1. Characteristic and Formation of Hydroxyapatite Synthesized from Heat Treatment of Cuttlefish Bone
Kridsada Faksawat
Department of Physics, Faculty of Science,
King Mongkut’s University of Technology Thonburi

2. Outlines Experiment Objectives Conclusion Introduction2
Experiment
Introduction
Objectives
bone
Ball milling
Cuttlefish bone
hydroxyapatite
hydroxyapatite
Ball milling
Conclusion
Results & Discussion
My research will be in five section such as introduction……………………….

3. Introduction Nano scale Shape and Size Effect 1 to 100 nanometers3
Ref:
0 Dimension 1
Dimension 2 3
Ref:
Shape and Size Effect
Increasing of specific surface area
Nano scale
1 to 100 nanometers
Nanotechnology is science, engineering and technology conduct at the nanoscale, which is about 1 to 100 nanometers. The nanoscale occur to shape and size effect, which is increasing of specific surface area. Nanomaterial is well-known that the main types of nanostructure materials for example 0 dimension, 1dimension, 2dimension and 3dimension. The each nanostructure can use in several application such as quantum dot, solar cells for renewal energy, fiber optics for communication, electronic devices.

4. Introduction Hydroxyapatite Bone Enhanced resorbability4
Enhanced resorbability
Improved densification
sinter ability
Improved cell proliferation
Improved cellular activity related to bone growth
Bone
Especially, application in medical is very importance. Scientist and doctor use nanomaterial in surgery and bone replacement. The nanomaterial is hydroxyapatite. The hydroxyapatite in nanoscale enhanced resorbability, improved densification, sinter ability, improved cell proliferation, and improved cellular activity related to bone growth. Hydroxyapatite found in the real bone more to 70%. Where is it in part of bone?
Ref:
Hydroxyapatite
Ref:

5. Introduction Inorganic mineral Hexagonal Bio ceramics Hydroxyapatite5
Collagen triple helix
Osteon
Mineralized fibrils
Spongy bone
Osteonic canal
Hydroxyapatite
Compact bone
Microscopic view
Ca5(PO4)3OH
Lamellae
Hexagonal
The bone consist of spongy bone and compact bone and other calcified tissues can be considered as natural anisotropic composites consisting of bio minerals embedded in a protein Lamellae. In the lamellae is bio mineral phase, which is one or more types of calcium phosphates, comprises 65–70% of bone and the organic phase, which is primarily in the form of collagen, accounts for the remaining portion. The collagen, which gives the bone its elastic resistance, acts as a matrix for the deposition and growth of minerals. Among the CaP salts ,hydroxyapatite this formula is (Ca10(PO4)6(OH)2, HAp). The structure of Hap is hexagonal. It is bio ceramic and inorganic mineral.
Bio ceramics
Collagen fiber
Inorganic mineral

6. Introduction Hydroxyapatite 90-95% of Ca
How do you synthesized hydroxyapatite? 6
Natural material
Ca source
90-95% of Ca
Hydroxyapatite
Hydroxyapatite is synthesized from precursors that are ca and phosphate source. Calcium source can found from various natural materials such as bovine bone or pig bone, coral, eggshells and seashells especially cuttlefish bone consists of 90-95% of Ca. The phosphate source can found from commercial material such as K2HPO4, (NH4)2HPO4, Ca2P2O7 and Na2HPO4. Researchers synthesized hydroxyapatite by each PO4 sources. They found that after the synthesized HAp by K2HPO4 found trace of K in HAp. In the synthesized Hap by (NH4)2HPO4 found odor problem. Ca2P2O7 found B-TCP phase in HAp and then Na2HPO4 found trace of Na but lessness cuttlefish bone for precursor in Hap synthesis.
Chemical material
PO4 source
K2HPO4
Ca2P2O7
Na2HPO4
(NH4)2HPO4
Trace of K
Found β -TCP
Trace of Na
Odor problem

7. Introduction Hydroxyapatite How to synthesis of hydroxyapatite 7
gelation
H2O/EtOH
Sol gel
Solid state
Hydroxyapatite
Ball milling
PO43- solution
Ca2+ solution
Precipitation
In the synthesized Hydroxyapatite. It is synthesized by several method for instance, the first is solid state, Ball milling, microwave irradiation, Sol gel and Precipitation method
The each of method have different disadvantages and advantages. And the method is the most famous. These are Ball milling and precipitation. So we pick the both method for this work.
Microwave
irradiation

8. Objectives 8
To study phase transformation of cuttlefish bone by various sintering temperature.
To study characteristics of hydroxyapatite synthesized from various heated cuttlefish bone by ball milling method.
To study crystal structure, functional group and morphology of synthesized hydroxyapatite by ball milling method.
Then in this work, I would like to studies in 3 part. The first is ….

9. Part 2. Hydroxyapatite synthesis
Experiment 9
200 to 1300 ˚C
Ball milling method
Vary precursor
Part 2. Hydroxyapatite synthesis
Part 1. Temperature effect on cuttlefish bone
room
temperature
500 °C
The experiment can divide in 2 part
900 °C

10. Experiment: Temperature effect on cuttlefish bone Part 1 Part 110
Cuttlefish bone
Cuttlefish bone powder
ground
Part 1
Temperature effect on cuttlefish bone
1300 °C
200 °C
Characterization by XRD
First step, the cuttlefish bone is washed with water and dried. Next step, the dry cuttlefish bone is ground to powder by Agate mortal and heated at 200 to 1300 degree Celsius for 4 hour .and these are results after heated it. the color of heated sample at 200 degree Celsius is yellow, sample at 500 degree Celsius is black and over the 900 degree Celsius is white. Finally the all sample is characterized by XRD.
Hydroxyapatite synthesis
Heated at 200 to °C
500 °C
1100 °C
900 °C

11. Results & Discussion: Part 1 Temperature effect on cuttlefish bone11
Temperature effect on cuttlefish bone
Orthorhombic
CaO (lime)
CaCO3 (calcite)
CaCO3 (aragonite)
Before heated
T 900 °C
T 500 °C
Intensity (a.u.)
2theta (degree)
room
temperature
a ≠ b ≠ c
α = β = γ = 90 
Rhombohedral
500 °C
a = b = c
α ≠ β ≠ γ ≠ 90 
Cubic
900 °C
a = b = c
α = β = γ = 90 
The results of temperature effect on cuttlefish bone show that the XRD pattern of cuttlefish bone at various temperature in fig 1. the initial phase of cuttlefish bone is CaCO3 that is aragonite phase following green star at graph and structure of aragonite is orthorhombic.
When the cuttlefish bone is heated at 500 degree celsius, the aragonite phase transform to Calcite phase completely which is polymorph of calcium carbonate following blue rectangle and structure of calcite phase is rhombohedral.
Then, the cuttlefish bone is heated at over 900 degree Celsius, the calcite change to lime phase that component is calcium oxide following red circle and the structure of lime phase is cubic.
The results summarized that high temperature occur to change structure of cuttlefish bone. Moreover structure is high crystallinity. So, the this changing of phase and structure is called thermal decomposition of calcium carbonate. The this effect is calcaium carbonate is decomposed to calcium oxide and carbon dioxide by heat treatment
Thermal decomposition of CaCO3
CaCO CaO CO2
Heated
900 °C
Fig 1 XRD pattern of temperature effect on cuttlefish bone at various temperature

12. Experiment: Ball Milling Part 2 5CaO + 3(NH4)2HPO4 + H2O12
5CaO + 3(NH4)2HPO4 + H2O
Ca5(PO4)3(OH) + 5H2O + 6NH3
Vary calcium sources
CaO (lime phase) (for example)
CaCO3 (aragonite phase)
CaCO3 at 500 °C (calcite phase)
CaO 5M
(NH4)2HPO4 3 M
DI water
Milled 5 to 120 min
Stainless steel
container
the second method is ball milling in Hap synthesis.
Let’s begin to CaO is mixed with di ammonium orthophosphate at molar ratio 5 to 3 and DI water is solvent in the mixture. The mixture is milled with steel ball in aluminum container. In this ball milling method we vary precursor following this ……Then the sample is milled by high energy ball miller for 5 to 120 minutes. And this is reaction of the sample which is milling that.
steel ball

13. Experiment: Ball Milling Part 2 dried for 48 h sample Characterization13
Ground until powder
0090 °C
dried for 48 h
Hydroxyapatite
After that, the milled sample is poured with palette glass. Then, the sample is dried at 90 degree Celsius by oven for 48 hours. Next, the sample is ground until to powder by agate mortar. Final step, the powder is characterized by XRD, FTIR and SEM.
X-ray diffractometer: XRD
Fourier transform infrared spectrometer: FTIR
Scanning electron microscopy: SEM
Characterization
sample

14. Results & Discussion: Part 2 Hydroxyapatite synthesis by ball milling14
Hydroxyapatite synthesis by ball milling
hydroxyapatite
CaCO3 (aragonite)
2theta (degree)
CaCO3 (aragonite)
Ball milled 5 min
Ball milled 30 min
Ball milled 60 min
Ball milled 120 min
Intensity (a.u.)
a) Aragonite phase precursor
5CaCO3 + 3(NH4)2HPO4 + H2O
Ca5(PO4)3OH + 4H2O + 5CO2 + 3NH3
HAp =
Pure hydroxyapatite phase at milling time 60 min
Hexagonal
According to
JCPDS file NO
Hydroxyapatite phase
JCPDS file NO
the fig on the left hand side, the xrd pattern of synthesized hydroxyapatite from Calcium carbonate phase showed green rhombus, (NH4)2HPO4 showed blue rectangle and aragonite phase showed orange circle.
The result show that Hap peak appear the sample milled at 5 min but remain initial phase.
When the increasing of ball milling time of Hap synthesis be the cause of Hap completely at 60 min follow JCPDS file No and the structure of Hap is hexagonal. This is chemical reaction in ball milling method
Fig 2 XRD pattern of synthesized hydroxyapatite from cuttlefish bone at different phase precursor by ball milling method

15. Hydroxyapatite and CaCO3 phase appeared
Part 2
Results & Discussion: 15
Hydroxyapatite synthesis by ball milling
hydroxyapatite
CaCO3 (calcite)
2theta (degree)
CaCO3 (calcite)
Ball milled 5 min
Ball milled 30 min
Ball milled 60 min
Ball milled 120 min
Intensity (a.u.)
b) Calcite precursor
5CaCO3 + 3(NH4)2HPO4 + H2O
Ca5(PO4)3OH + 4H2O + 5CO2 + 3NH3
HAp =
Hydroxyapatite and CaCO3 phase appeared
at milling time 5 to120 min
Hexagonal
According to
JCPDS file NO
Hydroxyapatite phase
JCPDS file NO
the fig on the left hand side, the xrd pattern of synthesized hydroxyapatite from Calcium carbonate phase showed green rhombus, (NH4)2HPO4 showed blue rectangle and calcite phase showed Blue circle.
The result show that Hap peak appear the sample milled at 5 min but remain initial phase.
When the increasing of ball milling time of Hap synthesis be the cause of initial phase peak decrease and the Hap peak follow JCPDS file No and the structure of Hap is hexagonal. This is chemical reaction in ball milling method
Fig 2 XRD pattern of synthesized hydroxyapatite from cuttlefish bone at different phase precursor by ball milling method

16. Results & Discussion: Part 2 Hydroxyapatite synthesis by ball milling16
Hydroxyapatite synthesis by ball milling
hydroxyapatite
CaO (lime)
2theta (degree)
CaO (lime)
Ball milled 5 min
Ball milled 30 min
Ball milled 60 min
Ball milled 120 min
Intensity (a.u.)
c) Lime phase precursor
5CaO + 3(NH4)2HPO4 + H2O
HAp =
Ca5(PO4)3(OH) + 5H2O + 6NH3
Pure hydroxyapatite phase at milling time 5 min
Hexagonal
According to
JCPDS file NO
Hydroxyapatite phase
JCPDS file NO
the fig on the left hand side, the xrd pattern of synthesized hydroxyapatite from Calcium oxide phase showed green rhombus, (NH4)2HPO4 showed blue rectangle and CaO phase showed violet circle.
The result show that Hap peak appear the sample milled at 5 min but remain initial phase.
When the increasing of ball milling time of Hap synthesis be the cause of Hap completely at 60 min follow JCPDS file No and the structure of Hap is hexagonal. This is chemical reaction in ball milling method
Fig 2 XRD pattern of synthesized hydroxyapatite from cuttlefish bone at different phase precursor by ball milling method

17. Results & Discussion: Part 2 Hydroxyapatite synthesis by ball milling17
Hydroxyapatite synthesis by ball milling
Synthesis process
%Transmittance
%Transmittance
%Transmittance
CO32- group
the FTIR spectra of Hap, the spectra appear in wavenumber and that is phosphate group and 636 that is hydroxyl group
The spectra band at 1400 to 1700 result of synthesis process and band at result of water molecule in Hap particle.
4000
3800
3600
2500
2000
1500
1000
500
4000
3800
3600
2500
2000
1500
1000
500
4000
3800
3600
2500
2000
1500
1000
500
Wavenumber (cm-1)
Wavenumber (cm-1)
Wavenumber (cm-1)
a) Aragonite phase precursor
b) Calcite precursor
c) Lime phase precursor
Fig 3 FTIR spectra of synthesized hydroxyapatite from cuttlefish bone at various different phase precursor by ball milling method

18. Average particle size 60-70 nm
Results & Discussion: 18
Hydroxyapatite synthesis by ball milling
Ball milling time
a) Aragonite phase precursor
b) Calcite precursor
c) Lime phase precursor
120 min
500 nm
500 nm
500 nm
Average particle size nm
Fig. 4 SEM image of synthesized hydroxyapatite from cuttlefish bone at various different phase precursor by ball milling method

19. Conclusions Conclusions Conclusions Conclusions Conclusions19
Conclusions
Conclusions
From results of temperature effect on cuttlefish bone
Aragonite phase change to calcite phase completely and calcite phase transform to lime phase completely at temperature 500 °C and 900 °C respectively.
Conclusions
From results of hydroxyapatite synthesis
Conclusions
Hydroxyapatite phase appear at milling 5 minutes
Hydroxyapatite phase appear completely at 60 minutes in CaCO3 (aragonite phase) precursor.
Hydroxyapatite phase appear completely at more than 120 minutes in CaCO3 (calcite phase) precursor
Hydroxyapatite phase appear completely at 5 minutes in CaO (lime) precursor
Conclusions
So, from the result can summarize in 3 main point. The first is …..
Conclusions
Conclusions
Conclusions

20. References
2020
Amin, S., Bekhit, A.E., Azam, A. and Zhifa, S., 2015, “Synthesis of Nano- Hydroxyapatite (nHA) from Waste Mussel Shells Using a Rapid Microwave Method”, Materials Chemistry and Physics, Vol , pp
Mehdi, S.S., Khorasani, M.T., Ehsan, D.K. and Ahmad, J., 2013,“Synthesis Methods for Nanosized Hydroxyapatite Indiverse Structures ”, Acta Biomaterialia, Vol. 4, pp
These is references….
Liu, J., Li ,K., Wang, H., Zhu, M. and Yan, H., 2014, “Rapid Formation of Hydroxyapatite Nanostructures by Microwave Irradiation”, Chemical Physical, Vol. 396, pp

21. Thank you for your attention