Crystallinity of Hydroxyapatite Powders from Acid Washed and Thermally-Treated Tilapia Bones

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CMUJS Vol.21, No 1 (2017) 9-15


Bienvenido M. Butanas, Jr.
Materials Science Laboratory, Department of Physics, Central Mindanao University, University Town, Musuan, Maramag, 8710, Bukidnon, Philippines



This paper presents the production of quality hydroxyapatite (HAp) powders from tilapia bones using thermal treatment. Hydroxyapatite was successfully synthesized by calcination of 0.5M HCl pre-treated tilapia bones. Powder X-ray diffraction (XRD) revealed the nano-crystalline hexagonal structure of HAp powders. Peaks associated with impurities such as hydrates and carbonates decreased and vanished along with the coalescence of particles as calcination temperature increased. XRD results also showed better crystal formation at 800 and 900oC. The synthesized HAp powder in this study conformed well to the properties of biological apatites.

Keywords: Bioceramic, Biomaterial, HAp, Nano-crystalline material




Developing ceramic materials to replace broken bones, teeth, and hard tissues is one of the latest trends of research works by material scientists for the past two decades. There have been extensive studies on bioceramic materials (Park, Condrate, & Hoelzer 1998; Park et al., 1998), which reported that the most suitable material as a substitute for bones is the mineral hydroxyapatite (HAp) (Hench, 1998). It is chemically represented as Ca10(PO4)6(OH)2 and is the principal component of bones and teeth (Afshar, Ghorbani, Ehsani, Saeri, & Sorrel, 2003). This is mainly applied as a substitute material for hard tissue implantation due to its excellent biocompatibility, bioactivity, and osteoconductivity (Park et al., 1998; Park et al., 1998; Hench, 1998).

Hydroxyapatite has diverse applications in the following fields: chromatography (Morales, Burgues, Boix, Frails, & Clemente, 2000), solid state ionics (Aizawa, Hanazawa, Itatani, Howell, & Kishioka, 1999), catalyst (Sugiyama et al., 1999; Tanaka, Chikazawa, Kandori, & Ishikawa, 2000), drug delivery system (Panda, Ming-Fa, Chung, & Chin, 2001) and fuel cells (Gross, Berdt, Stephens, & Dinnebier, 1998; Verges et al., 2000) and has promising application as a chemical gas sensor (Gross et al., 1998; Verges et al., 2000; Nagar, Nishino, & Saeki, 1988; Mahabole, & Aiver, 2005). Because of these, various techniques have been developed to synthesize HAp bioceramic materials including hydrothermal processing (Yoshimura, Suda, Okamoto, & Ioku, 1994), microwave route (Vaidhayanathan & Rao, 1996), ultrasonic spray pyrolysis (Aizawa et al., 1999), precipitation routes (Ikoma & Yamazaki, (1999; Sugiyama et al., 1999), emulsion system (Furuzono, Walsh, Sato, Sonoda, & Tanaka, 2001), sonochemical synthesis (Kim & Saito, 2001) and sol-gel method (Jillavenkatesa & Condrate Sr, 1998). The main goal of these techniques is to produce Hap powders with high specific surface area and fine grain size with as little particle agglomeration as possible (Sung et al., 2004). Although promising, the use of these techniques in large-scale operations is still very costly (Vijayalaksmi &  Rajeswari, 2006), so there is a need to develop new techniques to synthesize HAp powder at significantly lower costs. Fortunately, there are promising results of HAp powder synthesis from eggshells (Sasikumar & Vijayaraghavan, 2006) and fish bones (Prabakaran & Rajeswari, 2006; 2006; Butanas, 2010; Butanas & Vequizo, 2013; Fara & Abdullah, 2015) using the thermal treatment method. Eggshells and fish bones are ideal starting materials. Eggshells are rich in calcium while fishbones naturally contain appatite-like substances (Currey, 2001). Previous studies (Prabakaran & Rajeswari, 2006; Butanas, 2010; Butanas & Vequizo, 2013; Fara & Abdullah, 2015) have shown that formation of HAp typically occurred at 900oC. Moreover, Butanas and Vequizo (2013) reported that pre-treatment by acid washing produced well-crystallized HAp as suggested by their scanning electron microscopy (SEM) photomicrographs. However, further characterization of the synthesized HAp powder is necessary for it to have any potential Osseo applications.

This paper presents the X-ray powder diffraction (XRD) characterization of HAp’s synthesized by acid pre-treatment with 0.5 M HCl and thermal treatment of tilapia bones. Tilapia ranks third among the most important fish in the world, in inland fisheries after carps with production reaching 723,000 tons in 2006 (FAO, 2009). The Philippines is very fortunate since it is considered as a major producer of marine and inland fisheries in the world (FAO, 2009) having tilapia as the second most cultured fish next to milkfish (BFAR, 2007). Making use of tilapia bones as a raw material is an advantage given the fact that the Philippines, specifically Bukidnon, has an abundant supply of it. Typically, these bones are dumped as a waste product during fish processing and food consumption thus allowing the researchers to produce high-quality HAp powders from a relatively cheaper source.




HAp Synthesis

The fish bones were collected during fish processing as a waste product at the central  market of Iligan City, Lanao del Norte, Philippines and then boiled in distilled water for 1 hour to soften the attached meat. The fish bones were then washed using strong flowing water from the faucet to remove the remaining fish meat. These were then washed with reagent grade 0.5M hydrochloric acid (HCl) (Merck, Philippines) to eliminate organic residues further. The bones were then thoroughly washed with ethanol and deionized water to remove residual HCl and other contaminants. After drying at 150oC for three hours the bones were crushed in a blender and calcined at various temperatures (i.e., 700oC, 800oC, 900oC and 1000oC) for 5 hours using a furnace (ThermolyneTM Industrial Benchtop Muffle Furnace, Thermo Fisher Scientific Inc., USA). The calcined powders were then ground to <50 µm using an agate mortar and pestle prior to the XRD analysis.

XRD Characterization

The crystal structure and crystallinity of HAp powders were determined using XRD. The X-ray diffraction patterns of samples calcined at various temperatures were obtained in the range 2θ = 5o-90o at a scan speed of 0.02o/ min. with an X-ray diffractometer (XRD-7000, Shimadzu Corporation, Japan) operating at 40kV and 30mA generating CuKα with a wavelength of 0.154 nm. For determination of diffraction line positions (2θ-values and d2θ values), the prepared samples were mixed with pre-ground and annealed ZnO powders (100oC) that serve as an internal standard for corrections. The obtained raw data were plotted. The plotted XRD patterns were corrected using the standards 2θ-values of ZnO then compared to Powders Diffraction Files of ZnO, PDF # 361451, and HAp (Marcovic, Fowler, & Tung,  2004).




Calcination Mass Loss

Figure 2 shows that there was a higher loss of mass that occurred as the calcination temperature increases. This trend was to be expected because of the more extensive desorption of water molecules and decomposition of organic materials in the raw bone sample at a higher temperature. At 700oC, about 35% of mass loss was observed. This was attributed to the decomposition of organic compounds of the raw bone structure (Ozawa & Suzuki, 2002) such as collagens, fats, etc. There was also mass loss due to removal of physisorbed and chemisorbed water (Marcovic et al., 2004).

A slight mass loss increase of about 1% was observed as the samples were calcined at 800o1000oC. This was probably due to the further removal of remaining traces of carbonates.

Figure 2: Average mass loss of tilapia bone powders calcined at 700oC-1000oC.


XRD Analyses

Figure 3 shows the XRD pattern of tilapia bone powder calcined at 700oC, 800oC, 900oC, and 1000oC. Notice that we only considered the peaks in 2θ = 5o – 60o since our standard reference material only uses this range. As shown in Figure 3, peaks that correspond to crystalline ZnO were easily identified through PDF # 36-1451. Around 2θ = 31.76o, overlapping of (211) plane associated with HAp and (100) plane of ZnO occurred. The (211) plane was the strongest peak for HAp as depicted by the broad peak around (100) ZnO plane.

Calcination had a strong effect on the tilapia bone powders at 7000C. More peaks associated with the crystal planes of HAp appeared (i.e., (100), (111), (102), (300), (310), (311), (222), (321), (410), (402), and (004)). The increasing crystallinity of HAp was apparent as the (002), (210), and (213) peaks became more intense and narrower. These results suggested that the powders are at a submicron level and consistent with the FTIR spectra and SEM photomicrographs obtained by Butanas and Vequizo (2013).

Tilapia powders calcined at 8000C exhibited a greater crystallinity of HAp crystals as suggested by the appearance of additional peaks (212), (302), (113), (312), (213), (501), and (420). The major peaks, i.e., (002), (211), (213), (222), (310), (004), (210), (321) and (312), (501), and (420). The major peaks, i.e., (002), (211), (213), (222), (310), (004), (210), (321), and (312), of HAp also became more distinct, narrower, and more intense compared with crystal formed at 700oC. Broadening becomes less, and this was likely due to decomposition of remaining traces of volatile material at 800oC. Peaks greater than 2θ = 60o were beyond the range of the HAp’s standard 2θ range and did not belong to ZnO peaks.

More distinct peaks of HAp were observed at 9000C. The crystallinity of calcined product at this temperature was less ambiguous than those synthesized at 700, 800, and 10000C, which indicates better HAp crystal formation at higher temperatures.

Calcination of tilapia bone powders at 10000C also formed crystalline HAp with negligible amounts of tricalcium phosphates (TCP). This confirmed the absence of the TCP absorption bands in the FTIR results of Butanas and Vequizo (2013).

Figure 3: XRD patterns of HAp samples calcined at temperatures 700oC, 800oC, 900oC and 1000oC.



Crystalline HAp powders were successfully synthesized from Tilapia bones using acid washing and thermal treatment. XRD results revealed that the best calcination temperature for the formation of well-crystallized HAp was around 800oC. As calcination temperature increased, tilapia bone powder continued to transform into a well-crystallized and crystalline HAp. Finally, the characterization results conform to the hexagonal structure of HAp as well as to the characteristics and composition of biological apatites (LeGeros, 1991).



The author would like to thank Central Mindanao University for the financial support through research fund R-030 of 2013 and likewise to the National Institute of Geological Science (NIGS) of UP-Diliman for the XRD characterization.



Afshar, A., Ghorbani, M., Ehsani, N., Saeri, M. R., & Sorrell, C. C. (2003). Some important factors in the wet precipitation process of hydroxyapatite. Materials and Design, 24,  197-202. doi:10.1016/S0261-3069(03)00003-7.

Aizawa, M., Hanazawa, T., Itatani, R., Howell, F. S., & Kishioka, A. J. (1999). Characterization of hydroxyapatite powders prepared by ultrasonic spray-pyrolisis technique. Journal of Materials Science, 34, 28652873. doi:10.1023/A:1004635418655.

Bureau of Fisheries and Aquatic Resources. (n.d.). Philippine fisheries profile 2007. Retrieved July, 2013, from http://www.bfar.

Butanas Jr., B. M., & Vequizo, R. (2013). Morphological and chemical properties of  biological apatite powders from 0.5 hydrochloric acid (HCL) pre-treated and thermally treated tilapia bones. Paper presented at the 35th Annual Scientific Meeting –National Academy of Science and Technology, Manila Hotel, Metro Manila,                          Philippines.

Butanas Jr., B. M. (2010). Structural and morphological properties of hydroxyapatite powders from thermally treated tilapia bones for biomedical applications (Unpublished master’s thesis). Mindanao State University Iligan Institute of Technology, Iligan City, Philippines.

Currey, J. (2001). Biomaterials: sacrificial bonds heal bone. Nature, 414, 699. DOI:10.1038/414699a.

Fara, A. N. K., & Abdullah, H. Z. (2015). Characterization of derived natural hydroxyapatite  (HAp) obtained from different types of tilapia fish bones and scales. AIP Conference Proceedings, 1669, 020077. doi:10.1063/1.4919215.

Food and Agriculture Organization of the United Nations. Food and Agriculture Organization Fisheries and Aquaculture Department. The State of World Fisheries and Aquacul ture 2008. Retrieved from

Furuzono, T., Walsh, D., Sato, K., Sonoda, K., & Tanaka, J. J. (2001). Effect of reaction temperature on the morphology and size of hydroxyapatite nanoparticles in an emulsion system. Journal of Materials Science                    Letters, 20, 111-114. DOI:10.1023/A:1006725931450.

Gross, K. A., Berdt, C. C., Stephens, P., & Dinnebier, R. (1998). Oxyapatite in hydroxyapatite coatings. Journal of Materials Science, 33, 39853991. DOI:10.1023/A:1004605014652.

Hench, L. (1998). Biomaterials: a forecast for the future. Biomaterials, 19, 1419-1423. doi:10.1016/S01429612(98)00133-1.

Ikoma, T., & Yamazaki, A. J. (1999). Preparation and structure refinement of monoclinic  hydroxyapatite. Journal of Solid State Chemistry, 144, 272-276. DOI:10.1006/ jssc.1998.8120.

Jillavenkatesa, A., & Condrate, Sr, R. A. (1998). Sol-gel processing of hydroxyapatite. Journal of Materials Science 33, 41114119.DOI:10.1023/A:1004436732282.

Kim, W., & Saito, F. (2001). Sonochemical synthesis of hydroxyapatite from H3PO4 solution with Ca(OH)2. Ultrasonics Sonochemistry, 8, 85-88. DOI:10.1016/S13504177(00)00034-1.

LeGeros, R. Z. (1991). Calcium phosphates in oral biology and medicine. Monographs in Oral Science, DOI:10.1159/isbn.978-3318-04021-0

Mahabole, M. P., Aiyer, R. C., Ramakrishna, C. V., Sreedhar, B., & Khairnar, R. S. (2005). Synthesis characterization, and gas sensing property of hydroxyapatite ceramic. Bulletin of Materials Science, 28 (6), 535545.

Marcovic, M., Fowler, B. O., & Tung, M. S. (2004). Preparation and comprehensive characterization of a calcium hydroxyapatite reference material. Journal Research of the National Institute of Standards and Technology, 109, 553-568. DOI:10.6028/ jres.109.042.

Morales, J. G., Burgues, J. J., Boix, T., Frails, J., & Clemente, R. R. (2000). Precipitation of stoichiometric hydroxyapatite by a continuous method. Crystal Research and Technology, 36, 15-26.

Nagar, M., Nishino, T., & Saeki, T. (1988). A new type of CO2 gas sensor comprising porous hydroxyapatite ceramics. Sensors and Actuators, 15, 145-151. DOI:10.1016/02506874(88)87004-5.

Ozawa, M., & Suzuki, S. (2002). Microstructural development of natural hydroxyapatite originated from fish-bone waste through heat treatment. Communications of the American Ceramic Society, 85, 1315-1317.

Panda, R. N., Ming-Fa, H., Chung, R. J., & Chin, T. S. (2001). X-ray diffractometry and x-ray photoelectron spectroscopy investigations of nanocrystalline hydroxyapatite synthesized by hydroxide gel technique. Japanese Journal of Applied Physics, 40, 5030. doi:10.1143/JJAP.40.5030.

Park, E., Condrate, Sr., R. A., & Lee, D. (1998). Infrared spectral investigation of plasma spray coated hydroxyapatite. Materials Letters, 36, 38-43. DOI:10.1016/420 S0167577X(97)00287-5.

Park,  E., Condrate Sr,  R. A. & Hoelzer, D. T. (1998). Interfacial characterization of plasma spray coated calcium phosphate on Ti-6Al-4V. Journal of Materials Science: Materials in Medicine, 9, 643-649. DOI:10.1023/A:1008931508465.

Prabakaran, K., & Rajeswari, S. (2006). Development of hydroxyapatite from natural fish bone through heat treatment. Trends Biomaterials Artificial Organs, 20, 20-23.

Sasikumar, S., & Vijayaraghavan, R. (2006). Low temperature synthesis of nanocrystalline hydroxyapatite from eggshells by combustion method. Trends Biomaterials Artificial Organs, 19, 70-73.

Sugiyama, S., Nakanishi, T., Ishimura, T., Morigi, T., Hayashi, H., Shigemoto, N., & Moffat, J. B. (1999). Preparation, characterization and thermal stability of lead hydroxyapatite. Journal of State of chemistry, 143, 296302. DOI:10.1006/jssc.1998.8126.

Sugiyama, S., Yasutomi, T., Moriga, T., Hayashi, M., & Moffat, J. B. (1999). Preparative enhancement of the thermal stability of calcium hydroxyapatite. Journal of Solid State Chemistry, 142, 319-324. DOI:10.1006/jssc.1998.8040.

Sung, Y., Lee, Y., & Yang, J. (2004). Crystalization and sintering characteristics of chemically precipitate hydroxyapatite nanopowder. Journal of Crystal Growth, 262, 467-472. DOI:10.1016/j.jcrysgro.2003.10.001.

Tanaka, H., Chikazawa, M., Kandori, K., & Ishikawa, T. (2000). Influence of thermal treatment on the structure of calcium hydroxyapatite. Physical Chemistry Chemical Physics, 2, 2647-2650. DOI:10.1039/ B001877P.

Vaidhayanathan, B., & Rao, K. J. (1996). Rapid microwave assisted synthesis of hydroxyapatite. Bulletin of Material Science, 19, 1163-1165.

Verges, M. A., Gonzalez, C. F., Gallego, M. Solier, J., Cachadina, I., & Matijevic, E. (2000).  A new route for the synthesis of calcium-deficient hydroxyapatites with low Ca/P ratio: Both spectroscopic and electric characterization. Journal of Materials Research, 15, 2526-2533. DOI:10.1557/ JMR.2000.0362.

Vijayalaksmi, U., & Rajeswari, S. (2006). Preparation and characterization of microcrystalline hydroxyapatite using sol-gel method. Trends Biomaterials Artificial Organs, 19, 57-62.

Yoshimura, M., Suda, H., Okamoto, K., & Ioku, K. J. (1994). Hydrothermal synthesis of biocompatible whiskers. Journal of Materials Science, 29, 3399-3402. DOI:10.1007/ BF00352039.



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