Synthesis and Characterization of Hydroxyapatite from Duck Eggshell by Wet Precipitation Process

Introduction

Hydroxyapatite, Ca10(PO4)6(OH)2, is the main inorganic compound present in hard tissues such as human bone. Consequently, it is readily considered as a bioactive material for artificial bone and teeth substitution because of its biocompatibility, osteoconductivity, chemical and biological affinity with human bone tissues and teeth. Furthermore, it is widely used in biomedical application as filler, coating on bone and dental implants.

Hydroxyapatite is derived from natural resources rich in calcium carbonate (CaCO3) like limestone [1], cockle shells [2], egg shells [3,4], animal bone [5] or coral [6,7]. In this study, it was synthesized from duck egg shells using precipitation method. The selection of duck egg shells as a source of calcium was due to its abundance in Indonesia. Buasri et al (2013) reported that the content of calcium in the form of calcium oxide (CaO) in duck egg shells was 98,925%.

There are quite number of methods that can be used to produce synthetic nanoparticle HAp such as : precipitation [8,9], hydrothermal [2,6], mechanochemical [10] and sol-gel [3,11]. The precipitation method was chosen in this study due to its simple and low-cost processing technique in industrial scale. This method generally produces particles in the nano-scale region and less than 100nm [12]. However, the hydroxyapatite obtained using this method contains considerable contaminant. Therefore, to solve this problem, HAp was synthesized from duck egg shells through the formation of Precipitated Calcium Carbonate (PCC) using modified carbonation method.

In our previous research, hydroxyapatite was synthesized from cockle shells and eggshells PCC’s using hydrothermal and sol gel methods [2,3]. PCC is a calcium carbonate compound (CaCO3), which is processed from natural resources containing calcium carbonate through a series of chemical reactions. Its particles are homogenous i.e. same size with micro-scale particles and have high purity (99.8%). Azis et al, [2] obtained a highly purified hydroxyapatite from PCC cockle shells without any other crystalline phase by using hydrothermal method. Meanwhile, synthesis of hydroxyapatite from PCC duck egg shells using precipitation method has never been previously reported.

Materials and methods Materials preparation

The duck egg shells (Figure 1) were collected from Pekanbaru, Indonesia. Other materials used include diammonium hydrogen phosphate ((NH4)2HPO4), (Merck), 2M nitric acid (HNO3), 65% ammonium hydroxide (NH4OH) (Merck), CO2 and aquadest. The duck egg shell samples were thoroughly washed, cleaned and air-dried for two days. Afterwards, they were crushed and grounded to fine powder consistency using a blender.

Figure 1. Duck Egg Shells

Process of forming precipitated calcium carbonate (PCC) from duck egg shells

The procedure for the formation of PCC from duck egg shells was carried out using the modified carbonation method, referred from Azis et al. [2]. PCC is the main material used in synthesizing hydroxyapatite via precipitation method. The reaction for the formation of PCC using modified carbonation method is represented by the following equations [13].

Calcinaton

\begin{equation} 2CaCO_{3}+Heat\rightarrow 2CaO+2CO_{2} \tag{1} \end{equation}

Hydration

\begin{equation} CaO+2HNO_{3}\rightarrow 2Ca\left ( NO_{3} \right )_{2}+H_{2}O \tag{2} \end{equation}

\begin{equation} Ca\left ( NO_{3} \right )_{2}+2NH_{4}O_{3}\rightarrow Ca\left ( OH \right )_{2}+2NH_{4}NO_{3} \tag{3} \end{equation}

Precipitation

\begin{equation} Ca\left ( OH \right )_{2}+CO_{2}\rightarrow CaCO_{3}+H_{2}O \tag{4} \end{equation}

Synthesis of hydroxyapatite from PCC duck egg shells using precipitation method

Because the solubility of PCC in water is very low, its usually dissolved in mineral acid. Therefore, in this study 5gram of PCC powder from duck egg shells were dissolved in 200ml of 0.3M HNO3 solution. Furthermore, 360ml of (NH4)2HPO4 solution was prepared by varying the molar ratio of Ca and P reactant by 1.67; 1.77 and 1.87. Afterwards, it was added in drops to the PCC solution at a rate of 6ml/min for 24h and stirred at 300rpm. The pH was monitored at 10-11 using 33% NH4OH. The precipitate object was stirred by using magnetic stirrer at 200, 250 and 300rpm for 24h at room temperature (27oC) and aged for 24h. Subsequently, it was filtered with a filter paper and repeatedly washed with aquadest until the pH of solution was 7. Afterwards, the precipitates were dried at 110oC for 24h and sintered at 500oC for 1h.

The chemical equations for the reaction are shown below:

\begin{equation} CaCO_{3(s)}\left ( PCC \right )+HNO_{3(aq)}\rightarrow Ca(NO_{3})_{2(aq)} \tag{5} \end{equation}

\begin{equation} 10Ca(NO_{3})_{2}4H_{2}O+6\left ( NH_{4} \right )_{2}HPO_{4}+8NH_{4}OH\rightarrow Ca_{10}(PO_{4})_{6}(OH)_{2}+20NH_{4}NO_{3}+20H_{2}O \tag{6} \end{equation}

Characterization

The hydroxyapatite powder was characterized using X-ray diffraction (X’Pert Powder DY 3688) with Cu Kα radiation. Meanwhile, the Fourier Transform Infrared Spectroscopy (FTIR, Perkin Elmer Spectrometer Frontier) was used to analyze the bonding structure of the samples. The surface morphology was probed using scanning electron microscopy (SEM) linked to energy dispersive X-ray microanalysis (EDX) (JEOL JED 2300). Furthermore, the surface area of the hydroxyapatite was characterized using Brunauer–Emmett–Teller (BET) measurements and Surface Area Analyzer (SAA, Quantachrome NovaWin2).

 Results and discussion

The FTIR spectrum of HAp powder (Figure 2 a,b,c) showed the sharpen bands of PO4-3 at 1025-1029 cm-1. According to Stanciu et al. [14], the sharpen peaks of PO4-3 implies that the crystallinity of the hydroxyapatite powder is good. Unfortunately, from Figure 2(a) and (b), it is seen that the broad bands at 3370 and 3379cm-1 were ascribed to the N-H asymmetric stretching mode [15]. However, it was assumed that O-H stretching mode corresponded to H2O at bands 3800-2500cm-1.

Figure 2. FTIR spectra of the HAp powder at ratio of Ca/P reactant 1.67 and stirring speed (a) 200, (b) 250, (c) 300 rpm

The variation of stirring speed in HAp synthesis did not have a significant influence on the XRD pattern of hydroxyapatite powder (Figure 3). The XRD pattern of synthesized hydroxyapatite powder was compared to the standard hydroxyapatite ICDD 01-074-4172 (Figure 3d). The intensity peak was observed at an angle of 2θ: 25.8o, 28.9o 31.9o, 32.2o, 32.9o, 33.9o and 39.7o, which was very close to the standard ICDD 01-074-4172. Furthermore, the XRD pattern of the synthesized hydroxyapatite powder (Figure 3a,b,c) did not contain any other crystalline phase.

Figure 3. XRD pattern of HAp powder at stirring speed a) 300, b) 250, c) 200 rpm, d) HAp standard ICDD 01-074-4172

The morphology and content of element present in the synthesized hydroxyapatite at molar ratio of Ca and P reactant : 1.67; 1.77 and 1.87 and stirring speed of 200rpm, were confirmed from the SEM micrograph data as shown in Figure 4.

Figure 4. SEM micrograph of hydroxyapatite powder at 200 rpm and the molar ratio of Ca and P reactant: a) 1.67 b) 1.77 and c) 1.87

The diameter of hydroxyapatite crystal was calculated using the Scherrer’s equation. The value obtained at the variation of Ca and P molar ratio for 200 rpm stirring speed, is shown at Table 1.

Table 1. The diameter of hydroxyapatite crystal for 200 rpm stirring speed

No Variable
Ca/P reactant Diameter of HAp crystal (nm)
1 1.67 23.086
2 1.77 23.062
3 1.87 40.356

The best result of the synthesized hydroxyapatite at stirring speed 200rpm and molar ratio of Ca and P reactant 1.77, has crystalline size about 23.062 nm. The measurement of elemental composition (Ca, P and microelement) are summarized in Table 2.

Table 2. Ca, P and microelement content and Ca and P ratio in HAp product

Element Measured content (wt %) Ca and P ratio HAp product
P 17.55 1.699
Ca 29.83
Al 0.83

The ratio molar of Ca and P hydroxyapatite was synthesized from PCC of duck egg shells was 1.699. The surface area of hydroxyapatite powder was characterized using BET analysis and the value obtained was 55.929m2/g.

Conclusion

In this study, hydroxyapatite nanoparticle powder was successfully synthesized from the PCC of duck egg shells using precipitation method. The variation of stirring speed did not have a significant influence on the synthesized hydroxyapatite. The best hydroxyapatite powder was synthesized at stirring speed 200rpm and molar ratio of Ca/P 1.77. The XRD pattern showed the high purity of hydroxyapatite and the nano hydroxyapatite crystalline size obtained was 23.062nm.

 Acknowledgements

The authors would like to acknowledge the Kemenristekdikti and Universitas Riau for their financial support under Dana Penelitian Berbasis Kompetensi DRPM and Dana DIPA UNRI.

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