Effect of physical and chemical parameters on the β-Tricalcium phosphate synthesized by the wet chemical method

In this paper, the synthesis of nanosized powders β -Tricalcium phosphate β -Ca3(PO4)2 was studied by the wet chemical method at different values of reaction temperature, initial concentration of diammonium hydrogen phosphate (NH4)2HPO4, calcium nitrate tetrahydrate Ca(NO3)2·4H2O addition rate, pH of reaction and aging time. All these parameters have a great impact on the properties of the resulting β-TCP nano powders. Analysis results of morphology, structure of TCP powder from infrared (IR) spectra, X-ray diffraction (XRD) and transmission electron microscopy (TEM) indicated that the synthesized TCP powder had spherical crystal shape with crystallite size, calculated by XRD method, less than 60 nm, mono and biphasic structure composed of βTCP with pyrophosphate calcium or hydroxyapatite. The variation of the synthesis conditions did not affect the morphology, but it affect the size of crystallites and particles.


Introduction
Tricalcium phosphate (TCP) is one of the variations of the calcium phosphate compounds with more applications in bone tissue regeneration [1][2][3] , due to its chemical composition Ca3(PO4)2 being similar to the natural bone tissue 4 .TCP is widely used in the biomedical field because of excellent biocompatibility, high bioactivity, non-toxicity and non-inflammatory behaviour and non-immunogenic properties.It can exist in two possible forms, α and β, and normally β form gets converted into α with annealing at temperatures higher than 1250-1300°C 5 .The β-TCP is bioresorbable, and bioresorption occurs through osteoclastic activity 6,7 .It has good biodegradability and higher dissolution rate in the body's environment after implantation, which is absorbed and replaced by new bone 8 .Many methods are used to synthesize of such biomaterials including wet-chemical method 9,10 , solid-state process 11,12 and microwave irradiation 13,14 , sol-gel, etc 15,16 .The most conventional is the precipitation in aqueous medium starting from Ca(NO3)2 and (NH4)2HPO4 as raw materials.It is noteworthy that β-TCP cannot be precipitated from solution, but only be prepared by calcination, e.g. of Apatitic tricalcium phosphate Ca9(HPO4)(PO4)5(OH) at temperatures over 750°C 17 .9 Ca(NO3)2+ 6 (NH4)2HPO4 + 6 NH4OH → Ca9(HPO4)(PO4)5OH + 18 NH4NO3 +5 H2O Ca9(HPO4)(PO4)5OH → 3 Ca3(PO4)2 + H2O However, the synthesis of a pure β -TCP by this method requires close control of many parameters such as reaction pH, temperature, the stoichiometry of the raw materials, ageing time.A slight variation of these experimental parameters can generate drastic variations in the composition of the final product 18 and reveal the pyrophosphate calcium phase (Ca2P2O7 or CPP) or the hydroxyapatite phase (Ca10(PO4)6(OH)2 or HAp).These conditions remain specific and should be controlled by synthesis preparation parameters 13,19 .Therefore, it is crucial for biomedical applications to control some parameters of TCP particles such as crystallite size, morphology and phase composition.Bioactivity of calcium phosphate materials depends on many factors during the synthesis procedure including precursor reagents, impurity contents, crystal size and morphology, concentration and mixture order of reagents, pH and temperature.Such conditions are application specific and should be controlled by synthesis preparation parameters 13,19 .Therefore, controlling crystallite size, morphology and phase composition of TCP is very important for biomedical applications.
In this paper, we present some results of β -TCP nanopowder synthesized by the wet chemical method and the effects of synthetic conditions, i.e. reaction temperature, ageing time, pH of mixture solution, (NH4)2HPO4 addition rate and reactant concentration on crystallite size, fraction of crystallinity and morphology.

Materials and Methods
Tricalcium phosphate was synthesized by the wet chemical method using (NH4)2HPO4 solution at the various (NH4)2HPO4 (Riedel-de Haën, Germany) concentrations 0.15, 0.20, 0.25M in distilled water.The Ca(NO3)2•4H2O (Scharlau, Spain) concentration was set at 0.36M to obtain the Ca/P molar ratio of 1.50.Its solution was added dropwise into (NH4)2HPO4 solution with addition rate of 3, 30 and 300 ml min -1 under a constant stirring condition.During the synthesizing process, the pH value varies between 7 and 9.The pH of the solution was adjusted by the addition of ammonium hydroxide NH4OH.The reaction was conducted at different temperatures (30, 40, 50, 60 and 70°C).This precipitated solution was stirred for 2 h and aged at room temperature (RT) for different times (2, 24, 48 and 72h).The precipitate was filtered and washed repeatedly using distilled water to remove NH4 + and NO3 -ions.The resultant precipitate was dried at 70° C for 24 h in a dry oven and then crushed in a mortar.The powders were calcined at 800°C for 1 h.
The phase purity and crystallinity of the TCP powder were analyzed by X-ray diffraction (XRD) (XPERT-PROPW3050/60 (θ/θ) using CuKα radiation λ = 1.54056Å and operating at 45 kV and 40 mA, step angle of 0.03° and 2θ in range of 15-60°.Crystalline phases detected in the patterns were identified by comparison to the standard patterns from the ICDD-PDF (International Center for Diffraction Data-Powder Diffraction Files).The crystallite dimensions (D) were calculated using Debye-Scherrer Eq. (1): Where D is the crystallite size (nm), λ the wavelength of X-ray beam (0.15406 nm for Cu-Kα radiation), FWHM the full width at half maximum for the diffraction peak under consideration (rad), and θ is the diffraction angle (°).The crystallinity noted by Xc corresponds to the fraction of crystalline β-TCP phase in the investigated volume of powdered sample, evaluated by the Eq (2): Where I0210 is the intensity of (0 2 10) reflection of β-TCP structure and V300/0210 is the intensity of the hollow between (3 0 0) and (0 2 10) reflections 20,21 .
The characteristic functional groups of β-TCP were identified by Fourier transform infrared (FTIR) spectroscopy, VERTEX 70, Genesis Series (400-4000 cm-1, resolution 4, scans 20).For this 1% of the powder was mixed and ground with 99% KBr and the spectrum was taken in the range of 400 to 4000 cm -1 .
The size and morphology of fine TCP powder were observed on a transmission electron microscope (Philips CM10, Eindhoven, The Netherlands) that operated at the acceleration voltage of 100 kV.

Results and Discussion
The reaction temperature was an important factor that could affect the morphology, the phase structure and the crystallinity of the synthesized TCP powder.(Fig. 1) and Table 1 show the FTIR spectra and the bonds of the functional groups of the TCP powder synthesized using Ca(NO3)2.4H2Oand (NH4)2HPO4 solution at the different reaction temperatures, with (NH4)2HPO4 concentration of 0.15M and at an addition rate of about 300 ml/min, at an aging time of 2 h and at a pH of about 9. The IR spectra of all synthesized samples showed the characteristic peaks corresponding to β-TCP 22 .The bands at 1043 and 1078 cm -1 corresponded to the triple degenerate ν3 antisymmetric stretching vibration of PO4 3-.968 cm -1 band was assigned to ν1, the symmetric stretching vibration of PO4 3-.The bands at 607 and 559 cm -1 corresponded to the triple degenerate ν4 antisymmetric stretching mode.The peak and the broad band of the single molecule of adsorbed water were also discerned at 1633 and between 3700 and 3100 cm -1 respectively.Moreover, the FTIR had shown the presence, at 729 and 1218 cm -1 , of P2O7 4- group, which is characteristic to the calcium pyrophosphate phase β-Ca2P2O7 (JCPDF 9-346).With the temperature rising, these bands become narrower and low then gradually disappear.The TCP crystal diameter calculated from the Scherrer equation showed that the crystal diameter and degree of crystallinity increased from 20 to 59 nm and from 93 to 99 on increasing the reaction temperature from 30 to 70°C, respectively Table 2. Increasing the temperature results in faster motion of molecules, so there was an increased chance of therm colliding with each other; the TCP particles concentrated to form larger particles.At 30°C, the TCP particle had the smallest diameter.It has been found that the control of the crystallinity of calcium phosphates is necessary for their biological applications 23 .Since calcium phosphates with lowlevel of crystallinity show high osteoconductivity, the synthesized powders can be used to promote osseointegration or used as a coating to promote bone in growth into prosthetic implants 24 .

Effect of reactant concentration
By varying the precursor concentration, the kinetics of TCP precursor synthesis can be affected.By increasing the precursor concentration, the solubility limit at a given pH is more rapidly exceeded, creating a burst of primary nuclei for crystal growth.However, as the reactants are continually added, the primary nuclei continue to grow rapidly high precursor concentrations resulted in larger crystallite and particle sizes.(Fig. 3) presents the IR spectra of the TCP powder obtained at various initial concentrations of (NH4)2HPO4 solution at room temperature, at an addition rate of about 300 ml/min, at an ageing time of 48 h and at a pH of about 9. It could be found that the IR spectra of the TCP samples had the same shapes and the characteristic peaks of the functional groups of TCP are shown.The very weak band of the secondary phase, P2O7 at 728 cm -1 , gradually disappeared during the decrease in the concentration of (NH4)2HPO4 solution from 0.25 to 0.15.   3 introduce XRD spectra and XRDcalculated crystal diameters and crystallinity, respectively.In general, XRD patterns of the TCP samples had the same shapes and had only the characteristics peaks of the TCP molecule.The insignificant amount of Ca2P2O7 detected by FTIR in the samples synthesized with 0.25 and 0.2M is undetectable by XRD.For powder prepared at 0.15 M, besides the β-TCP, an additional small peak is detected at 2θ = 31.8°such peak corresponds to Hap (JCPDS 9-0432) phase.The crystal diameter was in a range from 40 to 51 nm.XRD analysis also shows a weak effect of reacting concentration (in the abovementioned range) on the TCP crystal diameter.

Effect of Ca(NO3)2 addition rate
The adding rate of Ca(NO3)2 affects the morphology, structure and size of formed TCP crystals.By varying the precursor addition rate, nucleation and crystal growth rates can be controlled.Rapid addition of precursors results in localized high concentrations of precursors, exceeding the solubility of TCP in those regions, which favors nucleation and formation of small crystals 25 .(Fig. 4) presents the IR spectra of the samples synthesized with the dropping rates of 3, 30 and 300 ml.min -1 at RT, at an ageing time of 48 h and at a pH of about 9.All IR spectra of the samples have similar shapes and the specific peaks corresponding to functional groups in the β-TCP molecule.The bands at 725 and 1200 cm -1 indicated the presence of P2O7 4-groups.4 introduce XRD spectra and XRD-calculated crystal diameters and crystallinity, respectively.XRD patterns of the β-TCP samples had the same shapes and had all the characteristics peaks of the molecule.The diffractograms of the samples prepared with a precursor addition rate of 30 ml/min and 3 ml/min show additional peaks relative to the group P2O7.From this table, it was found that when Ca(NO3)2 adding rate increased the crystal diameter decreases and crystallinity increases.To confirm the XRD results, the morphology of TCP powder was further analyzed by TEM images.(Fig. 7) and Table 4 present the TEM images and TEM-based calculated average particle sizes.TCP particle had a spherical shape and highly agglomerated.It has been found that nanoparticles with spherical morphology are better than other irregular morphologies due to the good space fillings and the low percentage of voids in the final product 26 .However, when Ca(NO3)2 adding rate increased, particle size decreased.These results allowed concluding that the slow addition of precursors results in a regime favouring crystal growth and formation of larger particles.

Effect of pH of the reaction
Two parameters govern which phase will form for a given calcium phosphate: the initial calcium to phosphate ratio of the reactants and the pH at which the reaction occurs.We synthesized TCP powder by dropping slowly Ca(NO3)2.4H2Osolutioninto (NH4)2HPO4 solution at room temperature with various pH 7, 8 and 9 at an ageing time of 24 h.The IR spectra of TCP powder synthesized at three pH values are shown in (Fig. 8).By comparing these IR results with data in Table 1, it was found that the obtained TCP had all the characteristic peaks of TCP.The very weak bands of the secondary phase, Ca2P2O7 at 727 and 1213 cm-1 appear in the sample synthesized with pH=7.Also, the XRD results indicated the characteristic diffraction peaks of TCP, and no other phases of calcium phosphate were detected (Fig. 9) except for the spectrum of the sample synthesized with pH=7 shows additional peak characteristic of the β-Ca2P2O7 phase.The data in Table 5 shows that the degree of crystallinity increase and become stable at pH = 8, XRD analyses also demonstrate a weak effect of pH (in the above-mentioned range) on the TCP crystal diameter, when the ph increased from 7 to 9 the crystallite size did not increase significantly, only from 40 to 43 nm.(Fig. 10) and Table 5 show the TEM images and average particle size of TCP calculated from the TEM images, respectively.The pH did not affect the morphology of TCP powder.As for the TEM results, it could be found that the TCP crystals had a spherical shape.

Effect of ageing time
The crystallinity and structural development of TCP is also affected by varying the ageing time of the precipitate.Longer ageing times ensure that reagents are fully reacted and precipitate out of the solution.The IR spectra of TCP samples prepared at 25°C, pH = 9 with various ageing times 2, 24, 48 and 72 hours are shown in (Fig. 11).By comparing these IR results with data in Table 1, it was found that the spectrums are similar to that of β-TCP, also, at 727, 1190 and 1213 cm -1 , we can observe the presence of P2O7 4- group.
The XRD spectra of TCP powder prepared at different ageing times are represented in (Fig. 12).The characteristic diffraction peaks of TCP are indicated.The results showed that TCP synthesized at the different ageing time had the main crystalline phase present.The powder 72h presents a slight shift in XRD peaks towards lower theta value; this may be due to the elongation in the crystal structure.Also, a minor amount of pyrophosphate was also found.However, the crystallite size and the degree of crystallinity increase with ageing time and become stable after 48h for both (Table 6) The TEM images of TCP powder prepared at ageing times 2h and 72 h are presented in (Fig. 13), At different ageing times, all obtained TCP had almost spherical and strongly agglomerated particles.The data in Table 6 shows that the particle size slightly increased with increasing ageing time.

Conclusion
Nano-sized β -Tricalcium phosphate powder was synthesized by the wet chemical method, by varying process parameter, followed by calcination, using calcium nitrate, diammonium phosphate solution as reactants, and ammonia as adjusting agent.The obtained results showed that the synthetic conditions were important in controlling the quality, shape and size of the TCP powder, but had significant effects on morphology.The calcined powders show the presence of two distinguishable crystalline phases β -Tricalcium phosphate and β -pyrophosphate or hydroxyapatite, spherical shape, crystallite size less than 60nm and a high degree of crystallinity.All the results obtained allow determining the optimal conditions for synthesizing pure nanosized β-TCP with a high degree of crystallinity and a crystalline size of less than 50 nm.Indeed, a synthesis temperature of 50 °C, a dropping rate of 300ml.min - , ageing time of 48h and a pH of about 9 are considered as the best synthesis conditions.

Figure 1 .
Figure 1.IR spectra of TCP powder synthesized at different temperatures.

Figure 2 .
Figure 2. XRD spectra of TCP powder synthesized at different temperatures.The XRD spectra of the TCP powder synthesized at the various temperatures are shown in (Fig.2).All XRD patterns show diffraction peaks characteristics of β-Tricalcium phosphate presents in standards and in literature.The major phase, as expected, is β-TCP, which is confirmed by comparing data obtained with the ICDD PDF 009-0169.Powders exhibited sharp

Figure 3 .
Figure 3. IR spectra of the TCP samples synthesized at different initial concentrations of (NH4)2HPO4 solution

Figure 4 .
Figure 4. IR spectra of the TCP samples synthesized at different initial concentrations of (NH4)2HPO4 solution

Figure 8 .Figure 9 .
Figure 8. IR spectra of TCP powder synthesized at different pH values

Figure 11 .Figure 12 .
Figure 11.IR spectra of TCP powder synthesized at different ageing times

Table 1 .
Wave numbers for the functional groups of TCP.

Table 2 .
The crystal diameter and crystallinity of TCP powder synthesized at different temperatures.

Table 3 .
The crystal diameter and crystallinity of TCP powder synthesized at various (NH4)2HPO4 concentrations.

Table 4 .
The crystal diameter, crystallinity and average particle size of TCP powder synthesized at different Ca(NO3)2•4H2O addition rates.

Table 5 .
The crystal diameter, crystallinity and average particle size of TCP powder synthesized at different pH values.

Table 6 .
The crystal diameter, crystallinity and average particle size of TCP powder synthesized at the different ageing time.