1. INTRODUCTION:

Hydroxyapatite, HA_P [Ca₁₀(Po₄)₆(OH)₂] is a typical bioactive material. Due to its excellent Osteoconductivity, biocompatibility, bioactivity, chemical, the biological similarity with the mineral constituents of human bones, teeth, it is used as bone cement, drug delivery, toothpaste additive and dental implants [1-6]. The size of HA_P crystal in natural human bone is in nano range. The constituent elements of HA_P are primarily calcium and phosphorus with a stoichiometric Ca/P ratio of 1.667.

HA_P is primarily composed of calcium, phosphorous and hydroxide ions that are eliminated at elevated temperatures. HA_P and other related calcium phosphate minerals have been utilized extensively as implant materials for a decade due to its excellent biocompatibility and bone bonding ability [7-9]. The porous structure of biomaterial allows the flow of extracellular fluid through the inner structure of biomedical devices. Hence it has adequate osteoconductivity, mechanical interlocking for firm fixation of material and enhances the adhesion between the natural and synthetic bone by the formation of an apatite layer [10,11]. The characteristics of HA_P are a high specific surface area, fine grain size distribution and small particle agglomeration depend on the method of preparing HA_P. For the preparation of HA_P different processes had been developed, including wet-chemical precipitation route [12-18], solid state reaction [19, 20] and hydrothermal methods etc. [21].

Among these processes, wet chemical co-precipitation technique is the most promising method because of its simple operations, low operating temperature and high yields of pure products [22]. During the reactions, the reaction media involves no foreign elements except water, the only by-product. For this reason, it is of great importance to developing inexpensive HA_P synthesis methods focused on the precise control of particle size, morphology and chemical composition [23-25]. However, it needs highly qualified and controlled parameters like pH of solutions, temperature, working atmosphere, the composition of the starting materials, reagent concentration, addition rate in reaction, stirring rate, maturation and presence of impurities to obtain HA monophase [26,27]. In this present study, we aimed to enrich the properties of wet-chemically prepared HA_P at different concentrations of the starting solution were refluxed and aging for a longer time to increase the quantity of HA.

2.MATERIALS AND METHODS:

2.1. Synthesis of pure HA_P:

In this study, three samples of nanohydroxyapatite were prepared by wet-chemical precipitation method using calcium chloride dihydrate (CaCl₂.2H₂O) and disodium hydrogen phosphate (Na₂HPO₄) (AR grade reagents Merck India) taken as the calcium and phosphorus precursors respectively and are dissolved separately in de-ionized water. The pH of both solutions was maintained at 11 by adding 1 M of NaOH drop by drop. The Na₂HPO₄ solution was added in dropwise to the CaCl₂.2H₂O solution while the solution was vigorously stirred and maintained at a temperature of 60˚C for 2h. After that, the same solution was further vigorously stirred for 1 h until to obtain white precipitation. The resulting precipitated was washed three times with hot water and then filtered. The filtered samples were dried at 100˚C for 12 h then milled by using mortar and pestle. The chemical reagent and starting materials concentrations are presented in table 1 and illustrated the preparation procedure in figure 1.The prepared samples are analysed by using XRD, FTIR, FESEM, EDX, and micro Raman.

Table 1. Chemical compositions of the prepared sample.

Sample	CaCl₂.2H₂O (mole)	Na₂HPO₄ (mole)
HA₁	0.5	0.3
HA₂	1.0	0.6
HA₃	1.5	0.9

X-ray diffraction (XRD) pattern was obtained at room temperature using the X-pert Pro PAN powder analytic (with Cu-Kα radiation, λ=1.54060A˚) at 40 KV and 30mA and the scan 2 (between 10˚ to 80˚. FTIR spectra were obtained by Magna 550 Ni colet IR spectrometer in the region 4000-400 cm^-1.

Fig. 1. A flow chart of HAp preparation using co-precipitation technique

The morphology of the synthesized HA_P-1, 2,3 nanopowders was examined by Field Emission Scanning electron microscopic (FE-SEM) analysis was carried out using Zeiss EVO 18-EDX special edition machine compatible with EDX machine. Micro Raman spectra of the samples were recorded at room temperature using a micro-Raman spectrometer at 514.5 nm (Princeton instrument Acton sp 2500).

3. RESULTS AND DISCUSSION:

3.1. X-ray diffraction (XRD) Analysis:

Figure 2 shows the XRD patterns of all as-prepared HAp samples through which phase identification was done and the obtained reflections are in good agreement with ICDD card no 74-0565 and none of the peaks appeared indicating that the samples are single phased HAp. All the diffractions of the samples are attributed to hydroxyapatite whose symmetry is hexagonal. From the XRD patterns, the crystallite size, strain, dislocation density were estimated for as-prepared samples, respectively [28-31] whose values are tabulated in table 2. The sharp and broad peak (211) in HA₂ indicates the better crystallinity compared to the other as-prepared samples. As the concentration increases the crystalline size increase the strain, dislocation density decreases.

Fig. 2. XRD patterns of as-prepared HA powders (HA₁) Ca/P = 0.5/0.3, (HA₂) Ca/P=1.0 /0.6 and (HA₃) Ca/P = 1.5/0.9.

Table 2. Crystalline size, dislocation density and strain for as-prepared HA nanoparticles

Sample	Crystalline size (nm)	Dislocation density x 10¹⁴ (lines/m²⁾	Strain x 10^-3 (lines^-2.m^-4)	d-spacing (Å)
HA₁	14.02	50.87	2.47	2.78
HA₂	23.62	17.92	1.46	2.80
HA₃	33.07	9.14	1.04	2.80

3.2 FTIR Analysis:

Fig.3. FTIR patterns of the HA_pprepared at various concentration ratios (Ca/P):(HA₁) 0.5/0.3, (HA₂) 1.0/0.6 and (HA₃) 1.5/0.9.

Functional groups associated with as-prepared hydroxyapatite powder were identified by FTIR spectroscopic analysis. All the samples exhibited the main vibration bands normally attributed to HA_P as shown in figure 3 [32]. The band between 565 and 605 cm^-1 belongs to triply degenerate O-P-O anti-symmetric bending mode in the phosphate group, which occupies two sites in the crystal lattice. As the crystallization was carried out in the alkaline range, the dissolving of atmospheric CO₂ yields CO₃^2- based on peaks from 1384 to 1423 cm^-1 and from 868 to 873 cm^-1. The band from 868 to 873 cm^-1indicates bending mode of CO₃^2- group and suggest a B type carbonate substitution. The asymmetric stretching mode of CO₃^2-group was also observed from 1384 to 1423 cm^-1 in the FTIR spectra. The peak at 1634cm^-1 is the signature of the bending mode of the hydroxyl group in the adsorbed water. The strong and broadband at 3441 is assigned to the intermolecular stretching vibration of the hydroxyl group in the crystal structure of HAp. The peak at 3441cm^-1 is assigned to a free O-H stretching mode of vibration due to surface water in the crystallites. Carbonate ions can substitute for either OH^- (or) PO₄^3- ions in the apatite structure. Since carbonates are constituents of bone structures, the presence of CO₃^2-may improve the bioactivity of HAp rather than being a cause of concern. These results are confirmed by previous reports of HA nanoparticles [33-35].

3.3. Micro Raman analysis

The micro Raman spectra of pure HAp powder at different concentrations are shown in figure 4. Whose corresponding frequency assignments are shown in table 4. All the examined samples exhibited the main vibration bands normally attributed to HA_P. The PO₄ (P-O)band at 961 is characteristic of HA_P and it is present in all the samples. This mode is associated with the totally symmetric stretching mode of the free tetrahedral phosphate ion [36]. The phosphate modes at 1123, 1056 and 994 cm^-1 are due to P-O asymmetric stretching. The intensity of the characteristic band 962 cm^-1 is very weak in HA₁ while moderate in HA₃ but it is very high in HA₂. So, among the three samples, HA₂ shows better crystallinity. This result has good agreement with the XRD result. The assignments are given in Table 4

Table 3. Frequency assignments of HA nanoparticles

Wave number (cm^-1)	Functional groups	Assignment	References
3441	O-H	Stretching mode	33
1634	O-H	Bending mode	35
1384	CO₃^2-	Asymmetric stretching mode	35
1032, 1058	PO₄^3-	Asymmetric stretching mode	35
868, 870	CO₃^2-	Bending mode	35
565, 602	PO₄^3-	Anti symmetric Bending mode	34

Table 4. Micro Raman frequency assignments of HA nanoparticle.

Wave number

(cm^-1)

Assignments

Reference

1123,1056, 994

Asymmetric stretching mode (ν₃) of the PO₄ group (P-O bond)

962

Symmetric stretching mode (ν₁) of the PO₄ group (P-O bond)

34, 36

Field emission scanning electron microscopy has been employed to ascertain the morphological analysis of the synthesized HA_P-1, 2,3 nanopowder. The grain size the synthesized HA_P is estimated to be about 30 nm for HA₁ and HA₃ samples but of HA₂ it is ranging from 30 to 50 nm. The uniform grain size with a narrow size distribution shows the good crystallinity. The formation mode of HA_Pparticles appears as spherical particles agglomerated together as in figure 5. The elements present in HA_P phase such as O, P and Ca were detected by EDX for the samples which indicate the purity of the resultants HA_P 1,2,3 nanopowder. The representative SEM image and EDX spectra are shown in figures 5& 6.

4. CONCLUSION:

Nano-HAp has been successfully synthesized using co-precipitation method. The formation of HAp nanoparticles was confirmed by XRD, FTIR and Micro Raman. The crystalline size of as-prepared nanoHAp ranges from 14 to 33 nm. As the concentration increases the crystallite size increases while the strain, dislocation density decreases. In XRD and micro Raman spectrum HA₂ has greater intensity than the remaining samples so we ascertained that (HA₂) [(Ca/P) 1.0/0.6]has bettercrystallinity. No characteristic peaks of impurities were observed, indicating that the product has high purity. So this technique is more suitable for laboratory purpose.

The vibrational frequencies were also found using FTIR and Raman spectroscopy is good agreement with previous reports.

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Received on 11.04.2018 Modified on 20.04.2018

Asian J. Research Chem. 2018; 11(3):545-550.

DOI:10.5958/0974-4150.2018.00097.4