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

Outlines Experiment Objectives Conclusion Introduction 2 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……………………….

Introduction Nano scale Shape and Size Effect 1 to 100 nanometers 3 Ref: http://eng.thesaurus.rusnano.com 0 Dimension 1 Dimension 2 3 Ref: http://commonsensecanadian.com 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.

Introduction Hydroxyapatite Bone Enhanced resorbability 4 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: http://onco-info.ru Hydroxyapatite Ref: http://i.vimeocdn.com

Introduction Inorganic mineral Hexagonal Bio ceramics Hydroxyapatite 5 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

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

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

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 ….

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

Experiment: Temperature effect on cuttlefish bone Part 1 Part 1 10 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 1300 °C 500 °C 1100 °C 900 °C

Results & Discussion: Part 1 Temperature effect on cuttlefish bone 11 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 CaCO3 CaO + CO2 Heated 900 °C Fig 1 XRD pattern of temperature effect on cuttlefish bone at various temperature

Experiment: Ball Milling Part 2 5CaO + 3(NH4)2HPO4 + H2O 12 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

Experiment: Ball Milling Part 2 dried for 48 h sample Characterization 13 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

Results & Discussion: Part 2 Hydroxyapatite synthesis by ball milling 14 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. 09-0432 Hydroxyapatite phase JCPDS file NO. 09-0432 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 09-0432 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

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. 09-0432 Hydroxyapatite phase JCPDS file NO. 09-0432 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 09-0432 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

Results & Discussion: Part 2 Hydroxyapatite synthesis by ball milling 16 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. 09-0432 Hydroxyapatite phase JCPDS file NO. 09-0432 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 09-0432 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

Results & Discussion: Part 2 Hydroxyapatite synthesis by ball milling 17 Hydroxyapatite synthesis by ball milling Synthesis process %Transmittance %Transmittance %Transmittance CO32- group the FTIR spectra of Hap, the spectra appear in wavenumber 565 603 964 and 1100 that is phosphate group and 636 that is hydroxyl group The spectra band at 1400 to 1700 result of synthesis process and band at 3400-3800 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

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 60-70 nm Fig. 4 SEM image of synthesized hydroxyapatite from cuttlefish bone at various different phase precursor by ball milling method

Conclusions Conclusions Conclusions Conclusions Conclusions 19 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

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. 149-150, pp. 607-616. 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. 281-312. 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. 429-432..

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