Wet chemical synthesis of nanocrystalline hydroxyapatite flakes: effect of pH and sintering temperature on structural and morphological properties

Wet chemical synthesis of hydroxyapatite (HAp) nanostructures was carried out with different solution pH values (9, 10 and 11) and sintering temperatures (300°C, 500°C, 700°C and 900°C). The effects of pH and sintering temperature on the structural and morphological properties of nanocrystalline HAp powders were presented. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) analysis were performed to obtain the crystalline structure, chemical composition, morphology and particle size of the HAp powders. The TEM analysis is used in order to observe the rod- and flake-like HAp structures. XRD confirms the presence of both HAp hexagonal and monetite phases, although the monetite phase was less abundant in the resultant powders. Increase in pH reduced the monetite phase and enhanced Ca/P ratio from 1.7 to 1.83. Additionally, an increment in sintering temperature increased the crystallite size from 20 to 56 nm. The SEM analysis revealed the formation of semi-spherical and flake-like HAp structures with preferential flake morphology. An increase in pH and sintering temperature resulted in the growth and coalescence of crystals resulting in a porous capsular morphology. The FTIR analysis confirmed the reduction of carbonate stretching modes with an increase in pH and H–O–H antisymmetric stretching mode is eliminated for powders sintered at 900°C confirming the formation of stable and porous HAp powders.


Introduction
Currently, the biomaterial technologies are widely used in the healthcare industry, cell biology and drug delivery systems [1]. Furthermore, the research in this field is continually under development to meet the future needs of the market, mostly in bioimplants [2 -4], which makes the innovation of new biomaterial synthesis extremely important. One of the most relevant ceramic biomaterials from the calcium phosphate family is hydroxyapatite (HAp), which is widely used in the fabrication of implants due to its high biocompatibility with soft tissues, muscles and skin. Additionally, its similar composition to minerals in the human bones and teeth makes the union simple and effective with the bones in a natural way [5]. A great and dedicated effort has also been made to control the shape, grain size, chemical purity and crystalline degree of HAp through several alternative routes [6 -12].
One of the most used methods to obtain synthetic HAp is the 'conventional solid-state method', in which the particles obtained maintain the shape of the phosphate precursor particles [5]. On the other hand, the 'wet chemical method' is performed using aqueous solutions at relatively low temperatures like those in living beings [13], and the particles obtained by this method usually are porous and exhibit an inhomogeneous chemical composition [14,15]. But in the 'hydrolysis reaction' method, the chemical reaction of calcium phosphate with water is performed at 1008C [16]. Moreover, the 'hydrothermal method', which includes chemical reactions of aqueous solutions with the precursor materials, allows obtaining well-crystallized powders with homogeneous chemical composition [17][18][19][20][21][22][23]. Besides the works mentioned above, there are many other chemical synthesis methods for preparing micro-and nano-HAp structures [24][25][26][27][28], but the results show that there are still serious difficulties to control the particle size and crystalline degree of HAp. In addition, the final performance of the biomaterials is degraded by the secondary products of the chemical reactions and/or unreacted species during the synthesis. Physical methods to synthesize biomaterials, such as selective laser sintering, present similar difficulties to obtain a final product with desirable characteristics [29,30].
Recently, there has been a great demand for large quantities of inexpensive HAp nanoparticles with well-defined morphology and crystalline phase for diverse commercial applications such as threedimensional printing. In this regard, the wet chemical precipitation technique has various advantages such as simplicity, low reaction time and temperatures, high purity and cost-effectiveness. Principally, scalability and reproducibility using wet chemical process are potential [24,27,31].
The best way to obtain a synthetic biomaterial with desirable characteristics is to keep control of the synthesis parameters like precursor concentration, sintering time, temperature, acidity (or pH) and volume of the solvent. In general, improvement of the bioactivity and biocompatibility is related to a smaller HAp particle size with a stoichiometric Ca/P ratio (approx. 1.6). This can be achieved by varying pH and the sintering process which subsequently control the shape and size distribution of the resulting biomaterials [18,20,32,33]. In this work, the wet precipitation method is used to obtain homogeneous and porous HAp powders with nano-and micrometre structures with ammonium hydroxide as precipitation agent by varying pH and the sintering temperature. A detailed discussion about the change in morphology, chemical structure (functional groups), Ca/P ratio and crystallite size by using combined precipitation-sintering method is presented.

Synthesis of hydroxyapatite powders
All the chemicals used in this work were purchased from Sigma Aldrich with high purity (greater than 99%

Materials' characterization
The morphological characterization of the HAp powders was performed using low-vacuum scanning electron microscopy (SEM, JEOL JSM5900-LV) equipped with an Oxford energy-dispersive X-ray spectroscope (EDS), operated at 20 kV. The X-ray diffraction analysis was carried out by a Siemens D5000 diffractometer powder technique in the range of 208 -608 in 2u (8) to identify the crystalline phases. To identify the functional groups in HAp samples, an infrared spectroscopy analysis was performed using Nicolet Nexus 670 FTIR operated at room conditions in the range of 4000-400 cm 21 .

Results
3.1. Scanning electron microscopy and energy-dispersive X-ray spectroscopy analysis SEM was employed to obtain the morphological characteristics of the HAp powders. Figure 1 shows the SEM images of the HAp powders sintered at 3008C, 5008C, 7008C and 9008C at pH values 9 (figure 1a-d), 10 (figure 1e-h) and 11 (figure 1i-l). pH = 9 pH = 10 pH = 11 SEM micrographs show an important influence of the sintering temperature on the morphology of HAp. Increase in sintering temperature changed completely the HAp morphology from semi-spherical nanoparticles to flake-like structures. As the sintering temperature increases, the evaporation of the residues makes the morphology as an interconnected flake-like porous network (figure 1d,h,l). Figures 2 and 3 show the particle size histograms and the variation of particle size with respect to the sintering temperature. From figures 3 and 4, it is evident that the increase in the sintering temperature induced the growth of the particle size from tens of nanometres to some micrometres for all pH values. This may be due to the diffusion of the particles at higher temperatures and formation of flake-like structures with interconnections [35,36].
Apparent influence of the pH on the shape of the HAp agglomerates is revealed by a careful comparison of figure 1a,e,i, in which semi-spherical particles ranging from 30 to 50 nm, rod-like structures approximately 40 -80 nm and flake-like structures approximately 80-150 nm are observed. Also, for pH 10, the formation of interconnections started at lower temperatures of approximately 5008C (figure 1f ), whereas for pH 9 and 11, the formation of interconnections started at 7008C (figure 1c,k). In order to calculate the Ca/P ratios, the EDS analysis of all the samples was performed and figure 4a-c shows the EDS spectra of HAp powders calcinated at 9008C for pH 9, 10 and 11. The Ca/P ratios obtained for all samples using the EDS analysis were from 1.66 to 1.83 and are presented in table 1. For being used in bioactivity, a pure stoichiometric HAp powder Ca/P ratio should be 1.67 [37,38] but only the samples synthesized at pH 10 possess the Ca/P ratio close to the stoichiometric HAp. The detailed growth mechanism is explained in §3.5 relating XRD and FTIR results.

Transmission electron microscopy analysis
Transmission electron microscopy (TEM) was employed to observe the rod-like structures that were observed in the SEM analysis for HAp powders calcinated at 3008C. Figure 5 shows the TEM images of the HAp powders sintered at 3008C and at pH 11. Detailed TEM analysis was impossible due to the agglomeration of the HAp structures. From figure 5, it is observed that the powders contain a mixture of semi-spherical, rod-and flake-like morphological structures. The rod-like morphology is predominant compared to remaining structures. Owing to the overlapping of the structures, it is difficult to analyse the exact structure of the rods, but a minimum diameter of approximately 20 nm and a minimum length of approximately 40 nm were observed.

X-ray diffraction analysis
The XRD patterns of as-prepared and sintered HAp powders obtained at pH 9, 10 and 11 are shown in figure 6a-c, respectively. A glance at the XRD spectra shows the formation of two crystalline phases of calcium phosphates: hydroxyapatite (HAp) and monetite (CaHPO 4 ) with a preferential orientation in (211) plane. The main diffraction peaks in this pattern match with two JCPDS cards: no. 46-0905 and no. 09-0080, corresponding to HAp and monetite, respectively [13,39] The crystallite size for HAp was calculated from X-ray diffractogram of strong reflections with intensity % by measuring the full width at half maximum (FWHM). The Scherrer equation for calculating the crystallite size is given as follows: where K is the Scherrer constant, l is the wavelength of X-ray for diffraction, b hkl is the FWHM of the corresponding plane and u hkl is the angle measured of the same plane [13,40]. The constant K in the above formula generally takes the value 0.9, assuming the shape of crystallites. According to the Scherrer equation, the crystallite size obtained for HAp was in the range of 20-56 nm ( figure 7). After comparing all the XRD patterns of figure 6, it is observed that all the diffraction peaks were more narrow with the increase in the sintering temperature. This implies that the samples improved their crystallinity and crystallite size (figure 7) due to the reduction in strain and defect density of the crystals. The presence of CaHPO 4 was observed only for samples sintered at 7008C and 9008C, and it is due to the increase in the number of chemical reactions between the residual species [41], which resulted in agglomeration and formation of new compounds [42,43].  Figure 2. Histograms of the obtained particle size distribution of the HAp powders sintered at 3008C, 5008C, 7008C and 9008C with pH 9 (a-d), 10 (e-h) and 11 (i-l).
rsos.royalsocietypublishing.org R. Soc. open sci. 5: 180962 By comparing figures 6 and 7, it is observed that for the samples with pH 10, the crystallite sizes were higher for samples sintered at 5008C, 7008C and 9008C. It is possible to note that the intense peak at 32.928 in 2u, which is associated with (300) plane for HAp is lower. As explained in §3.1, release of Ca 2þ , PO 4 32 and OH 2 ions restrict the growth in (300) plane [44].
We believe that the presence of adequate OH 2 ions for the pH 10 resulted in optimum Ca/P ratio (table 1) which subsequently improved the HAp plane, for example (211) and (300), and reduced the monetite peak intensities compared to other pH values. Therefore, from XRD and SEM analyses, it can be reasoned out that the pH 10 is optimum to obtain more pure HAp with desirable crystallite size and interconnected flake morphology for biomedical applications [45 -48]. All the above-mentioned phosphate bands are observed at all pH values, but these bands become stronger and narrow with the increasing sintering temperature, which confirms the growth of phosphate ions and crystallization of HAp at higher temperatures.

Fourier transform infrared spectroscopy analysis
Additionally, two pronounced peaks in the IR spectra at 629 and 3570 cm 21 were identified and associated with the symmetric stretching mode of hydroxyl group. The hydroxyl stretching modes at 629 and 3570 cm 21 become available continuously with the increase in temperature from room temperature to 9008C, showing that HAp was obtained from the beginning; sintering temperature only modified the crystalline degree of HAp, according to XRD patterns ( §3.3). The OH 2 band at 3570 cm 21 shows a weak intensity for samples at pH 9 and goes on improving from samples at pH 10 and 11, which makes it clear that the amount of OH 2 ions present is increasing for higher pH values; this result is in good agreement with the SEM and XRD results. As the pH of the solution increases, the presence of the OH 2 ions also increases and this leads to the growth in desired planes like (300)  bands at 1321 and 1417 cm 21 are present for all pH values. An ideal and pure HAp structure should not present any carbonate vibrational modes, but its presence at 1321 and 1417 cm 21 may be due to its atmospheric adsorption after the synthesis, which is unavoidable in chemical synthesis techniques, whereas the presence of carbonate band at 873 could be associated with the synthesis product but it diminishes by increasing the pH. Increasing of the sintering temperature also diminishes the presence of the carbonate band intensities, which is in good agreement with XRD and SEM results. The broad envelope observed in all HAp powders in the IR spectra at 3464 cm 21 is assigned to n 1 -stretching mode of water (H 2 O) molecule and its intensity decreases with the increase in sintering temperature, and this peak is eliminated at 9008C, which indicates the evaporation of the solvents during the sintering process. Also, two bands at 1630 and 1650 cm 21 correspond to n 2 bending modes of H-O -H in H 2 O lattice ( figure 8 and table 2). At pH value 9 (figure 8a), the 1650 cm 21 band appears only for as-prepared samples and sintered for 3008C and 5008C. But, for samples sintered at 7008C and 9008C, 1650 cm 21 band vanishes and a band at 1630 cm 21 appears. A contrary behaviour is observed for samples with pH values 10 and 11 (figure 8b,c). This transition of water molecule bending modes at temperature 7008C and at pH 10 resulted in optimum Ca/P values (table 1)    samples with pH 9 (figure 8a) and at 2390 cm 21 for samples with pH 10 and 11 and are attributed to the C-H stretching modes originated due to the presence of the monetite phase, which is also confirmed in the XRD analysis. We believe that the presence of monetite peaks is corroborated by the partial decomposition of the HAp phase due to its sintering in air [51,52].

Discussion
As the solution pH is increased by the addition of NH 4 OH, the Ca 2þ , (PO 4 ) 32 and OH 2 ions are liberated and the OH 2 ions are situated on the facets of the formed nuclei [44,53]. Later, the growth occurs only on the planes without OH 2 ions on their surface. For pH 9, semi-spherical particle with deviated Ca/P ratio is obtained (figure 1a) and when the pH is increased to 10, OH 2 ion concentration also increases on the facets, which restricts the growth in that facet and allows the growth in the facets without OH 2 ions, which is the preferential orientation. This resulted in the flake-like structures with adequate Ca/P ratio (marked in red circles in figure 1e).
Furthermore, with the increase in the pH to 11, Ca/P ratio increases and restricts the growth due to the presence of more OH 2 ions, which resulted in rod-like structure (marked with blue circles in figure 1i). Therefore, increased pH restricted the growth in some planes and resulted in flake-and rod-like HAp nanostructures. Additionally, increasing pH value improved the solubility which subsequently fastened the precipitation and agglomeration of particles [54][55][56]. Thus, pH is an important parameter to acquire the desired morphology. Figure 9 shows the morphological transformation of synthesized HAp particles with respect to the pH and sintering temperature. It is evident from figure 9 that the increase in pH affected the HAp morphology, whereas increase in the sintering temperature resulted in coalescence of particles.
Another key factor that is affected due to the change in pH and sintering temperature is Ca/P ratio (amount of calcium and phosphate phases) of HAp. For a stoichiometric HAp powder, Ca/P ratio is 1.67, which is observed for samples prepared with pH 10 and sintered at 3008C and 5008C. But XRD confirms a mixture of both HAp and monetite phases, which is like commercially available HAp with identical XRD spectrum of stoichiometric HAp (Ca/P ¼ 1.67) [51,[57][58][59][60]. We believe that the presence of adequate OH 2 ions for the pH 10 resulted in optimum Ca/P ratio (  improved the HAp plane, for example (211) and (300), and reduced the monetite peak intensities compared to other pH values. Therefore, based on FTIR, SEM and XRD analyses, we can say that the pH and the sintering temperature play an important role on the purity, morphology and the crystallinity of the HAp structures and also it can be reasoned out that the pH 10 is optimum to obtain relatively stoichiometric HAp with desirable crystallite size and interconnected flake morphology for biomedical applications [45 -48]. As we mentioned previously in the Introduction section, HAp obtained in this work can be used as an implantation material, to repair injured bones and teeth. However, before being used as biocompatible material, the HAp must have satisfied requirements associated with the physicochemical characteristics such as shape, size, chemical purity and crystalline degree. Therefore, our methodology is relevant to obtain HAp with size-and shape-controlled nanostructures by varying pH or sintering temperature which are adequate for medical uses. Additionally, these results open the possibility of applying this methodology to synthesize HAp for other different applications. For example, non-medical applications of HAp include packing media for column chromatography, gas sensors, catalyst and host materials; where each application requires different and specific characteristics of HAp.

Conclusion
Wet precipitation synthesis of HAp powders was successfully performed, and the results obtained indicated that the morphological, crystalline and chemical characteristics of the HAp have a strong dependency on synthesis parameters, mainly the pH value during the synthesis and the sintering temperature. For samples at pH 9, spherical particles around 30 -50 nm were observed. At pH 10, HAp flakes approximately 150 nm were identified and at pH 11 resulted in a combination of HAp rods, particle and flakes. Furthermore, the sintering temperature plays a crucial role in the morphology of the HAp nanoparticles. Irrespective of the pH values, as the sintering temperature increases, an effect of densification was found which results in the gathering of those nanostructures synthesized at room temperature and resulted in a flake-like interconnected HAp grain which is an indication of coarsening of the HAp particle size. The TEM analysis confirms the formation of rod-and flake-like HAp structures. XRD analysis revealed the formation of HAp and monetite phases with crystallite size ranging from 20 to 56 nm. It was noted that an increment of the pH leads to a decrease in the monetite and carbonate products, but also a widened Ca/P ratio with an optimum for pH 10. The FTIR analysis showed the presence of the stretching and bending vibrational modes of carbonate, phosphate, water molecule and hydroxyl ion groups. The FTIR analysis also demonstrates the effect of pH by the removal of carbonate vibrational modes resulting in pure HAp phase powders. From the obtained results, we can conclude that an appropriate combination of pH and sintering temperature allow controlling the morphology, crystallinity and chemical characteristics of the HAp for a specific application as biomaterial.
Data accessibility. All the data files of XRD spectra, crystallite size and FTIR spectra reported in this article have been uploaded as part of the electronic supplementary material.