Facile Chemical Processing of Geloina coaxans Shell and Sodium dihydrogen Phosphate as Precursors to Produce Hydroxyapatite

This paper reports the synthesis of hydroxyapatite from Geloina coaxans shell and sodium dihydrogen phosphate as precursors by facile and simple method. Firstly, Geloina coaxans shell calcined at 1000 o C for 12 hours to produce lime or CaO. The chemical composition of Geloina coaxans shell calcined was analyzed using the XRF technique, and the result shows that the main component of Geloina coaxans shell calcined was calcium. Synthesis of hydroxyapatite has been done with stirring and aging time parameters and molar ratio of precursors was 1,67. The XRD patterns showed a specific peak of hydroxyapatite that appeared at 2θ=31.8 o and based on the XRD results on the aging time indicated that the rate of growth of HAp crystals was better and faster at aging for 12 hours. The crystal size of HAp obtained was 37 nm. The FTIR spectra of hydroxyapatite showed characteristics of hydroxyl and phosphate bands in sample spectra. The morphology of hydroxyapatite synthesized also was examined by SEM and the results showed spherical morphology.


INTRODUCTION
Hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ], or HAp is one of calcium phosphate with ratio Ca/P 1.67. It's a bioactive and biocompatible bioceramic (Hui et al., 2010) that having similar minerals phase in the natural bone. This biomaterial has many applications that cause many works in these research fields. The specific physical and chemical properties such as crystalline size, particle size, and morphology of HAp nanoparticles be a major topic and play a key role in several applications as adsorbent, catalyst (Bouka et al., 2016), drug delivery, and implant application. Further, it is important to note that crystalline size, particle size, surface area, and morphology of HAp nanoparticles play a key role.
Synthesis of hydroxyapatite can be conducted with several methods and types of precursors. Recently, the optimization of natural resources has attracted attention due to several reasons, such as abundant materials, non-toxic, easy to process with simple processing, and good results. Natural resources such as limestone, mollusk shells, animal bone have been converted as starting material on synthesis biomaterial hydroxyapatite. Many of wastes of Mollusk shell have been processed as calcium precursor on synthesis hydroxyapatite. Shavandi et al., (2014) has synthesized nano-hydroxyapatite from mussel shell with microwave method, oyster shells also have processed to produce hydroxyapatite with the wet method (Rujitanapanich et al., 2014), Macoma balthica (Felsen et al., 2015), and abalone shell (Chen et al., 2015) This paper presents, utilization of mollusk shell Geloina coaxans shell from Riau Province as source calcium combined with NaH 2 PO 4 to produce synthetic nanohydroxyapatite. NaH 2 PO 4 is one of phosphate source can be used on synthesis HAp. The coprecipitation method was used due to simple, easy, and can be done at room temperature. High yields, low energy consumption, homogenous mixing, and nanophase particle size control was also a reason to choose this method. Reaction parameters such as pH, concentration, physical parameters like stirring time, and aging time very influence the results of the sample synthesized. In this work, stirring time and aging time were reaction parameters chosen to learn the crystallinity of hydroxyapatite that synthesis at pH reaction was 11.

Synthesis Procedure
Sample Geloina coaxans shell or GCS was calcined at 1000 °C for 12 hours to produce lime. The calcined GCS was treated using DDW to produce 1 M of Ca(OH) 2 . The next step, NaH 2 PO 4 or sodium dihydrogen phosphate solution 0.6 M was added dropwise to Ca (OH) 2 . pH was adjusted at 11 with NH 4 OH solution and the stirring rate was adjusted at 300 rpm for one hour. The obtained suspension was kept for 24 hours at room temperature. The next step, obtained suspension was filtered and dried for 2 hours at 105 °C. The prepared powders were calcined for 1 hour at 900 o C. The same procedures were also done for stirring time 30, 120, 240, and 300 minutes and aging time for 12, 36, and 48 hours. Synthesized samples were characterized using XRD, FTIR, and SEM respectively.

RESULTS AND DISCUSSION
Calcination is the thermal decomposition of minerals to produce its metal oxide below of it's melting point. Geloina coaxans shell was calcined at 1000 o C for 12 hours to produce lime or CaO. Data of XRD analysis were collected in the 2 with range 5 o to 80 o using Cu-K radiation ( = 1.5406 Å). XRD analysis was done to learn the mineral phases of the sample. The obtained data were compared to standard patterns or JCPDS. The crystallite sizes were calculated by using the Debye Scherrer equation. The result of XRD is shown in Figure. 1.
Based on Figure 1, the calcination process of Geloina coaxans shell after sintering 1000 o C for 12 hours has produced lime or CaO. The XRD pattern has shown a specific peak of lime. The highest peak appeared at 2 = 37,5 o compared to JCPDS (No. 37-1497), other peaks of lime also seen at 2 = 54.0 o ; 32.3 o ; 67.4 o , and 64.3 o .The low peaks at 34.2 o and 18.1 o has also proved appearance another phase portlandite or Ca (OH) 2 in minor percentage. These peaks are suitable with JCPDS (No 44-148). These minerals can be caused by interaction of lime with water vapor. This phenomenon is related to the hygroscopic properties and less stability of lime to room temperature and pressure, that can react spontaneously at air moisture. Previous research, Zhong et al. (2017) calcined abalone shell at 1000 o C while mussel shell calcined to produce lime at 1000 o C for 3 hours (Edralin et al., 2017). Rujitanapanich et al. (2014) also converted oyster shells to lime with several calcination temperatures at 1000, 1100, and 1200 o C for 2 hours, and concluded that sintering at 1200 o C for 2 hours have produced lime successfully. Yoosuk et al. (2010) also explained CaCO 3 can be decomposed at 800 o C to produce lime.
The chemical compositions analysis of Geloina coaxans shell calcined was also done by using the XRF technique. The XRF analysis was conducted to estimate the qualitative and quantitatively chemical composition of GCS calcined. The results of the XRF analysis can be seen in Table 1.
The result show that the major component of Geloina coaxans shell calcined was calcium oxides and also consist of several elements. The high component of CaO is related to the presence of calcium carbonate, (CaCO 3 ) which is the major component of the GCS confirmed by the XRD results and there are no characteristic peaks of CaCO 3 . The high intensity and narrow peak of XRD results proved the decomposition has occurred athough there is another phase portlandite seen with minor percentage. Previous research, the CaCO 3 of Mussel shell was also converted to pure calcium oxide after calcined at 900 o C. The results proved by measured with XRD technique (Shavandi et al., 2014)

Synthesis of Hydroxyapatite
Precipitation method synthesis is the simple and frequently used on synthesis hydroxyapatite or HAp. The peak of the XRD pattern shown the result of the chemical reaction of calcium and phosphate precursors at pH reaction was 11 at several of stirring time. The pH and micro-environment of synthesis are usually carried out at alkaline pH. The common pH is 10. The chemical processing of precursors phosphate or PO 4 and calcium or Ca is a very influenced crystallinity of hydroxyapatite. Several reaction parameters also influence the formation of hydroxyapatite.
Crystallinity and purity of hydroxyapatite synthesized are variable. In the first analysis, the prepared sample was analyzed by XRD to learn the chemical phase, crystallinity, and purity. The analysis was carried out by using monochromatic Cu-K radiation (= 1.154060 nm). The XRD result of synthesized hydroxyapatite was shown in Figures 2 and 3 for stirring time and aging time respectively. The XRD pattern in Figure 2 shows the effect of stirring time on synthesis hydroxyapatite at 30, 60, 120, 240, and 360 minutes. All of the samples describe the specific peaks of hydroxyapatite and have similar trends but with different intensities.  . represents of XRD pattern of hydroxyapatite synthesized by aging time at 12, 24, 36, and 48 hours. Aging time was another factor influence the formation of hydroxyapatite. The XRD result of aging time for 12 hours shows the intensity is higher than others and revealed a characteristic peak of HAp at 2θ = 31.7º are suitable with JCPDS (No 09-0432) and others peak at 2θ = 32.8 o ; 49.4 o ; 34.0º; and 25.8º. The percentage of HAp produced was 95,83 %. Based on the XRD pattern, another chemical phase of calcium phosphate also detected with minor intensity. β-TCP (β-tricalcium phosphate) phases appeared at 2θ = 21.7 o and 28.8 o , correspond to JCPDS of β-TCP (No 09-0169). The percentage of crystallinity aging time for 24, 36, and 48 hours was 95.07; 94.90; and 94,91%. These results explain that the percentage of hydroxyapatite decreased with increasing aging time, and the results indicate that the rate of growth of HA crystals was better and faster at aging for 12 hours. The data and observations proved the increase of the aging time led to the smaller crystallite size. Bang and Othman (2014) explained the aging time for 24 hours was an excellent condition to obtain thermal stability of hydroxyapatite which is calcined at 1200°C for one hour.
The mean crystallite size of the sample was calculated by Debye Schererr formula in Equation below In this formula K is the Schererr constant which is taken as 0.89,  is the wavelength of Cu-Ka, while is for the fullwidth at half maximum (FWHM) and is stands for diffraction angle . The average the crystallite size at stirring time for 60 minutes and aging time for 24 hours was 37 nm while hydroxyapatite prepared for aging time 12 hours was 27 nm. Abidi and Murtaza (2014) found the crystallite size of HAp powder around 8.47 -24.47 nm.
Reaction of this process shown below: NaH 2 PO 4 (s)+ H 2 O(l) NaH 2 PO 4 (aq) 10Ca(OH) 2 (aq)+6NaH 2 PO 4 (aq)+NH 4 OH(aq) Ca 10 (PO 4 ) 6 (OH) 2 (s) + NH 4 OH(l) + 6 NaOH(l) (3) FTIR spectroscopy was examined to learn functional groups of hydroxyapatite and support the data obtained with the XRD instrument. The results of FTIR analysis can be seen in (Figure 4 and Table 2). The result of FTIR at stirring time for 60 minutes is shown in Figure 4. Phosphate group appeared at 570, 962, 1041, and 1084 cm -1 while the vibration of the hydroxyl group assigned with a peak at 3642 cm -1 . Mohandes et al., (2014) explained that the vibration of 557-568 cm −1 are attributed to the bending vibrations of phosphate groups. Candidato et al. (2015) also illustrated the appearance of phosphates at 567 and 1036 cm -1 , while the hydroxyl band at 3570 cm -1 . he band modes of hydroxyl group were also detected at 3567 and 3420 cm -1 respectively (Chen et al., 2015;Swain and Sarkar, 2011). Additionally, the band at 1400 cm -1 was assigned to the vibration of CO 3 2-. The vibration is caused by the reaction process without a vacuum condition. Edralin et al., (2017); Sasikumar (2010) also detected carbonate group at 1417 cm -1 and 1500 cm -1 .   Figure 5. Previous research Swain and Sarkar (2011) also demonstrates the spherical powders of prepared hydroxyapatite after aging one hour.
Morphology of particle can influence the activity and application of hydroxyapatite. Previous research has shown that shapes of hydroxyapatite play crucial roles in several applications, such as adsorbent or drug delivery. The shape of particle hydroxyapatite can influence the activity of cells.
Recently, the usability of HA nanoparticles (n-HA) against several type of cancer cells was reported. Hydroxyapatite (HAp) powder with various morphologies has been successfully synthesized and examined their influence on human colon cancer HCT116 cell inhibition. The results showed that the inhibition of cellular activity reached 80 percent of nanoHAp in the form of rods. The data revealed the morphology of hydroxyapatite play crucial role in on cellular activities (Dey et al., 2014).

CONCLUSION
In this research, physical parameters such as stirring time and aging time can influence the crystallinity and purity of hydroxyapatite synthesized. The results showed that both reaction parameters are effective in increasing the reaction rate on the formation of hydroxyapatite powder. Based on

Functional Group
Wavenumber ( the XRD results on the aging time indicated that the rate of growth of HA crystals was better and faster at aging for 12 hours. The data and observations also indicated the shorter the aging time led to the smaller crystallite size was 37 nm. Functional group analysis with FTIR instrument proved the specific band of hydroxyapatite appeared at a characteristic peak of hydroxyapatite while SEM analysis showed agglomeration and quite uniform sphericalparticles.