Synthesis of Apatite - Chitosan Composite Using Duck Eggshell and Chitosan with Temperature and pH Control

SYNTHESIS OF APATITE - CHITOSAN COMPOSITE USING
DUCK EGGSHELL AND CHITOSAN WITH TEMPERATURE
AND pH CONTROL

CUCU SUKMAYA

DEPARTMENT OF PHYSICS
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
2010

SYNTHESIS OF APATITE - CHITOSAN COMPOSITE USING
DUCK EGGSHELL AND CHITOSAN WITH TEMPERATURE
AND pH CONTROL

CUCU SUKMAYA

DEPARTMENT OF PHYSICS
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
2010

And say:
“Work (righteousness): Soon will Allah observe your work, and
His Messenger, and the Believers: Soon will ye be brought back
to the knower of what is hidden and what is open: then will He
show you the truth of all that ye did.”

I dedicated this small-thesis to my parents and my family

ABSTRACT

CUCU SUKMAYA. Synthesis of Apatite-Chitosan Composite Using Duck Eggshell and Chitosan with Temperature
and pH Control. Supervised by AKHIRUDDIN MADDU and SETYANTO TRI W.
Composite of apatite-chitosan was prepared by using chitosan as polymer matrix. Calcium phosphate was prepared
from CaO and (NH4)2HPO4, which the ratio Ca : P had to be 1.67. Chitosan is produced by deacetilase from
biopolymer chitin. Duck eggshell (95% CaCO3) as starting material was converted to be calcium oxide (CaO)
through calcinations with temperature 1000oC for 5 hours. In-situ and ex-situ methods were prepared from apatite
and chitosan (dissolved with acetate acid 2%). Calcium phosphate prepared for bone implantation, but it is too
fragile and brittle to be implant into the body. So addition of chitosan can decrease crystalinity and increase
mechanic characteristic (tougher). Composite apatite-chitosan was resulted by fast precipitation. Fast precipitation

was held in a breaker glass with nitrogen atmosphere, under controlled temperature at 37oC and pH 7, in order to
increase amount of hydroxyapatite. Precipitation with CaO which was dropped by (NH4)2HPO4. Hydroxyapatite and
chitosan were appearing at samples with in-situ and ex-situ methods which indicating by peak matching of XRD
(closed lattice parameter, crystal size), overlapping at FTIR spectra and supporting by SEM picture (homogenous
and agglomerated particle). However, it still needs further research to examine biocompatibility osteoconductive
degree of sample by in-vivo and in-vitro test.
Keyword: calcium phosphate, chitosan, temperature, pH

Title

:

Synthesis of Apatite - Chitosan Composite Using
Chitosan with Temperature and pH Control

Name

:

Cucu Sukmaya

NIM

:

G74051483

Duck Eggshell and

Approved by:
1st Supervisor,

2nd Supervisor,

(Dr. Akhiruddin Maddu)
NIP : 19660907 199802 1 006

(Setyanto Tri W, M.Si.)
NIP : 19760731 200501 1 003

Known by:
Head of Physics Department

(Dr. Ir. Irzaman, M.Si)
NIP : 19630708 199512 1 001

Graduation date:

SYNTHESIS OF APATITE - CHITOSAN COMPOSITE USING
DUCK EGGSHELL AND CHITOSAN WITH TEMPERATURE
AND pH CONTROL

Thesis
a paper submitted in partial fulfillment
of requirement for bachelor degree
Faculty of Mathematics and Natural Sciences
Bogor Agricultural University

CUCU SUKMAYA

DEPARTMENT OF PHYSICS
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
2010

PREFACE
Alhamdulillah, I could finish this thesis successfully, which is one of biophysics project of Physics
Department funded by Hibah A2 Bersaing Dikti 2008. This research synthesizes biocomposite of hydroxyapatitechitosan for using in medical application as material of bone implantation.
The realization of this project would not have been possible without the dedicated helping of
Dr. Akhiruddin Maddu, and Mr. Setyanto Tri W, M.Si as my research supervisors. To all of these people, I owe its
whole-hearted gratitude that impossible to describe. Nonetheless, I also would like thank to Mr. Sulistyoso Giat MT
and Mr. Wisnu from BATAN for XRD analysis, Mr. Wikanda and Mr. Wawan from Geology Laboratories Bandung
for SEM-EDXA characterization.
I am also thankful for Ummi, Abi (alm), Ayu, Ende, Ujang, Mr. Henky, Leti and whole families,
Hydroxyapatite team work, for the pray and support. To my entire lecturer thank you for all guidance and
dedication. For all of Physics 42 and for my entire friend who stand beside me, it is pleasure to be here with you all.
Thank you so much.

Bogor, May 2010

CURRICULUM VITAE
Cucu Sukmaya was born in Ciamis on 1 September 1988 as the first child from the couple of
Elon Sunardi (Alm) and Lilis Rosmiati.
He was a student in TK Miftahul Huda at 1991-1993, SD Negeri 1 Gereba at 1993-1999,
SMP Negeri 1 Kawali at 1999-2002, and SMA Negeri 1 Kawali at 2002-2005.
He was accepted at IPB in 2005 by means of USMI as department of physics student.
During studying, the writer was an active student in staff of Student Executive Board
Organization IPB at 2005-2007, had ever been a chair of Student Executive Board Organization
MIPA faculty at 2008, in HIMAFI at 2007-2008. The writer had ever been a laboratory assistant
of Elementary Physics at 2005-2008, assistant of Islamic Education for TPB 2007-2009, teaches in International
SAHID Boarding School 2009-2010 and teaches in Islamic of Junior High School at 2008-now.

CONTENTS
Page
FIGURE LIST ......................................................................................................................................viii
TABLE LIST ......................................................................................................................................... ix
APPENDIX LIST ................................................................................................................................... x
INTRODUCTION
Background..................................................................................................................................... 1
Objective of Research ..................................................................................................................... 1

Hypothesis ...................................................................................................................................... 1
Time and Place of Research ........................................................................................................... 1
THEORY
Human Bone Structure ................................................................................................................... 1
Apatite ............................................................................................................................................ 2
Hydroxyapatite ............................................................................................................................... 3
Chitin and Chitosan ........................................................................................................................ 3
Duck Eggshell ................................................................................................................................ 4
Apatite-Chitosan Composite ........................................................................................................... 4
Sample Characterization ................................................................................................................. 5
Atomic Absorption Spectroscopy (AAS) ....................................................................................... 5
X-RAY Diffraction (XRD) ............................................................................................................. 5
Scanning Electron Microscopy (SEM) ........................................................................................... 5
Fourier Transform-Infra Red (FT-IR)............................................................................................. 6
MATERIALS AND METHODS
Materials and Equipments .............................................................................................................. 6
Experimental Methods .................................................................................................................... 6
Duck Eggshell Calcinations ...................................................................................................... 6
Precipitation and Composite Synthesis ..................................................................................... 6
RESULT AND DISCUSSION

Characteristic of Calcined Duck Eggshell .............................................................................................. 7
Characteristic of Apatite-Chitosan Composite ........................................................................................ 9
X-Ray Diffraction (XRD) ............................................................................................................... 9
Fourier Transform-Infra Red (FTIR) ............................................................................................ 10
Scanning Electron Microscopy (SEM) ......................................................................................... 11
CONCLUSION ..................................................................................................................................... 11
REFERENCES ..................................................................................................................................... 12
APPENDIX ........................................................................................................................................... 14

FIGURE LIST
Page
1. The crystal structure of hydroxyapatite ................................................................................................... 3
2. Shrimps as one of the chitin resource ..................................................................................................... 3
3. Structure of chitosan ............................................................................................................................... 4
4. Ducks and Duck Eggshell ....................................................................................................................... 4
5. Atomic Absorption Spectroscopy processes ........................................................................................... 5
6. X-Ray Diffraction processes ................................................................................................................... 5
7. Scanning Electron Microscopy processes ............................................................................................... 6
8. Fourier Transform Infra-Red processes .................................................................................................. 6
9. FTIR spectra of heated duck eggshell at 1000oC within (a) 5 hours (b) 10 hours................................... 8

10. XRD pattern of heated duck eggshell at 1000oC within (a) 5 hours (b) 10 hours ................................... 8
11. XRD patterns of A1, A2 and A3 ............................................................................................................. 9
12. XRD patterns of B1, B2 and B3............................................................................................................ 10
13. FTIR spectra sample A1, A2, A3 and Chitosan .................................................................................... 10
14. SEM picture of sample A1, A2, A3 and chitosan ................................................................................. 11

TABLE LIST
1.
2.
3.
4.
5.
6.
7.
8.
9.

Page
Contain of bone minerals ........................................................................................................................ 2
Composite material for use in the body .................................................................................................. 2

Duck eggshell major composition ........................................................................................................... 4
Samples code .......................................................................................................................................... 7
Percent of calcium calcined duck eggshell by AAS................................................................................ 8
Percent transmission of carbonate groups ............................................................................................... 8
Crystallinity of sample ............................................................................................................................ 9
Lattice parameter of sample ................................................................................................................... 9
Crystal size of sample ........................................................................................................................... 10

APPENDIX LIST
1.
2.
3.
4.
5.
6.
7.
8.
9.

Page

Experimental Flow Chart ...................................................................................................................... 14
XRD pattern of calcined duck eggshell ................................................................................................ 15
Characterization Devices ...................................................................................................................... 16
Calculation of Chemical Reaction ........................................................................................................ 18
Calculation of maximum XRD peak of samples ................................................................................... 19
JCPDS Reference .................................................................................................................................. 25
Lattice parameter calculation match to hidroxyapatite ......................................................................... 28
Crystal size of samples .......................................................................................................................... 40
AAS of duck eggshell calcination ......................................................................................................... 41

1

INTRODUCTION
Background
A biomaterial is a synthetic material
used to replace part of a living system or in
function to intimate contact with living tissue
[1]. A biomaterial is different from a
biological material such as bone that is
produced by a biological system, but it has
resembled structure and function. Bones are
rigid organs that form part of the endoskeleton
of vertebrates. They function to move,
support, and protect the various organs of the
body, produce red and white blood cells and
store minerals. Damage in bone caused by
accidental case and suffering of diseases, can
be heal with implant of hard tissue. Fractured
bone case has different effect depending on
age levels. Bone Specialists have researched
about precise biomaterial referred to
biocompatibility
degree
and
prices.
Biocompatibility is a material characteristic
that non-toxic and compatible for the body,
which has been proved by in vivo and in vitro
test [2].
Clinical research in producing bone
material has produced interesting result, for
example auto graft, allograft and xenograft.
An ideal biomaterial precise selection for
implantation process must be biocompatible,
bioactive and overflow. Based on ways to
make it, substitution biomaterial classified to
synthetic biomaterial and natural biomaterial.
Synthetic biomaterials are biomaterial which
use synthetic reagents, for example CaCl2 and
Ca(OH)2 whereas natural biomaterials are
biomaterial which use natural reagents, for
example eggshell, coral reef and algae.
One of ways to increase biocompatibility
degree of biomaterial is using natural
reagents, such as Duck Eggshell as source of
calcium precursor. Beside to increase
biocompatibility degree, using duck eggshell
also has economic value (in some area,
eggshell included as organic waste). The other
reason in using duck eggshell is that highest
percentage than eggshell chicken. Moreover,
as we knew that calcium is the important
compound in bone minerals.
In the recent time has developed a new
kind of natural biomaterial that hydroxyapatite
with formula Ca10(PO4)6(OH)2, it has good
characteristic such as pores, resorption,
bioactive, non-corrosion, inert and worn-out
endure, but in other side hydroxyapatite have
weakness likes brittle and fracture, that
become constraint in design. To solve that
constraint, added strongest material, elastic,

biocompatible and cheap and in this research,
we use chitosan as added material for apatite
be apatite-chitosan composite. As we knew
that chitosan is a kind of natural polymers as
product of processing shrimp shell waste,
beside it cheap, strong and elastic, chitosan
has characteristic non-toxic and overflow [3].
Objective of Research
This research is conducted in order to
synthesis and characterize apatite-chitosan
composite based on calcium from duck
eggshell and chitosan from shrimp shell
waste, so that relevant in implantation
process. Synthesis is based on in-situ and exsitu methods, while characterization is
performed through X-Ray Diffraction (XRD),
Fourier Transform-Infra Red (FTIR) and
Scanning Electron Microscopy (SEM).
Hypothesis
Precipitation
of
apatite-chitosan
composite in the body normal condition, body
temperature (~37oC) and body pH (~ 7) are
able to increase quantity of hydroxyapatite
compound that appear.
Time and Place of Research
This research was conducted from August
2008 through January 2009, which took place
in IPB Biophysics Laboratory. The sample
analysis performed in Pusat Pengembangan
Ilmu
Pengetahuan
dan
Teknologi
(PUSPITEK) BATAN Serpong Tanggerang,
Pusat
Penelitian
dan
Pengembangan
(PUSLITBANG) Kehutanan Bogor and
Geology Laboratory Bandung.

THEORY
Human bone structure
Bones are rigid organs that form part of
the endoskeleton of vertebrates. They function
to move, support, and protect the various
organs of the body, produce red and white
blood cells and store minerals. Bone tissue is a
type of dense connective tissue. Because
bones come in a variety of shapes and have a
complex internal and external structure, they
are lightweight, yet strong and hard, in
addition to fulfilling their many other
functions. One of the types of tissue that
makes up bone is the mineralized osseous
tissue, also called bone tissue, which gives it
rigidity and a honeycomb like threedimensional internal structure. Other types of
tissue found in bones include marrow,
endosteum and periosteum, nerves, blood

2

vessels and cartilage. There are 206 bones in
the adult human body and about 270 in an
infant.
Bones have some main functions:
Protection — Bones can serve to protect
internal organs, such as the skull protecting
the brain or the ribs protecting the heart and
lungs; Mineral storage — Bones act as
reserves of minerals important for the body,
most notably calcium and phosphorus;
Movement — Bones, skeletal muscles,
tendons, ligaments and joints function
together to generate and transfer forces so that
individual body parts or the whole body can
be manipulated in three-dimensional space.
The interaction between bone and muscle is
studied in biomechanics, etc [4].
The primary tissue of bone, osseous
tissue, is a relatively hard and lightweight
composite material, formed mostly of calcium
phosphate in the chemical arrangement termed
calcium hydroxyapatite (this is the osseous
tissue that gives bones their rigidity). It has
relatively high compressive strength but poor
tensile strength of 104-121 MPa, meaning it
resists pushing forces well, but not pulling
forces. While bone is essentially brittle, it
does have a significant degree of elasticity,
contributed chiefly by collagen. All bones
consist of living cells embedded in the
mineralized organic matrix that makes up the
osseous tissue [5].
The matrix is the major constituent of
bone, surrounding the cells. It has inorganic
and organic parts. The inorganic is mainly
crystalline mineral salts and calcium, which is
present in the form of hydroxyapatite. The
matrix is initially laid down as unmineralised
osteoid (manufactured by osteoblasts).
Mineralization
involves
osteoblasts
secreting
vesicles
containing
alkaline
phosphates. This cleaves the phosphate groups
and acts as the foci for calcium and phosphate
deposition. The vesicles then rupture and act
as a centre for crystals to grow on.
Table 1 Contain of bone minerals
Element
Contain (wt. %)
Ca
34
P
15
Mg
0.5
Na
0.8
K
0.2
C
1.6
Other elements
47.9
The organic part of matrix is mainly
composed of collagen. This is synthesized
intracellular as tropocollagen and then

exported, forming fibrils. The organic part is
also composed of various growth factors, the
functions of which are not fully known. These
factors present include glycosaminoglycans,
osteocalcin, osteonectin, bone seal protein and
Cell Attachment Factor. One of the main
things that distinguish the matrix of a bone
from that of another cell is that the matrix in
bone is hard.
Table 2 Composite material for use in the
body [5]
Material
Polymers
Nylon
PTFE
Polyester
Silicone
Metals
Ti and its
alloys
Co-Cr
alloys
Stainless
steels Au,
Ag, Pt
Ceramics
Carbon
Aluminum
oxide

Advantage Disadvantage

Application

Ductile,
Not
strong,
light, easy prone
to
to fabricate creep,
degradable

Suture, vascular
prosthesis,
accetabular cup,
artificial
ligament

Ductile,
strong,
tough

Biocompati
ble, inert or
bioactive,
strong
in
Hydroxyapa compressio
tite
n, stiff
Composite
CarbonStrong,
carbon
stiff, tailorMetalmade,
distinctive
PMMA
HA-HDPE properties

Prone
to Artificial joint,
corrosion,
bone plate and
unwanted ion screw,
dental
release
root
implant,
pacer,
suture
wire
Brittle, weak
in
tension,
sometimes
fragile

Cardiovascular
device, dental
prosthesis, joint
prosthesis,
orthopedic
implant

Difficult
to Joint implant,
make,
high heart
valve,
production
bone cement
cost

Apatite
Apatite is a group of phosphate minerals,
usually
referring
to
hydroxyapatite,
fluorapatite, and chlorapatite, named for high
concentrations of OH−, F−, or Cl− ions,
respectively, in the crystal. The formula of the
admixture of the three most common end
members is written as Ca5(PO4)3(OH, F, Cl),
and the formulae of the individual minerals
are written as Ca5(PO4)3(OH), Ca5(PO4)3F and
Ca5(PO4)3Cl, respectively [20].
Apatite is one of few minerals that are
produced and used by biological microenvironmental systems. Apatite has a Moh's
Scale hardness of five. Hydroxyapatite is the
major component of tooth enamel. A
relatively rare form of apatite in which most
of the OH groups are absent and containing
many carbonate and acid phosphate
substitutions is a large component of bone
material.
Fluor apatite (or fluoroapatite) is more
resistant to acid attack than is hydroxyapatite.

3

For this reason, toothpaste typically contains a
source of fluoride anions (e.g. sodium
fluoride,
sodium monofluorophosphate).
Similarly, fluoridated water allows exchange
in the teeth of fluoride ions for hydroxyl
groups in apatite. Too much fluoride results in
dental fluorosis and or skeletal fluorosis. [6]
Phosphorite
is
a
phosphate-rich
sedimentary rock, which contains between
18% and 40% P2O5. The apatite in
phosphorite is present as cryptocrystalline
masses referred to as cellophane [7].
Hydroxyapatite
Hydroxyapatite belongs to the apatite
family. Apatite is a general term crystalline
calcium phosphate mineral. There are many
apatite compound, including flouroapatite,
chloroapatite,
carbonate-apatite,
and
hydroxyapatite. Hydroxyapatite chemical
formula is Ca10(PO4)6(OH)2. Hydroxyapatite
is a calcium phosphate including hydroxide
and has Ca/P (molar ratio) 1.67.
Hydroxyapatite is one of few material that is
classed as bioactive material, so that it will
support bone ingrowths without breaking
down or dissolving when be used for
implantation in human body [8].

for bone implantation. Hydroxyapatite has
been utilized as a fertilizer, a fluorescent
substance, an absorbent, a catalyst, and many
kind of biomaterial. Biomaterial based on
hydroxyapatite has been applied to dental,
orthopedic, and other medical uses [2].
The various methods for preparing
hydroxyapatite have been wet method that use
solution reaction (from solution to solid), dry
method that use solid reaction (from solid to
solid), hydrothermal method that use
hydrothermal reaction (from solution to solid),
alkoxide method that use hydrolysis reaction
(from solution to solid), and flux method that
use fused salt reaction (from melt to solid).
In hydroxyapatite structure, carbonate can
substitute OH- ion, and form carbonate apatite
type A, and if substitute PO43- ion will form
carbonate apatite type B. Generally,
precipitation at low temperature will form
carbonate apatite type B, while apatite from
dry reaction at high temperature will produce
carbonate apatite type A. Biological apatite is
predominance of carbonate apatite type B and
a few of carbonate apatite type A [9].
Chitin and Chitosan
Chitin
and
chitosan
are
aminoglucopyranans composed of GlcNAc
and GlcN residues. These polysaccharides are
renewable resources which are currently being
explored intensively by an increasing number
of academic and industrial research groups
[10].

Figure 1 The crystal structure of
hydroxyapatite
(Hanson, Bob. 2005. 150000000:1 Model of
Hydroxyapatite.
www.stolaf.edu/people/hanson)
Hydroxyapatite is the most stable calcium
phosphate phase at normal temperature and
pH between 4.2 and 12. Hydroxyapatite
crystal unit has hexagonal structure which
lattice parameter a = b = 9.432 Å and c =
6.881 Å (Fig. 1). It does not have the
mechanical strength to enable it to succeed in
long-term load bearing application. It is the
most commonly used calcium phosphate in
the medical field, as it possesses excellent
biocompatibility and is osteoconductive.
Hydroxyapatite in human body usually
named biological hydroxyapatite. It found
mainly in human or animal teeth and bones. In
medical application, hydroxyapatite is used

Figure 2 shrimps as one of the chitin resource
It is usually understood that chitin
(Chemical Abstracts Registry (CAS) 1398-614) is the polymer of β-1, 4 linked N-acetyl
glucosamine (2-aceta mido-2-deoxy-β-Dglucopyranose, GlcNAc)) whereas chitosan
(CAS 9012-76-4) is corresponding polymer of
glucosamine (GlcN). However, neither chitin
nor chitosan are homo polymers, as both
contain varying fractions of GlcNAc and
GlcN residues [21]. The polymers may be
distinguished by their solubility in 1 %
aqueous acetic acid. Chitin, containing ca >
40% GlcNAc residues (FA > 0.4) is insoluble,

4

whereas soluble polymers are named chitosan.
Chitosan is prepared from suitable chitinous
raw materials, mostly by a sequence of
deproteinization,
demineralization,
and
chemical deacetylation procedures. The
molecular weight of chitosan depends on the
source of the biological material, as well as on
the condition of the acetylation process [11].

Figure 3 structure of chitosan
Low toxicity, antimicrobial activity and
physic-chemical functionality of chitosan
offer a great potential for medical
applications, as documented in several review
(Muzzarelli, 1997b; Muzzarelli et al., 1997a;
Paul and Sharma, 2000). Chitosan is currently
not registered as a drug for the treatment of
disease, but GlcN – either as the
hydrochloride or the sulfate – is used widely
for the relief of pain in musculoskeletal
rheumatoid disease, including arthrosis,
arthritis or osteoporosis [14].
Duck Eggshell
The generalized eggshell structure, which
varies widely among species, is a protein
matrix lined with mineral crystals, usually of a
calcium compound such as calcium carbonate.
It is calcium build-up and is not made of cells.
Harder eggs are more mineralized than softer
eggs.
Calcium (Ca) as precursor in apatite
mineral synthesis consists in Duck Eggshell in
a large scale. Duck Eggshell is kind of biomineral composite ceramic which contained
95 % calcium carbonate (CaCO3), and for last
5 % are calcium phosphate, magnesium
carbonate, and solutes protein.

Figure 4 Ducks and Duck Eggshells
Without protein, crystal structure is
unstable to keep that shape. Matrix compound
has control functions to mineralization,
crystallographic texture and biomechanical
characterization [12].

Table 3 Duck Eggshell major composition
[What are eggshell made of?
http://FredSenese senese@antoine.
frostburg.edu]
Major
Contents Melting point
composition (%)
Water
29 - 35
Protein
1.4 - 4
Calcium
95
828°C
carbonate
Calcium
837 - 841oC
Magnesium
0.37 - 0.4 648 - 649.3 oC
Eggshell color is caused by pigment
deposition during egg formation in the oviduct
and can vary according to species and breed,
from the more common white or brown to
pink or speckled blue-green. Although there is
no significant link between shell color and
nutritional value, there is often a cultural
preference for one color over another [13].
Apatite-chitosan Composite
Composite material is combination two or
more material phase, either macro or different
microform or it is the chemical composition to
obtain equilibrium of character applied in
wide application. In general, expansion of
composite technology is to increase structural
efficiency and material character characteristic
significant, like for the application of light
material but very strong [5].
Ceramics, polymer, metal and composite
material, advantage and disadvantage owned
by it, developed to overcome bone problems.
Polymer haves the power of low mechanic
compared to bone, metal haves the power of
big mechanic but very corrosive, than
ceramics are brittle and it is hardness is low
come easy break. Best approach is when
producing all the character from polymer,
metal and ceramic in the form of composite
materials.
Nature composite formed from most of
ceramics (hydroxyapatite) and polymer
(collagen),
with
level
of
complex
microstructure enables to be imitated causing
gives mechanical property at high bone. Many
researches which has been done substitution
of bone to from composite material formed
from
hydroxyapatite
and
polymer.
Hydroxyapatite measures up to a real good
like bioactive, biocompatible, nontoxic and
osteoconductive but have low hardness
(fragile). Chitosan, form of deacetil from
chitin is abundance nature polymer, many
found in crustacean. Chitosan measures up to
biocompatible and bio-reabsorbed, nontoxic

5

and hardly easy to dissolve in dilution of acid.
Some studies at composite apatite-chitosan
that partially is biodegradable become an
advantage. When matrix polymer is
reabsorbed, new bone can grow around of
hydroxyapatite particles.
Atomic Absorption Spectroscopy (AAS)
Atomic Absorption Spectroscopy is a
technique for determining the concentration of
a particular metal element in a sample [1].
Atomic absorption spectroscopy can be used
to analyze the concentration of over 62
different metals in a solution.

Figure 5 Atomic Absorption Spectroscopy
processes
The technique makes use of absorption
spectrometry to assess the concentration of an
analyte in a sample. It relies therefore heavily
on Beer-Lambert law.
In short, the electrons of the atoms in the
atomizer can be promoted to higher orbitals
for an instant by absorbing a set quantity of
energy (i.e. light of a given wavelength). This
amount of energy (or wavelength) is specific
to a particular electron transition in a
particular element, and in general, each
wavelength corresponds to only one element.
This gives the technique its elemental
selectivity.
As the quantity of energy (the power) put
into the flame is known, and the quantity
remaining at the other side (at the detector)
can be measured, it is possible, from BeerLambert law, to calculate how many of these
transitions took place, and thus get a signal
that is proportional to the concentration of the
element being measured [15].
X-Ray Diffraction (XRD)
X-ray Diffraction analysis makes use of Xray emission resulting from collision between
electron and target that can be Cr, Fe, Co, Cu,
Mo or W. X-ray emission is continuously
specific distributed for each certain
wavelength of target. This process has side
effect that is the change of kinetic energy of
electron become heat, therefore the X-ray
quantities influenced by melt point and
thermal conductivity of target. XRD analysis

could inform us the structure of sample, such
as crystal system, lattice parameter, and
preferred orientation. XRD result also can
inform volume fraction and crystalline of
sample. It is also useful to identify a mixture
that is referred to as semi quantitative
identification of sample phase. Then X-ray is
transmitted through sample that will be
characterized, so x-ray will be transformed
into varied type of energy and absorbed some.

Figure 6 X-Ray Diffraction processes
Interaction of X-rays with sample creates
secondary diffracted beams of X-rays related
to inter-planar spacing in the crystalline
powder according to a mathematical relation
called Bragg’s Law below [13]:
n λ =2 d sin θ
n is an integer, λ is the wavelength of the Xrays, d is the interplanar spacing generating
the diffraction, and θ is the diffraction angle. λ
and d are measured in the same units, usually
angstrom [15].
Scanning Electron Microscopy (SEM)
The image in scanning electron
microscope is formed and displayed by
making use of electrons. In scanning electron
microscope, the surface of a solid sample is
scanned in raster pattern with a beam of
energetic electrons [14].
The column of an SEM contains an
electron gun for producing electrons and
electromagnetic lenses corresponding to the
condenser system. However, these lenses are
operated in such a way as to produce a very
fine electron beam, which is focused on the
surface of the sample [15]. At any given
moment, the sample is bombarded with
electrons over a very small area. They may be
elastically reflected from the sample or
absorbed by the sample and give rise to
secondary electrons of very low energy,
together with X- rays.
They may be absorbed and give rise to the
emission of visible light. In addition, they may
give rise to electric currents within the
sample. All these effects can be used to

6

produce an image. By far the most common,
however, is image formation by means of the
low-energy secondary electrons.

This allows multiple samples to be collected
and averaged together resulting in an
improvement in sensitivity. Because of its
various advantages, virtually all modern
infrared spectrometers are FTIR instruments
[16].

MATERIALS AND METHODS

Figure 7 Scanning Electron Microscopy
processes
An Energy Dispersive X-Ray Analyzer
(EDXA) is a common accessory which is
gives the scanning electron microscope (SEM)
a very valuable capability for elemental
analysis [15].
Fourier Transform Infra Red (FT-IR)
Fourier transform infrared (FTIR)
spectroscopy is a measurement technique for
collecting infrared spectra. Instead of
recording the amount of energy absorbed
when the frequency of the infra-red light is
varied (monochromator), the IR light is
guided through an interferometer. After
passing the sample the measured signal is the
interferogram. Performing a mathematical
Fourier transform on this signal results in a
spectrum identical to that from conventional
(dispersive) infrared spectroscopy.

Materials and Equipments
The materials which use in this research
are CaO, pro-analyze (NH4)2HPO4.2H2O,
Chitosan, aquades, and aquabides.
The equipments are beaker glass, mortar,
crucible, aluminum foil, pipette Mohr,
magnetic
stirrer,
centrifuge,
hotplate,
analytical scales, furnace, incubator, digital
thermometer, pH meter, whatman filter paper,
X-Ray Diffraction (XRD), Fourier Transform
Infra Red (FT-IR) and Scanning Electron
Microscopy (SEM).
Experimental Methods
Duck Eggshell Calcinations
Duck Eggshell as precursor has to be
washed at first, than removing its inner
membrane would be easier, before heating it
by using furnace in order to remove organic
composition of its content and decompose
calcium carbonate into calcium oxide at
1000oC for 5 hours. This treatment has the
best duration using furnace indicated the
largest amount calcium oxide or the least
calcium carbonate and efficiently electricity
[18]. So after calcinations of Duck Eggshell
was finished, powder of eggshell (CaO) will
be characterized by X-Ray Diffraction (XRD)
to know amount of calcium oxide and calcium
carbonate
and
Atomic
Absorption
Spectroscopy (AAS) to know presentation of
calcium in sample.
Precipitation and Composite Synthesis

Figure 8 Fourier Transform Infra-Red
processes
FTIR spectrometers are cheaper than
conventional spectrometers because building
of interferometers is easier than the
fabrication of a monochromator. In addition,
measurement of a single spectrum is faster for
the FTIR technique because the information at
all frequencies is collected simultaneously.

Control Preparation of Composite
Apatite is obtained by dissolving Duck
Eggshell (CaO) which has been calcination in
100 ml aquabides in a beaker glass is
continued with addition of (NH4)2HPO4
dissolved in 100 ml aquabides is done with
dropper from burette with speed 60ml/minute.
Calculation of number of Duck Eggshell and
(NH4)2HPO4 based on result from ratio of
concentration Ca/P 1.67. Calcium Content
from Duck Eggshell follows result of AAS.
Precipitation done at specific situation
(nitrogen atmosphere, temperature about 37oC

7

with or without pH controlled at about 7).
Aging sample during 24 hours at incubator
with temperature 37oC. Precipitate then is
filtered using centrifuge. Draining of
precipitate is done by using incubator at
temperature 50oC during 45 hours.
In-situ Preparation of Composite
Treatment of in-situ same as control but at
making of sample in-situ Duck Eggshell
(CaO) which has been dissolved in 100 ml
aquabides is added by chitosan which has
been dissolved applies CH3COOH 2%.
Amount of chitosan applied through
comparison with control result which has been
obtained before all 55:35 (55 is result of
apatite from control and 35 is amount of
chitosan used). CH3COOH 2% which in
adding as according to the many chitosan
which will be dissolved (applies comparison
of volume). Continued by addition of
(NH4)2HPO4 dissolved in 100 ml aquabides
with dropper from burette with speed
60ml/minute.
Precipitation done at specific situation
(nitrogen atmosphere, temperature about 37oC
with or without pH controlled at about 7).
Aging sample during 24 hours at incubator
with temperature 37oC. Precipitate then is
filtered using centrifuge. Draining of
precipitate is done by using incubator at
temperature 50oC during 45 hours.
Ex-situ Preparation of Composite
Treatment of ex-situ same as control,
dissolves Duck Eggshell (CaO) which has
been calcination in 100 ml aquabides in a
beaker glass is continued with addition of
(NH4)2HPO4 which dissolved in 100 ml
aquabides is done with dropper from burette,
with speed 60ml/minute. Addition of chitosan
which has been dissolved using CH3COOH
2% is done by after precipitation completed
before precipitate in aging, dropped by using
pipette. Amount of chitosan applied through
comparison with control result which has been
obtained before all 55:35 (55 is result of
apatite from control and 35 is amount of
chitosan used). CH3COOH 2% which in
adding as according to amount of chitosan
which will be dissolved (used comparison of
volume).
Precipitation done at specific situation
(nitrogen atmosphere, temperature about 37oC
with or without pH controlled at about 7).
Aging sample during 24 hours at incubator
with temperature 37oC. Precipitate then is
filtered applies centrifuge. Draining of

precipitate is done by using incubator at
temperature 50oC during 45 hours.
Tabel 4 Samples code
Sample Code

Reagent

Addition of
Chitosan

Controled
Temperature
and pH

A1 (Control)

CaO+
(NH4)2HPO4

-

A2 (In-situ)

CaO+
(NH4)2HPO4

Before
Precipitation

A3 (Ex-situ)

CaO+
(NH4)2HPO4

After
Precipitation

B1 (Control)

CaO+
(NH4)2HPO4

-

Temperature

B2 (In-situ)

CaO+
(NH4)2HPO4

Before
Precipitation

Temperature

B3 (Ex-situ)

CaO+
(NH4)2HPO4

After
Precipitation

Temperature

Temperature
and pH
Temperature
and pH

XRD Characterization
Equipment XRD used is Shimidzu XRD
7000, source of target of CuKα (λ= 1.54056
Angstroms). Before characterized, sample
blended until become powder, and then about
1 gram then is packed into holder which is
fairish 2x2 cm2 at diffract meter.
SEM/EDXA Characterization
Sample is put down in aluminum which
has plate two sides then is arranged in layers
with auriferous formation as thick 48 nm.
Sample which has been arranged in layers
observed to applies SEM (Scanning Electron
Microscopy) with strain 22 kVs and
magnification 5000x, 10000x and 20000x.
Characterization with Energy Dispersive XRay Analysis (EDXA) is a set with SEM.
FTIR Characterization
Precipitate which has been dried and
blended becomes powder characterized by
FTIR
spectroscopy.
Two
milligrams
precipitate mixed with 100 magnesium’s KBr,
made infrared pellet (IR) then is tested with
wave number reach 4000-400 cm-1, KBr
always is figured in each gauging to eliminate
adsorption of background.

RESULT AND DISCUSSION
Characteristic of Calcined Duck Eggshell
There are the result of yielded pure
calcium oxide from calcinations of duck
eggshell, which influenced by temperature
and duration of calcinations. Then, those

8

samples were characterized by AAS, XRD
and FTIR.
Duck eggshell contain calcium carbonate
(CaCO3). When it is heated, CO2 release into
the air to form calcium oxide.
Table 5 Percent of calcium calcined
duck eggshell by AAS
Sample
Calcium (%)
1000ºC, 5 hours
83.96
1000ºC, 10 hours
85.90

Although sample B has the least content of
carbonate than sample A, but in this research,
we use sample A of which heated at 1000oC
for 5 hours, because of electricity efficiency
of sample A better than sample B. Besides
that, the difference percent transmission
between sample A and sample B is not
significant.

a
= CaO

Result of FTIR characterization show
compound of calcium carbonate.
Table 6 Percent transmission of carbonate
groups
Percent transmission
Sample
1450 cm-1 875 cm-1
1000ºC, 5 hours
6
7
1000ºC, 10 hours
7
8
According to FTIR spectra (Figure 10),
still there is carbonate content in all samples
which is indicated by the presence of IR
carbonate’s group bands around wave number
of 1450 cm-1 and 875 cm-1. Although all
samples have carbonates bands, each of them
diverse in their transmission (Table 7).
The higher percent transmission means the
lower carbonates content that exist in the
samples. Among two samples, sample B of
which heated at 1000oC for 10 hours has the
least content of carbonate, 8% at 875cm-1 and
7% at 1450 cm-1.

a

b

Figure 10 FTIR spectra of heated duck
Eggshell at 1000oC within
(a) 5 hours, (b) 10 hours

b
= CaO

Figure 11 XRD pattern of heated duck
Eggshell at 1000oC within
(a) 5 hours, (b) 10 hours
This finding is emphasizing by XRD result
(figure 11).
According to JCPDS data base, sample A
is dominated by CaO (JCPDS 82-1691),
although there is also minor appearance of
CaCO3 peak (JCPDS 47-1743).
Figure 11 shown sample A (1200) has
intensity (arb. unit) higher than sample B
(1000). it is mean that increasing of
absorption value of CaO, followed by
increasing amount of CaO at the sample.
The best duration of Duck Eggshell
heating treatment is 5 hours, because of
calcinations process that liberate a molecule
of carbon dioxide (CO2) leaving CaCO3 be
CaO is reversible. Once the calcium oxide
product has cooled, it immediately begins to
absorb carbon dioxide from the air. After a
certain time, it is completely reconverted into
calcium carbonate, since Gibbs free energy
decrease as chemical ion reacted [23].

9

Experimentally, cooling process after
calcinations is conducted rapidly, as the
increasing duration of heating treatment was
indeed result much more of reconverted
calcium carbonate. Therefore, for further
process, 1000oC for 5 hours (sample A) set up
is chosen.
Characteristic
Composite

of

that exist in sample. Crystallinity is calculated
by comparing crystalline area fraction to the
total of crystalline area fraction and
amorphous area fraction [25].

= Hydroxyapatite

Apatite-chitosan

X-Ray Diffraction (XRD)
X-Ray Diffraction pattern (Figure 12)
shows that the majority of precipitates
correspond to hydroxyapatite (JCPDS 090432). Lattice parameter determination using
Cohen’s methods is calculated from the value
of d for hexagonal structure, the distance
between adjacent planes in the set (hkl), as the
following equation [24].

= Hydroxyapatite

Table 7 Crystallinity of sample
Sample
Crystallinity (%)
A1
83.22
A2
65.00
A3
62.33
B1
85.13
B2
67.19
B3
65.32
Lattice parameter obtained by calculation
is in assumption that those peaks are
correspondence to hydroxyapatite as the
tendency shows that sample consist of this
compound. Peak of XRD pattern is
determined by calculation of 2θ accuracy
(appendix 7).
Table 8 Lattice parameter of sample
Sample
a (Å)
Reference
9.418
A1
9.445
A2
9.653
A3
9.835
B1
9.518
B2
9.916
B3
10.011

c (Å)
6.884
6.885
6.998
7.035
6.985
7.068
7.123

The presence of chitosan in sample
influences lattice parameter.
Phase that has the highest accuracy is
determined as the sample phase. However,
there still a probability for that peak be used
by more than one phase. Characterization was
performed in order to get crystallinity degree
that explains the fraction of crystalline phase

= Hydroxyapatite

Figure 12 XRD patterns of A1, A2 and A3
Lattice parameter of samples (table 8)
influence by pH. Sample A1, A2 and A3 has
high accuracy of lattice parameter than sample
B1, B2 and B3. Lattice parameter accuracy
obtained by calculation in assumption that
those peak correspond to hydroxyapatite
emphasize the tendency that samples are
composed of hydroxyapatite, detailed
calculation as shown on appendix 7.
Crystallinity of samples (Table 10) is less
than 85% but more than 60% and decrease as
the addition of chitosan into sample.
Crystallinity of sample A1, which normal
process (without addition of chitosan) is the
highest than sample A2 and A3.

10

The data gives information that chitosan as
natural polymer; addition into calcium
phosphate will reduce its crystallinity. X-Ray
Diffraction pattern shown the presence of
chitosan at sample A2 at 2θ = 10.57, A3 at 2θ
=10.42 and 19.72. And then sample B2 at 2θ
=10.64 and B3 at 2θ =10.40.
= Hydroxyapatite

= Hydroxyapatite

= Hydroxyapatite

Crystallite
size
is
calculated
as
demonstrated by Scherer’s equation below at
002 planes (appendix 8).

β is FWHM (full width at half maximum) of
the broadened diffraction line on the 2θ scale
(radians), λ is wavelength, which is
1.54060x10-10 m and k is constant, which is
0.94 for biological material. Crystal size
varied among addition of chitosan and pH
controlled. Comparing to the crystal size of
human bone, that in range of 18-23 nm, so the
closest crystal size are sample A1, A2, and
A3. So, the analysis considering amount of
hydroxyapatite, lattice parameter, crystal size
and crystallinity, indicated sample A1, A2,
and A3 are better, since it is closest crystal
size to that human bone and lattice parameter
to hydroxyapatite, low crystallinity, and high
amount of hydroxyapatite.
Fourier Transform Infra Red (FTIR)
These FTIR spectra below record
wavelength versus percent transmission, as
shown in detailed on figure 14. The size of the
peak or through in the spectrum is a direct
indication of the amount of material presence.
All IR spectra show the stretching and
vibration modes of OH- groups appear at 3565
cm-1 and 635 cm-1, whereas the bands derived
from 635 cm-1 is indicated groups of
hydroxyapatite [15].

Figure 13 XRD patterns of B1, B2 and B3
Based on XRD pattern, A1, A2, and A3
has the higher amount of hydroxyapatite than
B1, B2, and B3 which shown with high
intensity peak in every sample.
Table 9 Crystal size of sample
Sample code

d002 (nm)

A1
A2
A3
B1
B2
B3

29.7133
27.9246
27.4797
25.9294
29.1900
26.5918

Figure 14 FTIR spectra sample A1, A2, A3
and Chitosan
Analysis of FTIR shows that formed of
apatite at sample A and B with functional
group appearance PO4, OH and CO3.
Functional Group NH2, C-H and amide I and
amide II is characteristic from chitosan which
appear at sample A2 and A3, it is mean that at
sample A2 and A3 has been formed apatitechitosan composite.

11

At this sample also seen happened
overlapping some wavelength like functional
group NH2 that overlap with functional group
OH. Happened overlapping at some
wavelength numbers owned by chitosan and
apatite, show already happened bond between
chitosan with hydroxyapatite.
Identification of functional group NH2 and
C-H, amide I and amide II at sample A2 and
A3, proves that bond chitosan and calcium
phosphate were happen. It is mean apatitechitosan have successfully is formed. Method
in-situ and ex-situ show the difference at
appear of chitosan. However, functional
groups appear in both methods applied same,
only different from its transmittance and
wavelength.
Scanning Electron Microscopy (SEM)
As seen in figure 15, there are SEM
picture of all sample with 20.000 times
magnification. It is shown that there is
microcrystalline particle as figured of
arranged particle orderly. As figured as like a
huge number of small ball densely, it is
indicating apatite is formed in the samples.
From the sample, we can see that there is
indicating hydroxyapatite in spherical phase at
samples. Almost all samples have its
agglomerated particles, which can be seen on
sample A3. Moreover, it can be seen that
sample A1 and A2 have more homogenous
and densely arrange particles, indicated by the
least of agglomerated and differences element
on sample surface.
Particle hydroxyapatite in composite
disseminates uniform, it can see through
chitosan matrix, which has interacted between
cells. Pores has different form compared to
hydroxyapatite its self, in pure chitosan
sample of pore more flatly and