGahren, G. A. Perkinson, J.A. Growich, R.A. Leese, G.A. Ellestad, ‘Chitosan by Fermentation,’ Process Research and Development
Section of Medical Research, A division of the American Cyanamid Company, N. Y., 1983 (report). 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. D. Horton and D. R. Lineback, Meth. Carbohydr. Chem., 1995, 5, 405. V. E. Tikhonov, L. A. Radigina and Y. A. Yamskov, Carbohydr. Res., 1996, 290, 33. Reference 8, p 214. Reference 8, p 248. Reference 8, p 249. Reference 8, p 183. Reference 8, p 263. Reference 8, p 231. Reference 8, p 233. Reference 8, p 235. Reference 8, p 236. Reference 8, p 238. Reference 8, p 244. Reference 8, p 246. W. Schlotzhauer, O. Chortyk, P. Austin, J. Agric. Food Chem., 1976, 24 (1), 177. Reference 8, p 247. Reference 8, p 259. Reference 8, p 260. M. Kawakami, Jpn. Kokai Tokkyo Koho, JP06, 24, 934, 1994, CA 121: 65303n R.E. Harmon, K.K. De and S.K. Gupta, Carbohyd. Res.,1973, 31, 408. N. Nishikawa, Jpn Kokai Tokkyo Koho, JP05, 271, 094, 1995, CA 122, 32015n
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ISOLATION OF CHITIN: COMPOSITION AND CHARACTERISTICS OF THE EXOSKELETON OF SOME JAMAICAN ARTHROPODS
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.
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
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 and detection limits in the range 10 -3 to 10 g
. 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.
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,
Ca + 1n = 49Ca + γ…………………………………………Equation 2.1
If the radioactive isotope (e.g. Ca
) 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
allows standards to be done once for a batch of samples. Samples and standards
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.
Figure 2.1 SCHEMATIC DIAGRAM OF SAMPLE FLOW
FROM IRRADIATION TO COUNTING STAGE
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
11 A = N ϕ σ (1 – e-λti) e-λtd ………………………………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
element) in barns (1 barn = 10-24 cm2); λ = Radioactive decay constant; ti = irradiation time; td = decay time (time from end of irradiation to start of count); Because the SLOWPOKE-2 reactor used for INAA at ICENS has exceptional neutron flux stability. If the same irradiation times are used for both standard and sample, it follows that Asam / Astd = Nsam / Nstd ×e-λtdsam/ e-λtdstd…………Equation 2.3 Where, Asam = activity induced in sample; Astd = activity induced in standard; Nsam = number of atoms of element in sample; Nstd = number of atoms of element in sample; However, the ratio of the concentration in the sample Csam to that in the standard Cstd is Csam / Cstd = Nsam / Nstd x Wstd / Wsam………………Equation 2.4
Where, Wsam = weight of sample; Wstd = weight of standard; Therefore, the concentration in the sample is Csam = Cstd x (Asam / Astd) x (Wstd/Wsam) x (e-λtdstd / e-λtd sam) ………………………………………………………………………Equation 2.5 Experimentally, count rate (which is directly proportional to activity) is usually measured instead of activity. A high count rate of decay is desirable to minimise the duration of the counting period. However, high count rates can cause ‘pulse pile up’ in the detector as the electronics can only process a certain number of gamma rays per second. If counting rates exceed the resolving time of the detector, a correction must be made to account for the difference between elapsed time (clock) and live (available counting) time 13. Good reproducibility is essential for all analytical techniques. Imprecise results are not always due to the method but are often due to inhomogenous distribution of the element of interest in the matrix being analysed. INAA results are not usually affected by matrix effects. Because of this, it is often applied as an independent check on a new analytical method to make sure no systematic error is affecting the technique 14.
Accuracy of the INAA technique is excellent, depending mainly on the accuracy of the standards being used for comparison with the sample. The principal errors that arise during INAA analysis are due to self shielding, unequal neutron flux for sample and standard, counting uncertainties and errors in counting due to scattering, absorption and differences in counting or irradiation geometry between sample and standard. The errors from these causes usually can be reduced to less than 10% by acknowledging routine quality control methods. Uncertainties in the range of 1 - 3% are frequently obtained. Only a few milligrams of the samples are required and as little as 10 -5 µg of several elements can be detected. The sensitivity of the method is limited by the sensitivity of the detector, the decrease in activity at the time of counting, the time available for counting and the magnitude of the background count rate relative to the count rate of the analyte. Many authors overestimate the sensitivity of a favoured technique, but sensitivity of a method can be dependent on the sample matrix 15. INAA has been used routinely to measure trace element concentrations in complex matrices such as human and animal tissue, coal, fly ash, petroleum, river sediments, urine, faeces, blood etc. More than 25 elements can be analysed at the same time 10. Analysis can be performed without destroying the sample 9, 10 and it is therefore popular in forensic science. Other techniques used in elemental analysis include atomic emission, absorption or fluorescence spectrometry and mass spectroscopy. No single
technique presents a general answer to the large variety of problems involved in elemental analysis .
DETERMINATION OF PERCENTAGE CALCIUM IN SOME JAMAICAN CRUSTACEAN SHELLS
INTRODUCTION To determine the acid best suited for the digestion process, lobster shells
were digested with five different acids over varying times and the loss in weight calculated. In addition, INAA was applied to the digested lobster shells for the determination of percentage by weight of residual calcium present, expressed as calcium carbonate. The results from the weight loss and INAA experiments were compared, which allowed the efficiency of the acid digestion to be determined. The best acid (most efficient) was then used to digest all the crustacean shells and the percentage residual calcium (as calcium carbonate) determined by comparison with the total percentage calcium (as calcium carbonate) also determined by INAA. Experimental details are given in Appendix one. 2.3.2 DIGESTION OF LOBSTER SHELLS WITH DIFFERENT ACIDS OVER VARYING
OPTIMISING OF DIGESTION CONDITIONS BY
PERCENTAGES AND (b) INAA
Weight loss In an effort to assess the efficiency of calcium removal by acid digestion a
series of experiments was designed using lobster shells and different acids over varying digestion times and the results compared. The best acid was expected to
be associated with the largest weight loss and percentage weight loss associated with percentage calcium (present as calcium carbonate). Lobster shells were chosen on the basis that they were most readily available and because their texture was intermediate between the prawn shells (soft) and the crab shells (hard, coarse). Lobster shells obtained from fishermen at Port Henderson beach, St. Catherine, Jamaica, were cleaned, oven dried, crushed, redried and weighed. Accurately weighed samples of the shells were treated with aliquots of the hydrochloric acid, nitric acid, trichloroacetic acid, acetic acid and sulphuric acid (all 2 M) in round bottom flasks contained in ice baths of temperatures between 0 – 4 °C. Digestion times used were 1, 6 and 48 h. The undigested portion of the shell, called the chitin-protein residue (complex), was collected by filtration after the digestion period was complete, washed with water until neutral (as indicated by filter paper), air-dried, weighed and the percentage weight loss determined (Table 2.1).
Table 2.1 PERCENTAGE WEIGHT LOSS ON DIGESTION OF
LOBSTER SHELLS WITH DIFFERENT ACIDS OVER DIFFERENT DIGESTION TIMES
Weight loss /% Time /h 1
Acids (2 M)
HCl HNO3 CCl3COOH CH3COOH
52 54 53 42
56 57 55 42
57 56 60 50
Progress of the digestion was evident by the frothing of the solution associated with the production of carbon dioxide. In a digestion time of 1 hour weight loss percentages obtained for HCl, HNO3 and CCl3COOH, did not differ significantly. They were 52%, 54% and 53% respectively, all higher than the 42% obtained after using CH3COOH. When the digestion time was increased to 6 hour the weight loss percentages increased with the use of HCl, HNO3 and CCl3COOH; the values were 56%, 57% and 55%, respectively. The weight loss percentage obtained from using CH3COOH remained unchanged. (Table 2.1).
Increasing the digestion time to 48 hours generally increased the weight loss percentages over those obtained for 6 hour period. There was a 1% increase with HCl digestion, a 4% increase with CCl3COOH and an 8% increase with CH3COOH. However, a decrease in weight loss percentage was obtained with the HNO3 digestion where, the percentage changed from 57% (6 hour) to 56% (48 hour) (Table 2.1). Weight loss percentages obtained for sulphuric acid could not be determined conclusively because of the formation of calcium sulphate, which is sparingly soluble in water. The weight loss percentages obtained were too small for such a strong acid. Overall, HCl, HNO3 and CCl3COOH all appeared suitable for digestion of shells over the 1, 6, or 48 hour periods. The weight loss percentages obtained were expected to be related to the amount of calcium salts that had gone into solution, which may be interpreted as percentage calcium carbonate (calcium carbonate is the main calcium compound found in crustacean shells) 2. However, there was the possibility of the digestion of organic polymers that form a significant part of the shells. To determine conclusively the percentage calcium carbonate hence the efficiency of the calcium carbonate digestion process, INAA was used.
INAA INAA was used to confirm the best conditions required for digestion as
well as address the matter of efficiency of digestion. Approximately 0.25 g of each of the chitin-protein residues obtained by digestion with the different acids outlined in 2.3.1 a, was weighed out in polythene vials. INAA was used to determine the percentage calcium using the OMNIGAM Neutron Activation Analysis software package (EG&G Ortec, Oakridge Tennesse). To assess analytical accuracy, the concentration of calcium in reference materials was also determined in the same manner. The results obtained from the INAA experiments are shown in Figure 2.3. Digestion of lobster shells with 2 M acids over a 1 hour period left behind a residue containing 14, 9, 8 and 25% calcium (as calcium carbonate) with use of HCl, HNO3, CCl3COOH and CH3COOH respectively (Figure 2.3). In the 6 hour digestion period HCl, HNO3, CCl3COOH and CH3COOH were ineffective in removing 7, 3.9, 5 and 19% respectively calcium (as carbonate) from the lobster shell samples. With a 48 hour digestion period, only 1% calcium (as carbonate) remained in the residue after applying HCl. In addition, 2, 3 and 16% calcium (as carbonate) were left undigested when the acids HNO3, CCl3COOH, and CH3COOH, respectively, were used (Figure 2.3). Based on the findings of the optimisation experiments, 2M HCl was the most effective acid for the digestion of lobster shells. By using a digestion time of
48 hour and keeping the reaction medium between 0 and 4 °C, a complex with 1% residual calcium carbonate was produced.The gypsum reference material studied had percentage calcium expressed as carbonate 55% compared to 54 % the value calculated from the manufacturer thus indicating the accuracy of the results. FIGURE 2.3 INAA RESULTS AFTER DIGESTION OF
LOBSTER SHELLS WITH DIFFERENT ACIDS OVER DIFFERENT TIMES
CALCIUM CARBONATE IN RESIDUE / %
20 15 10 5 0
hydrochloric nitric trichloroacetic ACIDS acetic
1h 6h 48 h
9 7 3.9 1 2
8 5 3
Gypsum reference material: calcium expressed as calcium carbonate (manufacturer's value) calcium expressed as calcium carbonate experimental value 54%, 55%
OPTIMISED ACID DIGESTION CONDITIONS LOSS
AS DETERMINED BY WEIGHT
The results obtained in the optimisation study indicated that digestion was optimum when 2M HCl was used for 48 h. Consequently, it became the acid of choice for digestion of calcium from lobster, land crab blue crab and prawn shells. Lobster shells from the same batch used in the optimisation study, were treated with 2M HCl for 48 hour in ice bath maintained at 0– 4 °C. Land crab shells (obtained from Port Henderson beach, St. Catherine, Jamaica); blue crab shells (obtained from Port Royal, Kingston, Jamaica) and prawn shells (obtained from prawn bred by Best Dressed Chicken, Barton Isle, St. Elizabeth), were oven dried, crushed and weighed. Samples of each of the shells were accurately weighed and treated with 2M HCl for 48 h, the temperature of the reaction medium kept between 0 and 4 °C. The undigested portion of the shells (the chitin-protein residue) was filtered from the solution, washed with wate, dried weighed and the weight loss percentages calculated. Tables 2.2 - 2.5 show the weight loss percentages obtained from the lobster, land crab, blue crab and prawn shells.
Table 2.2 PRELIMINARY WEIGHT LOSS RESULTS OF
DIGESTION OF LOBSTER SHELLS WITH 2M HCl
Shell sample name RGf/1/25a RGf/1/25b RGf/1/25c RGf/1/25d RGf/1/25e RGf/1/25f RGf/1/25g
Weight of shell /g 1.00 1.03 1.02 1.06 1.00 1.01 1.01
Weight of residue /g 0.44 0.46 0.45 0.46 0.44 0.44 0.43 0.45
Weight loss /% 56 56 56 57 56 57 58 57
Table 2.3 PRELIMINARY WEIGHT LOSS RESULTS OF
DIGESTION OF LAND CRAB SHELLS WITH 2M HCl
Shell sample name RGf/1/28a RGf/1/28b RGf/128c RGf/1/28d RGf/1/28e RGf/1/28f RGf/1/28g
Weight of shell /g 1.0140 1.0144 1.0198 1.0064 1.0146 1.0004 1.0136
Weight of residue /g 0.5139 0.5396 0.5039 0.5102 0.4832 0.5100 0.5333 0.5134
Weight loss /% 49 47 51 49 52 49 47 49
Table 2.4 PRELIMINARY WEIGHT LOSS RESULTS OF
DIGESTION OF BLUE CRAB SHELLS WITH 2M HCl
Shell sample name RGf/1/29a RGf/1/29b RGf/1/29c RGf/1/29d
Weight of shell /g 1.0166 1.0107 0.9435 0.9487
Weight of Residue /g 0.5163 0.4779 0.4329 0.4126 0.4599
Weight loss /% 49 53 54 57 53
Table 2.5 PRELIMINARY WEIGHT LOSS RESULTS OF
DIGESTION OF PRAWN SHELLS WITH 2M HCl
Shell sample name RGf/1/30a RGf/1/30b RGf/1/30c RGf/1/30d
Weight of shell /g 0.9888 1.0043 1.0072 0.9959
Weight of Residue /g 0.4944 0.5544 0.5352 0.5440 0.5320
Weight loss /% 50 45 49 45 47
Effervescence associated with the production of carbon dioxide accompanied the digestion of the shells. The reaction involving the lobster shells was the most vigorous followed by the prawn shells. The land crab shells were the least active. The results show weight loss averaging 57, 49, 53, and 47% for lobster, land crab, blue crab, and prawn, respectively (Tables 2.2, 2.3, 2.4 and 2.5). Therefore, assuming that all except 1% of the calcium salts was dissolved by acid digestion (based on optimisation/INAA results), lobster shells were expected to have the most calcium present as calcium carbonate, followed by the blue crab, land crab and prawn. The validity of the assumption was explored by the use of INAA in the next section.
CARBONATE CONTENT OF
CRUSTACEAN SHELLS AND
(b AND c) CHITIN-PROTEIN RESIDUE - AS DETERMINED BY INAA The weight loss percentages obtained in section 2.3.3 may not have been equal to the total percentage of calcium salts that were present in the crustacean shells. The aim of this investigation was to determine firstly the total percentage calcium (as carbonate) that were present in the shells, by the use of INAA (a) and secondly the percentage calcium (as carbonate) that remained in the chitin protein residue after digestion with 2M HCl (b). Thus, the effectiveness of the acid digestion process in the production of chitin from the lobster, land crab, blue crab and prawn shells could be determined.
CRUSTACEAN SHELLS The total percentage of calcium expressed as calcium carbonate in lobster,
land crab, blue crab and prawn shells was determined by INAA. Samples of dried and ground shells were weighed out in polythene vials and irradiated in the nuclear reactor the gamma radiation emitted counted and the percent calcium determined using the OMNIGAM neutron activation software package. The results of the analysis are shown in Table 2.6. Table 2.6 RESULTS OF ANALYSIS OF CRUSTACEAN SHELLS FOR CALCIUM BY INAA Source Lobster Land crab Blue crab Prawn (batch 1) Prawn (batch 2) Gypsum (experimental) Gypsum (manufacturer's) Empty
ND = Calcium not detected
Calcium /% 16.7 27.8 25.8 14.8 18.8 21.9 21.7 ND
Calcium carbonate /% 42 70 65 37 47 55 54 ND
The land crab shells contained the most calcium (as carbonate) (70%) followed by blue crab shells (65%), lobster shells (42%) and prawn shells (37%).
A second batch of prawn shells obtained and irradiated had 47% calcium (as carbonate) (Table 2.6). This difference in percentages between the two different batches of prawn may have been due to their differing ages, as the calcium carbonate content of the shell may vary with the stage of crustacean development. The percentages of calcium (as carbonate) found by INAA in the shells of the four crustacean species investigated varied significantly from the calcium carbonate levels determined by weight loss. A comparison of the percentage calcium carbonate determined in the shells (by INAA) and the average weight loss percentage are shown in Table 2.7. Table 2.7 COMPARISON OF PERCENTAGE CALCIUM (AS CALCIUM CARBONATE) DETERMINED BY INAA AND AVERAGE WEIGHT LOSS Source Average weight loss /% 57 49 53 47 Calcium carbonate in shells /% 42 70 65 37
Lobster Land crab Blue crab Prawn
For lobster and prawn shells, weight loss percentages were higher than the percentage calcium (as carbonate) present in these shells, as determined by INAA. However, the weight loss percentages of the shells obtained for the two species of crab were less than the percentage of calcium (as carbonate) determined by INAA
(Table 2.7). The lower percentage for the lobster and prawn (by INAA) compared with weight loss suggested that all the calcium present as calcium carbonate was dissolved from these shells, along with a small amount of the other main portion of the shell 1, the organic portion (hydrolysis). The higher percentages for the crabs (by INAA) compared with the weight loss percentage, indicated that the calcium salts present as calcium carbonate were not totally digested from the shells. There was also the possibility of hydrolysis of the organic polymers in the crab shells although this was not indicated in these results. The variation in the percentage calcium as calcium carbonate (by INAA) and the weight loss percentages prompted a further investigation of the effectiveness of 2M HCl digestion of all the crustacean shells studied. Thus, the calcium contents of the chitin-protein residues were determined. (b) (i) CHITIN-PROTEIN RESIDUE A comparison of the weight loss percentages and percent calcium as calcium carbonate determined by INAA suggested that there was incomplete removal of the calcium salts from the land crab and blue crab shells. On the contrary, more material than the calcium carbonate present in the lobster and prawn shells appeared to be removed by this digestion. In addition, there could have been incomplete calcium carbonate digestion in the lobster and prawn shells. The effectiveness of the acid digestion was investigated in this section by determining the percentage calcium as calcium carbonate that remained in all the chitinprotein residues after digestion of the crustacean shells with HCl (2M).
A portion of chitin-protein residues produced (Section 2.3.3) was analysed by INAA and the percentage calcium as calcium carbonate determined. The possibility of calcium being present in the sample vials was investigated by analysing an empty vial.The results of these experiments are summarised in Table 2.8. Table 2.8 RESULTS OF ANALYSIS OF CHITIN-PROTEIN RESIDUE
OBTAINED FROM 2M HCl DIGESTED SHELLS FOR CALCIUM BY INAA
Calcium /% 3.5 24.8 21.8 0.59 21.5 ND
Calcium carbonate in residue /% 9 62 55 2 54 ND
Percentage extraction /% 79 11 15 96 -
lobster land crab blue crab prawn gypsum (manufacturer's) empty vial
ND = Calcium not detected
The chitin-protein residues had varying levels of residual calcium carbonate. The samples obtained from lobster and prawn shells had low levels of residual calcium (as calcium carbonate), 9 and 2%, respectively, equivalent to 79 and 96% extraction efficiency as determined by Equation 2.6. Extraction efficiency (%) = (CaS – CaR)/CaS × 100…………………Equation 2.6
Where, CaS = calcium carbonate in shells by INAA (%); CaR = calcium carbonate in residue by INAA (%); The residues obtained from digestion of land crab and blue crab shells however had high levels of residual calcium as calcium carbonate. The percentages obtained were 62% (for land crab) and 55% (for blue crab) corresponding to 11% and 15% extraction efficiency (Table 2.8). Therefore, calcium carbonate was not being totally removed from the shells after digestion with 2M HCl for 48 h. Calcium was not detected (ND) in the empty vial. A comparison of percentage weight loss, the total percentage calcium as calcium carbonate in the shells and the percent calcium as calcium carbonate present in the chitin-protein residues (by INAA) were made. In the lobster, shells the 57% weight loss compared with total 42% calcium carbonate in shells and 9% calcium carbonate in chitin protein residue supported the suspicion that the organic portion of the shell was hydrolysed during acid digestion. The 47% weight loss, 47% calcium carbonate in shells and 2% calcium carbonate in chitinprotein residue suggested that there was almost complete digestion of calcium from the prawn shells. Land crab and blue crab shells showed 49% and 53% weight loss respectively, compared with 70% and 65% total calcium carbonate in the shells. This showed that, particularly in the land crab, the calcium was not being effectively removed by acid digestion as the residue still contained 62% and 55% calcium carbonate.
CALCIUM CARBONATE CONTENT OF CHITIN-PROTEIN RESIDUE REDUCED The percentage calcium carbonate measured for residues of lobster shells
after 2M HCl digestion during the optimisation studies, was 1%. When this was repeated in section 2.3.4b, (Table 2.8), 9% calcium carbonate remained undigested. This observation prompted a repeat of the experiment, vigorously shaking the reaction vessel during the 48 hour digestion period and the residues thoroughly washing in water before drying and weighing. The samples were then analysed by INAA to determine the percentage calcium as calcium carbonate. The weight loss percentages were also determined. These new results obtained by INAA were recorded in Table 2.9. A graphical view of the improvements in digestion made for each type of crustacean shell is shown in Figure 2.4. The weight losses obtained after digestion with HCl (2M) are shown in Table 2.10. Table 2.9 NEW RESULTS OF ANALYSIS OF 2M HCl
FOR CALCIUM (AS CALCIUM CARBONATE) DETERMINED BY INAA
Source lobster land crab blue crab prawn gypsum standard
Average calcium carbonate /% <1 52 43 <1 24.4 (24)
Figure 2.4 PERCENT CALCIUM PRESENT IN CRUSTACEAN SHELLS
BEFORE AND AFTER DIGESTION WITH 2M HCl AS DETERMINED BY INAA
CALCIUM CARBONATE / %
100 90 80 70 60 50 40 30 20 10 0 9 1 2 1 42 70 62 54 65 55 43 47
before digestion after digestion after digestion (repeat)
Table 2.10 NEW WEIGHT LOSS PERCENTAGES AFTER 2M HCl DIGESTION OF CRUSTACEAN SHELLS Source Average weight Average weight Average weight of shells of chitin-protein loss /g residue /% /g 4.9 4.8 4.8 4.9 2.1 2.0 1.7 2.1 57 58 64 58
Lobster Land Crab Blue Crab Prawn
The percentage calcium (as carbonate) that remained after digestion was
less than 1% for lobster shells which was consistent with the results of the optimisation study. The chitin-protein residue obtained from digesting the prawn shells also contained less than 1% calcium (as carbonate) (Table 2.9). Thus, there was improvement in the efficiency of digestion of these two types of crustacean shells. Improvement in the level of calcium carbonate digestion was also evident for the crab shells. The percentages went from 62 to 52% in the land crab and from 55 to 43% in the blue crab shells (Figure 2.4). In the case of the lobster and prawn shells, the calcium carbonate could be efficiently extracted by acid digestion. In the case of the crab species the shells appeared to be very resistant to acid digestion as the residues contained high calcium concentrations. The weight loss results in Table 2.10 were in agreement with what was previously observed. That is, hydrolysis of organic polymers occurred during acid digestion. These effect was more pronounced for the blue crab shells. The shells contained 65% calcium (as carbonate). After acid digestion the residue contained 43% calcium (as carbonate), yet weight loss percentage averaged 64% (Table 2.10). A comparison of the average weight loss percentages obtained before and the new average weight loss percentages were made. The weight loss percentages generally increased with the improvement in the percentage calcium carbonate removed, as expected. They went from 47% to 58% (2% to < 1% CaCO3) with the prawn shells; 49% to 58% (62% to 54% CaCO3) with the land crabs and from 53% to 64% (55% to 43% CaCO3) in the blue crabs. The weight loss for the lobster shells remained constant at 57% although the residual calcium carbonate
was brought to less than 1% by weight from 9%. Improving the efficiency of the digestion of calcium carbonate may to some extent affect the quantity of chitin produced because of the hydrolysis of chitin16, which can occur.
ATOMIC ABSORPTION SPECTROSCOPY (AAS) AAS is an alternative technique to INAA that was used in this work for analysing shells for their calcium content. INAA is the better technique since AAS requires dissolution of the sample whereas INAA is a direct solid sample analysis and is less prone to matrix interferences 14. The foundation of atomic absorption dates back to 1802 when Wollaston 17 discovered black lines in the spectrum of the sun, which were later investigated by Fraunhofer 17. Brewster
postulated that absorption processes in
the atmosphere of the sun caused these lines. Kirchhoff and Bunsen
investigating the spectra of alkali and alkaline earth metals demonstrated that the typical yellow line emitted by sodium salts in a flame is identical to the black line in the spectrum of the sun. When a gaseous atom in its ground state absorbs a specific quantum of energy from an external source of radiation, it can attain an excited state in which electrons surrounding the atom occupy higher energy levels than usual. This is an unstable state and the atom quickly and spontaneously returns to its ground state as the electrons return to their original orbital position. The exact amount of energy that was absorbed during the excitation process is emitted during this decay process.
The amount of the analyte element present is determined by
measuring a parameter called Absorbance
which is related to the reduction in
the intensity of the beam of radiation passing through the gaseous sample
(Equation 2.7) 20. A = log (I0/I)……………………………………….Equation 2.7 Where, A = Absorbance; I0 = Intensity of radiation projected into sample; I = Intensity of radiation passed through sample. Quantitative measurements in atomic absorption are based on Beers’ law 21 which states that concentration is proportional to Absorbance, where, A = abc……………………………………………Equation 2.8 Where, a = Absorption coefficient, a constant which is a characteristic of the absorbing species at a particular wavelength; b = Length of the radiation path intercepted by the absorption species in the absorption cell; c = Concentration of the absorbing species; Absorbances of standard solutions containing known concentrations of analyte are measured and the absorbance data are plotted against concentration. Ideally, this should be a straight line as indicated by Beer’s law 21, and this is usually observed
at lower concentrations and absorbances. As concentration and absorbance increase however non ideal behavior in the absorption process causes deviation from linearity. The absorbance of the sample is measured and the concentration of the analyte determined from the calibration curve. Modern atomic absorption instruments have the ability to perform automatic curve correction, calibrate, and compute concentrations using absorbance data from linear and non-linear curves 22. Initially the sample being analysed is atomised in a cell by a flame or an electrically powered graphite furnace. Air - acetylene is the preferred flame for the determination of many elements in atomic absorption, producing temperatures of about 2300 °C 23. The external radiation required for excitation is delivered by line sources, for example, the hollow cathode lamp which are manufactured for individual elements. Radiation passes from the source through the atomised sample to a monochromater that disperses it and isolates a specific wavelength that is passed directly to a detector, usually a photomultiplier tube (PMT). The PMT produces an electrical current, the magnitude of which depends on the intensity of the radiation falling on it. Comparison with known standards and the use of Beers Law 21 enables the concentration of the analyte in the sample to be determined 24. The above description is for a single beam spectrophotometer. In a double beam spectrophotometer the light from its source is divided into a sample beam, which is focused through the sample cell, and a reference beam, this is directed
around the sample cell. The actual readings obtained represent a ratio of the sample and reference beams. The result is that fluctuations in source intensity are not reflected in the read out obtained. No lamp warm up period is required in contrast to the single beam spectrophotometer 25.
CALCIUM CARBONATE CONTENT - AS DETERMINED BY AAS
INTRODUCTION INAA was used to determine the percentage calcium as calcium carbonate
present in both shells and chitin-protein residues and was conclusive. AAS is a method which is cheaper, more readily available and was used to find the percentage calcium present as calcium carbonate in the solution that is obtained after acid digestion of the crustacean shells. Both the results of AAS experiments and INAA experiments were expected to compliment each other in that the following relationship was expected to hold: (CaR × WR) + (CaF × WS) ÷ WS = TCa = CaS………………..Equation 2.9 where,
CaR = Calcium carbonate in chitin-protein residue by INAA (%);
WR = Weight of chitin-protein residue (g);
CaF = Calcium Carbonate in filtrate by AAS (%);
WS = Weight of shells (g);
TCa = Total percent calcium carbonate in shell calculated (%); CaS = Calcium carbonate in shell by INAA (%).
AAS may therefore be used as a check, in conjunction with the information on weight loss on acid digestion. To determine the percentage of the shell that was
not calcium salt (organic polymers) that had dissolved, Equation 2.10 was used.
Weight loss (%) = CaF + OP…………………………………Equation 2.10
CaF = Calcium carbonate in filtrate (%); OP = Organic polymers (%).
RESULTS AND DISCUSSION OF CALCIUM CARBONATE
DETERMINATION BY AAS
Fresh samples of lobster and land crab shells were dried, weighed and digested for 48 hour with 2M HCl. The digestion product obtained was filtered and the residue washed with water, dried and collected for INAA to determine the percentage calcium as calcium carbonate. The washings that were combined with the filtrate were also collected and made up to 250 mL with distilled water. Diluted portions of these solutions were analysed by AAS and the percentage calcium, expressed as calcium carbonate determined. The results of both experiments are shown in Table 2.11.
TABLE 2.11 PERCENTAGE CALCIUM (AS CALCIUM CARBONATE)
DETERMINED BY AAS AND INAA
WR /g 1.235 0.3623
Weight loss /% 59 64
CaS /% 42 70
CaR /% 0.125 40
CaF /% 42 57
TCa /% 42 72
lobster land crab
CaR = Calcium carbonate in chitin-protein residue by INAA (%), WR = Weight of chitin-protein residue (g), CaF = Calcium Carbonate in filtrate by AAS (%), WS = Weight of shells (g), TCa = Total percent calcium carbonate in shell calculated (%), CaS = Calcium carbonate in shell by INAA (%)
For the lobster shell sample, the calculation showed that the total percentage calcium as calcium carbonate calculated (TCa) was 42%
(Equation 2.9). This was equal to the total calcium carbonate in the shells (CaS). The AAS determined percentage (CaF) was also equal to the latter, which suggested that all the calcium carbonate was digested Table 2.11. In addition, for the lobster sample 59% weight loss occurred to produce the chitin-protein residue. With 42% calcium as calcium carbonate in the solution then, organic polymers that were hydrolysed amounted to 17% of the shells (Equation 2.10). In the land crab shell sample where calcium carbonate in residue (CaR) was 40%, TCa was 72% (Equation 2.9). This was close to CaS Table 2.11. The two differed by 2%. Digestion of the land crab shells resulted in 64% weight loss. Therefore, organic polymer hydrolysed amounted to 7% (Equation 2.10). CaF
(57%) was less than CaS (70%) because the crab shell was incompletely digested. The percentage organic polymers digested, 17% for the lobster shells and 7% for the land crab shells, confirmed that crab shells are more resistant to acid than lobster shells. Overall, it was shown that AAS was able to determine conclusively the percentage calcium as calcium carbonate present in the shells of the more easily digested crustacean shells for example, the lobster shells. This method however was not sufficient for the harder, more acid resistant shells like the crab shells. AAS is however suitable for routine check analysis on the samples as digestion proceeds.
CHITIN CONTENT OF CRUSTACEAN SHELLS AS DETERMINED BY ALKALINE HYDROLYSIS
INTRODUCTION With the calcium present as calcium carbonate in crustacean shells
properly quantified, it became easier to determine their percentage of chitin. The first step to obtaining the percentage chitin was to boil the chitin–protein residue with sodium hydroxide and then weigh the unhydrolysed product (UHP) (Equation 2.11). Chitin-protein residue = UHP + Hydrolysed material…….Equation 2.11 Where, UHP = Unhydrolysed product. There may be reservations in calling the UHP, chitin, because of the existing possibility of impurities mainly calcium. Therefore, the UHP was analysed by INAA to determine if the hydrolysis process affected the percentage of undigested calcium carbonate, particularly in the crab shells. By considering the weight of UHP (WUHP) and the percentage calcium carbonate impurities (CaUHP) the percentage pure chitin was determined (Equation 2.12).
Chitin% = [WUHP – (CaUHP × WUHP)] / WS × 100………….Equation 2.12 Where, CaUHP = Calcium carbonate impurities in chitin (%); Ws = Weight of shells. An attempt was also made to determine the presence of and types of amino acids and proteins that were present in the hydrolysed product. This involved the use of Gas Chromatography – Mass Spectrometry (GC-MS), the ninhydrin test and electrophoresis 26. GC-MS along with a total elemental analysis aided the determination of the composition of the exoskeletons. 2.6.2 Percent unhydrolysed product (UHP%) after alkaline hydrolysis The chitin-protein residues obtained after acid digestion were boiled with 1M NaOH for 48 hours. The unhydrolysed product (UHP) obtained was filtered washed repeatedly with water until neutral, dried and then weighed. The percentage UHP was calculated with Equation 2.13 and the results obtained after duplicate experiments are shown in Table 2.12. UHP% = WUHP / WS × 100…………………………….…..Equation 2.13 Where, UHP% = unhydrolysed product (%); WUHP = Weight of Unhydrolysed product; WS = Weight of shells.
Table 2.12 ALKALINE HYDROLYSIS OF CRUSTACEAN
SHELLS – PERCENTAGE UNHYDROLYSED PRODUCT
Average weight of shells /g
Average Average Average weight of weight of unhydrolysed Chitin-protein unhydrolysed product residue product /% /g calculated /g 2.1 2.0 1.8 2.1 1.0 1.7 1.7 1.7 21 35 36 35
lobster land crab blue crab prawn
4.9 4.8 4.8 4.9
Lobster shell samples had overall 21% unhydrolysed product after alkaline hydrolysis. The land crab and prawn shell samples had on average 35%, unhydrolysed product whilst the blue crab had 36%, after hydrolysis. These results on their own suggested that the lobster shells would contain the least amount of chitin, and the other three samples would contain about the same as each other. This however did not take into account impurities in the UHP, which will be discussed next. 2.6.3 PERCENT CALCIUM CARBONATE IMPURITIES
IN UNHYDROLYSED PRODUCT
In the land crab and blue crab shells, a large amount of calcium carbonate was present after acid digestion and was expected to be present after the alkaline hydrolysis process. It was therefore necessary to determine the percentage
calcium (as carbonate) in the unhydrolysed product in order to determine the percent chitin present in the crustacean shells. INAA was used to determine the percent calcium (as carbonate) present in the UHP. A sample of practical grade crab chitin obtained from Sigma Co. was also irradiated for comparison. The percentages are shown in Table 2.13. Table 2.13 CALCIUM CARBONATE CONTENT OF UNHYDROLYSED PRODUCT Source Average calcium /% < 0.5 20 19 < 0.5 0.02 22.1 (22) 22.9 (22) Average calcium carbonate /% <1 49 49 <1 0.05 26
Lobster Land Crab Blue Crab Prawn Crab (Sigma Co.) Gypsum Calcium std.
In addition, GC-MS, the ninhydrin test and electrophoresis
used to determine the presence of, and the type of amino acids and proteins that were hydrolysed in the solution. A portion of the solution obtained from alkaline hydrolysis of the chitinprotein residues was filtered made more alkaline and extracted with a mixture of dichloromethane. A diethyl ether extraction was also carried out after acidifying a
fresh portion of the solution. Extractions were done to obtain samples for GC-MS the polypeptides and amino acids present. Another portion of the sample was analysed using the ninhydrin and the electrophoresis 26 test. UHP obtained from the prawn and lobster shells had less than 1% calcium (as calcium carbonate) (Table 2.13). These were white compared to the brown colour of the chitin-protein residue (Photograph 2.1). On average, 49% of the UHP obtained from the land crab and blue crab shells was calcium carbonate (CaUHP). The sample of practical grade crab chitin obtained from Sigma Co, when analysed was shown to contain 0.05% calcium as calcium carbonate.
Photograph 2.1 CHITIN AND CHITOSAN SAMPLE OF PRAWN (LEFT) AND LOBSTER (RIGHT)
↓ CHITIN ↓ CHITOSAN ↓ CHITIN ↓ CHITOSAN
The CaUHP for the lobster and prawn were expected since the percentage calcium (as calcium carbonate) present after acid digestion was very small (less than 1%). For the land crab and blue crab samples, higher if not the same percentages of calcium carbonate were expected after alkaline hydrolysis, since the percentages were calculated with respect to the weight of the unhydrolysed products (smaller weight compared with chitin-protein residue). The percentage calcium carbonate in the land crab was 54% in the chitin-protein residue and 49% in the UHP. In the blue crab it was 43% in the chitin-protein residue compared to 49% in the UHP, a reasonable change. A small increase in the percentage residual calcium carbonate may be due to the small amount of protein that was present in the chitin-protein residue of the crab shells. The ninhydrin test, electrophoresis 26 and GC-MS suggested the absence of any significant amount of protein in the solution obtained after alkaline hydrolysis. The ninhydrin test indicated the presence of aminoacids, by the characteristic blue colour obtained by heating ninhydrin and solution on filter paper. However, the gel electrophoresis
that followed this test was negative.
The characteristic blue bands a positive sign for the presence of polypeptides and amino acids were absent. The GC-MS indicated very few amino acids, for example, glycine was present.
COMPOSITION OF THE EXOSKELETON The exoskeleton is composed of chitin, calcium and other metals and non-
metals, proteins and other organic substances. Their final percentages are stated
below. The percentage chitin was determined using Equation 2.12. The percentages of metals and nonmetals were determined by INAA and the organic substances, excluding chitin were determined by GC-MS.
Percentage chitin The isolation of chitin involved two clear steps. These were digestion of
calcium present as calcium carbonate and hydrolysis of the chitin-protein residue obtained. The percentage of UHp of all the crustacean shells were determined based on the weight of the shells used. These were 21 and 35% in the lobster and prawn shells. With less than 1% calcium as calcium carbonate present in these UHP, it was concluded that the percentage chitin present in the lobster and prawn shells were a minimum of 21 and 35%, respectively. The percentage chitin in crab shells was calculated by considering the percentage calcium carbonate in the UHP (Table 2.12 and Table 2.13), and applying Equation 2.12. Therefore, for the land crab and blue crab shells, percentage chitin was at least 18 and 19%, respectively.
Elemental composition by INAA The other elements apart from calcium that were present in the crustacean
shells, were determined. The shells were found to contain small quantities of metals e.g. Na, K, Mg, Al and Mn.; and non-metals e.g., Br and Cl (Tables 2.14).
Tables 2.14 ELEMENTAL COMPOSITION OF SHELLS Shells /% Na K MgO 0.31 0.035 2.8 0.65 0.17 1.0 0.35 0.23 2.9 0.13 0.12 Land Crab Blue Crab Lobster Prawn
Shells /mg/kg Br Al2O3 Mn Cl
31.0 ND 70.0 140.0
105.0 445.0 137.0 776.0
390.0 ND 11.0 476.0
221.0 276.0 42.0 293.0
ND = Not detected in shell
The quantities varied with species and may be an indication of variation in the animals’ diets or habitats. For example, the land crab, which is not a marine dweller, contained less of the halogens than the other species. The presence of these elements coupled with the organic materials, make crustacean shells a possible source of fertiliser. Many of these substances may not be eliminated during acid and base hydrolysis and will remain as contaminants in chitin.
OTHER ORGANIC SUBSTANCES
The other organic materials present in the crustacean shells were determined using GC-MS. The solutions obtained after alkaline hydrolysis of the chitin protein residues were divided into two portions, one of which was made more alkaline and the other acidic. The alkaline solution was extracted with dichloromethane and the acidic solution with methylene chloride. The solvents containing the components being analysed were then evaporated to dryness, derivatised with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and analysed by GC-MS and a Pfleger/Maurer/Weber MS drug library used to determine its constituents. The GC-MS and library revealed the presence of a variety of compounds: aromatic as well as aliphatic amines, high molecular weight carboxylic acids and alkanes.
REMOVAL OF CALCIUM FROM CRUSTACEAN SHELL BY COMPLEXATION The harsh conditions of acid digestion followed by base hydrolysis can
affect the isolation efficiency of chitin. Under these conditions chitin may be deacetylated to chitosan or hydrolysed into its N-acetyl monomeric units
Complexation is a mild alternative for the removal of calcium from lobster shells 28. Any weight loss obtained from using complexing agents is expected to be the result of removal of calcium without any effect on the organic polymers. Thus the effectiveness of the complexation method was compared with the acid digestion method on the basis of weight loss only. 2.7.1 REMOVAL
OF CALCIUM FROM CRUSTACEAN SHELL BY COMPLEXATION
[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.
TABLE 2.15 PERCENTAGE CALCIUM CARBONATE IN LOBSTER SHELLS OVER
DIFFERENT TIME PERIODS USING EDTA SOLUTION AT ROOM TEMPERATURE
Time for digestion / min. 15 60 180
Weight loss /% 23 40 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
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
Chitinprotein residue /g 0.083 0.14
Weight loss /%
H2O, RT H2O, 8085 °C H2O, 8083°C ETOH, RT EtOH, pH 4, RT EtOH, pH 9.2, RT.
0.12 0.12 0.12
0.12 0.13 0.13
0.10 0.11 0.11
17 8 8
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
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.
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 /% 15 17 Chitin /% 24 28
Addition of acid to the exoskeleton of the Blaberus cockroach did not
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.
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
(a) lobster shell
(b) prawn s hell
calcium carbonate 42%
c hitin 35%
c alc ium c arbonate 47% protein and other materials 18%
protein and other materials 37%
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.
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.
REFERENCES FOR CHAPTER TWO 1. P.W. Kent, and M.W. Whitehouse, “Biochemistry of the Amino Sugars,” Butterworths Scientific Publication, London, 1955, p 94.
Reference 1, p 92. De Soete, R. Gigbels and J. Hoste, “Neutron Activation Analysis,” John Wiley and Sons, London, 1972, Vol 34, p 1. D.A. Skoog and J.J. Leary, “Principles of Instrumental Analysis,” A. Harcourt Brace Janovich College Publishing, N. Y., 1992, p 410. Reference 4, p 411. J.C. Kotz and K.F. Purcell, “Chemistry and Chemical Reactivity,” Saunders College Publishing, New York, 1987, p 1009. G. C. Lalor, R. Rattray, H. Robotham, Jamaica Journal of Science and Technology, 1990, 1 (1), 65. Reference 3, p 4. Reference 4, p 413. Nuclear Engineering Teaching Laboratory, Department of Mechanical Engineering, University of Texas, Austin, 1995. Reference 4, p 412.
8. 9. 10.
Reference 4, p 414.
Reference 3, p12
Reference 3, Vol 34, p 7.
Reference 3, Vol 34, p 8.
Reference 1, p 92.
B. Welz, “Atomic Absorption Spectroscopy,” Verlag Chemie GmbH, D6940 Weinheim, 1976, p 1.
R. D. Beaty, J. D. Kerber, “Concepts Instrumentation and Techniques in Atomic Absorption Spectrophotometry,” Perkin Elmer Co-orporation, Norwalk, 1993, p 1-1. Reference 18, p 1-5. Reference 18, p 1-6.
Perkin Elmer, “Analytical Methods for Atomic Absorption Spectrometry,” 1994, p 16.
Reference 21, p 17.
Reference 21, p 13.
Reference 21, p 4.
Reference 21, p 6. K. D. Golden, M Phil. Thesis, Beta galactosidase (beta-Dgalactohydrolase) (E. C. 18.104.22.168) from Coffea arabica, its possible role in fruit ripening and ethylene synthesis, Biochemistry Department, UWI, Mona, 1991, p 46. R. A. A. Muzzarelli, Chitin, Pergamon Press N.Y., 1976, p 90. Reference 27, p 91.
CHAPTER THREE CHARACTERISATION OF CHITIN
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.
C 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-2deoxy-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 2amino-2-deoxy D-glucose.
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
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 losstemperature 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).
Figure 3.1 TGA CURVES OF PRAWN (CPWN2a) AND LOBSTER(CLOB2a) CHITIN
Figure 3.2 DSC CURVE OF LOBSTER CHITIN
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
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.
SCANNING ELECTRON MICROSCOPY (SEM) Sir Charles W. Oatley
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 (Photograph 3.1).
of the compound as shown by position s on the photograph
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, 10µM) µ
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 =
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.
Photograph 3.4 SEM OF CHITIN FROM BLABERUS COCKROACH LEG (HIGHER MAGNIFICATION, SCALE BAR = 10µM) µ
Photograph 3.5 SEM OF CHITIN FROM BLABERUS
(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.
Photograph 3.6 SEM of chitin from Blaberus cockroach wings (higher magnification, scale bar = 10µm) µ
C NMR ANALYSIS OF CHITIN MONOMER 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)
biosynthetic chitin (called artificial chitin) (8) (cross polarisation magic angle spinning - CP / MAS) 11.
4 H, OH HO
6 CH2OH 5 3 2
O H, OH NH C O C H3 1
4 HO HO 3
6 CH2OH 5
2 OH OH
(6) CHITIN MONOMER
HO O HOH2C
6 HOH2C NHCOCH3 4 O HO 3
8 NHCOCH3 7
(8) BIOSYNTHETIC (ARTIFICIAL CHITIN)
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
200 instrument. The chemical shifts for each carbon were then determined and compared with glucose and biosynthetic chitin from the literature (Table 3.1).
C DATA FOR HYDROLYSED CHITIN
GLUCOSE AND CHITOSAN HYDROCHLORIDE
Literature values C Glucose (D2O/TMS) / δ ppm 1 2 3 4 5 6 7 (C=O) 8 (CH3) 93.6 73.2 74.5 71.4 73.0 62.3 Artificial chitin CP/MAS solid / δ ppm 105.0 56.2 74.3 84.4 76.9 61.9 175.0 23.8 Hydrolysed chitin (D2O/TMS) / δ ppm 99.9 61.7 76.9 96.4. 83.4 67.7 183.9 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
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.
IR SPECTRAL ANALYSIS – FUNCTIONAL GROUP ANALYSIS AND % N-ACETYLATION DETERMINATION.
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.
Figure 3.4 IR SPECTRUM OF UNPURIFIED CRAB CHITIN OBTAINED FROM SIGMA CO.
55 45 35 25 3500 2500
Wavenumber cm -1
Figure 3.5 IR SPECTRUM OF SAMPLE CHITIN FROM LOBSTER SHELLS
65 60 55 50 45 40 3550 3050 2550 2050 1550 1050
Wavenumber / cm -1
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.
Figure 3.6 IR SPECTRUM OF SKIN-LIKE MATERIAL OBTAINED
FROM THE WING OF AN ADULT BLABERUS COCKROACH AFTER NAOH DIGESTION
100 90 80 70
60 50 40 30 20 10 0 3950 3450 2950 2450 1950 1450 950 450
Wavenumber / cm -1
IR SPECTRUM OF POWDERED MATERIAL OBTAINED
FROM THE LEG OF AN ADULT BLABERUS COCKROACH AFTER NAOH DIGESTION
80 60 40 20 0 4400 3900 3400 2900 2400 1900
Wavenumber / cm
Figure 3.8 IR SPECTRUM OF THE WING OF AN ADULT BLABERUS COCKROACH
100 % Transmittance 80 60 40 20 0 3950 3450 2950 2450 1950 1450 Wavenumber / cm -1
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
, 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.
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 Nacetylation of chitosan was proposed by G.K. Moore and G.A. Roberts (1955)
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
as an internal standard to correct for film
thickness or for differences in chitosan concentration if a KBr disc was used. Domzy and Roberts
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
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 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
-1 2867 cm ,
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
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
true values at higher levels; the comparison made with the titration method of Broussignac 15. Using A cm-1 / A3450 cm-1 (Domzy and Roberts
) and a different
baseline proposed by Miya et. al
, 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
the new baseline proposed by Miya et. al was put together by Baxter et. al (1992)
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
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) and the Σ method of Baxter et. al
which required the use of Equation 3.2 the baselines
labeled (Ω). The absorbances at 1655 cm-1 and 3450 cm-1 were determined from Ω the specified baselines.
Figure 3.9 IR SPECTRUM OF UNPURIFIED CRAB
CHITOSAN OBTAINED FROM SIGMA CO.
0.5 0.45 0.4 Absorbance 0.35 0.3 0.25 0.2 0.15 3550 3050 2550 2050 1550
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.
IN THE CHARACTERISATION OF CHITIN AND OF
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 % Nacetylation was determined for the samples using the method of Domzy and Roberts
and by the method of Baxter et. al
. The percentages obtained are
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.
Table 3.2 PERCENTAGE N-ACETYLATION OF CHITIN AND CHITOSAN SAMPLES Sample N-acetyl N-acetyl (A1655 cm-1/A3450 cm-1) (A1655 cm-1/A3450 cm-1) × (100/1.33) × 115 /% /%
8.7/16.3 × (100/1.33) = 40 69 2.5/16.3 × 115 = 18 (≤ 15) 51
crab chitosan from Sigma Co., RGf/1/113a crab chitin from Sigma Co., RGf/1/116a lobster chitin, RGf/1/105b lobster chitin RGf/1/21a-c lobster chitin clob lobster chitin, clob2c prawn chitin, cpwn prawn chitin, cpwn2b land crab chitin, clc lobster crude chitosan, RGf/1/80 N-Ac. chitosan, RGf/1/82a N-Ac. chitosan, RGf/1/82c N-Ac. chitosan, RGf/1/82d N-Ac. chitosan, RGf/1/82e lobster chitosan RGf/1/90 lobster chitosan RGf/1/97a lobster chitosan RGf/1/114a lobster chitosan, RGf/1/115b lobster chitosan RGf/1/102
63 61 61 66 60 60 48 40
54 57 42 67 61 42 60 30
49 55 59 61 90 56 37 40 62.
31 37 45 57 39 13 26 21 18
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)
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.
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 % NAc (13%, 18%) were soluble in 10% acetic acid and hence showed a successful conversion of chitin to chitosan.
REFERENCES FOR CHAPTER 3
W.W.M. Wendhandt, “Thermal Methods of Analysis,” John Wiley and Sons, New York, 1974, Vol 19, p 6. Reference 1, p 193. Reference 1, p 134. Reference 1, p 212. Reference 1, p 215. Reference 1, p 242. R. E. Lee, “Scanning Electron Microscopy and X-ray Analysis,” PTR Prentice-Hall Inc., New Jersey, 1993, p 9. O. C. Wells, “Scanning Electron Microscopy,” McGraw-Hill Inc., New York, 1974, p 2. E. Cohen, Ann. Rev. Entomol, 1987, 32, 72. T.E. Walker, R.E. London, T.W. Whaley, R. Barker and N.A. Matwiyoff, J. Am. Chem. Soc, 1976, 98:19,5808. J. N. Bemiller, Meth. Carbohyd. Chem., 1965, 5, 103. Y. Shigemasa, H. Matsurra and H.Saimoto, International Journal of Biological Molecules, 1966, 18, 237. 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. G. Domszy, G. A. F. Roberts, Makromol. Chem., 1985, 186, 1671. A. Baxter, M. Dillon, K.D.A. Taylor and G.A.F. Roberts, Int. J. Macromol., 1992, 14, 166.
2. 3. 4. 5. 6. 7.
9. 10 11. 12.
CHITIN AND ECONOMICS
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)
Unpurified chitin from crab shell (10 g)
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,
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.
EXPERIMENTAL DETAILS FOR CHAPTER TWO
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).
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.
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
as calcium carbonate determined by INAA. (Figure 2.3).
CALCIUM CARBONATE CONTENT OF CRUSTACEAN SHELLS - AS DETERMINED BY
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
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
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.
CHITIN CONTENT OF CRUSTACEAN SHELLS AS DETERMINED BY ALKALINE
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
A drop of the filtrate obtained from lobster and prawn sample after
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.
The filtrates obtained from lobster and prawn samples after alkaline hydrolysis (60 L) were added to 60 L 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 L) and tracking dye (5 L, bromothymol blue (1%)) were then added to the sample. The sample (20 L) 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
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).
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).
FOR CHAPTER THREE
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).
C 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
determined using a Bruker AC 200 NMR spectrometer instrument (Table 3.1).
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 Nacetylated 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
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 was determined for crab chitosan obtained from Sigma Co. (RGf/1/113a), crab chitin from Sigma Co. (RGf/1/116a), lobster chitin (RGf/1/105b), lobster chitosan (RGf/1/115b), lobster chitin (RGf/1/21a-c), lobster chitin (clob), lobster chitin (clob2c), lobster chitin (clob2c), prawn chitin (cpwn),
prawn chitin (cpwn2b), land crab chitin (clc), lobster crude chitosan (RGf/1/80), homogenous N-Ac. Chitosan (RGf/1/82a), homogenous N-Ac. Chitosan (RGf/1/82c), homogenous N-Ac. Chitosan (RGf/1/82d),homogenous N-Ac. Chitosan (RGf/1/82e), lobster chitosan (RGf/1/90), lobster chitosan (RGf/1/97a), lobster chitosan (RGf/1/102), lobster chitosan (RGf/1/101), and lobster chitosan (RGf/1/114a). The chitin samples not obtained from sigma were prepared by acid digestion followed by alkaline hydrolysis of crustacean shells.
Dried samples of the chitin and chitosan samples were blended with KBr to form KBr discs. These were then placed into a Spectrum 1000 Perkin Elmer FTIR Spectrometer, previously standardised with polystyrene, to determine the absorbances of the functional groups present in the compounds. From the spectra, the % N-acetylation were determined using the method of Domzy and Roberts
and Baxter et. al 1, the absorbances at 1655 and 3450 cm-1 and the baselines labeled (Σ) and (Ω), shown in Figure 3.9 (crab chitosan sample, RGf/1/113a)
The method of Domzy and Roberts 2 and Baxter et. al 1 required the use of the baseline labeled (Σ) and Equation 3.1, and baselines labeled (Ω) and
Equation 3.2 respectively.
% N-acetylation =(A1655 cm-1/A3450 cm-1) × (100/1.33)……………Equation 3.1
% N-acetylation = (A1650 ÷ A3450) × 115 ………………………….Equation 3.2)
The absorbances at 1655 cm-1 and 3450 cm-1 were determined from these specified baselines. The percentages obtained are shown in Table 3.2.