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66 Chapter 5 Extraction, Characterization and Standardization of Chitosan

5. EXTRACTION, CHARACTERIZATION AND STANDARDIZATION OF

CHITOSAN

5.1. Background

Although many gastric retentive devices have been developed using chitosan,

few were successful in improving gastric residence time. Many of them were failure

in some cases are briefly discussed as follows,

Floating drug delivery systems developed with Chitosan having internal

cavities were prepared by deacidification. When added to acidic (pH 1.2) and neutral

(deionized water) media, these granules were immediately buoyant and provided a

controlled release of the candidate drug prednisolone. Laminated preparations

prepared by coating the chitosan granule’s layer with chitosan membranes were also

immediately buoyant and provided controlled release profile of the drug. According to

the authors, this might be explained by the fact that the laminated preparation has

higher inter-subject variation and higher pH-dependency of the drug release than the

chitosan granules. However, both the chitosan granules and the laminated

preparations could be helpful in developing drug delivery systems that will reduce the

effect of gastrointestinal transit time152

.

The release rate of indomethacin from both chitosan granules and

conventional commercial indomethacin capsules was evaluated. While the con-

ventional capsules disintegrated immediately and released the drug rapidly in the

acidic medium, chitosan granules released the drug rapidly at acidic medium and

slowly at neutral or alkaline medium. The authors think that good gel-formation of

chitosan at low pH and poor gel-formation at high pH might explain the pH-

dependence of the release of the drug. The diffusion of the drug molecules out of the


chitosan granules was enhanced because of the swelling and gel-formation at low pH.

For the chitosan granules, the area under the plasma concentration–time curve

(AUC0→8h) was 10.65 mg.h/mL, while for the commercial capsule it was 9.39

mg.h/mL slower release rate from chitosan granules and longer residence time in the

stomach could explain the slight increase in AUC152

.

Floating systems have a density lower than the density of the gastric juice.

Thus gastric residence time and hence the bioavailability of drugs that are absorbed in

the upper part of the GI tract will be improved. Intragastric floating dosage forms are

useful for the administration of drugs that have a specific absorption site, are insoluble

in the intestinal fluid, are used for the treatment of gastric diseases. From the above

statement it was well understood that chitosan can be used as suitable polymer for the

development of floating drug delivery system because of its poor solubility at acidic

environment, swellability and low density, moreover chitosan is biocompatible and

biodegradable.

5.1.1. Introduction

Chitosan can be obtained by the alkaline deacetylation of chitin. Chitosan is

non-toxic, biocompatible and biodegradable substance. These properties make

chitosan as a good candidate for the development of novel gastrointestinal (GI)

floating drug delivery systems. Chitin is the most abundant cellulose polysaccharides

in nature. Chitin can be isolated from the exoskeletons of crustaceans and more

particularly from shrimps and prawns where chitin is produced. Crabs are another

important source of chitosan which was found to be more amenable for deacetylation.

It has different solubility characteristics based up on its degree of deacetylation. High

degree of deacetylation shows higher solubility and low degree of deacetylation

shows poor solubility. It has swelling characteristics due to much weaker


intermolecular hydrogen bonding ascribable to the parallel arrangement of the main

chains. This swelling property enables floating and drug diffusion mechanism of the

drug delivery system153

.

5.1.2. Physicochemical and biological properties of chitosan

The word chitosan refers to a large number of polymers, which differ in their

degree of N- deacetylation (40–98%) and molecular weight (50,000–2,000,000 Da).

These two characteristics are very important to the physicochemical properties of the

chitosan and hence, they have a major effect on the biological properties. Chitosan is

a weak base with a pKa value of the D-glucosamine residue of about 6.2–7.0 and,

therefore, is poorly soluble at acidic, neutral and strong alkaline pH values. However,

it makes salts with inorganic and organic acids such as hydrochloric acid, acetic acid,

glutamic acid, and lactic acid. In acidic medium, the amine groups of the polymer are

protonated resulting in a soluble, positively charged polysaccharide that has a high

charge density (one charge for each D-glucosamine unit). Chitosan can form gels by

interacting with different types of divalent and polyvalent anions, which help in

diffusion mechanism of drug from its dosage forms.

Increasing the degree of deacetylation increases the viscosity. This can be

explained by the fact that high and low deacetylated chitosan have different

conformations in aqueous solution. Chitosan has an extended conformation with a

more flexible chain when it is highly deacetylated, because of the charge repulsion in

the molecule. However, the chitosan molecule has a rod-like shape or coiled shape at

low degree of deacetylation due to the low charge density in polymer chain. The

viscosity of chitosan solution is also affected by factors such as concentration and

temperature. As the chitosan concentration increases and the temperature decreases,

the viscosity increases.


According to Chen et al.154

the degree of deacetylation of chitosan, which will

determine the number of intermolecular hydrogen bonds, was found to affect the

rigidity of the polymer. For chitosan hydrogels, the extent of dissociation of the

hydrogen bonding may affect the swelling kinetics of the gels. At low pH, the

hydrogen bonding dissociates due to the protonation of the amine groups leading to

faster swelling. When the salt form of chitosan is neutralized, it can form

interpenetrating polymer networks with other neutral polymers, e.g. poly ethylene

oxide, poly vinylpyrrolidone, and poly vinyl alcohol, by inter molecular hydrogen

bonding. Because chitosan has favorable biological properties such as

biodegradability and biocompatibility155

, it has attracted a lot of attention in the

pharmaceutical and medical fields. Chitosan has low oral toxicity with an LD in rats

of 16 g/ kg152

. Toxicity of chitosan might depend on different factors such as degree

of deacetylation, molecular weight, purity, and route of administration. Further studies

of toxicity need to be carried out for particular applications of the polymer. The

chemical, biological and pharmaceutical properties of chitosan are summarized in

Tables 5.1.


Table 5.1: Chemical, Biological and Pharmaceutical properties of chitosan

Chemical properties of

chitosan*

Biological properties of

chitosan*

Pharmaceutical

properties of chitosan*

Cationic polyamine Biocompatibility particle size <30 µm

High charge density at pH<6.5 Natural polymer density between 0.95 and

1.40 g/cm3

Adheres to negatively charged

surfaces

Biodegradable to normal

body constituents pH 6.5–7.5

Forms gels with polyanions Safe and non-toxic insoluble in water

High molecular weight linear

polyelectrolyte

Hemostatic, bacteriostatic,

and fungistatic partially soluble in acids

Viscosity, high to low Spermicidal Swellable by absorbing

water

Chelates certain transitional

metals Anticancerogen

Amiable to chemical

modification Anticholesteremic

Reactive amino/ hydroxyl groups Reasonable cost, Versatile

*Adapted from Ref. [155

]

5.1.3. Chemical methodology for the preparation of chitosan

Shrimp or crab shells proteins are removed by treatment with 3–5% (w/v)

NaOH aqueous solution at 80–90°C for a few hours or at room temperature overnight.

The inorganic constituents of the product are then removed by treatment with 3–5%

(w/v) HCl aqueous solution at room temperature giving a white to beige colored

sample of chitin. Treatment of the sample with 40–45% (w/v) NaOH solution at 90–

120°C for 4–5 h results in N- deacetylation of chitin. The insoluble precipitate is

washed with water to give a crude sample of chitosan. The conditions used for

deacetylation will determine the polymer molecular weight and the degree of


deacetylation. The crude sample is dissolved in 2% (w/ v) acetic acid, then the

insoluble material is removed giving a clear supernatant solution which is neutralized

with NaOH solution resulting in a purified sample of chitosan as a white

precipitate152

. Further purification may be necessary to prepare medical and

pharmaceutical grade chitosan.

5.1.4. Applications of Chitosan

The interest in chitosan, originates from the study of the behaviour and

chemical characteristics of lysosomes and enzymes present in the human body fluids.

It dissolves certain bacteria by cleaving the chitinous material of the cell walls. A

wide variety of medical applications for chitosan derivatives have been reported over

the last three decades. The poor solubility of chitosan is the major limiting factor in its

utilization and investigation of its properties and structure. Despite these limitations,

various applications of chitosan have been reported in the literature153

.

Due to its physical and chemical properties chitosan is being used in a vast

array of widely different products and applications, ranging from pharmaceutical

products and cosmetics products to water treatment and plant protection. In different

application, different properties of chitosan are required. These properties changes

with degree of deacetylation and molecular weight as well. It is already known that

compounds having a molecular weight lower than 2900Da derived from chitosan can

pass through membranes. Since chitosan do not cause any biological hazard and are

inexpensive, these polymers might be suitable for use in the preparation of dosage

forms of commercial drugs156

.

Controlled release technology emerged during the 1980s as a commercially

sound methodology. The achievement of predictable and reproducible release of an


agent into a specific environment over an extended period of time has many

significant merits. The most significant merit would be to create a desired

environment with optimal response, minimum side effect and prolonged efficacy.

This is relatively new technology and requires an interdisciplinary scientific approach.

Chitosan controlled delivery systems are at developing stage and being used for a

wide variety of reagents in several environments156

.

5.2. Methods of Extraction

5.2.1. Raw materials preparation

The different local resources used to extract chitosan are described in Table

5.2. The shells of these species were scraped free of loose tissue, washed thoroughly

free from sand and extraneous matter, then raw material was sun dried for up to 72

hours, and pulverized to make powder which was then passed through a sieve to get

particle size approximately 250µm then subjected to demineralization and

deproteinization157

. Schematic representation is shown in Figure 5.1.

Table 5.2: Different local resources used to extract chitosan

Chitosan source

(Latin name) English name Max. length (cm) Max. width (cm)

Penaeus indicus

(de Haan) Prawn 6 0.3

Metapenaeus affinis

(Milne-Edwards) Jinga Shrimp 15 2.5

Portunus pelagicus

(Linne) Blue Crab-Male 20 27

Portunus pelagicus

(Linne) Blue Crab-Female 20 26


Figure 5.1: Scheme for preparation of Chitosan

Washing, drying, grinding & sieving

Shell powder

Particle size = 250µm

Chitin + CaCO3 + proteins

Demineralized shell

Chitin + proteins

Chitin

Demineralization

0.25-1M HCl

- CaCO3

Deproteinization

1M NaOH, 70°C

24 hrs, - Proteins

Washed with ethanol

Purified by boiling

with Acetone for

15min

Microwave radiation for

20min at 900w Temp

Chitosan

Marine Species

Shell waste from food processing

Treated with 45%

NaOH for 24 hrs at

room temp


5.2.2. Demineralization

The mineral content in the exoskeleton of crustacean was removed by

demineralization. Demineralization was carried out in dilute HCl solution. All species

were treated with 0.25 M HCl solution at ambient temperature with a solution-to-solid

ratio of 40 mL/g. Steps involved in demineralization process are given in following

expression.

𝐷𝑟𝑖𝑒𝑑 𝑆𝑕𝑒𝑙𝑙𝑠 0.25𝑀 𝐻𝐶𝑙

𝐷𝑒𝑚𝑖𝑛𝑒𝑟𝑖𝑙𝑖𝑧𝑎𝑡𝑖𝑜𝑛 ↑ 𝐶𝑂2 → 𝐷𝑒𝑚𝑖𝑛𝑒𝑟𝑖𝑙𝑖𝑧𝑒𝑑 𝑆𝑜𝑙𝑖𝑑 (𝐶𝑕𝑖𝑡𝑖𝑛)

The resulting solid was washed with distilled water until neutral. Then, the

demineralized samples were dried and weighed. The number of baths and their

duration (15-180 min) were dependent on the species. It was observed that the emission

of CO2 gas was more or less important according to the studied species. It also depends

upon the mineral content of different species and penetration of the shells by

hydrochloric acid. It was found that the larger amount of mineral content the greater

gas emission. The CO2 emission was stronger in case of blue crab than other

species157

.

5.2.3. Deproteinization

Deproteinization of chitin was carried out using 1M NaOH (20 mL/g of chitin)

at 70°C. The treatment was repeated several times. The absence of proteins was

indicated by the absence of color of the medium at the last treatment, which was left

overnight. The resulting solution then washed to neutrality. Finally, it was washed

with ethanol (10 mL/g of chitin) and later boiled for 15min in acetone to remove any

impurities. The purified chitin was then dried. The chitin content was determined

from the weight differences of the raw materials and that of the chitin obtained after

acid and alkaline treatments. Ash content of dried chitin was determined by burning


the samples at 600°C in a muffle furnace157

. Steps involved in deproteinization

process are given in following expression.

𝐶𝑕𝑖𝑡𝑖𝑛 1𝑀 𝑁𝑎𝑂𝐻

𝑎𝑡 70°𝐶→ 𝐷𝑒𝑝𝑟𝑜𝑡𝑖𝑛𝑖𝑧𝑒𝑑 𝐶𝑕𝑖𝑡𝑖𝑛 → 𝑊𝑎𝑕𝑒𝑑 𝑤𝑖𝑡𝑕 𝑒𝑡𝑕𝑎𝑛𝑜𝑙 →

𝐵𝑜𝑖𝑙𝑒𝑑 𝑤𝑖𝑡𝑕 𝑎𝑐𝑒𝑡𝑜𝑛𝑒 𝑡𝑜 𝑟𝑒𝑚𝑜𝑣𝑒 𝑎𝑛𝑦 𝑖𝑚𝑝𝑢𝑟𝑖𝑡𝑖𝑒𝑠 → 𝐷𝑟𝑖𝑒𝑑 𝑝𝑢𝑟𝑖𝑓𝑖𝑒𝑑 𝑐𝑕𝑖𝑡𝑖𝑛

5.2.4. Deacetylation

As per literature the conventional method used preparation of chitosan

involves long processing and more time consuming. In order to decrease the long

processing times typically required to achieve N-deacetylation, an alternative

microwave method has been used. Deacetylation of chitin can be highly facilitated by

steeping in strong sodium hydroxide at room temperature before heating. This

steeping method has been adapted to samples for one day before conversion by

microwave radiation method. A mixture of chitin and 45% NaOH was placed in a

conical flask, covered tightly with cotton, and then subjected to microwave radiation.

The mixture then cooled with cold water and after filtration chitosan was washed to

neutral pH and freeze dried using VIRTIS Freezemobile 5EL with sentry

microprocessor control freeze dryer157

. Steps involved in demineralization process are

given in following expression.

𝑃𝑢𝑟𝑖𝑓𝑖𝑒𝑑 𝐶𝑕𝑖𝑡𝑖𝑛 → 𝑇𝑟𝑒𝑎𝑡𝑒𝑑 𝑤𝑖𝑡𝑕 45% 𝑁𝑎𝑂𝐻 𝑎𝑡 𝑟𝑜𝑜𝑚 𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑒 𝑓𝑜𝑟 24 𝑕

→ 𝑀𝑖𝑐𝑟𝑜𝑤𝑎𝑣𝑒 𝑟𝑎𝑑𝑖𝑎𝑡𝑖𝑜𝑛 𝑓𝑜𝑟15 min𝑎𝑡 900𝑊 𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒

→ 𝐹𝑟𝑒𝑒𝑧𝑒 𝑑𝑟𝑖𝑒𝑑 𝑡𝑜 𝑔𝑒𝑡 𝑠𝑜𝑙𝑖𝑑 𝑐𝑕𝑖𝑡𝑜𝑠𝑎𝑛

The deacetylation kinetics was followed by monitoring the DDA% as a

function of time. In microwave heating method the duration of subjecting microwave

radiation to chitin/NaOH mixture was 6, 8, 10, 12 and 15min at 900W.


5.2.5. Determination of degree of deacetylation

The deacetylation degree of chitosan was determined by the potentiometric

titration method using Calomel and silver/silver-chloride electrodes158

. 0.5g of

chitosan was dissolved in 25mL of 0.1M standard aqueous HCl solution. The solution

was then made up to 100mL with distilled water and calculated amount of KCl was

added to adjust the ionic strength to 0.1. The titrant was a solution of 0.05M NaOH.

pH was measured using pH meter under continuous stirring. The titrant was added

until the pH values reached 1, the standard NaOH was then added stepwise and the

pH values of solution were recorded and a curve with two inflection points were

obtained. The difference between the volumes of these two inflection points

corresponded to the acid consumption for the salification of amine groups of chitosan

and permitted the determination of degree of acetylation, through the following

Equation,

DDA% =12.1(V2-V1) × Mb/W

Where (V1 and V2) are the base volumes referred to first and second inflection

points respectively in mL, (Mb) is the base molarity in g/mol, and (W) is the original

weight of the polymer in g.

The data obtained for the degree of deacetylation of chitosan is given in Table.

5.3. The DDA% is shown in Figure 5.2 which was obtained by considering time in min

on x-axis and DDA% on y-axis.

The chitosan samples were obtained in different time interval of 6, 8, 10, 12, 14,

16, 18 and 20min with specific DDA of 21, 33, 45, 56, 65, 72, 79 and 88% respectively.

The samples were subjected for the determination of bulk density, swelling index,


viscosity and solubility to find out the suitability as these properties are important for

floatation. The results obtained for these parameters are presented in Table. 5.3.

Table 5.3: Degree of deacetylation of chitosan at various time interval and results

obtained for the evaluation of preliminary parameters

Time in

min DDA%

Density

g/cm3

Swelling index

%

Viscosity

cps

Solubility in

0.1N HCl

6 21 4.26 ± 1.25 22.74 ± 2.58 124 ± 4.65 Insoluble

8 33 3.68 ± 2.15 28.64 ± 4.62 259 ± 5.54 Insoluble

10 45 3.25 ± 0.58 32.96 ± 1.25 354 ± 6.65 Insoluble

12 56 2.68 ± 0.97 45.15 ± 3.62 346 ± 4.65 Insoluble

14 65 1.45 ± 0.64 74.52 ± 2.56 385 ± 4.23 Slightly soluble

16 72 0.95 ± 0.65 97.58 ± 0.54 462 ± 7.56 Poorly soluble

18 79 0.96 ± 2.56 95.94 ± 1.64 476 ± 8.62 Sparingly soluble

20 88 0.92 ± 1.85 82.68 ± 6.67 514 ± 3.45 Freely soluble

Figure 5.2: Degree of deacetylation

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16 18 20

Time in min

DD

A%


5.2.6. Percentage yield of chitosan

Microwave method has been used to extract chitosan this method is an

effective than the conventional heating method. The percentage yield was calculated

from the ratio between the initial weight and the final yield multiplied by 100. The

percentage yield of chitosan is given in Table 5.4 and depicted as Figure 5.3.

Table 5.4: Percentage yield of chitosan

Source Percentage yield of chitosan

Prawn 7.85

Jinga Shrimp 5.68

Blue Crab-Male 12.64

Blue Crab-Female 9.88

Figure 5.3: Percentage yield of chitosan

0

2

4

6

8

10

12

14

Prawn Jinga Shrimp Blue Crab-

Male

Blue Crab-

Female

% Y

ield

of

chit

osa

n


5.3. Characterization

5.3.1. Fourier Transform Infrared Spectroscopy (FTIR)

Infrared spectra was measured by KBr-supported sample of chitosan over the

frequency range 4400-400 cm-1

at resolution of 4 cm-1

using a model 2000 Perkins-

Elmer spectrometer performed at Laila Impex research centre, Vijayawada. The

sample was thoroughly mixed with KBr, the dried mixture was then pressed to result

in a homogeneous sample/KBr disc159

. The FTIR spectra was recorded and

interpreted to find out the functional groups presents which were compared with

reported reference standard. FTIR spectrum is depicted as Figure 5.4 and

interpretation of spectral data are given in Table 5.5.

Figure 5.4: FTIR Spectra of chitosan-I


Table 5.5: Interpretation of FTIR spectral data of Chitosan

424.76, 542.94, 607.41, 637.70 C-Br stretch

689.69, 851.64 C-H out of plane

777.77, 902.41 C-Cl stretch, P-OR esters

982.60, 1009.99, 1116.68, 1147.15 P-H bend, C-N stretch, C-O stretch

1202.38 C-H stretch

1252.79, 1286.24, 1329.75 C-O stretch

1427.66, 1491.13 C-C ring stretch

1534.23, 1600.68, 1639.54 N=O nitroso, C-O stretch, C=C stretch

2937.08, 3103.74, 3237.83, 3376.69 CH stretch

3400.94, 3505.71 OH stretch

5.3.2. Differential scanning calorimetry (DSC)

DSC was performed using a 10mg sample from ambient to 350°C at a heating rate

of 10°C/min in a dynamic (50 mL/min) synthetic air atmosphere using DSC-50 Shimadzu

automatic analyzer performed at Diya Labs, Mumbai. The optimum temperature to melt the

chitosan sample was found to be 137.4°C at -4.16(W/g)157

. DSC spectrum is depicted as

Figure 5.5.

Figure 5.5: DSC Thermogram of Chitosan-I


5.3.3. X-ray powder diffractometry (XRD)

The XRD measurement on powder sample was carried out to detect the

crystallinity of the extracted sample of chitosan. XRD study was performed by using a

model D500 Siemens diffractometer at Diya Labs, Mumbai. The diffractometer was

operated between 2θ angles of 5° and 45°. Ni-filtered Cu Kα radiation was used as the

X-ray source157

. The relative crystallinity of the polymer was calculated by dividing

the area of the crystalline peaks by the total area under the curve. The percentage

crystallinity at different angles are given in Table. 5.6 and XRD pattern of chitosan is

depicted as Figure 5.6.

Figure 5.6: XRD patterns of Chitosan-I

Table 5.6: Interpretation of XRD pattern of Chitosan

Peak at 2θ angle % crystallinity

10.4° 62.4

20.8° 44.6

30.2° 31.4

34.1° 27.5

41.4° 28.4


5.3.4. Scanning electron microscopy (SEM)

The surface morphology of chitosan was observed using SEM. The dried

sample of chitosan was ground and then coated with gold under vacuum using a

sputter coater. The scanning electron microscopy (SEM) was conducted using a JEOL

JSM-630 J scanning electron microscope operated at 5.0 kV157

. The observed SEM

images at different magnification are depicted as Figure 5.7.

Figure 5.7 SEM micrographs of Chitosan-I


5.3.5. Nuclear magnetic resonance 13

C NMR

NMR spectra were recorded using Bruker II 600 spectrometer in 2%

deuterated acetic acid in D2O solution. The experiments were run at 70°C,

temperature at which the solvent (HOD) peak does not interfere with any chitosan

peaks. After dissolution, approximately 1 mL of the chitosan sample solution was

transferred to 5 mm NMR tube. The sample tube was inserted in the magnet and

allowed to reach thermal equilibrium for 10 min before performing the experiment.

Then measured NMR spectrum was interpreted and compared with reported reference

standard to conform the presence of chitosan compound157

. Interpretation of NMR

spectral data is given in Table. 5.7. The NMR spectrum is depicted as Figure. 5.8.

Figure 5.8: 13

C NMR Spectrum of Chitosan-I


Table 5.7: Interpretation of NMR spectrum of Chitosan

Location Ppm Intensity Comment

O

H

OH

H2N

O

O OH

H

HH

H74.24

80.62

70.86

107.53

64.35

52.24

Predicted structure of Chitosan based

on Interpretation

1 107.53 662 Tetrahydropyran

2 52.24 776 Aliphatic




6 64.35 984 Aliphatic

Chemical formula C8H17NO5

Chemical Name 3-amino-tetrahydro-6-(hydroxymethyl)-

2,5-dimethoxy-2H-pyran-4-ol

5.3.6. Mass spectroscopy study (MS)

MALDI-TOF MS, Matrix Assisted Laser Desorption Ionization-Time Of

Flight MS, involves mixing the sample with a matrix, which is then coated on to a

plate or probe, and subjected to a collimated focused laser beam, causing ionization

and desorption. MALDI enables analysis of molecules up to approximately 500kDa

and has the advantage of producing large mass ions, with high sensitivity and little

fragmentation. MALDI is now commonly coupled with Delayed Extraction (DE-

MALDI), causing the produced ions to enter the Time of Flight analyzer at the same

time. MALDI-TOF MS is used to determine molecular weight and may be employed

in a large number of biochemical applications159

. Based on the interpretation of mass

spectra the molecular weight of chitosan was found to be 207.44. The mass spectrum

is depicted as Figure 5.9.


Figure 5.9: Mass Spectrum of Chitosan-I

5.4. Standardization of chitosan159, 160

Partial deacetylation of chitin results in the production of chitosan, which is a

polysaccharide comprising copolymers of glucosamine and N-acetylglucosamine.

Chitosan is the term applied to deacetylated chitins in various stages of deacetylation

and depolymerization and it is therefore not easily defined in terms of its exact

chemical composition. A clear nomenclature with respect to the different degrees of

N-deacetylation between chitin and chitosan has not been defined, and as such

chitosan is not one chemical entity but varies in composition depending on the

manufacturer. In essence, chitosan is chitin sufficiently deacetylated to form soluble

Amine salts. The degree of deacetylation necessary to obtain a soluble product must

be greater than 80–85%. Chitosan is commercially available in several types and

grades that vary in molecular weight by 10,000–1,000,000, and vary in degree of

deacetylation and viscosity.


5.4.1. General properties

The isolated chitosan was white to light yellow flakes. There was no

characteristics odour and taste.

5.4.2. Solubility

Sparingly soluble in water; poorly soluble in 0.1N HCl and simulated gastric

fluid; practically insoluble in ethanol (95%), other organic solvents, and neutral or

alkali solutions at pH above approximately 6.5. Chitosan dissolves readily in dilute

and concentrated solutions of most organic acids and to some extent, in mineral

inorganic acids (except phosphoric and sulfuric acids). Upon dissolution, amine

groups of the polymer become protonated, resulting in a positively charged

polysaccharide and chitosan salts (chloride, glutamate, etc.) that are soluble in water;

the solubility is affected by the degree of deacetylation.

5.4.3. Viscosity

A wide range of viscosity types is commercially available. Owing to its high

molecular weight and linear, unbranched structure, chitosan is an excellent viscosity

enhancing agent in an acidic environment. It acts as a pseudo-plastic material,

exhibiting a decrease in viscosity with increasing rates of shear. The viscosity of

Chitosan solution increases with increasing chitosan concentration, decreasing

temperature, and increasing degree of deacetylation. Viscosity of chitosan (1%w/v)

was performed by using Ostwald viscometer by dissolving the chitosan in 2% acetic

acid.

5.4.4. Particle size distribution

Particle size of powdered chitosan was determined by sieve analysis. Sieves

40-180 were used and checked properly for their integrity. The particle size of

chitosan was found to be <30 mm.


5.4.5. Determination of loss on drying

Loss on drying is the loss in weight in % w/w resulting from water and

volatile matter of any kind that can be driven off under specified conditions. The test

is carried out on a well-mixed sample of the substance using IR moisture analyser. If

the substance is in the form of large crystals, reduce the size by rapid crushing to a

powder.

Weigh a glass-stoppered, shallow weighing bottle that has been dried under

the same conditions to be employed in the determination. Transfer to the bottle the

quantity of the sample specified in the individual monograph, cover it and accurately

weigh the bottle and the contents. Distribute the sample as evenly as practicable by

gentle sidewise shaking to a depth not exceeding 10mm. Place the loaded bottle in the

drying chamber (oven or desiccator) as directed in the monograph, remove the stopper

and leave it also in the chamber. Dry the sample to constant weight or for the

specified time and at the temperature indicated in the monograph. After drying is

completed, open the drying chamber, close the bottle promptly and allow it to cool to

room temperature (where applicable) in a desiccator before weighing. Weigh the

bottle and the contents.

5.4.6. Determination of sulphated ash

Silica was heated to redness for 10 minutes, allowed to cool in a desiccator

and weighed. The crucible 1g of the substance being examined was transferred and

contents were weighed. The sample was ignited until the substance was thoroughly

charred. The residue was moistened with 1mL of sulphuric acid, heated gently until

the white fumes are no longer evolved and ignite at 800°C ± 25°C until all black

particles have disappeared. The crucible was allowed to cool, few drops of sulphuric


acid was added and heated. The operation was repeated until two successive weighing

do not differ by more than 0.5 mg.

5.4.7. Glass Transition Temperature

The glass transition temperature (Tg) of the dry material was determined using

Model DSC-50 Shimadzu automatic analyzer. Sample size varied between 5 and

10mg, heating rate was maintained at 40K min-1

.

The temperature interval was set to -50 to 150°C. The Tg was determined by

calculating the temperature of the half step height.

5.4.8. Limit test for chlorides

Chitosan solution was prepared as directed in the monograph and transfer to a

Nessler cylinder. 10 mL of dilute nitric acid was added to this, further diluted to 50

mL with water and 1 mL of 0.1M silver nitrate was added. The solution was stirred

immediately with a glass rod and allowed to stand for 5 minutes and protected from

light. When viewed transversely against a black background any opalescence

produced is not more intense than that obtained by treating a mixture of 10.0 mL of

chloride standard solution (25 ppm cl) and 5 mL of water in the same manner.

5.4.9. Limit test for heavy metals

The limit for heavy metals is indicated in the individual monographs in terms

of ppm, i.e., the parts of lead, Pb, per million parts (by weight) of the substance being

examined.

Standard solution: 1mL of lead standard solution (20 ppm Pb) was pipetted and

transferred in to 50mL Nessler cylinder and diluted with water to 25 mL. pH was

adjusted with dilute acetic acid between 3.0 and 4.0.


Test solution: 25 mL of the test solution was prepared in 50mL Nessler cylinder as

directed in the monograph by dissolving the specified quantity of the substance being

examined in sufficient water to produce 25mL. pH was adjusted with dilute acetic

acid between 3.0 and 4.0, dilute with water to about 35 mL and mixed well.

Procedure: 10 mL of freshly prepared hydrogen sulphide solution was added to each

of the cylinders containing the standard solution and test solution respectively, mixed

well, diluted to 50 mL with water, allowed to stand for 5 minutes and viewed

downwards over a white surface; the colour produced with the test solution was not

more intense than that produced with the standard solution.

5.4.10. Limit test for arsenic

Limit test of arsenic was performed to determine the presence of trace

amounts of arsenic by converting the arsenic in a substance under test to arsine, which

was then passed through a solution of silver diethyl dithiocarbonate to form a red

complex. The red complex so produced is compared visually, to the colour produced

similarly in a control containing an amount of arsenic equivalent to the limit given in

the individual monograph. The test was performed with arsenic test apparatus.

Procedure: Standard was prepared by pipetting 3mL of standard arsenic solution in

to a generator flask and diluted to 35mL with water. Test was prepared as per

individual monograph. 20mL of sulfuric acid, 2mL of potassium iodide, 0.5mL of

strong acid stannous chloride and 1mL of isopropyl alcohol were mixed with standard

and test solutions separately. Allowed to stand at room temperature for 30min. Then

3mL of silver diethyl dithio carbonate and 3g of granular zinc were added to the

mixture and allowed to evolution of hydrogen and the colour development to proceed


at room temperature for 45min. Any red colour produced by the test preparation does

not exceed that produced by the standard preparation.

5.4.11. Sterility test

The sterility test was carried out as per Indian pharmacopoeia. Sterility test

was intended for detecting the presence of viable forms of bacteria, fungi and yeasts

in the sample. The test was performed based on the principle that if bacteria or fungi

placed in a medium which provides nutritive material and water and kept at room

temperature, the organism will grow and their presence can be indicated by turbidity

in the clear medium.

A simple direct inoculation method was used to carry out sterility test. In this

method the specified quantity of sample was introduced under aseptic condition into a

test tube of culture medium. Then it was incubated at 25°C and 45%RH for 14 days

and the growth of micro organisms in the medium was observed. The culture medium

was examined during and at the end of incubation period to find out any microbial

growth.

5.4.12. Determination of Bulk density and tap density

Apparent bulk density (b) was determined by pouring the material into a

graduated cylinder. The bulk volume (Vb) and weight of the powder (M) was

determined. The bulk density was calculated using the formula.

b = M/Vb

The measuring cylinder containing a known mass of powder was tapped for a

fixed time. The minimum volume (Vt) occupied in the cylinder and the weight (M) of

the blend was measured. The tapped density (t) was calculated using the following

formula,


t = M/Vt

5.4.13. Compressibility Index

The simplest way for measurement of free flow of powder is compressibility,

a indication of the ease with which a material can be induced to flow is given by

compressibility index (I) which is calculated as

I = (t - o/t) 100

t = tapped density

o = initial bulk density

The value below 15% indicates a powder which usually give rise to good flow

characteristics whereas above 25% indicate poor flowability.

5.4.14. Stability and Storage Conditions

Chitosan powder is a stable material at room temperature, although it is

hygroscopic after drying. Chitosan was stored in a tightly closed container in a cool,

dry place.

The results of all above standardization parameters were compared with

reference standard of reported articles are given in Table 5.8.


Table 5.8: Results of standardization of chitosan

Parameters evaluated Reference standard Results obtained

Appearance White to light yellow flake White to light yellow flake

Odour and Taste None No characteristic odour and

taste observed

Solubility Insoluble to freely soluble

depends on %DDA

Poorly soluble at 72%

DDA

Viscosity Less than 100cps 464 ± 6.62 cps

Particle size distribution (µm) 40-80 mesh 60 mesh

Incompatibility Nil Nil

Loss on trying Less than 10% 8%

Ash content <1.5％ 0.8％

Glass transition temperature NA 76.4°C

Limit test for chloride <25ppm <25ppm

Limit test for heavy metals <20ppm <20ppm

Limit test for Arsenic <10ppm <10ppm

Acidity/Alkalinity (pH) 4-6 6.5

Sterility test Absence of viable content Absence of viable content

Molecular weight 150-450 207.44

Degree of deacetylation % 50-120% 72%

Bulk density (g/cm3) 0.366 ± 0.006 (g/cm

2) 0.245 ± 0.006 (g/cm

2)

Tap density (g/cm3) 0.334 ± 0.027 (g/cm

2) 0.289 ± 0.027 (g/cm

2)

Compressibility Index (%) 24.70 ± 1.52 (%) 15.76 ± 1.24 (%)

Stability Stable at room temperature Stable at room temperature

Storage condition

Chitosan should be stored in

a tightly closed container in

a cool, dry place.

Chitosan should be stored

in a tightly closed

container in a cool, dry

place.


5.5. Results and discussion

5.5.1. Percentage yield of chitosan

Chitosan was isolated from four sources, two kinds of marine crab female and

crab male shells, shrimp and prawn shells, all from the India, Tuticorin region of the

Bay of Bengal. All these species were identified and authenticated by Dr. Santhanam,

Dean, Fisheries and animal science university, Thuthukudi, Tamil Nadu. Different

local resources used to extract chitin are shown in Table 5.2. Microwave method has

been used to extract chitosan. The percentage yield was found to be lowest in shrimp

5.68%, in prawn 7.85% and highest in the crab male shell contains higher chitosan

12.64% than crab female 9.88%. The percentage yield of chitosan is given in Table

5.4 and depicted as Figure 5.3.

5.5.2. Deacetylation of chitosan

To avoid long heating times, chitosan was prepared from chitin by

deacetylation in 45% sodium hydroxide solution using microwave radiation

technology. Microwave heating, as an alternative to conventional heating techniques,

has been proved more rapid and efficient for chemical reactions. To speed up the

process, the chitin was steeped in 45% sodium hydroxide for 24 h at room

temperature before subjecting chitin to microwave radiation. The degree of

deacetylation for poorly soluble chitosan was determined by potentiometric titration

method. DDA% was determined at various time intervals of 6, 8, 10, 12, 14, 16, 18

and 20min have shown 21, 33, 45, 56, 65, 72, 79 and 88% respectively. Effect of

time on the DDA% under microwave heating method for chitosan found to be

increasing with time. The deacetylation percentage above 70 was obtained after

15min in microwave heating. 72% of DDA was obtained at 16min, at this stage the


chitosan is having all desired properties required for FDDS, which is confirmed by

determining density, swelling index, viscosity, solubility. The results obtained for

these parameters are given in Table 5.3. The DDA% with time is shown in Figure 5.2.

5.5.3. FTIR analysis

IR Spectra of chitosan is shown in Figure 5.4. A single band was observed in

spectrum at 3441.56cm-1

which is commonly assigned to the stretching of the CO

group hydrogen bonded to amide group of the neighboring intra-sheet chain. The

strong band at 1430cm-1

is seen in the spectrum while a distinct band at 1416cm-1

occurs in the spectrum of chitosan. The band due NH stretching at 3264cm-1

and

3107cm-1

can be seen clearly in the spectrum assigned these bands to CO-NH

intermolecular bonding and H bonded NH group. OH-out-of plane bending at 703cm-1

and NH-out-of plane bending at 750cm-1

can be observed in the spectrum. This is due

to a relatively low crystalline and loosely ordered structure showing weaker inter- and

intramolecular hydrogen bonding in chitosan. Interpretations of IR spectral data are

given in Table. 5.5.

5.5.4. Differential Scanning calorimetric analysis (DSC)

A DSC curve of chitosan was shown in Figure 5.5. The curve shows that

weight loss occurs in two stages. The first stage starts around 91.6°C and the second

stage starts around 316°C. The first stage is assigned to the loss of water because

polysaccharides usually have a strong affinity for water and therefore may be easily

hydrated.

The second one corresponds to the thermal decomposition of chitosan. The

decomposition temperature of chitosan was found to be 316°C. This result indicates

that chitosan exists as a stable structure toward thermal decomposition.


5.5.5. X-Ray powder diffractometry of chitin

XRD analysis was applied to detect the crystallinity of the isolated chitosan.

The XRD pattern of chitosan, show five sharp crystalline reflections at 10.4°, 20.8°,

30.2°, 34.1° and 41.2°.

X-ray diffraction exposed the characteristics of chitosan more clearly due to

the different arrangements adopted by these polymorphs. Figure 5.6 shows XRD pat-

terns of chitosan. The XRD profile of chitosan exhibits well resolved and intense

peaks while a broad diffuse scattering and less intense peaks were found for chitosan

at 10.4° and 20.8°, crystallinity percent was found to be 62.4 and 44.6 respectively.

This indicates that chitosan is a partially crystalline polymorph because of its

antiparallel compact structure.

The crystallinity percentage was calculated by dividing the area of the

crystalline peaks by the total area under the curve. The X-ray diffraction data are

presented in Table 5.6.

5.5.6. Scanning electron microscopy (SEM)

Figure 5.7 shows SEM photographs of powder chitosan. A very uniform with

a lamellar organization and dense structure was observed clearly for chitosan.

5.5.7. 13

C NMR analysis

Chemical composition of chitosan was obtained by 13

C NMR spectrum using

2% deuterated acetic acid in D2O as solvent. Figure 5.8 shows the 13

C NMR spectrum

(600 MHz) of chitosan. The absence of methyl proton resonance from protein

between 1.0 and 1.5 in 13

C NMR spectrum gives a good indication to the purity of

chitosan sample. Interpretations of NMR data are given in Table 5.7.


5.5.8. Mass spectroscopy

Figure 5.9 shows the mass spectrum of chitosan was measured by MALDI,

which enables analysis of molecules approximately up to 500KDa. Molecular weight

of the chitosan was calculated by interpreting the mass spectrum was found to be

207.44.

5.5.9. Standardization of chitosan powder

The isolated chitosan powder was subjected for standardization using many

parameters such as appearance, odour, taste, solubility, viscosity, particle size

distribution, incompatibility, loss on trying, ash content, glass transition temperature,

limit tests, pH, sterility test, density, and degree of deacetylation.

The appearance of isolated chitosan was white to light yellow flake it does not

have any characteristic odour and taste. The pH was found to be 6.5, which indicates

more compatible with stomach environment. Density was low as 0.95g/mL which

may be helpful in the development of floating system.

Chitosan was poorly soluble in water; practically insoluble in ethanol (95%),

other organic solvents, and neutral or alkali solutions at pH above approximately 6.5.

Chitosan dissolved readily in dilute and concentrated solutions of acetic acid and

sparingly soluble in hydrochloric acid.

The viscosity of chitosan (1% w/v) was performed by using Ostwald

viscometer. Viscosity was found to be 464 ± 6.64cps which confirms chitosan is an

excellent viscosity enhancing agent in an acidic environment.

Chitosan was complied all other evaluated parameters such as viscosity, limit

test for chloride heavy metals and arsenic, loss on trying and ash content were found

to be 464 ± 6.62 cps, <25ppm, <20ppm, <10ppm, 8% and 0.8% respectively.


The apparent particle, tap and bulk densities of chitosan were shown within

the limit. The apparent density increases slightly with higher N-acetylation. Bulk and

tap density are dependent on particle density which indicated good flowability of the

powder and hence while using both these values the Carr index was calculated. The

lower Carr index indicates the better flowability of the powder. These was no physical

changes observed during storage condition which indicates the isolated chitosan is

more stable at room temperature

The sterility test indicated no evidence of growth hence the chitosan sample

being examined was passes the test for sterility. Results obtained for all

standardization parameters are given in Table 5.8.

5.6. Conclusion

Chitosan has been isolated from local marine sources of Thuthukudi, Tamil

Nadu, India, by treatment with dilute HCl solution for demineralization, and dilute

NaOH for deproteinization. In FTIR spectra, the amide band is split for chitosan. The

XRD, SEM results indicate that chitosan is a crystalline polymorph because of its

parallel structure.

Chitosan was hydrolyzed using microwave heating method. Chitosan

produced from microwave heating reduced the time of deacetylation within few min-

utes (~16 min) to reach to the DDA% of 72%.

Chitosan is an abundant natural polymer, obtained by alkaline N-deacetylation

of chitin. The physical and chemical properties of chitosan, such as inter-and

intramolecular hydrogen bonding and the cationic charge in acidic medium, makes

this polymer attractive for the development of floating drug delivery system. Being a

natural polymer and having all desired properties, chitosan can be used as a good


candidate for oral floating drug delivery. Also, because of its favorable biological

properties such as non-toxicity, biocompatibility, and biodegradability, chitosan is a

promising candidate for the enhancement of absorption of drugs through floatation

using floating drug delivery system. Chitosan acts as an absorption enhancer in the

intestine by increasing the residence time of dosage forms at mucosal sites. As a result

of the physical, chemical, and biological properties, chitosan can be used in many

different formulations for drug delivery in the GI tract.

Based on the results obtained from characterization and standardization it can

be concluded that the isolated chitosan has all desired properties that are required to

develop FDDS. These properties allow for the application of chitosan to be used for

proposed floating drug delivery system.

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