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Effect of Calcium Precursors and pH on the Precipitation of Carbonated Hydroxyapatite
Available online at www.sciencedirect.com
ScienceDirect Procedia Chemistry 19 (2016) 539 – 545
5th International Conference on Recent Advances in Materials, Minerals and Environment (RAMM) & 2nd International Postgraduate Conference on Materials, Mineral and Polymer (MAMIP), 4-6 August 2015
Effect of calcium precursors and pH on the precipitation of carbonated hydroxyapatite Radzali Othmana,*, Zaleha Mustafaa, Chong Wee Loonb, Ahmad Fauzi Mohd Noorb a
Faculty of Manufacturing Engineering, Universiti Teknikal Malaysia Melaka, Hang Tuah Jaya, 76100 Durian Tunggal, Malacca, Malaysia b School of Materials & Mineral Resources Engineering, Universiti Sains Malaysia, Engineering Campus, 14300 Nibong Tebal, Penang, Malaysia
Abstract Three types of calcium precursors (nitrate, hydroxide and catbonate) were used in the synthesis of carbonated hydroxyapatite (cHA) using a precipitation method via a chemical reaction with di-ammonium hydrogen phosphate as the phosphate precursor. The precipitation method was chosen over many other methods due to its flexibility to changes in processing parameters to control the phases formed, the particle size, as well as, the morphology of the as-synthesized powders. The focus of the study was on cHA as it is deemed to mimic the composition of the human bone much closer as compared to the stoichiometric hydroxyapatite. When the chemical reaction was completed, the precipitate was dried, ground and characterized by x-ray diffraction (XRD), electron microscopy (both FESEM and TEM) and particle size analysis. Only the nitrate precursor produced a single-phase carbonated hydroxyapatite (cHA), whilst the other two precursors produced a secondary calcite phase or did not react fully. This is due to the low solubility of the calcium hydroxide and the incomplete reaction of the calcium carbonate. An increase in pH has been observed to lead to higher carbonate content in the synthesized cHA and a smaller crystallite size. © byby Elsevier B.V. This is an open access article under the CC BY-NC-ND license ©2016 2016The TheAuthors. Authors.Published Published Elsevier B.V. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia. Peer-review under responsibility of School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia Keywords: Hydroxyapatite; biocomposite; strength and toughness
* Corresponding author E-mail address:[email protected]
1. Introduction In recent decades, orthopaedic medication, such as bone repair and regeneration were rapidly studied to improve the health level in the society. Various artificial materials, such metallic, ceramic, polymeric and composite
1876-6196 © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia doi:10.1016/j.proche.2016.03.050
540
Radzali Othman et al. / Procedia Chemistry 19 (2016) 539 – 545
materials were used for orthopaedic repair and reconstruction 1. The materials used in orthopaedic application haveto be biocompatible, biodegradable and able to provide the function of tissue regeneration 2,3. Of all the choices of materials, calcium phosphates, especially hydroxyapatite (HA), is currently regarded as the best biomaterial to be used in bone repair and regeneration. This is due to the fact that natural bone is made up of an organic compound (collagen) as well as an inorganic compound (HA). The inorganic apatite compound offers osteoconductivity, osteoinductivity and a bone-bonding ability in the process of bone growth or bone healing 4-6. As HA is the main constituent of the bone, synthetic HA, with the chemical formula Ca10(PO4)6(OH)2, was introduced and widely applied in orthopaedic surgeries 7. Similarity in chemical composition to natural bone allows HA to form at the interface of the surrounding bone tissue, by mimicking the natural bone apatite phase 8. However, carbonated hydroxyapatite (cHA) was found to have a much closer compositional similarity to the mineral in natural bone. Carbonated hydroxyapatite (cHA) shows a higher bioactivity than HA, and the smaller particle size of cHA would inevitably bring about better tissue-implant interactions9,10. It has also been proven that nano-sized apatite particles, which have larger surface, lead to an enhancement in biological properties 11. The properties and reactivity of the apatite cement were found to have been influenced by both the calcium precursors and firing temperature12. They have found that identical synthesis history, morphology and crystallinity does not culminate in producing similar reactivity. Besides, the presence of impurities could hamper reactivity of the cement. Various synthesis methods had been introduced to produce HA or cHA, which included hydrothermal, sol-gel, precipitation, mechano-chemical, mechanical activation and other methods. Amongst these synthesis methods, the precipitation route is one of the most promising techniques to produce nano-sized apatite particles13,14. Morever, the precipitated particle sizes can be controlled by the synthesis temperature and pH conditions 15. It is the objective of this work to establish the processing parameters for cHA which have not been as intensively studied as HA. These would include the effects of using different calcium precursors as well as the effect of pH on the phase(s) formed, the changes in particle sizes and the particle morphologies as the pH is varied. 2. Experimental methods 2.1 Precursor materials Three different chemical grade calcium precursor materials were used, viz. calcium nitrate tetrahydrate, Ca(NO3)2.4H2O (Merck, ≥ 99.0% purity), calcium hydroxide, Ca(OH) 2 (Fluka, ≥96% purity), and calcium carbonate, CaCO3 (Sigma Aldrich, ≥99.0 purity). The calcium precursor was reacted dropwise with a phosphate precursor, viz. di-ammonium hydrogen phosphate, (NH4)2HPO4, (Fluka, ≥98.0 purity). In the reaction to produce cHA, sodium hydrogen carbonate (NaHCO3)was used as the source to supply CO32- whilst ammonium hydroxide (NH4OH) was used as a pH control for alkaline solutions. All solutions were prepared to an initial 1M concentration. 2.2 Flowchart for the synthesis of cHA Firstly, the solution containing the carbonate ions was added dropwise into the solution containing the phosphate ions at a rate of 40 drops per minute and continuous stirring. NH 4OH was also added dropwise to adjust the pH of the mixed solution. In this work, the pH used were 8.5, 9, 10 and 11. When all the carbonate-containing solution had been added, stirring was continued for another 30 minutes. The carbonate-phosphate mixture was then added dropwise (at a rate of 40 drops per minute) into the calcium ions solution whilst being continuously stirred. The colour of the mixed solution started to change slowly from transparent to milky white in colour. This change indicated that precipitation had taken place immediately when the dropwise process started. An additional 30 minutes of stirring was performed after the solutions were fully mixed to ensure complete reaction. The solution was then filtered using a Whatman 90mm filtration set with a 542 grade filter paper of average pore size 2.7 Pm. A vacuum pump was used to enhance the filtration rate. The white precipitate produced was washed three times with deionized water to remove any possible residue in the precipitate. The precipitate was finally dried in an oven at
Radzali Othman et al. / Procedia Chemistry 19 (2016) 539 – 545
541
100oC for 24 hours. The dried agglomerated powder was then ground with an agate pestle and mortar before being sieved through a 90 Pm sieve. The flowchart for the synthesis of cHA is shown in Figure 1. 2.3 Powder characterization The ground powders were then subsequently characterized using a number of techniques such as XRD (Bruker D8), FESEM (Zeiss Supra 55VP) and TEM (Philips CM12).
Fig. 1. Flowchart for the synthesis of cHA
3. Results and discussion 3.1 Phase analyses using XRD The phases formed upon using three different calcium precursors and four different pH at room temperature were determined by x-ray diffraction (XRD). The powders synthesized using the calcium carbonate precursor was found to exhibit mainly carbonate peaks and this is attributed to the reaction being unsuccessful using the parameters in this work. Henceforth, no other result was reported for this precursor. The phase analyses for the the calcium hydroxide and calcium nitrate precursors at four different pH are shown in Figures 2 and 3, respectively. In all the samples using the hydroxide precursor, calcite is observed as a secondary phase apart from the major cHA phase (Fig. 2). However, the intensity of the calcite phase is observed to decrease as the pH increases. This is attributed to the low solubility of Ca(OH) 2 in water which resulted in the CO32- from the mixture reacting with the remaining Ca(OH)2 in solution to form CaCO3. The presence of calcite CaCO3 was similarly reported by others17.
542
Radzali Othman et al. / Procedia Chemistry 19 (2016) 539 – 545 CaCO3 cHA
Fig. 2. Diffractograms of cHA powders synthesized using a calcium hydroxide precursor
On the other hand, upon using a calcium nitrate precursor, cHA is found to be the only phase formed at different pH (Fig.3). The results show that as the pH of the synthesis solution is increased, the peaks become more broadened, implying a decrease in crystallinity. The broadened peaks also reflect that the carbonate content in the cHA samples is increased with an increase in the synthesis pH 15. Some parts of the diffraction patterns also show broadened peaks where low intensity peaks are merged together. This indicates that the samples are partially amorphous and consist of nanosized cHA powders18. cHA
Fig. 3. Diffractograms of cHA powders synthesized using a calcium nitrate precursor
3.2 Morphology of particles The morphology of the cHA particles was first observed under the FESEM. However, the images of powders from both the nitrate-based and hydroxide-based precursors at four different pH values showed a heavily agglomerated structure, and hence not included for further discussion. Images observed under TEM (Figures 4 and 5) are foud to be more illustrative and are used for the measurement of size (in terms of length and width). From Fig. 4, it can be observed that as the synthesis pH increases, the size of the cHA paticle is decreased from about 30nm to 15nm. Consequently, the shapes of the particles appear to change from short rod-like to rice-like, indicating that the length of the particle has decreased. In this study, NH 4OH was used to adjust the synthesis pH, causing the concentration of OH- to increase. The high concentration of OH- could restrict the movement of Ca2+, PO43- and CO32- in the synthesis solution, thus the cHA nucleation process could be reduced, as reported in a previous
Radzali Othman et al. / Procedia Chemistry 19 (2016) 539 – 545
543
researchwork19. Hence, the growth of the particles is restricted and this results in smaller particles being formed when cHA is synthesized at higher pH using a nitrate-based precursor.
Fig. 4. TEM micrographs of cHA powders synthesized using a calcium nitrate precursor at different pH (a) 8.5; (b) 9.0; (c) 10.0 and (d) 11.0
The TEM images of the cHA particles synthesized using the hydroxide precursor are shown in Fig. 5. The shapes of the particles formed vary markedly from those produced using the nitrate precursor, i.e. the particles are irregularly shaped. Besides, the sizes of the particles range from 10nm to 25nm, regardless of the change in synthesis pH.
Fig. 5. TEM micrographs of cHA powders synthesized using a calcium hydroxide precursor at different pH (a) 8.5; (b) 9.0; (c) 10.0 and (d) 11.0
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Radzali Othman et al. / Procedia Chemistry 19 (2016) 539 – 545
3.3 Particle size analysis The average particle size of the cHA particles was measured from several TEM images for both nitrate- and hydroxide-derived cHA. The length and width of 40 particles were measured at various magnifications to obtain an average value and this was then computed to obtain the ratio of length to width (aspect ratio). The cHA’s derived from nitrate precursor show a decrease in aspect ratio (i.e. length) of the particles as the pH increases (Table 1). It had been suggested that a decrease in aspect ratio is caused by the higher substitution of carbonate into the phosphate site in the cell lattice20. On the other hand, there is no definite trend in the change of particle size for the hydroxide-based cHA. This is due to the fact that the hydroxide cHA also contained a calcite phase. The high carbonate content inhibited the growth of cHA particles, causing the particles to be smaller in size. Table 1. Aspect ratio (length/width) of cHA particles at different pH using 2 different precursors pH value / Precursor
Nitrate-based
Hydroxide-based
8.5
2.842
2.024
9.0
2.832
2.191
10.0
2.080
1.915
11.0
1.970
2.066
4. Conclusions It has been confirmed that a single-phase carbonated hydroxyapatite can only be successfully synthesized using a calcium nitrate tetrahydrate precursor as compared to two other precursors, viz. calcium hydroxide and calcium carbonate, under the conditions employed in this work. It has been observed that the morphology of the cHA particles differs markedly between those obtained using the nitrate and hydroxide precursors, being short rods in the former and irregularly shaped in the latter. Nonetheless, cHA particles from both precursors are in the nano size range, being slightly smaller in size when the carbonate content (in the cHA lattice as well as due to CaCO 3 presence) is higher. The particle size of cHA particles synthesized using the nitrate precursor shows a decrease in size as the pH is increased, whilst there is no significant change in size with pH for the particles synthesized using the hydroxide precursor. Acknowledgements The authors gratefully acknowledge the financial support from Universiti Teknikal Malaysia Melaka and Universiti Sains Malaysia (FRGS - 6071285). References 1. Yoganand CP, Selvarajan V, Cannillo V, Sola A, Roumell E, Goudouri OM, Paraskevopoulos KM, Rouabhia M. Characterization and invitro bioactivity of natural hydroxyapatite-bioglass ceramics synthesized by thermal plasma processing. Ceramics International, 2010, 36, 1757-1766. 2. He M, Chang C, Peng N, Zhang L. Structure and properties of hydroxyapatite/cellulose nanocomposite films. Carbohydrate Polymers, 2012, 87, 2512-2518 3. Ganghoffer JF. A contribution to the mechanics and thermodynamics of surface growth: application to bone external remodelling. International Journal of Engineering Science, 2012, 50, 166-191 4. Chen F, Tang QL, Zhu YJ, Wang KW, Zhang ML, Zhai WY, Chang J. Hydroxyapatite nanorods/pol(yvinyl pyrolidone) composite nanofibers, arrays and three-dimensional fabrics: electrospun preparation and transformation to hydroxyapatite nanostructures. Acta Biomaterialia, 2010, 6, 3013-3020. 5. Yang GL, He FM, Hu JA, Wang XX, Zhao SF. Biomechanical comparison of biomimetically and electrochemically deposited hydroxyapatite-coated porous titanium implants. Journal of Oral and Maxillofacial Surgery, 2010, 68, 420-427.
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6. Bang LT, Othman R. Aging time and synthesis parameters of nanocrystalline single phase hydroxyapatite produced by a precipitation method. Ceramics – Silikáty, 2014, 58, 157-164 7. Jayabalan M, Shalumon KT, Mitha MK, Ganesan K, Epple M. Effect of hydroxyapatite on the biodegradation and biomechanical stability of polyester nanocomposites for orthopaedic applications. Acta Biomaterialia, 2010, 6, 763-775. 8. Kusrini E, Sontang M. Characterization of x-ray diffraction and electron spin resonance: effects of ssintering time and temperature on bovine hydroxyapatite. Radiation Physics and Chemistry, 2012, 81, 118-225. 9. Jia N, Li SM, Zhu JF, Ma MG, Xu F, Wang B, Sun RC. Microwave-assisted synthesis and characterisation of cellulose-carbonated hydroxyapatite nanocomposites. Materials Letters, 2010, 64, 2223-2225. 10. Bang LT, Tsuru K, Munar M, Ishikawa K, Othman R. Mechanical behaviour and cell response of PCL coate D-TCP foam for cancellous-type bone replacement. Ceramics International, 2013, 39, 5631-5637. 11. Raucci MG, Guarino V, Ambrosio L. Hybrid composite scaffolds prepared by sol-gel method for bone regeneration. Composites Science and Technology, 2010a, 70, 1861-1868. 12. Cicek G, Aksoy E.A, DUrucan C, HAsirci N, Alpha tricalcium phosphate TCP: solid state synthesis fom different calcium precursors and the hydraulic reactivity, J. of Materials Science: Materials in Medicine, April 2011, Vol. 22, 4, pp 809-817 13. Islam M, Mishra CP, Patel R. Physicochemical characterization of hydroxyapatite and its application towards removal of nitrate from water. Journal of Environmental Management, 2010, 91, 1883-1891. 14. Salimi MN, Bridson RH, Grover LM, Leeke GA. Effect of processing conditions on the formation of hydroxyapatite nanoparticles. Powder Technology, 2012, 218, 109-118. 15. Raucci MG, D’Anto V, Guarino V, Sardella E, Zeppetelli S, Favia P, Ambrosio L. Biomineralized porous composite scaffolds prepared by chemical synthesis for bone tissue regeneration. Acta Biomaterialia, 2010b, 6, 4090-4099. 16. Pieters LY, Van der Vrrken NMF, Declercq HA, Cornelissen MJ, Verbeeck RMH. Carbonated apatites obtained by the hydrolysis of monetite: Influence of carbonate content on adhesion and proliferation of MC3T3-E1 osteoblastic cells, Acta Biomaterialia, 2010, 6, 15611568. 17. Barinov SM, Rau JV, CEsaro SN, Durisin J, Ferro D, Trionfetti G, Carbonate release from carbonated hydroxyapatite in the wide temperature range, J. Mat. Sc. : Material Med (2006) 17, pp 597-604 18. Kalita SJ, Verma S. Nanocrystalline hydroxyapatite bioceramic using microwave radiation: synthesis snd chsracterization. Materials Science and Engineering C, 2010, 30, 295-303. 19. Sanoshi KP, Chu MC, Balakrisnan A, Lee YJ, Kim TN,Cho SJ. Synthesis of nano hydroxyapatite powder that simulate teeth particle morphology and composition. Current Applioed Physics, 2009, 9, 1459-1462. 20. Wu YS, Lee YH, Chang HC. Preparation and characteristics of nanosized carbonated apatite by urea addition with co-precipitation method. Materials Science and Engineering C, 2009, 29, 237-241.
ScienceDirect Procedia Chemistry 19 (2016) 539 – 545
5th International Conference on Recent Advances in Materials, Minerals and Environment (RAMM) & 2nd International Postgraduate Conference on Materials, Mineral and Polymer (MAMIP), 4-6 August 2015
Effect of calcium precursors and pH on the precipitation of carbonated hydroxyapatite Radzali Othmana,*, Zaleha Mustafaa, Chong Wee Loonb, Ahmad Fauzi Mohd Noorb a
Faculty of Manufacturing Engineering, Universiti Teknikal Malaysia Melaka, Hang Tuah Jaya, 76100 Durian Tunggal, Malacca, Malaysia b School of Materials & Mineral Resources Engineering, Universiti Sains Malaysia, Engineering Campus, 14300 Nibong Tebal, Penang, Malaysia
Abstract Three types of calcium precursors (nitrate, hydroxide and catbonate) were used in the synthesis of carbonated hydroxyapatite (cHA) using a precipitation method via a chemical reaction with di-ammonium hydrogen phosphate as the phosphate precursor. The precipitation method was chosen over many other methods due to its flexibility to changes in processing parameters to control the phases formed, the particle size, as well as, the morphology of the as-synthesized powders. The focus of the study was on cHA as it is deemed to mimic the composition of the human bone much closer as compared to the stoichiometric hydroxyapatite. When the chemical reaction was completed, the precipitate was dried, ground and characterized by x-ray diffraction (XRD), electron microscopy (both FESEM and TEM) and particle size analysis. Only the nitrate precursor produced a single-phase carbonated hydroxyapatite (cHA), whilst the other two precursors produced a secondary calcite phase or did not react fully. This is due to the low solubility of the calcium hydroxide and the incomplete reaction of the calcium carbonate. An increase in pH has been observed to lead to higher carbonate content in the synthesized cHA and a smaller crystallite size. © byby Elsevier B.V. This is an open access article under the CC BY-NC-ND license ©2016 2016The TheAuthors. Authors.Published Published Elsevier B.V. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia. Peer-review under responsibility of School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia Keywords: Hydroxyapatite; biocomposite; strength and toughness
* Corresponding author E-mail address:[email protected]
1. Introduction In recent decades, orthopaedic medication, such as bone repair and regeneration were rapidly studied to improve the health level in the society. Various artificial materials, such metallic, ceramic, polymeric and composite
1876-6196 © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia doi:10.1016/j.proche.2016.03.050
540
Radzali Othman et al. / Procedia Chemistry 19 (2016) 539 – 545
materials were used for orthopaedic repair and reconstruction 1. The materials used in orthopaedic application haveto be biocompatible, biodegradable and able to provide the function of tissue regeneration 2,3. Of all the choices of materials, calcium phosphates, especially hydroxyapatite (HA), is currently regarded as the best biomaterial to be used in bone repair and regeneration. This is due to the fact that natural bone is made up of an organic compound (collagen) as well as an inorganic compound (HA). The inorganic apatite compound offers osteoconductivity, osteoinductivity and a bone-bonding ability in the process of bone growth or bone healing 4-6. As HA is the main constituent of the bone, synthetic HA, with the chemical formula Ca10(PO4)6(OH)2, was introduced and widely applied in orthopaedic surgeries 7. Similarity in chemical composition to natural bone allows HA to form at the interface of the surrounding bone tissue, by mimicking the natural bone apatite phase 8. However, carbonated hydroxyapatite (cHA) was found to have a much closer compositional similarity to the mineral in natural bone. Carbonated hydroxyapatite (cHA) shows a higher bioactivity than HA, and the smaller particle size of cHA would inevitably bring about better tissue-implant interactions9,10. It has also been proven that nano-sized apatite particles, which have larger surface, lead to an enhancement in biological properties 11. The properties and reactivity of the apatite cement were found to have been influenced by both the calcium precursors and firing temperature12. They have found that identical synthesis history, morphology and crystallinity does not culminate in producing similar reactivity. Besides, the presence of impurities could hamper reactivity of the cement. Various synthesis methods had been introduced to produce HA or cHA, which included hydrothermal, sol-gel, precipitation, mechano-chemical, mechanical activation and other methods. Amongst these synthesis methods, the precipitation route is one of the most promising techniques to produce nano-sized apatite particles13,14. Morever, the precipitated particle sizes can be controlled by the synthesis temperature and pH conditions 15. It is the objective of this work to establish the processing parameters for cHA which have not been as intensively studied as HA. These would include the effects of using different calcium precursors as well as the effect of pH on the phase(s) formed, the changes in particle sizes and the particle morphologies as the pH is varied. 2. Experimental methods 2.1 Precursor materials Three different chemical grade calcium precursor materials were used, viz. calcium nitrate tetrahydrate, Ca(NO3)2.4H2O (Merck, ≥ 99.0% purity), calcium hydroxide, Ca(OH) 2 (Fluka, ≥96% purity), and calcium carbonate, CaCO3 (Sigma Aldrich, ≥99.0 purity). The calcium precursor was reacted dropwise with a phosphate precursor, viz. di-ammonium hydrogen phosphate, (NH4)2HPO4, (Fluka, ≥98.0 purity). In the reaction to produce cHA, sodium hydrogen carbonate (NaHCO3)was used as the source to supply CO32- whilst ammonium hydroxide (NH4OH) was used as a pH control for alkaline solutions. All solutions were prepared to an initial 1M concentration. 2.2 Flowchart for the synthesis of cHA Firstly, the solution containing the carbonate ions was added dropwise into the solution containing the phosphate ions at a rate of 40 drops per minute and continuous stirring. NH 4OH was also added dropwise to adjust the pH of the mixed solution. In this work, the pH used were 8.5, 9, 10 and 11. When all the carbonate-containing solution had been added, stirring was continued for another 30 minutes. The carbonate-phosphate mixture was then added dropwise (at a rate of 40 drops per minute) into the calcium ions solution whilst being continuously stirred. The colour of the mixed solution started to change slowly from transparent to milky white in colour. This change indicated that precipitation had taken place immediately when the dropwise process started. An additional 30 minutes of stirring was performed after the solutions were fully mixed to ensure complete reaction. The solution was then filtered using a Whatman 90mm filtration set with a 542 grade filter paper of average pore size 2.7 Pm. A vacuum pump was used to enhance the filtration rate. The white precipitate produced was washed three times with deionized water to remove any possible residue in the precipitate. The precipitate was finally dried in an oven at
Radzali Othman et al. / Procedia Chemistry 19 (2016) 539 – 545
541
100oC for 24 hours. The dried agglomerated powder was then ground with an agate pestle and mortar before being sieved through a 90 Pm sieve. The flowchart for the synthesis of cHA is shown in Figure 1. 2.3 Powder characterization The ground powders were then subsequently characterized using a number of techniques such as XRD (Bruker D8), FESEM (Zeiss Supra 55VP) and TEM (Philips CM12).
Fig. 1. Flowchart for the synthesis of cHA
3. Results and discussion 3.1 Phase analyses using XRD The phases formed upon using three different calcium precursors and four different pH at room temperature were determined by x-ray diffraction (XRD). The powders synthesized using the calcium carbonate precursor was found to exhibit mainly carbonate peaks and this is attributed to the reaction being unsuccessful using the parameters in this work. Henceforth, no other result was reported for this precursor. The phase analyses for the the calcium hydroxide and calcium nitrate precursors at four different pH are shown in Figures 2 and 3, respectively. In all the samples using the hydroxide precursor, calcite is observed as a secondary phase apart from the major cHA phase (Fig. 2). However, the intensity of the calcite phase is observed to decrease as the pH increases. This is attributed to the low solubility of Ca(OH) 2 in water which resulted in the CO32- from the mixture reacting with the remaining Ca(OH)2 in solution to form CaCO3. The presence of calcite CaCO3 was similarly reported by others17.
542
Radzali Othman et al. / Procedia Chemistry 19 (2016) 539 – 545 CaCO3 cHA
Fig. 2. Diffractograms of cHA powders synthesized using a calcium hydroxide precursor
On the other hand, upon using a calcium nitrate precursor, cHA is found to be the only phase formed at different pH (Fig.3). The results show that as the pH of the synthesis solution is increased, the peaks become more broadened, implying a decrease in crystallinity. The broadened peaks also reflect that the carbonate content in the cHA samples is increased with an increase in the synthesis pH 15. Some parts of the diffraction patterns also show broadened peaks where low intensity peaks are merged together. This indicates that the samples are partially amorphous and consist of nanosized cHA powders18. cHA
Fig. 3. Diffractograms of cHA powders synthesized using a calcium nitrate precursor
3.2 Morphology of particles The morphology of the cHA particles was first observed under the FESEM. However, the images of powders from both the nitrate-based and hydroxide-based precursors at four different pH values showed a heavily agglomerated structure, and hence not included for further discussion. Images observed under TEM (Figures 4 and 5) are foud to be more illustrative and are used for the measurement of size (in terms of length and width). From Fig. 4, it can be observed that as the synthesis pH increases, the size of the cHA paticle is decreased from about 30nm to 15nm. Consequently, the shapes of the particles appear to change from short rod-like to rice-like, indicating that the length of the particle has decreased. In this study, NH 4OH was used to adjust the synthesis pH, causing the concentration of OH- to increase. The high concentration of OH- could restrict the movement of Ca2+, PO43- and CO32- in the synthesis solution, thus the cHA nucleation process could be reduced, as reported in a previous
Radzali Othman et al. / Procedia Chemistry 19 (2016) 539 – 545
543
researchwork19. Hence, the growth of the particles is restricted and this results in smaller particles being formed when cHA is synthesized at higher pH using a nitrate-based precursor.
Fig. 4. TEM micrographs of cHA powders synthesized using a calcium nitrate precursor at different pH (a) 8.5; (b) 9.0; (c) 10.0 and (d) 11.0
The TEM images of the cHA particles synthesized using the hydroxide precursor are shown in Fig. 5. The shapes of the particles formed vary markedly from those produced using the nitrate precursor, i.e. the particles are irregularly shaped. Besides, the sizes of the particles range from 10nm to 25nm, regardless of the change in synthesis pH.
Fig. 5. TEM micrographs of cHA powders synthesized using a calcium hydroxide precursor at different pH (a) 8.5; (b) 9.0; (c) 10.0 and (d) 11.0
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Radzali Othman et al. / Procedia Chemistry 19 (2016) 539 – 545
3.3 Particle size analysis The average particle size of the cHA particles was measured from several TEM images for both nitrate- and hydroxide-derived cHA. The length and width of 40 particles were measured at various magnifications to obtain an average value and this was then computed to obtain the ratio of length to width (aspect ratio). The cHA’s derived from nitrate precursor show a decrease in aspect ratio (i.e. length) of the particles as the pH increases (Table 1). It had been suggested that a decrease in aspect ratio is caused by the higher substitution of carbonate into the phosphate site in the cell lattice20. On the other hand, there is no definite trend in the change of particle size for the hydroxide-based cHA. This is due to the fact that the hydroxide cHA also contained a calcite phase. The high carbonate content inhibited the growth of cHA particles, causing the particles to be smaller in size. Table 1. Aspect ratio (length/width) of cHA particles at different pH using 2 different precursors pH value / Precursor
Nitrate-based
Hydroxide-based
8.5
2.842
2.024
9.0
2.832
2.191
10.0
2.080
1.915
11.0
1.970
2.066
4. Conclusions It has been confirmed that a single-phase carbonated hydroxyapatite can only be successfully synthesized using a calcium nitrate tetrahydrate precursor as compared to two other precursors, viz. calcium hydroxide and calcium carbonate, under the conditions employed in this work. It has been observed that the morphology of the cHA particles differs markedly between those obtained using the nitrate and hydroxide precursors, being short rods in the former and irregularly shaped in the latter. Nonetheless, cHA particles from both precursors are in the nano size range, being slightly smaller in size when the carbonate content (in the cHA lattice as well as due to CaCO 3 presence) is higher. The particle size of cHA particles synthesized using the nitrate precursor shows a decrease in size as the pH is increased, whilst there is no significant change in size with pH for the particles synthesized using the hydroxide precursor. Acknowledgements The authors gratefully acknowledge the financial support from Universiti Teknikal Malaysia Melaka and Universiti Sains Malaysia (FRGS - 6071285). References 1. Yoganand CP, Selvarajan V, Cannillo V, Sola A, Roumell E, Goudouri OM, Paraskevopoulos KM, Rouabhia M. Characterization and invitro bioactivity of natural hydroxyapatite-bioglass ceramics synthesized by thermal plasma processing. Ceramics International, 2010, 36, 1757-1766. 2. He M, Chang C, Peng N, Zhang L. Structure and properties of hydroxyapatite/cellulose nanocomposite films. Carbohydrate Polymers, 2012, 87, 2512-2518 3. Ganghoffer JF. A contribution to the mechanics and thermodynamics of surface growth: application to bone external remodelling. International Journal of Engineering Science, 2012, 50, 166-191 4. Chen F, Tang QL, Zhu YJ, Wang KW, Zhang ML, Zhai WY, Chang J. Hydroxyapatite nanorods/pol(yvinyl pyrolidone) composite nanofibers, arrays and three-dimensional fabrics: electrospun preparation and transformation to hydroxyapatite nanostructures. Acta Biomaterialia, 2010, 6, 3013-3020. 5. Yang GL, He FM, Hu JA, Wang XX, Zhao SF. Biomechanical comparison of biomimetically and electrochemically deposited hydroxyapatite-coated porous titanium implants. Journal of Oral and Maxillofacial Surgery, 2010, 68, 420-427.
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